Revisão Bibliográfica - Universidade Federal do Ceará

Transcription

RENORBIO
PROGRAMA DE PÓS-GRADUAÇÃO EM BIOTECNOLOGIA
EMPREGO DE TECNOLOGIAS EMERGENTES NO PROCESSAMENTO DE SUCO
DE LARANJA ADICIONADO DE FRUTO-OLIGOSSACARÍDEOS E SUCO
PREBIÓTICO DE LARANJA PRODUZIDO VIA SÍNTESE ENZIMÁTICA
FRANCISCA DIVA LIMA ALMEIDA
Fortaleza-Ce
2015
Tese submetida à Coordenação do Curso de
Pós-graduação em Biotecnologia - Renorbio, da
Universidade Federal do Ceará, como requisito
parcial para obtenção do grau de Doutor em
Biotecnologia.
Área de concentração: Biotecnologia Industrial
Ponto focal: Universidade Federal do Ceará
(UFC)
Orientadora: Profª Drª Sueli Rodrigues.
Fortaleza-Ce
2015
Dados Internacionais de Catalogação na Publicação
Universidade Federal do Ceará
Biblioteca de Ciências e Tecnologia
A446e
Almeida, Francisca Diva Lima.
Emprego de tecnologias emergentes no processamento de suco de laranja
adicionado de fruto-oligossacarídeos e suco prebiótico de laranja produzido via síntese
enzimática. / Francisca Diva Lima Almeida. – 2015.
108 f. : il. color.
Tese (Doutorado) – Universidade Federal do Ceará, Centro de Ciências Agrárias,
Programa de Pós-Graduação em Biotecnologia (Rede Nordeste de Biotecnologia),
Fortaleza, 2015.
Área de Concentração: Biotecnologia Industrial.
Orientação: Profa. Dra. Sueli Rodrigues.
1. Suco de laranja. 2. Oligossacarídeos. 3. Bioteconologia. I. Título.
CDD 660.6
Tese submetida à Coordenação do Curso de
Pós-graduação em Biotecnologia - Renorbio, da
Universidade Federal do Ceará, como requisito
parcial para obtenção do grau de Doutor em
Biotecnologia.
Aprovada em: 30/10/2015
BANCA EXAMINADORA
______________________________
Profa. Dr. Sueli Rodrigues (Orientadora)
Universidade Federal do Ceará (UFC)
______________________________
Profa. Dr. Ana Lúcia Fernandes Pereira
Universidade Federal do Maranhão (UFMA) - Membro
______________________________
Profa. Dr. Luciana Rocha Barros Gonçalves
Universidade Federal do Ceará (UFC) - Membro
______________________________
Dr. Edy Sousa de Brito
Empresa Brasileira de Pesquisa Agropecuária (EMBRAPA)
______________________________
Dr. Elenilson de Godoy Alves Filho
Universidade Federal do Ceará (UFC) - Membro
Dedico este trabalho,
ao meu anjinho particular, Heitor (in memoriam),
que intercede por mim junto a Deus.
AGRADECIMENTOS
Primeiramente a Deus por sempre ter me dado forças para continuar nessa
caminhada. Por me conduzir sempre nos melhores caminhos, por colocar pessoas tão
bondosas e cheias de luz em meu caminho.
Aos meus pais, José Eudes de Almeida e Maria de Fátima Lima Almeida, por
todo amor e carinho de sempre. Por confiarem e acreditarem nas minhas escolhas. Por
sempre me darem forças, pelas inúmeras vezes que me ligaram perguntando: “Como
está o doutorado? E o artigo?”. Pelas orações da minha mãe, por todo cuidado e zelo.
Amo vocês e, é por vocês todo o meu esforço.
À minha orientadora, Profa. Dra. Sueli Rodrigues, que me acolheu de braços
abertos desde o mestrado. Obrigada pelos inúmeros ensinamentos, por todas as
oportunidades, por estar sempre à disposição, por acreditar em mim, por cada ajuda e
puxão de orelha. Obrigada por ser esse exemplo de profissional.
Ao Leonardo Primo, presente de Deus na minha vida. Obrigada por toda
paciência durante esses anos que me acompanhou durante o doutorado. Obrigada por
toda ajuda, toda torcida, por todo amor, por me fazer confiar que no final tudo daria
certo. Obrigada pelo companheirismo, mesmo durante o período em que estávamos
afastados fisicamente.
À Rosane, minha dupla inseparável do doutorado e da vida. Obrigada pelos
agradáveis momentos que compartilhamos juntas, sorrimos juntas, choramos juntas.
Ao meu querido “Sexteto” do coração: Ana Lúcia, Virgínia Kelly, Gleison, Adalva
e Erbênia. Pelas conversas e alegrias compartilhadas, pela amizade sincera, pelos
conselhos e por tornar meus dias mais alegres.
Ao Dublin Institute of Technology (DIT), nas pessoas do Jesus Frias, Patrick
Cullen (PJ) e Paula Bourke, por terem aceitado a parceria e terem me acolhido e
orientado tão bem durante o período de doutorado sanduiche em Dublin-Irlanda.
Aos amigos queridos e companheiros de laboratório e sala do DIT: Richam,
Lubna, Sonal, Lu, Carmem, Shashi, NN, por toda ajuda, ensinamentos, momentos de
alegrias, por me acolherem tão bem na “Ilha de Esmeralda”.
Aos amigos do LABIOTEC: Niédila, Cristiane, Cláudia, Jonas, Elenylson,
Imilena, Soraya, Simone, Mariana, Nair, Lívia, às Thatis Nunes, Vidal e Cavalcante, por
todo carinho, em especial ao Wesley, por todo carinho e amizade e, também, por toda
ajuda na finalização deste trabalho.
Às minhas queridas irmãs, Virgínia, Clarissa e Cristiane por todo carinho e
força, por sempre torcerem por mim.
Aos meus queridos “Bioamigos”, em especial, Karine Pires, André Leandro,
Milena Esmeraldo, Eduardo Neves, Luís Neto, Anderson Weyne, Camila Salviano,
pelos momentos prazerosos que compartilhamos durante o período de doutorado.
Aos doutores Edy Brito e Elenilson Godoy e às professoras doutoras Ana Lúcia
Fernandes e Luciana Gonçalves por, gentilmente, aceitarem o convite para participar da
banca examinadora.
Aos meus tios Regina e Alfredo por me acolherem durante quase todo o
período do doutorado. Obrigada por todo carinho.
À CAPES peça concessão da bolsa de estudos e ao CNPQ pelo financiamento
do meu projeto de pesquisa.
Muito obrigada a todos, de coração!
RESUMO
ALMEIDA, Francisca Diva Lima Almeida. Emprego de tecnologias emergentes no
processamento de suco de laranja adicionado de fruto-oligossacarídeos e suco
prebiótico de laranja produzido via síntese enzimática. 2015. Tese – Programa de
Pós-graduação em Biotecnologia - Renorbio. Universidade Federal do Ceará, Fortaleza.
O objetivo desta pesquisa foi empregar tecnologias emergentes no processamento de
suco prebiótico de laranja adicionado de fruto-oligossacarídeos (FOS) e em suco
prebiótico de laranja produzido via síntese enzimática. A primeira etapa da pesquisa
consistiu em avaliar o efeito da aplicação das tecnologias de plasma e de alta pressão,
como métodos de conservação, em suco de laranja adicionado de 7% de FOS
comercial. O suco foi exposto direta e indiretamente ao processamento por plasma em
diferentes tempos: 15 30, 45 e 60 s. Para o processamento com alta pressão, o suco foi
tratado a uma pressão de 450 bars por 5 minutos. Após os tratamentos, a concentração
de fruto-oligossacarídeos foi quantificada pela técnica de cromatografia em camada
delgada (CCD), utilizando o equipamento densitômetro. Determinações de cor, pH e
concentração de ácidos orgânicos foram também realizadas. Ambos os processos não
degradaram os FOS presentes no suco. Ácidos orgânicos e a cor das amostras tratadas
também foram preservados. Na segunda etapa da pesquisa, foi avaliado o efeito da
aplicação dos tratamentos de plasma e ozônio em suco prebiótico de laranja produzido
via síntese enzimática. O suco foi exposto direta e indiretamente ao processamento por
plasma, a 70 kV, em diferentes tempos: 15 30, 45 e 60 s. Para o processamento com
ozônio, diferentes cargas (0,057, 0,128 e 0,230 mg/ O3.mL de suco) foram avaliadas.
Após os tratamentos, a concentração de oligossacarídeos foi determinada pela técnica
de HPLC. Os valores de pH, cor, conteúdo de fenólicos totais e atividade antioxidante
total também foram determinados. Ambos os processos promoveram uma degradação
parcial dos oligossacarídeos no suco. Contudo, o suco manteve uma quantidade
suficiente de oligossacarídeos para ser classificado como um alimento prebiótico. Os
demais parâmetros analisados foram preservados. Diante disso, sugere-se que os
tratamentos de plasma, alta pressão e ozônio são alternativas não térmicas adequadas
para o tratamento de suco de laranja prebiótico.
Palavras-chave:
Tecnologia
de
plasma.
Alta
Oligossacarídeos. Fenólicos. Capacidade antioxidante
pressão.
Ozônio.
Laranja.
ABSTRACT
ALMEIDA, Francisca Diva Lima Almeida. Emprego de tecnologias emergentes no
processamento de suco de laranja adicionado de fruto-oligossacarídeos e suco
prebiótico de laranja produzido via síntese enzimática. 2015. Tese – Programa de
Pós-graduação em Biotecnologia - Renorbio. Universidade Federal do Ceará, Fortaleza.
The aim of this research was to use emerging technologies on the processing of the
prebiotic orange juice added of fructo-oligosaccharides (FOS) and in prebiotic orange
juice produced by enzymatic synthesis. The first stage of the study was evaluated the
effect of atmospheric pressure cold plasma (ACP) and high pressure processing (HPP)
on the prebiotic orange juice added 7% commercial FOS. The orange juice was directly
and indirectly exposed to plasma discharge at 70 kV with processing times of 15, 30, 45
and 60 seconds. For high pressure processing, the juice containing the same
concentration of FOS was treated at 450 bars for 5 minutes. After the treatments, the
fructo-oligosaccharides were qualified and quantified by Thin Layer Chromatography
(TLC), using densitometer. The organic acids, color analysis and pH values were also
evaluated. Both processes did not degrade the FOS. The organic acids and the color of
the treated samples were also preserved. On the second stage of the study, the effect of
plasma and ozone treatments on prebiotic orange juice produced by enzymatic
synthesis were evaluated. The orange juice was directly and indirectly exposed to
plasma discharge at 70 kV with processing times of 15, 30, 45 and 60 seconds. For
ozone processing, different loads (0.057, 0.128 and 0.230 mg/ O 3.mL of juice) were
evaluated. After the treatments, the oligosaccharides were quantified by HPLC. The
juice pH, color, total phenolic content and total antioxidant activity were also determined.
Both processes promoted a partial degradation of the oligosaccharides in the juice.
However, the juice maintained an enough amount of oligosaccharides to be classified as
a prebiotic food. The other parameters analyzed were preserved. Thus, atmospheric
cold plasma and ozone are suitable non-thermal alternatives for prebiotic orange juice
treatment.
Keywords: Emerging technologies. Prebiotic. Orange. Oligosaccharides. Phenolics.
