here - IPB

Transcription

here - IPB

Annual
meeting
of
the
International
Honey
Commission
Book of abstracts of the
II International Symposium on Bee Products
Annual meeting of the International Honey Commission
September 9-12, 2012
School of Agriculture
Polytechnic Institute of Bragança
Bragança
Portugal
Edited by
Miguel Vilas-Boas
Luís Guimarães Dias
Luís Miguel Moreira
Bragança, Portugal, September 9-12, 2012
1
Annual
meeting
of
the
International
Honey
Commission
Title:
II International Symposium on Bee Products. Annual Meeting of IHC: book of abstracts
Editors:
Miguel Vilas-Boas, Luís Guimarães Dias, Luís Miguel Moreira
Photography:
Luís Miguel Moreira (Associação de Apicultores do Parque de Montesinho)
Helena Guedes (Associação de Apicultores do Parque de Montesinho)
Published by:
Instituto Politécnico de Bragança
Printed by:
Tipografia – Artegráfica Brigantina
Number of copies:
150
Cover Design:
Atilano Suarez, Serviços de Imagem do Instituto Politécnico de Bragança
Date:
September 2012
Legal deposit:
347901/12
ISBN:
987-972-745-140-1
2
Annual
meeting
of
the
International
Honey
Commission
Organizers
Federação Nacional
dos Apicultores
de Portugal
In collaboration with
Supporters
3
Sponsors
Annual
meeting
of
the
International
Honey
Commission
PLATINUM
O Mel do Parque de Montesinho de
cor escura, sabor intenso e rico em
sais minerais, é em grande parte o
resultado da vegetação, dominada
pelo Castanheiro, Carvalho negral e
por vastas áreas de urzes, carquejas,
rosmaninhos e estevas.
É nesta região do Nordeste
Transmontano, entre paisagens
naturais únicas, onde o lobo e as
suas presas silvestres, a majestosa
águia-real e muitas outras espécies
selvagens ocorrem, que é produzido
o Mel do Parque de Montesinho.
O modo de produção biológico
garante que as características do mel
se mantêm inalteradas, chegando
ao consumidor um produto impar,
provavelmente muito semelhante
àquele que se obtinha nos primórdios
desta actividade milenar.
The dark Honey of Parque
Montesinho, with intense flavor and
rich in minerals, is largely the result of
vegetation dominated by chestnut,
pyrenean oak and vast areas of
heather, gorse, lavandula and cistus.
It is in this region of “Nordeste
Transmontano“,
among
unique
natural landscapes, where the wolf
and their wild prey, the majestic royal
eagle and many other wildlife species
occur, which is produced the Honey
of Park Montesinho.
The organic production method
ensures that the characteristics of
honey remain unchanged, reaching
the consumer a unique product,
probably very similar to that obtained
in the early days of this ancient
activity.
Le Miel du Parc de Montesinho de
couleur foncé, gôut intensif et riche
en sais minéraux, est en grande partie
le résultat de la végétation dominée
par le châtaignier et par le chêne
negrais, et par d´énormes aires de
forêts de bruyères.
C’est dans cette région du Nordeste
Transmontano,
entre
paysages
naturels úniques où le loup et ses
prises sylvestres , le majestieux aigleroyal et plusieurs espèces sauvages
surgissent, où est produit le Miel du
Parc de Montesinho.
Le mode de production biologique
garantie que les caractéristiques
du miel se mantiennent inaltérées
arrivant au consommateur un produit
unique, très semblable à celui qui
sóbtenait dans les origines de cette
activitée millénaire.
Agrupamento de Produtores do "Mel do Parque", Lda
273 329
Casa
do Mel, Quinta das Fontaínhas
Agrupamento
de Produtores do+351
"Mel
do470
Parque", Lda
Apartado 231
+351 273 329 471
5301-903 Bragança — Portugal
+351 273 329 470
do Mel, Quinta das Fontaínhas [email protected]
Casa
4
Apartado 231
5301-903 Bragança — Portugal
+351 273 329 471
[email protected]
Annual
meeting
of
the
International
Honey
Sponsors
Commission
GOLD
658 Vet Registered Ad landscape_Layout 1 10/07/2012 10:35 Page 1
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Vita (Europe) Limited, Vita House, London Street,
Basingstoke, Hampshire, RG21 7PG, United Kingdom.
BRONZE
5
Sponsors
Annual
meeting
of
the
International
Honey
Commission
BRONZE
6
Annual
meeting
of
the
International
Honey
Commission
Welcome message
Gudrun Beckh
President of IHC
At the last IHC-meeting in Buenos Aires 2011, Miguel
Vilas-Boas invited the IHC-members to Portugal for the
annual IHC-meeting 2012.
I would like to thank him and all the involved people
from the organizing Committee, Isabel C.F.R. Ferreira,
Luís Guimarães Dias and Maria Alice Pinto, for all the
efforts and work they prepared to set up our meeting
together with an International Symposium on Bee
products. I also thank the organising Institutions in
Portugal (CIMO, IPB and FNAP) for their support.
As chair of the Symposium, I welcome all researchers
from different countries participating in this event.
I am looking forward to learn about latest scientific
findings and to face experience exchange between all
the people working on bee products worldwide.
7
Annual
meeting
of
the
International
Honey
Commission
Chairman
Gudrun Beckh, President of IHC, Quality Services International GmbH, Germany
Organizing Committee
Miguel Vilas-Boas (Co-ordinator), CIMO & IPB, Portugal
Isabel C.F.R. Ferreira, CIMO & IPB, Portugal
Luís Guimarães Dias, CIMO & IPB, Portugal
Maria Alice Pinto, CIMO & IPB, Portugal
Scientific Committee
Andreas Thrasyvoulou, Aristotle University of Thessanloniki, Greece
Anne Claire Martel, French Agency for Food Environmental and Occupational
health & safety, France
Gian Luigi Marcazzan, Honeybee and Silkworm Research Unit - CRA-API, Italy
Herve Casabianca, CNRS – Analytique Central Services, France
Jaume Cambra, University of Barcelona, Spain
Ligia Muridian, University of São Paulo, Brasil
Lutz Elflein, Intertek Food Services GmbH, Germany
Maria Campos, University of Coimbra, Portugal
Peter Gallmann, Swiss Bee Research Centre, Switzerland
Stefan Bogdanov, Bee Product Science, Switzerland
Teresa Sancho, University of Burgos, Spain
Vassya Bankova, Bulgarian Academy of Sciences, Bulgaria
Vikas Nanda, Sant Longowal Institute of Engineering and Technology, India
Werner v.d. Ohe, LAVES -Institut für Bienenkunde Celle, Germany
Volunteers
Soraia Falcão
Andreia Tomás
Joana Coelho
Mário João Gomes
Maria João Sousa
Hugo Froufe
8
Annual
meeting
of
the
International
Honey
Commission
International Honey Commission
History
The starting point of International Honey Commission (IHC) was in 1990 when a
European Honey Commission headed by Stefan Bogdnov, Switzerland, was formed in
order to harmonise existing standard methodology for honey analysis and international
standards within Europe. Already in 1995, 28 honey specialists from 13 countries were
working in this group. With the involvement of more researchers and experts from all
over the world, the group’s name was changed into International Honey Commission.
The chairman of IHC was Werner von der Ohe, Germany, from 1996 to 2010.
As first result from a collaborative study, the special issue of Apidologie “ Harmonised
methods of the European Honey Commission” was published in 1997. New honey
standards within the European Union and the Codex Alimentarius were worked out,
based on the proposals of the IHC.
The following subject of IHC focussed on the characterisation of European
monofloral honeys. More than 61000 data were collected and elaborated. The extra
issue on European unifloral honeys (Apidologie 35, 2004) is still the most important
basis for establishing trade specifications.
With the beginning of 2000, the residue problems were influencing the honey
trade, leading to a new challenge for improve analytical methodologies. Therefore
an IHC working group was formed in order to harmonise and validate residue test
methods for honey. At the same time further working groups e.g. sensory, adulteration,
geographical origin of honey, other beeproducts standardisation were established. In
2010 additional IHC-subgroups for different continents were proposed.
Who can participate?
Anybody who works in honey/beeproducts research or quality control as a longterm activity can become a member, provided that he/she is interested to participate
actively in the activity of the group. Every member is responsible for communication
of changes of his/her email to be web co-ordinator.
Objectives of the IHC
The IHC is a network, created for the enhancement of knowledge on quality and
research on honey. The main objective of this network is to improve analysis methods
of honey and other bee products and to propose new quality criteria. The specific
current objectives of the IHC are:
- to work out better and new analysis methods of honey and the other bee products;
- to inform members of the group on current aspects of quality and control of
honey and other bee products;
- to work out standards for other bee products besides honey.
9
Annual
meeting
of
the
International
Honey
Commission
Activities
Working groups have been established in 2001. The IHC meets annually on working
sessions, where the current activities of the group are discussed, new activities are
planned and ideas exchanged concerning honey quality and control.
Direction Board of IHC
Chair
Gudrun Beckh, Germany
Co-Chairs
Teresa Sancho, Spain
Ligia Bicudo, Brazil
Vikas Nanda, India
Working groups and Leaders
Geographical and
botanical origin
Honeydew
Andreas Thrasyvoulou
Werner von der Ohe
Residues
Melipona honey
Asian bee’s honey
Anne Claire Martel
Ligia Almeida
Vikas Nanda
Wax
Honey Analysis Methods
Good Production Practice
Hansjoachim Roth
Stefan Bogdanov
Teresa Sancho
Peter Gallmann
Melissopalynology
Sensory analysis
Organic honey
Werner von der Ohe
Lucia Piana
Jaume Cambra
Pollen
Royal Jelly
Propolis
Maria Campos
Hervé Casabianca
Vassya Bankova
Bee Venom
Apimedical Science
Honey Microbiology
Wassili Krylov
Josef Simuth
Stefan Bogdanov
Alejandra Vasquez
Tobias Olofsson
Adulteration
Lutz Elflein
10
Annual
meeting
of
the
International
Honey
Commission
Venues
The conference will take place at the Auditorium Dionísio Gonçalves in the
Campus Santa Apolónia- School of Agriculture of the Polytechnic Institute of Bragança
(September 9-11).
Internet and computer access
Wireless internet is available campus wide to participants of the conference with
Eduroam European network. A computer room will also be available to participants
of the conference (see the Map) for internet acess. A room for the speakers will be
available (see the Map).
Transportation
The organization will provide a bus from and to hotels recommended by the
conference (Hotel S. Lázaro, Estalagem Turismo, Estalagem Turismo, Pousada São
Bartolomeu and Pousada de Juventude). Check for schedules at your hotel and at
Information Desk.
Alternatively, taxi service is available upon request:
Praça de Táxis Av. João da Cruz, Telephone: 273 322 138
Praça de Táxis Estação Rodoviária, Telephone: 273 322 007 Food and Refreshments
A welcome reception will be served in the Domus Municipalis (in the Castle of
Bragança) in the 9th of September at 7 pm.
Lunches on monday (10th of September) and tuesday (11th of September) will be
served at the IPB Canteen (Cantina Alternativa do IPB), located at the Campus de Santa
Apolónia (see the following map).
Morning and afternoon coffee breaks will be served nearby the Auditório Dionísio
Gonçalves.
Social event for accompanying persons
Tuesday at 9.30 am. Guided tour to Bragança city.
11
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B – Reception Desk
D – Computer Room
F – Poster Session
Annual
meeting
of
the
International
Honey
Commission
Topics of the symposium
•
•
•
•
•
New challenges in honey quality and standardization
Innovative analytical tools for bee products evaluation
Standards and regulations for other bee products rather than honey
Non conventional uses of bee products
Biological and functional properties of bee products
Organizers
•
•
•
•
International Honey Commission
CIMO – Mountain Research Centre, Portugal
IPB – Polytechnic Institute of Bragança, Portugal
FNAP – National Federation of Portuguese Beekeepers
In collaboration with
•
•
•
•
International Bee Research Association
Municipality of Bragança
Montesinho Natural Park
CoraNE
13
Annual
meeting
of
the
International
Honey
Commission
Contacts
II International Symposium on Bee Products. Annual Meeting of IHC
Escola Superior Agrária
Instituto Politécnico de Bragança
Campus de Santa Apolónia, Apartado 1172
5301-855 BRAGANÇA
PORTUGAL
Email: [email protected]
Fax: (+351) 273 325 405
Telephone: (+351) 273 303 382
http://ipb.pt/ihc2012
14
Annual
meeting
of
the
International
Honey
Commission
History
Beekeeping has always been considered a
prominent economic activity in the northeastern
corner of Portugal, known as the “Trás-os-Montes”
region. In November 11th of 1514, the royal charter
granted to Bragança by King Manoel, already
pointed out some economic privileges of honey bee
products “wax, honey (...) above nine coins per batch
and for those who buy it for personal use, above one
coin, will be tax-free”. At the time, the wax was used
mostly for manufacturing candles, which were used
in religious ceremonials as a testimony of miracles
and graces received. Some dioceses applied a tithe
in wax and honey to local beekeepers.
In the early nineteenth century, honey bee products
had a considerable commercial value. The wax, for
example, was more valued than cereals, while honey was used in direct exchange
for olive oil. It is common to find local villages and places that retain ancient names
associated with the bees activity, as “Torre da colmeia” (Tower of the hive), “Colmeais”
(Apiaries), “Abelheira” (Bee nest), among others.
Currently, beekeeping in Northern Portugal is practiced by 4,854 beekeepers, who own
over 142,000 hives, with a honey annual production
of 3,000 tons, representing 25% of the national
production. Lavandula stoechas, Castanea sativa
and Erica sp. pl. honeys are the dominant unifloral
honeys in the region of “Trás-os-Montes”. These
honeys provide a wide menu able to satisfy any
customer desire: from the light amber honey with
a mild flavour to the dark mountain honeys with a
strong taste. The production of reddish honeydew
honeys, with high conductivity and low levels of
sugar, is another valuable resource of the “Trásos-Montes” region, where the dense oak forests of
the natural parks provide food to the honey bees
when flowers are scarce. Quality label honey from
“Trás-os-Montes” is one of the strengths of this
economic activity and the ideal tool to compete in
the global market with products from other regions of the world. The guarantee of this
quality requires deep and continuous research in the characteristics and properties
together with the innovation in equipment and methodologies suitable to perform
the analytical control. Those are the key challenges of the IHC in the search for new
knowledge, new approaches and overleap of old and new challenges in the science
of bee products. The symposium is a great opportunity to bring together bee product
researchers and to promote the interchange of information, experience and new ideas.
15
Annual
meeting
of
the
International
Honey
Commission
Conference Overview
Sunday (09-09-2012)
9H30 - 18H00
Registration
10H30 - 12H30
IHC Group Meetings (to be defined by group leaders)
Sensory group - Room G5-S3/01
Adulteration/Residues group - Room G5-S3/02
Pollen group - Room G3-S3/02
14H30 -15H00
Meeting of the Direction board of IHC
15H00 -16H15
IHC Meeting
16H15 -16H45
Coffee break
16H45-18H00
IHC Meeting
19H00
Welcome reception by the City Council - Domus Municipalis;
Cultural event - “Renascer das Tradições” from Póvoa, Miranda do Douro.”
16
Annual
meeting
of
the
International
Honey
Commission
Conference Overview
Monday (10-09-2012)
9H30 - 10H00
Open ceremony
10H00 -11H00
Plenary session – P1. Biological and functional properties of bee products for
medicinal purposes. Invited Speaker: Maria G Campos (Porugal)
11H00 -11H30
Coffee break
11H30 -11H45
OC1. Protein complexation by polyphenols during honey storage is linked with a
decline of its antibacterial activity.
11H45 -12H00
OC2. The physiological potential of honey based on immunostimulatory effects of
royal jelly proteins.
12H00 -12H15
OC3. Propolis: antimicrobial activity, phenolic compounds and role in the
inflammation.
12H15 -12H30
OC4. Solid state fermentation of bee-collected pollen induced by lactic acid starter
cultures with probiotic bacteria.
12H30 -14H00
Lunch
14H00 - 15H00
Plenary session – P2. Standards for bee product and for their analyses – the
BEEBOOK option. Invited Speaker: Peter Gallmann (Swiss)
15H00 -15H15
OC5. Pollen composition discrimination by FTIR-ATR spectroscopy - Ofélia Anjos,
Portugal
15H15 - 15H30
OC6. Determination of physicochemical characteristics of honey comparing
reference methods and fourier transform infrared spectroscopy performed in four
different laboratories.
15H30 - 15H45
OC7. Novel, direct, reagent-free method for detection of beeswax adulteration by
single reflection attenuated total reflectance mid-infrared spectrometry.
15H45 - 16H00
OC8. Slovenian honeys database: examples of the use of data.
16H00 - 16H30
Coffee break
16H30-16H45
OC9. Honey authenticity: overview of state-of-the-art methodology and new
analytical developments for the detection of honey adulteration with sugar syrups.
16H45 - 17H00
OC10. Development and validation of a liquid chromatographic - tandem mass
spectrometric method for the detection of fumagillin in honey: use in a stability
study.
17H00 - 17H15
OC11. Optimization of polarimetric method for specific rotation determination in
honeys.
17H15 - 17H30
OC12. Sugar analysis by a multi-sensor system: applying to honey samples.
17H30 -19H30
Visit to the natural park of “Montesinho”.
20H30
Symposium banquet
17
Annual
meeting
of
the
International
Honey
Commission
Conference Overview
Tuesday (11-09-2012)
9H00 -10H00
Plenary session – P3. Improvements of European legislation governing bee products.
Invited Speaker: Andreas Thrasyvoulou (Greece)
10H00 -11H00
Poster Session
11H00 -11H30
Coffee break
11H30 -11H45
OC13. Royal Jelly: Quality, Safety and Authenticity.
11H45 -12H00
OC14. Bee feeding influences royal jelly composition.
12H00 -12H15
OC15. Chemical composition of dehydrated bee pollen produced in Brazillian states
Paraná and Santa Catarina.
12H15 -12H30
OC16. A contribution to the establishment of bromatological reference values for
Colombian pollen in the context of Latin American regulations.
12H30 -14H00
Lunch
14H00 - 15H00
Plenary session – P4. Honey characterization, a useful tool for local honeys. Future
challenges. Invited Speaker: Antonio Bentabol
15H00 -15H15
OC17. A model project: research platform on honey and other bee products.
15H15 - 15H30
OC18. Physicochemical characteristics of Colombian pot-honey.
15H30 - 15H45
OC19. Characterization of the Serbian honey using modern analytical methods.
15H45 - 16H00
OC20. Chemical, sensory and melissopalynological features of Croatian common sage
(Salvia officinalis L.) honey.
16H00 - 16H15
OC21. Sensory profile of some of the main Italian unifloral honeys. Development and
possible uses.
16H15 - 16H45
Coffee break
16H45 - 17H00
OC22. The main honeydew producing insects in Greece.
17H00 - 17H15
OC23. Study of the perception of a liquid and translucent honey and creamed honey
in France.
17H15 - 17H30
OC24. Pyrrolizidine alkaloids in Swiss honey and bee Pollen.
17H30 -17H45
OC25. Effect of high cell density fermentations on the optimisation of mead
fermentation.
17H45 - 18H00
OC26. Monitoring the gravitational reflex of the ectoparasitic mite Varroa destructor:
a novel bioassay for assessing toxic effects of acaricides and possible acaricidal
properties of honeybee products.
18H00
Closing ceremony
18
Annual
meeting
of
the
International
Honey
Commission
PLENARY SESSIONS
Abst.
Author(s)
Title
P1.
Maria G. Campos; Danielle Aguiar;
João J.S. Sousa
Biological And Functional Properties Of Bee Products For
Medicinal Purposes.
P2.
Peter Gallmann
Standards for bee product and for their analyses – the
BEEBOOK option.
P3.
Andreas Thrasyvoulou;
Chrysoula Tananaki;
Maria Dimou; Emmanuel
Karazafiris; Georgios Goras;
Basilis Liolios; Dimitris Kanelis
Suggestion for improvements of European Legislation
governing Bee Products.
P4.
Antonio Bentabol
Honey characterization, a useful tool for local honeys.
Future challenges.
19
Annual
meeting
of
the
International
Honey
Commission
P1.BiologicalAndFunctionalPropertiesOfBeeProductsForMedicinalPurposes.
Maria G. Campos (1); Danielle Aguiar (1); João J.S. Sousa (2)
1: Drug Discovery Group, Center for Pharmaceutical Studies, Faculdade de Farmácia, Universidade de
Coimbra. 3000-548 Coimbra, Portugal
2: Drug Development, Center for Pharmaceutical Studies, Faculdade de Farmácia, Universidade de Coimbra.
3000-548 Coimbra, Portugal
Due to possible therapeutic applications of bee products in veterinary and humans is
important to be aware that a simplified registration as “Herbal medicinal products” and/or
“Traditional herbal medicinal products” can be done in a near future and add an increased value
to these products.
For that, the quality control of the crude materials collected by beekeepers needs an accurate
follow-up and the purification of extracts, when need, will obey to the European directives
established from European Medicines Agency.
The use of Bee Products for therapeutic and food purposes is ancestral and in the last decade
several studies have been developed in this direction. The investigated bioactivities in these
raw materials shown a strong indicating viability in different applications, however, variations in
floral sources can be determining the type of the therapeutic ranging from preventive activities,
for example, the antioxidant activity, prevention of prostate cancer, increased bone mass and
lowering the lipid profile to the anti-inflammatory in rheumatoid arthritis, antimicrobial activity
and others (Yamaguchi et al, 2006; Lustosa et al, 2008; Liu et al, 2008, Uzbekova et al, 2003).
From the research developed, markers will be developed for quantitative and qualitative
analysis of each product and/or extracts once they vary depending on the floral source as for
example bee pollen (Campos et al, 2008). Its inclusion in pharmaceutical formulations, even in
a simplified registration, need to be supported with a monograph that safeguards the Public
Health and maintains its bioactivities in-depth knowledge and consistent. Despite, the potential
is enormous and the work tend to be systematized, the standardization of quality control
standards for the more bioactive bee products as bee venom, propolis, royal jelly and bee pollen
still require studies of toxicity, that are still poorly studied.
Further studies consistent with this goal can provide a short-term development of a
document that guarantees the consumer a quality product and a risk assessment consistent
with the legislation.
The determination of toxic contaminants such as heavy metals and pesticides, among
others should be included in the final documents. Another aspect that can’t be neglected, and
that still needs some research, as there are few results available concerning is the contamination
of genetically modified plants whose impact is still unpredictable.
This open an important contribute to the profitability of bee products and also allows the
consumer the confidence that is crucial to guarantee good products on the market.
1) Campos M G; Bogdanov S; Almeida-Muradian L B; Szczesna T; Yanina M (2008) Standardisation of bee
products: pollen, Journal of Apicultural Research and Bee World 47 (2): 154–161
2) Liu XD; Zhang JL; Zheng HG; Liu FY; Chen Y (2008) Clinical randomized study of bee-sting therapy for
rheumatoid arthritis, Zhen Ci Yan Jiu. 33 (3):197-200
3) Lustosa S R; Galindo AB; Nunes LCC; Randau KP; Rolim Neto PJ (2008) Própolis: atualizações sobre a
química e a farmacologia, Rev Bras Farmacogn 18: 447-454.
4) Uzbekova DG; Makarova VG; Khvoynitskaya LG; Slepnev AA (2003) Evaluation of Bee-collected Pollen influence in lipid peroxidation, antioxidant system and liver function in old animals, J Hepatol 38: 703 Suppl.
2
5) Yamaguchi M; Hamamoto R; Uchiyama S; Ishiyama K; Hashimoto K (2006) Anabolic effects of Bee Pollen
Cistus ladaniferus extract on bone components in the femoral-diaphyseal and -metaphyseal tissues of
rats in vitro and in vivo, J Health Sci 52(1): 43-49.
* Presenting author: [email protected]
20
Annual
meeting
of
the
International
Honey
Commission
P2. Standards for bee product and for their analyses – the BEEBOOK option.
Peter Gallmann (1)*
1: Swiss Bee Research Centre, Agroscope
One of the main objectives of the IHC is to develop, improve and harmonize methods for
analyzing honey and other bee products and to establish and/or improve standards for bee
products. The difficulty of such tasks is keeping the harmonized methods and standards up to
date. With the rapid development of technologies and knowledge, traditional methods and
standard collections are of limited lifespan. Nevertheless in a globalized world comparison
between results worldwide is crucial. This is not only true for bee products but for all
disciplines of honeybee research. COLOSS, one of the largest honeybee research network
faced such problems of comparison of results across member nations. To solve it, a platform
for standardization was recently set up. The idea behind the BEEBOOK, a manual of standard
methodologies in Apis mellifera research, is to bring the specialists for each method together
and for them to agree on those to be established as standard. To launch this work, a panel of
senior authors has been chosen to invite specialists, coordinate the selection of methods and
their writing up in the manual. The researchers contributing to this task are rewarded in form
of publications. The International Bee Research Association (IBRA) has agreed to publish these
standards in a special issue, thus providing citable articles. Once the first edition of the manual
is released, an internet platform can be used to update the BEEBOOK in real time, as new or
improved standards become available. This contribution evaluates the advantages for IHC of
using the established structure and process of BEEBOOK to keep track with the rapid evolution
of bee product research.
21
Annual
22
meeting
of
the
International
Honey
Commission
Annual
meeting
of
the
International
Honey
Commission
P3. Suggestion for improvements of European Legislation governing
Bee Products.
Andreas Thrasyvoulou (1); Chrysoula Tananaki (1); Maria Dimou (1); Emmanuel Karazafiris (1);
Georgios Goras (1); Basilis Liolios (1); Dimitris Kanelis (1)
1: Lab. of Apiculture, School of Agriculture, Aristotle University, Thessaloniki, Greece
The Laboratory of Apiculture-Sericulture of AUTH, based on 20 years’ analyses of honey
from the market, makes suggestion for improvements of European legislation governing
the authenticity of the product. Council Directive 2001/110/EC relating to honey seek to
provide the fullest possible information about the botanical and geographical origin, but the
physicochemical, organoleptic and microscopic characteristics of monofloral honeys had not
yet been defined. Hence, the floral origin could not be legally ascertained. Greece, established its
own criteria to verify eight types of domestic monofloral honeys through European legislation.
Another important point to be issued is the declaration of the country of origin where the honey
has been harvested. This provision of Directive was totally replaced by the phrase “honey blends”
which means that the product originates from more than one member state or third country.
Some honey importers took advantage of the above provision and replaced entirely the “country
of origin” with the indication “mixtures” or “honey blends” giving inaccurate information to
consumers or concealing the real origin of the product. In some countries like Italy and Greece
National stipulation demanded the written reference of the country of harvesting regardless
of the Directive’s provision. Even in those cases the right of European law is above the National
ones and the national decisions are not applicable in practice.
The criteria based on the composition of honey should also be reconsidered since there are
monofloral honeys that are not included within the Directive. For example greek fir honey may
have less of 45g/100g fructose and glucose content, erica spp. honey more than 20% water
content, knot weed (Polygonum aviculare), cotton, Jerusalem thorn (Paliurus spina-christi) and
other floral honeys more than >0,8 mS/cm electrical conductivity, spring pine honey, lavender,
erica, robinia below 8 DN diastase. In honeys with natural low content of enzyme, the Directive
permits diastase up to 3 DN, but demands HMF lower than 15 mg/kg. This conjunction of HMF
to diastase causes unnecessary difficulties in the marketing of the product either as monofloral
or as a mixture. In addition the extreme variability of diastase in monofloral honeys and the
absence of a correct starting point of this enzyme should be considered.
Other Regulations like 37/2010 (MRLs in products of animal origin) 396/2005 (pesticide
residues from agricultural practices), 889/2008 (organic production and labeling of organic
products) and Directive 2000/13/EC (labeling presentation and advertise) are discussed and
criticized.
____________
23
Annual
meeting
of
the
International
Honey
Commission
P4. Honey characterization, a useful tool for local honeys. Future challenges.
Antonio Bentabol (1)*
1: Casa de la Miel, Cabildo of Tenerife; Tenerife, Spain
Honey is a food product known since the dawn of humanity, and nowadays is rising in its
acceptation by consumers as an interesting food to supply their diet, leading to the growth in
the honey world consumption. However, there is a real lack of information in a great part of the
population about honey quality, its markers and different attributes, which, many times, limit
the buying decision of consumers.
On the other hand, honey is a food product that is part of a global market, being acquired as
a commodity in large quantities in most of our markets (importing countries), making it difficult
for the honey local producers to sell their honey
To be competitive, the local producers are promoting their honey through the quality and
identifying the geographic origin, in order to show consumers the differences in their products.
But to change the way honey is acquired in the market, it is necessary to have got previously
detailed and exhaustive information about the physical-chemical, pollen, sensory, etc.
characteristics of these honeys, together with tools or elements that assure to the consumers the
type of honey, as well as establish systems that can guarantee the accuracy of the information
provided, so that the consumer who wants to search and find specific honeys, can achieve it.
Most studies on honeys focus on the evaluation of the chemical composition and the
melissopalinological analysis of honeys. Generally, the sensory component has been relegated
to second place or even forgotten. This work intends to show, considering the specific experience
with honeys from Tenerife (Spain), the importance of honey characterization as a previous step
for honey quality standardization, and the role of joint implementation of sensory techniques,
chemical and pollen as the most appropriate way to define them. Our experience shows that the
application of these three analytical tools allows the differentiation and honey identification,
particularly in honeys with hypo-represented pollen, where the pollen and physicochemical
analysis can lead to results that would mask the correct type of honey flowers.
The establishing of the necessary elements that allows to identify and define the quality of
honey, and the development of methods that can help to know and grade this quality, is one of
the main future challenges in honey standardization. Its proper implementation and definition
could influence the sustainability of the beekeeping industry of the importers countries in the
medium and long term.
________________
24
Annual
meeting
of
the
International
Honey
Commission
ORAL COMMUNICATIONS
Abst.
Author(s)
Title
OC1.
Liset Maldonado-Alvarez;
Katrina Brudzynski
Protein complexation by polyphenols during honey
storage is linked with a decline of its antibacterial
activity.
OC2.
Katarina Bilikova
The physiological potential of honey based on
immunostimulatory effects of royal jelly proteins.
OC3.
Ana Paula Pereira; Luís G. Dias;
João Carlos Silva; Leticia M. Estevinho
Propolis: antimicrobial activity, phenolic compounds
and role in the inflammation.
OC4.
Carlos Alberto Fuenmayor;
Amanda C. Díaz-Moreno;
Carlos M. Zuluaga-Domínguez;
Martha Cecilia Quicazán
Solid state fermentation of bee-collected pollen
induced by lactic acid starter cultures with probiotic
bacteria.
OC5.
Ofélia Anjos; Dinis Francisco;
Paulo Antunes; Maria da Graça Campos
Pollen composition discrimination by FTIR-ATR
spectroscopy - Ofélia Anjos, Portugal.
OC6.
Birgit Lichtenberg-Kraag;
Roberto Piro; Beatrix Brinkmann;
Lucia Piana; Patricia Beaune;
Verena Kilchenmann; Christina Kast
Determination of physicochemical characteristics
of honey comparing reference methods and fourier
transform infrared spectroscopy performed in four
OC7.
