thamiris renata martiny
TRANSCRIPT
UNIVERSIDADE FEDERAL DE SANTA MARIA
CENTRO DE TECNOLOGIA
PROGRAMA DE PÓS-GRADUAÇÃO EM ENGENHARIA QUÍMICA
Thamiris Renata Martiny
OBTENÇÃO DE EXTRATOS DE FOLHAS DE OLIVEIRA PARA
APLICAÇÃO COMO AGENTE ANTIMICROBIANO EM
EMBALAGENS BIODEGRADÁVEIS ATIVAS
Santa Maria, RS
2021
Thamiris Renata Martiny
OBTENÇÃO DE EXTRATOS DE FOLHAS DE OLIVEIRA PARA
APLICAÇÃO COMO AGENTE ANTIMICROBIANO EM
EMBALAGENS BIODEGRADÁVEIS ATIVAS
Tese apresentada ao programa de Pós-
graduação em Engenharia Química da
Universidade Federal de Santa Maria, como
requisito parcial para obtenção do Título de
Doutora em Engenharia Química.
Orientador: Prof. Dr. Guilherme Luiz Dotto
Santa Maria, RS
2021
DEDICATÓRIA
Dedico esse estudo a todos os profissionais que fazem ciência e pesquisa, que apesar dos
percalços seguem firmes na construção de um mundo melhor e que, em especial, nesse último
ano que se passou mostraram-se ainda mais imprescindíveis.
AGRADECIMENTOS
A Deus pela presença constante, me dando força em todos os momentos.
Aos meus pais, que sempre incentivam e apoiam a evolução da minha qualificação. Não há
palavra que descreva minha gratidão. Muito obrigada!
Aos meus irmãos, Renan e Ygor, pela parceria de sempre.
Ao meu amado companheiro de vida, Raimar, pela dedicação, compreensão, apoio, dicas e
imensa ajuda para realização desse trabalho, sobretudo na revisão do idioma estrangeiro.
Aos meus familiares, que sempre estiveram presentes na minha caminhada, ajudando com
extrema generosidade nos momentos em que precisei.
À Profª. Drª. Gabriela Silveira da Rosa, por sua orientação, dedicação, amizade, pela
confiança, pelos ensinamentos e pelo incentivo para que eu me aprimorasse permitindo meu
amadurecimento profissional.
Ao Prof. Dr. Guilherme Luiz Dotto por sua orientação, atenção, disponibilidade sempre e pela
liberdade no desenvolvimento deste trabalho.
Aos membros da banca, Profª. Drª. Caroline Costa Moraes, Profª. Drª. Mariana Agostini de
Moraes, Profª. Drª. Raquel Cristine Kuhn e Drª Kátia Regina Kuhn pela disponibilidade.
Aos técnicos dos laboratórios onde o trabalho foi desenvolvido, pela imensa ajuda e
colaboração, afinal o êxito também depende de vocês.
À estância Guarda Velha de Pinheiro Machado, na figura do Eng. Vinícius Leite e demais
colaboradores, pela disponibilidade das amostras.
Aos meus colegas de trabalho da Eletrobras CGTELETROSUL, pela compreensão, incentivo
e ajuda, em especial a Quelen, ao Giordâni, ao Nelson e ao Wagner.
À Camila e ao Theilor, que generosamente, me abrigaram em sua residência nos dias de aula
em Santa Maria.
Ao Grupo de Pesquisa Engenharia Processos e Sistemas Particulados (GPEPSP) pela parceria
e compartilhamento de saberes.
À Universidade Federal do Pampa (UNIPAMPA) pela infraestrutura e todo apoio de sempre.
À McGill University na figura do Professor Vijaya Raghavan pela oportunidade de realizar
uma parte importante dessa tese utilizando sua infraestrutura física e intelectual.
Ao Programa de Pós-Graduação em Engenharia Química da Universidade Federal de Santa
Maria (UFSM), o qual proporcionou meu desenvolvimento acadêmico oportunizando a
realização do Doutorado.