Antioxidant capacity
LISTA DE FIGURAS
CAPÍTULO 1
Figura 1 - Representação da reação do aceptor .......................................................... 27
Figura 2 - Esquema de montagem experimental para sistema de plasma frio. ............ 36
Figura 3 - Sistema esquemático de tratamento de ozônio. .......................................... 38
CAPÍTULO 2
Figure 1 - Degree of polymerization of the fructo-oligosaccharides in water (A) and
orange juice (B) after direct exposure of plasma treatment ........................................... 59
Figure 2 - Relative concentration from fructo-oligosaccharides in water (a) and in
orange juice (b) after direct plasma exposure (in).. ....................................................... 60
Figure 3 - Relative concentration from fructo-oligosaccharides in water (a) and in
orange juice (b) after indirect plasma exposure (out).. .................................................. 61
Figure 4 - Relative concentration from fructo-oligosaccharides in water and orange
juice after high pressure processing .............................................................................. 63
Figure 5 - Concentration (g/L and g/100mL) of organic acids in orange juice after
high pressure processing .............................................................................................. 68
Figure 6 - Concentration (g/L and g/100mL) of organic acids in orange juice after
direct and indirect plasma exposure... ........................................................................... 70
CAPÍTULO 3
Figure 1 – Experimental setup for plasma treatment. ................................................... 81
Figure 2 – Experimental setup for of ozone treatment .................................................. 82
Figure 3 – Degree of polymerization of the oligosaccharides in prebiotic orange juice
before (control) and after ozone treatment. ................................................................... 90
Figure 4 – Oligosaccharides quantification after direct exposure (A) and indirect
exposure (B) of the plasma treatment. The control sample (without plasma treatment)
corresponds to the time zero.. ....................................................................................... 92
Figure 5 – Oligosaccharides quantification after ozone processing. The control
sample (without ozone treatment) corresponds to the load zero ................................... 93
Figure 6 – Sugars concentration after plasma direct exposure (A) and indirect
exposure (B) and after ozone treatment ........................................................................ 95
Figure 7 – Total phenolic content after plasma (A) and ozone treatment (B) ............... 99
LISTA DE TABELAS
CAPÍTULO 2
Table 1 - Color parameters of the orange juice after direct exposure by plasma .......... 64
Table 2 - Color parameters of the orange juice after indirect exposure by plasma.. ..... 65
Table 3 - Color parameters of the orange juice treated by HPP.................................... 66
Table 4 - pH values in prebiotic orange juice after high pressure processing ............... 67
Table 5 - pH values in prebiotic orange juice after plasma treatment. .......................... 67
CAPÍTULO 3
Table 1 – pH values in prebiotic orange juice after ozone processing. ......................... 88
Table 2 – pH values in prebiotic orange juice after plasma treatment ........................... 89
Table 3 – Oligosaccharide concentration and loss after non-thermal treatment ........... 94
Table 4 – Color parameters of the prebiotic orange juice after ozone processing.. ...... 96
Table 5 – Color parameters of the prebiotic orange juice after plasma treatment ......... 97
Table 6 – ABTS antioxidant activity in prebiotic orange juice after plasma treatment . 101
Table 7 – ABTS Antioxidant activity in prebiotic orange juice after ozone treatment .. 102
LISTA DE ABREVIATURAS E SIGLAS
ABTS
2,2 – azinobis (ácido 3-etilbenzotiazolina- 6 – sulfônico)
DPPH
2,2 – difenil1 – picrilhidrazil
FOS
Fruto-oligossacarídeos
TLC
Thin Layer Chromatography
HPLC
High Performance Liquid Chromatography
DP
Degree of Polymerization
ACP
Atmospheric Cold Plasma
DBD
Descarga de barreira dielétrica
LAB
Lactic acid bacteria
GS
Glucansucrases
FS
Fructansucrases
HMF
5- Hidroximetil-2-furaldeído
SUMÁRIO
INTRODUÇÃO .............................................................................................................. 17
CAPÍTULO 1: REVISÃO BIBLIOGRÁFICA ................................................................. 20
1.1 Alimentos funcionais: uma visão geral .................................................................... 20
1.2 Oligossacarídeos prebióticos .................................................................................. 22
1.2.1 Oligossacarídeos prebióticos produzidos via síntese enzimática ......................... 25
1.3 Sucos de frutas como veículo de ingredientes funcionais. ...................................... 27
1.3.1 Suco de laranja como um alimento funcional ....................................................... 28
1.4 Métodos de processamentos de alimentos: convencionais X tecnologias
emergentes ................................................................................................................... 31
1.4.1 Tecnologia de plasma na conservação de alimentos ........................................... 34
1.4.2 Emprego do ozônio .............................................................................................. 37
1.4.3 Altas Pressões ..................................................................................................... 39
REFERÊNCIAS ............................................................................................................. 41
CHAPTER 2 - EFFECT OF ATMOSPHERIC COLD PLASMA AND HIGH PRESSURE
PROCESSING ON FRUCTO-OLIGOSACCHARIDES, ORGANIC ACIDS AND
ORANGE JUICE COLOR ............................................................................................. 50
2.1 Introduction.............................................................................................................. 51
2.2 Materials and Methods ............................................................................................ 53
2.2.1 Prebiotic orange juice preparation ........................................................................ 53
2.2.2 Plasma Treatment ................................................................................................ 53
2.2.3 High pressure processing ..................................................................................... 54
2.2.4 Carbohydrate analysis .......................................................................................... 55
2.2.4.1 Fructo-oligosaccharides degree of polymerization characterization .................. 55
2.2.5 Color measurement .............................................................................................. 56
2.2.6 Organic acid quantification ................................................................................... 57
2.2.7 Statistical analysis ................................................................................................ 57
2.3. Results and Discussion .......................................................................................... 58
2.3.1 Fructo-oligosaccharides quantification by TLC ..................................................... 58
2.3.2 Color parameters .................................................................................................. 64
2.3.3 Effect of ACP and HPP on the juice pH ................................................................ 67
2.3.4 Organic acids quantification by HPLC .................................................................. 68
2.4. Conclusions ............................................................................................................ 71
REFERENCES .............................................................................................................. 72
CAPÍTULO 3: EFFECTS OF ATMOSPHERIC COLD PLASMA AND OZONE ON
PREBIOTIC ORANGE JUICE....................................................................................... 75
3.1. Introduction............................................................................................................. 76
3.2. Materials and methods ........................................................................................... 78
3.2.1 Orange juice ......................................................................................................... 78
3.2.2 Dextransucrase production and enzyme activity determination ............................ 78
3.2.3 Prebiotic oligosaccharides synthesis in orange juice ............................................ 79
3.2.4 Plasma treatment on prebiotic orange juice ......................................................... 79
3.2.5 Ozone treatment on prebiotic orange juice ........................................................... 81
3.2.6 Carbohydrate analysis .......................................................................................... 82
3.2.6.1 Oligosaccharides degree of polymerization ....................................................... 82
3.2.6.2 Sugars and oligosaccharides quantification ...................................................... 83
3.2.7 Color analysis ....................................................................................................... 84
3.2.8 Total phenolic content .......................................................................................... 85
3.2.9 Antioxidant activity determination ......................................................................... 85
3.2.9.1 DPPH method ................................................................................................... 86
3.2.9.2 ABTS method .................................................................................................... 86
3.2.10 Statistical analysis .............................................................................................. 87
3.3 Results and discussions .......................................................................................... 87
3.3.1 Orange juice characterization ............................................................................... 87
3.3.2 Effect of plasma and ozone on the juice pH ......................................................... 88
3.3.3 Oligosaccharides characterization after plasma and ozone treatments................ 89
3.3.4 Oligosaccharides quantification by HPLC after plasma and ozone treatments. ... 91
3.3.5 Color analysis ....................................................................................................... 96
3.3.6 Total phenolic content .......................................................................................... 99
3.3.7 Total antioxidant activity ..................................................................................... 100
3.4. Conclusion............................................................................................................ 103
CONSIDERAÇÕES FINAIS ........................................................................................ 104
REFERENCES ............................................................................................................ 105
Introdução 17
INTRODUÇÃO
Um número crescente de consumidores está, cada vez mais, consciente da
importância dos alimentos funcionais devido aos benefícios adicionais de saúde que
estes oferecem. Diante disso, os alimentos funcionais, sendo uma das principais
categorias de alimentos do mercado de saúde e bem-estar global, estão se tornando
um grande foco de desenvolvimento de novos produtos na indústria de alimentos.
Dentro da categoria de alimentos funcionais, os prebióticos vêm ganhando um
destaque especial. Eles são ingredientes alimentares seletivamente fermentados que
permitem mudanças específicas, tanto na composição quanto na atividade da
microbiota gastrintestinal, conferindo benefícios de saúde e bem-estar ao hospedeiro.
Atualmente, a grande maioria dos alimentos contendo oligossacarídeos são
produtos à base de leite. Contudo, a produção de alimentos prebióticos não lácteos é
uma opção interessante e bastante promissora para a produção desse tipo de alimento
a baixo custo e em escala industrial.
Nesse contexto, sucos de frutas são considerados substratos adequados para
veicular esses ingredientes funcionais, uma vez que são importantes fontes de
compostos bioativos, como fenólicos, vitamina C e carotenóides. No entanto, as
tecnologias utilizadas para seu processamento e armazenamento podem causar
alterações no conteúdo nutritivo dos mesmos. Tratamentos térmicos são os métodos
comumente aplicados para prolongar a vida útil dos alimentos líquidos através da
inativação de micro-organismos e enzimas. Contudo, o calor transferido ao produto
pode provocar alterações bioquímicas e nutritivas irreversíveis, que afetam a qualidade
do suco e, muitas vezes, a aceitação do produto.
Introdução 18
Dessa forma, o desenvolvimento de alimentos funcionais vem estimulando a
inovação e o desenvolvimento de novos produtos na indústria de alimentos. Entretanto
a estabilização microbiológica do mesmo sem o uso de conservantes e processamento
térmico representa um desafio. Os desenvolvimentos na área de processamento de
alimentos são, geralmente, conduzidos de forma a combinar a inovação tecnológica
com os hábitos sociais e culturais, a fim de produzir alimentos que satisfaçam as
necessidades nutricionais, pessoais e sociais de todas as comunidades.
Diante disso, a tecnologia de plasma representa uma alternativa potencial aos
métodos tradicionais de processamento não térmico de alimentos. Enquadra-se, ainda,
entre as tecnologias avançadas não térmicas como uma abordagem alternativa para a
eliminação de micro-organismos deteriorantes e patógenos. Este processo apresenta
numerosas vantagens sobre os métodos convencionais, como: processo operacional de
baixo custo, tempo de tratamento curto a baixas temperaturas, natureza não tóxica e
aplicação para uma grande variedade de produtos (Ziuzina et al. 2013).
A aplicação da tecnologia de alta pressão (HPP), em alimentos, resulta em
produtos com qualidade sensorial superior quando comparados com produtos
submetidos a processamento térmico (Patras et al. 2009). O processamento utilizando
altas pressões é capaz, ainda, de ativar e inibir micro-organismos, ativar e inativar
enzimas a baixas temperaturas, enquanto que os compostos de baixo peso molecular,
tais como vitaminas e compostos relacionado com a pigmentação e aroma,
permanecem inalterados (Carnonell-Capella et al. 2013).
O uso do ozônio, por sua vez, tem se destacado como um método alternativo
aos processos térmicos, e é empregado na conservação de sucos de frutas e outros
Introdução 19
alimentos, com o intuito de atender a demanda dos consumidores por produtos prontos
para o consumo, que sejam frescos e seguros. O ozônio é um alótropo triatômico de
oxigênio e é caracterizado por um elevado potencial de oxidação, transmitindo
propriedades bactericidas e viricidas. O ozônio inativa os micro-organismos através de
oxidação e o residual de ozônio decompõe-se a produtos não tóxicos (isto é, oxigênio),
tornando-o um agente antimicrobiano ambientalmente amigável para utilização na
indústria de alimentos (Patil et al. 2009b).
Diante disso, o presente trabalho de tese teve por objetivo a aplicação, como
método de conservação, das tecnologias emergentes de plasma e alta pressão no
processamento não térmico de suco de laranja adicionado de fruto-oligossacarídeos
(FOS) e aplicação de plasma e ozônio em suco prebiótico de laranja produzido via
síntese enzimática. Após a aplicação dessas tecnologias, os sucos foram analisados
para avaliar o efeito da aplicação dessas tecnologias sob os compostos nutritivos e
seus ingredientes funcionais.
Capítulo 1 20
CAPÍTULO 1: REVISÃO BIBLIOGRÁFICA
1.1 Alimentos funcionais: uma visão geral
Os alimentos funcionais, sendo uma das principais categorias de alimentos do
mercado de saúde e bem-estar global, estão se tornando um grande foco de
desenvolvimento de novos produtos na indústria de alimentos (Khan et al. 2013). Esta
por sua vez, tem um papel central no sentido de facilitar as práticas alimentares mais
saudáveis através da prestação e promoção de alimentos que sejam saborosos,
visualmente atrativos e saudáveis (Nagpal, Kumar, and Kumar 2012; Saad et al. 2013).
Nesse contexto, grandes avanços vêm ocorrendo no desenvolvimento dos alimentos
funcionais.
Alimentos funcionais são definidos como alimentos que afetam beneficamente a
saúde de quem os consomem, além dos efeitos nutricionais adequados. Essa classe de
alimentos é similar aos alimentos convencionais com relação à aparência, contudo eles
vão além do papel tradicional de alimentos como provedor de nutrição. Eles fornecem
função específica, proporcionando efeitos adicionais de saúde sobre o consumo
sustentável (Lamsal 2012; Silva, Rabelo e Rodrigues 2012). Ingredientes alimentares
funcionais estão inclusos, principalmente, os probióticos, prebióticos, vitaminas e
minerais e são comumente aplicados em leites fermentados, iogurtes, bebidas
esportivas, alimentos para bebês, etc (Lamsal 2012).
Uma das estratégias para a produção de alimentos funcionais é a adição de
um composto bioativo em um alimento convencional, levando-se em conta diferentes
Capítulo 1 21
variáveis, tais como: a interação desse composto com a matriz alimentar, a estabilidade
ao processamento e a sua biodisponibilidade (Amigo-Benavent et al. 2013). A principal
motivação dos consumidores para a compra de alimentos funcionais é o crescente
desejo em prevenir doenças crônicas, como doença cardiovascular, Alzheimer e
osteoporose, ou para aperfeiçoar a saúde, por exemplo, aumentando a energia,
estimulando o sistema imunológico e geração de bem-estar (Bigliardi and Galati 2013;
Khan et al. 2013; Nobre, Suvarov e De Weireld 2014). No desenvolvimento de
alimentos funcionais é necessário compreender os impactos sensoriais de seus
componentes e determinar como a sua adição aos produtos pode influenciar a
preferência e aceitabilidade dos consumidores em termos de aparência, aroma, sabor e
textura, a fim do desenvolvimento e formulação direta dos produtos (Pimentel, Madrona
e Prudencio 2014).
Atualmente, existe um grande interesse na melhoria da saúde e função
intestinal, uma vez que a microbiota intestinal desempenha um papel fundamental na
saúde humana. Qualquer estresse sobre a microbiota intestinal devido a fatores bióticos
e abióticos pode levar a distúrbios microbiológicos resultando em um aumento do risco
de doenças (Sangwan et al. 2014). Esta melhoria da microbiota intestinal é comumente
conseguida pelo consumo de alimentos probióticos, os quais são suplementos
alimentares que contém micro-organismos vivos. Contudo, uma alternativa bastante
eficaz e que vêm ganhando destaque nos últimos tempos, é o consumo de ingredientes
alimentares denominados prebióticos (Tako et al. 2014).
Capítulo 1 22
1.2 Oligossacarídeos prebióticos
O termo “Prebiótico” foi definido pela primeira vez em 1995 por Gibson e
Roberfroid, como “ingredientes alimentares não digeríveis que afetam beneficamente o
hospedeiro por estimularem seletivamente o crescimento e/ou atividade de um ou de
um número limitado de espécies bacterianas já residentes no cólon” (Gibson and
Roberfroid, 1995). No entanto, a literatura recente não restringe o cólon como único
local de ação e, o termo prebiótico é atualmente definido como “um ingrediente
alimentar seletivamente fermentado que permite mudanças específicas, tanto na
composição quanto na atividade da microbiota gastrintestinal, conferindo benefícios de
saúde e bem-estar ao hospedeiro” (Figueroa-González et al. 2011; Roberfroid, 2010;
Saad et al. 2013).
Com exceção da inulina, que é uma mistura de fruto-oligossacarídeos e
polissacarídeos, os prebióticos são misturas de oligossacarídeos não digeríveis, que
consistem de 3-10 monômeros de carboidratos (Saad et al. 2013). Dentre estes, podem
ser citados os fruto-oligossacarídeos, glico-oligossacarídeos, isomalto-oligossacarídeos
(IMOS), galacto-oligossacarídeos, oligossacarídeos de soja, etc (Patel e Goyal 2010).
Estes oligossacarídeos são encontrados em diferentes fontes alimentares, tais como:
leite, mel, cana de açúcar, soja, alho, beterraba (Mussatto e Mancilha 2007).
Atualmente, existe um grande interesse no uso de prebióticos como
ingredientes de alimentos funcionais, pois eles têm a capacidade de modular a
composição da microbiota intestinal, uma vez que o consumo regular dos prebióticos
estimula, de forma seletiva, o crescimento e atividade das bactérias probióticas, as
Capítulo 1 23
quais atuam na melhoria da saúde, além de minimizar o crescimento de bactérias
patogênicas (Srinivasjois, Rao e Patole 2013).
As mudanças entre as colônias de espécies microbianas no intestino humano
produz uma ampla gama de efeitos positivos, incluindo o aumento da saciedade e da
absorção de cálcio da dieta, regulação da motilidade intestinal, produção de ácidos
graxos de cadeia curta, prevenção de diarreia e constipação (Johnson et al. 2013;
Pimentel et al. 2014). Além disso, o consumo regular de prebióticos alivia os sintomas
de constipação, estimula o sistema imunológico (Lee and Mazmanian 2010), pode
diminuir o risco de câncer de cólon e os fatores de risco associados à obesidade e
síndrome metabólica (Johnson et al. 2013).