Miguel Maia; Fernando M. Nunes
Novel, direct, reagent-free method for detection of
beeswax adulteration by single reflection attenuated
total reflectance mid-infrared spectrometry.
OC8.
Mojca Korošec; Terezija Golob;
Jasna Bertoncelj
Slovenian honeys database: examples of the use of
data.
OC9.
Lutz Elflein; Hartmut Wischmann;
Martin Linkogel; Torsten Peix;
Florian Rommerskirchen;
Hartje Müller; Sandra Piosek;
Dirk Klaus; Mandy Schmidt;
Lars Ganske; Bianca Eickermann;
Saskia Timmermann;
Kirsten Schneidermann
Honey authenticity: overview of state-of-the-art
methodology and new analytical developments
for the detection of honey adulteration with sugar
syrups.
OC10.
Els Daeseleire;
Wim Reybroeck
Development and validation of a liquid
chromatographic - tandem mass spectrometric
method for the detection of fumagillin in honey: use
in a stability study.
OC11.
S. Serrano; I. Rodríguez;
F. Rincón
Optimization of polarimetric method for specific
rotation determination in honeys.
OC12.
Luís G. Dias; Mara E.B.C. Sousa;
António M. Peres; Jorge Sá Morais;
Letícia Estevinho; A.A.S.C. Machado
Sugar analysis by a multi-sensor system: applying to
honey samples.
OC13.
Ayse Bakan; Ozlem Aslan;
Neşe Aslı Öncü; Banu Bahar;
Ebru Pelvan; Cesarettin Alasalvar;
Nihat Ozcan; Canan Dogan;
İlknur Demirtaş; Senem Akkuş
Çevikkalp; Mustafa Yaman;
Esra Agel; İmge Oktay;
Hayrettin Ozer; Aslı Elif Sunay:
Taylan Samancı; Tuğçe Daştan
Royal Jelly: Quality, Safety and Authenticity.
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Commission
OC14.
Wytrychowski Marine;
Daniele Gaelle; Batteau Magali;
Guibert Sylvie; Casabiance Hervé
Bee feeding influences royal jelly composition.
OC15.
José A. Gasparotto Sattler;
Adriane Melo; Ellen de Souza Soares;
Luciana Barbosa dos Santos;
Davi F. Sampaio Meira;
Ligia B. de Almeida-Muradian
Chemical composition of dehydrated bee pollen
produced in Brazillian states Paraná and Santa
Catarina.
OC16.
Martha Quicazán; Carlos Fuenmayor;
Consuelo Díaz; Carlos Zuluaga
A contribution to the establishment of
bromatological reference values for Colombian
pollen in the context of Latin American regulations.
OC17.
Aslı Elif Sunay;
Taylan Samancı
A model project: research platform on honey and
other bee products.
OC18.
Consuelo Díaz-Moreno;
Carlos Fuenmayor; Carlos Zuluaga;
Martha Quicazán
Physicochemical characteristics of Colombian pothoney.
OC19.
Dušanka Milojković-Opsenica;
Nebojša Nedić; Silvio Kečkeš;
Biljana Marošanović;
Filip Andrić; Maja Natić;
Živoslav Tešić
Characterization of the Serbian honey using modern
analytical methods.
OC20.
Dražen Lušić; Nada Vahčić;
Gian Luigi Marcazzan;
Vladimir Mićović; Darija Vukić Lušić;
Duška Ćurić;
Francesca-Vittoria Grillenzoni
Chemical, sensory and melissopalynological features
of Croatian common sage (Salvia officinalis L.)
honey.
OC21.
Massimiliano Magli; Lucia Piana;
Angela Savino; Mariassunta Stefano
Sensory profile of some of the main Italian unifloral
honeys. Development and possible uses.
OC22.
Sofia Gounari; George Goras;
Chysa Tananaki; Andreas Thrasyvoulou
The main honeydew producing insects in Greece.
OC23.
Robin Azemar; Valérie Nevers ;
Myriam Laurie; Benjamin Poirot ;
Alain Le Bail
Study of the perception of a liquid and translucent
honey and creamed honey in France.
OC24.
Christina Kast; Arne Dübecke;
Verena Kilchenmann;
Katharina Bieri; Gudrun Beckh;
Cord Lüllmann
Pyrrolizidine alkaloids in Swiss honey and bee
Pollen.
OC25.
A.P. Pereira; A. Mendes-Ferreira;
L.M. Estevinho; A. Mendes-Faia
Effect of high cell density fermentations on the
optimisation of mead fermentation.
OC26.
Alexandros Papachristoforou;
Chrisovalantis Papaefthimiou;
George Theophilidis
Monitoring the gravitational reflex of the
ectoparasitic mite Varroa destructor: a novel
bioassay for assessing toxic effects of acaricides and
possible acaricidal properties of honeybee products.
26
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Commission
OC1.Proteincomplexationbypolyphenolsduringhoneystorageislinkedwitha
decline of its antibacterial activity.
Liset Maldonado-Alvarez (1); Katrina Brudzynski (1,2)*
1: Department of Biological Sciences, Brock University, St. Catharines, Ontario, Canada
2: API-Medicals, St. Catharines, Ontario, Canada
The protein–polyphenol interactions in foods have important nutritional consequences due
to polyphenols’ ability to bind and precipitate proteins. Our previous studies have shown that
these interactions are responsible for formation of melanoidins, high molecular weight brown
complexes, consisting of polyphenols, proteins and sugars. The functional consequences of
the polyphenol-protein interactions with regard to honey antibacterial activity have not been
determined. The objective of the present study was (a) to investigate the effects of storage time
and storage temperature on the formation of protein-polyphenol complexes and (b) to correlate
their formation during storage with antibacterial activity of honeys. Freshly obtained honeys
(n=13) were divided onto three groups and stored at -20ºC, 4ºC and room temperature for a period
of 1 year. Every month, honey samples were precipitated by a mixture of three alcohols (80%
ethanol: 80% methanol: 70% propanol), and both soluble and precipitate fractions were tested
for the total protein concentration (Pierce method), total phenolic content (Folin-Ciocalteau
method), and protein profiles by SDS-PAGE. Initially, in the soluble fractions, the protein profiles
on SDS-gels ranged from 4 to 8 polypeptide bands and the total protein contents ranged
from 3344 μg/ml (buckwheat honeys) to 96.2 μg/ml (wild flower honey). During the first three
month of storage, the polypeptide bands on SDS-gels lost their sharp definition and showed
a significant decrease in intensity of band staining as indicated by semi-quantitative imaging
system. Concomitantly, a rapid, 70% reduction in protein content was observed (p<0.0001),
independently of storage temperature. In precipitate fractions, positive reactions with ferric
chloride revealed increasing formation of polyphenol-protein complexes. Storage beyond the
initial three-month period brought no significant decrease in protein concentrations. Honeys
kept at -20ºC, 4ºC showed slower rates of the decrease of protein content. In contrast to proteins,
the polyphenol concentration decreased only by 2% during three-month storage, however this
decrease was temperature-sensitive; lower for honeys stored at -20ºC versus room temperature
(p<0.001) and 4ºC versus -20ºC (p<0.01). The bacterial susceptibility assay against Escherichia coli
indicated that during three-month storage at room temperature the MICs of honeys decreased
by two to four doubling concentrations. In conclusion, storage time and storage temperature
reduced the content of unbound proteins due to increasing formation of protein-polyphenol
complexes in honeys. The formation of polyphenol-protein complexes coincided with the
reduction of growth inhibitory activities of honeys.
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the
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OC2.ThePhysiologicalPotentialofHoneyBasedonImmunostimulatoryEffects
of Royal Jelly Proteins.
Katarina Bilikova (1)*
1: Institute of Molecular Biology, Slovak Academy of Sciences, Bratislava, Slovakia
Until now the properties of honey have bee defined based exclusively on the content of plant
components in the nectar of geven plant. We have found that royal jelly (RJ) proteins presented in
honey are potential marker of authenticity of honey (Bíliková, 2010). The antimicrobial potential
of honey based on proteins of honeybee origin was tested by microtiter based assay. An inhibition
of the growth of P. larvae was observed only in protein fraction of same samples (cherry, rape and
honeydew honeys). It was not found an antimicrobial activity of honey proteins, neither from
colonies with AFB symptoms nor healthy colonies. Despite of this we have found for first time
that honey contained antibiotic microbial RJ peptide apisimin in all tested samples including
honeys from saccharoze syrup honeys (Bortolotti, 2010). Based on our previously experiences
of testing immunostimulatory effect of major royal jelly proteins and honey on production
of cytokine TNF-alfa by mouse macrophage we have prepare a new protocol for testing of
physiological properties of honey underlining authenticity of honey. Profiling expression of
cytokines stimulated by RJ proteins on a global scale will have significant impact not only in
human immunotherapy but also in characterization of immunopotential of honeybee nutrition.
The presence of specific cytokines, chemokines, interleukins and other proteins captured by the
antibody chip was detected using Streptavidin-conjugated dye as a reporter. RJ, honey, honey
proteins, apalbumin1 at concentartion 50-500 μg/ml stimulated mouse macrophages to release
cytokines as follows: G-CSF, GM-CSF, IFN-g, IGF-II, IL-12p40/p70, IL-12p70, IL-1a, IL-1b, IL- 6, IL9, Leptin, MCP1, PF-4, TIMP-1, TIMP-2, TNF-alfa, Thrombopoietin, VEGF and bFGF. Granulocytemacrophage colony-stimulating factor (GM-CSF) have role in local tissue defense. Monocyte
chemoattractant protein-1 (MCP-1) plays a major role in the recruitment of inflammatory cells
to the lungs of patients with chronic obstructive pulmonary disease. Serum platelet factor-4
(PF4) an endogenous pro-coagulant have a major role in inflammation. Presented simultaneous
detection of multiple cytokines undoubtedly provides a powerful tool for evaluation of quality
of honeybee nutrition and it is a contribution to nutritional immunology. The new experimental
knowledge on the similarities between the immune systems of insects and mammals confirmed
indirectly original empirical observations that royal jelly and honey could induce stimulation of
immune response in an animal, based on the RJ proteins as immunomodulators.
1) Bíliková, K., and Šimúth, J. (2010) New criterion for evaluation of honey: quantification of royal jelly protein
apalbumin 1 in honey by ELISA, J. Agric. Food Chem. 58: 8776-8781.
2) Bortolotti, L., Truchado, P., Allende, A., Kaatz, H.H., Bertelli D., Plessi, M., Bílikova, K., Šimúth, J., Moritz, R.F.A.,
Barbean, F.A.T., and Sabatini, A.G. (2010) In The quality of honey for bees and man (Behrens, D., and Moritz,
R.F.A., Eds.), pp. 6-62, Nova Science Publishers, New York.
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OC3. Propolis: antimicrobial activity, phenolic compounds and role in the
inflammation.
Ana Paula Pereira (1); Luís G. Dias (2); João Carlos Silva (2)*; Leticia M. Estevinho (2)
1: IBB-Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology,
Universidade de Trás-os-Montes e Alto Douro, Vila Real, Portugal
2: CIMO, Mountain Research Centre, Escola Superior Agrária, Instituto Politécnico de Bragança, Bragança,
Portugal
Propolis is a beehive product prepared by bees of the Apis mellifera species, using resinous
substances collected from various plants. These substances are mixed with β-glycosidase
enzyme of their saliva, partially digested and added to bee wax to form the final product. In the
present study, it was evaluated the antimicrobial activity against multi-resistant microorganisms
and the anti-inflammatory activity, assessed by the effect on the hyaluronidase enzyme, of
propolis samples from Portugal. Simultaneously, it was studied the effect of extraction solvents
on this biological activities. It was chosen the hydro-alcoholic extract because this was the
most effective for extracting phenolic compounds. The antimicrobial activity was accessed in
Gram-positive (Staphylococcus aureus) and Gram-negative bacteria (Pseudomonas aeruginosa,
Escherichia coli) and yeasts (Candida albicans), isolated from different biological fluids and the
results were then compared with the obtained for reference microorganisms. Regarding the
pollen profiles, marked differences were found among the samples from the different regions
under study. In the propolis from Bragança the dominant pollen was Erica sp., whereas the
dominant species in Coimbra and Beja were Populus tremula and Eucalyptus sp., respectively.
The propolis from Bragança was the one that possessed the highest polyphenols’ content.
It was verified that all the extracts inhibited the hyaluronidase enzyme in a dose-dependent
manner. The propolis that showed higher inhibitory activity was the one from Bragança and
the product from Beja was the less effective. Despite the differences amongst the polyphenols
concentrations, the anti-inflammatory activity of the samples did not differ significantly,
suggesting that polyphenols are not the only factor responsible for the bioactive properties
of this beehive product. Concerning the antimicrobial activity, Candida albicans was the
most resistant and Staphylococcus aureus the most sensitive. Propolis showed greater activity
against Gram-positive bacteria than Gram-negative ones, something that may be explained by
structural differences of the cell wall of these two types of bacteria. In addition, the reference
microorganisms were more sensitive than the ones isolated from biological fluids. This study
suggests that the simultaneous use of propolis and antibiotics may reduce the acquisition of
resistances and consequently avoid the use of more powerful therapies, even though further
studies are required.
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30
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Honey
Commission
OC4.SolidStateFermentationofBee-CollectedPollenInducedbyLacticAcid
Starter Cultures with Probiotic Bacteria.
Carlos Alberto Fuenmayor (1,2)*; Amanda Consuelo Díaz-Moreno (1); Carlos Mario ZuluagaDomínguez (1); Martha Cecilia Quicazán (1)
1: Instituto de Ciencia y Tecnología de Alimentos (ICTA), Universidad Nacional de Colombia, Bogotá,
Colombia
2: Department of Food Science and Technology, University of Milan, Milan, Italy
Bee-collected pollen has interesting nutritional features regarding its use for human
consumption. However, previous studies have revealed the limitations of digestive system
of monogastric animals to take advantage of some of its nutrients, partly because of the
exine, a resilient microstructure that protects pollen grains from environmental degradation.
Technological adequacy and induction of solid-sate fermentative processes were studied
as alternatives aimed at improving the nutrients bioavailability of dried pollen pellets. In an
initial phase, the effect of different heat treatments on microbial load, antioxidant capacity and
fatty acids profile of reconstituted (rehydrated) pollen, was evaluated, finding that a 121°C/15
min treatment succeeds in reducing microbial load without significantly affecting the other
characteristics assessed. In a second phase, four commercial LAB starters with probiotic features
(Lactobacillus acidophilus, Lactobacillus paracasei subsp. paracasei, and two mixed commercial
cultures: YOMIX 205 LYO and CHOOZIT MY 800 (Danisco, Denmark), were inoculated (108 UFC/g)
on the heat-treated reconstituted pollen and then incubated during 72 h at 35 ± 1°C in order
to promote fermentation; this process was monitored by pH, acidity, viable LAB account, sugars
and lactic acid concentration. L. acidophilus was shown to be the most promissory of all the
starters by acidifying capacity (0.16 g lactic acid/kg*h) and survival rate during incubation (108
UFC/g, after 24 h). SEM images analysis showed an appreciable effect of these processes on the
exine. Results demonstrate that it is feasible to obtain an innocuous fermented product with
the bromatological profile of pollen, a high number of viable LAB and an increased lactic acid
concentration.
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OC5. Pollen composition discrimination by ATR-FTIR Spectroscopy.
Ofélia Anjos (1,2)*; Dinis Francisco (1); Paulo Antunes (3); Maria da Graça Campos (4)
1: IPCB - Polytechnic Institute of Castelo Branco, Castelo Branco, Portugal
2: CERNAS –Natural Resources, Environment and Society Res. centre, Coimbra, Portugal
3: CATAA - Agri-food Technological Support Center, Castelo Branco, Portugal
4: Center of Pharmaceutical Studies, Laboratory of Pharmacognosy, Faculty of Pharmacy, University of
Coimbra, Coimbra, Portugal
Bee pollen is a mixture of different pollens grain collected in different flowers that improve
a different composition (Campos et al, 2008) and different colors. Bee pollen is known by its
rich nutritional composition, is composed of proteins, lipids, sugar, fiber, minerals, amino acids,
phenolic compounds and vitamins.
FTIR-ATR spectroscopy represents a useful technique for identifying chemical structures,
which is based on the analysis of absorption peaks at certain wave numbers (expressed in cm−1).
The aim of this study is compare different spectral regions of pollen from different floral
sources in order to predict differences in the chemical composition. 16 different bee pollen
samples with different botanical origin were used.
FTIR-ATR spectra were acquired with a Bruker FT-IR spectrometer (Alpha) using a diamond
single reflection attenuated total reflectance (ATR) device and a zero filling of 2. Duplicate
spectra per sample were obtained with 32 scans per spectrum at a spectral resolution of 4 cm-1
in the wavenumber range from 4000 to 400 cm-1. Principal component analyses (PCA) were
performed using OPUS Quant 2 (Bruker Optics, Ettlingen, Germany).
Second-derivatives of FTIR spectra are generally used to improve resolution in the original
FTIR spectra. So, in the spectral data analyze was used the second derivation and selected the
zones of proteins, lipids, carbohydrates and fibers absorption to better interpret the spectra.
The pollen spectra present significant differences in several spectral zones which indicate a
different chemical composition.
The pollen spectra present a band appeared between 3650 and 3000 cm-1 corresponding to
hydrogen bonded O–H stretching vibrations and two well defined peak at 2926 cm-1 due to C–H
stretching vibrations and 2500 cm-1. Between bands at 1200–970 cm-1 are mainly due to C–C and
C–O stretching in pyranoid ring and to C–O–C stretching of glycosidic bonds. There are also a
higher variability with different peaks on region between 1700 and 1200 cm-1 corresponding to
aromatic and sugar compounds. These spectral zones were already identified for these groups
of compounds by Coimbra et al. (1998) and Gómez-Ordóñez and Rupérez (2011).
The PCA analysis of the transformed spectra shows clearly the difference between samples.
It was possible to group the 16 samples in different classes concerning the peaks area and the
compound groups. This allows creating a classification model that identifies an unknown pollen
sample. In the future it will be possible to make the calibration with the standard chemical
methods in order to have a quantification of the bee pollen compounds by FTIR-ATR. This
analysis could be promising, because is need only a pollen grain as sample.
1) Campos MG, Bogdanov S, Muradian LB, Szczesna T, Mancebo Y, Frigerico C, Ferreira F, (2008) Pollen
composition and standardisation of analytical methods, Journal of apicultural Research and bee world 47
(2) 156-163.
2) Coimbra, MA; Barros, A; Barros, M; Rutledge, DN; Delgadillo, I (1998) Multivariate analysis of uronic acid
and neutral sugars in whole pectic samples by FT-IR, Carbohydrate Polymers 37 (3) 241–248.
3) Eva Gómez-Ordóñez, E; Rupérez, P (2011) FTIR-ATR spectroscopy as a tool for polysaccharide identification
in edible brown and red seaweeds, Food Hydrocolloids 6 (25) 1514–1520.
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Commission
OC6.DeterminationofphysicochemicalcharacteristicsofhoneycomparingReferenceMethodsandFourierTransformInfraredSpectroscopyperformedinfour
Birgit Lichtenberg-Kraag (1)*; Roberto Piro (2); Beatrix Brinkmann (3); Lucia Piana (4); Patricia
Beaune (5); Verena Kilchenmann (6); Christina Kast (6)*
1: LIB, Hohen Neuendorf, Germany
2: IZSLER, Brescia, Italy
3: LUA, Trier, Germany
4: Piana Ricerca e Consulenza, Castel San Pietro Terme BO, Italy
5: Société famille Michaud apicultures, Gan, France
6: ALP-Haras, Bern, Switzerland
Fourier Transform Infrared Spectroscopy (FT-IR) determines several physicochemical
measurands simultaneously and hence is faster and less laborious than the current reference
methods. FT-IR is the ordinary analytical method used for analysis of milk and other food
products. In several laboratories, FT-IR has recently been established as routine method for
honey analysis. With this study we aimed to evaluate if FT-IR analysis leads to comparable results
even if instrument types and sample preparation (diluted and undiluted samples) are different.
Hence we were interested to investigate if FT-IR can be considered equivalent to the established
methods.
Therefore, the measurands fructose, glucose, pH, electrical conductivity and free acidity
were determined by reference and FT-IR analysis in four different laboratories using Foss or
Bruker instrumentation and the measurands water and sucrose in two and three laboratories,
respectively. So far, we analyzed several honey samples from German ring trials, as well as six
different European unifloral honeys. The methods were analyzed and compared using statistical
z-score evaluation and other common statistical test (eg. Cochran).
The reference methods of all laboratories showed good accuracy for all the honey types.
Average values of the reference methods were subsequently used for the evaluation of the
quality of the FT-IR results. The accuracy of these results were related to the honey type and
differed among the measurands. Most measurands were comparable for FT-IR and reference
methods when acacia, rape, lavender, tilia and honeydew honeys were analyzed, however the
some measurands determined by FT-IR deviated a lot when chestnut honey was examined.
In general, fructose, glucose, pH and free acidity gave mostly comparable values with both
methods, while the values of the electrical conductivity diverged more from the reference
method.
Different results obtained from FT-IR and reference methods may be related to the honey
types used for FT-IR calibration in the various laboratories, rather than the different FT-IR
instrumentation or sample preparation. This first interlaboratory testing shows, that FT-IR is
a promising approach even for a product with a high variability of components like honey.
Knowing the outliers in the ring trials, each laboratory can improve or correct its specific
calibrations. Improved correlation should be verified in an additional forthcoming ring trial.
____________
* Presenting author: [email protected] and [email protected]
34
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OC7.Novel,direct,reagent-freemethodfordetectionofbeeswaxadulteration
by single reflection attenuated total reflectance mid-infrared spectrometry.
Miguel Maia (1); Fernando M. Nunes (2)*
1: Apismaia, Beekeeping Service. Estrada Municipal 1221, nº62 5000-027 Vila Real, Portugal
2: CQ – Chemistry Research Centre, Chemistry Department, University of Trás-os-Montes e Alto Douro,
5000-801 Vila Real, Portugal
In this work a novel, direct, reagent-free method for detection of beeswax adulteration
by paraffin, microcrystalline wax, tallow and stearic acid, by single reflection attenuated total
reflectance mid-infrared spectrometry was developed. The use of the absorbance ratios,
I1739cm-1/I2852cm-1, I1714cm-1/I2852cm-1, I1739cm-1/I1714cm-1 allows to detect a minimum
of 5% paraffin / microcrystalline wax and tallow adulteration and 0.5% stearic acid adulteration
of beeswax. Upper and lower critical limits for beeswax authenticity were established from
the analysis of virgin beeswax and validated by independent analysis of real sheet and comb
beeswax samples by high-temperature gas-chromatography with flame ionization detection.
Furthermore, besides it simplicity in sample handling, the amount of sample and the time
needed is far less than that of the previous described methods based on chemical analysis and
chromatographic techniques. These advantages result in time and cost savings and an increase
in the number of samples that can be analyzed and most importantly with only one method
there is possible detecting the main beeswax adulterants.
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Annual
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OC8. Slovenian honeys database: Examples of the use of data.
Mojca Korošec (1)*; Terezija Golob (1); Jasna Bertoncelj (1)
1: University of Ljubljana, Biotechnical Faculty, Ljubljana, Slovenia
Natural geographic conditions and climatic diversities in Slovenia along with appropriate
beekeeping technology enable production of different honey types regarding their predominant
botanical origin. Traditionally two blended (multifloral and honeydew honey) and several
monofloral types of honey are produced. The honey types are well recognized among Slovenian
consumers; nevertheless the average honey consumption is among the highest in Europe and is
slightly higher than the average amount of honey produced in Slovenia (0.9 kg/capita).
In the frames of different research projects since year 1999 extensive sampling has been
performed and about 2200 samples of honeys from all four Slovenian macroregions have
been collected directly from the beekeepers. The most typical samples of particular honey
type (in total 380 samples) were selected by the means of sensory assessment and submitted
to further analyses. Descriptive sensory analysis was applied for detailed characterisation of
sensory properties, and different physico-chemical analyses were used for determination of
legally defined quality parameters and other physico-chemical properties (content of protein,
amino acids, carbohydrates, elements, phenolic compounds, and antioxidant activity, colour
and isotopic parameters) of Slovenian robinia (Robinia pseudoacacia), linden (Tilia spp.),
chestnut (Castanea sativa Mill.), fir (Abies alba Mill.), spruce (Picea abies (L.) Karst.), multifloral and
honeydew honey.
Databases are reported to be important source of information in testing authenticity of food
products. Therefore the applicability of data on Slovenian honey composition was tested by
multivariate statistical methods (PCA and LDA). In the comparison of pure honey samples and
samples deliberately adulterated with 2-20 % sugar syrup a good discrimination was obtained.
Furthermore, the database proved to be applicable in verification of the geographical origin
of a honey sample from Slovenia (checking e.g. if a sample of chestnut honey is derived from
Kočevje region and may qualify as PDO honey Kočevski med). On the basis of physico-chemical
and isotopic properties a clear differentiation among Slovenian, commercial (originating from
EU and non-EU countries) and deliberately adulterated Slovenian honey samples, respectively,
was found as well.
Data from the database in combination with chemometrics proved to be a useful and reliable
tool for testing the botanical and geographical origin, and authenticity of honey produced in
Slovenia.
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OC9.HoneyAuthenticity:Overviewofstate-of-the-artmethodologyandnew
analytical developments for the detection of honey adulteration with sugar
syrups.
Lutz Elflein (1)*; Hartmut Wischmann (1); Martin Linkogel (1); Torsten Peix (1); Florian
Rommerskirchen (1); Hartje Müller (1); Sandra Piosek (1); Dirk Klaus (1); Mandy Schmidt (1); Lars
Ganske (1); Bianca Eickermann (1); Saskia Timmermann (1); Kirsten Schneidermann (1)
1: Intertek Food Services GmbH, Food Laboratory Bremen, Germany
A broad range of different types of sugar syrups occur as adulterants of honey. Nowadays,
sugar syrups made from C3 plant species (beet, rice, wheat, tapioca, and others) predominate
besides the well-known high fructose corn syrups and inverted sugar cane syrups of C4 plant
origin. While sugar syrups from C4 plant origin can be easily detected due to the large difference
in carbon 13C/12C isotopic ratios compared to authentic honey, the detection of sugar syrups
from C3 plant origin is an ongoing analytical challenge. According to the current state-of-theart, several complementary methodologies have to be used to detect all relevant adulterants
with reasonable sensitivity and accuracy, including EA/LC-IRMS, EA/LC-CRDS; LC-MS/MS, LCUV, LC-RI; LC-ELSD, ICP-MS and enzymatic assays. First of all, isotope ratio mass spectrometry
(IRMS) coupled to elemental analyzer (EA) and liquid chromatography (LC) is used as a nonspecific screening method to detect sugar syrups both from C4 and C3 plant origin. As a very
recent development, IRMS as detection technique can be replaced by optical detectors using
the principle of cavity ring-down spectroscopy (CRDS) to measure 13C/12C isotopic ratios with
the advantage of easier operation and maintenance. For the first time, the practical hyphenation
with LC is described and performance data of EA/LC-IRMS vs. EA/LC-CRDS reported. These
techniques represent a significant improvement compared to the well-known conventional
AOAC method for C4 sugars in honey (AOAC 998.12) in terms of detection sensitivity for C4
sugars and detection capability of C3 sugars. However, there is still a limitation for certain
mixtures of C3 sugars and honey for which no satisfactory level of detection can be realized
due to very similar isotopic values (e.g. rice syrup and rape honey). Therefore, additional and
complementary methods are necessary to improve detection capabilities in these cases.
Depending on the type of syrup, different solutions were developed. Inverted sugar syrups can
contain traces of the enzymes used for production from sucrose (beta-fructofuranosidase) or
starch (beta-/gamma-amylase), not occurring naturally in honey. Detection of these enzymes
is realized by incubation with an enzyme specific substrate followed by LC analysis of the
released reaction product with refractive index (RI) or UV detection. A very sensitive detection
of honey-foreign oligosaccharides (degree of polymerization n ≥ 4) indicating the presence of
sugar syrups produced from starch is achieved by gradient LC analysis with evaporative light
scattering detection (ELSD). Moreover, rice syrups contain specific marker compounds called
SM-R (specific marker - rice syrup, analysis by LC-MS/MS) and/or TM-R (trace marker - rice syrup,
analysis by ICP-MS) which allow for a sensitive detection in adulterated honey. Low temperature
acid inverted sugar syrups can be detected by determination of the citric acid, ascorbic acid or
sulfur dioxide content of honey. Finally, adulteration of honey with caramel color (E150c, E150d),
probably used to mask addition of sugar syrup or ultrafiltration of honey is detectable by LCMS/MS analysis. In addition to the analytical aspects, the interpretation of the measured values
and assessment of authenticity based on collected reference data from authentic samples is
illustrated.
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38
Annual
meeting
of
the
International
Honey
Commission
OC10.Developmentandvalidationofaliquidchromatographic-tandemmass
spectrometricmethodforthedetectionoffumagillininhoney:useinastability
study.
Els Daeseleire (1); Wim Reybroeck (1)*
1: Flemish Government, Institute for Agricultural and Fisheries Research (ILVO), Technology and Food
Science Unit, Brusselsesteenweg 370, 9090 Melle, Belgium
Nosemosis, a damaging adult bee disease caused by Nosema apis or N. ceranae was in the
past often treated with fumagillin, an antibiotic commercialized under the name Fumidil B®.
Despite the fact that the use of Fumidil® is presently not permitted, the product is still available
in the United Kingdom. In the past it was shown that residues of antibiotics can be present
in honey after treatment. In order to be able to screen honey samples on the presence of
fumagillin, a liquid chromatographic tandem mass spectrometric was developed and validated
according to Commission Decision 2002/657/EC. After addition of the internal standard
roxithromycin, 5 g of honey was dissolved in 10 ml of water and a clean-up was performed
on a C18-SPE column. Five µl of the final extract was injected into the LC-MS/MS system on a
Waters X-Terra® C8 column kept at 25°C. The instrument used was an Acquity UPLC (Waters)
system. Gradient elution was performed with a mobile phase mixture of methanol and 2 mM
ammonium formate in 0.01 % formic acid. Mass spectrometric detection was performed on a
Quattro Ultima Pt® instrument, following the transition of the precursor ion to three product
ions. For the internal standard, one transition was measured. Performance characteristics
evaluated were specificity, linearity, recovery, repeatability, intra-laboratory reproducibility,
decision limit, detection capability and expanded measurement uncertainty. For specificity,
honey was spiked with compounds belonging to the groups of the tetracyclines, sulfonamides,
nitro-imidazoles, phenicols and quinolones at 10 µg/kg. No interference was detected at the
retention time of fumagillin that could give rise to a false non-compliant result. For linearity, a
correlation coefficient of at least 0.9988 was obtained in honey. Eight blank honey samples were
spiked at three different concentrations being 3, 4.5 and 6 µg/kg. Quantification was performed
making use of a calibration curve in matrix. Recoveries ranged between 102 and 105 % and CVs
for repeatability were between 4 and 5 %. To determine the intra-laboratory reproducibility, the
above experiment was repeated two times and CVs ranged from 6 to 10 %. Decision limit was
determined as the concentration for which a signal to noise ratio of minimum 3 was obtained
for the three product ions and was determined at 3 µg/kg. Detection capability of the method is
3.3 µg/kg and the expanded measurement uncertainty is 24.6 %.
