AFS – Advances in Food Sciences es in Food Sciences

Transcription

AFS – Advances
Advances in Food Sciences
Continuation of CMTL founded by F. Drawert
Production by PSP – Parlar Scientific Publications, Angerstr. 12, 85354 Freising, Germany
in cooperation with Lehrstuhl für Chemisch-Technische Analyse und Lebensmitteltechnologie,
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Printed in GERMANY – ISSN 14311431-7737
© by PSP Volume 24 – No 3. 2002
1
AFSAFS- Editorial Board
Chief Editors:
Prof. Dr. H. Parlar
Institut für Lebensmitteltechnologie und Analytische Chemie, TU München 85350 Freising-Weihenstephan, Germany - E-mail: [email protected]
Dr. G. Leupold
Institut für Lebensmitteltechnologie und Analytische Chemie, TU München 85350 Freising-Weihenstephan, Germany - E-mail: [email protected]
CoCo-Editor:
Prof. Dr. R. G. Berger
Zentrum Angewandte Chemie, Institut für Lebensmittelchemie, Universität Hannover
Wunstorfer Straße 14, 30453 Hannover - E-mail: [email protected]
AFSAFS Advisory Board
E. Anklam, I
F. Coulston, USA
N. Fischer, D
A. Görg, D
D. Hainzl, P
D. Kotzias, I
M.G. Lindhauer, D
S. Nitz, D
M. Spiteller, D
R.F. Vogel, D
P. Werkhoff, D
M. Bahadir, D
J.M. de Man, CAN
S. Gäb, D
U. Gill, CAN
W.P. Hammes, D
F. Korte, D
B. Luckas, D
A.M Raichlmayr-Lais, D
H. Steinhart, D
R.P. Wallnöfer, D
Editorial ChiefChief-Officer:
Selma Parlar
PSP- Parlar Scientific Publications - Angerstr.12, 85354 Freising, Germany
E-Mail: [email protected] - www.psp-parlar.de
Marketing Chief Manager:
Max-Josef Kirchmaier
MASELL-Agency for Marketing & Communication, Public-Relations
Angerstr.12, 85354 Freising, Germany
E-Mail: [email protected] - www.masell.com
2
CONTENTS
ORIGINAL PAPERS
EFFECTS OF STERILANTS ON GROWTH OF Pleurotus Sajor-Caju ON CASSAVA PEELS
94
C.O. Adenipekun and I. O Fasidi
ANTIOXIDANT PROPERTIES OF OLIVE PHENOLIC COMPOUNDS
ON SUNFLOWER OIL STABILITY
99
R. S Farag, N. M. Abd-Elmoien and E. A. Mahmoud
GLYKOSIDISCH GEBUNDENE AROMASTOFFE IN HOPFEN (Humulus lupulus L.):
1. ENZYMATISCHE FREISETZUNG VON AGLYCONEN
106
H. Kollmannsberger und S. Nitz
MICROBIAL AND BIOCHEMICAL CHANGES OCCURRING DURING
FERMENTATION OF marula (Sclerocarya birrea subspecies caffra) JUICE
TO PRODUCE mukumbi, A TRADITIONAL ZIMBABWEAN WINE
116
A. Mpofu and R. Zvauya
COMPARATIVE STUDIES ON BIOSORPTION OF COBALT (II), NICKEL (II),
LEAD (II) AND MANGANESE (II) BY FOUR DIFFERENT FUNGI
121
M. H. Habibi, G. Emtiazi, Z. Khalesi and M. A. Haghighipour
SHORT COMMUNICATION
PHYSICOCHEMICAL ANALYSIS OF TOKAT REGION (TURKEY) HONEYS
125
M. Tüzen
BOOK REVIEWS – BÜCHERSCHAU
128
G. Leupold
INDEX
135
93
EFFECTS OF STERILANTS ON GROWTH OF
Pleurotus Sajor-Caju ON CASSAVA PEELS
C.O. Adenipekun and I. O Fasidi
Department of Botany and Microbiology, University of Ibadan, Ibadan, Nigeria
SUMMARY
The addition of 0.5% formalin as sterilant resulted in
the best mycelial growth of Pleurotus sajor-caju on cassava peel wastes and also served as a good biocide in
reducing the rate of contamination by other microorganisms. In all the series of experiments, in which calcium
sulphate, calcium carbonate, ammonium nitrate, ammonium sulphate and urea were added, the fungus exhibited
the best mycelial growth on the substrates containing
0.1% (w/v) of the salts, except for calcium carbonate,
where the best growth was attained only by addition of
1.0% (w/v).
cultivation consisted of sawdust (73-76%), chalk (12%), carbamide (0.3-0.5%), NPK (0.3 – 0.5%) and water
(2.0 – 2.5%).
Recent studies show that Pleurotus species can be
cultivated on sterilized straw compost [6]. Fasidi and
Kadiri [7] found that Lentinus subnudus, a Nigerian edible mushroom grew best on Andropogan tectorum
(Poaceae) straw supplemented with 30% rice bran or
milled cassava peels. Fructification also occurred on logs
of Spondias mombin and unfermented compost comprising straw, rice bran, horse dung and CaSO4.
However, the addition of ammonium salts resulted in
a decrease of mycelial growth with an increase in the salt
concentration. A mixture of various proportions of CaSO4
and CaCO3 to the substrate produced no significant effects on the growth of the fungus. It was also observed
that the addition of the insecticide Rogor L 40 and fungicide Brestan in concentrations of 0.5%, 1.0% and 2.0%
had highly significant inhibitory effects on growth.
In Nigeria, cassava peels are one of the important
wastes generated during the processing of cassava for
garri production. It is proposed to crush the peels and
allow this to ferment with the liquid squeezed out from
the cassava mash (second waste in garri production) having microorganisms capable of hydrolyzing the glucosides. The resulting product is dried and used as animal
feed. But if it will be useful as a substrate, then this will
be a direct way of cleaning the environment. Pleurotus
sajor-caju is an exotic and highly nutritive species and its
cultivation should be encouraged to supplement the protein requirements of Nigerians. To guarantee optimal
mycelial growth, also formalin was used as a sterilant,
because there are no up-to-date decrees or legislative
regulations concerning its use in food production. This
study, therefore, aims at investigating the growth of the
oyster mushroom using cassava peels, an agricultural
waste, as the main substrate.
KEYWORDS:
Pleurotus sajor-caju, cassava peels, fungicides, insecticides.
INTRODUCTION
In Nigeria mushrooms are eaten generally because of
their desirable flavour and food values. Kadiri and Fasidi [1]
have shown that P. tuber-regium is highly nutritive and
very rich in proteins but also in sugars such as galactose.
They are also consumed in various combinations of medicinal herbs and other ingredients with the intention to
cure headache, stomach ailments, colds and fever as well
as asthma, smallpox and high blood pressure [2, 3].
MATERIALS AND METHODS
The pure culture of Pleurotus sajor-caju was obtained from I. O. Fasidi, Department of Botany and Microbiology at the University of Ibadan. Fresh cultures
were got by regular subculturing on potato dextrose agar
medium.
The cultivation of Pleurotus species on tree stumps
and logs was first described by Falck [4]. Lozovoi [5]
conducted a study on Pleurotus ostreatus and other Pleurotus strains and found that the recommended substrate for
Cassava peels were collected fresh from the cassava
flour mill at Abadina, University of Ibadan, sun-dried for
94
were prepared in various concentrations, 0.1%, 0.5%,
1.0% and 2.0% (w/v).
a period of one hour and then macerated in a laboratory
mill into particles of about 5 mm in diameter.
15 g of ground cassava peels were weighed into each
petri dish, followed by addition of 10 ml of salt solution
with different concentrations and, finally, mixed thoroughly with the cassava peels as substrate.
Effects of sterilants
The effect of some sterilants on the rates of contamination and growth of P. sajor-caju mycelia on cassava peel
substrate was determined. The sterilants Chlorox and formalin were prepared in different concentrations (Chlorox,
10% and 20% (v/v); formalin, 0.5%, 1.0% and 2.0% (v/v)).
15 g of ground cassava peels were weighed into each petri
dish containing 10 ml of sterilant solution. Then the mixture was stirred until the substrates were well-moistened.
The plates were then cooked at 100 °C for a period of one
hour in a waterbath and steam-sterilized in an autoclave at
121 °C for 30 min or left on the bench unsterilized.
Also the effect of a mixture of two mineral salts, calcium carbonate and calcium sulphate, on the mycelial
growth of the fungus was tested. A fixed concentration of
CaSO4 (0.2%) with varying amounts of CaCO3 (0.2%,
0.4%, 2.0% and 4.0% w/v) was used. Into each petri
dish containing 15 g of ground cassava peels, 5 ml of
0.2% CaSO4 (w/v) was pipetted, followed by the addition of 5 ml each of the CaCO3 solutions in different
concentrations. Afterwards the solutions were mixed
thoroughly with the cassava peels.
Effect of biocides
Effect of additives
In the set-up, 10 ml distilled water served as control
solution and each treatment was carried out in triplicate.
The petri dishes were wrapped with aluminium foil and
autoclaved at 121 °C for 30 min. After cooling each of the
substrates was inoculated in the centre with an agar plug
(0.7 mm) obtained from a 7-day old pure culture of Pleurotus sajor-caju by means of a sterile cork borer. These
sets of plates were then incubated at 30 ± 2 °C and readings of linear growth of mycelia on each plate were taken
at 2-day intervals for a period of 8 days except for the
experiments with biocides where readings were taken
daily for a period of 7 days.
The effect of some mineral salts (calcium sulphate,
calcium carbonate, ammonium nitrate ammonium sulphate and urea) as additives on the rate of mycelial
growth on cassava peels was investigated. These salts
The ANOVA test was used to determine the effect
of the treatments on the growth of mycelia of the fungus
(P < 0.05 and P<0.01). The data were further analyzed
using the LSD test at (P<0.05 and P<0.01).
Two fungicides, Benlate and Brestan, an insecticide
roger 40 and sterilants fermalin and Aldrex T were prepared in different concentrations – 0.5%, 1.0% and 2.0%.
For Benlate solution, 10ml of absoluten alcohol was
added to dissolve the powder and the volume was made
up to 100ml with sterile distilled water. Fifteen grammes
of ground cassava peels were weighed into each isolate;
10ml of sterilant solution was pipetted into each plates
the mixture was well stirred so that the substrate is well
soaked.
TABLE 1 - Effectiveness of different methods of sterilization on rates of mycelial growth of Pleurotus sajor-caju
Substrates
CP + water + autoclaving (control)
CP + 10% chlorox and autoclaving
CP + 10% chlorox and cooking
CP + 10% chlorox and left unsterilized
CP + 20% chlorox and autoclaving
CP + 20% chlorox and cooking
CP + 20% chlorox and left unsterilzed
CP + 0.5% formalin and autoclaving
CP + 0.5% formalin and cooking
CP + 0.5% formalin and left unsterilized
Diameter of mycelial growth (mm)
6.38 ± 0.32
5.40 ± 0.65 ns
6.38 ± 0.38 ns
C
5.25 ± 0.25ns
4.63 ± 0.88ns
C
6.65 ± 0.20ns
C
C
4.28 ± 0.15ns
C
C
4.23 ± 0.13ns
0.70 ± 0.00
C
Each figure is a mean of 3 readings ± standard error taken on 8th day.
CP = cassava peels; C = No growth recorded due to contamination; ns = values not significant (P < 0.05, P < 0.001) by LSD test;
* = Significant (P<0.05) by LSD test; ** = Highly significant at (P < 0.05 and P < 0.01) by LSD test.
95
TABLE 2 - Effect of some biocides on mycelial growth of Pleurotus. sajor – caju on cassava peels.
Substrates
CP + water (control)
CP + 0.5% Aldrex T
CP + 1.0% Aldrex T
CP + 2.0% Aldrex T
CP + 0.5% Benlate
CP + 1.0% Benlate
CP + 2.0% Benlate
CP + 0.5 % Brestan concn
CP + 1.0% Brestan concn.
CP + 2.0% Brestan concn
CP + 0.5% formalin
CP + 1.0% formalin
CP + 2.0% formalin
CP + 0.5% Rogor 40
CP + 1.0% Rogor 40
CP + 2.0% Rogor 40
5.50 ± 0.06
5.05 ± 0.19ns
5.55 ± 0.00ns
4.85 ± 0.08ns
1.70 ± 0.10**
2.87 ± 0.13**
1.78 ± 0.46**
4.80 ± 0.46ns
4.75 ± 0.13ns
3.50 ± 0.06**
6.18 ± 0.09**
4.50 ± 0.00*
0.70 ± 0.00**
4.25 ± 0.25**
3.22 ± 0.53**
1.69 ± 0.14**
CP = cassava peels; ns = values not significant (P < 0.05, P < 0.001) by LSD test.
taminations. This is in agreement with findings of Genders [9] who used formalin as a sterilant before and during the process of composting either to prevent the spread
of brown plaster mould or to sterilize soil sometimes used
for casing where sterilization equipments are not available. In the series of experiments, when CaSO4, CaCO3,
NH4NO3, (NH4)2SO4 and urea were added, the fungus
showed the best mycelial growth on the substrate to
which 0.1% (w/v) concentration of the salts was added,
except CaCO3, where optimal growth was achieved at a
concentration of 1.0% (Table 3).
RESULTS AND DISCUSSION
In all the substrates to which sterilants (Chlorox or
formalin) were added followed by antoclaving, good mycelial growth was observed, even in the control, but the unsterilized substrate resulted in a serious culture contamination (Table 1). The LSD test showed no significant difference (P < 0.05 and P< 0.01) between control and all treatments except that of 2.0% formalin and cooking. Pizer [8]
reported that steam sterilization for one hour at 20 lbs pressure prior to inoculation with spawn, altered the physical
nature of the composts by dispersing starch and also
brought about coagulation of proteins. He also found that
the effect of sterilization on mycelial growth of fungus was
dependent on the nature of the compost, the previous
treatment and time of sterilization. Pleurotus sajor-caju
exhibited lower mycelial growth on most of the substrates,
to which biocides were added, compared to the control
(Table 1). Better mycelial growth, however, was recorded
on substrates to which 1.0% Aldrex T and 0.5% formalin
were added, the latter stimulating better growth (Table 2).
Lowest mycelial growth was recorded at 2.0% concentrations of the biocides with the lowest growth rate for formalin. Benlate and Rogor 40 at all concentrations had a highly
significant effect (P<0.01) on mycelial growth, the former
being a fungicide and the latter an insecticide. Brestan only
recorded a highly significant effect (P≤ 0.05) at 2.0% (w/v),
possibly its most efficacious concentration.
Duggar [10] found in his experiments that some slight
advantages resulted from the inclusion of calcium compounds. Pizer and Thompson [11] suggested that the addition of small amounts (0.5 parts of calcium per 100 parts
of dry compost) flocculated the manure and that this was
the most suitable for rapid, vigorous growth. Calcium has
also been found to be an indispensable nutrient, since it is
physiologically antagonistic to potassium and magnesium
and also overcomes the inhibitory effect of these elements
on the growth of the mycelium [12]. Calcium sulphate
(gypsum) is a ‘conditioner’ or ‘fertilizer’, which improves
the yield of mushrooms when cultivated in the laboratory
and on commercial basis, because of its indirect activity
by improving conditioning of manure compost overcoming greasiness, excessive moisture, excess ammonia and
excess alkalinity [13]. CaCO3 has been found to be less
effective at concentrations of 0.1% and 0.5% (w/v),
probably due to its low solubility under alkaline conditions or possibly that alkali carbonate is formed by base
exchange, increased pH and had a detrimental effect on
the structure of the mature culture [10]. Calcium has also
been used to adjust the levels of pH between 7 and 8 [13].
Formalin had highly significant effects (P ≤ 0.05) at
concentrations of 0.5% and 2.0% (v/v). The low concentration coupled with autoclaving might have provided a
selective substrate favourable for mycelia and other con-
96
TABLE 3 - Effect of additives on mycelial growth of P. sajor. caju on cassava peels.
Substrates
CP + water (control)
CP + 0.1% CaSO4
CP + 0.5 % CaSO4
CP + 1.0% CaSO4
CP + 2.0% CaSO4
CP + 0.1% CaCO3
CP + 0.5% CaCO3
CP + 1.0% CaCO3
CP + 2.0% CaCO3
CP + 0.1% NH4NO3
CP + 0.5% NH4NO3
CP + 1.0% NH4NO3
CP + 2.0% NH4NO3
CP + 0.1% (NH4)2SO4
CP + 0.5% (NH4)2SO4
CP + 1.0% (NH4)2SO4
CP + 2.0% (NH4)SO4
CP + 0.1% urea
CP + 0.5% urea
CP + 1.0% urea
CP + 2.0% urea
6.00 ± 0.00
6.60 ± 0.20ns
5.92 ± 0.69ns
4.63 ± 0.10*
4.63 ± 0.10*
6.52 ± 0.76ns
5.77 ± 0.02ns
7.07 ± 0.44*
6.08 ± 0.08ns
6.75 ± 0.75ns
4.60 ± 0.40**
4.51 ± 0.06**
5.14 + 0.39ns
7.75 ± 0.25**
6.25 ± 0.05ns
4.91 ± 0.05*
4.45 ± 0.20**
6.48 ± 0.38ns
6.28 ± 0.53ns
5.60 ± 0.10ns
5.30 ± 0.30ns
ns = values not significant (P < 0.05, P < 0.001) by LSD test
due to the fact that mushroom mycelium is high in nitrogen
(6.44% of its dry weight) with nearly half of this nitrogen in
a water-soluble form and that ammonium salts of strong
acids soon develop a highly acidic substratum that the mycelium is inhibited and eventually killed [15]. Zadrazil [16]
recorded that inorganic nitrogen (e.g. ammonium nitrate)
increases the yield of fruit bodies by about 30%. Addition of
urea at all concentrations had no significant effect on growth
of the fungus. This is in contrast to the findings of Styer [14]
who reported that urea has been recognized as a utilizable
nitrogen source and is sometimes more suitable than aminoacids in certain edible mushrooms.
Duggar [10] found in his experiments that some slight
advantages resulted from the inclusion of calcium compounds. Pizer and Thompson [11] suggested that the addition of small amounts (0.5 parts of calcium per 100 parts
of dry compost) flocculated the manure and that this was
the most suitable for rapid, vigorous growth. Calcium has
also been found to be an indispensable nutrient, since it is
physiologically antagonistic to potassium and magnesium
and also overcomes the inhibitory effect of these elements
on the growth of the mycelium [12]. Calcium sulphate
(gypsum) is a ‘conditioner’ or ‘fertilizer’, which improves
the yield of mushrooms when cultivated in the laboratory
and on commercial basis, because of its indirect activity
by improving conditioning of manure compost overcoming greasiness, excessive moisture, excess ammonia and
excess alkalinity [13]. CaCO3 has been found to be less
effective at concentrations of 0.1% and 0.5% (w/v),
probably due to its low solubility under alkaline conditions or possibly that alkali carbonate is formed by base
exchange, increased pH and had a detrimental effect on
the structure of the mature culture [10]. Calcium has also
been used to adjust the levels of pH between 7 and 8 [13].