A todos que colaboraram e me ajudaram direta ou indiretamente ao longo do doutorado e na
elaboração desse trabalho, o meu reconhecimento.
“Na vida, não existe nada a temer, mas a entender.”
(Marie Curie)
RESUMO
OBTENÇÃO DE EXTRATOS DE FOLHAS DE OLIVEIRA PARA
APLICAÇÃO COMO AGENTE ANTIMICROBIANO EM
EMBALAGENS BIODEGRADÁVEIS ATIVAS
AUTOR: Thamiris Renata Martiny
ORIENTADOR: Prof. Dr. Guilherme Luiz Dotto
COORIENTADORA: Profª. Drª. Gabriela Silveira da Rosa
Este trabalho teve como objetivo obter extratos de folhas de oliveira (EFO) através da técnica
de extração assistida por micro-ondas para posterior aplicação dos extratos obtidos na
elaboração de filmes biodegradáveis ativos. O processo de extração, assim como a influência
dos parâmetros do processo sobre o conteúdo de compostos bioativos presentes no extrato
aquoso foram estudados utilizando planejamento de experimentos. O conteúdo de compostos
fenólicos totais (FT), a atividade antioxidante (AA), a atividade antimicrobiana do EFO
contra a Escherichia coli (E. coli) e o conteúdo de oleuropeína obtidos sob condições ótimas
de extração foram às respostas avaliadas. Complementarmente, foram estudados os métodos
de extração por maceração e assistido por ultrassom para obtenção dos extratos de folha de
oliveira. Após, foram elaborados filmes à base de carragenana com e sem a adição de EFO
pelo método de casting. Os filmes foram avaliados quanto à cor, solubilidade, espessura,
permeabilidade ao vapor d’água e propriedades mecânicas. O efeito protetor dos filmes contra
a atividade antimicrobiana na embalagem de carne de cordeiro também foi avaliado. As
condições ótimas de extração assistida por micro-ondas, utilizando a ferramenta estatística da
desejabilidade, foram: 100°C, 2 min e pH 6. Nessas condições é possível obter AA de
92,87%, FT de 103,87 mgGAE.g–1 (b.s.), além de atividade antimicrobiana contra E. coli, com
valor de concentração inibitória mínima de 50 mg.mL-1 e conteúdo de oleuropeína de 11,59
mg.g-1 (b.s.). Quando comparada às tecnologias de extração por maceração e ultrassom, a
tecnologia de micro-ondas se mostrou mais eficiente, apresentando uma maior recuperação de
compostos bioativos de interesse. A incorporação do EFO na matriz de carragenana provocou
mudanças nas propriedades dos filmes, com o aumento na espessura, na elongação e na
permeabilidade ao vapor d’água, e a diminuição na tensão de ruptura e no módulo de
elasticidade do filme. A solubilidade não foi significativamente afetada pela adição do EFO,
porém a diferença de cor com a adição de EFO foi de 64,72%. Outro importante resultado foi
o da análise in vivo de psicrófilos, ou seja, quando aplicados na embalagem de carne de
cordeiro os filmes de carragenana com extrato apresentaram efeito inibitório na contagem de
psicrófilos. O estudo revelou os benefícios do extrato de folhas de oliveira, obtido por micro-
ondas incorporado em filmes à base de carragenana com potencial para a aplicação como
embalagens ativas.