The method was used to determine the stability of fumagillin in honey. Honey was spiked
at 20 µg/kg with fumagillin and was analysed immediately. The rest of the sample was kept at
room temperature in the dark. After two weeks the concentration of fumagillin was reduced
with 29 %, while after four weeks concentration was reduced with 52 %. The final results will be
discussed at the symposium.
The method was also used to analyse 20 honey samples from 2011 but as fumagillin is
unstable in honey, the negative results obtained have no significance. Samples of 2012 will
be analysed immediately after the harvest in order to check if fumagillin is no longer used in
practice.
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39
Annual
40
meeting
of
the
International
Honey
Commission
Annual
meeting
of
the
International
Honey
Commission
OC11.Optimizationofpolarimetricmethodforspecificrotationdeterminationin
honey.
S. Serrano (1)*; I. Rodríguez (1); F. Rincón (1).
1: Department of Food Science and Technology. University of Córdoba
The polarimetric method for determination of specific rotation in honeys (International
Honey Commission, 1997) was optimized using a Plackett-Burman experimental design
augmented to resolution IV via fold-over by increasing to thirty two runs, the following eleven
ACFs were investigated: origin of honey (blossom or honeydew), honey liquefying temperature,
honey liquefying time, pre-filtration, sample weight, water volume, Carrez 1 volume, Carrez 2
volume, filtration, polarimeter tube length, polarimeter type (analogical or digital).
This method originally published in 1997 (Apidology) was improved in 2002. At present
it does not show results on collaborative trials and precision of the method, repeatability and
reproducibility. These reasons demand new research and revision for this analytical method.
Identification of key factors and subsequently optimized conditions, ensuring enhanced
accuracy and precision when determining specific rotation in honeys are obtained.
____________
41
Annual
meeting
of
the
International
Honey
Commission
OC12. Sugar analysis by a multi-sensor system: applying to honey samples.
Luís G. Dias (1)*; Mara E.B.C. Sousa (1); António M. Peres (1,2); Jorge Sá Morais (1);
Letícia Estevinho (1); Adélio A.S.C. Machado (3)
1: CIMO - Escola Superior Agrária, Instituto Politécnico de Bragança, Bragança, Portugal
2: LSRE - Laboratory of Separation and Reaction Engineering - Associate Laboratory, LSRE/LCM, Escola
Superior Agrária, Instituto Politécnico de Bragança, Portugal
3: LAQUIPAI - Departamento Química, Faculdade de Ciências, Universidade do Porto, Portugal
One of the emerging approaches for analysis of liquid samples with complex matrices is the
Electronic Tongue (ET) since it allows evaluating tastes by calibration, mimicking the human
tongue. The ET records a pattern of signals that depends on the matrix solution composition,
which information is extracted into qualitative and quantitative information by multivariate
statistical methods.
The chemical sensors used in these devices, usually, differ from those of the traditional
chemical sensors because they have the ability to obtain global information about the solution
(cross-sensibility sensors, the signal results from sensitization to various substances). Selectiveions sensors (high selectivity for detecting substances) may also be included in the analytical
system, allowing cross-information as well as specific information about the sample matrix.
Recent works with ET showed the wide range of applications such as, classification of honey
(Dias, 2008), detection of milk adulterations (Dias, 2009), detection of protein levels (gliadins) in
different foodstuffs (Peres, 2011) and classification of soft drinks accordingly to different added
fruit juice contents (Dias, 2011).
Moreover, the analytical performance suggests that ETs could have a wider set of applications
as, quantification if the substances to be analysed are major compounds in the sample.
With the aim of test this hypothesis, an all-solid-state potentiometric ET was developed and
has being tested to quantify fructose and glucose contents, which are important constituents
of the food products, as an alternative tool for the quantification of these sugars in real samples.
Several multivariate data treatments for quantitative analysis of these two sugars (MLR, PLS
and others) are considered as well as, their application to the results obtained in honey samples
analysis.
Acknowledgements: Collaboration of the Portuguese National Beekeepers Federation in providing honey
samples is gratefully acknowledged.
1) Dias, L A; Peres, A M; Vilas-Boas, M; Rocha, M A; Estevinho, L; Machado, A A S C (2008) An electronic tongue
for honey classification, Microchim. Acta 163: 97-102.
2) Dias, L A; Peres, A M; Veloso, A C A; Reis, F S; Vilas-Boas, M; Machado, A A S C (2009) An electronic tongue
taste evaluation: identification of goat milk adulteration with bovine milk, Sensors & Actuators: B. Chemical
136: 209-217.
3) Peres, A M; Dias, L G; Veloso, A C A.; Meirinho, S G; Sá Morais, J; Machado, A A S C (2011) An electronic
tongue for gliadins semi-quantitative detection in foodstuffs, Talanta 83: 857–864.
4) Dias, L ;, Peres, A M; Barcelos, T P; Sá Morais, J; Machado, A A S C (2011) Semi-quantitative and quantitative
analysis of soft drinks using an electronic tongue, Sensors & Actuators: B, Chemical 154: 111–118.
______________
42
Annual
meeting
of
the
International
Honey
Commission
OC13. Royal Jelly: Quality, Safety and Authenticity.
Ayse Bakan (1)*; Ozlem Aslan (1); Neşe Aslı Öncü (1); Banu Bahar (1); Ebru Pelvan (1); Cesarettin
Alasalvar (1); Nihat Ozcan (1); Canan Dogan (1); İlknur Demirtaş (1); Senem Akkuş Çevikkalp (1);
Mustafa Yaman (1); Esra Agel (1); İmge Oktay (1); Hayrettin Ozer (1); Aslı Elif Sunay (2); Taylan
Samancı (2); Tuğçe Daştan (2)
1: TUBITAK MRC Food Institute P.O.Box:21 41470 Gebze Kocaeli, Turkey
2: Altıparmak Gıda San. ve Tic. Koll. Şti. Çavuşbaşı Cad. No:70 Çekmeköy 34782 İstanbul, Turkey
Royal jelly is a product that is secreted from the hypopharyngeal and mandibular glands of
nurse honeybees and has a complex composition of proteins, sugars, fatty acids, sterols, phenols,
minerals and other components. It has been widely used in commercial medical products,
healthy foods and cosmetics in many countries and it has a big potential in the economics of
the beekeeping sector. However, there are no standards at European level for the royal jelly,
like the other bee products except honey. This means that it is possible to find products in the
market under these labels without any quality and authenticity control. This might imply a risk
for both the consumer and the small and medium enterprises of the sector, which not only
have to compete with cheaper products produced in Eastern and South American countries but
also compete with a product that sometimes is not even royal jelly. APIFRESH, EU founding FP7
project, aims for the standardisation of royal jelly as well as pollen.
In this research, pure royal jelly samples which were produced for the project in European
countries (Italy, Greece, Bulgaria, and Turkey) were analyzed for determination of their quality,
safety and authenticity. Royal jelly samples from China were also analyzed to make a comparison.
The physicochemical composition of royal jelly samples was determined by analyzing
moisture, ash, protein, lipid, fructose, glucose and saccharose content, pH, acidity, colour as
well as 10-HDA which is one of the most important criteria of royal jelly authenticity. 13C/12C
isotopic ratio and pollen spectra of the samples were also defined for this purpose. In addition
these parameters, concentrations of trace (Bi, Cd, Pb, Sn, W, Sb, Cr, Co, Se) and mineral (Ca, Mg,
K, Na, Zn, Fe, Cu, Mn) elements were systematically investigated in botanically and geologically
defined royal jelly samples. The royal jelly samples were also analyzed for their sensorial (aroma,
taste and aftertaste) and microbiological properties ( yeast, mold, coliform, E.coli and S.aureus
load ).
Biological activities of royal jelly are mainly attributed to the phenolic compounds and
the antioxidant acitivity is basically due to the presence of these constituents. Therefore, total
phenolics and total antioxidant activity of royal jelly were defined. In addition, vitamins ( Vitamin
B1, Vitamin B2, Vitamin C And Vitamin B5), sterol composition (cholesterol, brassicasterol,
24-methylene cholesterol, campesterol, B-sitosterol, D5-avenasterol, 5,24-stigmastadienol, D7stigmastenol, D7-avenasterol) and fatty acid composition (palmitic acid, oleic acid, linoleic acid,
linolenic acid, stearic acid, heptadecanoic acid) were determined as other healthy compounds.
While palmitic acid, oleic acid, linoleic acid and linolenic acid were found as the predominant
fatty acids; campesterol and 24-methylene cholesterol were shown as two important sterols in
royal jelly samples respectively. Antimicrobial activity of royal jelly on the bacteria Salmonella,
E.coli and S. aureus was also studied. It was shown that a very small amount of royal jelly (0.03
g) showed clearly inhibitory effects against these bacteria (108 bacteria /1 ml). All results of
European RJ samples were compared with Chinese royal jelly.
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43
Annual
44
meeting
of
the
International
Honey
Commission
Annual
meeting
of
the
International
Honey
Commission
OC14. Bee Feeding Influences Royal Jelly Composition.
Wytrychowski Marine (1); Daniele Gaelle (1); Batteau Magali (1); Guibert Sylvie (1); Casabiance
Hervé (1)*
1: CNRS UMR 5280- Département SCA - Echangeur de Solaize - Chemin du Canal - 69360 Solaize - France
Feeding bees with artificial sugars and/or with exogenous proteins increase royal jelly (RJ)
production. Such beekeeping practices are currently observed all around the word. The aim of
this work is to define analytical criteria that allow detecting bee feeding practises.
We have developed and validated analytical methods to quantify various parameters in RJs:
water, protein, 10-HDA, amino acid, sugar contents. Moreover stable isotope ratios (δ13C/12C and
δ15N/14N) have been measured in RJ samples by isotope ratio mass spectrometry.
This study demonstrates, from the analysis of around 800 RJs, that bee feeding leads to a
RJ analytically different from a genuine one, obtained in more natural conditions namely from
nectar and pollen collected by the bees in the environment of the hives. Indeed some analytical
criteria of both kinds of RJs are different and allow distinguishing between them.
Natural samples (500) are obtained through a collaboration with French beekeepers
belonging to the GPGR (Groupement des Producteurs de Gelée Royale). GPGR is representative
of the French production, being responsible for more than 80% of it. Membership of this group
implies respecting a quality convention concerning the production, harvest, conservation
and trade of RJs. Feeding bees with artificial sugars is not allowed during RJ production ; only
honey and pollens can be used in the case of there being insufficient honeydew. The samples
have been collected in different French areas during the course of three harvesting seasons
representing various geographical and botanical origins.
In comparison, RJs from feeding experiments with artificial sugars and/or with exogenous
proteins have been provided by some apiarists in order to evaluate the impact of such a feeding
on the analytical composition of RJ. The feeding experiments have been carried out with
different C3 and C4 sugars (cane sugar, maize hydrolysate, beet, hydrolysed starch, inverted
syrups) and with 2 proteins (yeast and soy powders).
In addition, around 150 commercially available RJ samples and 50 RJs produced by Italian
beekeepers as representative materials produced and traded worldwide have been analyzed by
the same methodology.
Thanks to some parameters including isotopic measurements of δ13C/12C (Daniele, 2011) and
the contents of some particulate sugars (Daniele, 2012), we have succeeded in distinguishing
between RJs produced from natural sources and those obtained from sugar feeding
(Wytrychowski, 2012). We have demonstrated that RJs commercially available in France are
mainly produced by artificial feeding. Actually, no norm or recommendation for RJ definition,
production and quality control exist.
While an ISO working group proceeds to a draft since 3 years, no real consensus has been
obtained at this time. Contrary to honey (codex alimentarius), feed sources of bees are not
described for RJ, leading to different production procedures in the world. Those differences in
beekeeping procedures induce physicochemical modifications of the royal jelly produced.
1) G. Daniele, H. Casabianca, (2012) Sugar composition of French royal jelly for comparison with commercial
and artificial sugar sample, Food Chemistry, DOI 10.1016/ j.foodchem.2012.03.008.
2) G. Daniele, M. Wytrychowski, M. Batteau, S. Guibert, H. Casabianca, (2011) Stable isotope ratios
measurements of royal jelly samples for controlling production procedures, impact of sugar feeding,
Rapid Commun. Mass Spectrom. 25: 1929-1932.
3) M. Wytrychowski, G. Daniele, H. Casabianca, (2012) Combination of sugar analysis and stable isotope ratio
mass spectrometry measurement to detect artificial bee feeding for royal jelly production, Analytical and
Bioanalytical Chemistry, DOI: 10.1007/s00216-012-5934-6.
______________
45
Annual
meeting
of
the
International
Honey
Commission
OC15.ChemicalCompositionofDehydratedBeePollenProducedinBrazillian
States Paraná and Santa Catarina.
José Augusto Gasparotto Sattler (1)*; Adriane Melo (1); Ellen de Souza Soares (1); Luciana
Barbosa dos Santos(1); Davi Figueiredo Sampaio Meira(1); Ligia Bicudo de Almeida-Muradian(1)
1:University of São Paulo, São Paulo, Brazil
According to Instruction No. 3, January 19, 2001, from the Ministry of Agriculture, Livestock
and Supply, pollen is defined as the result of clumping of the pollen of flowers, made by worker
bees, by salivary substances and their nectar, which is collected in the hive entrance. According
to this standard dried bee pollen should have no more than 4% of moisture, minimum of 1.8%
in lipids, minimum of 8% in proteins and maximum of 4% in ashes. The aim of this study was to
determine the chemical composition of ten samples of dehydrated bee pollen from the south
region of Brazil and to compare with the limits established by Brazilian legislation. This region
has subtropical climate, differently from the rest of the country. Six samples were collected from
Parana and four from Santa Catarina state during the months of September and December of
2011. For the protein determination it was used the micro-Kjeldahl method, with the conversion
factor of 6.25. Lipid determination was made using the intermittent Soxhlet extractor using
diethyl ether as solvent. Ashes were determined gravimetrically after incineration of the sample
oven at 550°C until constant weight. For moisture determination the samples were dried
under infra-red radiation, in equipment composed of electronic precision balance Micronal
(B160), adapted with infra-red dryer Mettler Toledo (LP16), with adjustments to the intensity of
radiation emitted, so that the sample reaches 85 °C. The levels ranged from 1.0% to 4.05% for
moisture, 12.3% to 21.8% for proteins, 0.9% to 5.9% for fat and 1.34% to 2.0% for ashes. Among
the ten samples analyzed only three from the State of Rio Grande do Sul and one from the state
of Santa Catarina showed moisture values above the limit established by law. It is noted that
this sample may have been collected with a high percentage of moisture in nature and that the
dehydration process of the bee pollen was not enough to reduce the humidity to acceptable
value. All samples showed values appropriate for proteins. In the composition of lipids only two
samples had values below the minimum standard established by the Brazilian legislation, which
may be related to characteristics of the botanical origin of the samples. It is concluded that three
of ten samples of dehydrated bee pollen showed some parameter outside the limits established
by Brazilian legislation. There is no regulation in the European Union for bee pollen.
Acknowledgments: The authors acknowledge FAPESP for its financial support regarding research and
Doctorate scholarship granted to Adriane Alexandre Machado de Melo. CNPq for granting a Scientific Initiation
Sholarship to Davi Figueiredo Sampaio Meira and Productivity in Research Scholarship granted to Ligia Bicudo
de Almeida-Muradian. CAPES for the Master’s scholarship granted to José Augusto Gasparotto Sattler.
______________
46
Annual
meeting
of
the
International
Honey
Commission
OC16.AContributiontotheEstablishmentofBromatologicalReferenceValues
for Colombian Pollen in the Context of Latin American Regulations.
Martha Quicazán (1)*; Carlos Fuenmayor (1); Consuelo Díaz (1); Carlos Zuluaga (1)
1: Instituto de Ciencia y Tecnología de Alimentos, Universidad Nacional de Colombia, Bogotá D.C.
Standards for bee pollen are not established either by Codex Alimentarius, European
Union or FDA. Meanwhile, there is a more dynamic regulatory environment in Latin America;
Argentina, Brazil, Mexico, Salvador and Honduras have current regulations for pollen (Brasil,
2001; Almeida,2005). Colombia, because of its geographical location, has some areas with
higher annual production than the world average (6-7 kg / hive). Altiplano Cundiboyacense,
a central region located more than 2000 masl, produces about 40kg/ hive; this fact constitutes
an important advantage for alternative development. Colombian government drives different
strategies; one of them was focused on the elaboration of a technical standard project for
pollen, based on chemical characterization and contextualization of the results with existing
regulations in Latin America. Between 2008 and 2011, 196 pollen samples were collected in
Altiplano Cundiboyacense from Apis mellifera hives fitted with traps. The pollen was dried,
cleaned and milled previous to the analyses: moisture, pH, free acidity, protein, fat, ash, dietary
fiber and minerals (Na, K, Ca, Fe, Mg, Cu, Zn). There is no international consensus around moisture
content; Brazil, Salvador and Honduras accept 4% as maximum, while Mexico and Argentina set
a maximum of 8%; 72% of Colombian pollen samples agree with the first standard and 90% with
the second. About 96% of pollen samples had pH value between 4 and 6, which is consistent
with the requirements in other countries. Although Brazil, Salvador and Honduras allow 300
mEq/kg for free acidity, only 85% of Colombian samples meet this value. In consequence, it
is not recommended to establish yet a Colombian requirement for this parameter The protein
content of total Colombian samples is higher than the minimum included in Latin American
standards (18g/100g dry basis) and some of them exceed the maximum established by the
Mexican standard; 38% of the samples have more than 25g/100g dry basis (maximum value in
Honduras regulations). The standards of Mexico and Brazil set a minimum for fat (1.5% and 2.5%
respectively); all Colombian samples satisfy this requirement and 50% of them exceeded 6.5
g/100 g (maximum established in Mexican standard); therefore it is not appropriate to propose
a maximum lipid content and it could be set a minimum limit of 1.8g/100g dry basis for this
parameter. Only Brazilian and Mexican standards include crude fiber requirement and do not
consider dietary fiber and specific minerals values. Standards of Brazil, Argentina, Salvador and
Honduras establish a minimum of 4 g/100g, dry basis, for ash content; meanwhile, the Mexican
standard establishes a range between 1.5 and 2.2g/100g, dry basis. The results show that 100%
of the samples meet the first requirement and only 22% the second standard because they
exceed 2.2 g/100 g. Therefore, 3.5 g/100g for ash content is a permissible maximum.
1) Almeida-Muradian, L; Pamplona, L; Coimbra, S; Barth, O (2005) Chemical composition and botanical
evaluation of dried bee pollen pellets, Journal of Food Composition and Analysis 18: 105-111.
2) Brasil (2001). Regulamentos Técnicos de Identidade e Qualidade, de Apitoxina, de Cera de Abelha, de
Geléia Real Liofilizada, de Pólen Apícola, de Propólis, de Extrato de Própolis.Instrucao normativa n 3 de
2001. Brasilia, Brasil: Ministério da Saúde.
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47
Annual
48
meeting
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International
Honey
Commission
Annual
meeting
of
the
International
Honey
Commission
OC17.Amodelproject:ResearchPlatformonHoneyandOtherBeeProducts.
Aslı Elif Sunay (1)*; Taylan Samancı (1)
1: Honey Packers, Industrialists and Exporters Association
Cooperation between universities, industry and public bodies is very important for regional
and global competitiveness and sustainable development. A platform was formed by this
project between 17 parties that brings universities, industry and public bodies together to work
on bee products for the development of the sector.
The aim of this project is to produce and share information that will enhance the
development of scientific research, new products and systems about contribution of bees,
honey and other bee products on healthy living and environment by a platform (BEE PLATFORM)
consisting of universities, industry, non-governmental organizations and public bodies. Raising
social awareness and consumer consciousness by using audio-visual information technologies
on production, consumption and health effects of safe bee products is also aimed. Focusing
on economic activities that depend on know-how, Bee Platform intends to provoke nonconventional uses of bee products by increasing consumption of these products for their
biological activities and health effects.
In this project, 30 pollen samples (26 from Turkey, 4 from Spain), 8 propolis samples (3 from
Turkey, 4 from China, 1 from Argantina), 19 royal jelly samples (13 from China, 6 from Turkey)
and 66 honey samples (from Turkey) were analyzed for their chemical properties, residues,
contaminants, microbiological content, total phenolics, flavonoids, antioxidant capacity and
profile of phenolic substances. Bioavailability of bee products was also tested. 12 honey samples
with the highest antioxidant capacities and all propolis samples were used to test effects of
these products on different breast cancer races in vitro. Under the scope of the project, training
seminars on good hygiene and agricultural practices and health effects of bee products were
organized both for producers and packers. Also a documentary film was prepared for TV
describing the production stages of bee products, life of beekeepers and health effects of these
products besides contribution of bees to the environment. The story of the film was published as
a book with three other books on health effects of bee products, good hygiene and agricultural
practices and honey plants.
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49
Annual
meeting
of
the
International
Honey
Commission
OC18. Physicochemical characteristics of Colombian pot-honey.
Consuelo Díaz-Moreno (1)*; Carlos Fuenmayor (1); Carlos Zuluaga (1); Martha Quicazán (1)
1: Instituto de Ciencia y Tecnología de Alimentos, Universidad Nacional de Colombia, Bogotá D.C.,
Colombia
New data have been obtained about composition and physicochemical properties of
Colombian pot-honeys. The national technical standard for pot-honey was done considering the
data published by Souza et al. (2006) but this document has no information regarding Colombian
stingless bee honeys. This general lack of knowledge has had several consequences. First,
there are difficulties in regulating the adulteration and falsification of stingless bee pot-honey.
Second, the geographic location of Colombia and its mega-biodiversity have been identified as
advantages for meliponiculture, but so far there were not conducted studies on the quality of
pot-honey. This study analyzed pot-honey samples from different genera native from several
regions of Colombia. The number of analyzed honey samples varies according to the genus and
the species (Frieseomelitta sp.(6 samples), Melipona compressipes (12 samples), Melipona favosa
(7 samples), Melipona eburnea (7 samples), Melipona sp. (18 samples), Nannotrigona testaceicornis
(3 samples), Nannotrigona sp. (4 samples), Paratrigona sp. (4 samples), Partamona sp. (2 samples),
Plebeia sp. (1 sample), Scaptotrigona limae (2 samples), Scaptotrigona sp. (4 samples), Tetragona
sp. (21 samples) and Tetragonisca angustula). The largest number of samples corresponded to
the genus Melipona and Tetragonisca (45 samples) because there is wide breeding of those
species. Performed physicochemical analyses were: moisture, carbohydrates (glucose, fructose
and disaccharides), ash, minerals, color, pH, free acidity, diastase activity, HMF, conductivity and
specific rotation. This work summarizes existing information regarding the physicochemical
properties and quality of Colombian pot-honey. The ranges for physicochemical features of
Melipona genus were: moisture (24.8 – 27.6 g/100g), fructose (36.7 – 39.3 g/100g), glucose (30.9
– 38.5 g/100g), disaccharides (3.1 – 6 g/100g), ash (0.01 – 0.20 g/100g), color (34.4 – 80 mm
Pfund) and the ranges for physicochemical features of Tetragonisca angustula were: moisture
(22 – 26.6 g/100 g), fructose (24.7 – 35.6 g/100g), glucose (17.1 – 29.9 g/100g), disaccharides (1.8
– 6.4 g/100g), ash (0.21 – 0.28 g/100g), color (49 – 70 mm Pfund), pH (4.2 – 4.5), free acidity (39,2
– 62.1 meq/kg), diastase activity (16.7 – 25.9 DN), HMF (1.3 – 3.3 mg/kg), conductivity (601 - 715
µS/cm), specific rotation (1.3 – 3.9). It is necessary to continue the characterization process that
leads to a better knowledge of this valuable product.
1) Souza B, Roubik D, Barth O, Heard T, Enríquez E, Carvalho C, Villas-Bôas J, Locateli J, Persano-Oddo L,
Almeida-Muradian L, Bogdanov S, Vit P. (2006) Composition of stingless bee honey: setting quality
standards. Interciencia, Interciencia 31: 867-875.
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50
Annual
meeting
of
the
International
Honey
Commission
OC19.CharacterizationoftheSerbianHoneyUsingModernAnalyticalMethods.
Dušanka Milojković-Opsenica (1); Nebojša Nedić (2); Silvio Kečkeš (3); Biljana Marošanović (4);
Filip Andrić (1); Maja Natić (1); Živoslav Tešić (1)*
1: Faculty of Chemistry, University of Belgrade, Belgrade, Serbia
2: Faculty of Agriculture, University of Belgrade, Belgrade, Serbia
3: Analysis, Belgrade, Serbia
4: SP Laboratory, Bečej, Serbia
The analytical characterization of honey in order to detect adulteration or to verify
geographical or botanical origin has been a scientific long-time preoccupation. Despite the long
tradition of Serbian honey production, the issues of physical, chemical and sensory properties of
this important naturally product have not been so far systematically studied. Although Serbia is
not a big country, differences in climate, soil and plants provide a solid base for the production
of different types of honey, such as those most often produced: acacia (Robinia pseudoacacia),
sunflower (Helianthus annuus) and linden (Tilia cordata) honey. In the scope of this work a total
of 380 different honeys from three unifloral botanical origins (acacia, sunflower, and linden), as
well as multifloral honey from various regions of Serbia have been characterized and classified
by applying modern analytical (ICPMS, IC, HPLC-Orbitrap, IRMS) and chemometrical methods.
Besides to basic physicochemical parameters (water content, pH, free acidity, conductivity
and optical rotation), content of minerals (K, Ca, Mg, Na, Cd, Ni, Cr, Zn, Cu and Mn), sugar
profile (glucose, fructose, sacharose, trehalose, maltose, isomaltose, gentiobiose, turanose,
melesitose and isomaltotriose), amino acid contents (Ala, Arg, Asp, Cys, Glu, Gly, His, Leu, Ile,
Met, Lys, Phe, Pro, Ser, Thr, Tyr, and Val), as well as polyphenols contents have been determined.
Spectrofluorimetric data have been recorded for each sample with excitation wavelengths in
the range of 250-500 nm, and C-13 stable isotope ratio has been determined. On the basis of
the results obtained, Serbian honey was compared with honeys from different countries. All
results obtained are in the ranges proposed by the European legislative. By application of
different chemometric methods (Principal Component Analysis, Cluster Analysis and Linear
Discriminant Analysis) the most important criteria for authenticity assessment have been
established. Physicochemical data are sufficient to provide good differentiation among three
botanical species of honeys: linden, acacia and sunflower. Multifloral honey was successfully
separated from all three unifloral types by the means of spectrofluorimetric data. All samples
have strong excitation bands in the range of 250-300nm, that are missing in all three unifloral
species. Specific excitation spectra originates from the presence of tryptophan which further
influenced by differences in chemical composition.
Also, specific group of polyfloral honey samples originating from Zlatibor area can be
distinguished from the rest of studied samples based primarily on mineral content as well as
some physicochemical parameters. The serpentine silicate soil mostly characteristic for Zlatibor
area, which is rich in magnesium and nickel compounds, strongly influences specific flora, and in
the same way provides specific mineral fingerprint of collected multifloral honeys.
Acknowledgements: This work has been supported by the Ministry of Education and Science of Serbia, Grant
No. 172017 and by FP7 RegPot project “Reinforcement of the FCUB towards becoming a centre of excellence in
the region of WB for molecular biotechnology and food research” (FCUB-ERA GA No. 256716). The authors are
grateful to The Association of the Beekeeping Organizations of Serbia for kindly collection of honey samples.
______________
51
Annual
52
meeting
of
the
International
Honey
Commission
Annual
meeting
of
the
International
Honey
Commission
OC20.Chemical,SensoryandMelissopalynologicalFeaturesofCroatianCommon
Sage (Salvia officinalis L.) Honey.
Dražen Lušić (1,4)*; Nada Vahčić (2); Gian Luigi Marcazzan (3); Vladimir Mićović (1); Darija Vukić
Lušić (1); Duška Ćurić (2); Francesca-Vittoria Grillenzoni (3)
1: Department of Environmental Health, Faculty of Medicine, Rijeka, Croatia
2: Faculty of Food Technology and Biotechnology, Zagreb, Croatia
3: CRA-API, Bologna, Italy
4: Croatian Apitherapy Society
Total of 49 representative honey samples were covered by the study of specific features
and characterization of common sage honey (Salvia officinalis L.) deriving from the North
Croatian littoral area and produced during the period of three consecutive years (2005-2007).
Honey samples were submitted to melissopalynological assessment in order to obtain detailed
characterization of common sage honey melissoplynological profile comprising representation
of sage pollen in honey as well as pollen spectra of both nectarous and non nectarous
botanical species, including also the presence of honeydew elements. Chemical and physical
analyses were carried out comprising 11 analytical quality parameters: moisture, pH, acidity,
electrical conductivity, reducing sugars, sucrose, invertase, diastase, specific optical rotation,
hydroxymethylfurfural and proline. Sensory analysis was completed too and sensory parameters
of common sage honey (visual, odour, taste and tactile characteristics) has been reviewed and
described in details with particular accent to the presence or absence of defects in honey.
Research of specific features of common sage honey was complemented with detailed analysis
of climatic elements that prevailed on apiary sites during blossoming of the Salvia officinalis L.
and expressed impact of climate elements on quality features of honey samples was detected.
Characteristic underrepresentation of common sage pollen was determined; furthermore 106
necatrous and 35 non nectarous botanical species and families were identified, all sharing
physical areal and time of flowering. Characteristic ranges of common sage honey analytical
parameters were determined in comparison to different unifloral honeys: lower values for
sucrose and invertase, medium values for electrical conductivity, pH, proline and specific optical
rotation and highly expressed values for reducing sugars, acidity and diastase activity. Based
on the results of sensory analysis, detailed sensory profile was proposed. Significant honeydew
influence was noticed, strongly influenced by climatic elements prevailing on apiary sites during
the sage blossoming period; however honey samples still detaining their nectarous character.
____________
53
Annual
meeting
of
the
International
Honey
Commission
OC21.SensoryprofileofsomeofthemainItalianunifloralhoneys.Development
and possible uses.
Gian Luigi Marcazzan (1)*; Massimiliano Magli (2); Lucia Piana (3); Angela Savino (4);
Mariassunta Stefano (5)
1: CRA-API, Agricultural Research Council, Honeybee and Silkworm Research Unit, Via di Saliceto 80,
40128-Bologna, Italy
2: Istituto di Biometeorologia, Consiglio Nazionale delle Ricerche, Via Gobetti 101, I-40129 Bologna, Italy
3: Piana Ricerca e Consulenza - via Emilia levante 840 I-40024 Castel San Pietro Terme BO, Italy
4: ICQRF, Ispettorato centrale della tutela della qualità e repressione frodi dei prodotti agroalimentari, Via
Irno 11, 84098 Salerno, Italy
5: Centro Agrochimico Regionale – A.S.S.A.M.- Via Roncaglia, 20 - 60035 Jesi AN, Italy
Sensory evaluation enables us to distinguish the botanical origin of honey and to identify
and quantify certain defects (fermentation, impurities, off-odours and flavours). It also plays an
important role in defining product standards and in the related controls regarding botanical
denominations or other specific labels. Moreover, it is an essential part of consumer preference/
aversion studies (Piana L. et al 2004).