Pleurotus sajor-caju showed better mycelial growth
than the control on all the substrates to which a mixture of
mineral salts was added. No significant difference (P<0.05,
P<0.01) was observed when the individual treatments were
compared with the control by means of the LSD test.
From these results it is clear that P. sajor-caju, an exotic species, can be cultivated in the tropical laboratory or
in large quantities in the field on composts of cassava
peels with 0.1% (w/v) of calcium sulphate, ammonium
nitrate, ammonium sulphate, urea and 1.0% of calcium
carbonate. The addition of biocides such as formalin at
0.5% as a sterilant followed by autoclaving to reduce the
high rate of contamination by other fungi and microorganisms will ultimately result in better fruit body production
of P. sajor-caju on cassava peels.
The addition of ammonium salts at 0.1% (w/v) levels
resulted in better mycelial growth, compared to the control.
Styer [14] reported ammonium salts as useful sources of
nitrogen, the most complex being the most effective. A
decrease in mycelial growth of the fungus with increase in
the concentration of salts was also observed. This might be
97
TABLE 4 - Effect of a mixture of additives on growth of P. sajor-caju on cassava peels.
Substrates
CP + water (Control)
CP + 0.2% CaS04 + 0.2% CaC03
CP + 0.2% CaS04 + 1.0% CaC03
CP + 0.2% CaS04 + 2.0% CaCo3
CP + 0.2% CaS04 + 4.0% Cac03
4.88 ± 0.19
4.88 ± 0.38ns
5.10 ± 0.10ns
5.32 ± 0.20ns
5.0 ± 0.29ns
ns = values not significant (P < 0.05, P < 0.001) by LSD test.
REFERENCES
[1]
Kadiri; M. and Fasidi, I. O. (1990a). Nig. J. Sci 24: 86 – 9.
[2]
Oso, B. A. (1977). Mycologia 69 (2): 271-279.
[3]
Fasidi, Isola O. and Olorunmaiye, Kehinde S. (1994). Food
Chem. 50, 397-401.
[4]
Falck, R. (1917). Z. forst. Jagdires 4: 159 – 165.
[5]
Lozovoi, V. D. (1980). Rastit Resur 16 (1): 38 – 45.
[6]
Chandrashekar, Y. R.; Bano, Z. and Rajarathnan, S. (1981).
Trans Brit. Mycol. Soc. 77 (3): 491 – 495.
[7]
Fasidi, I. O. and Kadiri, M. (1993). Rev. Biol. Trop. 4(3):
411 – 415.
[8]
Pizer, N. H. (1937). J. Agric Sci. 27: 349 – 376.
[9]
Genders, R. (1982). Mushroom growing for everyone. Ist ed.
Faber and Faber publication 115pp.
[10] Duggar, B. M. (1905). U.S. Dept. Agr. Bur. Pl. Ind. Bull. 85:
1 – 60.
[11] Pizer, N. H. and Thompson, A. J. (1938). J. Agric Sc. 28:
604 – 617.
Received for publication: March 14, 2002
Accepted for publication: September 10, 2002
[12] Treschow, C. (1944). Dansk. Botanisk Arkiv. 11: 1 – 180.
[13] Singer, R. C. (1961). Mushrooms and Truffles. Botany cultivation and utilization. World crops books 1st ed. interscience publishers (nc 272 pp).
CORRESPONDING AUTHOR
C.O. Adenipekun
Department of Botany and Microbiology
University of Ibadan
Ibadan - NIGERIA
[14] Styer, J. F. (1928). Amer. J. Bot. 15: 246 – 250.
[15] Waksman and Nissen, W. (1932). Amer. J. Bot. 19: 514 – 537.
e-mail: [email protected]
[16] Zadrazil, F. (1980). Eur. J. Appl. Microbiol. Biotechnicol 9:
31 – 35.
AFS/ Vol 24/ No 3/ 2002 – pages 94 - 98
98
ANTIOXIDANT PROPERTIES OF OLIVE PHENOLIC
COMPOUNDS ON SUNFLOWER OIL STABILITY
R. S Farag, N. M. Abd-Elmoien and E. A. Mahmoud
Biochemistry Department, Faculty of Agriculture, Cairo University, Giza, Egypt.
SUMMARY
The phenolic compounds from ripe leaves and fruits
of Kronakii olive variety were extracted and fractionated
into three major fractions, i. e., free, esterified and residual phenolic acids. These fractions were individually
mixed with sunflower oil in different concentrations (100,
200 and 400 ppm) to assess their antihydrolytic and antioxidant behaviour. Some measurements of rancidity were
conducted by estimating e.g., acid, peroxide and thiobarbituric acid values for sunflower oil alone and mixed with
phenolic components during storage at room temperature.
The antihydrolytic and antioxidant phenomena of olive
phenolic compounds were compared with BHT activity as
a common synthetic antioxidant.
Therefore, there is a great need for substituting the
aforementioned synthetic antioxidants by other natural
antioxidants [1, 5, 6].
Among the most important natural antioxidants are
tocopherols and ascorbic acid. Tocopherols are potent in
vivo inhibitors of lipid peroxidation but they are less effective than BHA or BHT as food antioxidants [6].
Melted beeswax and its unsaponifiable constituents were
mixed with butter oil or refined cottonseed oil to study the
hydrolytic and oxidative effects of the mixtures [7]. The
unsaponifiables at different levels exhibited antihydrolytic and antioxidant effects on butter oil and refined
cottonseed oil, respectively. Xinchu et al. [8] found that
petroleum ether, acetic acid, ether and alcohol (95%)
extracts of different parts of Salvia plebeia induced an
antioxidant activity. Thyme and cumin essential oils were
used to prevent cottonseed oil and butter rancidity during
storage at room temperature [1]. In addition, thyme and
clove essential oils are quite safe and can be applied practically as natural antioxidants for lipids [9]. Charai et al.
[10] studied the effect of essential oils obtained from
certain aromatic plants as natural antioxidants for olive
oil. Their results showed a wide variation in the antioxidant activity of the essential oils and the highest activity
was observed with Thymus broussonetti essential oil.
Total and free polyphenols obtained from both leaves
and fruits of Kronakii olive cultivar possessed antihydrolytic and antioxidant activities increasing with concentration. At 400 ppm level they exhibited remarkable effects,
superior to those of BHT, in retarding sunflower oil stability.
KEYWORDS: Polyphenols, olive fruits and leaves, sunflower oil,
quality assurance tests, rancidity.
The aim of the present work was to extract and fractionate the total polyphenols from the leaves and fruits of
Kronakii olive cultivar, i.e., free, esterified and residual
phenolic compounds. These fractions were individually
added to sunflower oil to increase its stability and to compare their antioxidant activity with BHT.
INTRODUCTION
Lipid peroxidation causes various damages not only
in living organisms but also in foods. To retard undesirable changes in lipids due to oxidation it is necessary to
add antioxidants to food products before use [1, 2]. The
most common antioxidants are tocopherols and synthetic
phenolic compounds such as butylated hydroxy anisole
(BHA) and butylated hydroxy toluene (BHT). The use of
BHT or BHA in food has been decreased because of their
suspected action as promoters of carcinogenesis, as well
as the general consumer rejection of synthetic food additives [3]. In addition, BHA and BHT are characterized by
high volatility and instability at elevated temperatures [4].
Source of olive leaves and fruits
The leaves and ripe olive fruits of Kronakii cultivar
were collected during the season 2000 from the Horticulture Research Institute, Ministry of Agriculture, Giza,
Cairo, Egypt.
99
Solvents and Reagents
All solvents were distilled before use. Butylated hydroxy toluene (BHT) and thiobarbituric acid (TBA) were
purchased from Sigma Chemical Co., St Louis, MO,
USA; and Gerbsaure Chemical Co. Ltd., Germany, respectively.
Sunflower oil
Refined sunflower oil was obtained from Cairo Oil
and Soap Co., El-Ayat, Giza, Egypt. The oil peroxide and
acid values were 2.0 meq/kg and 0.4 mg KOH/1g oil,
respectively.
Extraction of olive polyphenols:
The polyphenols of olive fruits and leaves were extracted with ethanol followed by centrifugation at 1,500 g
for 15 min. The ethanolic extracts were dried over anhydrous sodium sulfate and evaporated to dryness [11].
Fractionation of polyphenols
Free, esterified and residual phenolic fractions were
separated from olive fruits and leaves of Kronakii cultivar
according to the method of Dabrowski and Sosulski [12].
Free phenolic acids were initially extracted with tetrahydrofuran containing NaBH4 (0.5%), followed by extraction of soluble phenolic esters with a mixture of methanol: acetone: water (7:7:6, v/v/v). Alkaline hydrolysis was
employed, followed by extraction with a mixture of diethyl ether: ethyl acetate: tetrahydrofuran (1:1:1, v/v/v) to
obtain the insoluble bound phenolic acids.
Oxidation systems
Different concentrations of total, free, esterified and
residual phenolic compounds (100, 200, 400 ppm) and
BHT (200 ppm) were individually added to sunflower oil.
The antihydrolytic and antioxidant activities of each phenolic fraction were examined by the acid, peroxide and
thiobarbituric acid tests daily over a period of 18 days.
These values were used to compare the effectiveness of
the phenolic fractions on sunflower oil stability.
Quality assurance methods
The acid and peroxide values were determined using
Standard American Oil Chemists Society methods (A. O.
C. S. [13]). The secondary oxidation products were determined by the thiobarbituric acid (TBA) test [14]. Three
replications were run for each parameter during sunflower
oil storage and the mean values are presented in the text.
Statistical analysis
The data of quality assurance tests were subjected to
analysis of variance with a randomised complete block
design to partition the effects of different parameters [15].
The simple regression coefficient (reaction slope) for acid
value was statistically calculated.
There is currently a great worldwide interest in finding new and safe antioxidants from natural sources to
prevent food rancidity. The present study was focused on
olive polyphenols which do not induce undesirable odour
or taste, when separated from olive leaves and fruits of
Kronakii variety (very cheap natural source) into 3 major
fractions, i.e., free, esterified and residual phenolic compounds. These fractions were added individually to sunflower oil at various concentrations besides the total polyphenols in order to extend its shelf-life.
The antioxidant and antihydrolytic activities of the
various olive phenolic components under study were
determined by comparing their efficiency with the most
commonly used synthetic antioxidants (BHT, BHA and PG
added to fats and oils at concentrations of 100-400 ppm to
suppress the development of peroxides during food storage (Allen and Hamilton, [16]). For this, in the experiment BHT (200 ppm) was mixed only with sunflower oil.
The phenolic fractions were added at concentrations of
100, 200 and 400 ppm. The changes in efficiency were
determined by the commonly used methods such as acid,
peroxide and thiobarbituric acid values.
Fig. 1 shows the changes in acid values of sunflower
oil mixed with phenolic fractions of olive leaves and
fruits of Kronakii variety, and BHT during storage at
room temperature. The acid values for sunflower oil
alone, sunflower oil mixed with BHT, and total, free,
esterified or residual phenolic fractions linearly increased
with the storage period. To evaluate the effectiveness of
the phenolic material added to sunflower oil, the reaction
slope of the acid value curves was used as a guide in this
context. Accordingly, the slope values for the acidity of
sunflower oil alone and mixed with BHT (200 ppm), and
total (100, 200, 400 ppm) and free (100, 200, 400 ppm)
phenolic fractions of Kronakii leaves were 0.5; 0.3; 0.4,
0.3, 0.1; and 0.4, 0.3, 0.2, respectively. The slope values
of the acid value curves representing the esterified (100,
200, 400 ppm) and residual (100, 200, 400 ppm) phenolic
fractions of Kronakii leaves were identical (0.5). Slope
values higher than 0.5 indicate pro-hydrolytic effects,
whereas those lower than 0.5 demonstrate anti-hydrolytic
activity. Hence, the systems containing BHT, total and
free phenolic fractions exhibited an anti-hydrolytic activity. Conversely, esterified and residual phenolic fractions
at various levels caused non-significant anti-hydrolytic
activity on sunflower oil.
The slope values of sunflower oil acidity using the
phenolic fractions of Kronakii fruits were nearly similar
to that obtained from the leaves. In general, total and free
polyphenols possessed an anti-hydrolytic activity, which
was increased by increasing their concentration. Also, one
has to point out that the use of the latter two fractions at
400 ppm level significantly exhibited anti-hydrolytic
activity and were superior to that of BHT in retarding
sunflower oil hydrolytic rancidity.
100
FIGURE 1 - Effect of total (T), free (F), residual (R) and esterified (E) polyphenolic compounds
of Kronakii olive fruits and leaves on the acid value of sunflower oil.
101
FIGURE 2 -Effect of total (T), free (F), residual (R) and esterified (E) polyphenolic compounds
of Kronakii olive fruits and leaves on peroxide value of sunflower oil.
102
FIGURE 3 – Effect of total (T), free (F), residual (R) and esterified (E) polyphenolic compounds
of Kronakii olive fruits and leaves on thiobarbituric acid (TBA) value of sunflower oil.
103
Fig. 2 shows the changes in peroxide values of sunflower oil mixed with phenolic fractions obtained for both
leaves and fruits of Kronakii olive cultivars during storage.
An autocatalytic chain reaction was induced, i.e., the rate of
hydroperoxide formation increased non-linearly with the
time. Since the curves in Fig. 2 show an increase in peroxide values with time, the peroxide values at the 14th day of
storage for all systems were divided by that of the control
(sunflower oil without any additives) to demonstrate the
effect on the stability of sunflower oil. Therefore, a value
higher than 1.0 indicates pro-oxidant effects while the
values lower demonstrate anti-oxidant activity. Sunflower
oil mixed only with BHT (200 ppm) was used as a control
guide to indicate the anti-oxidant or pro-oxidant activities.
The relative peroxide values for BHT (200 ppm), total
(100, 200, 400 ppm) and free (100, 200, 400 ppm) phenolic
componds of Kronakii leaves were 0.26; 0.40, 0.26, 0.12
and 0.40, 0.26, 0.12, respectively, and those for esterified
(100, 200, 400 ppm) and residual (100, 200, 400 ppm)
phenolic compounds of Kronakii leaves were all identical
(1.0). The relative values for all systems using phenolic
compounds from olive fruits were calculated as mentioned
before and the values were nearly similar to those obtained
from leaves of Kronakii olive cultivar.
The antioxidant values for the systems containing
200 ppm of total and free phenolic compounds from
Kronakii olive leaves and fruits exhibited antioxidant
activity similar to a system comprised of sunflower oil
and BHT (200 ppm). On the other hand, the addition of
esterified and residual phenolic compounds to sunflower
oil of both leaves and fruits at various concentrations
(100, 200, 400 ppm) did not exhibit any antioxidant activity on sunflower oil. It is worth-mentioning that 400 ppm
level of total and free phenolic compounds obtained from
both leaves and fruits produced superior antioxidant
power compared to that of BHT.
Fig. 3 shows the TBA values for the systems sunflower oil (control), sunflower oil plus BHT (200 ppm),
total (100, 200, 400 ppm), free (100, 200, 400 ppm), esterified (100, 200, 400 ppm) and residual (100, 200, 400 ppm)
phenolic compounds extracted from leaves and fruits of
Kronakii olive cultivar. The levels of secondary oxidation
products from sunflower oil were very low and gradually
increased with time. The addition of BHT and total or free
phenolic fractions of olive leaves and fruits to sunflower
oil significantly decreased the formation of secondary
oxidation products at all concentrations, with their decreasing tendency by increasing the levels of free and
total phenolic fractions. The content of secondary oxidation products at the 16th day of storage period for sunflower oil containing 100, 200, and 400 ppm of total and
free phenolic compounds extracted from Kronakii leaves
were 0.36, 0.21, 0.07 and 0.37, 0.23, 0.07, respectively,
and nearly identical to those using the fruit phenolic compounds (0.36, 0.21, 0.09 and 0.36, 0.21, 0.07). On the
other hand, the esterified and residual fractions of both
fruits and leaves did not cause any significant decrease of
the level of secondary oxidation products.
Several authors extracted various phenolic compounds from different plant sources and they generally
caused an increase in the shelf-life of some vegetable oils.
For instance, polyphenols were extracted from the olive
oil using hexane, acetone and ethanol in a simple sequential procedure yielding three fractions, A, B, and C, by
Fayad et al. [17]. Fractions B and C were found to contain
the highest ortho-diphenol concentrations (about 3%).
The addition of purified fraction B at a level of 100 ppm
to refined olive or soybean oils partially inhibited the
oxidative deterioration when the oils were stored in the
dark at 100 ºC. Also, Xing and White [18] reported that
the antioxidant activities of oat groats and hulls increased
with increasing concentrations. During 20 days of storage
the groat extract (0.3%) was not significantly different
from tertiary butyl hydroquinone (TBHQ) after day 16,
and hull extracts (0.2 and 0.3%) were not significantly
different from TBHQ on day 20. The antioxidative activity of the total and free polyphenolic fractions tested, in
general, could be attributed to the presence of hydroxyl
groups in the phenolic ring. This is supported by the powerful antioxiant activites of the well-known synthetic
BHT and the natural antioxidant thymol [19, 20]. The
antioxidant activity to BHT or thymol is related to the
inhibition of hydroperoxide formation. The first step in
lipid oxidation is the abstraction of a hydrogen atom from
a fatty acid and oxygen involvement gives a peroxy radical. Generally, the antioxidants suppress the abstraction of
hydrogen atoms from a fatty acid moiety which leads to
the decrease of hydroperoxide formation. It is well-known
that the phenolic compounds act as hydrogen donors in
this reaction mixture and, therefore, the formation of
hydroperoxides is decreased. The results of the present
study are in agreement with these statements. The phenolic OH groups have to be in the free form and, if attached
to other groups (such as glycosidic residues), it would
prevent their antioxidant power due to the lack of hydrogen atoms donated to fatty acid radicals. This hypothesis is
supported by the fact that the total and free phenolic compounds induced powerful antioxidant effect, while the
esterified and residual phenolic compounds exhibited only
a low effect on retarding sunflower oil oxidative rancidity.
104
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[2]
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[3]
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[4]
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[5]
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[6]
Tsimidou, M. and Boskou, D. (1994). Antioxidant activity of
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[7]
Farag, R. S.; Hassan, M. N. A. and Ali, F. M. (1993). Beeswax and its unsaponifiable components as natural preservatives for butter and cottonseed oils. J. Food Sci. & Nutr., 44:
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[8]
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[9]
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clove essential oils as natural antioxidants. African J. of Agricultural Sciences, 18:168-176.
[10] Charai, M.; Faid, M. and Chaouch, A. (1999). Essential oils
from aromatic plants (Thysmus broussonetti Boiss., Origanum compactum Benth., and citrus limon (L.) N. L. Burm.)
as natural antioxidants for olive oil. J. Essen. Oil Res., 11 :
517-521.
[11] Kanner, J.; Edwin, F., Rina, G.; Bruce, G. and John, E.
(1994). Natural antioxidants in graps and wines. J. Agric.
Food. Chem., 42, 64-69.
[12] Dabrowski, K. J. and Sosulski, F. W. (1984). Composition of
free and hydrolyzable phenolic acids in defatted flours of ten
oilseeds. J. Agric Food Chem. 32: 128-130.