Palavras-chaves: Olea europea, carragenana, micro-ondas, filme biodegradável
ABSTRACT
OBTAINING OLIVE LEAF EXTRACTS FOR APPLICATION AS AN
ANTIMICROBIAL AGENT IN ACTIVE BIODEGRADABLE PACKAGING
AUTHOR: Thamiris Renata Martiny
ADVISOR: Pro. Dr. Guilherme Luiz Dotto
COADVISOR: Profª. Drª. Gabriela Silveira da Rosa
This work had as objective to obtain extracts of olive leaves (OLE) through the technique of
extraction assisted by microwaves for later application of the extracts obtained in the
elaboration of active biodegradable films. The extraction process, as well as the influence of
the process parameters on the content of bioactive compounds present in the aqueous extract,
were studied using design of experiments. The content of total phenolic compounds (PC), the
antioxidant activity (AA), the antimicrobial activity of OLE against Escherichia coli (E. coli)
and the content of oleuropein obtained under optimal conditions of extraction were the
responses evaluated. In addition, methods of extraction by maceration and assisted by
ultrasound to obtain olive leaf extracts were studied. Afterwards, carrageenan films were
made with and without the addition of OLE by the casting method. The films were evaluated
for color, solubility, thickness, water vapor permeability and mechanical properties. The
protective effect of films against antimicrobial activity on lamb meat packaging was also
evaluated. The optimal conditions for microwave assisted extraction, using the desirability
statistical tool, were: 100°C, 2 min and pH 6. Under these conditions it is possible to obtain
AA of 92.87%, PC of 103.87 mgGAE.g–1 (d.b), in addition to antimicrobial activity against E.
coli, with a minimum inhibitory concentration value of 50 mg.mL-1 and oleuropein content of
11.59 mg.g-1 (d.b). When compared to the extraction technologies by maceration and
ultrasound, the microwave technology proved to be more efficient, presenting a greater
recovery of bioactive compounds of interest. The incorporation of OLE in the carrageenan
matrix caused changes in the properties of the films, with an increase in thickness, elongation
and permeability to water vapor, and a decrease in the tensile strength and elasticity modulus
of the film. The solubility was not significantly affected by the addition of OLE, however the
color difference with the addition of OLE was 64.72%. Another important result was the in
vivo analysis of psychrophiles, that is, when applied to the packaging of lamb meat,
carrageenan films with extract had an inhibitory effect on the count of psychrophiles. The
study revealed the benefits of olive leaf extract, obtained by microwave incorporated in films
based on carrageenan with potential for application as active packaging.
Keywords: Olea europea, carrageenan, microwave, biodegradable film
LISTA DE ILUSTRAÇÕES
CAPÍTULO 1
Figura 1 - Estrutura da Tese ..................................................................................................... 21
CAPÍTULO 2
Figura 1 - Apresentação da planta ............................................................................................ 27
Figura 2 - Folhas de oliveira ..................................................................................................... 29
Figura 3 - Estruturas básicas das carragenanas ........................................................................ 39
CAPÍTULO 3
Fig. 1. Kinetic growth profiles of E. coli against olive leaf extracts. MAE=microwave-assisted
extraction; UAE=ultrasound-assisted extraction; NC=negative control; PC=positive
control.......................................................................................................................................58
CAPÍTULO 4
Fig. 1. Olive leaves. .................................................................................................................. 71
Fig. 2. Central rotational composite design for microwave-assisted extraction (MAE) with
experimental values and coded levels of independent variables. ............................................. 72
Fig. 3. Extracts obtained from microwave extraction. The numbers correspond to each extract
obtained in the respective extraction condition (1-17) of Table 1. ........................................... 75
Fig. 4. Pareto chart of the responses (a) total phenolic compounds and (b) antioxidant activity
.................................................................................................................................................. 76
Fig. 5. Profile of predicted values and desirability for antioxidant activity and total phenolic
compounds for microwave extraction. ..................................................................................... 79
CAPÍTULO 5
Figure 1. Samples of lamb meat that were packed using different films: (A) polyvinyl chloride
(PVC) film, (B) carrageenan control film (CAR-C), and (C) carrageenan film with olive leaf
extract (CAR-OLE). ............................................................................................................... 100 Figure 2. Visual appearance of the carrageenan films: (A) CAR-C and (B) CAR-OLE. CAR–
C: carrageenan film, CAR–OLE: carrageenan film with olive leaf extract. .......................... 101
LISTA DE TABELAS
CAPÍTULO 2
Tabela 1 - Aplicação de filmes biodegradáveis incorporados com antimicrobianos ou
antioxidantes. ............................................................................................................................ 41
CAPÍTULO 3
Table 1. Total phenolic (TP), antioxidant activity (AA), oleuropein (OP) and hydroxytyrosol
(HT). ......................................................................................................................................... 55 Table 2. Antimicrobial Inhibition (AI) effect of extracts obtained from MAE and UAE against
E. coli. ....................................................................................................................................... 59
CAPÍTULO 4
Table 1. Total phenolic compounds (T.phenolics) and antioxidant activity (Antioxidant.A) of
extracts obtained by microwave. .............................................................................................. 75 Table 2. Analysis of variance for phenolic compounds. .......................................................... 77 Table 3. Analysis of variance for antioxidant activity.............................................................. 78
Table 4. Parameters used in the simultaneous optimization of microwave extraction responses.