In this work we have focused our attention on botanical denomination and we have studied
the sensory profiles of five principal unifloral Italian honeys: chestnut honey (Castanea sativa
Miller); citrus honey (Citrus spp.); eucalyptus honey (Eucalyptus spp.); black locust honey (Robinia
pseudoacacia L.) and honeydew honey.
The aim of the study is to propose an alternative method (“profile method”) congruent
with modern techniques through which it can be proven as to whether a honey conforms to its
declared botanical origin.
In earlier studies conducted by the sensory group within the IHC (International Honey
Commission), a harmonized glossary was developed (Piana L. et al 2004) and two methods
of evaluation proposed: the routine method and the “yes/no” classification method. These
methods are based on the ability of trained assessors to evaluate the correspondence of a
declared monofloral honey to a standard that they have memorized. The alternative method
we are going to propose takes into consideration the attributes and their intensity. For each
unifloral honey type the most significant attributes have been selected and estimated. In this
way a typical profile has been produced. Consequently, in unifloral honey assessment if the
profile is within the limits of the expected profile it is considered to conform to the botanical
declaration of origin.
In our study the unifloral honey profile was developed by a panel of specialized expert
assessors. The preparation of the samples and the olfactory and olfactory-gustatory
characteristics have been performed as described by Piana (Piana L. et al 2004).
Each taster smelled and tasted the honey under consideration, in order to analyse the
olfactory and gustatory characteristics. Twelve attributes were evaluated. Attributes were
assessed by marking a 10 cm line scale and quantified measuring of the location of the mark
from the origin. The data obtained for the 12 attributes were used to define the sensory profile
of each sample using the median values.
The number of samples studied varied between 9 and 11 for each unifloral typology and
were used in order to define the unifloral sensory profile that would include not only the best
representative unifloral honey, but also the acceptable “commercial” unifloral honey.
Each unifloral profile has highlighted specific characteristics. Principal component analysis
(PCA) and linear discriminate analysis (LDA) clearly permit the discrimination of the studied
unifloral honey.
1) Piana M.L.; Persano Oddo L.; Bentabol A.; Bruneau E.; Bogdanov S.; Guyot Declerck C. (2004) Sensory
analysis applied to honey: state of the art, Apidologie 35 special issue, 26-37.
____________
54
Annual
meeting
of
the
International
Honey
Commission
OC22. The main honeydew producing insects in Greece.
Sofia Gounari (1)*, George Goras (2), Chysa Tananaki (2) , Andreas Thrasyvoulou (2)
1: Lab. of Bee Pathology, Institute of Veterinary Research, NAGREF, Neapoleos 25, 15341, Athens, Greece
2: Lab. of Apiculture – Sericulture, Dep. Of Agriculture, Aristotle University of Thessaloniki, Greece
It is estimated that at least 70% of the annual production of honey in Greece derives from
honeydew.
Concretely 60% derives from the honeydew produced from Pinus halepensis and Pinus
brutia. Another 10% from the honeydew producing insects parasite on fir trees, both on Abies
cephalonica and Abies borisii. More than that in specific years beekeepers take a good honey
harvest from oak trees also, especially from Q. frainetto and Q. cerris.
The most important honeydew producing insect is Marchalina hellenica (Coccoidea:
Marchalinidae), which parasites on pine trees. It has one generation per year. Overwinters as
3rd instar nymph and produces large quantities of honeydew from March to April, before its
last mould to female adult. The crawlers of the new generation appear on the trees in June.
Marchalina attach to the tree and feed sucking the phloem sup. As the insect grows the droplets
of honeydew become bigger. Beekeepers transfer their beehives again in pine forests from
August to October.
Five honeydew-producing insects had been found on fir trees. Three aphids, Cinara abieticola
(Cholodkovsky), Cinara pectinatae (Nördl.) and Mindarus abietinus (Koch) and two scale insects
Physokermes spp.and Eulecanium sericeum (Lindiger). The most important of these is the genus
Physokermes which until recently believed that it has 3 species in Greece, Ph. hemicryphus, picea
and innopinatus. In course of the recent survey of scale insects in Greece, a new for science
species was found and described as Physokermes hellenicus Gounari and Kozár, sp. n.(in press).
By this new data, the species number in Physokermes genus increased to 12 and to 8 species in
the Palaearctic Region now. By four Physokermes species Greece has most species in the western
part of the Plaearctic Region. Physokermes spp. has one generation per year. Overwinters as a 2nd
instar nymph and produce large quantities of honeydew from the end of May until the first ten
days of July. Then the female adult starts to ovulates under its shell.
The main honeydew producing insects on oak trees are one scale insect Parthenolecanium
rufulum and two aphids Lachnus roboris roboris and Tuberculoides annulatus. The main periods
for honeydew exploitation from honeybees are early spring (March) and then summer (August).
____________
55
Annual
56
meeting
of
the
International
Honey
Commission
Annual
meeting
of
the
International
Honey
Commission
OC23.Studyoftheperceptionofaliquidandtranslucenthoneyandcreamed
honey in France.
Robin Azemar (1)*; Valérie Nevers (1); Myriam Laurie (1); Benjamin Poirot (1); Alain Le Bail (2)
1: APINOV, La Rochelle France
2: ONIRIS, Nantes, France
Honey is principally eaten under two forms: liquid and creamed. In France, sales for each of
them represent 50% (Meneau 2011). Creamed honey, stored in jars, is usually melt in tea or coffee,
eaten on bread and directly on spoon. Packing honey under liquid form makes its use easier
and/or original (plastic squeezer, straw, etc.). In many countries (Turkey, India, etc.) professionals
of the beekeeping industry declare that their consumers prefer eating liquid honey. Since 2007,
APINOV Company is developing a process which enables to store honey in liquid form for a long
period (12 months minimum) without affecting its organoleptic properties (Poirot 2011). The
aim of this study is to assess, thanks to a sensory analysis, the interest that can represent -or notliquid honey instead of creamed honey on a sample group composed of sixty people (minimal
number of participants needed to ensure statistical validity of the study according to the French
norm AFNOR XP V09-501). In order to carry out this study, five descriptors were submitted to the
judgment of the subjects through a hedonic test. Samples were composed of a raw sunflower
creamed honey and then of the same honey transformed into liquid and translucent form
thanks to APINOV process. First of all, two visual descriptors were tested: color and consistency.
The aim was to compare the attraction for the two samples before their consumption in order
to detect which one of the two products consumers would be visually interested in before
tasting them. Then, taste and mouth feel were submitted to the judges. The objective here was
to understand consumers’ feeling after having tested the two products. Finally, people had to
grade the samples on a global assessment. For each descriptor, a two-factor analysis of variance
without replication was realized on the results. Results of statistical tests did not show significant
product effect on any descriptors. Thus, consumers do not have any preference for a product.
In opposition, the subject effect (consumer effect) was significant for 4 descriptors out of 5. It
means that French consumers do not have the same expectations when they eat honey. These
conclusions corroborate the theory that the two phases are eaten in France (50/50 for each
phase). Feelings of national beekeeping professionals are thus validated by this study.
1) Meneau, C (2011) Etude de consommation du miel en France. Etude de marché, APINOV.
2) Poirot, B (2011) Procédé de traitement du miel pour obtenir un miel liquide et limpide, Patent n° EP 2 294
929 (A1).
____________
57
Annual
meeting
of
the
International
Honey
Commission
OC24. Pyrrolizidine Alkaloids in Swiss honey and bee pollen.
Christina Kast (1)*; Arne Dübecke (2); Verena Kilchenmann (1); Katharina Bieri (3); Gudrun
Beckh (2); Cord Lüllmann (2)
1: Zentrum für Bienenforschung, Agroscope Liebefeld-Posieux ALP-Haras, Schwarzenburgstrasse 161, 3003
Bern, Switzerland
2: Quality Services International GmbH, 28199 Bremen, Germany
3: Institut für Pollenanalyse, Talstrasse 23, 3122 Kehrsatz, Switzerland
Pyrrolizidine alkaloids (PAs) are toxic plant products that have been found in various foods
including bee products. Recent studies indicate, that the level of contamination with PAs can
vary depending on the geographical and botanical origin of the product. The geographically
central location of Switzerland within Europe accounts for various different climate zones and
botanical settings comparable to the surrounding countries. Therefore, Switzerland serves as a
model to study PA contamination of honey and bee pollen produced in different climate zones.
Honeys were collected from five different climate zones during the production years 2009
to 2011. Our study comprised 47 floral honeys composed of polyfloral or mixtures of blossom/
honeydew honeys, as well as several unifloral honeys, such as rape, tilia, acacia and chestnut
honeys. Furthermore, we included 14 alpine mountain flower honeys and 10 honeydew honeys.
Bee pollen of the production years 2010 and 2011was obtained from the Swiss association for
the production of bee pollen. 22 bee pollen samples originated mainly from regions north of
the Alps and the alpine region. They were mixed samples collected in spring time between the
months of April and June.
The PA concentration was determined by target analysis using an HPLC-MS/MS-system,
allowing the detection of 18 different PAs and PA-N-oxides that have been found mainly in the
genera Echium, Eupatorium and Senecio. Qualitative (DIN 10760:2002-05, Berlin) and quantitative
pollen analysis confirmed the presence of Echium pollen in PA positive samples.
Our study suggests, that PAs are found more frequently in honeys from Swiss alpine regions
and areas south of the alps as compared to honeys from areas north of the Alps. PAs found
in the genera Echium were the most frequently found PAs in honey, followed by PAs found in
the genera Senecio and probably Eupatorium. In bee pollen, we most frequently detected PAs
originating from Echium plants, as well as PAs, that may originate from Eupatorium plants. Such
plants should be avoided around the beehive in order to prevent contamination of bee product
with toxic PAs.
In further studies with daily collected bee pollen, we plan to investigate the temporal course
of PA contamination. Furthermore, we are interested in identifying further plant species that
contribute to the PA contamination of bee products.
____________
* Presenting author: Christina. [email protected]
58
Annual
meeting
of
the
International
Honey
Commission
OC25.Effectofhighcelldensityfermentationsontheoptimisationofmeadfermentation.
A.P. Pereira (1,2)*; A. Mendes-Ferreira (1); L.M. Estevinho (2); A. Mendes-Faia (1)
1: IBB-Institute for Biotechnology and Bioengineering, Centre of Genomics and Biotechnology,
Universidade de Trás-os-Montes e Alto Douro, Vila Real, Portugal
Portugal
Mead is a traditional drink, containing 8-18% (v/v) of ethanol, which results from the
alcoholic fermentation of diluted honey performed by yeasts. It has been reported that
mead fermentation is a time-consuming process, often taking several months to complete,
depending on the type of honey, yeast strain and honey-must’s composition. An important
objective of mead makers is to reduce the fermentation time without decreasing the quality
of the end product. It has been shown that significant time can be saved in the fermentation
process by increasing the pitching rate, i.e., the amount of suspended yeast cells added to a
batch fermenter. Therefore, the aim of this study was to determine the adequate inoculum size
of two commercial winemaking strains of Saccharomyces cerevisiae (Lalvin QA23 and Lalvin ICV
D47) for the optimisation of mead fermentation. Honey must was prepared according to the
recipe developed by our team, supplemented with potassium tartrate, pH adjusted to 3.7 with
malic acid and the nitrogen concentration adjusted to 267 mg/L with diammonium phosphate.
The appropriate amounts of inoculum were pitched into the honey-must to obtain five different
pitching rates. Several parameters were determined during the fermentation to evaluate the
effect of the inoculum size on yeast growth, fermentation profile and mead composition. Minor
differences between the two strains in respect to growth kinetics were detected. As expected
the increasing of the inoculum size resulted in significant increases in cell biomass and CFUs’
numbers but also decreased the yeast net growth. The time required to reach the same stage
of fermentation ranged from 24 to 96 hours depending on the inoculum size. In accordance to
the results obtained the strain ICV D47 appears to be more suitable for the production of high
quality meads, although the strain QA23 provided better fermentation profile. However, sugars
were not fully consumed and about 25 mg/L of assimilable nitrogen remained at the end of all
fermentations. This is the first study of the effect of inoculum size on the optimisation of mead
fermentation, however further research is needed to improve its quality.
____________
59
Annual
60
meeting
of
the
International
Honey
Commission
Annual
meeting
of
the
International
Honey
Commission
OC26.Monitoringthegravitationalreflexoftheectoparasiticmite Varroadestructor:Anovelbioassayforassessingtoxiceffectsofacaricidesandpossible
acaricidal properties of honeybees’ products.
Alexandros Papachristoforou (1)*; Chrisovalantis Papaefthimiou (1); George Theophilidis (1)
1: Laboratory of Animal Physiology, School of Biology, Aristotle University of Thessaloniki, 54 124
Thessaloniki, Greece
In an effort to develop an innovating method for accurate evaluation of the efficacy of
acaricide compounds against Varroa destructor, we recorded and analyzed its gravitational
reflex (attempt to return to an upright position when fixed in a dorsal side-down position.
To monitor the force generated by the movement of Varroa’s sternal shield, a sensitive
isometric force displacement transducer was used. The transducer was mounted on a
micromanipulator, while its probe was extended to a micropin. The transducer was gently
attached either on the surface of Varroa’s sternal shield.
The pulses generated by the force of the rhythmic expansions had an average duration
of 3.11 s, force (amplitude) of 73 µN, and frequency of 0.228 Hz. Surprisingly, Varroa’s sternal
expansions, generated by this gravitational reflex were lasting for over 20 h and the parameters
examined (duration, force and frequency) remained constant for the first 10 h f recording, whilst
significant changes occurred only after 15 h.
Through these recordings we were able to investigate not only the effect of acaricides
applied through contact or evaporation but also some honeybee products, such as propolis
solutions or honeybees’ pheromones for their possible action against Varroa destructor.
____________
61
Annual
meeting
of
the
International
Honey
Commission
POSTER COMMUNICATIONS
Abst.
Author(s)
Title
PC1.
Carla Pereira; Alexandra Silva;
Lillian Barros; Miguel Vilas-Boas;
Isabel C.F.R. Ferreira
Honey as a potentiator in the bioactivity of lemon
flavoured black tea.
PC2.
Lillian Barros; Montserrat
Dueñas; Ana Maria Carvalho;
Celestino Santos-Buelga;
Isabel C.F.R. Ferreira
Phenolic compounds as markers for the botanical origin
of unifloral honeys from Castanea sativa flowers.
PC3.
Melia V. González-Porto;
Cristina Pardo
Healthy and nutricional components on bee pollen.
PC4.
Ahmad Al- Ghamdi;
Mohammad Javed Ansari;
Yehya Al-Attal
Inhibition of Candida albicans Biofilm Through The Use of
Some Saudi Arabian Honey.
PC5.
Einar Etzold;
Birgit Lichtenberg-Kraag
Big city or biosphere reserve: Authentification of regional
honeys.
PC6.
Rail Khismatullin;
Vassya Bankova; Natalya
Gavrilova; Galina Legotkina
Botanical origin and antimicrobial activity of propolis of
Perm Territory.
PC7.
Rail Khismatullin;
Roman Kaygorodov;
Elena Zubova
Influence of the honey’s botanical origin on the content
of vitamins C, В3 and В6.
PC8.
Adriane A. Machado de Melo;
José A. Gasparotto Sattler;
Antioxidant Activity of Dehydrated Bee Pollen Produced
in Rio Grande do Sul State, Brazil.
PC9.
Igor Jerković;
Zvonimir Marijanović;
Dragan Bubalo
Preliminary Research of Organic Extractives from Satureja
spp. Honey (Croatia).
PC10.
Teresa Szczęsna; Krystyna
Pohorecka; Ewa Was;
Monika Pytlak;
Helena Rybak-Chmielewska;
Katarzyna Jaśkiewicz
Acaricide residues determination in honey and beeswax.
PC11.
Jelena Kečkeš; Jelena Trifković;
Ljubiša Stanisavljević;
Kristina Lazarević; Milica Jovetić;
Živoslav Tešić;
Dušanka Milojković-Opsenica
Classification of Serbian Unifloral Honey on the Basis of
Their Amino Acids Profiles.
PC12.
Benjamin Poirot; Valérie Nevers;
Régis Brunet;
Marie-Jeanne Gourmaud;
Frédéric Bataille
Development of a decontamination process for beeswax.
PC13.
Capucine Meneau;
Benjamin Poirot ;
Myriam Laurie
Honey consumption in France: opinion survey on french
average.
PC14.
Beata Madras-Majewska;
Elżbieta Rosiak
Evaluation of chosen quality parameters of honey from
the Polish market.
62
Annual
meeting
of
the
International
Honey
Commission
PC15.
Vanilda A. Soares de Arruda;
Alexandre Vieira dos Santos;
Ortrud Monika Barth;
Alex da Silva de Freitas;
Phenolic compounds, flavonoid content and antioxidant
activity of dried bee pollen samples collected in Sao
Paulo, Brazil.
PC16.
Tananaki Chrysoula;
Ioanna Chekimian;
Thrasyvoulou Andreas
Propolis’s extraction techniques and antioxidant activity
of the extracts.
PC17.
Ewa Waś; Teresa Szczęsna;
Helena Rybak-Chmielewska;
Piotr Semkiw; Piotr Skubida
Determination of alkanes in beeswax from comb
foundation adulterated with paraffin after rebuilding by
bees using GC-MS technique.
PC18.
Massimiliano Valentini;
Caterina Cafiero;
Silvia Gardini
HRMAS-NMR (High Resolution Magic Angle Spinning
NMR) technique applied to the characterization of
botanical definition and geographical origin of honey.
PC19.
Escuredo Olga; Dobre Irina;
Rodríguez-Flores M. Shantal;
Seijo M. Carmen
Sugar composition and crystallization rates of different
honey types.
PC20.
Escuredo Olga;
Rodríguez-Flores M. Shantal;
Seijo M. Carmen
Antioxidant activity of honeys from Galicia (NW Spain).
PC21.
Ana Pascual-Maté;
Silvia Gardini; Miguel Ángel
Fernández-Muiño;
Maria Teresa Sancho
Sugar Profiles of Honeys from Castilla y León (Spain).
PC22.
Ofélia Anjos; Fátima Peres;
Paulo Antunes; José Rodrigues
ATR-FTIR Spectroscopy for determination of free acidity,
electrical conductivity, ash and total polyphenol content
in honey.
PC23.
Silvia Cristina Ferreira Iop;
Marcus Vinícius de Pinho Pires
Standardization and regulations of stingless bees honey
in Brazil: challenges and advances.
PC24.
Dalibor Titera; Michal Bednar;
Josef Stich
Are some enzymes in honey foreign or regular?
PC25.
Dalibor Titera; Michal Bednar;
Hana Vinsova
Are oligosaccharides in honey foreign or regular?
PC26.
X. Feás; J.A. Seijas;
M.P. Vázquez-Tato; T. Dias;
L.M. Estevinho
Evaluation of the effect of Hydrogen Peroxide (H2O2) in
the antimicrobial activity of honey.
PC27.
Ana Pascual-Maté; M. Teresa
Sancho; Miguel A. FernándezMuiño; Rosires Deliza;
Patricia Vit
Understanding sensory information of Melipona favosa
pot-honey.
PC28.
Miguel Ángel Fernández-Muiño;
Ana Pascual-Maté;
Maria Teresa Sancho
Volatile and semivolatile compounds of heather
(Ericaceae) honeys from Burgos (North Spain).
PC29.
Nair Alua; Ana Balola;
Celeste Serra
Antioxidant and Physicochemical Properties of Honeys
from Algarve Region.
63
Annual
meeting
of
the
International
Honey
Commission
PC30.
Mara E.B.C. Sousa;
Luís G. Dias; António M. Peres;
Letícia Estevinho;
Adélio A.S.C. Machado
Evaluation of an electronic tongue for honey
classification according to its pollen analysis.
PC31.
Patricia Combarros-Fuertes;
Mª Eugenia Tornadijo;
José María Castro; Leticia M.
Estevinho; José María Fresno
Antimicrobial activity of quality brands Spanish honeys.
PC32.
Isabel Valença; Nelma PértegaGomes; Marta Cunha;
Carmen Jerónimo;
Cristina Almeida Aguiar; Fátima
Baltazar
Portuguese propolis decreases PC-3 cell glycolytic
metabolism.
PC33.
Márcia Cruz; Ana Cunha; Rui
Oliveira;
Cristina Almeida Aguiar
Antigenotoxic potential of Portuguese propolis.
PC34.
André Santos; Miguel Vilas Boas;
Mª João Sousa
Bee products as natural compounds in Cosmetics.
PC35.
Joana Coelho; Soraia I. Falcão;
Alexandre Bera;
Renato Januario Sousa
Ligia B. Almeida-Muradian
Miguel Vilas-Boas
Phenolic composition of Brazilian propolis from Minas
Gerais.
PC36.
Soraia I. Falcão; Nuno Vale;
Susana M. Cardoso; Cristina
Freire; Miguel Vilas-Boas
Cistus Ladanifer L. secreted flavonoids as marker
compounds for differentiating Portuguese propolis types.
PC37.
Andreia Tomás; Soraia I. Falcão;
Miguel Vilas-Boas
Phenolic content of bee bread from Northeast of
Portugal.
PC38.
Mário Gomes; João Casaca;
Luís G. Dias;
Miguel Vilas-Boas
Trade barriers and economic impact of organic
beekeeping in Portugal.
PC39.
Veronika Kmecl; Aleš Gregorc;
Marinka Kregar; Romana Rutar;
Maja Smodiš Škerl;
Helena Baša Česnik
Quality control and authenticity review of honey from
Slovene market in the period 2007 - 2011.
PC40.
Lourdes Corredera;
Consuelo Pérez-Arquillué
Susana Bayarri; Regina Lázaro;
Antonio Herrera
Determination of colour and trace metal levels in honeys
from different environmental origins.
PC41.
Josipa Vlainić; Ivan Kosalec;
Silvija Zlatar; Toni Vlainić;
Dražen Lušić
Ivana Tlak Gajger
Antibacterial Activity of Various Honey Types Against
Different Pathogens.
PC42.
Kačániová Miroslava;
Hleba Lukáš; Chlebo Róbert;
Vukovič Nenad
Antibacterial activity of bee collected pollen.
PC43.
Kačániová Miroslava;
Hleba Lukáš; Chlebo Róbert;
Vukovič Nenad
The antimicrobial effect of honey against selected
antibiotic resistant bacteria.
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PC44.
Valerie Gaudin;
Celine Hedou;
Eric Verdon
Evaluation and Validation of a Biochip Multi-Array
technology for the screening of antibiotic residues in
honey according to the European guideline for the
validation of screening methods.
PC45.
Sayed Mazaher Sayedi;
M.B.Farshineh Adl; Ali Reza
Abassian; Mahmoud Salesi;
Mohammad Behjatian Esfahani;
Shahaboddin Mosharaf;
S.M.K.Deylami
Study of effect of various processing procedures on some
of the honey characteristics.
PC46.
M. Graça Miguel;
M. Dulce Antunes;
Smail Aazza; Joana Duarte;
M. Leonor Faleiro
Antioxidant and antimicrobial activities of “água-mel”
from Portugal
PC47.
Adrian Bugeja Douglas;
Everaldo Attard
Physicochemical of Maltese honey: an attempt to
determine seasonal variations in honey
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PC1. Honey as a potentiator in the bioactivity of lemon flavoured black tea.
Carla Pereira (1); Alexandra Silva (1); Lillian Barros (1); Miguel Vilas-Boas (1); Isabel C.F.R. Ferreira (1)*
1: CIMO-ESA, Instituto Politécnico de Bragança, Campus de Santa Apolónia, Apartado 1172, 5301-855
Bragança, Portugal
Honey plays an important role in human health by combating damage caused by oxidizing
agents, namely reducing the risk of heart disease, cancer, immune-system decline, cataracts,
and inflammatory processes, among others. The antioxidant activity of honey has been
extensively reported and attributed to the antioxidants present that include both enzymatic
and non-enzymatic substances (Ferreira et al., 2009). The same properties have been attributed
to black tea (Camellia sinensis) and lemon (Citrus limon), which contain powerful antioxidant
compounds such as flavonoids and vitamin C, respectively (Milasiene et al., 2007; Guimarães
et al., 2010). Although it has already been demonstrated that black tea, lemon and honey
have antioxidant activity, nothing is reported about the effects of their concomitant use. In
the present study, those effects were evaluated in infusions of lemon flavoured black tea with
three different kinds of honey (light amber, amber and dark amber) from Lavandula stoechas,
Erica sp. pl. and other indigenous floral species from Northeast Portugal, a region with high
amounts of this food product. The antioxidant properties were measured by different in vitro
chemical and biochemical assays: scavenging effects on DPPH (2,2-diphenyl-1-picrylhydrazyl)
radicals, reducing power, inhibition of β-carotene bleaching and inhibition of lipid peroxidation
in brain cell homogenates by TBARS (thiobarbituric acid reactive substances) assay. Antioxidant
compounds such as ascorbic acid, total phenolics and flavonoids were also quantified. Data
obtained showed that the use of honey (dark amber>amber>light amber) potentiates the
antioxidant activity of lemon flavoured black tea, which is already a mixture of two powerful
antioxidant matrixes (lemon and tea), increasing reducing power and lipid peroxidation
inhibition properties, but also the antioxidants content such as phenolics, flavonoids and
ascorbic acid. Moreover, the mixture of honey and black tea is much more favourable in the
antioxidants point of view than the use of honey alone.
Acknowledgements: Foundation for Science and Technology (FCT, Portugal)- strategic project PEst-OE/AGR/
UI0690/2011 to CIMO and SFRH/BPD/4609/ 2008 grant to L. Barros.
1) Ferreira, I C F R; Aires, E; Barreira, J C M; Estevinho, L M (2009) Antioxidant activity of Portuguese honey
samples: different contribution of the entire honey and phenolic extract, Food Chemistry 114: 1438-1443.
2) Guimarães, R; Barros, L; Barreira, J C M; Sousa, M J; Carvalho, A M; Ferreira, I C F R (2010) Targeting excessive
free radicals with peels and juices of citrus fruits: grapefruit, lemon, lime and orange, Food and Chemical
Toxicology 48: 99-106.
3) Milasiene, R; Sawicka, K; Kornysova, O; Ligor, M; Maruska, A; Buszewski, B (2007) Evaluation of antioxidant
activity of green and black tea (Camellia sinensis) and rooibos (Aspalathus linearis) tea extracts by means
of HPLC with reaction detector, Archieves in Separatoria Acta 5: 27-33.
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PC2.Phenoliccompoundsasmarkersforthebotanicaloriginofunifloralhoneys
from Castanea sativa flowers.
Lillian Barros (1); Montserrat Dueñas (2); Ana Maria Carvalho (1); Celestino Santos-Buelga (2);
Isabel C.F.R. Ferreira (1)*
Bragança, Portugal
2:Grupo de Investigación en Polifenoles (GIP-USAL), Facultad de Farmacia, Universidad de Salamanca,
Campus Miguel de Unamuno, 37007 Salamanca, Spain
The determination of the plant origin for honey and other bee products is of great interest.
One of the procedures used to determine the floral origin is pollen analysis, but nowadays other
analytical methods that could complement that approach are also available. In this perspective
there have been many studies that correlate plant source with the presence of certain
compounds (Soler et al., 1995; Tomás-Barberán et al., 2001). Recent studies have revealed that
the analysis of flavonoids and other phenolic compounds might be a very promising technique
to study the plant origin of honey and other bee products.
Chestnut trees (Castanea sativa Miller) are of great importance in several Portuguese
northeastern areas where chestnut honey is considered of high quality. The flowers of both
sexes (sometimes only male flowers which mature first) arranged in long upright catkins appear
in late June to August. The ripe pollen produces a characteristic heavy sweet odour.
In the present work, an exhaustive characterization of the phenolic compounds present in C.
sativa flowers was carried out by HPLC-DAD-ESI/MS following procedures previously described
(Barros et al., 2012). The phenolic profile obtained could be used to select some patterns that
may possibly be useful to determine honey botanical source.
C. sativa flowers presented high levels of total phenolic compounds (18.973 ± 40 mg/Kg,
fresh weight), being hydrolysable tannins (14.873 ± 110 mg/Kg) the most abundant group found.
A trigalloyl HHDP glucoside was one of the main hydrolysable tannins present. Flavonols such
as myricetin, quercetin, kaempferol and isorhamnetin derivatives were also found in C. sativa,
being quercetin-3-O-glucoside and quercetin-3-O-glucuronide the major flavonols present.
The obtained profile should be compared to other honey floral origins in order to point some
phenolic compounds specific of Castanea sativa, which could be used as botanical markers for
unifloral chestnut honeys.
Acknowledgements: Strategic project PEst-OE/AGR/UI0690/2011- CIMO; FCT, POPH-QREN and FSE (SFRH/
BPD/4609/2008- L. Barros). Consolider-Ingenio 2010 Programme (FUN-C-FOOD, CSD2007-00063)- GIP-USAL;
Spanish “Ramón y Cajal” Programme- M. Dueñas.
1) Barros, L; Dueñas, M; Carvalho, A M; Ferreira, I C F R F, Santos-Buelga C (2012) Characterization of phenolic
compounds in flowers of wild medicinal plants from Northeastern Portugal, Food Chemical and Toxicology
50: 1576-1582.
2) Soler, C; Gil, M; García-Viguera, C; Tomás-Barberán F A (1995) Flavonoid patterns of French honeys with
different floral origin, Apidologie 26: 53-60.
3) Tomás-Barberán, F A; Martos, I; Ferreres, F; Radovic, B S; Anklam, E (2001) HPLC flavonoid profiles as markers
for the botanical origin of European unifloral honeys, Journal of the Science of Food and Agriculture 81:485496.
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PC3. Healthy and nutricional components on bee pollen.
Melia V. González-Porto (1); Cristina Pardo (2)*
1: Centro Agrario de Marchamalo (CAR), Guadalajara, Spain
2: Universidad Complutense de Madrid, Spain
Bee pollen is an important natural product used therapeutically in medicine, pharmaceutical
industries and as nourishment. We have studied and characterized bee pollen samples from the
Iberian Peninsula hives. Our analysis includes: 1) Palynological identification by optical microscopy;
2) Healthy components content in fresh bee pollen as antioxidant components, polyphenols, by
Folin-Cicalteu method and vitamin C by HPLC-UV techniques; 3) Nutritional values in dry bee
pollen, as proteins by Kjeldahl method and sugars by HPLC techniques. The main results are:
1) We have found that pollen from families Cistaceae (Cistus, C. ladanifer, Helianthemun) and
Rosaceae (Prunus, Crataegus, Rosa) appears in the majority of the samples; pollen from Quercus
t. and Papilionaceae (Cytisus, Retama, Ornithopus, Anagallis, Vicia), Brassicaceae (Brassica,
Raphanus, Capsella), Asteraceae (Carduus, Crepis, Bidens, Centaurea, Cichoroideae), Ericaceae
(Erica, Calluna, Arbutus), Liliaceae and Capanulaceae families has a lower presence; and pollen
from Lamiaceae (Rosmarinus, Lavandula, Teucrium) and Cariophyllaceae families and Echium,
Juglans, Olea and Plantago appears occasionally. 2) The average of the polyphenols content
found is 1.20 g gallic acid/100 g pollen (range 0.48 - 2.08). We have observed a vitamin C content
average of 30.88 mg/100 g pollen, (range 4.90 - 77.64). In bee pollen samples dried at 40 ºC, the
vitamin C content drops strongly. For both variables, content of polyphenols and vitamin C, the
highest values were found in Rosaceae, Papilionaceae and Quercus t., whereas Cistaceae shows
lowest values. 3) The protein values have been analysed first using a portion of the whole pollen
sample and then the main pollen taxa has been analysed individually. The results in both cases
are very similar, 18.95% (range 13.4 - 26.96) and 19.93% (range15.82 - 23.46) respectively. Finally,
the main sugars found are glucose, 19.92% (range 13.33 - 24.15) and fructose 20.00% (range
14.71 - 23.65). Generally, sucrose is not detectable and other sugars, as turanose or maltose, are
in very low concentration. Similarly to the others components, the protein and sugars contents
show higher values for Rosaceae, Papilionaceae and Quercus t. From the above results we can
conclude that bee pollen from plants with succulent fruits or seeds, as Rosaceae, Papilionaceae
and Quercus t., have most healthy and nutritive components than bee pollen from plants with
dried fruits and seeds, as Cistaceae. The majority of Cistaceae bee pollen samples come from
Central Spain while the Rosaceae bee pollen samples are mostly from North-Central Spain, a
rich fruit trees region.