[13] A.O.C.S. (1985). Official and Tentative Methods of the
American Oil Chemists Society, 3rd ed. American Oil Chemists Society, Champaign, IL.
[14] Ottolenghi, A. (1959). Interaction of ascorbic acid and
mitochondrial lipids. Arch. Biochem. Biophys. 79:355-363.
[15] Steel, R. G. D., Torrie, J.H. (1980). Principles and procedures
of Statistics, 3rd edn. McGraw-Hill, New York, U.S.A.
[16] Allen, J. C. and Hamilton. R. J. (1983). Rancidity in Food.,
pp. 85-173. London and New York. Applied Science Publishers.
[17] Fayad, Z.; Sheabar, A. and Neemann, I. (1989). Separation
and concentration of natural antioxidants from the rape of olives., 65:990-993.
[18] Xing, Y. and White, P. (1997). Identification and function of
antioxidants from oat groats and hulls. J. Am. Oil Chem. Soc.
74 :303-307.
[19] Farag, R. S. and El-Khowas, K. H. M. M. (1989). Influence
of γ-irradiation and microwaves on the antioxidant property
of some essential oils. Ibid, 49:109-115
[20] Topallar. H., Bayrak, Y. and Iscan, M. J. (1997). A kinetic
study of the autooxidantion of sunflower seed oil. J. Am. Oil
Chem. Soc. 74: 1323-1327.
105
Received for publication: May 21, 2002
Accepted for publication: July 17, 2002
R. S. Farag
Biochemistry Department
Faculty of Agriculture
Cairo University
P.O.Box 12613
Giza - EGYPT
AFS/ Vol 24/ No 3/ 2002 – pages 99 - 105
GLYKOSIDISCH GEBUNDENE AROMASTOFFE
IN HOPFEN (Humulus lupulus L.):
1. ENZYMATISCHE FREISETZUNG VON AGLYCONEN
H. Kollmannsberger und S. Nitz
Department Lebensmittel und Ernährung, Lehrstuhl für Chem.-Techn. Analyse u. Chem. Lebensmitteltechnologie; Technische Universität München,
Wissenschaftszentrum Weihenstephan für Ernährung, Landnutzung und Umwelt, Weihenstephaner Steig 23, D-85350 Freising-Weihenstephan, FRG
GLYCOSIDICALLY BOUND FLAVOUR COMPOUNDS
IN HOP (Humulus lupulus L.): 1. ENZYMATICAL
LIBARATION OF AGLYCONES.
SUMMARY
The efficiency of different enzyme preparations (almond ß-glucosidase, glucosidase from Aspergillus niger,
pectinase, hesperidinase, α-amylase, a amylase-mixture, a
hemicellulase preparation) for the cleavage of glycosidically
bound flavour compounds of hops (Humulus lupulus L.)
was investigated. Enzymes were added to either synthetic
ß-D-glucosides (phenyl- and octyl-) or hop extracts. The
hop extracts were made by extraction with a watermethanol mixture, or by hot-water extraction and subsequent adsorption on an Amberlite XAD-2 resin. The isolated aglycones were investigated by gas chromatography
- mass spectrometry. Main compounds among the aglycones were 3-methylbutan-2-ol, 3- and 2-methyl-butan-1ol, 3-methyl-2-buten-1-ol, 3-methylpentan-2-ol, 3(Z)hexenol, hexanol, 1-octen-3-ol, benzylalcohol, 2-phenylethanol, linalool, α-terpineol, methylsalicylate, 2,6-dimethylocta-2,7-dien-1,6-diol, 3-hydroxy-7,8-dihydro-ßionol, 3-hydroxy-5,6-epoxy-ß-ionol, vomifoliol and 7,8dihydro-vomifoliol. Additionally small amounts of 3-hydroxy-ß-damascone, a precursor of the sensorial important ß-damascenone could be found among the aglycones.
Best yields of aglycones could be achieved with glucosidase from Aspergillus niger and with rapidase (a hemicellulase preparation with glycosidic activities). Commercially available α−amylase was found to be not suitable
for hydrolysis of hop glycosides.
KEYWORDS: Humulus lupulus L., hop flavour, glycosidically
bound volatiles, glycosides, aglycones, enzymatic hydrolysis.
ZUSAMMENFASSUNG
Verschiedene Enzympräparate (ß-Glucosidase aus
Mandeln, Glucosidase aus Aspergillus niger, Pectinase,
Hesperidinase, α-Amylase, ein Amylase-Gemisch, ein
Hemicellulase-Präparat) wurden auf ihre Eignung zur
Spaltung von glycosidisch gebundenen Aromastoffen des
Hopfens (Humulus lupulus L.) untersucht. Dazu wurden
synthetische ß-D-Glucoside (Phenyl- und Octyl-), wässrig-methanolische Hopfenextrakte und säulenchromatographisch über Amberlite XAD-2 aufgereinigte Hopfenextrakte mit den Enzymen versetzt und die extraktiv
abgetrennten Aglycone gaschromatographisch-massenspektrometrisch untersucht. Als mengenmäßig dominante
Aglycone fanden sich 3-Methylbutan-2-ol, 3- und 2-Methylbutan-1-ol, 3-Methyl-2-buten-1-ol, 3-Methylpentan-2ol, 3(Z)-Hexenol, Hexanol, 1-Octen-3-ol, Benzylalkohol,
2-Phenylethanol, Linalool, α-Terpineol, Methylsalicylat,
(E)-2,6-Dimethyl-Octa-2,7-dien-1,6-diol, 3-Hydroxy-7,8dihydro-ß-Ionol, 3-Hydroxy-5,6-epoxy-ß-Ionol, Vomifoliol und 7,8-Dihydro-Vomifoliol. 3-Hydroxy-ß-Damascon, aus dem durch Dehydratisierung der starke Geruchsstoff ß-Damascenon gebildet werden kann, war ebenfalls
in geringen Mengen unter den Aglyconen nachweisbar.
Die besten Ausbeuten an Aglyconen ergaben sich mit
Glucoside aus Aspergillus niger und mit Rapidase (ein
Hemicellulase-Präparat mit glycosidischen Nebenaktivitäten). Handelsübliche α−Amylase eignet sich nicht zur
Spaltung von Hopfenglycosiden.
EINLEITUNG
Glycoside (früher „Heteroside“ genannt) sind eine
umfangreiche Gruppe von Pflanzeninhaltsstoffen, welche
sich aus einem oder mehreren Zuckern und einem oder
mehreren Aglyconen (= Nicht-Zucker-Molekül-Anteilen)
zusammensetzen. Das Aglycon ist dabei über ein Sauerstoffatom (Ether-Bindung) an ein Halbacetal-Kohlenstoffatom eines Zuckers gebunden. Der am häufigsten natür-
106
lich vorkommende Zuckerrest der Glycoside ist die in ßStellung verknüpfte D-Glucose. Man nennt diese Glycoside dann ß-D-Glucoside. Auch Disaccharid-Glycoside
(z.B. Glucosyl-, Arabinosyl-, Rhamnosyl-, Xylosyl- und
Apiosyl-Gucoside) kommen in der Natur vor [1].
Schon früh wurde die Bedeutung von Glycosiden als
Vorstufen für wertgebende Aromastoffe in Vanille [2],
Rosenblättern [3] und Weintrauben [4] erkannt. Seither
konnte eine Vielzahl von Glycosiden mit über 200 unterschiedlichen Aglycon-Strukturen in fast 170 Pflanzenarten aus etwa 50 Pflanzenfamilien nachgewiesen werden
[1, 5]. Auch in den Rückständen der CO2-Hochdruckextraktion von Hopfen (Humulus lupulus L.) wurde kürzlich das Vorkommen glycosidisch gebundener Aromastoffe beschrieben [6,7].
Glycoside werden als Speicher- bzw. Transportform
von Aromastoffen angesehen [1]. Möglicherweise kommt
ihnen auch eine gewisse Schutzwirkung zu. Empfindliche
Zellmembranen könnten von hohen Gehalten an freien
phenolischen oder terpenoiden Alkoholen geschädigt
werden [5]. Glycoside sind geruchlos, entsprechende Aromastoffe lassen sich daraus jedoch durch Erhitzen im Sauren oder durch enzymatische Hydrolyse freisetzen. Empfindliche Aglyconstrukturen können bei der Hydrolyse der
Glycoside strukturell verändert werden [8 - 10]. Beispiele
dafür sind die Bildung des äußerst geruchsaktiven ßDamascenons aus 3-Hydroxy-ß-Damascon und 3-Hydroxy7,8-Dehydro-ß-Jonol [11], die Bildung der Rosenoxide aus
2,6-Dimethyl-3-Octen-2,8-diol [12] oder die Bildung von
Linalool und α-Terpineol aus Neryl-glycosid [9].
Glycosidextrakte können nach entsprechender Derivatisierung auch direkt gaschromatographisch untersucht
werden [1]. Meist begnügt man sich jedoch damit, nur die
durch gezielte enzymatische Hydrolyse freisetzbaren
Aglykone zu messen. Hierzu müssen die Glycoside zunächst aus dem Pflanzenmaterial isoliert werden. Bei
Blattmaterial wird üblicherweise mit kochendem Wasser
[13,14], Methanol [15] oder einem Wasser-MethanolGemisch (80:20 v/v) [6] extrahiert. Methanolzusatz bewirkt dabei auch eine Fällung von Protein (und damit eine
Inaktivierung von Enzymen) [13]. Andere störende Bestandteile wie Polyphenole können durch Bindung an
Polyvinylpyrrolidon eliminiert werden [13]. Dann folgt
meist eine säulenchromatographische Aufreinigung an
RP-18 [3,16,17] oder Amberlite XAD-2 [11,13,18,19]).
Um freie Zucker auszuwaschen wird zunächst mit Wasser
eluiert. Dem folgt meist eine Elution freier Aromastoffe
mit Pentan [11,18,19], Pentan-Ether [13], Ether [6] oder
Pentan-Dichlormethan [20,21], bevor die Glycoside mit
Methanol [13,19,21,22] oder Ethylacetat [18,20] abgelöst
werden. Die so gewonnene Glycosidfraktion kann nun
(nach Entfernen des Methanols) unter definierten Bedingungen (optimaler pH-Wert und Temperatur) einer enzymatischen Hydrolyse unterworfen werden. Nach entsprechender Inkubationszeit (8h [14,15], 12h [18], 16h [8,19],
24h [6,23,24] 48 h [17], 72h [22,25]) werden die Aglycone
isoliert und gaschromatographisch-massenspektrometrisch
nachgewiesen. Einige Schnellmethoden verzichten auf die
säulenchromatographische Aufreinigung [14], wobei hier
eine Glycosidhydrolyse nur mit einer, gegenüber störenden
Bestandteilen toleranten Glucosidase gelingt [26].
Zur enzymatischen Spaltung der Glycoside verwendet man meist ß-Glucosidase aus Mandeln („Emulsin“,
EC 3.2.1.21) [22]. Pectinasen (Pectinol [14], Rohapect C
[20] Pectinase aus A.niger [19]) können bessere Ergebnisse liefern. Pectinol VR ist weniger geeignet [27]. Die
Effizienz der Hydrolyse hängt nicht nur von der Inkubationsdauer und dem pH-Wert, sondern auch von der Struktur der Aglycone und dem Ursprung des Enzymes ab [1,
28, 29]. Tertiäre Alkohole wie Linalool und α-Terpineol
werden besser durch Glucosidase aus Aspergilus niger als
durch Glucosidase aus Mandeln gespalten [28]. Die
Unspezifität gegenüber tertiären Alkoholen ist vielen
pflanzlichen Glycosidasen eigen [26]. Zur Spaltung von
Glycosiden des Vomifoliols und anderer nor-Carotinoide
eignet sich kommerziell erhältliche Hesperidinase [23].
Zur Analyse von Aglyconen hat sich auch ein Hemicellulase-Präparat der Fa. Gist-Brocades (Seclin, Frankreich)
bewährt [21].
Mittels einer Appatatur zur kontinuierlichen Freisetzung von Aglyconen durch mehrmalige enzymatische
Spaltungen der selben Probe (Simultane Enzym Katalyse
Extraktion, SECE) [23] konnte beobachtet werden, daß
sich die Konzentration der freigesetzten Aglycone mit der
Zeit ändert. Die sensorisch interessanten C13-norIsoprenoide wurden dabei erst in späteren Hydrolysestufen in größeren Mengen freigesetzt [23].
Problematisch ist auch die Hydrolyse von Disaccharid-Glycosiden, da hier zuerst der an die Glucose gebundene andere Zuckerrest abgespalten werden muß, um
dann der ß-Glucosidase als Substrat dienen zu können
[30]. Entsprechende Enzymnebenaktivitäten sind hierbei
für eine möglichst vollständige Glycosidspaltung unerläßlich. Dies läßt sich auch durch die kombinierte Verwendung mehrerer Enzympräparate erreichen [21].
Im Rahmen unserer Untersuchungen über glycosidisch gebundene Aromastoffe des Hopfens berichten wir
hiermit über die Eignung verschiedener Enzympräparate
zur Freisetzung von Aglyconen aus Hopfenglycosiden.
MATERIAL UND METHODEN
Probenmaterial: getrocknete Hopfendolden (gerntet im
Jahr 2000) der Sorten Hallertauer Mittelfrüh (HHA),
Hallertauer Tradition (HHT), Hallertauer Magnum
(HHM), Hallertauer Hersbrucker (HHE), Czechischer
Saazer (CSA) bezogen über Fa. Hopsteiner (HHV), D84048 Mainburg. Phenyl-ß-D-Glucosid und Octyl-ß-D-
107
Glucosid von Sigma-Aldrich, Chromatographie-Harz
Amberlite XAD-2 von Sigma-Aldrich, Methanol puriss
p.a. (99,8% GC) von Fluka, Polyclar AT pract von Serva.
Enzyme: ß-D-Glucosidase aus Mandeln (Emulsin, EC
3.2.1.21; 6,3 U/mg), Glucosidase aus Aspergillus niger
(76 U/g), Pectinase aus Aspergillus niger (EC 3.2.1.15;
2,5 U/mg), Hesperidinase (7 U/g mit α-L-Rhamnosidase
und ß-D-Glucosidase Activität) von Sigma-Aldrich. αAmylase (Termamyl-120L) und Amylase-Mix (SAN
Super 240L mit α-Amylase, Gluco-Amylase, Protease,
stabilisiert mit Benzoe- und Sorbinsäure) von Novozymes
DK-2880 Bagsvaerd. Ein nicht komerziell verfügbares
Hemicellulase-Präparat mit glycosidischen Nebenaktivitäten („Rapidase F-64“) wurde uns dankenswerterweise von
Herrn Dipl.-Ing. B. Heimann, DSM Food Specialities, D44319 Dortmund zur Verfügung gestellt (Dieses Präparat
entspricht der in der Literatur [21] beschriebenen Hemicellulase REG2, von Gist-Brocades, Frankreich).
dard-Lösung (Methyl-Octanoat 1g/l) und Isolierung der
freigesetzten Aglycone durch 4-maliges Ausschütteln mit
50 ml Diethylether. Trocknung der Extrakte über Na2SO4
und Aufkonzentrierung an einer Vigreuxkolonne (40°C).
Säulenchromatographische Reinigung an XAD-2: jeweils
25 g fein zerriebene Hopfendolden (HHA, HHT, HHE,
HHM, CSA) mit 5 µM Phenyl-ß-D-Glucosid versetzen und
mit kochendem Wasser extrahieren. Nach Klärung mit
Polyclar und Proteinfällung mit Methanol, zur Trockne
einengen und in Wasser gelöst auf eine XAD-Säule aufbringen. Elution mit 1 Liter H2O (6 ml/min), 500 ml Pentan-Dichlormethan (2:1 v/v, 7 ml/min) und 500 ml Methanol
(5ml/min). Methanolisches Eluat zur Trockne einengen und
in 50 ml Phosphat-Citrat-Puffer pH 5,0 aufnehmen. Jeweils
25 ml ohne Zusatz und 25 ml mit 25 mg ß-Glucosidase, 24 h
bei 40°C unter Rühren inkubieren (vgl. [31]). Extraktion
mit Diethylether wie oben.
Gaschromatographie-Massenspektrometrie (GC-MS):
Modell-Lösung: jeweils 2,44 µM Phenyl-ß-D-Glucosid
und 1,62 µM Octyl-ß-D-Glucosid in 25 ml PhosphatCitrat-Puffer (21 g Citronensäure-monohydrat und 71,5 g
Na2HPO4 x 12 H2O pro Liter H2O dest., pH 5,0) lösen.
Hopfen-Extraktion: ca. 80 g Hopfendolden (Gemisch
aus HHA, HHE und CSA) im Mörser fein zerreiben und
nach Zusatz von 19,5 µM Phenyl-ß-D-Glucosid mit einer
Mischung aus 800 ml Methanol und 200 ml H2O 16 h
rühren. Flüssige Phase über Glaswolle abdekantieren, 10
min mit 40 g Polyclar rühren und filtrieren. Am Rotationsverdampfer Methanol abziehen (40°C, 250-70 mbar).
Wässrige Lösung (ca. 150 ml) 3mal mit 100 ml Diethylether ausschütteln. Anschließend zur Trockne einengen
und Rückstand in 200 ml Phosphat-Citrat-Puffer, pH 5,0
aufnehmen und auf 8 Inkubationsgefäße verteilen. Jeder
Ansatz enthält demnach 2,44µM Phenyl-ß-D-Glucosid
und ca. 10 g Hopfen in 25 ml Puffer.
Enzym
keines
α-Amylase
Amylase-Mix
Pectinase
Hesperidinase
Mandel-Glucosidase
Asp. niger-Glucosidase
Hemicellulase
Siemens SiChromat II GC direkt gekoppelt mit Finnigan
8222 Magnetsektorfeld-MS EI-Modus, 70 eV; 35-600 amu,
Injektor 250 °C; Transferleitung 200 °C Ionenquelle 180 °C;
Sniffing-Modul 250 °C; Temperaturprogramm 100°C, mit
5min auf 250°C bzw. 60°C mit 5°C/min auf 250°C. Trägergas Helium (3 ml/min bei 100 °C); Split 1:10;
Trennsäule TS2: SE54 (Supelco SPB-5); 30 m x 0,53
mm i.D., df 1,5 µm. Der Trägergasstrom wird am Ende der
Kapillartrennsäule über ein Live-T-Stück aufgesplittet zum
MS und zur mit angefeuchteter Preßluft gespülten SniffingMaske. (Splitverhältnis ca. 1:1)
Finnigan 9600 GC direkt gekoppelt mit Finnigan
4500 Quadrupole-MS. EI-Modus, 70 eV, 33-400 amu,
Injektor 200°C, Transferleitung 200 °C, Ionenquelle
150°C, Temperaturprogramm 60°C (10min) mit 2°C/min
auf 200°C. Trägergas Helium 1 ml/min, Split 1:10,
Trennsäule TS3: CW20M (Permabond, M&N) 50 m x
0,25 mm i.D., df 0,25 µm.
TABELLE 1 - Enzymzusatz bei den Inkubationsversuchen.