Contudo, para que os prebióticos possam atuar como ingredientes funcionais,
eles devem ser quimicamente estáveis às etapas de processamento de alimentos,
como calor, baixo pH e condições da reação de Maillard (Huebner et al. 2008). Esses
ingredientes devem, ainda, apresentar alguns critérios importantes: serem resistentes à
ação de enzimas salivares e intestinais e estimular o crescimento de bactérias
benéficas no intestino (Lamsal 2012).
A ingestão excessiva de ingredientes prebióticos pode causar desconforto
intestinal (Yang et al. 2011). Diante disso, a Agência Nacional de Vigilância Sanitária
(ANVISA) define o valor de referência dietética para fibras alimentares e frutooligossacarídeos (FOS) de no mínimo 3 g/dia de fibras ou FOS se o alimento for sólido
ou 1,5 g se o alimento for líquido, para que o mesmo apresente alegações com
propriedades funcionais, de acordo com o item 3.3 da Resolução nº 18/1999. É
recomendada, ainda, uma ingestão máxima de 30 g/ dia de fibras ou FOS (BRASIL,
Capítulo 1 24
2014). O consume de 200 mL de suco prebiótico de laranja corresponde a uma
ingestão de 14 g de FOS.
A European Food Standard Agency define os valores de referência para fibra
dietética a 25 g por dia para adultos de 18 anos de idade ou mais, para sustentar a
função normal do intestino (European Food Safety Authority, 2010). No entanto, não
existem recomendações oficiais com relação ao consumo de prebióticos. Enquanto
isso, muitos investigadores têm oferecido algumas sugestões: 10 g por dia de FOS
(Bouhnik et al. 1999) e 7 g por dia de galato-oligosacarídeos (Silk et al. 2009).
Diversos estudos envolvendo a adição de ingredientes prebióticos em alimentos
têm relatado efeitos positivos, uma vez que esses ingredientes apresentam a
capacidade de favorecer o crescimento de micro-organismos probióticos (Tako et al.
2014), bem como melhorias nos atributos físico-químicos, reológicos e sensoriais dos
produtos (Cruz et al. 2013; Liu et al. 2014; Freitas, Amancio e Morais 2012; Tako et al.
2014).
Os efeitos positivos das diversas pesquisas envolvendo a aplicação de
ingredientes prebióticos e os efeitos benéficos causados pelos mesmos, têm
despertado o interesse por parte de muitos grupos de pesquisas e, até mesmo, da
indústria alimentícia, de realizar novos estudos nessa área com o intuito de buscar o
desenvolvimento de produtos e expandir a oferta de alimentos com propriedades
funcionais.
Capítulo 1 25
1.2.1 Oligossacarídeos prebióticos produzidos via síntese enzimática
Nas últimas décadas, muitos estudos têm investigado a produção de prebióticos
como ingredientes alimentares capazes de promover um estado de bem-estar e saúde
aos consumidores. A maioria dos ingredientes prebióticos identificados atualmente são
obtidos por extração direta a partir de fontes naturais, ou produzidos por meio de
processos químicos que hidrolisam polissacarídeos, ou ainda, pela síntese química
através de reações de transglicosilação de carboidratos de baixo grau de polimerização
e síntese enzimática de dissacarídeos (Charalampopoulos e Rastall 2012; FigueroaGonzález et al. 2011; Saad et al. 2013).
Diversos trabalhos têm demonstrado a obtenção de ingredientes prebióticos por
diferentes vias. Sangwan et al. (2014) estudaram a produção de galactooligossacarídeos (GOS) a partir da síntese de β-galactosidase, produzida pelo
Streptococcus thermophilus. Silva et al. (2013b) investigaram a produção de frutooligossacarídeos (FOS), em meio aquoso, a partir da atividade enzimática da inulase
imobilizada. Praznik et al. (2013) avaliaram a estrutura de fruto-oligossacarídeos obtidos
de folhas e caules da cultivar Agave tequilana. Silva, Rabelo and Rodrigues (2012)
produziram oligossacarídeos prebióticos por processo enzimático, através da adição da
enzima dextranasacarase em suco de caju clarificado.
Oligossacarídeos
prebióticos
podem
ser
produzidos
por
processo
de
fermentação, contudo, a síntese enzimática apresenta uma maior produtividade e fácil
controle de processo (Rabelo et al. 2006). Enzimas da família glicosiltransferases (EC
2.4.x.y) são comumente usadas como ferramentas biotecnológicas para a síntese de
Capítulo 1 26
oligossacarídeos. Elas são responsáveis em catalisar a transferência de unidades de
glicose, a partir de uma molécula doadora, para um receptor específico (Rabelo,
Fontes, and Rodrigues 2009a; Rodrigues, Lona, and Franco 2005, 2006; Saad et al.
2013).
Bactérias ácido-lácticas apresentam a capacidade de produzir uma ampla
variedade de um grupo particular de glicosiltransferases, as quais são utilizadas para a
produção de oligossacarídeos (Rabelo, Honorato, e Rodrigues 2009b). Lactobacillus
spp. e Leuconostoc spp. estão envolvidos na síntese de importantes produtos para a
saúde humana, devido suas propriedades prebióticas e atividade imunomoduladora
(Bivolarski et al. 2013). Quando a estirpe Leuconostoc mesenteroides NRRL B512 é
cultivado em meio contendo sacarose como fonte de carbono, uma fonte de nitrogênio e
minerais, ele fermenta os açúcares do meio produzindo a enzima dextranasacarase
(Rodrigues, Lona, and Franco 2003).
Dextranasacarase é uma enzima bacteriana extracelular que catalisa a
formação de dextrana quando o meio contém sacarose como único substrato. Contudo,
em um meio contendo um aceptor como substrato (frutose, glicose, maltose, etc.), além
da sacarose como segundo substrato, a enzima apresenta a capacidade de produzir
oligossacarídeos prebióticos. Esta reação é conhecida como reação do aceptor e a
proporção sacarose: aceptor está diretamente relacionado com o rendimento e grau de
polimerização da dextrana e oligossacarídeos formados (Rodrigues, Lona and Franco
2006).
A reação do aceptor acontece devido à adição de unidades de glicose (a partir
da quebra da sacarose) na molécula aceptora, ou seja, essas unidades de glicose são
Capítulo 1 27
desviadas da cadeia de dextrana para a formação do produto do aceptor (Rabelo et al.
2006). Como as unidades de glicose são incorporadas na cadeia de oligossacarídeos, o
grau de polimerização dos mesmos tende a aumentar. Graus de polimerização com até
10 unidades de glicose, com as mesmas sendo incorporadas por síntese enzimática,
através de ligações do tipo α-1,6, são conhecidos como oligossacarídeos prebióticos
(Silva, and Rodrigues 2012). A figura a seguir representa um esquema da reação do
aceptor:
Dextranasacarase
Sacarose
Dextrana + Frutose
Dextranasacarase
Sacarose + Aceptor
Oligossacarídeos prebióticos + Frutose
Figura 1 – Representação da reação do aceptor. Fonte: Silva (2013a)
1.3 Sucos de frutas como veículo de ingredientes funcionais.
Atualmente, a grande maioria dos alimentos contendo oligossacarídeos
prebióticos são produtos à base de leite (Cruz et al. 2013). Entretanto, alguns
consumidores evitam o consumo destes produtos devido a problemas de alergia ao
leite, intolerância a lactose ou vegetarianismos (Araújo et al. 2014). Dessa forma, a
produção de alimentos prebióticos em escala industrial vem enfrentando grandes
desafios. A produção de oligossacarídeos não lácteos é uma opção interessante e
Capítulo 1 28
bastante promissora para a produção de alimentos prebióticos a baixo custo e em
escala industrial (Figueroa-González et al. 2011).
Nesse contexto, sucos de frutas são considerados substratos adequados para
veicular esses ingredientes funcionais, uma vez que são alimentos ricos em nutrientes,
como vitaminas, fibras, minerais e antioxidantes. Além disso, eles possuem perfis de
sabor agradável e são alimentos consumidos com regularidade por todas as faixas
etárias, devido seu sabor refrescante e seu status de alimento saudável (Pereira 2013;
Pimentel et al. 2014). Vários trabalhos têm reportado o uso de suco de frutas como
substrato para a incorporação de ingredientes prebióticos.
Araújo et al. (2014) e Coelho (2013) desenvolveram um novo produto funcional
a partir da desidratação, em leito de jorro, de suco de acerola e limão, respectivamente
contendo oligossacarídeos prebióticos, produzidos a partir da reação de aceptor da
dextranasacarase.
Silva (2013a) utilizou diferentes tipos de sucos de frutas: cajá, jambo e siriguela
para sintetizar oligossacarídeos prebióticos como forma de elaboração de novos
produtos funcionais. Silva, Rabelo and Rodrigues (2012) produziram oligossacarídeos
prebióticos por processo enzimático, adicionando a enzima dextranasacarase em suco
de caju clarificado.
1.3.1 Suco de laranja como um alimento funcional
Em todo o mundo, cerca de 55 milhões de toneladas de laranjas doces são
produzidas, com destaque para o Brasil como maior produtor (Okino-Delgado e Fleuri
Capítulo 1 29
2014). O suco de laranja fresco é um dos sucos mais consumidos mundialmente, com
uma participação de 35% do mercado de bebidas de frutas e, isso é devido ao seu rico
conteúdo em vitamina C e valor nutritivo (Agcam, Akyıldız, e Evrendilek 2014; Ferreira
et al. 2013), além da perfeita combinação dos seus atributos sensoriais, tais como a cor,
sabor e aroma (Vervoort et al. 2011). Ele é uma importante fonte de compostos
bioativos, como ácidos orgânicos, compostos fenólicos, carotenoides, os quais têm
mostrado serem bons contribuidores para a capacidade antioxidante total (CarbonellCapella et al. 2013; Zulueta et al. 2013).
Devido à riqueza nutricional que o suco de laranja apresenta, ele tem sido
considerado um substrato promissor para a elaboração de novos produtos com
propriedades funcionais.
Diante disso, diversos estudos têm demonstrado a potencialidade do suco de
laranja e de seus subprodutos como veículo para a entrega de ingredientes funcionais.
Nesse contexto, Manderson et al. (2005) avaliaram as propriedades prebióticas de
oligossacarídeos extraídos da casca de subprodutos da fabricação de suco de laranja,
bem como estudaram uma forma mais eficaz em termos de custos para a produção
desses oligossacarídeos.
Fontes (2012) avaliou a produção de oligossacarídeos prebióticos em sucos de
frutas, incluindo, dentre estes, o suco de laranja, seguido da secagem desses produtos
pela técnica de spray-drying. De acordo com os resultados obtidos para o suco de
laranja, a autora concluiu que o mesmo apresentou-se como um excelente substrato
para a síntese de oligossacarídeos prebióticos com elevados graus de polimerização.
Capítulo 1 30
Com relação à aceitação sensorial dos sucos contendo ingredientes funcionais,
diferentes estudos têm sido realizados com essa finalidade e os resultados ainda
apresentam algumas contradições. Luckow e Delahunty (2004) estudaram o impacto
sensorial de ingredientes funcionais probióticos no sabor e aroma de suco de laranja.
Os resultados desse estudo mostraram que sucos de laranja contendo propriedades
funcionais são associados a um sabor não característico do suco. Os consumidores
preferiram as características sensoriais do suco de laranja convencional ao suco
funcional.
Por outro lado, Coelho (2009) realizou um estudo envolvendo a aceitação
sensorial de suco de laranja probiótico e adoçado. Os resultados mostraram uma
aceitação global de 84% dos consumidores. A autora relata que a adição de açúcar
pode ser uma estratégia utilizada para melhorar a aceitação do suco de laranja
fermentado. Contudo, a informação ao consumidor acerca dos benefícios que esses
alimentos promovem à saúde, leva a uma melhoria da aceitação do produto.
Apesar do seu baixo pH, a estabilidade do suco de laranja é bastante limitada
durante a estocagem, devido um importante número de reações de deterioração, como
por exemplo, a degradação do ácido ascórbico, deterioração microbiana, ação de
enzimas, desenvolvimento de off-flavour, alterações na cor, textura e aparência (Zulueta
et al. 2013).
Tratamentos térmicos são os métodos comumente aplicados para prolongar a
vida útil dos alimentos líquidos através da inativação de micro-organismos e enzimas.
Contudo, o calor transferido ao produto pode provocar alterações bioquímicas e
Capítulo 1 31
nutritivas irreversíveis, que afeta a qualidade do suco e, muitas vezes, a aceitação do
produto (Demirdöven and Baysal 2014; Vervoort et al. 2011).
1.4 Métodos de processamentos de alimentos: convencionais X tecnologias emergentes
Sob o ponto de vista tecnológico, os principais objetivos dos processos de
preservação e conservação consistem em prolongar a vida útil dos produtos
alimentícios, oferecendo aos consumidores produtos que não somente apresentem boa
qualidade nutritiva e sensorial, mas também, alimentos seguros, isentos de microorganismos e suas toxinas. A escolha do método de conservação adequado irá
depender da origem do alimento, seu estado físico (sólido, líquido, emulsionante), o
tempo de conservação necessário e o destino final do produto (Fellows 2006).
Dentre os inúmeros métodos de conservação disponíveis, o tratamento térmico
continua sendo um dos métodos mais importantes empregados no processamento e
conservação dos alimentos, principalmente pelos efeitos de conservação através da
inativação de enzimas e micro-organismos. Contudo, a aplicação do calor pode resultar
em alterações dos componentes dos alimentos, responsáveis por suas características
sensoriais, bem como em alterações do conteúdo nutritivos dos mesmos (Demirdöven e
Baysal 2014).
Em sucos de frutas, o tratamento térmico é utilizado na preservação e no
processo de fabricação dos mesmos. Porém, as reações de escurecimento não
enzimático, resultante do aquecimento, causam alteração de cor, perdas de açúcar e
vitamina C e formação de 5- Hidroximetil-2-furaldeído (HMF), afetando assim, a
Capítulo 1 32
qualidade dos sucos de frutas. No entanto, conseguir a estabilização microbiológica
desses alimentos sem o uso de conservantes e aplicação de um processamento
térmico ainda é um grande desafio (Damasceno et al. 2008).