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PC4. Inhibition of Candida albicans BiofilmThroughThe Use of Some Saudi
Arabian Honey.
Ahmad Al- Ghamdi (1); Mohammad Javed Ansari (1)*; Yehya Al-Attal (1)
1: Chair of Eng. Abdullah Baqshan for Bee Research, Department of Plant Protection, College of food and
Agricultural sciences, King Saud University Riyadh, Kingdom of Saudi Arabia
Candida species, especially, Candida albicans, is a major human fungal pathogen. It is
a dimorphic fungus capable of causing superficial mucosal infections, as well as systemic
infections, in immunocompromised individuals. The factors responsible for its pathogenesis
are still not fully understood and increasing resistance to commonly used antifungal agents
necessitates the search for new formulations. The antifungal activity of honey against planktonic
C. albicans cells is well established. However, the effects of honey on C. albicans biofilm are yet
to be elucidated.
We investigated the effect of five different Saudi honeys from different plant sources on
biofilm formation in Candida albicans ATCC 10231. C. albicans biofilm were formed for 24 h at
37⁰ C in the 96 wells of microtitre plates. This biofilm inhibition by honey was concentration and
time dependent. The effect of honey was estimated using XTT reduction assay and the kinetics
of biofilm reduction was visualized by scaning electron microscopy (SEM) and fluorescence
microscopy (CSLM).
Of the five honey type tested. Sidr (Ziziphus) honey was found to be effective and four were
moderately effective.incubation with sidr honey lead to a clear reduction in size and structure
of mature biofilms. Concentration of sidr honey 30% (w/v) interfered with the C. albicans biofilm
formation.
Biofilm growth of C. albicans are highly resistant to antifungal agent such as azole derivatives
like fluconazole, as well as nystatin, chlorhexidine and amphotercin, and are known to have side
effect. Our findings indicate that different honey types are effective against C. albicans biofilms
inhibition. The significant antifungal activity of these honeys suggests that they could serve as a
source of compounds with therapeutic potential against Candida-related infections.
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PC5. Big city or biosphere reserve: Authentification of regional honeys.
Einar Etzold (1); Birgit Lichtenberg-Kraag (1)*
1: Institute for Bee Research, Hohen Neuendorf, Germany
Honey is characterized by the plants the bees have collected the nectar from. Hence one can
determine the botanical origin by extraction and identification of the pollen grains of a honey
sample. The fact that pollen analysis is also used for determination of the geographical origin
was used for characterization of regional honeys since certain areas differ in terms of landscape
and floral diversity.
In some regions local trademarks have already been established. Local trademarks support
the identification of the consumers with natural and cultural landscapes and assist them in
orientation for proven quality. The demands on quality of such regional products are usually
higher than given by legislation. This increases the image of the area.
In this project a biosphere reserve as well as the urban area of Berlin were chosen as
candidates for authentification.
The honeys from these regions were organoleptically and physico-chemically analysed. The
pollen spectrum was determined. Based on these investigations the typical floral sources of
these honeys were elaborated to create a profile for each area.
The first profile was worked out for the urban area of Berlin over a period of 7 years.
Depending on the season three categories can be distinguished by their typical pollen spectrum:
springtime, early summer and summer. Usually the pollen of five different plants made up 4565% of the total of the honey. The so called “marker-pollen“, which was found in every sample,
was robinia pseudoacacia in early summer and tilia spp. in summer. The main nectar sources in
Berlin were trees. Honey from each category contained pollen from myosotis. The fraction of
brassica pollen was very low (10-15% on average). The pollen diversity was high. Honey of the
summer category showed characteristic physical-chemical properties.
The profile of the honey from the biosphere reserve “Spreewald” was determined over a
period of 4 years. In contrast to the urban region of Berlin the main honey flow in this area was
characterized by wildflowers with trees and bushes being less represented. The pollen diversity
was high. In springtime, as expected, the fraction of nectar from Brassica was higher than in the
Berlin region. In summer the most important nectar sources were centaurea cyanus and trifolium
repens.
Based on the data of the project, the „Spreewälder Stiftungshonig“ was created by the
Bürgerstiftung Kulturlandschaft Spreewald e.V.. This provided support to local beekeepers in
respect to marketing their honey.
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PC6. Botanical origin and antimicrobial activity of propolis of PermTerritory.
Rail Khismatullin (1); Vassya Bankova (2); Natalya Gavrilova (1)*, Galina Legotkina (3)
1: Tentorium, Perm, Russia
2: Institute of Organic Chemistry with Centre of Phytochemistry, Bulgarian Academy of Sciences, Sofia,
Bulgaria
3: Center for Research and Certification Federal, Perm, Russia
Introduction. Antimicrobial activity is an important biological effect of propolis associated
with its chemical composition and botanical origin. The region under research is middle taiga,
north-eastern boundary of the honeybee’s habitat (Apis mellifera L.) with severe weather
conditions.
The purpose of our study is to determine the main plant sources of propolis of Perm Territory
and study the antimicrobial activity against Staphylococcus aureus.
Materials and methods. The studied samples of propolis were collected in different areas
of Perm Territory in 2010. Ethanol extracts (70%) of propolis (EEP) 1:10 were prepared in the
traditional way. The test culture was Staphylococcus aureus ATCC 6538P (105CFU/ml) incubated
on Mueller-Hinton agar containing EEP at 37 °C/24 h. The method of chromatography-mass
spectrometry has been used to study the chemical composition of propolis and its sources; the
identification of compounds was carried out according to the mass-spectral library NIST98 MS
and comparison of reference samples with spectra.
Results. More than sixty compounds have been identified in the samples. The chemical
composition is represented by flavanones, dihydroflavonols, flavones, flavonols, aromatic acids
and their esters, mono- and disaccharides. Specific taxonomic markers have been pointed out
for tree species: Betula pendula- sakuranetin, pectolinarigenin, Populus tremula - p-coumaric acid,
benzyl p-coumarate, di-p-coumaroyl acetyl glycerol, Populus nigra - pinobanskin 3-O-acetate,
dimethylallyl caffeate , 3-methyl-3-butenyl caffeate. Antimicrobial activity (on a dry basis) for
sample № 1 was 133.5 µg/ml, № 2 - 255.0 µg/ml, № 3 - 328.5 µg/ml, № 4 - 343.5 µg/ml , № 5 235.0 µg/ml.
Conclusions. The obtained data on the composition of propolis samples makes it possible
to characterize their origin from three sources – silver birch (Betula pendula), trembling poplar
(Populus tremula) and black poplar (Populus nigra). Results of antimicrobial activity correlate
with the total content in the samples of polyphenolic compounds and flavonoids.
Appendix. The research on the topic is still going on.
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PC7. Influence of the honey’s botanical origin on the content of vitamins C, В3
and В6.
Rail Khismatullin (1); Roman Kaygorodov (2)*; Elena Zubova (3)
1: The “Tentorium” Apicompany, Perm, Russia
2: Perm State National Research University, Perm, Russia
3: Center for Research and Certification Federal, Perm, Russia
Water-soluble vitamins C, B3 and B6 play specific and vital functions in human metabolism,
and their lack or excess can cause health problems. Vitamin C (ascorbic acid) carries out biological
functions of a reducer and coenzyme some metabolic processes, is an antioxidant. Vitamin B3
(nicotinic acid) participates in many oxidizing reactions of a cell, in metabolism of fats, fibers,
amino acids, fabric breath, processes of biosynthesis. Vitamin B6 (pyridoxine hydrochloride)
participates in formation of erythrocyte, is responsible for mastering by nervous cells of glucose,
is necessary for an albuminous exchange and takes part in an exchange of fats. Blossom honey
is produced by bees during collection and processing of nectar and pollen. Plant components
of honey are valuable food sources for bees and, along with sugars; they contain a wide range
of biologically active compounds, including amino acids, enzymes, vitamins, flavonoids and
others. According to different data sources (Bogdanov et al., 2006, 2008) honey has been found
to contain vitamins of B, K, E, A and C groups. The differences in honey flow conditions are
to be reflected in the composition of vitamins and functional properties of honey. There has
been a study on the content of vitamins C, B3 and B6 in the honeys of different botanical origin.
The vitamin content was determined in aqueous solutions of honey by reversed-phase HPLC
method with Ultimate 3000 (Dionex, Germany), column: Acclaim® C18; 3 μm; 120 Ǻ; 2,1×210
mm. As eluents have been used acetonitrile and 25 mМ КН2РО4 (рН 3,6). The chromatographic
conditions (content of mobile phase, gradient, flow rate) have been developed by the authors
of the article. For detection of vitamins were chosen wavelengths of 245 nm (C), 254 nm (B3)
and 280 nm (B6). Linden honey (from Tilia cordata Mill.), clover honey (from Trifolium repens
L.) and tributory honey (from Melilotus spp. L.) from different areas of the Perm Territory (Ural,
Russia) have been studied. In the analysis were used fresh the honeys collected in 2011. Each
type of honey has been studied in four multiple frequencies. Statistical data processing spent
with use of the program SigmaPlot 11.0 with Fischer-Test and method of the least significant
difference (LSD). Some regularities of the botanical factor’s influence on the formation of the
vitamin composition have been determined. The reason for differences in vitamin content can
be physiological and biochemical characteristics of different plant species. In linden honey was
significant lower content of vitamins C und B6 detected. Clover honey was characterized by
significant highest maintenance of vitamin B3. Honeys of the same botanical origin collected in
different parts of the Perm Territory had similar composition of vitamins, as proved by low values
of the standard deviation in the measurements. The work is aimed at the fundamental study
on the factors in melliferous landscapes and the development of technology of efficient use of
honey in the production of biologically active supplements, foods, cosmetics and medicines.
1) Bogdanov, S; Gallmann, P; Stangaciu S; Cherbuliez, T (2006) Bienenprodukte und Gesundheit, AlpForum
41: 3-50.
2) Bogdanov, S; Jurendic, T; Sieber, R; Gallmann, P. (2008) Honey for Nutrition and Health: A Review, Journal
of the American College of Nutrition 27: 677-689.
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PC8.AntioxidantActivityofDehydratedBeePollenProducedinRioGrandedoSul
State, Brazil.
Adriane Alexandre Machado de Melo (1)*; Davi Figueiredo Sampaio Meira (1); José Augusto
Gasparotto Sattler (1); Ligia Bicudo de Almeida-Muradian (1)
1: University of São Paulo, São Paulo, Brazil.
Bee pollen is a mix of bee-collected floral pollens with nectar and their salivary secretions.
It contains sugar, fiber, essential amino acids, saturated and unsaturated fatty acids, minerals,
vitamins and bioactive plant-derived secondary metabolites. Brazil is a vast country with a great
diversity of plants, and different types of bee pollen are produced due to the large variation
of the flower pollen sources. In recent years a number of studies have been conducted, but
the antioxidant activity of many samples remains unknown. The objective of this study was to
determine the antioxidant activity of eight dehydrated bee pollen samples produced in five
apiaries in Rio Grande do Sul State, Brazil. Samples were collected during August to December
2011. Antioxidant compounds were extracted with 70% ethanol, diluted to 1 mg/mL (bee
pollen/70% ethanol) and aliquots were evaluated using DPPH assay system. The decrease in
absorbance was determined at 517 nm at 30min and the antioxidant activity was expressed
as percentage of DPPH inhibition. The antioxidant activity in bee pollen samples ranged from
21.6% to 89.6%. Considering that the samples were collected from five different apiaries within
a five-month time interval, variations regarding antioxidant activity were expected as the
composition of bee pollen is straightly related to the plants visited by the bees for the collection
of flower pollen, its main constituent. Samples A and B, both collected in October, showed higher
antioxidant activity among the analyzed pollen being 89.6% and 89.5% respectively. Such activity
was statistically superior to the other dehydrated bee pollens (p<0.05). Two other samples (C and
D), both collected in September and at the same apiary where sample B was collected, showed
antioxidant activity of 76.7% and 61.8%. Samples E and F, the first collected in October and the
second in November, both at the same apiary, presented 21.6% and 45.1%, respectively. Sample
E presented the lowest antioxidant activity among the other dehydrated bee pollens (p<0.05).
It was observed that in the studied region, variations concerning vegetation occurred not only
from one apiary to the other, but also in the surrounding areas of a same apiary throughout
the year. Lastly, samples G and H, collected from different apiaries, in August (G) and December
(H) presented 80.8% and 47.5% respectively. The results are considerably relevant because the
samples were completely unknown and it is noticeable that in the assessed concentration, some
presented activity close to antioxidant standards such as BHA (96.7%) and alpha-tocopherol
(96.6%) in a concentration of 0.09 mg/mL.
Acknowledgments: The authors acknowledge FAPESP for its financial support regarding research and
Doctorate scholarship granted to Adriane Alexandre Machado de Melo. CNPq for granting a Scientific Initiation
scholarship to Davi Figueiredo Sampaio Meira and Productivity in Research Scholarship granted to Ligia Bicudo
de Almeida-Muradian. CAPES for the Master’s scholarship granted to José Augusto Gasparotto Sattler.
The opinions, hypothesis and conclusions or recommendations expressed in the article are those of the authors
and do not necessarily coincide with those of FAPESP.
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PC9. Preliminary Research of Organic Extractives from Satureja spp. Honey
(Croatia).
Igor Jerković (1); Zvonimir Marijanović (2); Dragan Bubalo (3)*
1: Faculty of Chemistry and Technology, University of Split, Split, Croatia
2: Polytechnic Marko Marulić in Knin, Knin, Croatia
3: University of Zagreb, Faculty of Agriculture, Zagreb, Croatia
The search for honey chemical markers indicating floral origin has been focus of many
studies in last 15 years in order to specify organic compounds (particularly headspace, volatile
and semi-volatile compounds) that are more closely associated with a particular honey type
and consequently would be helpful for a fast and reliable identification of its botanical source
(Kaškonienè, 2010). The isolation method may also have an effect on the results of analysis
of honey volatiles (Alissandrakis, 2005). In our previous study (Jerković, 2007) the headspace
chemical profile of S. montana honey was determined by headspace solid-phase microextraction
(HS-SPME). The main headspace volatiles were: ethyl benzoate, hotrienol, cis- and translinalool oxides followed by minor quantity of acetoin, 2-phenylethanol, phenylacetaldehyde,
β-phenylacetate, linalool and others. Hotrienol is probably formed during honey ripening.
Phenylacetaldehyde, 2-phenylethanol and benzaldehyde are ubiquitous in many honey types.
In the present research, ultrasonic solvent extraction (with the mixture pentane : diethyl ether
1 : 2 v/v (solvent A) and dichloromethane (solvent B)) was applied for the isolation of Satureja.
spp. honey volatile and semi-volatile compounds. Obtained extracts were analysed by gas
chromatography and mass spectrometry (GC-FID and GC-MS). Chromatographic profiles were
dominated by methyl syringate (61.2-68.8% (solvent A); 44.9-59.0% (solvent B)), not found
with HS-SPME due to its low volatility (the levels of methyl syringate are usually determined
by GC after methylation). Methyl syringate has already been reported as chemical marker of
Asphodel (Asphodelus microcarpus Salzm. et Viv.) honey with highest level (Tuberoso, 2009). It
is interesting to note that methyl syringate was detected in honeys obtained from plants of
different botanical families: black locust, rape, sweet chestnut, clover, lime, dandelion, sunflower,
thyme and manuka. In addition, methyl syringate is contributor of the honey antibacterial
activity since the honey peroxide-generating system does not account for all of the observed
antimicrobial activity.
1) Alissandrakis, E: Tarantilis P A; Harizanis P C; Polissiou M (2005) Evaluation of four isolation techniques for
honey aroma compounds, Journal of the Science of Food and Agriculture 85: 91-97.
2) Jerković, I; Marijanović, Z; Jelić, M (2007) Headspace volatiles from unifloral honeys of Satureja montana L.
and Salvia officinalis L. of Croatian origin isolated by solid phase microextraction (SPME). 55th International
Congress and Annual Meeting of the Society for Medicinal Plant Research, Planta medica 73: 896.
3) Kaškonienè, V; Venskutonis, P R (2010) Floral markers in honey of various botanical and geographical
origins: review, Comprehensive Reviews in Food Science and Food Safety 9: 620-634.
4) Tuberoso, C I G; Bifulco,E; Jerković, I; Caboni, P; Cabras, P; Floris, I (2009) Methyl Syringate: A Chemical Marker
of Asphodel (Asphodelus microcarpus Salzm. et Viv.) Monofloral Honey, Honey Journal of Agricultural and
Food Chemistry 57: 3895–3900.
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International
Honey
Commission
PC10. Acaricide residues determination in honey and beeswax.
Teresa Szczęsna (1)*; Krystyna Pohorecka (1,2); Ewa Was (1); Monika Pytlak (1); Helena RybakChmielewska (1); Katarzyna Jaśkiewicz (1)
1: Research Institute of Horticulture, Apiculture Division, Kazimierska 2 Str., 24-100 Puławy, Poland
2: National Veterinary Research Institute, Aleja Partyzantów 57 Str., 24-100 Puławy, Poland
Analytical procedures for acaricide residues determination in honey and beeswax were
elaborated. The study included the following substances: 2.4 – dimethylphenylformamide
(DMF), tau-fluvalinate, flumethrin, bromopropylate, acrinathrin, coumaphos, deltamethrin
- ingredients of active veterinary agents, which are most frequently used (for over 30 years)
for Varroa control. Since DMF is the major degradation product of amitraz, this substance was
selected for amitraz residues monitoring in honey and beeswax.
Solid Phase Extraction (SPE) and liquid-liquid techniques were adopted for acaricide
residues extraction from honey and beeswax, DMF determinations were performed using gas
chromatography with mass spectrometer (GC/MS) and other acaricides were determined by gas
chromatography with electron capture detector (GC/ECD).
Quantification limits for acaricide residues determination in honey and beeswax for DMF
was calculated on 0,05 mg/kg, for flumethrin – 1 mg/kg, and 0,5 mg/kg for other analysed
substances. Working range varied from 0,05 to 5 mg/kg for DMF, from 1 to 10 mg/kg for
flumethrin, and from 0,5 to 5 mg/kg for others. Correlation coefficient of linear dependence
of acaricide residues concentration on peak area in the working range received value over
0,995 for all analysed substances. Recovery ranged from 65 (for DMF) to 96% (for coumaphos),
75% on average for honey, and from 67 (for DMF) to 98% (for fluvalinate), 81% on average for
beeswax. Serial analysis of secondary reference material (honey and beeswax samples with
known quantities of analysed substances) show that coefficient of variation of repeatability for
all analysed substances was below 10% for both bee products, and day-to-day reproducibility
was below 15% for honey, and below 20% for beeswax.
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78
Annual
meeting
of
the
International
Honey
Commission
PC11.ClassificationofSerbianUnifloralHoneyontheBasisofTheirAminoAcids
Profiles.
Jelena Kečkeš (1); Jelena Trifković (2); Ljubiša Stanisavljević (3); Kristina Lazarević (4); Milica
Jovetić (4); Živoslav Tešić (2); Dušanka Milojković-Opsenica (2)*
1: Institute of Veterinary Medicine of Serbia, Belgrade, Serbia
2: Faculty of Chemistry, University of Belgrade, Belgrade, Serbia
3: Faculty of Biology, University of Belgrade, Belgrade, Serbia
4: Centre for Food Analysis, Belgrade, Serbia
Serbia has a very long tradition of beekeeping due to good geographical and climate
conditions and variety of botanical species. Acacia, linden and sunflower honey are the most
common types of honeys produced widely in different regions of Serbia, while basil and
rape honeys are produced in smaller quantities, as well as goldenrod and buckwheat honeys
as unique and very rare types. Regardless to beekeeping tradition and annual production of
honey amounting to about 5000 tones, chemical composition of Serbian honey has not been
systematically investigated so far. In our previous publication (Lazarević, 2012), classification
of three most important Serbian unifloral honeys based on their primary physicochemical
characteristics was described. In continuation of these studies, a free amino acids profile of
192 samples of seven different floral types of honey: acacia (Robinia pseudoacacia), linden
(Tilia cordata), sunflower (Helianthus annuus), rape (Brassica napus), basil (Ocimum basilicum),
giant goldenrod (Solidago virgaurea) and buckwheat (Fagopyrum esculentum) from six different
regions of Serbia, was performed in order to distinguish honeys by their botanical origin.
The content of free amino acids was determined by reversed-phase high-performance liquid
chromatography. A significant difference in content of different amino acids among botanical
species has been observed. The major amino acids present in all honey samples were prolin,
phenylalanine, alanine, arginine, tryptophan, and serine. Aspartic acid, glutamic acid, glycine,
and tyrosine were present in smaller quantities. It can be seen that the mean phenylalanine
content is much higher in samples of basil honey, comparing to sunflower, rape, buckwheat
and giant goldenrod, acacia and linden honey with very low percentage concentration. On the
basis of the established amino acid profiles, some important differences have been identified
among studied honey samples relying on the basic descriptive statistics data, and confirmed
by the multivariate chemometric methods. The Principal Component Analysis (PCA) has been
performed on the entire data set, in order to reveal the most important factors influencing
the grouping pattern among the several honey species. According to PCA, basil, acacia, rape
and sunflower honeys were fairly separated, while other three varieties cannot be satisfactory
distinguished. Basil honey samples were formed a small, well defined, cluster imposed with
phenylalanine, tyrosine, and leucine content. The model obtained by linear discriminant
analysis (LDA) could be used to unambiguously distinguish basil honeys from the rest of the
samples, and has moderate predictive power to separate genuine acacia, linden, sunflower, and
rape honeys.
Acknowledgements: This work has been supported by the Ministry of Education and Science of Serbia, Grant
No. 172017 and by FP7 RegPot project “Reinforcement of the FCUB towards becoming a centre of excellence in
the region of WB for molecular biotechnology and food research” (FCUB-ERA GA No. 256716). The authors are
grateful to The Association of the Beekeeping Organizations of Serbia for kindly collection of honey samples.
1) Lazarević, K B; Andrić, F; Trifković, J; Tešić, Ž; Milojković-Opsenica, D (2012) Characterisation of Serbian
unifloral honeys according to their physicochemical parameters, Food Chemistry 132: 2060–2064.
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Commission
Annual
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International
Honey
Commission
PC12. Development of a decontamination process for beeswax.
Benjamin Poirot (1); Valérie Nevers (1)*; Régis Brunet (2); Marie-Jeanne Gourmaud (2); Frédéric
Bataille (3)
1: APINOV, La Rochelle, France
2: Institut d’Analyse et d’Essais en Chimie de l’Ouest (IANESCO), Poitiers, France
3: VALAGRO Carbone Renouvelable Poitou-Charentes, Poitiers, France
Several scientific researches has highlighted that beeswax can be contaminated by medicines
used by beekeepers to overcome diseases (acaricides in particular) and by phytosanitary
products present in the environment (Bogdanov, 2006; Tene, 2009, Johnson, 2010). These
different compounds represent a threat for bees, which are very sensitive to them, but also for
human health because all bee products (honey, pollen, propolis, etc.) can be potentially polluted
through wax. This communication will deal with the development of a decontamination
process for beeswax. Firstly, a bibliographic study enabled to identify pollutants to eliminate
from beeswax by order of priority (depending on their detection frequency and concentration
in waxes). Then, an analysis method for 30 molecules considered as a priority was developed
by IANESCO: amitraz, atrazine, bifenthrin, bromoprophylate, chloramphenicol, chlorfenvinphos,
chlorothalonil, chlotianidin, coumaphos, cyhalothrin, cymiazole, cypermethrin, dialiphos,
α-endosulfan, fipronil, flumethrin, imidacloprid, malathion, methiocarb, parathion methyl,
p-dichlorobenzene, piperonyl butoxide, pyretrins I and II, streptomycin, sulfathiazole, taufluvalinate, tetracycline, thiametoxam, thymol, vinclozolin. 80% of them were successfully
analyzed by Gas Chromatography and/or High Performance Liquid Chromatography. In parallel,
two decontamination techniques for beeswax were tested by VALAGRO thanks to a central
composite design experimental plan: liquid/liquid extraction and deodorization (details of the
methods used are confidential). Results of these tests showed that deodorization method is
more promising than liquid/liquid extraction for the development of a decontamination process
for beeswax at industrial scale. Details about the percentage of residue elimination in beeswax
with these developed processes will be presented. For example, more than 98% of thymol can
be eliminated.
1) Johnson, R.M; Ellis, M.D; Mullin, C.A; Frazier, F. (2010) Pesticides and honey bee toxicity-USA, Apidologie
41: 312-331.
2) Tene, N; Vetillard, A;Treilhou, M (2009) Recherche de résidus de pesticides dans la cire d’abeille:
comparaison entre les cires de corps et d’opercules, Jean-Marie Barbançon et Monique l’Hostis, Ed., Journée
Scientifique Apicole, Saint Avold, pp. 97.
3) Bogdanov, S (2006) Contaminants of bee products, Apidologie 37: 1-18.
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81
Annual
meeting
of
the
International
Honey
Commission
PC13. Honey consumption in France: opinion survey on french average.
Capucine Meneau (1)*; Benjamin Poirot (1); Myriam Laurie (1)
1: APINOV, La Rochelle, France
The French beekeeping industry, as in many other countries, suffers a lack of statistics and
precise figures on national or regional production and consumption. No public surveys about
honey consumers’ habits are available either and the little information usable is now dating
from 2005. So, the aim of this survey was to fill this lack of data and make use of it for a better
comprehension of French consumers. This survey was conducted from September 24th 2010
to February 18th 2011 on 722 French average consumers. The questionnaire was composed of
23 questions (single and multiple choice, free expression and ranking questions) and available
from a link on the Internet. The maximal margin of error is of 3.65 percent. This work can thus
be qualified as reliant, such as national surveys conducted by French statistic institutes. First
results revealed that 90 percent of the participants eat honey. The principal places of purchase
represented (multiple answers were authorized) were hypermarkets and supermarkets (52
percent), beekeeper’s store (48 percent) and local markets (40 percent). Moreover, 57 percent
declared eating less than 5 medium-sized jars per year, and 18 percent more than 8 jars per year.
Participants were all asked their social and geographical situations, which enable to segment
results by age, profession and region depending on the need.
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82
Annual
meeting
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the
International
Honey
Commission
PC14.Evaluationofchosen qualityparameters ofhoney fromthe Polish market.
Beata Madras-Majewska (1)*; Elżbieta Rosiak (2)
1: Warsaw University of Life Sciences, Apiculture Division, Warsaw, Poland
2: Warsaw University of Life Sciences, Department of Food Hygiene and Quality Management, Warsaw,
Poland
Honey placed by bees in the honeycomb cells is usually characterized by the presence
of large number of microorganisms. They penetrate into the honey mainly from nectar or
honeydew. In many cases the reason may be water pollution and microbial contamination of the
esophagus and crop of bees. In addition, a frequent source of contamination of bee products
is the lack of hygiene of staff working on the collection, storage and transport of honey. Data
on the survival of saprophytic and pathogenic microflora in the honey bee is fragmentary and
incomplete (Snowdown and Cliver, 1996; Iojrisz, 1976, Salyers and Whitt, 2001).
The aim of this study was to evaluate the microbiological quality and selected physicochemical
parameters of bee products. Due to the widespread consumption and availability in the market
to assess were chosen nectar and honeydew honeys. The experimental material was honeys
available on the Warsaw market. There were examined 21 samples of honey: acacia, buckwheat,
rape, polyfloral, lime, honeydew, heather. By the purchasing source honeys were divided into
three categories: purchased in supermarkets, apiaries, and organic food stores.
Microbiological analyzes were performed using the classic plate method. Markings were
made on the total number of microorganisms, yeasts and molds as well as Bacillus sp. bacteria.
Three replicate were taken for every sample of honey. In addition, an assessment of the activity
of water with a use of water activity meter series 4TEV of Decagon.
On the basis of analyzes results the most microbiologically contaminated among the tested
honey samples were found lime honey. Contamination by the total number of microorganisms
was calculated as log 3,26 cfu/ml. The value of the number of yeasts and molds was 2,69 log
cfu/ml. In the case of Bacillus bacteria were counted acidifying bacteria of this kind. As with the
total number of microorganisms and the number of yeast and mold contamination by bacteria
Bacillus sp. was the highest in the case of lime honey. The analyzes of water activity using the
dew point method ranged 0,51-0,64 and were comparable with results obtained by other
authors (Gomes et al., 2010). Water activity above the value of 0,6 allows the activation of the
osmiophillic yeast and problems associated with fermentation during storage of honey.
1) Iojrisz N P. (1976) Produkty pczełowodstwa i ich ispolzowanie, Rosselchozizdat, Moskwa 44-46.
2) Gomes S; Dias L G; Moreira L L; Rodrigues P; Estevinho L (2010) Physicochemical, microbiological and
antymicrobial propertis of commerrcial honeys from Portugal, Food and Chemical Toxicology 48 (3) 544548.
3) Salyers A A; Whitt D D. (2001) Microbiology, diversity, disease, and the environment, Fitzgerald Science
Press, Bethesda.
4) Snowdown J A; Cliver D O. (1996) Microorganosms in honey, International Journal of Food Microbiology
21: 247-252.
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84
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International
Honey
Commission
Annual
meeting
of
the
International
Honey
Commission
PC15.Phenoliccompounds,flavonoidcontentandantioxidantactivityofdried
bee pollen samples collected in Sao Paulo, Brazil.
Vanilda Aparecida Soares de Arruda (1)*; Alexandre Vieira dos Santos (1); Davi Figueiredo
Sampaio Meira (1); Ortrud Monika Barth (2); Alex da Silva de Freitas (2); Ligia Bicudo de
Almeida-Muradian (1)
1: University of São Paulo, São Paulo, Brazil
2: Instituto Oswaldo Cruz (Fiocruz), Brazil
Recently, many investigations have been concerned with antioxidant properties of different
food products. The antioxidants are commonly obtained from food and include vitamins C and
E, β-carotene, and a variety of phenolic compounds, including flavonoids. Studies have shown
that the biological action of the bee pollen is due to the presence of phenolic compounds
such as flavonoids, phenolic acids and phenolic diterpenes which has also antioxidant activity.