Probe
BW
AMY
AMX
PCT
HSP
GLM
GLA
HEM
HP 5890 Ser. II Gaschromatograph direkt gekoppelt
mit Finnigan 8200 Magnetsektorfeld-Massenspektrometer. EI-Modus, 70 eV, 33-400 amu, Injektor 250 °C,
Transferleitung 230 °C, Ionenquelle 240 °C Temperaturprogramm 60°C (5min), mit 2°C/min auf 260°C. Trägergas Helium (1,15 ml/min bei 60°C), Split 1:10. Trennsäule
TS1: SE 54 (DB 5 J&W), 30m x 0,25mm i.D., df 0,25 µm.
Menge
keine
2,5 ml
2,5 ml
100 mg
250 mg
25 mg
50 mg
25 mg
Vergleich der Enzymaktivitäten: Die Modell-Lösungen
bzw. die Hopfenansätze jeweils mit Enzympräparat versetzen (siehe Tab. 1) und 66 h im Wasserbad (40°C) unter
Rühren inkubieren. Anschließend Zugabe von 0,5 ml Stan-
Identifizierung: Die Identifizierung erfolgte durch
Vergleich von Massenspektren (MS) und Retentionsindices (RITS1, RITS2 und RITS3) mit Daten von authentischen Referenzsubstanzen bzw. entsprechend abgesicherten Daten einer unter denselben GC-MS-Bedingungen
erstellten MS/RI-Bibliothek (Kollmannsberger, Weihenstephan). Wo keine Vergleichssubstanz zur Verfügung
stand, wurde zur Identifizierung auf entsprechende Literaturangaben zurückgegriffen.
108
ERGEBNISSE UND DISKUSSION
In Tabelle 2 sind die, mit den verschiedenen Enzympräparaten aus synthetischen Glucosiden freisetzbaren
Gehalte der Aglyconen wiedergegeben. Es fällt auf, daß
bei 66-stündiger Inkubation bereits im Blindwert ein Umsatz von 10 % (Octyl-Glucosid) bzw. 24 % (PhenylGlucosid) stattfindet. Bei 24-stündiger Inkubation liegen
diese Werte noch deutlich niedriger (3-7% der theoretischen Menge).
TABELLE 2 - Freisetzung von Aglyconen (µg/Ansatz) aus
synthetischem ß-D-Phenyl- (PHE) und ß-D-Octyl-Glucosid (OCT)
durch verschiedene Enzympräparate (Inkubation 66 h, 40°C)
PHE a)
229
54
134
251
191
200
201
231
233
Probe:
theoret. c)
BW
AMY
AMX
PCT
HSP
GLM
GLA
HEM
(+/-)b)
OCT a)
210
22
62
196
174
166
200
216
180
3
3
18
4
2
9
10
11
(+/-)b)
2
4
15
5
2
7
19
7
In Tabelle 3 sind die, mit den verschiedenen Enzympräparaten aus einer Hopfenprobe freisetzbaren Aglycone
zusammengestellt. Die Ausbeuten einiger typischer Aglycone sind in Abb. 2 graphisch dargestellt. Wiederum
bestätigt sich die geringe Aktivität der α-Amylase
(AMY), sowie die hohe Spezifität der A. niger Glucosidase (GLA) und der Hemicellulase (HEM) für das auch hier
zugesetzte Phenyl-ß-D-Glucosid. Das Amylase-Gemisch
(AMX) zeigt in dem nicht säulenchromatographisch aufgereinigten Hopfenextrakt eine deutlich geringere Hydrolyse-Effizienz für das Phenylglucosid als in der ModellLösung (Abb. 1). Möglicherweise wirken hier nicht abgetrennte Hopfenbestandteile inhibierend.
a)
Mittelwert aus Doppelbestimmung
Abweichung bei Doppelbestimmung
c)
theroretisch erzielbarer Wert
b)
120
100
110
PHE
101
83
80
87
102
88
59
60
40
Sehr gute Ausbeuten an allen Aglyconen erzielt man
mit der A. niger Glucosidase (GLA), während die anderen
Enzympräparate offensichtlich eine starke Substratspezifität aufweisen.
24
20
0
BW
120
AMY
OCT
AMX
93
100
PCT
HSP
GLM
95
83
79
PCT
HSP
GLA
HEM
1-Octen-3-ol wird am besten durch die beiden Glucosidasen (GLM, GLA) freigesetzt (Abb. 2). Bei den tertiären Alkoholen α-Terpineol und Linalool lassen sich mit
dem Hemicellulase-Präparat (HEM) die besten Ergebnisse erzielen. Hesperidase (HSP) liefert nur bei einigen norCarotinoid-Derivaten brauchbare Resultate. Mit dem
Amylase-Gemisch (AMX) werden die höchsten Werte für
3-Methyl-2-Pentanol und Vomifoliol erzielt (Abb. 2).
Während mit Mandel-Glucosidase (GLM) nur vergleichsweise wenig Vomifoliol gefunden wird, läßt sich
damit der höchste Gehalt an 3-Hydroxy-5,6-epoxy-ßJonol freisetzen (Tab.3).
103
86
80
60
30
40
20
10
0
BW
AMY
AMX
GLM
GLA
erstaunlich hohe Werte liefert. Die beiden Glucosidasen
(GLM, GLA) zeigen in den eingesetzten Konzentrationen
eine höhere Spezifität für das aliphatische Octyl-ß-DGlucosid. Das Hemicellulase-Präparat (HEM) und die
Hesperidinase (HSP) scheinen das Phenyl-ß-D-Glucosid
als Substrat zu bevorzugen. In Abb.1 sind die prozentualen Ausbeuten an Aglyconen bezogen auf den theoretisch
erreichbaren Wert dargestellt. Bei nur 24-stündiger Inkubation mit ß-Glucosidase bzw. Hemicellulase betragen die
Ausbeuten bei beiden Aglyconen etwa 76-78 % des nach
66-stündiger Inkubation erzielbaren Wertes. Neben der
Inkubationsdauer ist natürlich auch die Konzentration an
Enzym von entscheidenden Einfluß auf die Effizienz der
Hydrolyse. Bei Inkubation von synthetischem Phenyl-ß-DGlucopyranosid mit nur 25 mg A. niger Glucosidase wurden nur 73 % des mit 50 mg der Glucosidase unter ansonsten gleichen Bedingungen freigesetzten Phenols gemessen.
HEM
ABBILDUNG 1 - Hydrolyse (%) von synthetischem ß-D-Phenyl(PHE) und ß-D-Octyl-Glucosid (OCT) durch verschiedene Enzympräparate (Inkubation 66 h, 40°C).
Die schlechteste Ausbeute an Aglyconen erhält man
mit dem α-Amylase-Präparat (AMY), während das Amylase-Gemisch (AMX) mit den synthetischen Glucosiden
2,6-Dimethyl-2,7-Octadien-1,6-diol (= 8-HydroxyLinalool) verhält sich sehr ähnlich wie andere primäre
Alkohole (Z.B. 3-Methylbutanol, 3Z-Hexenol und Benzylalkohol). Dies könnte ein Hinweis darauf sein, daß es
über die endständige und nicht über die tertiäre OHGruppe glykosidisch verknüpft ist.
109
Phenol
Phenol
300
250
200
170
200
192
3-Z-Hexenol
150
253
140
225
204
100
98
87
80
70
150
100
50
7
41
50
26
0
0
BW AMY AMX PCT HSP GLM GLA HEM
BW
34
3
0
59
PCT
HSP GLM GLA
175
150
74
55
84
100
21
HEM
235
Benzylalkohol
200
124
150
50
AMY AMX
250
1-Octen-3-ol
100
0
0
117
95
44
50
9
1
0
0
2,6-Dimethyl-2,7-Octadien-1,6-diol 2
22
200
15
10
0
0
0
3
5
237
250
Linalool
30
20
6
7
AMY AMX
PCT
HSP GLM GLA
HEM
142
124
77
100
BW
134
124
150
46
50
18
0
3-Hydroxy-ß-Damascon
6
6
5
4
3
2
1
0
5
5
Vomifoliol
80
4
60
60
3
40
1
0
20
0
BW
AMY AMX
PCT
HSP GLM GLA
HEM
40
30
40
20
15
12
PCT
HSP GLM GLA
6
0
BW
AMY AMX
HEM
7,8-dihydro-Vomifoliol
3-Hydroxy-7,8-dihydro-ß-Jonol
150
100
45
50
0
43
58
59
9
10
74
HSP GLM GLA
HEM
3
0
0
1
0
BW
AMY AMX PCT HSP GLM GLA HEM
ABBILDUNG 2 - Freisetzung von Aglyconen aus Hopfenextrakten durch verschiedene Enzympräparate
110
10
6
2
0
9
8
4
5
BW AMY AMX PCT
11
12
132
TABELLE 3
Freisetzung von Aglyconen aus einem Hopfenextrakt durch verschiedene Enzympräparate (Inkubation 66 h, 40°C)
(Peak-Flächenwerte ausgewählter Massenfragmente - bezogen auf Standard Methyloctanoat)
Phenol (Standard)
3-Methylbutan-1-ol
3-Methyl-2-Buten-1-ol
3-Methylpentan-2-ol
3-(Z)-Hexen-1-ol
1-Octen-3-ol
4,6-Dimethylheptan-2-ol ?
Benzylalcohol
2-Phenylethanol
Methylsalicylat
Linalool
α-Terpineol
(Z)-2,6-Dimethyl-2,7-Octadien-1,6-diol
(E)-2,6-Dimethyl-2,7-Octadien-1,6-diol
3-Hydroxy-7,8-dihydro-ß-Jonol
3-Hydroxy-5,6-epoxy-ß-Jonol
3-Hydroxy-ß-Damascon
Vomifoliol
7,8-dihydro-Vomifoliol
BW
7
1
0
0
0
0
0
1
0
1
0
0
5
18
0
0
0
6
0
AMY
26
5
56
17
0
3
0
9
0
6
0
22
8
46
5
0
0
30
0
Tabelle 4 enthält alle in den 5 Hopfensorten nach säulenchromatographischer Vorreinigung und enzymatischer
Spaltung identifizierten Aglycone. Die Identifizierung
erfolgte durch Vergleich von Massenspektrum (MS) und
Retentionszeit (RT) mit einer institutseigenen ReferenzDatei bzw. über Literaturdaten (siehe Tab. 4) Die wichtigsten Strukturformeln sind in den Abbildungen 3-6
wiedergegeben. In einigen Proben waren auch geringe
Mengen an 3Z-Hexenal, Benzaldehyd und Phenylacetaldehyd nachweisbar. Diese Carbonylverbindungen sind
Oxidationsprodukte der korrespondierenden glycosidisch
gebundenen Alkohole und wurden nicht eigens aufgeführt. Hydroxy-Benzoesäure, Vanillinsäure, HydroxyZimtsäure und Ferulasäure konnten besonders nach enzymatischer Spaltung mit dem Hemicellulase-Präparat
Rapidase in größeren Mengen nachgewiesen werden.
2-(2-Butenyliden)-3,3-Dimethyl-5-(2-oxopropyl)-Tetrahydrofuran ist ein bekanntes Umwandlungsprodukt des 3Hydroxy-5,6-epoxy-ß-Jonols [33] und stellt daher ein
Artefakt dar. ß-Damascenon trat bei Sniffing-GC-MSAnalysen durch seinen typischen Geruch zur entsprechenden Retentionszeit hervor, die vorhandene Konzentration
reichte jedoch in den meisten Proben nicht für einen gesicherten massenspektrometrischen Nachweis aus.
Mittels Sniffing-GC-MS konnte, neben einigen noch
nicht identifizierten Substanzen, besonders den in Tab. 5
aufgeführten Bestandteilen der Aglycon-Fraktion ein
eindeutiger Geruch zugeordnet werden. Darüberhinaus
AMX
170
18
34
171
80
34
7
84
16
24
3
25
15
124
45
13
1
59
3
PCT
200
16
51
16
87
21
9
95
29
14
5
20
17
134
43
5
4
16
1
HSP
192
8
44
38
41
59
5
44
10
13
6
33
5
77
58
10
5
40
11
GLM
204
18
65
37
70
74
9
117
20
30
7
36
14
124
59
25
3
12
9
GLA
253
35
57
61
140
124
16
235
37
49
15
35
26
237
132
16
6
39
9
HEM
225
27
75
58
98
55
12
175
35
31
22
44
14
142
74
12
5
21
10
wird ein „muffig-ranziger“ Geruch bei 3- und 2Methylbuttersäure wahrgenommen. Da diese Säuren auch
im nicht mit Glucosidase versetzten Blindwert auftreten,
wurde ihnen keine weitere Beachtung geschenkt.
Unterzieht man einen Glycosidextrakt aus Hopfen einer sauren Hydrolyse (Kochen unter Rückfluß; 1 h bei pH
2,7) lassen sich massenspektrometrisch unter anderen αTerpineol, die beiden furanosiden Linalooloxide, Linalool, Limonen, p-Menth-1-en-9-al und ß-Damascenon
nachweisen. Das äußerst geruchsaktive ß-Damascenon
dürfte dabei aus dem glycosidisch gebundenem 3Hydroxy-ß-Damascon hervorgehen [11].
Der Nachweis von Glycosiden im Hopfen dürfte vor
allem für die Bierbereitung von Interesse sein [6,7]. Da
die freien Aromastoffe des Hopfens beim Würzekochen
weitgehend verloren gehen, stellen glycosidisch gebundene Aromastoffe eine zusätzliche Quelle zur Ausbildung
des Hopfenaromas im Bier dar. In Fortführung dieser
Arbeiten konnten wir zeigen, das die Glycoside den
Brauprozeß überstehen und sich im gehopften Jungbier
dieselben Aglycone freisetzen lassen wie im Hopfen.
Besonders Linalool und ß-Damascenon treten bei Sniffing-GC-MS-Analysen von gehopftem Bier geruchlich in
Erscheinung während sie in ungehopftem Bier praktisch
ohne Bedeutung sind. Inwiefern diese Geruchsstoffe zum
Hopfenaroma des Bieres einen Beitrag leisten, soll Gegenstand weiterer Untersuchungen sein.
111
TABELLE 4 - Durch enzymatische Spaltung freisetzbare Aglycone in säulenchromatographisch aufgereinigten wässrigen Extrakten aus verschiedenen Hopfensorten
Nr.
Verbindung
RITS1
RITS2
RITS3
m/e
Identifizierung
1
Butan-2-ol
613
1027
45,59-41,43,73
MS,RT
2
622
1041
43,71,59-53,86
MS,RT
3
2-Methyl-Propan-1-ol
632
1097
43-43,41,42,33,74
MS,RT
4
Butan-1-ol
674
1154
56-41,43,42,73
MS,RT
5
3-Methyl-Butan-2-ol
689
1096
45,55,73-43,87
MS,RT
6
696
1179
43,71-41,45,53,58,86
MS,RT
7
Pentan-3-ol ?
1114
59-41-55,58,57
MS,RT
8
Pentan-2-ol ?
1128
45-55,73,43
MS,RT
9
3-Methyl-Butan-1-ol
726
1217
55,42,70,41,43
MS,RT
10
2-Methyl-Butan-1-ol
730
1216
57,56,41,70
MS,RT
11
Pentan-1-ol ?
1263
42,55,70,41
MS,RT
12
4-Methyl-Pentan-2-ol
753
1175
45-43,69,84,87,57
MS,RT
13
793
772
1334
71,53,41,67,68,86
MS,RT
14
3-Methyl-Pentan-2-ol
789
1208
45-56,41,69,84,87
MS,RT
15
3Z-Hexenol
851
854
1393
67,41,82-55,69
MS,RT
16
Hexanol
865
864
1366
56-43,41,69,55,84
MS,RT
17
Cyclohexanol ?
887
898
1411
57,82-67,55,41
MS
18
1-Octen-3-ol
982
980
1461
57-72,43,81,85,99
MS,RT
19
Phenol (Standard)
983
990
2004
94,66,65,39
MS,RT
20
Benzylalkohol
1037
1050
1876
108,107,79,77
MS,RT
21
4,6-Dimethyl-heptan-2-ol ?
1055
1054
22
Linalool
1099
1106
23
Phenylethanol
1111
24
α-Terpineol
1192
25
Methylsalicylat
1188
26
4-Vinylphenol
1230
27
Geraniol
1253
28
(ein Monoterpenalkohol?)
29
30
31
E-2,6-Dimethyl-2,7-Octadien-1,6-diol
32
4-Hydroxy-Benzaldehyd ?
1417
121,122,65,103
MS
33
Vanillin
1441
151,152,81,109
MS,RT
107,138,77
MS,RT
59,79,94
[19]
43,57,69,85,45,87,126,144
MS
1555
71,93-41,55,80-121,136
MS,RT
1135
1910
91,92,122,65
MS,RT
1211
1699
59,93,121
MS,RT
1219
1774
120,152,92
MS,RT
1245
2395
120,91
MS,RT
1262
41,69-93,111,123
MS,RT
1272
1287
59-68,67,71,79,94,152
4-Vinylguajacol
1306
1339
2205
135,150-
MS,RT
Z-2,6-Dimethyl-2,7-Octadien-1,6-diol
1343
1361
2268
43,71,67,55,68
[32]
1363
1379
2308
43,71,67,55,68
[32]
34
Tyrosol
35
p-Menth-1-en-7,8-diol
36
Hydroxy-Benzoesäure
1590
121,138,93,65
MS
37
1613
168,153,97,125
MS
1589
1626
43,125,109,82,208,95,151
[33]
39
Vanillinsäure
2-(2-Butenyliden)-3,3-Dimethyl-5-2oxopropyl-Tetrahydrofuran
3-OH-ß-Damascon
1602
1637
2533
69,43,121,175,193,208
[34]
40
3-OH-7,8-dihydro-ß-Jonol
1651
1692
2627
121,43,119,93,105,136,-212
[22],[35]
41
3-OH-5,6-epoxy-ß-Jonol
1661
1703
43,125,109,82,208,107,166
[35]
42
3-OH-5,6-epoxy-ß-Jonon
1680
[36]
43
3-OH-ß-Jonon
44
Vomifoliol
45
p-Hydroxy-Zimtsäure
46
7,8-Dihydro-Vomifoliol
47
Ferulasäure
38
1473
1469
1780
1845
1504
2509
1715
123,43,109,95
1715
43,175,193
[37]
1837
124-43,79,135,150,168
[22],[38]
1869
164,147,119,91,65
MS
1898
43,110,111,152,96,68,170
[22]
1928
194,179,133,77,105
MS
112
HO
OH
1 OH
OH
OH
6 OH
2
4
3
13
OH
OH
OH
OH
OH
5
7
8
9
10
OH
OH
OH
OH
14
12
16
15
OH
OH
18
21
ABBILDUNG 3 - Enzymatisch freisetzbare aliphatische Alkohole des Hopfens (Nummerierung entspricht Tab. 4)
O
OH
OH
OH
O-Me
HO
20
23
34
25
CH3O
CHO
HO
HO
HO
26
CH3O
CHO
HO
32
29
OH
33
ABBILDUNG 4 - Enzymatisch freisetzbare aromatische und phenolische Hopfenaglycone (Nummerierung entspricht Tab. 4)
OH
OH
OH
OH
OH
27
22
OH
OH
HO
31
30
24
OH
35
ABBILDUNG 5 - Enzymatisch freisetzbare Monoterpene des Hopfens (Nummerierung entspricht Tab. 4)
113
OH
OH
O
HO
HO
OH
OH
O
46
O
OH
OH
O
38
41
40
O
O
O
-H2O
HO
39
44
ß-Damascenon
ABBILDUNG 6 - Enzymatisch freisetzbare Nor-Carotinoide des Hopfens (Nummerierung entspricht Tab. 4).