Vários trabalhos têm avaliado os efeitos do tratamento térmico convencional na
qualidade de sucos de frutas e alguns autores comparam seus impactos com a de um
processamento não térmico. Nesse contexto, Vervoort et al. (2011) compararam o
impacto do processamento térmico, tecnologia de altas pressões e o processo de
campo elétrico pulsado nos parâmetros de qualidade químico e bioquímico do suco de
laranja. Com relação aos parâmetros de qualidade investigados (açúcares, ácidos
orgânicos,
vitamina
C,
furfural,
carotenoides
e
HMF),
não
houve
impacto
significativamente diferente das três técnicas utilizadas.
Cortés, Esteve e Frígola (2008) estudando o efeito da estocagem refrigerada no
conteúdo de ácido ascórbico de suco de laranja submetido aos tratamentos de
pasteurização térmica e campo elétrico pulsado, relataram que, em temperatura de
estocagem a 2 ºC, a qualidade nutricional do suco de laranja (ácido ascórbico) foi
mantida por mais tempo no suco tratado por campo elétrico pulsado (277 dias de vida
de prateleira), enquanto que no suco tratado por pasteurização térmica convencional a
vida de prateleira só chegou até 90 dias. Patil et al. (2009a) e Tarazona-Díaz e Aguayo
(2013) avaliaram o efeito da pasteurização térmica em sucos de frutas e concluíram que
esse processo ocasionou degradação da cor dos produtos e redução dos compostos
bioativos, resultando na redução da qualidade sensorial e nutricional do suco.
Com relação à estabilidade química dos prebióticos às condições de
processamento de alimentos, inúmeros estudos têm reportado sobre isso. Matusek et
Capítulo 1 33
al. (2009), Klewicki (2007) e Huebner et al. (2008) estudaram a estabilidade de
ingredientes funcionais durante a pasteurização a baixos valores de pH, e concluíram
que os FOS são altamente susceptíveis à hidrólise nas condições de pasteurização de
sucos de frutas, dentre outras bebidas. A quantidade hidrolisada desses ingredientes é
maior quanto menor for o pH e mais longo for o tempo de pasteurização.
Diante disso, os prebióticos serão funcionalmente estáveis se a sua atividade
prebiótica, antes e após as condições de processamento de alimentos, aumentar ou
permanecer inalterada (Huebner et al. 2008). Dessa forma, um cuidado especial deve
ser observado durante a aplicação de um processo para a conservação e aumento da
vida útil de um produto com propriedades funcionais, visto que, o tratamento e suas
condições podem afetar as características do produto em questão.
Atualmente, as exigências dos consumidores por alimentos de melhor
qualidade aliado às deficiências das tecnologias existentes, estão estimulando, cada
vez mais, o desenvolvimento de novas abordagens na realização de novos estudos
nessa área com o intuito de buscar o desenvolvimento de novas tecnologias e produtos
e, assim, expandir a oferta de alimentos com propriedades funcionais (Misra et al.
2014b). Portanto, tecnologias não térmicas têm sido reportadas como excelente opção
para a obtenção de produtos com bons atributos de qualidade, seguros e preservando
seu conteúdo nutricional (Zulueta et al. 2013).
Capítulo 1 34
1.4.1 Tecnologia de plasma na conservação de alimentos
Plasma é um gás ionizado que pode ser produzido a partir da aplicação de uma
descarga elétrica a um gás inicialmente neutro. O processo pode ocorrer à temperatura
ambiente e pressão atmosférica, com um fluxo brilhoso composto de espécies químicas
com características antimicrobianas e bastante reativas: fótons UV, partículas
carregadas, superóxido, radicais hidroxila, óxido nítrico e ozônio (Bermúdez-Aguirre et
al. 2013; Rød et al. 2012).
Nesse contexto, quando o plasma é produzido sob as condições acima citadas,
é frequentemente chamado de “descargas luminescentes à pressão atmosférica” ou
ainda, “plasma de atmosfera fria (Atmosferic Cold Plasma – ACP)” para enfatizar o fato
de que as temperaturas dos gases estão próximas à temperatura ambiente (30- 60 °C)
(Fernández and Thompson 2012). Contudo, dependendo dos níveis relativos de energia
dos elétrons e espécies constituintes, os plasmas são classificados em plasmas
térmicos e não térmicos (Jiang et al. 2014).
Plasmas frios, à pressão atmosférica, caracterizam-se pelo desequilíbrio de
temperatura entre elétrons e íons, que podem ser gerados através de várias técnicas
(Pankaj et al. 2014b). Descarga de barreira dielétrica (DBD) é um método que oferece
versatilidade no seu modo de operação e configurações do sistema. Em DBD, o plasma
é gerado entre dois eletrodos, com uma grande diferença de potencial entre eles, e são
separados por uma ou mais barreiras dielétricas (Misra et al. 2015; Pankaj et al. 2013).
Particularmente, a técnica de plasma por descarga de barreira dielétrica oferece uma
grande vantagem, pois permite o tratamento de produtos dentro de embalagens
Capítulo 1 35
seladas, o que elimina o risco de contaminação pós-processamento (Misra et al.
2014a).
O uso da tecnologia de plasma para a conservação de alimentos embalados
tem sido bem documentado (Pankaj et al. 2014a). Misra et al. (2014a) demonstraram,
recentemente, a aplicação de plasma frio na conservação de morangos embalados,
sem comprometer a qualidade dos mesmos. Pankaj et al. (2013), por sua vez,
avaliaram a cinética de inativação da enzima peroxidase na conservação de tomates
embalados.
A temperatura do plasma frio varia em torno de 30–60 ºC, e esta temperatura é
desejada pela indústria de alimentos, uma vez que baixa energia é requerida para a
formação do plasma, além de não afetar os compostos sensíveis a altas temperaturas
(Bermúdez-Aguirre et al. 2013).
Esta
tecnologia
apresenta
numerosas
vantagens
sobre
os
métodos
convencionais, uma vez que apresenta custos operacionais de processo reduzido,
utiliza tempo de tratamento reduzido a baixas temperaturas, apresenta natureza não
tóxica e é aplicado a uma grande variedade de produtos (Ziuzina et al. 2013).
A aplicação do tratamento por plasma envolve duas abordagens distintas
quanto ao tipo de exposição aos quais os produtos são submetidos, sendo estas
denominadas de exposição direta e indireta. Em tratamento direto, a amostra fica entre
os dois eletrodos, em contato direto com o plasma, enquanto que, por outro lado, na
exposição indireta, a amostra é colocada a uma distância adjacente da descarga de
plasma e só é exposta às espécies reativas formadas (Fernández and Thompson
2012). A figura 2 apresenta um esquema do sistema de plasma
Capítulo 1 36
Figura 2 – Esquema de montagem experimental para sistema de plasma frio.
Adaptação de Pankaj et al. (2013)
Diversos estudos têm reportado sobre a aplicação da tecnologia de plasma frio
na conservação de alimentos, por meio da inativação de micro-organismos e, estes
estudos têm mostrado resultados efetivos de inativação (Fernández et al. 2013; Misra et
al. 2015; Patil et al. 2014; Ziuzina et al. 2014). As condições de tratamento aplicadas no
presente estudo foram baseadas nesses trabalhos.
Contudo, até o presente momento, esta tecnologia vem sendo aplicada na
conservação de alimentos sólidos e em nível de superfície, não existindo estudos
voltados sobre a aplicação do plasma frio na conservação das características
nutricionais e de qualidade de alimentos líquidos com propriedades funcionais.
Capítulo 1 37
1.4.2 Emprego do ozônio
O uso do ozônio tem se destacado como um método alternativo aos processos
térmicos, e é empregado na conservação de sucos de frutas e outros alimentos, com o
intuito de atender a demanda dos consumidores por produtos prontos para o consumo,
que sejam frescos e seguros (Patil et al. 2009a).
O ozônio é um alótropo tri-atômico do oxigênio e é um agente antimicrobiano
capaz de inativar micro-organismos em geral, como fungos, vírus, protozoários, esporos
fúngicos e bactérias. Isso é possível graças ao potencial altamente oxidante que o
ozônio apresenta, resultando nessas propriedades bactericidas e viricidas que ele
transmite. Os resíduos de ozônio se decompõem em produtos não tóxicos, sendo
considerado, dessa forma, um agente antimicrobiano ambientalmente adequado para o
uso na indústria de alimentos (Patil et al. 2009b). As reações de oxidação são causadas
pelo ozônio molecular dissolvido ou pelas espécies de radicais livres formados durante
a decomposição do ozônio (Patil et al. 2010a). A figura 3 apresenta uma representação
do sistema de tratamento com ozônio.
Em 1997, a Food and Drug Administration (FDA) dos EUA, declarou o ozônio
como um ingrediente “Geralmente Reconhecido como Seguro” (GRAS) para o uso no
processamento de alimentos (Tiwari et al. 2008b). Subsequentemente, o ozônio ganhou
aprovação como um aditivo de uso direto em alimentos, o que têm provocado o
interesse na aplicação de ozônio, tanto por parte de pesquisadores acadêmicos, quanto
por parte da indústria processadora de alimentos (Tiwari et al. 2008a; Tiwari et al. 2009;
Torres et al. 2011).
Capítulo 1 38
Figura 3: Sistema esquemático de tratamento de ozônio. Fonte: Tiwari et al. (2008a)
Tradicionalmente, o processamento por ozônio dentro da indústria de alimentos
tem se concentrado em alimentos sólidos, pela aplicação de qualquer tratamento
gasoso, ou ainda, pela lavagem de frutas e vegetais com água ozonizada. No entanto,
com a aprovação do ozônio como um aditivo direto à alimentação, surgiu a
possibilidade de aplicação do mesmo em alimentos líquidos (Patil et al. 2010a).
Diante disso, trabalhos recentes têm reportado a utilização do ozônio como um
método de conservação de alimentos, por meio da inativação de micro-organismos e,
estes estudos têm demonstrado resultados efetivos (Patil et al. 2009a; Patil et al. 2009b;
Patil et al. 2010b; Patil et al. 2010c). Contudo, apesar dos resultados efetivos, poucos
trabalhos têm sido realizados abordando os parâmetros sensoriais e nutritivos dos
sucos tratados. Até o momento, não existem estudos voltados à aplicação do ozônio na
Capítulo 1 39
conservação de sucos de frutas com propriedades prebióticas. Portanto, um dos
objetivos do atual trabalho de tese foi avaliar os efeitos do tratamento de ozônio nas
características funcionais de suco de laranja, além de avaliar as características
sensoriais e de qualidade do produto tratado.
1.4.3 Altas Pressões
O processamento de alta pressão (HPP) pode ser uma ferramenta útil para a
obtenção de produtos alimentícios mais saudáveis e seguros (Carnonell-Capella et al.
2013). Diante disso, esta tecnologia foi adaptada às necessidades específicas da
indústria de alimentos e, atualmente, uma gama de produtos tratados por pressão,
incluindo sucos de frutas e vegetais, molho de abacate, cozidos de presunto embalado,
arroz cozido e carne de frango marinado, já foram introduzidos na União Europeia e no
mercado americano (Patras et al. 2009).
No que diz respeito à qualidade de ingestão do produto acabado, a aplicação
da tecnologia de altas pressões pode apresentar vantagens sobre os processamentos
térmicos. Isso ocorre porque as moléculas pequenas, tais como compostos voláteis de
aroma e pigmentos relacionados com a qualidade sensorial dos alimentos não são
afetados pelo processamento de alta pressão (Patras et al. 2009).
Com esse tipo de processamento não-térmico é possível, ainda, ativar e inibir
micro-organismos, ativar e inativar enzimas a baixas temperaturas, enquanto que os
compostos de baixo peso molecular, tais como vitaminas e compostos relacionado com
a pigmentação e aroma, permanecem inalterados. Em alimentos fluidos, a pressão é
Capítulo 1 40
transmitida uniformemente e instantaneamente, isto é, não há gradientes. Ao contrário
do que acontece com os processos que envolve calor, a HPP é independente do
tamanho e geometria do produto, o que reduz o tempo necessário para processar
grande quantidade de alimentos (Carnonell-Capella et al. 2013).
Capítulo 1 41
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Ziuzina, D., Patil, S., Cullen, P. J., Keener, K. M., and Bourke, P. 2014. “Atmospheric
Cold Plasma Inactivation of Escherichia Coli, Salmonella Enterica Serovar
Typhimurium and Listeria Monocytogenes Inoculated on Fresh Produce.” Food
microbiology 42:109–16. Retrieved November 27, 2014
Zulueta, A., Barba, F.J., Esteve, M.J., and Frígola, A. 2013. “Changes in Quality and
Nutritional Parameters During Refrigerated Storage of an Orange Juice–Milk
Beverage Treated by Equivalent Thermal and Non-Thermal Processes for Mild
Pasteurization.” Food and Bioprocess Technology 6(8):2018–30. Retrieved
December 15, 2014 (http://link.springer.com/10.1007/s11947-012-0858-x).
Capítulo 2 50
CHAPTER 2 - EFFECT OF ATMOSPHERIC COLD PLASMA AND HIGH PRESSURE
PROCESSING ON FRUCTO-OLIGOSACCHARIDES, ORGANIC ACIDS AND
ORANGE JUICE COLOR
Abstract
In this study, the effect of atmospheric pressure cold plasma (ACP) and high pressure
processing on the prebiotic orange juice processing were evaluated. Orange juice
containing 7% (w/v) of commercial fructo-oligosaccharides (FOS) was directly and
indirectly exposed to plasma discharge at 70 kV for 15, 30, 45 and 60 seconds. For high
pressure processing, the juice containing the same concentration of FOS was treated at
450 bars for 5 minutes. After the treatments, the fructo-oligosaccharides were qualified
and quantified by Thin Layer Chromatography (TLC). The organic acids, color and pH
values were also evaluated. Both processes did not degrade the FOS, making them
suitable for the processing of functional foods containing FOS. The organic acids and
the color of the treated samples were also well preserved. Therefore, ACP and high
pressure processing can be used as non-thermal alternatives for the production of
prebiotic orange juice.
Keywords:
Non-thermal technologies,
chromatography
orange
juice,
organic acids,
thin
layer
Capítulo 2 51
2.1 Introduction
A growing number of consumers are aware of the importance of functional foods
due to their health benefits. Prebiotic oligosaccharides are among the functional
compounds that added to foods grant to them the desired functionality. Prebiotics are
defined as “non-digestible food ingredients that beneficially affects host by selectively
stimulating the growth and the activity of one or a limited number of bacteria in the
colon” (Hernandez-Hernandez et al. 2012). The beneficial health effects attributed to the
intake of prebiotic have been previously reported as well (Hess et al. 2011; Wichienchot
et al. 2010; Vergara et al. 2010).
Prebiotic compounds have been used in several food products. Fruits and fruit
juices can be used as vehicles for these compounds. Renuka et al. (2009) studied the
fortification of selected fruit juice beverages with fructo-oligosaccharides (FOS), a low
caloric prebiotic.