Fourteen samples of dehydrated bee pollen collected in São Paulo, Brazil during the years of
2010 and 2011 were analyzed for its botanical origin, phenolic compounds, flavonoids, and
antioxidant activity. Pollen loads were washed with 70% ethanol and identified using a 400x
magnification. The antioxidant activity was evaluated by the linking capacity of (i) the free
radical DPPH (2,2-Diphenyl-1-picrylhydrazyl), (ii) ORAC and (iii) system β-carotene/linoleic
acid. The simultaneous application of these three methods allows a more detailed description
of the antioxidative system of bee pollen. Bee pollen extracts were obtained using ethanol at
70%. They were diluted properly for each test. The experiments were performed in triplicate
and the results expressed as mean ± standard deviation. All statistical analysis were performed
using the program STATISTICA 8.0 and adopting the significance level of 5% (p<0.05). Each
dried bee pollen sample showed specific characteristics related to the plant species visited
by the bees. The samples were classified as monofloral or heterofloral, according to the wide
variability of pollen types of the Brazilian flora. It was found that 50% of the fourteen samples
analyzed were composed mainly of Mimosa caesalpiniaefolia (Fabaceae-Mimosoideae), 28.6%
of Cocos nucifera (Arecaceae) and 21.4% of Astrocaryum (Arecaceae) pollen grains. The presence
of phenolic compounds, flavonoid content and antioxidant activity indicates that bee pollen
samples collected in São Paulo, Brazil, may have potential biological activity. High antioxidant
activity were found for the three methods. The differences of the results obtained, using the
three methods, can be explained according to the different mechanisms involved, which are
a hydrogen atom transfer reaction (ORAC), an electron transfer (DPPH), and other ones which
evaluates the capacity of the antioxidants to inhibit the displacement of the β-carotene by
the reaction of free radicals formed during the oxidation of linoleic acid. The statistical results
ranged from 15.28 ± 2.46 to 32.14 ± 2,88 mg GAE/g of bee pollen for phenolic compounds; 2.51
± 0.28 to 9.62 ± 0.42 mg quercetin/g of pollen for flavonoids concentration; 1.07 ± 0.01 to 4.61
± 0.33 mg/mL for EC50; 176.29 ± 19.09 to 339.81 ± 10.06 µmols eq. Trolox/g for ORAC and 61.45
± 0.70 to 90.42 ± 1,45% for β-carotene/linoleic acid. In this way, it is expected that samples with
different phenolic compositions behave differently in vitro models.
Acknowledgements: The authors acknowledge FAPESP for financial support to a research project and a
doctoral scholarship grant to Vanilda Aparecida Soares de Arruda. Also to the CNPq for granting scientific
initiation scholarships to Alexandre Vieira dos Santos and Davi Figueiredo Sampaio Meira and roductivityResearch scholarships to Ligia Bicudo de Almeida-Muradian and Ortrud Monika Barth.
The opinions, hypothesis and conclusions or recommendations expressed in the article are those of the authors
and do not necessarily coincide with those of FAPESP.
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85
Annual
meeting
of
the
International
Honey
Commission
PC16.Propolis’sextractiontechniquesandantioxidantactivityoftheextracts.
Tananaki Chrysoula (1); Ioanna Chekimian (1); Thrasyvoulou Andreas (1)*
1: Laboratory of Apiculture-Sericulture, School of Agriculture, Aristotle University of Thessaloniki, Greece
Propolis is a sticky resinous substance produced by honeybees from various plant sources.
The composition of this product is very complex varying according to the botanical origin
and the geographic region. Bulk propolis consists of about 50% resin, 30% wax, 10% essential
oils, 5% pollen and 5% other substances. Regarding its the chemical composition it is mainly
composed of terpenes, flavonoids, aliphatic and aromatic acids and their esters. Bees prepare
this product to seal holes of their hive, to cover the internal walls and also to reduce the size
of the entrance. Nowadays there is a growing interest, mainly in the field of medicine, for its
antioxidant, antibacterial, antifungal, anti-inflammatory, antiviral activity.
The aim of this study was to investigate and compare different extraction methods for
propolis using different solvents. For this reason three different techniques - mix, stir, and
ultrasounds - were used and three solvents - ethanol, methanol and water - were tried. The
combination of these parameters were applied for three different periods of time. In the extracts
the antioxidant activity and the dry matter were determined using FRAP (Ferric Reducing
Antioxidant Power) and gravimetric methods respectively.
Statistically significant differences at a level of 5% (p < 0.05) were found in the antioxidant
activity of diluted extracts among the extraction techniques, but also among the extraction
mediums. The higher values were determined after treatment with ultrasounds for one hour.
In these cases the FRAP values ranged from 5671,7 to 5909 μΜ for the ethanolic extracts and
from 5020.7 to 5911.7 μΜ for the methanolic extracts. In all treatments the aqueous extracts had
low FRAP values (60.0 – 1577.9 μΜ). Statistically significant differences were also found in the
dry matter of the extracts among the different techniques. The average of the estimated values
for the ethanolic extracts was 12.5 ± 1.3%, for the methanolic extracts 14.0 5 ± 1.4% and for the
aqueous extracts 0.5 ±0.3%.
After the selection of the best technique with ultrasounds it was applied in propolis’ samples
collected from seven different regions of Greece. In these samples the antioxidant activity and
the dry matter were determined and compared in order to find differences due to geographical
origin.
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86
Annual
meeting
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the
International
Honey
Commission
PC17.Determinationofalkanesinbeeswaxfromcombfoundationadulterated
with paraffin after rebuilding by bees using GC-MS technique.
Ewa Waś (1)*; Teresa Szczęsna (1); Helena Rybak-Chmielewska (1); Piotr Semkiw (1); Piotr
Skubida (1)
1: Research Institute of Horticulture, Apiculture Division in Pulawy, Kazimierska 2 Str., 24-100 Pulawy,
Poland
The aim of the study was qualitative and quantitative analysis of alkanes in beeswax from
comb foundation adulterated with paraffin after rebuilding by bees. Another objective was to
verify ability to use gas chromatography with mass detector technique (GC-MS) for detection of
beeswax adulteration with paraffin.
The research material consisted of: technical paraffin obtained from Lotos company, comb
foundation made from pure beeswax, comb foundation with different addition of paraffin (10,
30 and 50%) and beeswax derived by melting of comb foundation adulterated with paraffin
after rebuilding by bees (10 samples from each batch).
Determination of alkanes in paraffin and beeswax used in the experiment, was done
according to procedure elaborated in the Bee Products Quality Testing Laboratory, Apiculture
Division in Pulawy. Analysis were carried out using gas chromatograph with mass detector
(GC-MS). Identification of studied compounds was done based on the mass spectra of the NIST
05 library and on the retention times. Quantitative analysis of alkanes was performed using
internal standard method with squalane (C30H62) as an internal standard and standard mixture
of individual alkanes (from C8H18 to C40H82). The coefficients of variation of repeatability and
within-laboratory reproducibility for total alkanes were 0.9 and 2.5, respectively. Uncertainty of
the method is 0.37% for total alkanes.
In the composition of paraffin long-chain alkanes, which contained in their molecules from
20 to 40 carbon atoms have been identified. Alkanes of natural beeswax had less than paraffin
carbon atoms in their molecules (from 20 to 35). The total content of alkanes in paraffin was
49.4%. Quantitative analysis of alkanes in the comb foundation used in the studies was also
performed. The content of alkanes determined in the individual batches of comb foundation
(before introduction to hives) was as follows: 1st (0% paraffin - 100%) - control - 9.4%; 2nd (10%
paraffin - 90% beeswax) - experimental - 14.9%; 3th (30% paraffin - 70% beeswax) - experimental
- 24.7%; 4th (50% paraffin - 50% beeswax) - experimental - 33.6%. After rebuilding of comb
foundation by bees the content of alkanes was decreased and in these batches was ranged,
respectively: 1st- from 9.0 to 9.4, on average 9.2%; 2nd - from 12.4 to 14.0% with the average
13.4%; 3th - from 18.8 to 20.7%, on average 19.9% and 4th - from 22.3 to 27.6%, with the average
at 25.2%.
Based on this experiment can be concluded that detection of beeswax adulteration
with hydrocarbons of alien origin (e.g. paraffin) is difficult because of the similar qualitative
composition of hydrocarbons in beeswax and paraffin. The other problem is, that we can detect
smaller amounts of adulterant after rebuilding of comb foundation by bees. However, GC-MS
technique allows for detection an addition of paraffin in beeswax. Higher amounts of alkanes
with even number of carbon atoms in the molecule and presence of alkanes, which contain
above 35 carbon atoms in the molecule may indicate beeswax adulteration with paraffin.
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87
Annual
88
meeting
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International
Honey
Commission
Annual
meeting
of
the
International
Honey
Commission
PC18.HRMAS-NMR(HighResolutionMagicAngleSpinningNMR)technique
appliedtothecharacterizationofbotanicaldefinitionandgeographicaloriginof
honey.
Massimiliano Valentini (1); Caterina Cafiero (1); Gian Luigi Marcazzan (2)*; Silvia Gardini (2)
1: CRA-RPS, Research Centre for the Soil-Plant System, Instrumental Centre of Tor Mancina, Strada della
Neve km. 1, 00015 Monterotondo (RM), Italy
2: CRA-API, Agricultural Research Council, Honeybee and Silkworm Research Unit, Via di Saliceto 80,
40128-Bologna, Italy
Characterization of honey’s botanical and geographical origins are determining factors for
its commercial value and for improving the image of honey to consumers. The description of
metabolic profiling is nowadays considered as an emerging technique which is increasingly
being used for the study of agri-food products. The potential of this technique consists in being
able to analyze small amounts of samples without preliminary treatment. For samples in gel
or suspension state, a well-defined NMR spectrum can be obtained from which it is possible
to derive the molecular markers which correlate with the properties we are interested in. In
addition, time employed for analysis is very short, about 5 minutes.
The aim of the study is to test the applicability of the HRMAS-NMR (High Resolution Magic
Angle Spinning NMR) technique on traceability of Italian honeys, combining data obtained
from NMR spectra and multivariate analysis. The samples were collected throughout Italy
from various climatic areas. Special attention was placed on the central area, Tuscany, where
the first protected designation of origin (POD) for an Italian honey was established. The honeys
were selected in advance using classical descriptors: sensory analysis, chemical-physical and
melissopalinological analyses for confirmation of botanical and geographical compliance.
The study focused at first on characterization, by assigning a HRMAS 1H-NMR spectrum
of the POD mark “Lunigiana” acacia honey, then 4 different varieties of Italian honeys (Citrus
spp. Eucalyptus sp. Castanea sativa Mill, Robinia pseudoacacia L.) were considered to identify
molecular markers and to evaluate traceability as a function of the botanical origin. The analysis
of a H-HRMAS NMR spectrum and the creation of the predictive model based on the analysis
of PCA of only two spectral regions related to the carbohydrates and aliphatic protons, led
to excellent discrimination of the four varieties of honey. The model could be improved and
expanded through the assignment of further NMR signals and also by analysis of honeys of
different botanical origin.
Finally 11 acacia honeys and 7 multifloral honeys from various Italian regions were used for
a study on traceability based on botanical origin according to their geographical origin, paying
particular attention to the honey produced in Liguria, Abruzzo and Calabria.
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89
Annual
meeting
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the
International
Honey
Commission
PC19. Sugar composition and crystallization rates of different honey types.
Escuredo Olga (1)*; Dobre Irina (2); Rodríguez-Flores M. Shantal (1); Seijo M. Carmen (1)
1: Department of Vegetal Biological and Soil Sciences, Faculty of Sciences, University of Vigo, Ourense,
Spain
2: Department of Biochemistry, University Dunarea de Jos, Faculty of Food Science and Engineering, Galati,
Romania
Honey is the natural sweet product elaborated by Apis mellifera from nectar of plants,
secretions of livings parts of plants or excretions of plant-sucking insects of the living part of
plants. The major compounds of the honey are the sugars, highlighting the fructose, glucose and
sucrose, followed by the water content. Both influence the physical and sensory characteristics
of the product. The relations between honey sugars and water content are used to predict
honey crystallization (Hadorn & Zuecher, 1974; Manikis & Thrasyvoulou, 2001).
Palynological characteristic, sugars composition, water content and the relations between
them were studied in some unifloral honeys of bramble, eucalyptus, heather, honeydew, lime,
rape, black locust, sunflower and sweet chestnut honeys in order to know the crystallization
tendency. Pollen spectrum was determined using the method of Louveaux et al. (1978). Sugars
were determined by anion exchange chromatography of high efficacy (HPAEC) using an ion
chromatograph DIONEX ICS-3000 with pulse amperometric detector (PAD) and water content
by a refractometric method. A total of 136 honeys were studied.
Bramble honeys had a mean value of Rubus pollen of 60%, eucalyptus honey had a mean
value of 77% of Eucalyptus pollen, heather unifloral a mean value of 37% of Erica pollen, lime
honey a mean percentage of Tilia pollen of 62%, rape honey a mean value of Brassica pollen
of 78%, acacia a mean content of Robinia pollen of 17% and sunflower a mean value of 62% of
Helianthus annuus pollen and chestnut a mean value of Castanea pollen of 78%. Robinia honeys
had the highest fructose concentration, with mean value of 42.3% while honeydew had the
lowest value (32.9%). Respect to glucose content, sunflower honeys and rape honeys had the
higher mean value (more than 37%), the lowest value correspond to honeydew honeys (23.2%).
The minor glucose content correspond to sweet chestnut honeys (26.1%). Acacia honeys had
the highest sucrose content with a mean value of 2.3%, whereas Rubus honeys had the highest
maltose content near 3%. Respect to reduced sugars rape and sunflower honeys had the highest
averages over 75%. Rape honey had the relation glucose/water higher than 2.0, fructose/
glucose minor than 1.14 and high relation (glucose-water)/fructose. Similar results was noted
for Helianthus honey. Also, the relation (glucose-water)/fructose was high in bramble honey.
On the contrary, honeydew, eucalyptus and sweet chestnut honeys had low glucose/water
and high fructose/glucose and low (glucose-water)/fructose. The ANOVA provides significant
differences in some of studied parameters according to the honeys types (P<0.05).
1) Hadorn H., Zuecher K. (1974) Spectrum of sugars and tendency to crystallization of honeys, Mitteilungen
aus den Gebiete der Lebensmitteluntersuchung und Hygiene 65 (4) 407-420.
2) Manikis I., Thrasyvoulou A. (2001) La relación entre las características físico-químicas de la miel y los
parámetros de sensibilidad a la cristalización, Apiacta 36 (2) 106-112.
3) Louveaux J., Maurizio A., Vorwohl G. (1978) Methods of melissopalynology, Bee World 59 (4) 139-157.
____________
90
Annual
meeting
of
the
International
Honey
Commission
PC20. Antioxidant activity of honeys from Galicia (NW Spain).
Escuredo Olga (1)*; Rodríguez-Flores M. Shantal (1), Seijo M. Carmen (1)
1: Department of Vegetal Biological and Soil Sciences, Faculty of Sciences, University of Vigo, Ourense,
Spain
Honey is a natural food to which are attributed many health benefits. The presence of
antioxidant compounds in the composition of honey confers it a good part of these properties.
Some of the compounds involved in the antioxidant activity of the honey are the phenols and
flavonoids. These are from plants and also have an important relation with the colour of the
product.
107 samples produced in Galicia (NW Spain) during the years 2008, 2009 and 2010 were
analyzed to determine the antioxidant activity and the relation with the botanical origin.
The determination of the botanical origin of honey was carried out in accordance to a
melissopalynological method (Louveaux et al. 1978). Antioxidant activity of the honey was
measured by the DPPH discoloration method (Sánchez-Moreno, 2002; Tabart et al., 2009). This
assay is based on the measurement of the scavenging ability of antioxidants over the stable
radical 2,2-diphenyl-1-picrylhydrazyl (DPPH) by a spectrophotometric method. It was calculated
by the percentage of RSA (radicals scavenging activity) and was also expressed as the mean
effective concentration (IC50) of an extract in mg/ml, which is the antioxidant content required
to reduce the initial concentration of DPPH to 50%. The concentration of the honey required
to scavenge 50% of DPPH (IC50) was calculated by a linear regression analysis using a standard
curve of Ascorbic acid.
The principal floral resources for honeybees in the studied area were Castanea sativa,
Rubus, Eucalyptus and Erica. The pollen of these plants was identified as dominant pollen in an
important number of the studied honeys and was the principal pollen types in more than 75%
of the samples. Other frequent pollen types as secondary pollens were Cytisus, Trifolium, Quercus
and Salix. The IC50 in studied honeys had an important variation, ranged from a minimum value
of 4.3 mg/mL to a maximum value of 36.2 mg/mL with a mean value of 11.1 mg/mL. Statistically
significant relationships between the principal pollen types and the IC50 were found. Eucalyptus
pollen showed high significant positive correlation with the IC50, while Castanea sativa pollen,
Erica pollen and Rubus pollen showed a significant negative correlation. Also it has been
checked the close relationship between the antioxidant activity (IC50) and the phenol content
and flavonoid content.
Acknowledgements: This study is financed by Conselleria de Medio Rural, Ministerio de Medio Ambiente, Medio
Rural y Marino and FEADER. Research project FEADER 2008-5.
1) Louveaux J, Maurizio A, Vorwohl G (1978) Methods of melissopalynology, Bee World 59 (4) 139-157.
2) Sánchez-Moreno C (2002) Review: Methods used to evaluate the free radical scavenging activity in foods
and biological systems, Food Science and Technology International 8 (3) 121-137.
3) Tabart J, Kevers C, Pincemail J, Defraigne J O, Dommes J (2009) Comparative antioxidant capacities of
phenolic compounds measured by various tests, Food Chemistry 113 (4) 1226-1233.
____________
91
Annual
92
meeting
of
the
International
Honey
Commission
Annual
meeting
of
the
International
Honey
Commission
PC21. Sugar Profiles of Honeys from Castilla y León (Spain).
Ana Pascual-Maté (1)*; Gian Luigi Marcazzan (2); Silvia Gardini (2); Miguel Ángel FernándezMuiño (1); Maria Teresa Sancho (1)
1: University of Burgos. Faculty of Sciences. Department of Biotechnology and Food Science. Plaza Misael
Bañuelos s/n. 09001 Burgos, Spain
2: The Agricultural Research Council, (CRA) - Honeybee and Silkworm Research Unit. Via di Saliceto 80.
40100 Bologna, Italy
Castilla y León is the largest autonomous community of Spain, placed in the North-Central
area of the country. The purpose of this work was to study the sugar profiles of 57 honeys from
this region, being Ericaceae, Lavandula sp., Thymus sp., Castanea sativa, and honeydews the
most important unifloral honeys. A GC-FID method with previous derivatization was used to
separate and quantify the sugars. Thirteen sugars were identified. Their percentages ranged as
follows: fructose 32,69 - 43,38 %, glucose 24,14 – 35,31%, sucrose 0,01 – 6,96%, trehalose 0,03
– 0,30%, maltose 2,32 – 6,56%, gentiobiose 0,04 – 0,35%, isomaltose 0,29 – 3,12%, raffinose
0 – 0,24%, erlose 0,02 – 1,71%, melezitose 0 – 1,73%, maltotriose 0 – 0,28%, panose 0 – 0,41%,
isomaltotriose 0 – 0,22%, and maltotetraose 0 – 0,28%. Total sugars content ranged between
65,84% and 84,46%. Together with the results of other parameters, data of this study are
potentially useful to get a Protected Geographical Indication seal for honeys from Castilla y León.
____________
93
Annual
meeting
of
the
International
Honey
Commission
PC22.ATR-FTIRSpectroscopyfordeterminationoffreeacidity,electricalconductivity, ash and total polyphenol content in honey.
Ofélia Anjos (1,2)*; Fátima Peres (1); Paulo Antunes (3); José Rodrigues (4)
1: IPCB – Polytecnic Institute of Castelo Branco, Castelo Branco, Portugal
2: CERNAS – Natural Resources, Environment and Society Res. Center, Coimbra, Portugal
3: CATAA – Agri-food Technological Support Center, Castelo Branco, Portugal
4: IICT – Tropical Research Institute, Lisboa, Portugal
The attenuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) could
be used for food analysis determinations and food quality monitoring. This technique presents
advantage because doesn’t need an extensive sample preparation.
Several honey parameters are defined in standards and need to be measured in order to
evaluate the honey quality.
Different commercial honeys from Portugal and Spain, with different floral sources were
used in this study. All honey samples were tested for ash content, free acidity and electrical
conductivity. All determinations were done using methods adopted by the International Honey
Commission. Total phenol content was determined by a modification of the Folin–Ciocalteau
method and the results expressed as mg of gallic acid equivalents (GAE)/100 g of honey. A total
of 50 honey samples were measured
This study compares the results of Fourier transform infrared spectroscopy using an
attenuated total reflection window (ATR-FTIR) with the physico-chemical parameters for
different honey samples analyzed by reference methods.
ATR - FTIR spectra were acquired with a Bruker FT-IR spectrometer (Alpha) using a diamond
single reflection attenuated total reflectance (ATR) device and a zero filling of 2. Duplicate
spectra per sample were obtained with 32 scans per spectrum at a spectral resolution of 4
cm-1 in the wavenumber range from 4000 to 400 cm-1. Principal component analyses (PCA) and
partial least squares regression (PLS-R) modeling were performed using OPUS Quant 2 (Bruker
Optics, Ettlingen, Germany).
The mean values and standard deviation for analyzed parameters were: electrical
conductivity – 451(mS/cm) ± 287,4; ash content – 0,18 % ± 0,17; Free acidity – 28,3 meq/kg ±
9,3; Total phenols content – 36,20 mg GAE/100g ± 23,9; which covers a high variability and gives
consequently a better calibration model.
PLS-R modeling could be a good methodology to predict ash content, free acidity, electrical
conductivity and total phenol content in honey samples (Table 1), with the advantage of ease
of operation, speed, no sample pre-treatment and solvent free. Therefore, the technique is
an alternative to the standard methods for routine analysis or control at-line of production
processes.
Table 1 - Cross validation and validation results of the calculated models for honey samples
Parameters
Preprocess
Electrical conductivity
Cross validation
Validation
r2
RMSECV
RPD
r2
RMSECV
RPD
1st d+MSC
0.925
92.4
3.64
0.854
100
2,65
Ash content
1st d
0.970
0,028
4,02
0.934
0.036
4,02
Free acidity
Const of v
0.824
4.6
2.4
0.605
4.7
1.61
Phenols content
Min-max N
0.844
8.14
2.53
0.803
9.44
2.3
d –derivative; MSC – multiplicative scatter correction; N – normalization; Const of v – constant offset validation;
RMSECV – root mean square error of cross-validation; RPD - ratio of the standard deviation of the reference
values of the validation samples
____________
94
Annual
meeting
of
the
International
Honey
Commission
PC23. Standardizationandregulationsof stingless bees honey in Brazil: challenges and advances.
Silvia Cristina Ferreira Iop (1)*; Marcus Vinícius de Pinho Pires (2)
1: Universidade Federal de Santa Maria - RS – Brazil
2: Colégio Militar de Curitiba - PR - Brazil
The growing concern about environment and awareness of nature conservation in general,
had led to the return of the exploration of native bees. The attractiveness of stingless bees
creation as a business has increased since the publication, in 2004, of the Regulation of the
stingless bees creation in Brazil. It is understood by meliponiculture, the rational creation of
stingless bees for commercial purposes. This activity can be integrated into forest plantations,
fruit plantations and short cicle crops and also may contribute through pollination, with the
increase of agricultural production and regeneration of natural vegetation. Consumption of
stingless bee honey is more widespread in northern and northeastern Brazil, although this
product is consumed in all regions. With the increasing of demand for differenciated honeys
, with medicinal properties, it is necessary the standardization and regulation this product to
ensure the quality offered. Because of lack data about the activities of native bees, most studies
related to physical-chemical analysis of honey from stingless bees in Brazil and around the world
using the methodology applied to honeys from Apis mellifera. In Brazil, several researchers are
leading studies in an attempt to characterize the stingless bee honey. The data obtained so far
show that there is a wide variation in measures considering honeys from the same genus and
between genera of stingless bees. The presence of studies on the physico-chemical composition
of stingless bees is a visible advance and can be observed in the range of articles published in
this regard. It should be noted, however, that the observed discrepancy could be due to lack
standardization in: a) collection of samples (T, rainfall, technology, collection way, packaging
and storage), b) time elapsed between collection and analyze c) type of treatment that this
product had before to arrive the lab, d) geographical information about the locality of honey
production and, e) minimum number of samples per region, for example. Some researchers
have suggested reference parameters for the characterization of honey from stingless bees in
Brazil, however, a comparison between them shows that there is no consensus in this regard.
The patterns suggested regarded all stingless bees, unlike suggested to Guatemala, Mexico
and Venezuela, which consider the division Melipona, and Trigona Scaptotrigona. It should be
noted that the lack of data about time of maturation of the honey of stingless bees can also
hinder the standardization since there is differences between honeys same location and same
genus. Considering the above, it is believed that the standardization of the quality parameters
of stingless bees honey in Brazil is still in the infancy and that the creation of a working group,
incorporating several public and private institutions interested in the subject, can be a way to
accelerate the standartization, by obtaining data on the physico-chemical and microbiologycal
characteristics of stingless bees honeys of different species, obtained under conditions of
rational production, as well as the appropriateness of technologies for extration equipments,
creation and storage, appropriate to the pecualirities of this product.
1) Iop, S. C. F.; Pires, M.V.P. (2012) Standardization and regulation of stingless bees in Brazil: challenges and
advances. Journal of Apicultural Research.
____________
95
Annual
96
meeting
of
the
International
Honey
Commission
Annual
meeting
of
the
International
Honey
Commission
PC24. Are some enzymes in honey foreign or regular?
Dalibor Titera (1)*; Michal Bednar (1); Josef Stich (2)
1: Vyzkumny ustav vcelarsky, Dol, Libcice nad Vltavou, Czechia
2: Österreichischer Erwerbsimkerbund, Mannhartsbrunn, Austria
Determination of foreign enzymes is used as a tool for mark of adulterated honeys.
Some probes of sunflower honey from Austria have the beta/gamma -amylase activity
higher than limit (5 units/kg of honey). This honey probes come direct from bee farms. No syrup
feeding of colonies was applicated on those farms. The reason of high activity of amylases is
unknown.
We collected set of sunflower honeys of Czech origin from the small private apiaries with
good beekeeping practice. No syrup feeding was made on those bee farms. No other sources
of foreign enzymes, like empty hives with store combs or bee farms with syrup feeders, were
detected in the flight distance of tested apiaries. The samples were analyzed in the same
laboratory as honey samples from Austrian colleagues. The analyzes of honeys discovered the
higher beta/gamma -amylase than used limit (5 units/kg of honey) activity again.
How wide is the natural range of beta/gamma - amylase in authentic honeys?
Where is the source of “foreign” enzyme? Feeding? Robbing? Sunflower? Metcalfa? Other
reasons?
The possible reasons are discussed.
____________
97
Annual
meeting
of
the
International
Honey
Commission
PC25. Are oligosaccharides in honey foreign or regular?
Dalibor Titera (1)*; Michal Bednar (1); Hana Vinsova (1)
1: Vyzkumny ustav vcelarsky [Bee Research Institute], Dol, CZ 252 66 Libcice nad Vltavou
The modified method of dextrin determination (Fetzer et al., 1933) was used for description
of some commercial products and honeys. This method is old but very simple, cheap and quick,
independent on special laboratory equipment.
If dextrins are presents in the investigated solution, precipitated with ethanol in an acidic
conditions White turbidity (ring) precipitated dextrins occurs at the interface of water and
alcohol phases. The more intense and wider, it suggests a higher content of foreign substances
in honey. Authentic nectar honeys have negative or very low reaction. The results of this reaction
are independent on HMF content in honey.
We compared set of inverted syrups, syrups based on starch hydrolysates, authentic honeys,
so called honeys based on summer feeding of colonies, various mixtures and other matrices.
Except this alcohol dextrin test response, values of monosaccharides, oligosaccharides, HMF,
conductivity and water content were recorded.
The market has bee feeding syrups based on inverted sucrose syrup and starch hydrolysates
based. Both products have a similar appearance, color and consistency. This method is
suitable for easy distinguish between inverted sucrose syrup and starch hydrolyzated syrup.
We discuss the possibility to use this method for discovery of bad beekeepers practice - massive
feeding of bees by cheap syrup in summer season to obtain more production and/or honey
adulteration in manufacture.
1) Fetzer W R; Evans J W; Longenecker J B (1933) Determination of Dextrin, Maltose, and Dextrose in Corn
Sirup, Industrial and Ingeneering Chemistry 15: 81-84.
____________
98
Annual
meeting
of
the
International
Honey
Commission
PC26.EvaluationoftheeffectofHydrogenPeroxide(H2O2)intheantimicrobial
activity of honey.
X. Feás (1); J.A. Seijas (1); M.P. Vázquez-Tato (1); T. Dias (1); L.M. Estevinho (2)*
1: Departament of Organic Chemistry, School of Sciences, University of Santiago de Compostela, E-27002
Lugo, Spain
2: CIMO, Mountain Research Centre, School of Agriculture, Polytechnic Institute of Bragança, Portugal
Bee hive products such as honey, propolis and royal jelly have been extensively used in the
past. Their use in Medicine dates back at least 4000 years, to Ancient Egypt where it was used for
the treatment of wounds among other conditions. With the discovery of modern antibiotics in the
early 20th century, the use of many effective products of traditional medicine was discontinued.
Although modern antibiotics use has meant a decrease in mortality, its widespread use has led
to the emergence of antibiotic-resistant bacteria and fungi decreasing the treatment options.
This led to an increase research of antimicrobial activity of honey as possible alternatives at least
for dermatological or wound applications.
Honey is a complex substance made up of hundreds of different compounds. Honey’s
antimicrobial activity was initially attributed to the high sugar content and low pH and later to
the activity of glucose oxidase which transforms glucose and water into hydrogen peroxide and
gluconic acid upon honey dilution, which is responsible for the antimicrobial activity in most
honeys.
Monofloral heather (Erica sp.) honey samples harvested in Portugal according to European
organic apiculture standards, were analyzed to test antibacterial activity against Bacillus cereus,
Escherichia coli, Pseudomonas aeruginosa and Staphylococcus aureus. A catalase solution was
added to honey samples to inactive the hydrogen peroxide and then the honeys were tested
to see if there was still any antibacterial activity. It was found out that the presence of catalase
induced a significant increase of the Minimum Inhibitory Concentration, suggesting that the
antimicrobial activity of honey was mainly due to the presence of peroxide. The non-peroxide
antimicrobial activity may be related with the concentration of phenolic compounds. In
addition it was verified a significant interaction between the catalase effect and the different
microorganisms.
Since antimicrobial properties of honey depend strongly on plant source and geographic
origin, together with other factors such as climatic conditions, soil type, and beekeeper activities,
the characterization of antimicrobial properties of honeys of diverse origins still appears to be
a sound research priority to obtain a reliable data on this valuable beehive product for medical
purposes.
____________
99
Annual
100
meeting
of
the
International
Honey
Commission
Annual
meeting
of
the
International
Honey
Commission
PC27. Understanding sensory information of Melipona favosa pot-honey.