TABELLE 5 - Mittels Sniffing-GC-MS in enzymatisch hydrolysierten Hopfenextrakten eindeutig zugeordnete Geruchsstoffe.
Verbindung
3Z-Hexenal
3Z-Hexenol
1-Octen-3-ol
2-Phenylacetaldehyd
Linalool
2-Phenylethanol
Geraniol
ß-Damascenon
Vanillin
Geruch
grün-grasig
grün-grasig
Champignon
süß-wachsig
blumig-citrus
blumig, Wein
citrusartig
blumig-fruchtig, Apfelsaft
Vanille
DANKSAGUNG
Für die Bereitstellung der Hopfenproben danken wir
Herrn Dr. M. Biendl, HHV D-84048 Mainburg. Für die
Überlasung eines Hemicellulase-Präparates („Rapidase F64“) danken wir Herrn Dipl.-Ing. B. Heimann, DSM Food
Specialities, D-44319 Dortmund. Frau E. Schütz danken
wir für ihre bewährte analytische Mitarbeit.
[4]
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R.A.; Studies on the Hydrolysis of vitis vinifera monoterpene
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rationalizing the monoterpene composition of grapes; J. Agric. Food Chem. 30: 1219-1223 (1982)
[10] Sefton, M.A., Williams, P.J., Generation of Oxidation Artifacts during the hydrolysis of norisoprenoid glycosides by
fungal enzyme preparations. J. Agric. Food Chem. 39: 19941997 (1991)
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Agric. Food Chem. 39: 1830-1832 (1991)
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[12] Knapp, H., Straubinger, M., Fornari, S., Oka, N., Watanabe,
N., Winterhalter P., S-3,7-Dimethyl-5-octene-1,7-diol and related oxygenated monoterpenoids from petals of rosa damascena Mill., J. Agric Food Cem. 46: 1966-1970 (1998)
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synthesis in rose flowers, Phytochemistry 8: 1339-1347
(1969)
[13] Wang, D., Yoshimura, T., Kubota, K., Kobayashi, A.; Analysis of glycosidically bound aroma precursors in tea leaves.
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[14] van den Dries, J.M.A., Baerheim Svendsen, A., A simple method for detection of glycosidic bound monoterpenes and other volatile compounds occuring in fresh plant material, Flavour and fragrance Journal 4: 59-61 (1989)
[29] Decker,C.H., Visser, J., Schreier, P. ß-Glucosidases from five
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from sloe tree (Prunus spinosa L.) leaves. J. Agric. Food
Chem. 40: 1898-1901 (1992)
[30] Günata, Y.Z., Bitteur, S., Brillouet, J.M., Bayonove, C.L.,
Cordonnier, R.E., Sequential enzymic hydrolysis of potentially aromatic glycosides from grapes. Carbohydr. Res. 134:
139-149 (1988)
[16] Williams, P.J., Strauss, C.R., Wilson, B., Massy-Westropp,
R.A.; Use of C18 reversed-Phase liquid Chromatography for
the isolation of monoterpene glycosides and nor-isoprenoid
precursors from grape juice and wines, J. Chromatogr. 235:
471-480 (1982)
[17] Winterhalter, P., Bound Terpenoids in the juice of the purple
passion fruit (Passiflora edulis Sims), J. Agric. Food Chem.
38: 452-455 (1990)
[18] Günata, Y.Z., Bayonove, C.L., Baumes, R.L., Cordonnier,
R.E., The aroma of grapes I. Extraction and determination of
free and glycosidically bound fractions of some grape aroma
components. J. Chromatogr. 331: 83-90 (1985)
[19] Sakho, M., Chassagne, D., Crouzet, J., African Mango glycosidically bound volatile compounds, J. Agic. Food Chem. 45:
883-888 (1997)
[20] Versini, G., Dalla Serra, A., Dell´Eva, M, Scienza, A., Rapp,
A., Evidence of some glycosidically bound new monoterpenes and norisoprenoids in grapes, in Bioflavour´87 (editor:
Schreier, P.), deGruyter, Berlin, p 161-170 (1988)
[21] Chassagne, D., Boulanger, R., Crouzet, J.; Enzymatic hydrolysis of edible Passiflora fruit glycosides; Food Chemistry
66: 281-288 (1999)
[22] Winterhalter, P., Schreier, P., Free and bound C13 norisoprenoids in Quince (Cydonia oblonga, Mill.) fruit. J. Agric.
Food Chem. 36: 1251-1256 (1988)
[23] Schwab, W., Schreier, P., Simultaneous Enzyme catalysis
Extraction: A versatile technique for the study of flavor precursors. J.Agric. Food Chem. 36: 1238-1242 (1988)
{24] Wilson, B, Strauss, C.R., Williams, P.J., Changes in free and
glycosidically bound monoterpenes in developing muscat
grapes; J. Agric. Food Chem. 32: 919-924 (1984)
[25] Wu, P., Kuo, M-C., Ho, C.T., Glycosidically bound aroma
compounds in ginger (Zingiber officinale Roscoe), J. Agric.
Food Chem. 38: 1553-1555 (1990)
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mit Trifluoracetat, Adv. Food Sci. (CMTL) in Vorbereitung
(2002)
[32] Winterhalter, P., Knapp, H., Straubinger, M., Fornari, S.,
Watanabe, N., Application of Countercurrent Chromatography to the Analysis of Aroma Precursors in Rose Flowers, in Mussinan, C.J., Morello, M.J. (Editors) Flavor Analysis, ACS Symposium Series 705: 181-192 (1998)
[33] Neugebauer, W., Winterhalter, P., Schreier, P.; 2-(2Butylidene)-3,3-dimethyl-5(2-oxopropyl)tetrahydrofuran: A
new degradation product of 3-Hydroxy-5,6-epoxy-ß-ionol. J.
Agric. Food Chem. 42: 2885-2888 (1994)
[34] Ohloff,G., Rautenstrauch, V., Schulte-Elte, Karl. H. Modellreaktionen zur Biosynthese von Verbindungen der Damascon-Reihe und ihre präparative Anwendung. Helv. Chim. Acta 56: 1503-1513 (1973)
[35] Humpf, H.U., Isolierung und Charakterisierung von Glykokonjungaten C13-norisoprenoider Aromastoffvor-stufen,
Dissertation Julius-Maximilian-Universität Würzburg (1992)
[36] Krammer, G., Winterhalter, P., Schwab, M., Schreier, P.,
Glycosidically bound Aroma Compounds in the fruits of prunus Species. J. Agric. Food Chem. 39: 778-781 (1991)
[37] Loeber, D.E., Russell, S.W., Toube, T.P., Weedon, B.C.L.,
Diment, J. Carotenoids and related compounds. Part XXVIII.
Synthesis of Zeaxanthin, ß-Cryptoxanthin and Zeinoxanthin
(a-Cryptoxanthin) J. Chem. Soc. (C) 404-408 (1971)
[38] Strauss, C.R., Wilson, B, Williams, P.J., 3-oxo-α-Ionol, Vomifoliol and Roseoside in Vitis vinifera fruit., Phytochemistry 26: 1995-1997 (1987)
Received for publication: July 29, 2002
Accepted for publication: August 15, 2002
[26] Aryan, A.P., Wilson, B., Strauss C.R., Williams, P.J., The properties of glycosidases of Vitis vinifera and a comparison of
their ß-glycosidase activity with that of exogenous enzymes.
An assessment of possible applications in enology. American
Journal of Enology and Viticulture 38: 182-188 (1987)
[27] Salles,C. Essaied, P., Chalier, P., Jallageas, J.C., Crouzet, J.
Evidence and characterization of glycosidically bound volatile components. Bioflavour´87 (editor: Schreier, P.), deGruyter, Berlin, p 145-160 (1988)
[28] Günata, Y.Z., Bayonove, C.L., Tapiero, C., Cordonnier, R.E.,
Hydrolysis of grape monoterpene ß-D-Glucosides by various
ß-Glucosidases, J. Agric. Food Chem. 38: 1232-1236 (1990)
115
H. Kollmannsberger/ S. Nitz
Lehrstuhl für Chem.-Techn. Analyse u. Chem. Lebensmitteltechnologie
Technische Universität München
Weihenstephaner Steig 23
85350 Freising-Weihenstephan - GERMANY
[email protected]
AFS/ Vol 24/ No 3/ 2002 – pages 106 - 115
MICROBIAL AND BIOCHEMICAL CHANGES OCCURRING DURING
FERMENTATION OF marula (Sclerocarya birrea subspecies caffra)
JUICE TO PRODUCE mukumbi, A TRADITIONAL ZIMBABWEAN WINE
1
Mpofu A. and 2Zvauya R.
1
Department of Soil Science and Agricultural Engineering, 2 Department of Biochemistry
University of Zimbabwe, Box MP 167, Mount Pleasant, Harare, Zimbabwe
SUMMARY
Traditional fermentation of marula juice from the
fruit of marula tree (Sclerocarya birrea subsp. caffra) to
produce mukumbi, a traditional Zimbabwean wine, was
investigated in the laboratory. Microbial and biochemical
changes were monitored throughout the 72 h fermentation
period. There was a ten-fold increase in both aerobic
mesophilic bacteria and lactic acid bacteria, and a hundred-fold increase in yeast and moulds during the fermentation period. The final pH and acidity values were 3.4 and
0.95 % lactic acid, respectively. The glucose concentration
increased from an initial value of 5.7 g/l to 8.6 g /l after 36 h,
and then gradually decreased to a value of 0.4 g/l. Citric
acid decreased from an initial value of 51.0 g/l to 5.5 g/l at
72 h. The microorganisms used citrate at first, then glucose
and fructose as carbon and energy sources. The maximum
alcohol level produced was 2.3 % (v/v) after 60 hrs of
fermentation.
KEYWORDS:
Alcoholic fermantation, yeasts, mukumbi, wine, marula.
INTRODUCTION
The majority of research on African fermented foods
has been done in West Africa [1-3]. Due to the extensive
studies done, some of the products have been
commercialized e.g. gari, dawa dawa, and ogi [4]. East
African fermented foods have also been studied to a
greater extent than those of Central Africa [5]. The
traditional fermented foods of Zimbabwe have not been
systematically studied, except for some studies on
fermented milk products [6], mahewu [7,8], masvusvu and
mangisi [9].
The marula fruit is one of the most commonly utilized
wild fruits of Southern Africa. Archeological evidence
indicates that the marula fruit was known and consumed by
mankind in Africa since 9,000- 10,000 years BC [10]. The
plant is widely spread in Africa especially in semi arid
regions [11]. The marula has characteristics which offer
remarkable opportunities to the development of agriculturally based industries in Africa, which include drought resistance, exceptionally high yielding of fruit per tree, the
possibility to utilize both the fruit and nut contained within
the seed, ease of harvesting tall trees, exotic flavor and
nutritive value of the fruit. Marula fruit can be consumed
as a fresh fruit. The fruit has been processed into products
such as mukumbi, wine, beer, jelly or jam. Limited amounts
of juice are utilized industrially for flavor enhancement in
the production of liquor in the Republic of South Africa,
for example “Amarula Cream”, which is nuttier than other
chocolate coffee liquors in the genre. Mukumbi is a popular
beverage prepared from the ripe fruit in many villages of
Zimbabwe. It is central to the most valued personal and
social ceremonies of both highly literate and less literate
societies. The green physiologically mature marula fruits
fall to the ground and ripen (turn yellow in color). They are
then harvested and processed to mukumbi. Fermentation
procedures vary in different parts of Southern and Central
Africa. Fermentation time varies from household to household, but is usually 72 hrs. In certain regions of Zimbabwe
the whole process is carried out under a tree. Mukumbi is
yellow in color with a tart-sour taste and a slightly turpentine-like aroma. The alcohol content of the beverage varies
from producer to producer and depends on the time of
fermentation.
To the best knowledge of the researchers, there is no
published scientific literature on microbial and chemical
changes occurring during preparation of wines from traditionally fermented fruits of Zimbabwe. This work was,
therefore, aimed at evaluating these aspects of a mukumbi
preparation.
116
Source of marula fruits
Fresh marula fruits were obtained from Mataga village in Mberengwa, a hot semi-arid communal area in
Southern Zimbabwe. Fruits were brought to the laboratory
overnight and immediately processed to mukumbi.
centration was determined as described by Lawrence [12].
Sucrose, glucose and fructose levels were determined with
kits using the U.V method according to the manufacturer’s
instruction (Boehringer Mannheim kit, Cat.No. 716 260).
Ethanol concentration was measured by gas chromatography (Shimadzu GC 4CM) using a 3 % Carbowax on Chromosorb column. The oven temperature was 55 oC, injection
temperature 80 oC and the carrier gas flow rate 5 ml/min.
Laboratory preparation of mukumbi
In general, rapid microbial and biochemical changes
were recorded in the alcoholic fermenting marula juice
during the first 48 h. The changes were less significant
during the last 24 h. The final product, mukumbi had a
slightly sweet sour taste, a fruity aroma and a mildly alcoholic flavor.
9
Log 10 (cfu / ml) .
Mukumbi was prepared in the laboratory in a manner
similar to the traditional procedure. Ripe marula fruits,
(10 kg) were pierced with a wooden spoon and the juice
together with the seed were squeezed out of the skins. The
skins were discarded. Seeds, which are coated by a mucilaginous flesh, were pounded in a wooden mortar and
pestle to completely extract the juice and the flesh. The
slurry mixture composed of the flesh and the juice was
poured into a 5 l earthenware pot. An equal volume of
water was added and the pot was covered with a wooden
plate. The slurry mixture was left to ferment naturally at
room temperature (25 oC) for 72 hrs. Fermented slurry
was filtered through a 435 µm sieve and, thereafter, ready
for consumption. A thick residue formed mainly from the
flesh was collected on the sieve and discarded. The experiment was repeated two more times to check the reproducibility of the fermentation.
Sampling
Samples (10 ml) for microbial and biochemical
analyses were collected during fermentation at 6 h intervals. A subsample (5 ml) was centrifuged at 12 000 rpm
for 10 min. The supernatant was kept frozen at -20 oC in 1 ml
Eppendorf tubes until biochemical assays.
Biochemical analysis
All assays were done in triplicate. The pH of the fermenting juice was measured immediately after sampling
using a Jenway 3010 pH meter. Titratable acidity was
measured immediately as described elsewhere [9]. Citric
acid concentration was determined with kits using the U.V
method according to the manufacturers´ instructions (Boehringer Mannheim kit, Cat.No. 139 076). Lactic acid con-
7
6
5
0
Microbiological analysis
Standard microbial analysis methods were used. Each
sample (1 ml) of the fermenting juice was serially diluted
in 9 ml of peptone water and spread in triplicates onto
selective media for microbial analysis. Total aerobic
mesophilic bacterial counts were done using plate count
agar (OXOID) and the plates were incubated at 30 oC for
48 hrs. Lactic acid bacterial counts were done using de
Man Rogosa Sharpe (MRS) agar (OXOID) and plates
were incubated at 37 oC for 48 hrs. Yeast and mould
counts were done using wort agar (OXOID) and plates
were incubated at 30 oC for 48 hrs. The microbial load
was expressed as colony forming units per milliliter
(cfu/ml) of fermenting juice.
8
6 12 18 24 30 36 42 48 54 60 66 72 78
Yeast and moulds
Aerobic mesophilic bacteria
Lactic acid bacteria
FIGURE 1
Microbial changes during fermentation of marula juice.
Microbial Analysis
Figure 1 shows the microbial changes occurring
(cfu/ml) during natural fermentation of marula juice to
produce mukumbi. The initial microbial load of the juice
was already high and diverse (i.e. 3.14 x 107 lactic acid
bacteria, 2.10 x 107 aerobic mesophilic bacteria and 2.05 x
106 yeasts and moulds), when the laboratory fermentation
started. The green physiological mature marula fruits fall,
and are collected from the ground to be processed to mukumbi. On falling, the skin of some of the fruits ruptures
exposing the juice to microbes on the fruit surface. Bees,
wasps and fruit flies introduce yeasts into fruits during
juice suckling. Yeasts survive from year to year in the
intestines of bees and wasps and they are readily transferred during the crushing season by fruit flies (Droso-
117
phila melanogaster). In a warm atmosphere, spontaneous
fermentation occurs amongst other biochemical reactions
induced by microbial activity. It is likely that fermentation of marula juice starts whilst the fruit is still on the
ground. The extent of the fermentation depends on the
time the fruit stays on the ground before processing.
Therefore, the juice which is finally fermented in the
laboratory to produce mukumbi was extracted from fruits
at different stages of fermentation.
sulted in a rise in pH. The microorganisms probably utilized citrate before the sugars as a carbon and energy
source. The pH starts to fall when more than 50 % of the
initial citrate concentration is used up. This decrease in pH
correlated well with a steady rise in titratable acidity from
0.35 to 0. 95 % lactic acid until the end of fermentation.
60
The highest total microbial load was 1.03 x 109 cfu/ml at
18 hrs. The total microbial load was constant from 42 hrs
until the end of the fermentation period, probably because
the microbes had entered the stationary phase of growth.
Figure 1 shows that lactic acid bacteria increased ten-fold
during the fermentation period. Lactic acid bacteria became predominant after 24 hrs and reached a maximum at
66 hrs. Lactic acid bacteria were the most abundant microorganisms during the fermentation. The aerobic mesophilic bacteria were the predominating group during the
first 24 hrs and increased ten-fold in 30 hrs. The yeasts
and moulds were not a predominant group at any stage,
but had the highest increase in numbers, ten-fold within
24 hrs and a hundred-fold within 42 hrs (Figure 1).
4.6
4.4
Citric acid (g/l)
50
4.2
40
4
30
3.8
pH
3.6
20
3.4
10
3.2
0
3
0
6
12
18
24
30
36
42
48
54
60
66
72
Time (h)
Citric acid
pH
FIGURE 3 - Changes in citric acid and pH
during fermentation of marula juice.
1
0.6
4
pH
0.4
0.2
3
0
-0.2
2
0,5
0,45
0,4
Lactic acid % (w / w)
0.8
Titratable acidity
5
-0.4
0
6 12 18 24 30 36 42 48 54 60 66 72 78
Time (h)
pH
0,35
0,3
0,25
0,2
0,15
0,1
Titratable acidity
0,05
0
FIGURE 2 - Changes in pH and titratable acidity
0
6 12 18 24 30 36 42 48 54 60 66 72
Time (h)
Lactic acid
Biochemical changes
Figure 2 shows changes in pH and titratable acidity during the 72 h fermentation period. The pH, increased in the
first 24 hrs, unlike results from work carried out by other
workers on traditional fruit fermentations [4, 5]. It then
gradually decreased to 3.50. This rise in pH was probably a
result of microorganisms, which metabolize citrate [13].