According to ANVISA (National Health Surveillance Agency, Brazil), the
recommended daily intake (RDI) for foods containing prebiotic compounds should
provide at least 3 g of FOS in solid foods or 1.5 g in liquid foods. Besides, the FOS
consumption should not exceed 30 g per day in products ready for consumption. The
consumption of 200 mL of the prebiotic orange juice corresponds to the intake of 14 g of
FOS.
Thermal technologies are still the golden standard processing for fruit juices
preservation. However, thermal processing may cause changes in functional molecules,
vitamins and other nutrients (Vervoort et al. 2011). Thermal pasteurization of orange
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juice can cause off-flavors and the degradation of the product's quality due to nonenzymatic browning (Patil et al. 2009).
Nowadays, the consumers are looking for safe food products with high quality
retention. The preservation of the nutraceutical properties of functional foods, of nonthermal innovative technologies to produce foods with a minimum of nutritional,
physicochemical, and organoleptic changes and without degradation of the prebiotic
compounds (Esteve and Frígola 2007).
Among these technologies, atmospheric cold plasma (ACP) and high pressure
technologies have been reported as a good non-thermal food processing that
guarantees food preservation at safe standards maintaining, as much as possible, the
fresh-like characteristics of foods (Ramos et al. 2013). Studies involving the application
of atmospheric cold plasma and high pressure on fruits and vegetables reported a
positive effect on pathogens and enzyme inactivation (Misra et al. 2014; Ziuzina et al.
2013; Patterson et al. 2005).
However, there is no published data showing the advantages and limitations of
these technologies, when applied to fructo-oligosaccharides (FOS), the most studied
and used functional carbohydrate in food industry. The evaluation of a non-thermal
treatment for fruit juice processing containing FOS is important because a previous
study showed thermal degradation of FOS due to thermal treatment (Matusek et al.
2009; Klewicki 2007). Orange juice is largely consumed due to its vitamin C content.
In this context, this study evaluated the effect of atmospheric cold plasma (ACP)
and high pressure processing on the integrity of functional fructo-oligosaccharides in
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orange juice. The influence of ACP and HPP processing on the organic acid profile and
on the juice color was also evaluated.
2.2 Materials and Methods
2.2.1 Prebiotic orange juice preparation
Orange juice (Squeez©, Fruit Juices Ltd, Ireland) was purchased from a local
supermarket (Dunnes, Dublin - Ireland). Orange juice containing prebiotic ingredients
was prepared by adding of 7% (w/v) of commercial fructo-oligosaccharide (ORAFTI©
P95, Beneo GmbH, Mann, Germany) in a liter of the juice. The pH of the juice was
determined by direct measurement in a 420A pHmeter, (Orion research Inc, Beverly,
MA. US). The pHmeter was calibrated before use with buffer solutions of pH 4.0, 7.0 and
10.0.
2.2.2 Plasma Treatment
The plasma treatment was carried out using a plasma generator system (DBDACP), model: 6CP120/60-7.5 (Phenix Technologies). The system consists of a variable
high voltage transformer with an input voltage of 230 V at 50 Hz and a maximum high
voltage output of 70 kV at 50 Hz. The two 15-cm-diameter aluminum disc electrodes
were separated by a polypropylene container, which served as a sample holder and as a
dielectric barrier with wall thickness of 1.2 mm. The distance between the two electrodes
Capítulo 2 54
was 22 mm and equal to the height of the container. Voltage was monitored using an
InfiniVision 2000 X-Series Oscilloscope (Agilent Technologies Inc., Santa Clara, CA,
USA). All experiments were performed at 70 kV peak to peak at ambient air and
atmospheric pressure conditions.
A volume of 20 mL of orange juice samples containing 7% (w/v) of fructooligosaccharide was transferred to an open Petri dish, which was placed in a
polypropylene box, sealed with a polymeric film of 50 µm thickness (Cryovac BB3050).
This film served as an additional layer of dielectric barrier (Pankaj et al. 2014). Then,
samples were treated with atmospheric cold plasma (ACP) with processing times of 15,
30, 45 and 60 seconds and different exposure kinds: direct plasma field (in) and indirect
plasma field (out). These treatment times were selected based on a previous study
carried for pathogens inactivation in orange juice (Ziuzina et al. 2013). These authors
achieved a complete bacterial inactivation after 20 s of direct and 45 s of indirect plasma
treatment. Treated samples of prebiotic orange juice were stored at room temperature
(25 ºC) for 24 h before opening the container. A control sample, containing the same
concentration of FOS dissolved in water was prepared and packed and treated in the
same way that the prebiotic orange juice
2.2.3 High pressure processing
High pressure processing (HPP) was carried out using an industrial equipment
(Hiberbaric), model: 300. Prebiotic orange juice was packed at 250 mL polyethylene
bottles, which were placed in polyethylene plastic bags, and vacuum sealed for the high
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pressure processing. A control sample, containing the same concentration of FOS
dissolved in water was prepared and packed and treated in the same way that the
prebiotic orange juice. The processing was done at the industrial conditions: 5 minutes
at 450 bars as recommended by HPP Toolings Business (Dublin, Ireland) for fruit juices.
The juice processing in industrial equipment is a differential of this study because almost
all published studies on fruit juice processing by HPP are done in small equipment with
small volumes. After the treatment, quality analyses were carried out on the final
product.
2.2.4 Carbohydrate analysis
2.2.4.1 Fructo-oligosaccharides degree of polymerization characterization
Thin Layer Chromatography (TLC) analysis was used to qualify and quantify the
FOS. The prebiotic orange juice samples were diluted (1:2), filtered, using glass fiber
prefilters AP25 13 mm diameter (Merck Millipore Ltd.) and cleaned on a C-18 SPE
cartridge, followed by filtration, using a syringe (2.0 mL) and a support comprising a
membrane (HA membrane cellulose ester 0,45 uM, 13 mm of diameter). The samples
were analyzed by TLC, using a silica gel on TLC plates SIGMA-ALDRICH (20x20 cm, 60
Å medium pore diameter; product number: 99570-25EA). Samples of 1 μL were applied
on the plate at 1 cm from the bottom and at a separation distance of 1.0 cm from each
other. The plates were disposed into the TLC chamber pre-conditioned at room
temperature (25 ºC). The solvent system used to separate the carbohydrate mixture was
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composed of n-butanol/2-propanol/H2O (10:5:4 [vol/vol/vol]) mixture (Shiomi, Onodera
and Sakai 1997). The TLC plate was irrigated by the solvent systems three times. To
visualize the separated carbohydrates in the plates, a fine spray containing 1butanol/water (80% w/w) as solvent, phosphoric acid (6.78 mL), urea (3 g) and ethanol
(8 mL) in 100 mL was used. The plates were oven heated at 120 °C for 10 min. For the
quantification of the oligosaccharides, a TLC scanner CAMAG 4 20x20 cm densitometer
was used, using the Planar winCATS Chromatografy Manager software. The
wavelength used was 450 nm. The analyses were performed in triplicate.
2.2.5 Color measurement
The color of the samples was measured using a colorimeter (Color Quest XE
Hunter Lab, Northants, UK). The instrument operates on CIELAB (L*, a* and b*) and it
was calibrated using white (L*= 93.97, a*= 0.88 and b*= 1.21) standard. The L, a, b
parameters were used to calculate the chromaticity, the hue angle and the ∆E according
to equations 1, 2 and 3, respectively:
Chroma =
(1)
Hue = tan-1
(2)
∆E= {(∆L2) + (∆a2) + (∆b2)}1/2
(3)
Where, Lo, ao and bo are the color values of the non-processed juice.
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2.2.6 Organic acid quantification
High Performance Liquid Chromatographic (HPLC) analysis was used to
quantify the organic acids. The prebiotic orange juice samples were diluted (1:4),
filtered, using glass fiber pre-filters AP25 13 mm diameter (Merck Millipore Ltd.) and
cleaned on a C-18 SPE cartridge, followed by filtration, using a syringe (2.0 mL) and a
support comprising a membrane (HA membrane cellulose ester 0.45 uM, 13 mm of
diameter). A HPLC system (Agilent Technologies 1260 Infinity) equipped with a pump
system and a UV-DAD detector. Organic acids were detected at 210 nm. Organic acids
were separated in an Aminex HPX-87H column (300 × 7.8 mm) (Bio-Rad) at 50 °C. The
isocratic elution was performed with 0.01M sulfuric acid in deionized water as mobile
phase for 30 min at 0.6 mL/min. The analyses were performed in triplicate.
2.2.7 Statistical analysis
Statistical analyses were performed using the statistical software Statistica
(Statsoft) version 10.0. The results were compared by Tukey test at a 95.0% confidence
level.
Capítulo 2 58
2.3. Results and Discussion
2.3.1 Fructo-oligosaccharides quantification by TLC
The thin layer chromatography (TLC) allowed the evaluation of the prebiotic
fructo-oligosaccharides concentration along with their degree of polymerization (DP).
The results were expressed as the relative concentration obtained by densitometry
(Robyt 2000). In the mixture of fructo-oligosaccharides some, mono- and disaccharides
such as glucose, fructose and sucrose, were observed and quantified besides the target
oligosaccharides such as kestose (DP3), nystose (DP4), 1-fructofuranosyl-nystose
(DP5), and FOS with higher with DP6 and DP7. Fig 1 shows the TLC results of the
fructo-oligosaccharides in water (A) and orange juice (B) after direct exposure of plasma
treatment.
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Fig 1 – Degree of polymerization of the fructo-oligosaccharides in water (A) and orange
juice (B) after direct exposure of plasma treatment
According to the manufacturer (ORAFT), the commercial FOS, used in this
study (P95) presents about 6% of mono and di-saccharides, which corresponds to the
upper clear spots in the first plate on Fig 1. These spots are darker in orange juice due
to the higher amount of sugars present in fruit juices. Fig 2 and Fig 3 shows the relative
amounts of FOS in water (a) and in orange juice (b), after direct and indirect plasma
exposure, respectively.
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Fig 2 – Relative concentration of fructo-oligosaccharides in water (a) and in orange juice
(b) after direct plasma exposure (IN). DP means degree of polymerization. It was
compared the effect of each process time on the FOS concentration.
* Average ± standard deviation of triplicate. Averages with different letters, the samples are statistically
different (p≤0.05)
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Fig 3 – Relative concentration of fructo-oligosaccharides in water (a) and in orange juice
(b) after indirect plasma exposure (OUT). DP means degree of polymerization. It was
compared the effect of each process time on the FOS concentration
*Average ± standard deviation of three replicates. Averages with different letters, the samples are
statistically different (p≤0.05)
The FOS profile regarding the time (15, 30, 45 and 60 s) and kind of exposure to
ACP (direct or indirect exposure) did not present a high variation in the percentage of
each degree of polymerization. The high similarity in the relative amount of the FOS in
water after direct and in indirect ACP treatment indicates that the kind of exposure had
no substantial effect on the FOS DP distribution.
The Fig 2 and Fig 3 show that the percentage of DP-7, DP-6, 1-FFN, nystose
and kestose in water are similar to the percentages of these fructo-oligosaccharides in
orange juice, proving that there were no degradation of FOS. Unlike some reported
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studies in which it was shown that the plasma can degrade active compounds, and
some carbohydrates, FOS were not degraded after plasma treatment (Park et al. 2007;
Benoit et al. 2011).
For the analysis of the samples obtained from high pressure treatment, it was
also used a FOS solution in water for comparison purposes. On the orange juice
analysis, it was also observed a high amount of sucrose, glucose and fructose
compared to the samples of FOS in water, due to the presence of these sugars in the
juice. Then, quantification was based only on the fructo-oligosaccharide, as done for the
plasma treatment.
It was observed that the oligosaccharides percentage of each degrees of
polymerization in water were similar to ones found. Fig 4 shows the fructooligosaccharides in water and orange juice after the high pressure processing (HPP).
Capítulo 2 63
Fig 4 – Relative concentration from fructo-oligosaccharides in water (W HPP) and
orange juice (OJ HPP) after high pressure processing. DP means degree of
polymerization. It was compared the effect of each treatment on the FOS concentration
statically different (p≤0.05)
Previous studies demonstrated the efficiency of HPP on the preservation of
interesting compounds as anthocyanins, phenolic compounds and ascorbic acid in
strawberry and blackberry purees (Patras et al. 2009). Other studies reported a
considerable retention of bioactive compounds and its physical-chemical properties in
fruit juices after high pressure processing (Ferrari, Maresca and Ciccarone 2010; Chen
et al. 2015; Cao et al. 2012).
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On the other hand, several studies have reported regarding the chemical
stability of the prebiotics to food process conditions. Matusek et al. (2009) and Klewicki
(2007) studied the stability of functional ingredients during pasteurization at lower pH
values. These authors concluded the FOS is highly susceptible to hydrolysis in
pasteurization conditions of fruit juices and drinks.
2.3.2 Color parameters
The Tables 1 and 2 shows the color analysis of the ACP treated orange juice.
The control sample (reference) was the non-treated juice containing FOS. The L*
parameter, that indicates the luminosity of the product, presented values from 55.96 ±
0.01 (non-treated juice) to 56.12 ± 0.01. The results showed statistical differences due to
low standard deviation. However, the highest difference between the samples was lower
than 0.5% (Lmax = 0.16).
Table 1 – Color parameters of the orange juice after direct exposure by plasma
Chroma
ho
∆E
Orange juice (control) 55.96 ± 0.01d
30.58 ± 0.02a
95.29 ± 0.02a
--
15s
56.02 ± 0.01c
30.27 ± 0.01b
94.68 ± 0.02b
0.46 ± 0.00c
30s
56.00 ± 0.01c
29.92 ± 0.03c
94.01 ± 0.01c
0.95 ± 0.01b
45s
56.05 ± 0.01b
29.82 ± 0.01d
93.83 ± 0.02d
1.08 ± 0.01a
60s
56.10 ± 0.01a
29.85 ± 0.02d
93.77 ± 0.01e
1.09 ± 0.01 a
Samples
L*
statically different (p≤0.05).
Capítulo 2 65
According to Tiwari et al. (2008) perceptible differences in color parameters can
be analytically classified as follows: very different (ΔE> 3); distinct (1.5 <ΔE <3) and
slightly different (ΔE <1.5). Thus, the results of ΔE of the samples after direct exposure
(table 2) and indirect exposure (table 3) to plasma treatment show that they were all
slightly different when compared with control juice.