Ana Pascual-Maté (1); M. Teresa Sancho (1)*; Miguel A. Fernández-Muiño (1); Rosires Deliza (2),
Patricia Vit (3,4)
1: University of Burgos. Department of Biotechnology and Food Science. Nutrition and Food Science
Division. Faculty of Sciences. Burgos. Spain
2: Embrapa Agroindústria de Alimentos, Rio de Janeiro - RJ, Brazil
3: Apiterapia y Bioactividad, Universidad de Los Andes, Mérida, Venezuela
4: Cancer Research Group, The University of Sydney, Lidcombe, Australia
Melipona favosa Fabricius 1798 thrives in the Venezuelan plains and produces a honey
relished since pre-Columbian times, and mentioned in the novel “Doña Bárbara” written by
Rómulo Gallegos. The pot-honey produced by this bee is known as ‘erica honey’. This peculiar
honey has higher water content and free acidity than Apis mellifera honey, but diastase activity
is very low. It is a delicate honey that keeps the scent of the flowers. Initial approaches of
descriptive sensory analysis were done with a panel of assessors who perceived floral-fruity
odor-aroma with a bouquet of the erica nest. Further acceptance tests with Spanish assessors
using a 10-cm unstructured line scale, positioned this honey in the highest average, compared
to Australian, Bolivian, Brazilian and Mexican pot-honeys. The analytical approach to correlate
sensory characteristics of Melipona favosa honey was carried out by aroma GC-MS and nonaromatic organic acid analysis. Preliminary results consist in 18 major aromatic components.
The analysis of seven erica honey samples showed that contents of L-malic and total citric acids
were similar to those of Apis mellifera, but D-gluconic acid was ten times higher (Sancho et
al., 2012), and this could explain the sour taste. Besides the identification of the floral aromas,
an interesting proposal for chemical studies would be to extract aromatic components of the
cerumen pots to identify if they are also present in the honey to confer its distinctive flavor.
1) Sancho MT, Mato I, Huidobro JF, Fernández-Muiño MA, Pascual A (2012) Non-aromatic organic acids of
honeys. In P Vit, SRM Pedro, DW Roubik. Pot-honey: A legacy of stingless bees. Springer, USA (in press).
____________
* Presenting author: [email protected];
101
Annual
meeting
of
the
International
Honey
Commission
PC28.Volatileandsemivolatilecompoundsofheather(Ericaceae)honeysfrom
Burgos (North Spain).
Miguel Ángel Fernández-Muiño (1)*; Ana Pascual-Maté (1); Maria Teresa Sancho (1)
1: University of Burgos. Faculty of Sciences. Department of Biotechnology and Food Science. Plaza Misael
Bañuelos s/n. 09001 Burgos, Spain
Volatiles have proved being useful as reliable markers in order to identify the floral
sources of honeys. This work aimed to analize the volatile and semivolatile components of 20
samples honeys with Ericaceae nectars, most of them Ericaceae unifloral honeys, collected
from individual apiaries in Burgos, a Spaniard Province placed in the North of Spain, in order
to identify possible floral markers. Volatile and semivolatile components have been isolated by
ethyl acetate extraction (D’Arcy, 1997) which avoids excessive heating. Extractives were later
analyzed by GC-MS. Identification of components was based on mass-spectrometric data and
in their retention kovats index. 87 natural volatiles and semivolatiles grouped in 7 chemical
families were detected and identified in the unmethylated extracts from the 20 studied heather
(Ericaceae) honey samples. The most relevant aliphatic compounds detected were levo- and
meso-butane-2,3-diol, 2-hydroxycyclopenten-2-en-1-one and isopropanol. Main monoterpenes
were alpha gurjunene, cadinene, loliolide and hotrienol. The benzene derivatives were the
most represented compounds including phenyllactic acid, methyl 4-methoxymandelate,
methyl 4-methoxyphenylacetate, phenylacetic acid, benzoic acid, methyl ester homoveratric
acid, 1-methoxy-4-propylbenzene, pyrocatechol, phenylacetonitrile and phenylacetaldehyde.
Norisoprenoids were also important being the most relevant (E)-4-(r-1’, t-2’, c-4’-trihydroxy-2’,6’,6’trimethylcyclohexyl) but-3-en-2-one and dehydrovomifoliol. Other norisoprenoid compounds
found in lesser proportion were: megastigmatrienone 1, 3-oxo-alpha-ionol, 3,4-dihydro-betaionone, 2,6,6-trimethyl-cyclohexene-1,4-dione, and α and β-isophorone. Finally, some flavonoids
such as pinocembrin, tectochrysin, chrysin and galanguin, many heterocyclic compounds and
Maillard reaction products were also identified.
____________
102
Annual
meeting
of
the
International
Honey
Commission
PC29.AntioxidantandPhysicochemicalPropertiesofHoneysfromAlgarveRegion.
Nair Alua (1,2); Ana Balola (1); Celeste Serra (1,2,3)*
1: Área Departamental de Engenharia Química, ISEL, Lisboa, Portugal
2: Centro de Estudos de Engenharia Química, ISEL, Lisboa, Portugal
3: Centro de Investigação em Engenharia Química e Biotecnologia, ISEL, Lisboa, Portugal
In the last years, a lot of studies were carried out on the antioxidant properties of different
foods and natural products. The published work aims to improve the knowledge about the
effects of these products on human health and develop new preservatives for food industry.
Honey is traditionally used as a sweetener agent with well-known health benefits. This
natural product contains a variety of compounds such as flavonoids and other polyphenols,
enzymes, vitamins and free amino acids as minor components, some of them with antioxidant
activity. Thus, the evaluation of honey antioxidant properties might promote the use of this
product as a natural dietary source of antioxidants. In this context, the purposes of the present
work were the evaluation of antioxidant capacity of portuguese honeys collected in Algarve
region and its physicochemical characterization by ash, moisture, pH, free acidity, refractive
index, color and electrical conductivity. Five Honey samples from Monchique and Aljezur regions
were submitted to physicochemical characterization according to the Harmonized Methods of
IHC and by the method proposed by Beretta et al. for colour evaluation. Four of these samples
had a predominant floral origin in Arbustus unedo L., Helianthus annuus L., Ceratonia siliquia L.
and Citrus sinensis L. and one sample was a multifloral honey. The Folin-Ciocalteau assay was
used to determine the total phenolic content with a standard curve generated by galic acid.
The antioxidant capacity was evaluated from the scavenging activity of honey samples for the
radicals 2,2 diphenyl-1-picrylhidrazyl (DPPH), following the absorbance decrease for the IC50
calculation.
The results showed that physicochemical parameters of all samples are according to the
quality standards for honey, except the sample from Arbustus unedo L that present an ash
content slightly higher (0.7%) than the recommended value. Moreover and as it would be
expected, this honey had high electrical conductivity (0.68 mS/cm). Honey samples with a
predominant floral origin in Helianthus annuus L., showed a low content of polyphenols (44
mg GAE/100g honey). For the other samples, the total phenolic values were within the range
obtained in our previous work related with the antioxidant activity of several honeys collected
in other portuguese regions, namely, Lousã and Açores (48 – 270 mg GAE/100g honey). Similar
behaviour was found in antioxidant activity results, as IC50 values obtained in this work also
varied between the range found for the other portuguese honeys (4 – 63 mg/ml). Correlation
studies between the properties of all honey samples were also performed.
1) Alvarez-Suarez, J M, Tulipani, S, Rumandini S, Bertoli, E, Battino, M (2010) Contribution of Honey in
Nutritionon and Human Health: A review, Mediterranean Journal of Nutrition and Metabolism 3 (1) 15-23.
2) Harmonized Methods of International Honey Commission (2002).
3) Beretta, G, Granata, P, Ferrero, M, Orioli, M, Facino, M (2005) Standardization of antioxidant properties of
honey by a combination of spectrophotometric/fluorimetric assays and chemometrics, Analytica Chimica
Acta 523: 185-191.
4) Serra, M C, Rodrigues, V, Taveira, V, Alua, N (2007) Antioxidant Potential of Portuguese Honeys from Lousã
region, 3rd International Symposium on Recent Advances in Food Analysis, Prague.
____________
103
Annual
104
meeting
of
the
International
Honey
Commission
Annual
meeting
of
the
International
Honey
Commission
PC30.Evaluationofanelectronictongueforhoneyclassificationaccordingtoits
pollen analysis.
Mara E.B.C. Sousa (1)*; Luís G. Dias (1); António M. Peres (1,2); Letícia Estevinho (1); Adélio A.S.C.
Machado (3)
1: CIMO - Escola Superior Agrária, Instituto Politécnico de Bragança, Bragança, Portugal
2: LSRE - Laboratory of Separation and Reaction Engineering - Associate Laboratory, LSRE/LCM, Escola
Superior Agrária, Instituto Politécnico de Bragança, Portugal
3: LAQUIPAI - Departamento Química, Faculdade de Ciências, Universidade do Porto, Portugal
Electronic tongues (ET) have attracted great interest due to its potential to obtain global
information from complex samples that could hardly be obtained by traditional instrumental
methods of analysis. These multi-sensor arrays provide a huge amount of sample information
which, by applying chemometric methods, allows sample identification/classification, taste
evaluation as well as, multicomponent analysis. The method of operation consists in obtaining
a signal pattern which corresponds to the overall information on the sample using chemical
sensors with high stability and cross sensitivity to different species in solution.
In this work, a potenciometric electronic tongue or taste sensor array was used. The device
had 20 sensors, based on all-solid-state electrodes with lipid polymeric membranes formed on
solid conducting silver supports.
This analytical system was used to analyse unifloral honeys, which honey pollen profiles were
obtained by pollinic analysis, that are representative of eight main types of pollens: Castanea sp.,
Echium sp., Erica sp., Eucalyptus sp., Lavandula sp., Prunus sp., Rubus sp. and Trifolium sp..
The signal profile information obtained from the ET analysis of the honey samples was
related with the pollinic analysis, using linear discriminant analysis. The results showed that ET
could be used for classifying the type of honey according to their pollen profile, when the main
pollen is in great abundance, being a possible alternative to traditional honey classification
techniques that are time consuming and require expert labour. The influence of the second
main pollen showed to be relevant in honey classification.
Acknowledgements: Collaboration of the Portuguese National Beekeepers Federation in providing honey
samples is gratefully acknowledged.
____________
105
Annual
meeting
of
the
International
Honey
Commission
PC31. Antimicrobial activity of quality brands Spanish honeys.
Patricia Combarros-Fuertes (1)*; Mª Eugenia Tornadijo (1); José María Castro (1); Leticia M.
Estevinho (2); José María Fresno (1)
1: Faculty of Veterinary, University of León, León, Spain
Portugal
80-90% of patients with head and neck cancer who receives radiation and/or chemotherapy
have oral mucositis. Oral mucositis is a complex process involving oxidative damage of oral
mucosal, inflammation, mucosal colonization by pathogenic microorganisms and direct
damage to DNA. The development of oral mucositis led to the interruption of the treatments,
periods of hospitalization, dysphagia with malnutrition in patients and increased healthcare
costs in treatments which have isolated mechanisms of action and are not fully satisfactory.
As honey has antioxidant, anti-inflammatory, antimicrobial and healing properties, it can be
used effectively in the prevention and treatment of oral mucositis by acting simultaneously on
different pathophysiological mechanisms. The aim of this study was to evaluate the antimicrobial
activity of honeys from different floral sources and to evaluate the possible differences that may
happen in such capacity between similar honeys of different years of production. We selected
six types of Spanish honey from quality brands avocado honey (Persea sp.) and chestnut honey
(Castanea sp.) from DOP Miel de Granada; lavender honey (Lavandula sp.) and rosemary honey
(Rosmarinus officinalis) from DOP Miel de La Alcarria; blackberry honey (Rubus sp.) and eucalyptus
honey (Eucalyptus sp) from PGI Miel de Galicia and two organic honey, thyme honey (Thymus
sp.) and heather honey (Erica sp.), collected during two consecutive harvests (2010 and 2011).
We studied antimicrobial activity against microorganisms isolated from oropharyngeal mucosa
of patients who suffer oral mucositis, Gram-positive (Staphylococcus aureus and Streptococcus
pyogenes) and Gram-negative bacteria (Pseudomonas aeruginosa and Escherichia coli) and yeasts
(Candida albicans and Candida glabrata). These assays gave us accurate information about
what would be expected from the application of honey in those patients. Antimicrobial test
were carried out according to Silva et al. (2012) using Nutrient Broth or Brain Heart Infusion to
bacterias or Yeasts Peptone Dextrose to yeasts on microplates (96 wells). Honey was diluted in
water and transferred into the first well. Serial dilutions were performed. 20 µl of a solution of 1%
TTC was used for detect antimicrobial activity
Candida albicans and Candida glabrata showed higher resistance and their growth was
not inhibited at the honey concentrations used. Further studies using higher concentrations
of honey are required. Among bacteria all the tested honeys were able to inhibit their growth
but showed different antimicrobial activity depending on the microorganism tested. Minimum
inhibitory concentration ranged from 3.9 to 250 mg/ml. Generally gram-negative bacteria were
less resistant, something that can be explained by the variability that exists in the resistance in
bacterial strains within a specie. Differences were also observed in the antimicrobial activity of
similar honeys from different years of production. Being a natural product, honey’s composition
is in a constant state of change, depending on factors such as climatology which affect plant
species available.
1) Silva, J.C; Rodrigues, S; Feás, X; Estevinho, L.M. (2012) Antimicrobial activity, phenolic profile and role in the
inflammation of propolis, Food and Chemical Toxicology 50: 1790-1795.
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PC32. Portuguese propolis decreases PC-3 cell glycolytic metabolism.
I. Valença (1,2,3); N. Pértega-Gomes (1,2); M. Cunha (1,2); C. Jerónimo (4); C. Almeida Aguiar (3)*;
F. Baltazar (1,2)
1: Life and Health Sciences Research Institute (ICVS), School of Health Sciences, University of Minho, 4710057 Braga, Portugal
2: ICVS/3B’s - PT Government Associate Laboratory, Braga/Guimarães, Portugal
3: Molecular and Environmental Biology Centre, Biology Department, University of Minho, 4710-057 Braga,
Portugal
4: Department of Genetics Portuguese Oncology Institute (IPO), Porto, Portugal
Prostate cancer remains the most frequent cancer in males. Multidrug resistance and tumor
metastasis remains the main cause of treatment failure and mortality in prostate cancer patients.
Therefore, it is important to discover new agents with antitumor properties. Natural products
have been explored as alternatives or complements to classical chemotherapy. Propolis is a bee
product composed by several compounds, such as, wax, resin, essential oils and other organic
compounds. Over the last decades, several studies have shown antimicrobial, antioxidant, antiinflammatory, antitumor propolis activities, among others.
The aim of this work was to evaluate the in vitro activity of a Portuguese propolis extract on
the human metastatic prostate cancer cell line PC-3. For this purpose, we analyzed the effect of
propolis on cell viability, proliferation and metabolism. Also, we studied the expression of key
glycolytic metabolic proteins, namely monocarboxylate transporters (MCTs) 1 and 4, before and
after treatment, to infer about the possible effect of propolis on these transporters responsible
for lactate efflux in cancer cells.
Cell viability, proliferation and metabolism were assessed with different concentrations
of propolis up to 72 hours. Cell viability was assessed by the Sulforhodamine B assay, cell
proliferation by BrdU incorporation and glycolytic metabolism by estimation of glucose
uptake and lactate efflux rates. Effect of propolis on the expression of MCTs was assessed by
immunocytochemistry, using specific antibodies.
Our results showed that Portuguese propolis inhibits cell viability, proliferation and
glycolytic metabolism (glucose consumption and lactate production) of PC3, in a dose- and
time-dependent way. We also observed that the expression of MCT1 and MCT4 in PC3 cells
appears to decrease after treatment with propolis, as assessed by immunocytochemistry.
This work shows that Portuguese propolis decreases the viability, proliferation and glycolytic
metabolism of the metastatic prostate cancer cell line PC3 and suggests that the alteration in
metabolism could be related to MCT1 and MCT4 expression inhibition, which are essential
proteins involved in the lactate efflux from glycolytic cells.
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PC33. Antigenotoxic potential of Portuguese propolis.
M. Cruz (1); A. Cunha (1,2); R. Oliveira (1,3); C. Almeida Aguiar (1,3)*
1: Biology Department, University of Minho, 4710-057 Braga, Portugal
2: Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB), Biology
Department, University of Minho University, 4710-057 Braga, Portugal
3: Molecular and Environmental Biology Centre (CBMA), Biology Department, University of Minho, 4710057 Braga, Portugal
Propolis is a waxy substance produced by bees (Apis mellifera L.) from collected plant buds
and barks that are subsequently masticated and mixed with the salivary enzyme β-glucosidase.
Bees use propolis in their combs to repair structural damage, to build aseptic locals for the
eggs of the queen, and also as a thermal insulator. The chemical composition of propolis varies
geographically, with the available flora, the time of collection and the bees´ race. Different groups
of compounds can be found in propolis, such as polyphenols, terpenoids, steroids and amino
acids. These compounds have been associated with diverse biological activities: antimicrobial,
antioxidant and scavenger of free radicals, antitumour, and antimutagenic, among others.
Portuguese propolis has been very poorly studied, hampering its economic valorization by
scientific support of the biological activities commonly assigned to samples from other origins.
Thus, our objective relates to the analysis and study of Portuguese propolis, particularly in
what concerns the evaluation of an array of biological activities. A sample collected in 2010
in Beira Alta (C, from Côa) was used to prepare an ethanol extract (EE) and this extract – C.10.
EE - was tested in different assays, using Saccharomyces cerevisiae as biological model. Propolis
protective effect on yeast cells was assessed by viability assays in the presence of hydrogen
peroxide (H2O2). Comet assay was performed to evaluate the antigenotoxic effect of C.10.EE.
Viability has improved when propolis was assayed, either by pre-incubation or co-incubation,
with yeast cells challenged with the oxidant agent H2O2. However, prolonged incubation of cells
with high concentrations of C.10.EE promoted a decrease of viability. Results obtained by the
comet assay suggest an antigenotoxic activity - which protects the genome against oxidative
stress - and also a genotoxic effect - when only propolis was incubated with yeast cells. In this
way, besides the antioxidant activity, our results suggest a prooxidant activity of C.10.EE.
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PC34. Bee products as natural compounds in Cosmetics.
André Santos (1); Miguel Vilas-Boas (1); Mª João Sousa (1)*
Bragança, Portugal
Since ancient times, among the Greeks, the Egyptians and later the Romans, honey and other
different bee products were considered essential for health and well-being [1]. The ancient Greek
society used them widely in medicine, and later associate them with the concept of wellness
and feminine beauty, in what can be considered the embryo of modern day cosmetics. Most
bee products can be consumed, or used in its original format, but there are many additional
applications when these products become an ingredient of a more complex product and, for
that, require transformation.
Based on the characteristics of each bee product (honey, beeswax, propolis or pollen), we
intended to develop new cosmetics products through the combination of them with natural
compounds obtained from plants, such as extracts, essential oils and dyes. The main goal is to
set a new range of formulations, based on both scientific and ancestral knowledge, allowing
the creation of new products and new possibilities for both beekeepers and consumers. Natural
products have been developed using the ability of honey as a moisturizing and smoothing
product to use in cosmetics, like shampoos and conditioners, in association with extracted
compounds from aromatic and medicinal plants. Propolis, due to its disinfectant, antibacterial
and anti-inflammatory capacities is a very promising raw material for the development of
creams and gels to combat acne [2]. The use of pollen in soaps and exfoliating products
allowed formulations to be enriched with natural proteins and minerals. Pollen, on the other
hand, can be used as natural pigment, allowing a pleasant color in both gels and creams. In
the preparation of soaps with different vegetable oils, the oils were chosen based on their fatty
acid composition, and recurring to tests of saponification. In the same oil we can find saturated
and unsaturated fatty acids in various percentages. Saturated fatty acids can accelerate the
hydrolysis and produce a hard soap, foamy, with more capacity to dissolve grease and that take
longer to oxidize. Unsaturated fatty acids can slow saponification, and originate a milder soap
that melts more easily but is less efficient dissolving fat and that oxidizes more easily. In the
group of saturated fatty acids, stearic and palmitic acids are those which originate a harder soap,
as obtained in our tests with mixtures of oil and beeswax. Lauric and myristic acids, like those
found in olive oil and palm oil, were also tested in our products, originating soaps with a greater
capacity to dissolve grease. The first step for the development of natural cosmetics began under
the European project EuropeAid/128139/L/ACT/GW which promotes the development of
beekeeping in Guinea-Bissau. Different soaps based on local coconut, palm and palm kernel oil
mixed with honey and beeswax are now produced in the “Associação dos Apicultores do Leste”,
along with fatty creams for body and lips protection.
Acknowledgements: The authors are grateful to the European Union for the financial support given to this
work EuropeAid/128139/L/ACT/GW
1) Charlton and Jane Newdick Jane: In Praise of honey (2005) ed. Boyne Valley Honey Company.
2) Tecnologia Farmacêutica vol.II (2008) ed. Fundação Calouste Gulbenkian.
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PC35. Phenolic composition of Brazilian propolis from Minas Gerais.
Joana Coelho (1)*; Soraia I. Falcão (1,2); Alexandre Bera (3); Renato Januario Sousa (3); Ligia B.
Almeida-Muradian (3); Miguel Vilas-Boas (1)
1: CIMO/IPB - Instituto Politécnico de Bragança, Portugal
2: REQUIMTE/FCUP – Faculdade de Ciências, Universidade do Porto, Portugal
3: Faculdade de Ciências Farmacêuticas, Universidade de São Paulo, São Paulo, Brasil
Propolis is the name for the resinous substance collected by honeybees (Apis mellifera) from
various plant sources and used in the construction, repair and protection of their hives. It is an
important apicultural product widely used in folk medicine due to several pharmacological and
nutritional applications (Bankova, 2000). Propolis chemical composition is extremely complex
and varies greatly with the plant origin. In regions of temperate climate bees obtain resins mainly
from the buds of Populus spp. being mainly composed by flavonoids, phenolic acids and their
esters. On the other hand in tropical areas the chemical composition of propolis shows a greater
regional variation. The green propolis, with origin in the leaves of Baccharis spp., is one of the
most studied types. It is mainly composed by prenylated p-coumaric acids and caffeoylquinic
acids (Bankova, 2000). This work outlines the study of the phenolic profile of propolis samples
from the state of Minas Gerais, Brazil. For that, seventeen samples of propolis were extracted
and analyzed according to our previous work (Falcão, 2010). Through HPLC-DAD it was possible
to identify two types of propolis: one with a common green propolis composition and another
similar to poplar propolis. Between propolis types, the composition was similar although
significant differences were found in their concentrations. Sixteen samples were assigned as
green type propolis with fourteen phenolic compounds quantified, being the most abundant
compounds dicaffeoylquinic acid in a range of 2.8 to 61.8 mg/g extract, its isomer in a range of
8.6 to 80.6 mg/g extract and artepillin C in the range of 2.6-51.1 mg/g extract. Only one sample
presented the typical composition of poplar propolis mainly composed by pinocembrin (37.7
mg/g extract), chrysin (32.3 mg/g extract) and pinobanksin-3-O-acetate (41.0 mg/g extract).
The different phenolic profiles found are correlated with different flora sources available in the
vicinity of the hives.
1) Bankova VS; De Castro SL; Marcucci MC (2000) Propolis: recent advances in chemistry and plant origin
Apidologie 31: 3-15.
2) Falcão SI; Vilas-Boas M; Estevinho LM; Barros C; Domingues MRM; Cardoso SM (2010) Phenolic
characterization of Northeast Portuguese Propolis: usual and usual compounds, Analytical and
Bioanalytical Chemistry 396: 887-897.
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PC36.CistusLadaniferL.secretedflavonoidsasmarkercompoundsfordifferentiating Portuguese propolis types.
Soraia I. Falcão (1,2)*; Nuno Vale (3); Susana M. Cardoso (4); Cristina Freire (2); Miguel Vilas-Boas (1)
3: CIQUP/FCUP – Faculdade de Ciências, Universidade do Porto, Portugal
4: CERNAS/IPC – Instituto Politécnico de Coimbra, Portugal
Propolis is a natural product derived from plant resins collected by honeybees and used in
the construction, repair and protection of their hives. Because of its broad spectrum of biological
activities and uses in health food as well as in folk medicine, there is a renewed interest in the
composition of propolis and its biological activities. The plant origin of propolis, bee glue,
determines its chemical diversity. Its chemical composition depends on the specificity of the
flora at the site of collection and thus on the geographic and climate conditions of this site. In
temperate regions, the resinous exudates of the buds from Populus species are the main source
of propolis and phenolic compounds like flavonoids, phenolic acids and its methoxylated and
esterified derivatives are the main compounds (Falcão, 2010). Our studies in the physicochemical
parameters, phenolic composition and antioxidant activity revealed that Portugal is a country of
propolis diversity and establish different two types of propolis.
The objective of the present work is to determine a possible phenolic association between
the flora and the dark green colour propolis from specific regions of Portugal. This type of
propolis, clearly different from the poplar propolis type, was characterized for the phenolic
content according with our previous work (Falcão, 2010) and compared with Cistus ladanifer
L. resin. This plant is a potential floral source due to its abundance in the hive neighborhood,
the typical odor of Cistus species that can be recognize in this propolis type and the fact that
local beekeepers frequently associate both materials. The chromatographic and spectrometry
methods used in this work enable to establish a correlation between the phenolic profile of
the propolis samples and Cistus ladanifer L. resin. Both present a phenolic composition rich in
kaempferol derivatives, highlighting the presence of a kaempferol-dimethyl-ether, a compound
absent in the poplar type and therefore a potential marker substance for origin discrimination
between this two types of propolis.
1) Falcão SI; Vilas-Boas M; Estevinho LM; Barros C; Domingues MRM; Cardoso SM (2010) Phenolic
characterization of Northeast Portuguese Propolis: usual and usual compounds, Analytical and
Bioanalytical Chemistry 396: 887-897.
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PC37. Phenolic content of bee bread from Northeast of Portugal.
Andreia Tomás (1)*; Soraia I. Falcão (1,2); Miguel Vilas-Boas (1)
Bee bread is the name given to the collected bee pollen which has been mixed with digestive
enzymes, transported to the hive in small pellets and stored in the combs with a tiny bit of
honey and bee wax. Pollen stored in this way undergoes lactic acid fermentation due to the
action of different enzymes, micro-organisms, moisture and temperature. The bee stored pollen
is the main source of nutrients for the bees, rich in proteins, lipids, vitamins and microelements.
Its composition differs from fresh bee-collected pollen due to the fermentation stage, which
is responsible for higher stability of the product and lead to chemical changes that increase
digestibility and nutritive value for the bee (Herbert, 1978). Due to its high nutritional value a
bee bread is considered to be a good complement in a healthy diet.
The aim of this work was to evaluate the phenolic content of bee bread samples from the
Northeast Portuguese region. For that, an ethanolic extract was prepared and the content in
total phenolics, flavones/flavonols and flavanones/dihydroflavonols was determined by UV-Vis
spectrometry according to previous work, (Nunes, 2011). The total phenolics content, expressed
as gallic acid equivalents, varied from 29-55 mg/g extract. Flavones/flavonols, expressed as
quercetin equivalents, range between 1.6 and 3.5 mg/g extract and the content of flavanones/
dihydroflavonols expressed, as pinocembrin equivalents, varied from 36 to 59 mg/g extract. The
phenolic content of bee bread is richer than those described in pollen of the same region which
is a good quality parameter to explore for further studies of this bee product.
1) Herbert, E; Shimanuki, H (1978) Chemical composition and nutritive value of bee collected and bee-stored
pollen, Apidologie 9: 33-40.
2) Nunes, L; Batista, V; Falcão, S I; Vilas-Boas, M; Cardoso, S M (2011) 1º Congresso Ibérico de ApiculturaAbstract book, pp 87, Instituto Politécnico de Castelo Branco, Castelo Branco, Portugal.
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PC38.TradebarriersandeconomicimpactoforganicbeekeepinginPortugal.
Mário Gomes (1)*; João Casaca (1); Paula Cabo (2); Luís G. Dias (2); Miguel Vilas-Boas (2)
1: Federação Nacional dos Apicultores de Portugal, Av. do Colégio Militar, Lote 1786 - 1549-012 Lisboa,
Portugal
2: CIMO-ESA, Instituto Politécnico de Bragança, Portugal
In Portugal, according to the official data provided by the Portuguese Veterinary Authority),
available in the Report of the National Beekeeping Program 2010, beekeeping is carried out by
17.291 beekeepers, which own a total of 562.557 colonies. According to the Research Institute
of Organic Agriculture (FiBL) this represents only 3,6% of the economic value of the Portuguese
organic production, given that only 119 beekeepers were registered as organic operators, with
a total of 15.927 colonies. Portuguese organic beekeeping are still far below other European
countries average such as Italy (8%), and even if compared with the 5,8% of Portuguese organic
farming, which should lead to more than 32.628 hives. Additionally, the great potential that
Portugal has due to its soil, climate, but also environmental conditions (very suitable to organic
beekeeping) and the consumer’s appreciation of organic products are key factors to strength
the Portuguese organic beekeeping sector.
With this study, and considering that organic beekeeping is already established in Portugal,
we aim to encourage the conversion/initiation in organic beekeeping of a significant number of
producers, providing them with a true picture of the Portuguese organic beekeeping sector and
its potential, based on the experience of beekeepers already certified as organic producers. The
information is obtained recurring to individual questionnaires, made directly to organic certified
beekeepers and oriented for the evaluation of their beekeeping activity.
The preliminary results of this research allowed us to establish a preliminary profile of
beekeepers working under Organic Production Mode (OPM): only 24% of the respondents
have beekeeping as the main economic activity, and although the majority (92%) consider
that organic honey has added market value, problems were identified either in production
and marketing. The most related difficulty of beekeeping under OPM is the effectiveness of the
treatments against Varroa, pointing the need to obtain alternative and more efficient methods.
Most respondents (56%) reported that there is need to improve consumer’s recognition of
Organic Honey, which is considered to be an important step towards production increment and
product added value.
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PC39.QualitycontrolandauthenticityreviewofhoneyfromSlovenemarketin
the period 2007 - 2011.
Veronika Kmecl (1)*; Aleš Gregorc (1); Marinka Kregar (1); Romana Rutar (1); Maja Smodiš Škerl
(1); Helena Baša Česnik (1)
1: Agricultural Institute of Slovenia, Ljubljana, Slovenia
During the period from 2007 to 2011 Agricultural Institute of Slovenia has performed
quality control and authenticity review of honey from Slovene market. Samples were taken at
supermarkets, shops, marketplaces and beekeepers. 970 of 1030 samples were taken randomly,
for the rest 60 we performed targeted sampling. For targeted samples, it was assumed that they
were of low quality or adulterated. 77% of honey was of Slovene origin and 23% was from various
EU and Latin American countries. Among 580 randomly taken samples, key physical-chemical
parameters, sensory and pollen analysis were determined according Council Directive 2001/110.