.................................................................................................................................................. 78
CAPÍTULO 5
Table 1. Thickness, water vapor permeability (WVP), mechanical properties, solubility, and
optical properties of carrageenan films. ................................................................................. 103 Table 2. The psychrophiles population measured initially and after 2 days of storage. ........ 105
LISTA DE ABREVEATURAS E SIGLAS
AA - Atividade Antioxidante - Antioxidant Activity
A.activity - Antioxidant activity
AI - Antimicrobial Inhibition
ANOVA - Análise de variância
Antioxidant.A - Antioxidant Activity
ASTM -American Society for Testing and Materials
b.s - base seca
CAPES - Coordination for the Improvement of Higher Education Personnel and the Natural
Sciences
CAR-C - Carrageenan Control Film
CAR-OLE - Carrageenan film with olive leaf extract
CCRD - Central Composite Rotational Design
CNPq - Brazilian National Counsel of Technological and Scientific Development
CFU - Colony-Forming Unit
d.b - dry basis
DPPH - 2,2-diphenyl-1-picrylhydrazyl
DSC - Calorimetria exploratória diferencial
E. coli - Escherichia coli
EB - elongation at break
EFO - Extrato de Folhas de Oliveira
EM - Elastic Modulus
FT - Fenólicos Totais
GAE - Ácido Gálico
HPLC - High performance liquid chromatography
HT - Hydroxytyrosol
MAE - Microwave-Assisted Extraction
MIC - Minimum inhibitory concentration
Microbial.A - Antimicrobial Activity
NC - Negative Control
NSERC - Engineering Research Council of Canada
OLE - Olive Leaf Extracts
O.L.Extracts - Olive Leaves Extracts
OP - Oleuropein
PET - Polietileno Tereftalato
PC - Phenolic Compounds
PVA - Permeabilidade ao Vapor de Água
PVC - Policloreto de Vinila
TGA - Análise Termogravimétrica
TP - Total Phenolic
T.ph - Phenolic Compounds
T.phenolics - Total Phenolic Compounds
TS - Tensile Strength
UAE - Ultrasound-Assisted Extraction
UFSM - Universidade Federal de Santa Maria
UNIFESP - Universidade Federal de São Paulo
UNIPAMPA - Universidade Federal do Pampa
U.K. - United Kingdom
USA - United States of America
WS - Water Solubility
VWD - Variable wavelength detector
WVP - Water Vapor Permeability
LISTA DE SÍMBOLOS
a* standard CIE Lab [ - ]
a exposed film surface m²
AA antioxidant activity %
A. activity antioxidant activity %
𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙 absorbance of the blank solution [ - ]
𝐴C absorbance of the control [ - ]
𝐴OLE absorbance of the extract samples [ - ]
𝐴𝑠𝑎𝑚𝑝𝑙𝑒 absorbance of the extract solution [ - ]
AI antimicrobial inhibition %
𝐴1OLE initial extract absorbance [ - ]
𝐴2OLE final absorbance of the extract [ - ]
𝐴1C initial absorbance of the control [ - ]
𝐴2C final absorbance of the control [ - ]
b* standard CIE Lab [ - ]
e thickness m
EB elongation at break %
EM elastic modulus MPa
Microbial.A antimicrobial activity %
L* standard CIE Lab [ - ]
mi initial dry mass g
mf final dry mass g
𝑂𝐷𝑒𝑥𝑡2 optical density of the sample after the
incubation period [ - ]
𝑂𝐷𝑒𝑥𝑡1 optical density of the sample before the
incubation period [ - ]
𝑂𝐷𝑐𝑜𝑛𝑡𝑟𝑜𝑙2 optical density of the control after the
incubation period [ - ]
𝑂𝐷𝑐𝑜𝑛𝑡𝑟𝑜𝑙1 optical density of the control before the
incubation period [ - ]
t time s
TS tensile strength MPa
WVP water vapor permeability g.m-1.Pa-1.s-1
W absorbed moisture g
WS water solubility %
ΔE* color difference %
ΔP partial pressure difference Pa
𝛽0 constant coefficient [ - ]
𝛽𝑖 linear coefficient [ - ]
𝛽𝑖𝑖 quadratic coefficient [ - ]
𝛽𝑖𝑗 cross-product coefficient [ - ]
error [ - ]
Χ𝑖 non-coded values of independent
variables [ - ]
𝛶 predicted response [ - ]
SUMÁRIO
CAPÍTULO 1 - ........................................................................................................................ 19 ESTRUTURA DA TESE, INTRODUÇÃO E OBJETIVOS .............................................. 19 1.1 ESTRUTURA DA TESE ................................................................................................... 20 1.2 INTRODUÇÃO .................................................................................................................. 22
1.3 OBJETIVOS ....................................................................................................................... 25
1.3.1 Objetivo geral ................................................................................................................. 25 1.3.2 Objetivos específicos ...................................................................................................... 25 - CAPÍTULO 2 - ..................................................................................................................... 26 REVISÃO DA LITERATURA ............................................................................................. 26 2.1 A OLIVEIRA ..................................................................................................................... 27 2.2 AS FOLHAS DE OLIVEIRA E SEUS EXTRATOS ........................................................ 28