Besides ascorbic acid, citric acid is the most abundant of the
organic acids in marula juice (11.6 mg / 100 g) [11]. Citric
acid level decreased from 51.0 g/l to 21 .0 g/l within 18 h,
and pH rose from 3.65 to 4.39 within the same period as
shown in Figure 3. Thus the depletion of citric acid re-
FIGURE 4 - Changes in lactic acid concentration
Lactic acid concentration in fermenting marula juice
(Figure 4) was constant during the first 6 hrs at 0.07 % (w/w)
and there was a steady increase reaching a maximum of
0.47 % (w/w) within 54 hrs. Thereafter, the concentration
remained constant at 0.46 % (w/w) until the end of fermentation period. Increase in lactic acid was a result of
the activity of lactic acid bacteria and contributed to a
fall in pH.
118
The pattern followed by glucose, fructose and sucrose
during fermentation is shown in Figure 5. Glucose had an
initial value of 5.7 g/l and increased to 8.6 g/l within 6 hrs
of fermentation. There was a decrease in glucose levels
after 36 hrs from 8.7 g/l to 0.4 g /l at the end of the fermentation. There was a sharp decrease of sucrose concentration within 12 hrs, followed by a gradual decrease
until the end of the fermentation period to 0.83 g/l. The
increase in glucose up to the 30 h fermentation period was
due to hydrolytic action of invertase from the yeast cells
breaking down sucrose to glucose and fructose. Yeast
utilized glucose to produce ethanol. This was supported by
a corresponding rise in ethanol from 1.5 g/l to 2.3 % (v/v)
at fermentation time (Figure 6). Lactic acid bacteria are
also thought to have utilized the sugars to produce lactic
acid amongst other secondary products.
60
Sugars (g / l)
50
40
30
20
10
Fructose increased steadily from an initial concentration of 12.93 g/l to a maximum concentration of 19.13 g/l
within 12 hrs. However, the levels of fructose remained
constant and only started to drop after 48 h of fermentation. The results indicate that microorganisms present
used glucose before fructose as a carbon and energy
source. Low final concentration of glucose, fructose and
sucrose at the end of 72 hrs is thought to indicate that the
fermentation process was essentially complete.
Figure 6 shows changes in ethanol concentration in
fermenting marula juice. There was already some ethanol
(0.2 % (v/v)) when the fermentation was commenced in
earthenware pots, suggesting that spontaneous fermentation of marula starts whilst the fruit is still on the ground.
There was very little increase in ethanol observed for the
first 18 hrs. It is interesting that this period corresponds to
a rapid decrease in citrate. A noticeable increase was
observed from 0.3 to 2.3 % ethanol between the 18 h and
60 h fermentation period. Thereafter there was a gradual
decrease in ethanol concentration until the end of fermentation. The increase observed in ethanol concentration
may be attributed to the increasing fermentative activities
of yeast cells. The ethanol levels only start to decrease
slightly when all the sugars are used up at 66 hrs.
0
0
6 12 18 24 30 36 42 48 54 60 66 72
CONCLUSION
Time (h)
Sucrose
Fructose
Glucose
Ethanol (g / l)
FIGURE 5 - Changes in glucose, fructose and sucrose
during fermentation of marula juice
20
18
16
14
12
10
8
6
4
2
0
The final pH value of 3.4 and the final titratable acidity value of 0.95 % lactic acid makes mukumbi fall within
values recommended for sweet dessert wines by Amerine
& Ough [14]. The ethanol content of mukumbi is comparable to agadagidi from plantain fruits which has an ethanol content of less than 3 % [15]. However, in mukumbi
the final level of ethanol, the pH and titratable acidity
varies from brew to brew as this is an uncontrolled fermentation. The possibility of using starter yeast cultures
should be investigated. Since acceptance of a product
depends considerably on consumer preference, additional
studies with sensory evaluation should be conducted to
optimize the process for small-scale industrial production.
ACKNOWLEDGMENTS
0
6 12 18 24 30 36 42 48 54 60 66 72 78
Time (h)
FIGURE 6 - Changes in ethanol levels during ermentation
of marula juice during the preparation of mukumbi.
The authors would like to thank the Farm-level Applied Research Methods for East and Southern Africa
(FARMESA) and the Swedish Agency for Research Corporation with Developing Countries (SAREC) for funding. We also acknowledge the assistance of DV Chiuswa,
T Mugochi, W Parawira and Mapindu villagers of
Mataga, Mberengwa for their knowledge on the art of
mukumbi preparation. The assistance of Norbert Tsanyiwa
during sample collection is greatly appreciated.
119
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Accepted for publication: September 03, 2002
[12] Lawrence AJ (1975): Determination of lactic acid in cream.
Australian Journal of Dairy Technology, 30, 14-15.
[13] Hugenholtz J, Starrenburg MJC & Weerkamp AH (1994):
Diacetyl production by Lactococcus lactis Optimization and
metabolic engineering. In. Proceedings of the 6th European
Congress On Biotechnology, (Eds Albergina A, Frontali L &
Sensi P) ECB6, Florence Italy, Elsevier Science.
A. Mpofu
Department of Soil Science and Agricultural
Engineering
University of Zimbabwe
Box MP 167, Mount Pleasant
Harare - ZIMBABWE
[14] Amerine MA & Ough CS (Ed) (1980): Methods of Analysis
of Musts and Wines, p. 241. New York, John Wiley
[15] Sanni AI & Oso BA (1988): The production of Agadagidi, a
Nigerian fermented beverage. Die Nahrung. 32, 319-326.
120
Fax (263) (4) 308046
AFS/ Vol 24/ No 3/ 2002 – pages 116 - 120
COMPARATIVE STUDIES ON BIOSORPTION OF
COBALT (II), NICKEL (II), LEAD (II) AND MANGANESE (II)
BY FOUR DIFFERENT FUNGI
Mohammad H. Habibia, Giti Emtiazib, Zohreh Khalesib and Mohammad Ali Haghighipoura
a
Chemistry, and b Biology Department, Isfahan University, Isfahan 81745-117, Iran
ABSTRACT
Four different fungi, Aspergillus terreus, Aspergillus
niger, Trichoderma reesei and Phanerochaete chrysosporium, showed different capacities for biosorption of heavy
metals, e.g. Co, Ni, Pb, Mn. Fungal biosorption largely
depends on the growth media. Phanerochaete chrysosporium grown on whey water had the highest sorption capacity (Co(II), 10; Ni(II), 12.5; Pb(II), 27.6 and Mn(II),
35 mg/g removal). This research showed that fungal biosorption strongly influences the removal of heavy metal
ions from aquatic systems.
KEYWORDS: Biosorption, metal ions, cobalt, nickel, lead, manganese, fungi, Aspergillus terreus, Aspergillus niger, Trichoderma
reesei and Phanerochaete chrysosporium.
INTRODUCTION
Increased industrial and human activities have impact
on the environment through heavy metal-containing waste
disposal. Mine drainage, metal industries, refining, dye
and leather industries, landfill leachate, agricultural runoff
and domestic effluents contribute to such a kind of waste.
Especially, electroplating wastewater is one of the major
industrial contributors of heavy metal pollution in surface
waters [1, 2]. There has been a growing concern with
environmental protection, achieved either by decreasing
the afflux of pollutants or their removal from contaminated media. The former is a feasible choice only for
pollutants of anthropogenic origin, whereas the latter is
unavoidable, especially for those of natural origin. Since
cobalt, nickel, lead and manganese are among the most
toxic elements, numerous efforts have been made to lower
their presence in contaminated media to innocuous quantities [3, 4].
The interactions of microorganisms and metal ions in
aqueous media have been the focus of a growing number
of scientific studies in recent years. The characteristics of
passive microbial metal binding, commonly termed biosorption [5], have been investigated for a wide range of
simple metal/organism systems. In addition, competition
studies in solutions of multiple cations and/or anions have
underscored the complexity of the sorption interactions
involved as well as demonstrating the ability of certain
co-ions to either reverse the augment of metal toxicity or
uptake [6-8]. The principle exponents of metal-microorganism interactions for environmental purposes remain
traditionally biological waste treatment systems of the
activated sludge/biological filter type.
In the concept of biosorption, several chemical processes may be involved, such as absorption, ion exchange
and covalent bonding with biosorptive sites of the microorganisms including carboxyl, hydroxyl, sulphydryl,
amino and phosphate groups. Fungal cell walls and their
components play a major role in biosorption process.
Fungal biomass can also take up considerable quantities
of heavy metals from aqueous solutions by adsorption or
related processes, even in the absence of physiological
activity [9-14].
The purpose of this investigation was to study the use
of different fungi, e.g. Aspergillus terreus, Aspergillus
niger, Trichoderma reesei and Phanerochaete chrysosporium, as biosorbents for heavy metals from artificial
wastewaters and to determine the maximum biosorption
capacity of the fungal biomass.
EXPERIMENTAL
Potato dextrose agar (PDA) and brain-heart infusion
(BHI) agar were obtained from Oxoid Ltd. Noble agar
was from Difco Laboratories. The fungi, Aspergillus
121
terreus, Aspergillus niger, Trichoderma reesei and Phanerochaete chrysosporium used in this study were obtained
from Isfahan University Microbial Collection. Aspergillus
terreus was isolated from rotting wood and identified by
laboratory methods in basic mycology (13). These fungi
were maintained on PDA. The capability of fungi to absorb the metals in the appropriate media was investigated.
After inoculation of the fungi on a shaker at 30 °C, the
biomass was harvested by filtration through a Watman
Nr. 1 filter paper with distilled water and dried at 50 °C in
an oven for 24 hrs.
In the first experimental series, biosorption of Co(II),
Ni(II), Pb(II) and Mn(II) was investigated batchwise in
conical flasks. 10 ppm of metal ions using CoCl2, NiSO4,
Pb(CH3COO)2 and MnCl2 were added separately to BHI
medium. After inoculation of the different fungi in the
flasks and incubation at 30 °C for 3 days on a rotary
shaker (120 rpm), the biomass was harvested and the
heavy metal content of the supernatants was assayed. In
another experimental series, biosorption of Co(II), Ni(II),
Pb(II) and Mn(II) was investigated after the production of
biomass in PDA (200 g filtrated and boiled potato include
10 g glucose in 1L distilled water) at 30 °C within 3 days or
whey water (waste effluent from cheesemaking with 10 g/L
lactose and 2g/L casein) at pH 6. The cells were washed
and added separately to 10 ppm solutions of the different
metal ions in flasks. After filtration of the biomass, the
supernatant was assayed for metal ions.
General procedure for biosorption: Dry fungal biomass (1.2 g)
was transferred to the different metal ion solutions (10 mg l-1
water), and after biosorption the biomass was separated
by filtration. The concentration of the metal ions in the
supernatant was measured by using a flame atomic absorption spectrometer. The biosorption capacity was obtained by the following equation:
Bc = [(C0 – C) V]/mb
where Bc is the biosorption capacity of the fungus
(mg g-1), C0 and C are the concentrations of metal ions in
the solution (mgl –1) initially and after biosorption, V is
the volume of the medium (L) and mb is the amount of
biomass (g).
Biosorption of lead (II) ions
The results showed that Phanerochaete chrysosporium biosorbed 27.2 mg g-1 of lead ions when grown on
whey water or 9 mg g-1 when grown on PDA (Tables 1
and 2). Compared to the other fungi used in this study,
Phanerochaete chrysosporium had maximum biosorption
capacity grown on both potato broth and whey water. The
addition of 10 ppm lead ions to BHI media suppressed
especially the growth of Phanerochaete chrysosporium.
Only 5 ppm of lead ions remained in the supernatant
(Table 4).
Biosorption of cobalt (II) ions
Trichoderma reesei biosorbed 11.6 mg g-1 of cobalt
ions grown on PDA and Phanerochaete chrysosporium
10mg g-1 when grown on whey water (Tables 1 and 2).
The addition of 10 ppm cobalt ions to BHI media suppressed the growth of all fungi, which indicates that both
biomass and culture medium affect their biosorption capacity (Table 3). High percentages (60-88%) remained in
the supernatants (Table 4).
Biosorption of manganese (II) ions
Aspergills niger biosorbed 6.7 mg g-1 of Mn(II) ions on
PDA broth and Phanerochaete chrysosporium 35mg g-1,
when grown on whey water (Tables 1 and 2). The addition of 10 ppm manganese ions to the BHI media did not
affect the growth of Aspergillus terreus and Trichoderma
reesei, but suppressed that of Aspergillus niger and Phanerochaete chrysosporium (Table 3).
Biosorption of nickel (II) ions
In identical experiments it was observed that Phanerochaete chrysosporium biosorbed 9.7 mg g-1 of Ni(II)
ions grown on PDA (Table 1) and 12.5 mg g-1 on whey
water (Table 2). The addition of10ppm nickel ions to BHI
media suppressed the growth of all fungi, but again especially that of Phanerochaete chrysosporium and Aspergillus niger (Table 3). 86-90% of the nickel ions added remained in the supernatants as similarly observed with
Mn(II) ions (Table 4).
TABLE 1
Biosorption capacity (mg g-1) of Co(II), Ni(II), Pb(II) and Mn(II) by different fungi grown on potato dextroseagar (PDA).
Biosorption Capacity (mg g-1)
Fungi
Co(II)
Ni(II)
Pb(II)
Mn(II)
Aspergillus terreus
3
5.9
4.2
5.3
Aspergillus niger
2.4
3.4
8.2
6.7
Phanerochaete chrysosporium
3.9
9.2
9.0
2.8
Trichoderma reesei
11.6
3.5
1.0
5.0
122
TABLE 2
Biosorption capacity (mg g-1) of Co(II), Ni(II), Pb(II) and Mn(II) by different fungi grown on whey water.
Biosorption Capacity (mg g-1)
Fungi
Co(II)
Ni(II)
Pb(II)
Aspergillus terreus
5.0
7.7
6.5
Mn(II)
Aspergillus niger
4.3
5.6
11.
11.7
10.0
12.5
27.6
35
Trichoderma reesei
0.2
1.5
N.D
4.0
4.8
N.D = not detected
TABLE 3
Maximum biomass of the different fungi grown on BHI in the presence of different metal ions (10 ppm).
Maximum biomass (g L-1)
Fungi
Co(II)
Ni(II)
Pb(II)
Mn(II)
No metal
Aspergillus terreus
4.0
5.2
10.2
8.9
12
Aspergillus niger
0.3
0.18
8.8
0.3
10
0.2
0.3
5.3
Trichoderma reesei
0.2
6.3
9.0
0.4
8
12
13
TABLE 4
Addition of 10 ppm Mn(II) ,Pb(II), Co(II) or Ni(II) to the media of different fungi
grown on BHI and concentration of the metal ions remaining in the supernatant.
Fungi
Concentration of metal ions (ppm)
Co(II)
Ni(II)
Pb(II)
Aspergillus terreus
6.0
4.3
1.5
Aspergillus niger
6.8
8.6
2.5
9.4
8.8
9.0
5.0
9.4
Trichoderma reesei
8.8
3.8
2.5
1.0
The addition of 10 ppm cobalt, nickel or manganese
ions suppressed the growth of Aspergillus niger and
Phanerochaete chrysosporium. Therefore, these ions remain nearly totally in the supernatant. However, Phanerochaete chrysosporium grown on whey water exhibited the
highest biosorption capacities (10, 12.5, 27.6 and 35 mg of
Co(II), Ni(II), Pb(II) and Mn(II) ions per g of biomass),
when grown on whey water (Table 2). Cultivated on
PDA, this fungus biosorbed 9.2 and 9.0 mg of Ni(II) and
Pb(II) ions (Table 1).
ACKNOWLEDGMENT
We are grateful to Isfahan University Research
Council for financial support of this work under grant
number 780413.
123
Mn(II)
1.4
REFERENCES
[1]
M. Ajmal, A.M. Sulaiman and A.H. Khan, Wat. Air Soil Pollut. 68, 485 (1993).
[2]
A. Golomb, Plating 59, 316 (1972).
[3]
M.A. Ferro Garcia, J. Rivera Utrilla, I. Bautista Toledo and
M.D. Mingoranc, Carbon 28, 546 (1990).
[4]
E. Fourest, A. Serre and J.C. Roux, Toxic. Environ. Chem.
54, 1 (1996).
[5]
E. Fourest and B. Volesky, Appl. Biochem. Biotechnol. 67,
215 (1997).
[6]
G. M. Gadd and C. White, Biotechnol, 33, 592 (1989).
[7]
Golomb, Plating 59, 316 (1972).
[8]
N. Hafez, A.S. Abdel-Razek and M.B. Hafez, J. Chem.
Technol. Biotechnol. 68, 19 (1997).
[9]
G. Yan and T. Viraraghavan, Water SA 26, 119 (2000).
[10] Z.R. Holan, B. Volesky and I. Prasetyo, Biotechnol. Bioeng.
41 819 (1993).
[11] C. White and G.M. Gudd, J. Chem. Tech. Biotech. 46, 331
(1990).
[12] A. Kapoor and T. Viravaghavan, Biores. Technol. 61, 221
(1997).
[13] E.J. Baron and M.S. Finegold, Diagonistic Microbiology, 8th
ed., pp. 681-767. The C.V. Mosby Com., London 1990.
[14] J.M. Brady and J.M. Tubin, Enzyme Microb. Technol, 17,
791 (1978).
Accepted for publication: October 10, 2002
Mohammad H. Habibi
Chemistry Department
Isfahan University
Isfahan 81745-117 - IRAN
Fax: +98-311-6689732
AFS/ Vol 24/ No 3/ 2002 – pages 121 - 124
124
PHYSICOCHEMICAL ANALYSIS OF
TOKAT REGION (TURKEY) HONEYS
Mustafa Tüzen1 and Mustafa Duran2
1
Gaziosmanpaşa University, Faculty of Science and Arts, Chemistry Department, 60250 Tokat-Turkey
2
Gaziosmanpaşa University, Faculty of Science and Arts, Biology Department, 60250 Tokat-Turkey
SUMMARY
Standard methods were used for the determination of
physico-chemical properties of Tokat region honey samples. All samples were examined for pH, total acidity,
moisture, ash, electrical conductivity, HMF, total solids,
reducing sugars, total soluble solids, total sugars, and
sucrose. Metal contents of honey samples were determined by atomic absorption spectrometry. The physicochemical characteristics of the honey samples investigated
met all the compositional and quality criteria of the
“Turkish Standards Institute (TSE 3036)” and generally
agreed with the earlier published results.
and a rich vegetation, honey production is rather low
since advanced apiculture technology is not followed
adequately. In Tokat region, the climate is mild in winter,
rainy in spring and autumn and all over the year, especially in spring and summer, ideal for apiculture. Tokat
has 39% forested area, 13% meadowland and pasture,
33% agricultural land and 15% others. Vegetation in
Tokat region is characterized by clover, trefoil, deadnettle, daisy, poppy, centaury, hyacinth, blackthorn, tulip,
monk, chicory, blackberry (common monocotyledon),
apricot, cherry, apple, acacia, peach, plum, walnut and
almond (common dicotyledon). The blossoming periods
of various flowers last from April to August.
KEYWORDS:
Honey, pysico-chemical analysis, Tokat, Turkey.
The faculties of agriculture, vocational high schools
of agriculture, the Ministry of Agriculture, Forestry and
Village Affairs, and the Education Unit of Integrated
Apiculture Project of Development Foundation of Turkey
carry out research on the characteristics of bee races of
the country and on artificial insemination, and produce
controlled mother queen and swarm.