64 of 580 samples (11%) were discordant with regulations regarding electrical conductivity,
HMF, diastase activity, water, the sum of fructose and glucose. The majority of samples had
inadequate designation of botanical origin some samples were probably pre-heated or stored
for longer time. 450 of 970 randomly taken samples were tested on acaricide residues amitraz,
coumaphos, bromopropilate, tau-fluvalinate and flumethrin and they were mainly of Slovene
origin. None of 450 samples exceeded maximal residue limit (MRL). We confirmed positive results
for amitraz at 20% samples with the highest content of 80 ppb. Maximal coumaphos content
was 50 ppb and 10% of results were above the limit of quantification (LOQ). We did not found
any residues of bromopropilate, tau-fluvalinate and flumethrin. Among 60 targeted samples 18
samples (30%) were non-conformable with regulations regarding key quality parameters and
authenticity test. 13 samples (22%) were adulterated which was confirmed by the enzyme test
of beta-fructofuranosidase and beta-gamma amylase and 13C Isotopic Analysis (C4/C3 - Sugars).
Five of adulterated samples were Slovene origin. The results of this review indicate that intensive
control of honey in Slovene market would be required.
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PC40.Determinationofcolourandtracemetallevelsinhoneysfromdifferent
environmental origins.
Lourdes Corredera (1); Consuelo Pérez-Arquillué (1)*; Susana Bayarri (1); Regina Lázaro (1);
Antonio Herrera (1)
1: Department of Animal Production and Food Science, Faculty of Veterinary, University of Zaragoza.
Miguel Servet 177, 50013, Zaragoza, Spain
Honey contains a variety of metals. Major metals are primarily derived from soil and nectarproducing plants, but some trace metals may present hazards to human health and adversely
affect the quality and the safety of honey. On the other hand, colour in honeys has been
associated with the concentration of some elements.
The aim of this work was to contribute to the data about lead and cadmium content of
honeys from different geographical and botanical origins and secondly to evaluate if their
content in honeys correlates with their colour.
Contents of lead (Pb) and cadmium (Cd) were determined by inductively coupled plasma
mass spectroscopy (ICP-MS) in honey samples. A survey of 30 honey samples from different
origins, were collected directly from the beekeepers and from Spanish supermarkets. The sample
was digested with concentrated nitric acid in porcelain crucibles and heated until nearly dry. The
residue was then diluted to 25 ml with milli-Q water and measured by ICP-MS. Sample colour
was determined with a Hanna colorimeter, allowing the classification of samples according to
the U.S. Department of Agriculture colour Standards Designations. The levels of contamination
found in this work were low, similar in all the samples, and in the range of those published in
other European honeys. The average concentrations were 21.33 µg/kg for Pb and 6.72 µg/kg
for Cd, with maximum levels of 125.50 µg/kg and 36.80 µg/kg respectively. Pb was the most
frequent element, found in 80 % of the samples, whereas Cd was only detected in 46 % of the
honeys. Although there are not maximum levels established for honey, these concentrations
are below the maximum contents set by European legislation (Commission Regulation (EC) No
1881/2006) for other food matrices. No statistically significant differences were found at the 95%
confidence level for Cd, but darker honeys (dark amber and amber) have a higher content of Pb
and higher total mineral content (combination of Pb and Cd) than the pale ones.
Acknowledgements: The authors wish to thank the Government of Aragón for giving a scholarship to Lourdes
Corredera, and for its financial support (Grupo de Investigación Consolidado A01, Fondo Social Europeo and
Ayudas Apícolas).
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PC41.AntibacterialActivityofVariousHoneyTypesAgainstDifferentPathogens.
Josipa Vlainić (1); Ivan Kosalec (2); Silvija Zlatar (3); Toni Vlainić (1); Dražen Lušić (4,5)*; Ivana Tlak
Gajger (3,4)
1: Rudjer Boskovic Institute, Department of Molecular Medicine, Zagreb, Croatia
2: Faculty of Pharmacy and Biochemistry, Department of Microbiology, Zagreb, Croatia
3: Faculty of Veterinary Medicine, Department for Biology and Pathology of Fish and Bees, Zagreb, Croatia
4: Croatian Apitherapy Society, Rijeka, Croatia
5: Faculty of Medicine, Department of Environmental Health, Rijeka, Croatia
Because of the remarkable therapeutic potential, honey has been used as a remedy for
thousands of years. Consequently, the apitherapy has recently entered into the focus of attention
as an unconventional method for treating certain conditions and diseases, especially infections
as well as promotion of the health and human well-being. Antimicrobial activity of honey has
been attributed to hydrogen-peroxide, which is produced by glucose-oxidase and phenolic
compounds. The total polyphenol content in honey partially reflect their antioxidant capacity.
The presence and concentration of phytochemicals, especially flavonoids as well as phenolic
acid, can vary and depend significantly upon honey origin. In order to assess the antimicrobial
activity of various honeys, total of 23 samples representing fewer honey types deriving from
different floral sources and geographical locations in Croatia were evaluated for their ability to
inhibit the growth of nine gram (+) and gram (-) bacteria and two moulds. The bioassay applied for
determination of the antimicrobial effects took up the well-agar diffusion method (preliminary
screening method). The micro-dilution method with TTC (2,3,5-triphenyl tetrazolium chloride)
indicated the viability of bacteria and resulted with the Minimum inhibitory concentration (MIC)
values. The total phenolic content was screened by the modified Folin-Ciocalteu method and the
antioxidant activity was assessed by means of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical
scavenging system. In addition, the color and pH of honey samples were analyzed. All honey
samples showed antimicrobial activity at variable levels, depending upon the floral source and
the geographical origin. A well-agar diffusion assay revealed the development of inhibition
zones depending on the type and concentration of honey involved as well as the test pathogen.
Gram (-) bacteria were found to be more susceptible as compared to gram (+). The majority of
tested pathogens showed MIC values in the range of 12-20% of honey. Generally, dark colored
honeys showed more inhibitory properties than light colored. Phenolic content, expressed as
gallic acid equivalent, ranged from 34.2 to 261.4 mg/kg and was highly correlated to the values
of honey antioxidant capacity. Higher antioxidative and scavenging activity was observed for
dark honey samples, too. This study showed that honey samples possess certain antibacterial
activity against test microorganisms that can be of certain use when considering an alternative
therapy in some conditions. Moreover, it gave the characterization of the antioxidant potential
as well as of the phenolic content of honey samples with some regard to the therapeutic efficacy
and sanitary benefits for different purposes. Moreover, the results of the study imply that specific
honeys tend to have certain capacity for the food spoilage prevention as the result of the growth
inhibition of food-borne microorganisms. It suggests that they could be potentially considered
for the use as food preservatives for some foods and under appropriate conditions what should
be further proved by additional research.
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PC42. Antibacterial activity of bee collected pollen.
Kačániová Miroslava (1); Hleba Lukáš (1); Chlebo Róbert (2)*; Vukovič Nenad (3)
1: Department of microbiology, Faculty of biotechnology and food sciences, Slovak University of
Agriculture in Nitra, Slovak Republic
2: Department of Poultry Science and Small Animals Husbandry, Faculty of Agrobiology and Food
Resources, Slovak University of Agriculture in Nitra, Slovak Republic
3: Department of Chemistry, Faculty of Science, University of Kragujevac, Serbia
Flower pollen, bee’s primary food source, contains concentrations of phytochemicals and
nutrients and is rich in carotenoids, flavonoids and phytosterols. Also, bee pollen has antimicrobial
effects. The in vitro antibacterial activities of Slovak pollen extracts were investigated against 3
different species of bacterial pathogens including Bacillus cereus CCM 2010, Escherichia coli CCM
3988 and Pseudomonas aeroginosa CCM 1960. Multifloral pollen was collected in the Nitra region
of the Slovakia. The freshly collected bee pollen was dried at 40 °C with protection from light
and ground into powder. A 150 g sample of pollen was extracted with 125 ml pure methanol
for 48 h. The crude extract was pooled and concentrated in a rotary evaporator completely.
Residues were kept in a hermetically-closed color glass vial at 4 °C until using. Stock cultures of
bacteria were grown in Nutrient Broth at 37 °C for 24 h. Final cell concentrations were 106 cfu.
ml-1. The disc diffusion method was applied for the determination of antimicrobial activities of
the 50 % ethanol extracts. Sterilized filter paper discs (6 mm) were put in the middle of Petri
plates containing 20 ml of Muller Hinton agar inoculated with 100 µl of the each bacterial
suspension. The absolute ethanol used as a negative control. The plates were incubated at 37 °C
and observed after 24 h. At the end of the period, inhibition zones around the paper disc were
measured. All tests were done sevenfold. The antimicrobial activity against Bacillus cereus CCM
2010 ranged between 0 to 3 mm, Escherichia coli CCM 3988 ranged between 0 to 5.5 mm and
against Pseudomonas aeroginosa CCM 1960 between 0 to 3.5 mm. The high antimicrobial effect
was found against Escherichia coli CCM 3988 (2.5±2.17 mm) and the lowest antimicrobial effect
was found against Bacillus cereus CCM 2010 (0.75±1.39 mm).
____________
121
Annual
meeting
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the
International
Honey
Commission
PC43.The antimicrobial effect of honey against selected antibiotic resistant
bacteria.
Kačániová Miroslava (1); Hleba Lukáš (1); Chlebo Róbert (2)*; Vukovič Nenad (3)
1: Department of microbiology, Faculty of biotechnology and food sciences, Slovak University of
Agriculture in Nitra, Slovak Republic
2: Department of Poultry Science and Small Animals Husbandry, Faculty of Agrobiology and Food
Resources, Slovak University of Agriculture in Nitra, Slovak Republic
3: Department of Chemistry, Faculty of Science, University of Kragujevac, Serbia
The antimicrobial effect of honey was described in many studies, but very important is
synergisms of antibiotic resistant bacteria and antimicrobial action of honey against these
bacteria. Therefore we have focused on antibacterial potential of natural Slovak honeydew
honey against isolated bacterial resistance strains. Honeydew honey solutions were prepared
in three concentrations: 50, 25 and 15.5 % (by mass per volume). The samples of each honey (10
g) and sterile water were stored at 37 °C for 30 min before mixing to facilitate homogenization.
The 50 % (by mass per volume) solutions were diluted to 25 %, than to 12.5 % and 6.25 %.
The samples were assayed immediately after dilution. For testing antimicrobial activity bacterial
isolates from animal products were used. For cultivation of bacterial strains Mac Conkey agar
was used. Cultivation of Enterobacteriaceae genera was done at 37 °C during 24 hours. For
recultivation and identification of Enterobacteriaceae strains Chromogenic coliform agar was
used. Biochemical identification of bacteria was done by ENTERO test 24. For identification of
Enterobacteriaceae strains were used MALDI-TOF MS Biotyper. We identified three Escherichia coli
strains, one Proteus mirabilis and one Klebsiella oxytoca strain. The sensitivity of all strains were
tested against ampicillin (AMP 10) 10 µg.disc-1, chloramphenicol (C 30) 30 µg.disc-1, meropenem
(MEM 10) 10 µg.disc-1, ceftriaxone (CRO 30) 30 µg.disc-1, ofloxacin (OFX 5) 5 µg.disc-1 and
oxytetracycline (OT 30) 30 µg.disc-1. For antibiotic susceptibility testing was used disc diffusion
method according CLSI (Clinical and Laboratory Standard Institute). The antimicrobial effect of
the natural honey was tested in experiments by using the agar well diffusion method. All plates
were incubated at 37 °C and inhibition zones were measured after 24 hours. After incubation, the
zones of inhibition of the growth of the bacteria around the disks were measured. The statistical
processing of the data obtained from all studies was implemented by means with StatgraphicS
5 software. The inhibition zones of honey sample varied from 3.33 mm by E. coli to 1.67 mm by
P. mirabilis. The best inhibition effect of honeydew honey was found in 50 % concentration of
honey against Escherichia coli in 2 strains (3.33 mm) and Klebsiella oxytoca (3.33 mm) and lower
antibacterial effect was found in 6.25 % concentration of honey against Proteus mirabilis.
____________
122
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meeting
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the
International
Honey
Commission
PC44. Evaluation andValidation of a Biochip Multi-Array technology for the
screeningofantibioticresiduesinhoneyaccordingtotheEuropeanguidelinefor
the validation of screening methods.
Valerie Gaudin (1)*; Celine Hedou (1); Eric Verdon (1)
1: Anses, Laboratory of Fougeres, European Union Reference Laboratory (EU-RL) for Antimicrobial and Dye
Residue Control in Food-Producing Animals, La Haute Marche-Javené, 35302 Fougères, France
The main chemicals are acaricides against varoa and antibiotics used for the control of bee
bacterial diseases, mainly tetracyclines, streptomycins, sulfonamides and chloramphenicol. No
maximum residue limits (MRLs) have been set for any antibiotics in honey. Therefore, in the
European Union, minimum Recommended Concentrations (RC) for the analytical performance
of methods to control a certain set of these non-authorized chemicals in honey have been
published by the EU Reference Laboratory (EU-RL) in 2007 [1]. Concerning the strategy of
control for antibiotic residues in honey, several screening tests are often used in parallel for the
detection of tetracyclines, macrolides, sulphonamides and/or streptomycins. So far there is still
a great need for a cheap and single multi-residue method.
Biochip Array Technology is an innovative assay technology for multi-analyte screening
of biological samples in a rapid and easy to use format. A multi-array system, called Evidence
Investigator™ (Randox, UK) was evaluated in our laboratory. The Evidence Investigator® system
is a biochip system, semi-automated, designed for research, clinical applications and veterinary
use. A competitive chemiluminescent immunoassay is employed for detection of antimicrobial
(AM) arrays. The kit MicroArray II (AM II) dedicated to the screening of 6 different families of
antibiotic residues was validated according to the European guideline for the validation of
screening methods [2]. Different performance characteristics were determined: specificity,
detection capability and applicability to different kinds of honey. Sixty blank samples of different
batches (floral origin, colour, texture) were analysed to determine specificity and applicability.
Two antibiotics per family were chosen to estimate the CCβ: the one which was announced
bearing 100 % of cross-reactions and the least sensitive antibiotic within the same family which
is satisfactorily detectable. The possible cross-reactions towards several compounds of the same
family and towards sulphonamides (another antibiotic family) were also checked.
The specificity was proven very satisfactory and the applicability to different kinds of
honey was demonstrated. The detection capabilities CCβ of the 12 different antibiotic residues
investigated during the study were determined below the Recommended Concentration of the
EU-RL [2] when it exits. The AM II kit is able to detect at least 6 quinolones, 4 tetracyclines and 3
of their 4-epimers, 3 aminoglycosides, 3 macrolides, the thiamphenicol, the florfenicol and the
ceftiofur along with one of its stabilized metabolites, the desfuroylceftiofurcysteine disulfide
(DCCD). The kit is not able to detect any of the 6 substances tested from the sulphonamide
family but another kit AM I plus is available for the screening of 14 sulphonamides and of
trimethoprim.
In conclusion, due to the low detection capabilities achieved, the MicroArray II could be
recommended for a reliable routine use for the screening in honey of representative substances
of 6 families of antibiotics. Accordingly and technically speaking, the AM II kit could probably
be satisfactorily replacing the use of 3 different kits (2 ELISA kits and 1 receptor test) currently
implemented in the French national control plan for screening antibiotics in honey.
1) CRL. Guidance paper of 7th December 2007. CRLs view on state of the art analytical methods for national
residue control plans. Community Reference Laboratories (CRLs) for residues according to Council
Directive 96/23/EC. 1-8.
2) CRL. 2010. Guideline for the validation of screening methods for residues of veterinary medicines (initial
validation and transfer).
____________
123
Annual
124
meeting
of
the
International
Honey
Commission
Annual
meeting
of
the
International
Honey
Commission
PC45. Study of effect of various processing procedures on some of the honey
characteristics.
Sayed Mazaher Sayedi (1)*; M.B. Farshineh Adl (3); Ali Reza Abassian (2); Mahmoud Salesi (1);
Mohammad Behjatian Esfahani (1); Shahaboddin Mosharaf (2); S.M.K. Deylami
1: Researcher of Agriculture and Natural Resources Research center, Isfahan Province
2: Scientific member of Agriculture and Natural Resources Research center, Isfahan Province
3: Member of Scientific Board Animal Science Research Institute, Karaj-Iran
Raw or untreated honey, in addition to carbohydrates, organic acids, minerals, pigments, wax
and pollen grain, contains active enzymes, and volatile chemical substances which are either
changed or denaturized by heat and processing. The aim of this research was to determine the
effect of heating and treating in the two normal (55c) and vacuum (40c) conditions on honey
and probability of modifications occurring in its qualities.
In this study, following collecting honey samples from various parts of the country,
destructive effect of heat in tow normal and under vacuum conditions was investigated.
Following to processing, such parameters as glucose, fructose, HMF and proline contents of raw
and processed honey were measured and compared.
In this research project, in addition to the normally assessed parameters, the proline level, a
free amino acid constituting 50 to 85 present of the total honey amino acids was measured as
well. Proline concentration was assessed employing spectrophotometer at 510nm. Data were
analyzed using SPSS software. Results of this study revealed that samples processed at a 55c
temperature suffered decreased proline and free acids and that, this difference compared with
raw honey and heating in vacuum at 40c was quite significant. The glucose, fructose and pH levels
showed no significant variations. The level of hydroxylmethylfurfural in the honeys processed of
55c was higher than in honey samples processed at either 40c at vacuum conditions and that of
two raw honey however, this difference was not significant.
Regarding the obtained results, it can be concluded that under vacuum conditions heating
inflicts less damages to the biological substances of honey.
____________
125
Annual
meeting
of
the
International
Honey
Commission
PC46. Antioxidant and antimicrobial activities of“água-mel”from Portugal
M. Graça Miguel (1)*; M. Dulce Antunes (1); Smail Aazza (1); Joana Duarte (2); M. Leonor Faleiro
(2)
1: Universidade do Algarve, Faculdade de Ciências e Tecnologia, Edifício 8, Instituto de Biotecnologia e
Bioengenharia, Centro de Biotecnologia Vegetal, Campus de Gambelas, 8005-139 Faro, Portugal
2: Universidade do Algarve, Faculdade de Ciências e Tecnologia, Edifício 8, Instituto de Biotecnologia
e Bioengenharia, Centro de Biomedicina Molecular e Estrutural, Campus de Gambelas, 8005-139 Faro,
Portugal
A honey-based product known as “água-mel” is produced in Portugal for ancient times.
This product has been used as sweetener in cakes, tea, and of great importance as natural
medicine on the alleviation of simple symptoms of upper respiratory tract. The evaluation of
the antimicrobial and antioxidant activities of “água-mel” is the main objective of the present
work. The correlation between antioxidant activity and the phenolic content of “água-mel”, its
colour and the Maillard reaction like products, such as melanoidins, possibly formed during the
“água-mel” production, is also addressed. The levels of polyphenols were closely dependent
on the producer and year of production, which may be due to the floral origin of the honey.
All samples presented antioxidant capacity when measured through by several methods, but
with diverse strength and dependent on the phenol content. In the method ORAC (oxygen
radical absorbance capacity), a positive correlation was found (r=0.799, p<0.01). Nevertheless,
such correlation was superior between melanoidin content assessed spectrophotometrically
as net absorbance at (A450-A720) and ORAC (r=0.901, p<0.01). The antimicrobial activity of three
different samples was determined against Gram-positive, Gram-negative bacteria and two
yeasts (Saccharaomyces cerevisae and Candida albicans). Both Gram positive and Gram negative
and also the two yeasts were more susceptible to one of the tested “água-mel” samples that can
be associated with higher levels of the polyphenol content. Enterobacter faecalis was the most
resistant bacterium being recovered from the highest concentration tested. The most active
“água-mel” sample was capable to successfully control the growth of two methicillin-resistant
Staphylococcus aureus strains being not recovered from all tested concentrations.
Acknowledgements: This study was funded by the Ministério da Agricultura, do Desenvolvimento Rural e das
Pescas, Portugal, under the Program PAN2011/2013, Medida A (Portugal).
____________
126
Annual
meeting
of
the
International
Honey
Commission
PC47. Physicochemical of Maltese honey: an attempt to determine seasonal
variations in honey
Adrian Bugeja Douglas(1)*; Everaldo Attard (1)
1: Division of Rural Sciences and Food Systems, Institute of Earth Systems, University of Malta Msida,
MSD2080, Malta
The Maltese Islands have been renowned for the production of high quality honey, since
ancient times. The Greeks called the island Μελίτη (Melite), meaning “honey-sweet,” a name that
was also used under the Roman rule. During the Arab occupation (870 - 1090 A.D.), the name
of was changed to Malta.
Although some sporadic studies were conducted, Maltese honey was never fully
characterized. Consequently, a public-funded project was developed and awarded, for
the scientific evaluation and characterization of Maltese honey. The aim of this study is to
characterize the physicochemical properties of Maltese honey. In this study, 104 samples were
analysed and 11 parameters were measured, including contents of colour index (CI), consistency,
brix, water content, pH, acidity, electrical conductivity (EC), HMF, diastase activity and proline.
The samples were collected from four harvests: spring 2011 (21), summer 2011 (33), autumn (22)
and spring 2012 (28). Honey colour distinctively demonstrate that late spring/summer honey is
predominantly of the thyme type (CI=1.739±0.269), while autumn honey is predominantly of the
carob type (CI=3.909±0.207). Most honey types are of the liquid to semi-solid type of consistency,
with moisture contents less than 18.958±0.528%, pH ranging between 3.74 and 4.01, and HMF
values usually not exceeding 17.615±3.857 mgkg-1. There was a positive correlation between
EC and proline content for all four harvests (r=0.975, n=82). The EC was exceptionally high for
the autumn samples (1894.727±143.623 µScm-1). Principal Component Analysis exhibited group
correlations within the first two factors. We conclude that most physicochemical parameters are
distinctively influenced by season.
____________
127
Annual
128
meeting
of
the
International
Honey
Commission
Annual
meeting
of
the
International
Honey
Commission
Index of authors
A
A. Mendes-Faia, 59
A. Mendes-Ferreira, 59
Adélio A.S.C. Machado, 42, 105
Adrian Bugeja Douglas, 127
Adriane A. Machado de Melo, 75
Adriane Melo, 46
Ahmad Al- Ghamdi, 70
Alain Le Bail, 57
Aleš Gregorc, 117
Alex da Silva de Freitas, 85
Alexandra Silva, 66
Alexandre Bera, 111
Alexandre Vieira dos Santos, 85
Alexandros Papachristoforou, 61
Ali Reza Abassian, 125
Amanda C. Díaz-Moreno, 31
Ana Balola, 103
Ana Cunha, 109
Ana Maria Carvalho, 67
Ana Pascual-Maté, 93, 101, 102
Ana Paula Pereira, 30, 59
André Santos, 110
Andreas Thrasyvoulou, 23, 55
Andreia Tomás, 114
Angela Savino, 54
Antonio Bentabol, 24
Antonio Herrera, 118
António M. Peres, 42, 105
Arne Dübecke, 58
Aslı Elif Sunay, 43, 49
Ayse Bakan, 43
B
Banu Bahar, 43
Basilis Liolios, 23
Batteau Magali, 45
Beata Madras-Majewska, 83
Beatrix Brinkmann, 34
Benjamin Poirot, 57, 81, 82
Bianca Eickermann, 38
Biljana Marošanović, 51
Birgit Lichtenberg-Kraag, 34, 71
C
Canan Dogan, 43
Capucine Meneau, 82
Carla Pereira, 66
Carlos Fuenmayor, 31, 47, 50
Carlos Zuluaga, 31, 47, 50
Carmen Jerónimo, 107
Casabiance Hervé, 45
Caterina Cafiero, 89
Celeste Serra, 103
Celestino Santos-Buelga, 67
Celine Hedou, 123
Cesarettin Alasalvar, 43
Chlebo Róbert, 121, 122
Chrisovalantis Papaefthimiou, 61
Christina Kast, 34, 58
Chrysoula Tananaki, 23
Chysa Tananaki, 55
Consuelo Díaz-Moreno, 50
Consuelo Díaz, 47
Consuelo Pérez-Arquillué, 118
Cord Lüllmann, 58
Cristina Almeida Aguiar, 107, 109
Cristina Freire, 113
Cristina Pardo, 69
D
Dalibor Titera, 97, 98
Daniele Gaelle, 45
Danielle Aguiar, 20
Darija Vukić Lušić, 53
Davi F. Sampaio Meira, 46, 75, 85
Dimitris Kanelis, 23
Dinis Francisco, 33
Dirk Klaus, 38
Dobre Irina, 90
Dragan Bubalo, 77
Dražen Lušić, 53, 119
Dušanka Milojković-Opsenica, 51, 79
Duška Ćurić, 53
E
Ebru Pelvan, 43
Einar Etzold, 71
Elena Zubova, 74
Ellen de Souza Soares, 46
Els Daeseleire, 39
Elżbieta Rosiak, 83
Emmanuel Karazafiris, 23
Eric Verdon, 123
Escuredo Olga, 90, 91
Esra Agel, 43
129
Annual
meeting
Everaldo Attard, 127
Ewa Was, 78, 87
F
F. Rincón, 41
Fátima Baltazar, 107
Fátima Peres, 94
Fernando M. Nunes, 35
Filip Andrić, 51
Florian Rommerskirchen, 38
Francesca-Vittoria Grillenzoni, 53
Frédéric Bataille, 81
G
Galina Legotkina, 73
George Goras, 55
George Theophilidis, 61
Georgios Goras, 23
Gian Luigi Marcazzan, 53, 54, 89, 93
Gudrun Beckh, 58
Guibert Sylvie, 45
H
Hana Vinsova, 98
Hartje Müller, 38
Hartmut Wischmann, 38
Hayrettin Ozer, 43
Helena Baša Česnik, 117
Helena Rybak-Chmielewska, 78, 87
Hleba Lukáš, 121, 122
I
I. Rodríguez, 41
Igor Jerković, 77
İlknur Demirtaş, 43
İmge Oktay, 43
Ioanna Chekimian, 86
Isabel C.F.R. Ferreira, 66, 67
Isabel Valença, 107
Ivan Kosalec, 119
Ivana Tlak Gajger, 119
J
J.A. Seijas, 99
Jasna Bertoncelj, 37
Jelena Kečkeš, 79
Jelena Trifković, 79
Joana Coelho, 111
Joana Duarte, 126
João Carlos Silva, 30
João Casaca, 115
130
of
the
International
Honey
Commission
João J.S. Sousa, 20
Jorge Sá Morais, 42
José A. Gasparotto Sattler, 46, 75
José María Castro, 106
José María Fresno, 106
José Rodrigues, 94
Josef Stich, 97
Josipa Vlainić, 119
K
Kačániová Miroslava, 121,
Katarina Bilikova, 29
Katarzyna Jaśkiewicz, 78
Katharina Bieri, 58
Katrina Brudzynski, 27
Kirsten Schneidermann, 38
Kristina Lazarević, 79
Krystyna Pohorecka, 78
L
L.M. Estevinho, 30, 42, 59, 99, 105, 106
Lars Ganske, 38
Ligia B. Almeida-Muradian, 46, 75, 85, 111
Lillian Barros, 66, 67
Liset Maldonado-Alvarez, 27
Ljubiša Stanisavljević, 79
Lourdes Corredera, 118
Lucia Piana, 34, 54
Luciana Barbosa dos Santos, 46
Luís G. Dias, 30, 42, 105, 115
Lutz Elflein, 38
M
M. Dulce Antunes, 126
M. Graça Miguel, 126
M. Leonor Faleiro, 126
M. Teresa Sancho, 101
M.B.Farshineh Adl, 125
M.P. Vázquez-Tato, 99
Mª Eugenia Tornadijo, 106
Mª João Sousa, 110
Mahmoud Salesi, 125
Maja Natić, 51
Maja Smodiš Škerl, 117
Mandy Schmidt, 38
Mara E.B.C. Sousa, 42, 105
Márcia Cruz, 109
Marcus Vinícius de Pinho Pires, 95
Maria da Graça Campos, 33
Maria Dimou, 23
Maria G. Campos, 20
Annual
meeting
of
the
International
Maria Teresa Sancho, 93, 102
Mariassunta Stefano, 54
Marie-Jeanne Gourmaud, 81
Marinka Kregar, 117
Mário Gomes, 115
Marta Cunha, 107
Martha Quicazán, 31, 47, 50
Martin Linkogel, 38
Massimiliano Magli, 54
Massimiliano Valentini, 89
Melia V. González-Porto, 69
Michal Bednar, 97, 98
Miguel A. Fernández-Muiño, 93, 101, 102
Miguel Maia, 35
Miguel Vilas-Boas, 66, 110, 111, 113, 114, 115
Milica Jovetić, 79
Miroslava, 122
Mohammad Behjatian Esfahani, 125
Mohammad Javed Ansari, 70
Mojca Korošec, 37
Monika Pytlak, 78
Montserrat Dueñas, 67
Mustafa Yaman, 43
Myriam Laurie, 57, 82
N
Nada Vahčić, 53
Nair Alua, 103
Natalya Gavrilova, 73
Nebojša Nedić, 51
Nelma Pértega-Gomes, 107
Neşe Aslı Öncü, 43
Nihat Ozcan, 43
Nuno Vale, 113
O
Ofélia Anjos, 33, 94
Ortrud Monika Barth, 85
Ozlem Aslan, 43
P
Patricia Beaune, 34
Patricia Combarros-Fuertes, 106
Patricia Vit, 101
Paulo Antunes, 33, 94
Peter Gallmann, 21
Piotr Semkiw, 87
Piotr Skubida, 87
R
Honey
Commission
Regina Lázaro, 118
Régis Brunet, 81
Renato Januario Sousa, 111
Roberto Piro, 34
Robin Azemar, 57
Rodríguez-Flores M. Shantal, 90, 91
Roman Kaygorodov, 74
Romana Rutar, 117
Rosires Deliza, 101
Rui Oliveira, 109
S
S. Serrano, 41
S.M.K. Deylami, 125
Sandra Piosek, 38
Saskia Timmermann, 38
Sayed Mazaher Sayedi, 125
Seijo M. Carmen, 90, 91
Senem Akkuş Çevikkalp, 43
Shahaboddin Mosharaf, 125
Silvia Cristina Ferreira Iop, 95
Silvia Gardini, 89, 93
Silvija Zlatar, 119
Silvio Kečkeš, 51
Smail Aazza, 126
Sofia Gounari, 55
Soraia I. Falcão, 111, 113, 114
Susana Bayarri, 118
Susana M. Cardoso, 113
T
T. Dias, 99
Tananaki Chrysoula, 86
Taylan Samancı, 43, 49
Teresa Szczęsna, 78, 87
Terezija Golob, 37
Thrasyvoulou Andreas, 86
Toni Vlainić, 119
Torsten Peix, 38
Tuğçe Daştan, 43
V
Valerie Gaudin, 123
Valérie Nevers, 57, 81
Vanilda A. Soares de Arruda, 85
Vassya Bankova, 73
Verena Kilchenmann, 34, 58
Veronika Kmecl, 117
Vladimir Mićović, 53
Vukovič Nenad, 121, 122
Rail Khismatullin, 73, 74
131
Annual
meeting
of
the
International
Honey
Commission
W
Wim Reybroeck, 39
Wytrychowski Marine, 45
X
X. Feás, 99
Y
Yehya Al-Attal, 70
Z
Živoslav Tešić, 51, 79
Zvonimir Marijanović, 77
132

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Transcription

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