2.3 MÉTODOS DE EXTRAÇÃO ............................................................................................ 30
2.3.1 Extração por Maceração ............................................................................................... 31 2.3.2 Extração assistida por ultrassom ................................................................................. 32 2.3.3 Extração assistida por micro-ondas ............................................................................. 33 2.4 SISTEMAS DE EMBALAGENS ...................................................................................... 34
2.4.1 Embalagens ativas ......................................................................................................... 34
2.4.2 Embalagens biodegradáveis ......................................................................................... 36 2.5 BIOPOLÍMEROS ............................................................................................................... 37
2.5.1 Carragenanas ................................................................................................................. 38 2.6 FILMES BIODEGRADÁVEIS .......................................................................................... 40
2.6.1 Técnicas para a preparação de filmes biodegradáveis ............................................... 41
2.6.2 Propriedades de filmes biodegradáveis ....................................................................... 43
- CAPÍTULO 3 - ..................................................................................................................... 47 MÉTODOS DE EXTRAÇÃO PARA PRODUÇÃO DO EXTRATO DE FOLHA DE
OLIVEIRA .............................................................................................................................. 47 3.1 INTRODUCTION .............................................................................................................. 50 3.2 MATERIALS AND METHODS ....................................................................................... 51
3.2.1 Reagents .......................................................................................................................... 51
3.2.2 Sample Preparation ....................................................................................................... 52 3.2.3 Extraction Procedure .................................................................................................... 52 3.2.4 Total Phenolics (TP) ...................................................................................................... 52
3.2.5 Antioxidant Activity (AA) ............................................................................................. 53 3.2.6 Oleuropein (OP) and Hydroxytyrosol (HT) ................................................................ 53
3.2.7 Antimicrobial Inhibition (AI) ....................................................................................... 53 3.2.8 Statistical analysis .......................................................................................................... 54 3.3 RESULTS AND DISCUSSION ......................................................................................... 54 3.4 CONCLUSION .................................................................................................................. 59
- CAPÍTULO 4 - ..................................................................................................................... 65 OTIMIZAÇÃO DA EXTRAÇÃO DE COMPOSTOS BIOATIVOS DA FOLHA DE
OLIVEIRA .............................................................................................................................. 65 4.1 INTRODUCTION .............................................................................................................. 68 4.2 MATERIALS AND METHODS ....................................................................................... 70
4.2.1. Olive leaves .................................................................................................................... 70 4.2.2 Chemicals ....................................................................................................................... 71 4.2.3 Apparatus and extraction ............................................................................................. 71 4.2.4 Experimental design ...................................................................................................... 72 4.2.5 Estimation of total phenolic compounds and antioxidant activity ............................ 73