INTRODUCTION
The climate and rich vegetation in Turkey provide a
very suitable environment for apiculture which is in a state
of expansion. Turkey is the third largest country, following
Soviet Union and USA, with regard to the number of hives.
In 1993, there were 3,686,000 hives representing a yearly
increase of 4.1%. The production of honey was 59.207 tons
in 1995 and increased to 80.000 tons in 1997 [1-2]. The
amount of exported honey increased (2.517,9 tons in 1988
and 5.000 tons in 1997), but it is still very low because
great amounts of honey produced are consumed within the
country. Production of honey in Tokat region (800 tons in
2001) is also quite low compared with the total honey
production in Turkey. There exist about 88 migratory and
1666 settled beekeepers and the number of hives is approximately 40.000. The common honeybee races are Apis
mellifera cacucasia, Apis mellifera anatilica and their
hybrid Apis mellifera cacucasia gorb. While the mean
production of honey is 20 kg per hive in the world, it is 16 kg
in Tokat [3]. Although there is a sufficient number of hives
However, only a few numbers of investigations have
been related to physical properties and chemical composition of Turkish honey samples [5-7]. In previous work,
some metals have been determined in Tokat region honeys by monitoring environmental pollution [4]. Therefore,
it is important to determine the essential composition and
quality factors of Tokat region honeys and physicochemical parameters were analyzed in this study using
various instrumental and analytical techniques.
MATERIAL AND METHODS
Sampling
Fourty liquid honey samples were collected from Tokat city, Turkey in 1999. The samples were preserved in
covered plastic containers and kept in the laboratory at
room temperature until analysis. The honey samples were
125
rich and light with a predominant sweet, clover-like flavour and an elegant floral aftertaste.
Physico-chemical analysis
The honey samples were analyzed according to the
methods of the Association of Official Analytical Chemists (AOAC) [8] and Turkish Standards Institute (TSE
3036) [9].
The pH was measured in a solution containing 5 g
honey in 10 ml distilled water. Total acidity was determined titrimetrically with 0.1 M NaOH (10 g honey dissolved in 75 ml distilled water). Moisture and total soluble solids were determined refractometrically (total solids
(%) calculated as 100 - moisture content). Ash percentage
was measured by calcination overnight at 550 ºC in a
furnace to constant mass. Electrical conductivity was
determined in a solution containing 2 g honey in 10 ml
distilled water at 20 ºC. Hydroxymethyl furfural (HMF)
was determined after dilution with distilled water and
addition of p-toluidine solution. Absorbance was measured at 550 nm using a 1 cm cell in a Jasco Model V-530
spectrophotometer. Total and reducing sugars were determined by titrimetric method (redox volumetry of Fehling reagent with methylene blue end point-detection).
Sucrose (%) was calculated as (total sugars-reducing
sugars) x 0.95. All determinations were carried out on wet
weight basis. Metal contents (Pb, Cd, Fe, Cu, Mn and Zn)
were determined according to the method earlier described using a Varian Spectr AA-220 atomic absorption
spectrometer equipped with a graphite furnace [4]. Ca,
Mg and Na, K were determined by flame atomic absorption spectrometry and flame photometry, respectively.
The chemical composition of the honey samples investigated was compared with the values recommended in
TSE 3036 (Table 1).
TABLE 1 - Chemical composition of Tokat honey samples
analyzed and values recommended for honey in TSE 3036.
Parameters
Mean
Range
Values in TSE 3036
pH
4.10
3.75-4.38
Total acidity (ml 0.1M NaOH/10 g honey)
3.26
1.20-4.85
Moisture (%)
16.10
15.30-18.26
0.26
0.12-0.50
max. 0.6%
Electrical conductivity (10 s cm )
5.37
3.25-7.62
-
HMF (mg/kg)
4.82
2.36-17.61
Ash (%)
-4
Total solids (%)
-1
83.90
81.74-84.70
max. 40 mmol/kg
max. 20%
max. 40 mg/kg
-
Reducing sugars (%)
74.48
70.05-78.54
Total soluble solids (%)
76.40
74.17-79.38
-
Total sugars (%)
77.56
71.63-86.45
-
2.93
1.50-7.66
Sucrose (%)
Sodium (mg/kg)
85±10
Potassium (mg/kg)
800±70
Calcium (mg/kg)
Magnesium (mg/kg)
min. 65%
max. 5%
30-100
-
400-1500
-
62±12
50-300
-
32±7
14-48
-
Copper (mg/kg)
0.75±0.09
0.30-1.45
-
Iron (mg/kg)
4.90±1.05
3.20-7.65
-
Manganese (mg/kg)
0.62±0.08
0.38-0.85
-
Zinc (mg/kg)
3.50±0.70
1.50-5.30
-
Lead (µg/kg)
54.6±5.8
39.30-64.50
-
5.45-10.76
-
Cadmium (µg/kg)
6.80±0.52
126
The results were also compared with several literature
data for other regions of Turkey. Values for moisture, pH,
sucrose, ash, HMF and reducing sugars are in agreement
with those of South-Eastern Anatolia honeys [10] and pH,
total acidity, moisture, reducing sugars, HMF, and sucrose with the quality parameters of Merin et al. [11].
Total acidity in Tokat honeys is lower than that of Saudi
honeys [5]. The Tokat honey samples are having pH values from 3.75 to 4.38. All samples fell within the Turkish
Legal Regulations for acidity, moisture, ash, reducing
sugars, sucrose and HMF values (TSE 3036) [9]. The
HMF content is the quality parameter indicating the state
of freshness of honey [12-13]. It is a by-product of fructose decay, formed during storage or heating. Thus, its
presence is considered to be the main indicator of honey
deterioration and it should not exceed 40-60 mg/kg after
processing and blending. HMF values varied between
2.36 and 17.61 mg/kg in the Tokat honeys and are lower
than those reported in Portuguese honeys [6], but in
agreement with other studies [5, 14]. Moisture, ash and
sucrose contents are lower, reducing sugar contents higher
and electrical conductivity values similar to those reported by Amin et al. [15].
[8]
AOAC. (1995) Official methods of analysis (16 th ed.). Association of Official Analytical Chemists, Washington, DC,
USA.
[9]
TSE 3036. (2002) The Turkish Standards Institute, Ankara,
Turkey.
[10] Yõlmaz, H., Yavuz, Ö. (1999) Content of some trace metals
in honey from south-eastern Anatolia. Food Chemistry, 65,
475-476.
[11] Merin, U., Bernstein, S., Rosenthal, I. (1998) A parameter
for quality of honey. Food Chemistry, 63(2), 241-242.
[12] Sancho, M.T., Muniategui, S., Huidorbo, J.F., Simal, J.
(1992) Aging of honey. Journal of Agricultural and Food
Chemistry, 40, 134-138.
[13] White, J.W. (1994) The role of HMF and diastase assays in
honey quality evolution. Bee World, 75(3), 104-117.
[14] Przybylowski, P., Wilczynska, A. (2001) Honey as an environmental marker. Food Chemistry, 74, 289-291.
[15] Amin, W.A., Safwat, M., El-Iraki, S. (1999) Quality criteria
of treacle (black honey). Food Chemistry, 67, 17-20.
Metal concentrations in the honeys analyzed are not
different from values reported in other honey samples of
Turkey [4, 10, 16]. The concentrations of K, Na, Cu, Mn and
Fe are found to be higher than those of Saudi honeys [5]. All
the honey samples contained insignificant amounts of
lead and cadmium.
[16] Üren, A., Şerifoğlu, A., Sarõkahya, Y. (1998) Distribution of
elements in honeys and effect of a thermoelectric power plant
on the element contents. Food Chemistry, 61, 185-190.
REFERENCES
[1]
Anon. (1995) Annual statistics of Republic of Turkey for
1995. DIE Ankara, Turkey.
[2]
Anon. (1997) Annual statistics of Republic of Turkey for
1997. DIE Ankara, Turkey.
[3]
Apicultural report, Tokat Agricultural Directorate, Turkey,
2002.
[4]
Tüzen, M. (2002) Determination of some metals in honey
samples for monitoring environmental pollution. Fresenius
Environmental Bulletin, 11(7), 366-370.
[5]
Al-Khalifa, A.S., Al-Arify, I.A. (1999) Physicochemical
characteristics and pollen spectrum of some Saudi honeys.
Food Chemistry, 67, 21-25.
[6]
Andrade, P.B., Amaral, M.T., Isabel, P., Carvalho, J.C.M.F.,
Seabra, R.M., Cunha, A.P. (1999) Physicochemical attributes
and pollen spectrum of Portuguese healter honeys. Food
Chemistry, 66, 503-510.
[7]
Perez-Arquillue, C., Conchello, P., Arino, A., Juan, T.,
Herrera, A. (1995) Physicochemical attributes and pollen
spectrum of some unifloral Spanish honeys. Food Chemistry,
54, 167-172.
Accepted for publication: October 10, 2002
M. Tüzen
Gaziosmanpaşa University
Faculty of Science and Arts
Chemistry Department,
60250 Tokat-TURKEY
Fax: +90 356 2521585
AFS/ Vol 24/ No 3/ 2002 – pages 125 - 127
127
AFS
Book Reviews - Bücherschau
Food, People and Society – A European
Perspective of Consumers` Food Choices
Frewer, L. J., Risvik, E., Schifferstein, H (Eds).
462 pages, 71 figures, 63 tables; Springer-Verlag Berlin –
Heidelberg – New York – London – Paris – Tokyo – Hong
Kong, 2001; ISBN 3-540-41521-1; Hardcover GBP 74.00,
US $ 109.00.
The exact determinants of food perception, liking and
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a group of scientists experienced in European crosscultural and interdisciplinary research in the special fields
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person who eats a particular food, whereas a part of the
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This broad spectrum of different approaches in this book
opens the opportunity to facilitate comprehension of the
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Therefore, this volume is essential for those engaged
in product development, market research and consumer
science in food and agro industries but also of great interest for students and academics interested in food perception and consumption, policy makers, health educators
and nutritionists.
FROM THE CONTENTS
Part I. Food: Introduction. Development and
acquisition of food likes. The food and I. Beliefs about
Fat: Why do we hold beliefs about fat. Product packaging
and branding. Effects of product beliefs on product
perception and liking. Consumer's quality perception.
ure of food attitudes. The origin of spices. Marketing
PDO and PGI. Effect of communication on sales of commodities. Food availability and the European consumer.
The economics of food choice. Food choice in Europe.
Beliefs associated with food production methods. Risk
perception, communication and trust. Risk perception and
food choice. Public Participation in developing policy
related to food issues. The future of European food
choice. Subject Index
NMR Spectroscopy: Data Acquisition
(CD-ROM included “Spectroscopic Techniques:
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Christian Schorn
347 pages; Wiley-VCH Weinheim – New York –
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The application of NMR spectroscopy in new fields
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fields NMR is, in the meanwhile, a part of industrial production and medicine, e.g. by MRI (magnetic resonance
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Processing Strategies
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Part II. People: Does taste determine consumption?
Understanding the psychology of food choice. Food
choice, phytochemicals and cancer prevention. Private
body consciousness. Food neophobia and variety seeking.
Convenience-oriented shopping. Food intake and the
elderly – Social Aspects. Food related lifestyle.
This complete series deals with all aspects of a standard
NMR investigation beginning with the definition of structural problems and ending with the unravelled structure.
Part III. Society: Cross-cultural differences in food
choice. Appropriateness as a cognitive-contextual meas-
Volume 1 “Processing Stzartegies” gives the theoretical background for all processing steps and demonstrates
128
the effects of different manipulations by means of suitable
examples. If you intend to operate a NMR spectrometer
yourself, or want to become more familiar with additional
powerful software tools to make the best of your NMR
data, you need this Volume 2 “Data Acquisition”.
This volume begins with the selection of the most appropriate pulse experiment(s) necessary to solve a structural problem by NMR analysis. Then the understanding
of the basic principles of the most common experiments
and being aware of the dependence of spectral quality on
the various experimental parameters as the most important prerequisites for a successful appli-cation of any
NMR experiment are explained. Spectral quality, on the
other hand, strongly determines the reliability of structural
information extracted in the subsequent steps of NMR
analysis. These facts are not only of interest for the NMR
operators but also for interpreters of spectral data. They
have also to be familiar with the interdependence of various experimental parameters such as acquisition time and
resolution, repetition rate, relaxation times and signal
intensities. Many mistakes made with the application of
modern NMR spectroscopy because of a lack of understanding of these basic principles may be avoided. This
volume covers all these aspects and explains them in an
interactive way. Using the Bruker software package
NMR-SIM together with 1D WIN-NMR and 2D WINNMR (included CD-ROM) allows the readers to simulate
routine NMR experiments and study the interdependence
of a number of NMR parameters to get an insight into
modern multiple pulse NMR experimental work.
Volume 3 “Modern Spectral Analysis” discussing the
strategies needed for efficient and competent extract of
the NMR parameters from the corresponding spectra and
Volume 4 “Intelligent Data Management” as an introduction to the computer-assisted interpretation of molecular spectra of organic compounds using Bruker software
(WIN-SPECEDIT, STRUKED together with 1D WINNMR and 2D WIN-NMR) are additionally recommended
to enable the user to evaluate NMR parameters, to generate and exploit dedicated databases and, finally, to establish molecular structures.
Meat refrigeration
S. J. James and C. James
Woodhead Publishing in Food Science and Technology
347 pages, numerous tables and figures; CRC Press Boca
Raton – Boston – New York – Washington D.C., published in Europe by Woodhead Publisihing Ltd, Abington
Hall, Abington, Cambridge, CB1 6AH, UK, 2002; ISBN
1-85573-442-7; Hardcover £ 135.00/€ 210.00 (plus p&p).
Chilling and freezing of meat remains one of the essential ways to extend shelf-life and maintain quality.
Based on the experiences and results of the internationally-renownded Food Refrigeration and Process Engineering Research Centre (FRPERC), this volume provides a recommendable guide either to the impact of
refrigeration on meat or the best practice in using it to
maximise meat quality for the consumer.
Part 1 considers the impact of refrigeration on meat
quality. First of all the microbiology of refrigerated meat
is explained including factors affecting the shelf-life of
refrigerated meat such as initial microbial levels, parameters like temperature and relative humidity but also other
considerations such as bone taint, cold and hot deboning.
Then drip production in meat refrigeration with chapters
on biochemistry of meat (structure of muscle, changes
after slaughter, water relationships in meat, ice formation
in muscle tissue), measurement of drip and factors affecting the amount of drip (animal factors, refrigeration factors, chilled storage) follows. The influence of refrigeration on texture of meat (muscle shortening, development
of ageing, influence of chilling, freezing and thawing) is
described in chapter 3 of part 1. In the last two chapters
an overview on colour changes in chilling, freezing and
storage of meat is given and the influence of refrigeration
on evaporative weight loss from meat is explained.
Part 2 examines the best practice in managing the cold
chain from carcass to consumer. The authors discuss primary chilling of red meat, freezing systems, tempering and
crust freezing, thawing, transportation of meat, chilled and
frozen storage, chilled and frozen retail display of wrapped
and unwrapped meat and meat products including overall
cabinet design and, finally, consumer handling.
The last part of this book summarizes the most
important aspects of process control with chapters on
thermophysical properties of meat (chilling: thermal
conductivity, specific heat, enthalpies, freezing, thawing
and tempering: ice content, heat extraction, thermal
conductivity, density), temperature measurements (instrumentation, calibration, measuring and interpretation),
specifying, designing and optimising of refrigeration
systems (Process: throughput, temperature requirements,
weight loss, plant design; Engineering: Environmental
conditions, room size, refrigeration loads and plant capacity, relative humidity, ambient design, defrosts; plant design and process definition) and secondary chilling of
meat and meat products (cooked meat, pastry products,
solid/liquid mixtures, process cooling, cook-chill).
At the end of each of the 16 chapters a comprehensive
list of references is included to give the interested reader
the possibility to get to know further details.
129
Food and Nutritional Supplements –
Their Role in Health and Disease.
Prüfmethoden für Chemikalien
J. K. Ransley, J.K. Donnelly, N. W. Read (Eds.)
Ulrich Schlottmann (Hrsg.)
197 pages, 13 figures, 24 tables; Springer-Verlag Berlin –
Heidelberg – New York – Barcelona – Hong Kong – London – Milan – Paris – Singapore – Tokyo, 2001; ISBN 3540-41737-0; Hardcover GBP 48.00, US $ 74.95.
unter Mitarbeit von R. Arndt, D. Kayser, R. Kanne, E.
Bruns, D. Brasse, B. Brumhard, N. Caspers, E. CremerSchlede, C. Haas, M. Liebsch, S. Madle und H.-B. Richter-Reichhelm
It is estimated that about 40% of people take nutritional supplements. This trend has risen dramatically in
recent years and is expected to increase further over the
next five years. In Western Europe the market for these
products is growing because consumers have been encouraged to take more responsibility for their own health.
Therefore, the purpose of this book is to elucidate the
phenomenon of self-medication with nutritional supplements from both a biological and psychological point of
view. Pharmacies, drug stores,health food shops but also
supermarkets stock a vast array of preparations including
vitamins, minerals, oils derived from flowers and fish,
tonics and herbal products which are also readily available by mail order and purchase over the Internet. What
are the reasons why so many people feel the need to take
these products?
Grundwerk + 3. Ergänzungslieferung zur 1. Auflage,
Stand: Januar 2002; 1326 Seiten.; S. Hirzel Verlag Stuttgart
- Leipzig, Wissenschaftl. Verlaggesellschaft mbH Stuttgart,
2002; ISBN 3-7776-1151-4; Loseblattsammlung, 2 Ringordner - Fortsetzungswerk € 148.00.
The book is divided into three main sections. In section 1 the phenomenal growth in the market for food and
nutritional supplements over the recent years is outlined.
It is explained why the body´s need for nutrients varies
over the lifecycle and during the course of an illness or
trauma. Other chapters deal with extending of the knowledge base of health professionals in the scientific field of
nutrition, feeling the need to self medicate with supplements (placebos) which can relieve suffering of patients.
The second part examines the scientific aspects behind the role key nutrients and components in food and
their role in prevention and treatment of disease. Vitamins, antioaxidants, phytoestrogens and probiotics are
considered in detail.
The last section practically evaluates two common
disease states for which nutrients may play a role in prevention and also treatment – coronary heart disease and
rheumatoid arthritis.
This book is recommended because each of the contributors has provided a concise but comprehensive coverage of the latest developments in our understanding of
nutrition in relation to food and nutritional supplements.
Therefore, an informed view has been created by the
editors as a basis when making decisions about healthy
eating or the use of dietary supplements.