Table 2 – Color parameters of the orange juice after indirect exposure by plasma
Samples
Orange juice (control)
L*
ho
Chroma
55.96 ± 0.01d 30.58 ± 0.02a
c
30.21 ± 0.01
b
∆E
95.29 ± 0.02a
94.56 ± 0.01
--
b
0.54 ± 0.01d
15s
56.03 ± 0.01
30s
56.07 ± 0.01b 30.10 ± 0.02c
94.36 ± 0.02c
0.70 ± 0.02c
45s
56.12 ± 0.01a 30.00 ± 0.01d
94.07 ± 0.03d
0.88 ± 0.01b
60s
56.11 ± 0.01a 29.83 ± 0.01e
93.92 ± 0.01e
1.05 ± 0.01 a
A small change in the Chroma and Hue values of ACP treated samples was also
observed. Again, the results were statistically different due to the small standard
deviation with maximum differences less than 3% for Chroma value (Cmax =0.75) and
less than 2% for hue value and hmax= 1.92). Thus, despite the instrumental statistical
difference the small change in the L*, Chroma and Hue value does not compromise the
product acceptance.
The Table 3 shows the color results of the samples treated by HPP.
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Table 3 – Color parameters of the orange juice treated by HPP
Samples
L*
Chroma
ho
Orange juice (control)
55.96 ± 0.01b 30.58 ± 0.02b
95.29 ± 0.02a
Prebiotic orange juice
58.49 ± 0.19a 41.71 ± 0.17a
86.17 ± 0.02b
The changes observed in the color parameters are higher than the observed for
ACP treatment. However, the changes due to HPP were positive and improved the color
of the final product. The L*-value in HPP treated sample increased from 55.96 ± 0.01 to
58.49 ± 0.19, which means that the sample became lighter. The Chroma value
increased from 30.58 ± 0.02 to 41.71 ± 0.17 indicating that the treated sample is more
vivid than the non-treated sample. The hue value decreased from 95.29 ± 0.02 to 86.17
± 0.02. The hue value represents the characteristic color of the juice. According to the
color wheel, the pure yellow corresponds to 90o. Orange juice characteristic color ranges
from orange to yellow depending on the fruit variety.
Despite the small variations in the instrumental color of the treated sample, the
changes did not compromise the characteristic color of the juice. After HPP treatment,
the color was enhanced becoming more vivid and clearer due to the increased Chroma
and L-value.
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2.3.3 Effect of ACP and HPP on the juice pH
The pH values of the juice samples submitted to non-thermal treatment are
presented in Tables 4 and 5.
Table 4 – pH values in prebiotic orange juice (OJ) after high pressure processing
Samples
pH values
Control
4.43±0.01a
OJ without FOS
3.90±0.00b
OJ with FOS
3.90±0.00b
Table 5 - pH values in prebiotic orange juice after plasma treatment
Treatment (time)
pH values
Direct exposure
Indirect exposure
Control
4.43±0.01a
4.43±0.01a
15s
3.90±0.10b
3.90±0.20b
30s
3.90±0.10b
4.00±0.10b
45s
4.00±0.10b
4.00±0.10b
60s
3.90±0.01b
3.90±0.10b
The non-thermal treatments (ACP and HPP) decreased the juice pH values. The
mean pH value obtained for the control was 4.43 and the pH values for treated orange
juice samples ranged from 3.9 to 4.0 (Table 5). Despite, the statistical differences, the
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plasma treated samples presented pH values in the expected range for orange juice.
This small decrease the pH value is associated with the increase in the concentration of
the main organic acids present in orange juice after the non-thermal processing.
2.3.4 Organic acids quantification by HPLC
Fig 5 shows the concentration of organic acids in orange juice after high
pressure processing.
Fig 5 – Concentration (g/ L and g/100 mL) of organic acids in orange juice after high
pressure processing. The control sample (without treatment) corresponds to the time
zero. It was compared the concentration of each acid on the different treatments
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In HPP treatment, it was possible to observe a significant increase in the
concentration of citric acid after HPP in the juice without FOS (10.60±0.04 g/ L) and for
the juice with FOS (10.42±0.02 g/ L), compared to the control (6.70±0.06 g/ L). Similarly,
the ascorbic acid (vitamin C) concentration in the juice with FOS (5.00±0.00 g/100 mL)
and without FOS (5.80±0.00 g/100 mL) increased after HPP, compared to the control
(from 3.5±0.00 g/100 mL). The control sample is the non-processed juice containing
FOS.
The organic acids in orange juice after ACP presented a similar behavior
compared to HPP, with an increased concentration of citric acid (from 6.70±0.06 g/ L to
8.18±0.02 – 9.00±0.08 g/ L). This increase was lower compared to the results obtained
after HPP. It was also observed a small increase in the concentration of ascorbic acid
(from 3.50±0.00 g/100 mL to 3.80±0.00 – 4.90±0.02 g/100 mL). Regarding the kind and
time of exposure some small and statistically significant differences were found (Fig 6).
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Fig 6 – Concentration (g/ L and g/100 mL) of organic acids in orange juice after direct
and indirect plasma exposure. It was compared the concentration of each acid on the
different process times and kinds of exposure. The control sample (without plasma
treatment) corresponds to the time zero
*Average ± standard deviation of triplicates. Averages with different letters, the samples are statically
different (p≤0.05)
This increase in concentration of organic acids may be related to disruption of
membranes after the processes as the orange juice contains pulp. Igual et al. (2010)
reported that thermal treatments, such as conventional pasteurization, degraded organic
acids in grape fruit. Their results showed that thermal treatment led to a significant
decrease in citric acid (from 1.538 to 1.478 mg/100 g) and ascorbic acid (from 36.0 to
34.3 mg/100 g). Cserhalmi et al. (2004) also reported a decrease of 11% on the vitamin
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C (ascorbic acid) content in raspeberry juice after pulsed electric field (PEF) treatment.
Estrada et al. (2013) evaluated the effect of the non-thermal process of ultra-high
pressure on bioactive compounds of orange juice. Their results showed that the
remaining content of L-ascorbic acid in the UHPH treated samples at any pressure was
significantly higher than in the thermal pasteurized one.
2.4. Conclusions
The application of plasma and high pressure processing showed to be
particularly interesting for orange juice containing prebiotic fructo-oligosaccharides. Both
processes do not degrade the FOS, making them suitable for the functional foods
processing containing FOS. The treated samples presented a slight variation on the
color parameters analyzed, in both treatments. However, human eyes are not able to
detect differences in the samples treated by plasma and HPP when compared with a
control sample. These processes also proved to be viable by the fact that not degrades
main organic acids in orange juice.
Acknowledgments
Authors thanks National Council of Technological and Scientific Development CNPq through the National Institute of Tropical Fruits (INCT-FT-CNPq) for the financial
support, Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES) for
the scholarship and Beneo-Orafiti for the FOS sample.
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Effect of Fermented Cashew Apple (Anacardium Occidentale L) Juice. LWT - Food
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Roig-Sagués, A. X. (2013). Influence of ultra high pressure homogenization processing
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National Health Surveillance Agency – ANVISA - Available in:
http://portal.anvisa.gov.br/wps/content/Anvisa+Portal/Anvisa/Inicio/Alimentos/Assuntos+
de+Interesse/Alimentos+Com+Alegacoes+de+Propriedades+Funcionais+e+ou+de+Sau
de/Alegacoes+de+propriedade+funcional+aprovadas
Acessed: 14/10/2014
Capítulo 3 75
CAPÍTULO 3: EFFECTS OF ATMOSPHERIC COLD PLASMA AND OZONE ON
PREBIOTIC ORANGE JUICE
Abstract
In this study, the effect of plasma and ozone treatments on the quality of orange juice
was evaluated. The juice was directly and indirectly exposed to a plasma field at 70 kV
for different treatment times: 15, 30, 45 and 60 seconds. For ozone processing, different
loads (0.057, 0.128 and 0.230 mg/ O3.mL of juice) were evaluated. The oligosaccharides
were quantified, in the juices, by HPLC. The juice pH, color, total phenolic content and
total antioxidant activity were also determined. Both processes promoted a partial
degradation of the oligosaccharides in the juice. However, the juice maintained an
enough amount of oligosaccharides to be classified as a prebiotic food. The phenolic
content and antioxidant capacity of the treated samples were also well preserved as the
pH and color. Thus, atmospheric cold plasma and ozone are suitable non-thermal
alternatives for prebiotic orange juice treatment.
Keywords: Emerging technologies, orange juice, phenolic, antioxidant activity
Capítulo 3 76
3.1. Introduction
Nowadays, there is a great interest in the use of prebiotics as functional food
ingredients to improve the human health (Liu et al. 2014). The beneficial health effects
attributed to the intake of prebiotics have been reported in several studies (Moreno-Vilet
et al. 2014; Tako et al. 2014). Prebiotic oligosaccharides are usually obtained by
polysaccharide hydrolysis or enzyme synthesis.
Lactic acid bacteria (LAB) are able to produce a wide variety of glucansucrases
(GS) and fructansucrases (FS), which synthesize glucans and fructans, respectively,
from sucrose with food and nutritional applications (Bivolarski et al. 2013). Leuconostoc
mesenteroides NRRL B512F produces the enzyme dextransucrase when cultivated in a
medium containing sucrose as substrate, a nitrogen source and minerals (Rodrigues et
al. 2003). Dextransucrase (EC 2.4.1.5) is a bacterial extra cellular enzyme, which uses
sucrose as a substrate to promote the synthesis of dextran releasing the fructose
moieties from sucrose (Rodrigues, 2005). When another substrate (acceptor) is also
present, besides sucrose, dextransucrase produces prebiotic oligosaccharides in a
reaction called acceptor reaction due to the addition of glucose units from sucrose to the
acceptor molecule (maltose, glucose, fructose) instead of into the dextran chain (Rabelo
et al. 2006). Oligosaccharides are well documented as effective prebiotic ingredients
that modulate the intestinal microbiota and provide other beneficial health effects such
as stool improvement, weight management, allergy alleviation (Johnson et al. 2013;
Sangwan et al. 2014) and mineral absorption (Tako et al. 2014).
Capítulo 3 77
Our group has successfully studied the oligosaccharides synthesis in fruit juices
thought the dextransucrase acceptor reaction. The oligosaccharides synthesized by L.
mesenteriodes B-512F dextransucrase present α-1,6 glycosidic bonds, which are not
digested in the stomach (Araújo et al. 2014; Rabelo et al. 2009; Silva et al. 2012).
Orange juice is one of the most worldwide consumed juice due to its high
nutritional value and pleasant taste (Agcam et al. 2014). Commonly, thermal treatments
are used in the preservation of fruit juices in order to extend their shelf life. However,
oligosaccharides are liable to hydrolysis in the pasteurization temperatures of fruit juices
and drinks (Matusek et al. 2009).
Nowadays, the consumer’s demand and the shortcomings of the existing
technologies are stimulating the development of alternative non-thermal approaches in
food processing (Misra et al. 2014a). In this context, atmosphere cold plasma (ACP) and
ozone processing are emerging technologies that offer many potential applications.
Recently, these technologies emerged as powerful tools for surface decontamination of
both foodstuffs and food packaging materials (Pankaj et al. 2014a). Several studies
have been focused on the application of ozone and plasma such as good alternative
processing technologies for decontamination of foods (Misra et al. 2014b; Ziuzina et al.
2013). However, there are no available data and studies concerning the effects of ozone
and cold plasma on the quality characteristics of functional foods.
In this context, this paper aims to evaluate the effect of the atmospheric cold
plasma and ozone processing on the quality of orange juice containing prebiotic
oligosaccharides.
Capítulo 3 78
3.2. Materials and methods
3.2.1 Orange juice
Orange juice (Squeez©, Fruit Juices Ltd, Ireland) was purchased from a local
supermarket (Dunnes, Dublin 1- Ireland). The pH of the juice was determined by direct
measurement in a 420A pHmeter, (Orion research Inc, Beverly, MA. US). The pHmeter
was calibrated before use with buffer solutions of pH 4.0, 7.0 and 10.0.
Reducing sugars were measured by high-performance liquid chromatography
(HPLC) analysis using an Agilent Technologies System. Separation was achieved in a
Supelcogel-Ca column at 80 °C. Ultrapure water (MilliQ System, Millipore, Bilberica, MA,
USA) at 0.5 mL/ min was used as eluent and the detector temperature was 35 °C. All
samples were analyzed in duplicate.
3.2.2 Dextransucrase production and enzyme activity determination
The dextransucrase production was carried out according to Rodrigues et al.
(2003). The enzyme produced was stocked frozen at -20 ºC prior to use. The enzymatic
activity of the dextransucrase was determined by quantifying the released fructose by
the DNS (3.5 dinitrosalicylic acid) method (Miller, 1959) using as substrate a 10% (w/v)
sucrose solution in sodium acetate (20mM, pH 5.2) at 30 oC (Rabelo et al. 2009;
Rodrigues et al. 2005). Dextransucrase activity was expressed in IU/ mL. One
Capítulo 3 79
international unit (IU) is the amount of enzyme that releases 1 mol of fructose per
minute under the assay condition.
3.2.3 Prebiotic oligosaccharides synthesis in orange juice
Oligosaccharides synthesis was carried out using the partially purified enzyme
and the optimum synthesis conditions (30 °C and pH 5.2). Synthesis was carried out in
1000 mL Erlenmeyer flask at 30 °C for 24 h (Rabelo et al. 2006) containing 800 mL of
the orange juice and using enzyme with 0.05 IU/ mL. The amount of enzyme was
modulated to avoid the oligosaccharides overproduction, which can cause intestinal
discomfort and or diarrhea.
3.2.4 Plasma treatment on prebiotic orange juice
The plasma treatment was carried out using a plasma generation (DBD-ACP),
model: 6CP120/60-7.5 - Phenix Technologies (Fig. 1). The system consisted of a
variable high voltage transformer with an input voltage of 230 V at 50 Hz and a
maximum high voltage output of 70 kV at 50 Hz. The two 15 cm diameter aluminum disc
electrodes were separated by a polypropylene container, which served both as a sample
holder and as a dielectric barrier with wall thickness of 1.2 mm. The distance between
the two electrodes was 22 mm, equal to the height of the container. Voltage was
monitored using an InfiniVision 2000 X-Series Oscilloscope (Agilent Technologies Inc.,
Capítulo 3 80
Santa Clara, CA, USA). All experiments were performed at 70 kV peak to peak at
ambient air and atmospheric pressure conditions.
An aliquot of 20 mL of the prebiotic orange juice was transferred to an open
Petri dish, which was placed in a polypropylene box, sealed with a polymeric film with 50
µm of thicknesses (Cryovac BB3050). This film served as an additional layer of dielectric
barrier (Pankaj et al. 2014). Then, the samples were treated with atmospheric cold
plasma (ACP) with processing times of 15, 30, 45 and 60 seconds and different
exposure kinds: direct plasma field (in) and indirect plasma field (out). These treatment
times were selected based on previously study carried for pathogens inactivation
(Ziuzina et al. 2013), where a complete bacterial inactivation was achieved after 20
seconds of direct exposure and 45 seconds of indirect plasma treatment. In the present
work, lower and higher exposure times to ACP were evaluated. Treated samples of
prebiotic orange juice were stored at room temperature (25 °C) for 24 h, after that, the
samples were analyzed. Treatments were done in duplicates and analyzes were
performed in triplicate.
Capítulo 3 81
Fig.1- Experimental setup for plasma treatment. (Adapted from Pankaj, Misra, & Cullen,
2013)
3.2.5 Ozone treatment on prebiotic orange juice
The prebiotic orange juice was also submitted to ozone treatment. Ozone was
generated using an ozone generator (Model OL80F, Ozone services, Burton, B.C.,
Canada – Fig. 2) and produced by a corona discharge generator. Pure oxygen was
supplied via an oxygen cylinder (Air Products Ltd., Dublin, Ireland) and the ﬂow rate was
controlled using a gas ﬂow regulator. The excess of ozone was destroyed by an ozone
destroyer unit. To prevent excess foaming, 3 drops of sterile anti-foaming agent
(Antifoam B emulsion, Sigma Aldrich, Ireland Ltd.) was added before the ozone
treatment. The treatment of the prebiotic orange juice samples was carried out using
different ozone loads: 0.057, 0.128 and 0.230 mg O 3/ mL of juice. These ozone loads
Capítulo 3 82
were based on the minimum load applied (0.075 mg O3/ mL) for 5 log CFU/mL of E. coli
inactivation in orange juice (Patil et al. 2009). Higher values were applied considering
the existence of more resistant microorganisms. Treatments were conducted in
duplicates and analyzes were performed in triplicate.
Fig.2- Experimental setup for of ozone treatment. (Adapted from Patil et al. 2009b)
3.2.6 Carbohydrate analysis
3.2.6.1 Oligosaccharides degree of polymerization
The prebiotic oligosaccharides produced in the orange juice were characterized
according to their degree of polymerization by Thin Layer Chromatography (TLC) on
Capítulo 3 83
Whatman K6 silica plates, 250-µm thickness (Whatman, Kent, UK). Diluted samples of
10 µL were absorbed onto the TLC plate by injection with a micropipette on a line at 1.5
cm above the lower plate edge and, at least 1.5 cm from each other. Then, the plate was
irrigated for two ascents in a solvent mixture composed of acetonitrile: ethy-1-acetate: 1propanol: H2O (85:20:50:90). To make sugars visible, the plates were revealed with a
solution containing 0.3% (w/v) of 1-naphthyl ethylenediamine dihydrochloride in
methanol with sulphuric acid 3% (v/v). The plates were then heated in an oven at 120 ºC
for 10 minutes.
3.2.6.2 Sugars and oligosaccharides quantification
After removing the dextran by ethanol precipitation, the supernatant containing
the oligosaccharides and the residual sugars was cleaned by SPE using the STRATA
C18 (SUPELCO) cartridge and filtered through a Nylon membrane (SUPELCO Acrodisc,
25 mm x 0.45 µm) to be analyzed by high-performance liquid chromatography (HPLC).
A Varian ProStar System (Varian, Palo Alto, CA, USA) equipped with two high
pressure pumps (Model 210), refractive index detector (Model 355) and a Timberline
column heating oven was used to carry out the assay. Oligosaccharides separation was
achieved in a Phenomenex Resex RSO column (10 x 300 mm) at 80 °C. Ultrapure water
(MilliQ System, Millipore, Bilberica, MA, USA) at 0.3 mL/min was used as eluent and the
detector temperature was 35 °C. All samples were analyzed in duplicate. The software
ProStar WS 6.0 was used to acquire and handle the data. The chromatographic
condition allowed the separation and quantification of simple sugars (sucrose, glucose
Capítulo 3 84
and fructose) and the oligosaccharides. The oligosaccharides were quantified using
maltotriose (DP3), maltotetraose (DP4), maltopentaose (DP5), maltohexaose (DP6) and
maltoheptaose
(DP7)
purchased
from
Sigma-Aldrich
(Sigma-Aldrich,
:St.
Louis, MO, USA).
3.2.7 Color analysis
The color of the samples was measured using a colorimeter (Colour Quest XE
Hunter Lab, Northants, UK). The instrument operates on CIELAB (L*, a* and b*) and it
was calibrated using white (L*= 93.97, a*= 0.88 and b*= 1.21) standard. The L, a, b
parameters were used to calculate the chromaticity, the hue angle and ∆E values
according to equations 1, 2 and 3, respectively:
Chroma =
(1)
Hue = tan-1
(2)
∆E= {(∆L2) + (∆a2) + (∆b2)}1/2
(3)
Where, Lo, ao and bo are colour values of control juice.
Capítulo 3 85
3.2.8 Total phenolic content
Total phenolic content was assayed by Folin-Ciocalteu method, developed by
Folin and Ciocalteu (1927). The method is based on the ability of phenolic compounds to
reduce a mixture of phosphomolybdic/phosphotungstic acid complexes (Folin reagent) in
alkaline medium. The analysis was carried out in a 96 well microtiter plate where 10 µL
of the sample (or water for blank) was mixed with 200 µL of Folin-Ciocalteu solution
(diluted in water 1:10). After 3 minutes of reaction and the development of the blue
colour, 10 µL of 20% (w/v) sodium carbonate solution was added. The reading was
taken at 765 nm using an Elisa reader spectrophotometer (Biotek Epoch, Winooski VT –
USA) using the software Gen 5 1.10 to handle the data. Gallic acid was the reference
standard and total phenolic was expressed as equivalents of gallic acid.
3.2.9 Antioxidant activity determination
The antioxidant activity of the prebiotic orange juice before and after ACP and
ozone treatment was assayed according to DPPH and ABTS methods, as further
described. DPPH and ABTS methods are recommended as easy and accurate assays
for measuring the antioxidant activity of orange juice (Kelebek et al. 2009)
Capítulo 3 86
3.2.9.1 DPPH method
The antioxidant activity of the prebiotic orange juice was determined
spectrophotometrically using DPPH• free radical method reported (Brand-Williams et al.
1995) with modifications. An aliquot of 30 µL of the orange juice (or water for blank)
diluted in methanol (1:10) was added to 1200 µL of DPPH• solution in methanol (0.06
mM). The absorbency was taken at 515 nm at 2 minutes intervals during 30 minutes.
The assays were carried out in a Thermo Fisher Scientific Spectrophotometer (model
Evolution 201 – USA). The antioxidant efficiency was expressed as EC50 determined as
the time when the concentration of substrate causes 50% loss in absorbance (DPPH •
activity).
3.2.9.2 ABTS method
The ABTS free radical capture reported by Re et al. (1999) was also used to
measure the antioxidant capacity of the prebiotic orange juice treated by ozone and
plasma. ABTS was dissolved in water to 7 mM concentration. ABTS radical (ABTS•+)
was produced by reacting 10 mL of the ABTS stock solution (7mM) with 176 µL of
potassium persulfate (140 mM) and the mixture was allowed to stand in the dark at room
temperature for 16 h before use. The ABTS•+ radical was diluted in ethanol to an
absorbance of 0.700 (±0.05) at 734 nm. An aliquot of 1.5 µL of the orange juice diluted
in methanol (1:7) was mixed to 1500 µL of ABTS •+ radical and the absorbance was
measured after 6 minutes against the blank using Thermo Fisher Scientific
Capítulo 3 87
Spectrophotometer (model Evolution 201 – USA). Trolox was the antioxidant standard.
Results were expressed as Trolox equivalent antioxidant activity per mL.
3.2.10 Statistical analysis
Statistical analyses were performed using the statistical software Statistica
(Statsoft) version 10.0. The results were compared by Tukey test at 95.0% confidence
level.
3.3 Results and discussions
3.3.1 Orange juice characterization
The concentration of sugar in the orange juice used as raw material was: 20.80
± 0.03 of sucrose; 15.41 ± 0.17 of fructose and 15.30 ± 0.36 of glucose. According to
Rabelo et al. (2009) and Rodrigues et al. (2006), the acceptor mechanisms requires high
concentration of reducing sugars since they are the acceptor of the reaction, which
promotes the deviation of glucosyl moieties to the acceptor molecule producing the
desired oligosaccharides. Thus, the level of sugars were adjusted to 75 g/ L of sucrose
and 75 g /L of reducing sugars (equimolar proportions) by adding external sucrose,
glucose and fructose. The enzyme synthesis resulted in 11.55 ± 0.25 g/ L of
oligosaccharides in the juice. This concentration is enough to consider the juice a
Capítulo 3 88
functional food. After the production of prebiotic oligosaccharides, the orange juice was
submitted to plasma and ozone treatments.
3.3.2 Effect of plasma and ozone on the juice pH
The pH values of the prebiotic orange juice after ozone processing ranged from
4.41 to 4.43. The pH of ozone treated samples (Table 1) was not statistically different
from the non-treated sample (control). Ozone processing did not affect the pH of
prebiotic orange juice after application of different ozone loads.
Table 1 – pH values in prebiotic orange juice after ozone processing
Charge of ozone (mg O3/ mL)
pH values
Control
4.43 ± 0.01a
0.057
4.41± 0.01a
0.128
4.42± 0.01a
0.230
4.43 ± 0.01a
Mean ± SD (n = 6). Means with the same letter are not significantly different by the Tukey test (p < 0.05).
This result agrees to the reported for other non-thermal processing of orange
juice such as sonication and pulsed electric field. (Tiwari et al. 2008a; Cortés, Esteve, &
Frígola, 2008). On the other hand, when prebiotic orange juice was plasma treated, the
pH of the treated samples was statistically different from the control (non-treated
sample). The mean pH value obtained for the control was 4.43 and the pH values for
treated orange juice samples ranged from 3.9 to 4.0 (Table 2).
Capítulo 3 89
Table 2 – pH values in prebiotic orange juice after plasma treatment
pH values
Treatment
(time)
Direct exposure
Indirect exposure
Control
4.43 ± 0.0 a
4.43 ± 0.01a
15s
3.90 ± 0.10b
3.90 ± 0.20b
30s
3.90 ± 0.10b
4.00 ± 0.10b
45s
4.00 ± 0.10b
4.00 ± 0.10b
60s
3.90 ± 0.01b
3.90 ± 0.10b
Mean ± SD (n = 6). Means with the same letter are not significantly different by the Tukey test (p < 0.05)
The orange juice pH depends on the harvest period (Sinclair & Ramsey, 1944).
As a citric fruit juice, the orange pH can reach values as low as 2.8 as reported by
Sinclair, Bartholomew & Ramsey (1945). Despite, the statistical differences, the plasma
treated samples presented pH values in the expected range for orange juice.
3.3.3 Oligosaccharides characterization after plasma and ozone treatments.
The prebiotic oligosaccharides produced in orange juice were characterized
according to degree of polymerization (DP). The DP of the treated samples were similar
to the non-treated sample (control). Figure 3 shows the Thin Layer Chromatography
(TLC) analysis of the produced oligosaccharides after ozone treatment, against the
control (non-treated sample).
Capítulo 3 90
Fig 3 – Degree of polymerization (DP) of the oligosaccharides in prebiotic orange juice
before (control) and after ozone treatment.
Oligosaccharides with DP up to 8 were obtained in all samples. Similar results
were obtained for ACP treatment. Regarding the oligosaccharides DP, the ACP and
ozone treatments did not affect the degree of polymerization of the oligosaccharides in
orange juice since the degree of polymerization of the treated samples was the same as
the untreated sample in both cases.
Capítulo 3 91
The oligosaccharides DP obtained in this study are in agreement to those
reported in previous studies on oligosaccharides synthesis in fruit juices. Rabelo et al.
(2009) and Vergara et al. (2010) evaluated the oligosaccharides production in fruit juices
by fermentation. These authors obtained oligosaccharides with degree of polymerization
up to 7 (DP7). Araújo et al. (2014) and Coelho et al. (2014) evaluated the enzyme
synthesis of oligosaccharides in acerola and lemon juice, respectively, and reported DP
up to 6 in both fruit juices.
Regarding to plasma and ozone treatments, it is not possible to compare our
results with other groups because so far, these treatments in juices containing functional
ingredients was never did before to our knowledge.
3.3.4 Oligosaccharides quantification by HPLC after plasma and ozone treatments.
Despite oligosaccharides with DP up to 8 were visualized on the TLC plates,
oligosaccharides with DP higher than 7 were below the quantification limit of the HPLC
method. The HPLC analysis showed the oligosaccharides concentration in prebiotic
orange juice before and after plasma and ozone treatments. The degradation of
oligosaccharides with higher degree of polymerization resulted in oligosaccharides with
lower degree of polymerization as presented in Figures 4 and 5. The results for direct
(A) and indirect plasma exposure (B) are presented in Figure 4.
Capítulo 3 92
Fig.4- Oligosaccharides quantification after direct exposure (A) and indirect exposure (B)
of the plasma treatment. The control sample (without plasma treatment) corresponds to
the time zero. DP means degree of polymerization.
Analyzing the concentration of oligosaccharides according to their degree of
polymerization (Figure 4A and 4B), it is clear that oligosaccharides with higher degree of
polymerization (DP4, DP5, and DP6) decreased when the treatment time was increased
on both kinds of exposure. On the other hand, the oligosaccharides with DP 3 (panose)
increased at the end of the plasma treatment. Before ACP treatment, the DP3
concentration was 2.50 ± 0.07 g /L and after 60 seconds of treatment, this concentration
was 3.17 ± 0.06 g /L and 3.09 ± 0.01 g/L for direct and indirect exposures, respectively.
The increase of panose (DP3) is due the degradation of higher DP oligosaccharides.
Capítulo 3 93
Figure 5 shows the oligosaccharides concentration according to degree of
polymerization obtained after ozone processing. Oligosaccharides with DP from 4 to 7
decreased with the increase of the ozone loads. On the other hand, panose (DP3)
increased after mild ozone loads and decreased after higher loads application.
Fig.5 - Oligosaccharides quantification after ozone processing. The control sample
(without ozone treatment) corresponds to the load zero. DP means degree of
polymerization.
Table 3 presents the total oligosaccharide loss for each non-thermal treatment.
The higher loss was observed for plasma treatment with indirect exposure and the
highest ozone load.
Capítulo 3 94
Table 3 – Oligosaccharide concentration and loss after non-thermal treatment.
Non-thermal
Oligosaccharide (g/ L) Oligosaccharide (g/ L) Oligosaccharide
treatment
Before treatment
After treatment
Loss (%)
Plasma IN (60s)
11.55 ± 0.25a
10.15 + 0.05b
12%
Plasma Out (60s)
11.55 ± 0.25a
9.07 ± 0.07b
22%
11.55 ± 0.25a
9.18 ± 0.04b
21%
Ozone
(0.230 mg O3/min.mL)
In ACP plasma ozone gas is formed in the head space. According to Wang et al.