Erstmalig sind hier in einer Loseblattsammlung
Prüfmethoden für Chemikalien (offizielle Texte der Methoden) sowie erläuternde Kommentare, Rechtsvorschriften (national und EU) einschließlich GLP und Risikobewertung zusammengestellt. Außerdem ist eine Einführung
ins Umfeld der Prüfmethoden (z. B. Chemikaliengesetz,
Verhältnis zu EU und OECD, Tierversuche, Erarbeitung
der Prüfmethoden) mit aufgenommen. Diese Methoden
werden weltweit zur Bestimmung der physikalischchemischen Eigenschaften, der Toxizität und der Ökotoxizität bei Prüfung und Anmeldung neuer Stoffe und bei
der Aufarbeitung von Altstoffen eingesetzt. Diese neugefasste und ergänzte Auflage umfasst 200 Seiten mehr als
die 1. Auflage. Es sind neue und revidierte Prüfmethoden
enthalten, weil inzwischen eine Anpassung der Richtlinie
Gefährliche Stoffe (67/548/EWG) - abgekürzt 22. AnpRL
erfolgte. Das Kapitel „Weitere Vorschriften“ wurde auf
den neuesten Stand gebracht und der Einfachheit halber
insgesamt ausgetauscht.
Diese Loseblattsammlung ist besonders für die Chemische Industrie, den Chemikalienhandel sowie für Chemische und Toxikologische Prüflabors bestimmt. Für
Chemiker und Toxikologen an Universitäten und Behörden sind diese kommentierten Texte unentbehrlich.
Grenzflächen und kolloid-disperse Systeme –
Physik und Chemie
Hans-Dieter Dörfler
989 Seiten, 579 Abbildungen, 88 Tabellen; SpringerVerlag Berlin – Heidelberg – New York – Barcelona –
Hong Kong – London – Milan – Paris – Singapore – Tokyo, 2002; ISBN 3-540-42547-0; Gebunden € 89.95 (gültig in Deutschland).
Endlich ein komprimiertes Lehrbuch für fortgeschrittene Studenten, Diplomanden und Doktoranden, aber
auch für Mitarbeiter in der einschlägigen Industrie, die
130
alle über die erforderlichen Grundkenntnisse verfügen,
um sich einführend mit den komplizierten Phänomenen
der Physik und Chemie der Grenzflächen und kolloiddispersen Systeme befassen zu können.
In den ersten 7 Kapiteln werden zunächst die wesentlichsten Eigenschaften der verschiedenen Grenzflächentypen behandelt. Erfreulicherweise ist dies eingebunden
in die messmethodischen Entwicklungen und Verfahren,
die von den behandelten Oberflächen und dabei auftretenden Grenzflächenphänomenen abgeleitet wurden.
In den folgenden Kapiteln 8-13 wird auf die Eigenschaften der grenzflächenaktiven Stoffe (Tensidchemie:
Adsorption, Mizellbildung, Bildung lyotroper Flüssigkristalle, Mikroemulsionen, Mechanismus des Waschprozesses) eingegangen. Auch hier ist die theoretische Beschreibung der auftretenden Phänomene mit den Anwendungen
derartiger Tensidsysteme in der chemischen Praxis verbunden. Besonders hervorzuheben ist hier die Beschreibung der Mizellbildung in Verbindung mit den Eigenschaften lyotroper Flüssigkristalle, weil die StrukturEigenschaftsbeziehungen der thermotropen Flüssigkristalle integriert wurden.
+
Die Kapitel 16-25 umfassen die praxisorientierte Darstellung der methodischen und theoretischen Aspekte, die
in der Grenzflächen- und Kolloidchemie eine Rolle spielen. In Kapitel 23 werden die rheologischen Messungen
vertieft, die bei der in den Kapiteln 8 und 15 vorgestellten
Struktur, Funktion und Anwendung von Membransystemen sowie Hydro- und Aerogelen zur näheren Ermittlung
der Eigenschaften dienen.
Insgesamt ein gelungenes Fachbuch, das durch Querverweise in den separat verständlichen einzelnen Kapiteln
den Bezug zu den anderen Kapiteln herstellt und das
Gesamtverständnis wesentlich erleichtert. Am Ende jedes
Kapitels kann der interessierte und kritische Leser anhand
eines zu beantwortenden Fragenkataloges überprüfen, ob
er die wichtigsten Aspekte auch verstanden hat. Der Autor hat, nicht zuletzt durch Einbeziehung von Fachkollegen, ein empfehlenswertes Lehrbuch auf dem neuesten
Stand der Forschung geschaffen.
DGF-Einheitsmethoden - Deutsche Einheitsmethoden zur Untersuchung von Fetten, Fettprodukten, Tensiden und verwandten Stoffen
Deutsche Gesellschaft für
Fettwissenschaft (DGF) e.V. (Hrsg.)
2. Aufl. einschließlich 8. Lieferung 2002; ca. 2300 Seiten;
Wissenschaftl. Ver-lagsgesellschaft mbH Stuttgart, 2002;
ISBN 3-8047-1938-4; Loseblattausgabe - 3 Ringordner/ Fortsetzungswerk € 158.00/sFr 252.80 (Vorzugspreis für DGF-Mitglieder und Mitglieder von EuroFedLipid: € 110.60/ sFr 177.00)
Die DGF-Einheitsmethoden brauchen nicht mehr eigens vorgestellt werden, da sie sich zu einem geschätzten
Standardwerk im Bereich der Fettanalytik entwickelt
haben und wegen ihrer Verständlichkeit und starken Orientierung an die Laborpraxis in Laboralltag, an Universitäten und Untersuchungsämtern allgemein durchgesetzt haben und genutzt werden. Die Verfahren sind vielfach in Ringtests geprüft und von namhaften Experten
entwickelt worden. Es wird hier der ständigen Weiterentwicklung in der Analytik durch jährliche und regelmäßige
Aktualisierung Rechnung getragen. International diskutierte und zur Zeit bereits normierte Verfahren werden bei
der Methodenerstellung berücksichtigt. Neben den mehr
als 350 analytischen Untersuchungsverfahren sind auch
statistische Methoden zur Überprüfung und Validierung
der ermittelten Meßdaten aufgenommen.
AUS DEM INHALT
Abt. A: Allgemeine Angaben; Abt. B: Fett-Rohstoffe;
Abt. C: Fette; Abt. D: Technische Fettsäuren; Abt. E:
Glycerin; Abt. F: Fettbegleitstoffe; Abt. G: Seifen und
Seifenerzeugnisse; Abt. H: Tenside; Abt. K: Fettreiche
Nahrungsmittel und Abt. M: Wachse.
Alles Bio oder was? –
Der schöne Traum vom natürlichen Essen
Hans-Ulrich Grimm
200 Seiten; S. Hirzel Verlag GmbH & Co., Stuttgart 2002;
ISBN 3-7776-1170-0; Kartoniert € 22.00.
In dieser völlig neu bearbeiteten, aktualisierten und ergänzten Ausgabe des 1999 erschienenen Werkes „Der BioBluff“ bezieht der Autor erneut Stellung zur Welt der in
Mode gekommenen Bio-Nahrung. Es ist natürlich auch für
Wissenschaftler einschlägiger Bereiche interessant, wie ein
früherer Journalist und Spiegel-Korrespondent, der heute als
freier Autor lebt und ein erklärter Anhänger von Biokost ist,
seine Erfahrungen, die er in zahlreichen Recherchen gesammelt hat, in diesem Buch kritisch aufarbeitet. In insgesamt 11 Kapiteln berichtet er über seine Entdeckungen in
Landwirtschaft und Industrie: Die Vorzüge der Naturkost –
Die Schattenseite des Bio-Booms: Konjunktur für Betrüger
– Legal, illegal: In den Grauzonen der Lebensmittelproduktion – Die Hochrisiko-Landwirtschaft – Der weltweite BioBoom – Blendende Geschäfte für Etikettenschwindler –
Zoff in der Szene – Das große Bio-Business – Die Industrialisierung der Biokost – Bio-Bluff in der Bäckerei – Der
Kampf um die Zukunft. In diesen Kapiteln beleuchtet er
nicht nur die Schattenseiten, sondern auch die Bio-Siegel,
auf die man sich unbedingt verlassen kann und die den
Traum vom natürlichen Essen Wirklichkeit werden lassen.
Das Buch wird schließlich nach dem Literaturverzeichnis
abgerundet durch einen Anhang mit dem Titel „Echt bio.
Was ist was im Bio-Land?“
131
Foodborne pathogens –
Hazards, risk analysis and control
Clive de W. Blackburn and Peter J. McClure (Eds.)
with contributions of Clive Blackburn, Peter McClure,
Roy Betts, David Legan, Mark Vandeven, Cynthia Stewart, Martin Cole, Tom Ross, Tom McMeekin, Mac Johnston, John Holah, Richard Thorpe, Martyn Brown, Sara
Mortimore, Tony Mayes, Chris Griffith, Chris Bell, Alec
Kyriakides, Jane Sutherland, Alan Varnam, Paul Gbbs,
Marion Koopmans, Rosely Nichols, Huw Smith, Maurice
Moss, Mansel Griffiths, Yasmine Motarjemi.
Hall, Abington, Cambridge, CB1 6AH, UK, Aug. 2002;
ISBN 1-85573-454-0; hardback £ 135.00/€ 235.00 +
P&P
In the last years trends in foodborne disease continue
to rise. Therefore, the identification and control of pathogens becomes more important for the food industry. The
editors have created an authoritive and practical guide
together with an international teams of experts in this
field which helps the practitioner to effective the control
mechanisms of individual food pathogens.
Part I deals with general techniques in assessing and
managing microbiological hazards beginning with a review of analytical methods. The following chapters describe pathogen behaviour and carrying out risk assessment, both a necessary basis for an effective food safety
management. Then good manufacturing practice in the
supply chain beginning with farm production is explained.
Details such as hygienic plant design, sanitation, safe
process design and operation are also discussed as important parts of the HACCP concept. The first part ends with
a chapter on safe practices for the consumers and also
food trade in the sectors of retail and catering.
In Part II the most important pathogens such as E.
coli, Salmonella, Listeria, Campylobacter, Aerobacter,
and enterotoxin-producing Staphylococcus, Shigella,
Yersinia, Vibrio, Aeromonas and Plesiomonas species are
characterized including their risk factors, detection methods and control procedures. This part is finished with a
chapter on spore-forming bacteria, e.g. Clostridium
botulinum or perfringens and Bacillus spp.
In the third part non-bacterial (viruses, parasites, toxigenic fungi) and emerging foodborne pathogens (e.g. Mycobacterium paratuberculosis) are introduced and described as the afore-mentioned bacteria. This part is concluded with an increasingly important chapter on chronic
disease. Microorganisms such as Aeromonas, Brucella
spp., Campylobacter spp., enterohaemorrhagic E. coli,
Enterobacter sakazakii, Helicobacter pylori, Listeria
monocytogenes, Mycobacterium paratuberculosis, nanobacteria, non-thypi Salmonella, Vibrio vulnificus, Yersinia
enterocolitica, Toxoplasma gondii, trematodes, Taenia
solium, Trichinella spiralis, and viral hepatitis A virus play
here an important role and some of them have contributed
to food- or water-borne outbreaks in the last years.
Meat processing – Improving quality
Joseph Kerry, John Kerry and David Ledward (Eds.)
with contributions of David Ledward, Tilman Becker, R.
K. Miller, Jennette Higgs, Feridoon Shahidi, Marianne
Jakobsen and Grete Bertelson, A. P. Moloney, Margit
Dall Aaslyng, Geoffrey R. Nute, H. J. Swatland, Peter
McClure, K. G. Rickert, Christian James, K. B. Madsen
and Jens Ulrich, Stephen J. James, Marie de LamballerieAnton, Peter Sheard, Ir Daniel Demeyer, A. M. Mullen
and H. M. Walsh.
Hall, Abington, Cambridge, CB1 6AH, UK, 2002; ISBN
1-85573-583-0; hardback £ 135.00/€ 210.00 + P&P
Meat has long been a central component of human
diet, both as major food in its own right and as an essential ingredient in many other food products. Concerns
such as safety have led to declining consumption of some
types of red meat, especially in regions such as the EU.
Therefore, this volume addresses questions of defining
meat quality in the mind of the consumers and of quality
enhancement during processing.
Part 1 considers the various aspects of meat quality.
There are chapters on what determines the quality of raw
meat, changing views of the nutritional quality of meat
and the factors determining such quality attributes as
colour and flavour.
Part 2 discusses how these aspects of quality are
measured, beginning with the identification of appropriate
quality indicators. It also includes chapters on both sensory analysis and instrumental methods including on-line
monitoring and microbiological analysis.
Part 3 reviews the range of new processing techniques that have been deployed at various stages in the
supply chain. Chapters include the use of modelling techniques to improve quality and productivity in beef cattle
132
production, new decontamination techniques after slaughter, automation of carcass processing, high pressure processing of meat, developments in modified atmosphere
packaging and chilling and freezing. There are also chapters on particular products such as restructured meat and
fermented meat products.
This new collection Meat Processing is recommendable to all interested practitioners in meat industry and
also scientists engaged in the field of food technology and
chemistry.
FROM THE CONTENTS
Chapter 1: Introduction; Chapter 2: Defining meat
quality (Introduction: what is quality? - Consumer perceptions of quality - Supplier perceptions of quality – Combining consumer and supplier perceptions: the quality
circle - Regulatory definitions of quality - Improving meat
and meat product quality – References)
Part 1: Analysing meat quality - Chapter 3: Factors
affecting the quality of raw meat (Introduction - Quality,
meat composition and structure - Breed and genetic effects on meat quality - Dietary influences on meat qualityRearing and meat quality - Slaughtering and meat quality Other influences on meat quality - Summary: ensuring
consistency in raw meat quality - Future trends - Sources of
further information and advice – References); Chapter 4:
The nutritional quality of meat (Introduction - Meat and
cancer - Meat, fat content and disease - Fatty acids in
meat - Protein in meat - Meat as a functional food - Meat
and micronutrients - Future trends - Conclusion –
References); Chapter 5: Lipid-derived flavours in meat
products (Introduction - The role of lipids in generating
meaty flavours - Lipid autoxidation and meat flavour
deterioration - The effect of ingredients on the flavour
quality of meat - The evaluation of aroma compounds and
flavour quality - Summary –References); Chapter 6:
Modelling colour stability in meat (Introduction - External factors affecting colour stability during packaging and
storage - Modelling dynamic changes in headspace composition - Modelling in practice: fresh beef - Modelling in
practice: cured ham - Internal factors affecting colour
stability - Validation of models - Future trends – References); Chapter 7: Maximising texture quality in meat
(Introduction - Pre-slaughter influences on texture - Postslaughter influences on texture - Measuring meat texture Future trends - Sources of further information and advice
–References).
Part 2: Measuring quality - Chapter 8: Quality indicators for raw meat (Introduction - Technological quality Eating quality - Determining eating quality - Sampling
procedures - Future trends - Acknowledgement – References); Chapter 9: Sensory analysis of meat (IntroductionThe sensory panel - Sensory tests - Category scales -
Sensory profile methods and comparisons with instrumental measurements - Comparisons between countries Conclusions – References); Chapter 10: On-line monitoring of meat quality (Introduction - Measuring electrical
impedance - Measuring pH - Analysing meat properties
using NIR spectrophotometry - Measuring meat colour and
other properties - Water-holding capacity - Sarcomere
length - Connective tissue - Marbling and fat content Meat flavour - Boar taint - Emulsions - Measuring
changes during cooking - Conclusions - Sources of further
information and advice – References); Chapter 11: Methods for the microbiological examination of meat and meat
products (Introduction - Sampling - Microbiological
methods - Quality assurance - Microbiological specifications - Future trends - Sources of further information and
advice – References).
Part 3: New techniques for improving quality - Chapter
12: Modelling beef cattle production to improve quality
(Introduction - Elements of beef cattle production - Challenges for modellers - Simple model of herd structure Future developments – References); Chapter 13: New
developments in decontaminating raw meat (Introduction Current decontamination techniques and their limitations Washing - The use of chemicals - New methods: steam Other new methods - Future trends – References); Chapter 14: Automated meat processing (Introduction - Current developments in robotics in the meat industry Automation in pig slaughtering - Case study: the evisceration process - Automation of secondary processes - Future
trends - References and further reading); Chapter 15: New
developments in the chilling and freezing of meat (Introduction - The impact of chilling and freezing on texture The impact of chilling and freezing on colour - The impact of chilling and freezing on drip loss and evaporative
weight loss - The cold chain - Temperature monitoring Optimising the design and operation of meat refrigeration Sources of further information and advice – References);
Chapter 16: High pressure processing of meat (Introduction: the principles of high pressure (HP) processing - The
effect of HP on food components - Effects on meat structure - Effects on enzyme release and activity - Effects on
texture and colour - Effects on lipid oxidation - Effects on
functional properties of meat proteins - Effects on microflora - Current applications and future developments Conclusions – References); Chapter 17: Processing and
quality control of restructured meat (Introduction - Product manufacture - Factors affecting product quality: temperature, ice content, particle size and mechanical properties - Factors affecting product quality: protein solubility
and related factors - Factors affecting product quality:
cooking distortion - Sensory and consumer testing - Future trends - Sources of further information and advice –
References); Chapter 18: Quality control of fermented
meat products (Introduction: the product - The quality
concept - Sensory quality and its measurement - Appearance and colour: measurement and development - Texture: measurement and development - Flavour: measure-
133
ment and development Taste and aroma: measurement
and development - The control and improvement of quality Future trends in quality development); Chapter 19:
The fat content of meat and meat products (Introduction Fat and the consumer - The fat content of meat - Animal
effects on the fat content and composition of meat - Dietary effects on the fat content and composition of meat Future trends - Sources of information and advice – References); Chapter 20: Quality control of low-fat meat
products (Introduction - The influence of fat on product
quality - Current trends in product development - Techniques for fat reduction in processed meats - Functional
ingredients used in low fat meat products - Other factors
influencing product quality - Future trends - Sources of
further information and advice –References); Chapter 21:
Packaging (Introduction - The use of modified atmosphere
packaging (MAP) - Developments in MAP systems - Active packaging - Future trends - Sources of further information and advice – References).
134
SUBJECT INDEX
A
Q
aglycones
alcoholic fermantation
Aspergillus niger
Aspergillus terreus
106
116
121
121
biosorption
book reviews
121
128
cassava peels
cobalt
94
121
enzymatic hydrolysis
106
fungi
fungicides
121
94
glycosides
glycosidically bound volatiles
106
106
honey
hop flavour
Humulus lupulus L.
125
106
106
B
quality assurance tests
99
rancidity
99
R
T
C
E
F
94
L
manganese
marula
metal ions
mukumbi
121
116
121
116
nickel
121
M
N
O
olive fruits
olive leaves
99
99
P
physico-chemical analysis
Pleurotus sajor-caju
polyphenols
121
125
94
99
S
sunflower oil
116
yeasts
116
subject-index
I
121
wine
Y
H
lead
125
121
125
W
G
insecticides
Tokat
Trichoderma reesei
Turkey
99
135
AUTHOR INDEX
A
Abd-Elmoien, N. M.
Adenipekun, C. O.
99
94
E
Emtiazi, G.
121
Farag, R. S.
Fasidi, I. O
99
94
F
H
Habibi, M. H.
Haghighipour, M. A.
121
121
Khalesi, Z.
Kollmannsberger, H.
121
106
Leupold, G.
128
Mahmoud, E. A.
Mpofu, A.
99
116
Nitz, S.
106
Tüzen, M.
125
Zvauya, R.
116
K
L
M
N
T
Z
author-index
136
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AFS – Advances in Food Sciences es in Food Sciences

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