(1999),
ozone
can
cause
polysaccharides
depolymerization
producing
other
polysaccharides with shorter chains as observed in the present study. Prebiotic
oligosaccharides synthesized by L. mesenteriodes B-512F dextransucrase present α-1,6
glycosidic bonds. Wang et al. (1999) also demonstrated that ozone process can degrade
polysaccharides with 1,6-linked α-D-glucose bonds.
Ben’ko, Manisova & Lunin (2013) reported that the main route of carbohydrate
ozonolysis is the cleavage of glycoside bonds, leading to depolymerization of the
macromolecule, and the oxidation of functional groups to form carbonyl and carboxyl
compounds, lactones, hydroperoxides and CO2. However, the mechanism of
oligosaccharides degradation due to the plasma and ozone application still needs to be
elucidated as this is an emerging field of investigation.
The amount of prebiotic oligosaccharides intake is a controversial issue as their
effect strongly depends on the oligosaccharide structure and the consumer itself (adults,
children or babies). The Brazilian Health Surveillance Agency (ANVISA, 2014) suggests
a prebiotic daily ingestion of about 1.5 g in liquid foods. Although some degradation of
Capítulo 3 95
oligosaccharides has occurred due to the non-thermal treatments, the minimum required
amount of oligosaccharides to be considered as a prebiotic food was maintained in both
plasma and ozone treated juice samples since a portion of 200 mL provides 1.8 g of
oligosaccharides.
Figure 6 shows the concentration of simple sugars after the direct (A) and
indirect (B) exposures of plasma processing and simple sugars after ozone (C)
processing.
Fig.6 - Sugars concentration after plasma direct exposure (A) and indirect exposure (B)
and after ozone treatment (C)
In both kinds of plasma exposure (Figure 6A and 6B) there was a decrease on
the fructose concentration and an increase on the sucrose along the treatment time. For
ozone processing (Figure 6C), the same behavior was observed with these sugars, after
the lowest ozone load.
Capítulo 3 96
Thus, the increase in sucrose concentration explains the decrease of the
oligosaccharides with a high degree of polymerization in prebiotic orange juice since the
degradation of oligosaccharides with higher degree of polymerization may also result on
the release of the simple sugar. Sucrose might have also been formed by the
combination of fructose and the glucose moiety released from the oligosaccharides.
3.3.5 Color analysis
Regarding the color parameters after ozone processing, the values of L* and
Chroma were not statistically different from the control (Table 4).
Table 4 – Color parameters of the prebiotic orange juice after ozone processing
Load (mg O3/ mL)
L*
Chroma
ho
∆E
Control
52.93 ± 0.02a
28.74 ± 0.07a
95.47 ± 0.02a
--
0.057
53.03 ± 0.02a
28.56 ± 0.03a
94.75 ± 0.05a,b 1.97 ± 0.01a
0.128
53.06 ± 0.01a
28.50 ± 0.02a
94.48 ± 0.02b
0.87 ± 0.01c
0.230
53.15 ± 0.02a
28.40 ± 0.02a
94.19 ± 0.02b
1.90 ± 0.01b
The Hue angle, in treated samples, presented a slightly, and statistically
significant reduction compared to the control sample. According to Tiwari et al. (2008)
perceptible differences in color parameters can be analytically classified as follows: very
different (ΔE> 3); distinct (1.5 <ΔE <3) and slightly different (ΔE <1.5). Thus, the ΔE
results of the samples after ozone treatment, show that only the sample submitted to
Capítulo 3 97
second charge, it was slightly different when compared with control juice. The other
samples were distinct whn compared to the control sample.
Table 5 presents the results obtained for plasma treated samples.
Table 5 – Color parameters of the prebiotic orange juice after plasma treatment
Direct exposure (in)
Treatment
(time)
L*
Chroma
ho
∆E
Control
52.93 ± 0.02d
28.74 ± 0.07d
95.47 ± 0.02a
--
15
56.02 ± 0.01b,c
30.27 ± 0.01a
94.68 ± 0.02b
0.46 ± 0.00c
30
56.00 ± 0.01c
29.92 ± 0.03b
94.01 ± 0.01c
0.95 ± 0.01b
45
56.05 ± 0.01b
29.82 ± 0.01c
93.83 ± 0.02d
1.08 ± 0.01a
60
56.10 ± 0.01a
29.85 ± 0.02b,c
93.77 ± 0.01e
1.09 ± 0.01 a
Treatment
Indirect exposure (out)
(time)
L*
Chroma
ho
∆E
Control
52.93 ± 0.02d
28.74 ± 0.07e
95.47 ± 0.02a
--
15
56.03 ± 0.01c
30.21 ± 0.01a
94.56 ± 0.01b
0.54 ± 0.01d
30
56.07 ± 0.01b
30.10 ± 0.02b
94.36 ± 0.02c
0.70 ± 0.02c
45
56.12 ± 0.01a
30.00 ± 0.01c
94.07 ± 0.03d
0.88 ± 0.01b
60
56.11 ± 0.01a
29.83 ± 0.01d
93.92 ± 0.01e
1.05 ± 0.01 a
The L* (lightness) values were in the range of 56.02 - 56.10 and 56.03 - 56.11
for direct and indirect plasma exposure, respectively, while the control sample (nontreated juice) was 52.93 ± 0.02. In all plasma treated samples the L* value was
statistically different from the control. Misra et al. (2014b) also observed a change in the
L* color parameter in plasma treated strawberries. Regarding the Chroma and the Hue
angle, a slight reduction in these values were observed in both kind of plasma exposure.
Capítulo 3 98
The L* is the luminous intensity of a color and reflects if the color is dark or light.
This parameter ranges from 0 (black) to 100 (white). The plasma treatment slightly
increased the juice L* parameter by about 3 units. The Chroma reflects the vividness or
the color saturation, which means how close the color is either the gray or the pure hue.
An increase in Chroma means that the color gets closer to the pure hue, becoming more
vivid. The Chroma values in plasma treated samples increased slightly, which means
that the treatment resulted in a more vivid instrumental color. The same behavior was
observed for pasteurized orange juice (Lee & Coates, 2003). The Hue angle is the
characteristic color of the sample. According to the color wheel, the pure yellow
corresponds to 90º. Orange juice characteristic color ranges from orange to yellow
depending on the fruit variety. The control sample Hue angle was 95.47º, which is close
to the pure yellow (90º) and is in agreement to the reported by Lee & Coates (2003) for
fresh orange juice (95.66º). The maximal decrease of the Hue angle observed in plasma
treated sample corresponds to less than 2º, which is very low and approximates the
juice color to pure yellow color. In pasteurized orange juice the Hue angle increased by
about 1.2 units compared to the control (Lee & Coates, 2003).
Despite
some
color
parameters
presented
statistical
difference,
the
characteristic color of the juice was kept in the expected ranger for orange juice with a
slightly increase in Chroma and lightness indicating that the juice became lighter and
more vivid. Thus, the ozone and plasma treatment did not compromise the product
color. Regarding to the ΔE results, the samples after direct and indirect exposure to
plasma treatment, show that they were all slightly different when compared with control
juice.
Capítulo 3 99
3.3.6 Total phenolic content
Figure 7A show the concentration of phenolic compounds after plasma
exposure. The values decreased from 2.52 ± 0.20 to 2.37 ± 0.10 g /L and 1.93 ± 0.12 g
/L for direct and indirect exposure, respectively. When the prebiotic orange juice was
processed under plasma indirect exposure, the total phenolic content was affected
(p<0.05) only after 60 seconds of treatment.
Figure 7B shows the total phenolic compounds concentration in prebiotic orange
juice after ozone treatment. Before ozone processing, the phenolic concentration in the
juice was 2.52 ± 0.20 g /L and at the end of processing this value decreased to 2.33 ±
0.07 g /L. After ozone treatment, the phenolic concentration was statistically the same of
the control sample.
3,5
3,0
3,0
2,5
a
a
a
a
a
a
a
a
a
a
2,0
a
a
1,5
1,0
in
out
0,5
Phenolics (mg/L)
Phenolics (mg/ L)
2,5
a
a
2,0
1,5
1,0
0,5
0,0
0,0
0
10
20
30
Time (s)
40
50
60
0,00
0,05
0,10
0,15
Ozone load (mg O3/mL)
Fig.7 - Total phenolic content after plasma (A) and ozone (B) treatment.
0,20
0,25
Capítulo 3 100
The plasma discharge generates energetic electrons that dissociate oxygen
molecules by direct impact. The single oxygen atom from the dissociation combines with
oxygen molecule (O2) to form ozone gas (Misra et al. 2014a). Phenolics compounds are
particularly susceptible to ozone attack (Stalter et al. 2011). According to Perez et al.
(2002), ozone reacts very efficiently on degradation of aromatic compounds of the
phenolic compounds. The molecular ozone action on the aromatic compound favors the
formation of hydroxylated and quinone compounds, because the formation of aliphatic
compounds is originated from rupture of the aromatic ring. Despite the phenolics
compounds are susceptible to ozone attack, only the indirect plasma exposure by 60 s
caused a significant change on the phenolics contents. However, after plasma indirect
exposure, 76% of the initial phenolic content was preserved in the treated juice.
In addition, our results showed higher concentration of phenolic compounds
when compared with the Kelebek et al. (2009) and Gil-izquierdo et al. (2001) results,
which presented the phenolic concentrations of 0.317 and 0.839 g /L, respectively to
different variety of oranges.
3.3.7 Total antioxidant activity
Total antioxidant capacity of prebiotic orange juice was evaluated using DPPH •
and ABTS• free radical methods. DPPH did not show any significant changes among the
treated samples and the control (results not shown). On the other hand, when
antioxidant activity was measured by the ABTS• free radical method, a reduction in the
antioxidant capacity of the prebiotic orange juice was observed after plasma and ozone
Capítulo 3 101
processing. The results obtained suggest that the ABTS• method is more sensitive since
there was a reaction between the ABTS radical and the antioxidants compounds of the
prebiotic orange juice not observed with the DPPH.
The antioxidant capacity results according to ABTS method of the prebiotic
orange juice treated with plasma are showed in Table 6.
Table 6 – ABTS antioxidant activity in prebiotic orange juice after plasma treatment
Treatment
ABTS (µM de trolox / mL)
(times)
Direct exposure
Indirect exposure
Control
4.97 ± 0.30a
4.97 ± 0.30a
15
3.11 ± 0.28b
3.61 ± 0.69a
30
3.18 ± 0.21b
4.61 ± 1.82a
45
3.31 ± 1.02b
4.35 ± 1.16a
60
2.63 ± 0.23b
4.40 ± 1.02a
When prebiotic orange juice was directly exposed to plasma there was a slight
reduction on the antioxidant capacity of the juice, however the reduction was
significantly different when compared to the control sample. After 60 s of direct exposure
to plasma field, the juice lost about 50% of the antioxidant capacity measured by ABTS.
On the other hand, when prebiotic orange juice was submitted indirectly to the plasma
field, the antioxidant compounds of all samples were statistically equal. According to
Ziuzina et al. (2014), 20 s of ACP treatment is enough to inactivate 5 log of E.coli in
orange juice. This processing time would not affect the antioxidant capacity of the
product according to the ABTS methodology.
Capítulo 3 102
The results related to the antioxidant capacity of the prebiotic orange juice after
ozone processing are showed in Table 7.
Table 7 – ABTS Antioxidant activity in prebiotic orange juice after ozone treatment.
Load (mg O3/mL)
ABTS (µM de trolox / mL)
0
4.97 ± 0.3a
0.057
5.46 ± 0.13a
0.128
5.17 ± 0.25a
0.230
4.08 ± 0.26b
After the application of the first and second loads, there was no change on the
antioxidant activity of the juice when compared to the control sample. These loads were
applied based on the minimum load applied (0.075 mg O 3/ mL) for 5 log CFU/mL
inactivation of E. coli in orange juice (Patil et al. 2009a).
On the other hand, when 0.230 mg O3/mL was applied, a statistical significant
reduction in the antioxidant capacity of the juice was observed. However, this value
corresponds to a loss of about 18% of the antioxidant capacity compared to the control
sample. In addition, this load is much higher than necessary to the pathogens
inactivation.
The results obtained for antioxidant capacity by ABTS method showed that the
ACP and ozone treatment of orange juice at the same conditions previously studied for
pathogens inactivation (Ziuzina et al. 2014; Patil et al. 2009a) does not affect the product
antioxidant capacity.
Capítulo 3 103
3.4. Conclusion
This work has shown that plasma and ozone treatments can be used as
emerging technologies for prebiotic orange juice processing without affecting the quality
of the juice regarding the phenolic content, antioxidant capacity and the product color.
Ozone treatment resulted in a higher reduction in the juice oligosaccharides content
compared to plasma processing. Nevertheless, at the end of both treatments, the juice
maintains the minimum required amount of the oligosaccharides to be classified as a
prebiotic food since a portion of 200 mL provides at least 1.5 g of oligosaccharides. The
elucidation of the oligosaccharides degradation mechanism by atmosphere cold plasma
and ozone processing is subject of further studies.
Acknowledgments
Authors thanks CNPq through the National Institute of Tropical Fruits (INCT-FT-CNPq)
for the financial support, CAPES for the scholarship.
Capítulo 3 104
CONSIDERAÇÕES FINAIS
As tecnologias de plasma e alta pressão foram efetivas no tratamento de suco
de laranja contendo fruto-oligossacarídeos (FOS) comerciais, uma vez que as mesmas
não ocasionam degradação dos FOS. Esses mesmos processamentos promovem uma
ligeira alteração nos parâmetros de cor. Contudo, essa mudança não afeta a qualidade
do produto, uma vez que não é possível detectar alterações visuais quando se compara
o suco tratado com uma amostra controle.
A aplicação do tratamento de ozônio no suco prebiótico de laranja proporciona
uma redução na concentração de oligossacarídeos em comparação com o
processamento de plasma. Contudo, as tecnologias não térmicas estudadas nesse
trabalho apresentam-se como alternativas adequadas para o processamento de suco
de laranja prebiótico, uma vez que, o suco tratado manteve a quantidade mínima
estabelecida de oligossacarídeos para ser designado como alimento funcional
prebiótico, além de não afetar os demais compostos bioativos presentes no produto.
Estudos posteriores devem ser conduzidos no intuito de elucidar os
mecanismos de degradação dos oligossacarídeos submetidos ao tratamento com
plasma e ozônio.
Capítulo 3 105
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Revisão Bibliográfica - Universidade Federal do Ceará

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