4.2.6 Potential of the leaf extract as a food preservative ..................................................... 73
4.2.7 Statistical Analysis ......................................................................................................... 74 4.3 RESULTS AND DISCUSSION ......................................................................................... 74
4.3.1 Optimization of microwave-assisted extraction conditions ....................................... 74 4.3.2 Oleuropein content and antimicrobial action of the O.L.Extracts ........................... 81 4.4 CONCLUSIONS ................................................................................................................ 83
- CAPÍTULO 5 - ..................................................................................................................... 90 EXTRATO DE FOLHA DE OLIVEIRA PARA OBTENÇÃO DE EMBALAGEM
ATIVA ..................................................................................................................................... 90 5.1 INTRODUCTION .............................................................................................................. 93 5.2 MATERIALS AND METHODS ....................................................................................... 95
5.2.1 Materials ......................................................................................................................... 95 5.2.1.1 Olive Leaves ................................................................................................................. 95
5.2.1.2 Chemicals ..................................................................................................................... 95
5.2.1.3 Bacterial Isolates .......................................................................................................... 95
5.2.2 Preparation of the Plant Extract .................................................................................. 96 5.2.3 Extract Characterization .............................................................................................. 96
5.2.4 Carrageenan Biodegradable Films .............................................................................. 97 5.2.4.1 Film Preparation .......................................................................................................... 97 5.2.4.2 Film Properties ............................................................................................................. 98
5.2.5 Storage Study: Inhibition of Psychrophiles in Lamb Meat ....................................... 99 5.2.6 Statistical Analysis ....................................................................................................... 100 5.3 RESULTS AND DISCUSSION ....................................................................................... 100
5.3.1 Olive Leaf Extract ....................................................................................................... 100
5.3.2 Film Evaluation ........................................................................................................... 101
5.3.3 Storage Study ............................................................................................................... 105 5.4 CONCLUSIONS .............................................................................................................. 106
- CAPÍTULO 6 - ................................................................................................................... 114 DISCUSSÃO, CONCLUSÃO GERAL E SUGESTÕES PARA TRABALHOS
FUTUROS ............................................................................................................................. 114 6.1 DISCUSSÃO .................................................................................................................... 115
6.2 CONCLUSÃO GERAL ................................................................................................... 118 6.3 SUGESTÕES PARA TRABALHOS FUTUROS ............................................................ 118
REFERÊNCIAS ................................................................................................................... 120
APÊNDICE – PÁGINAS INICIAIS DOS ARTIGOS PUBLICADOS ........................... 129
19
- CAPÍTULO 1 -
ESTRUTURA DA TESE, INTRODUÇÃO E OBJETIVOS
20
1.1 ESTRUTURA DA TESE
Nessa tese, as etapas de desenvolvimento da pesquisa estão apresentadas em 6
capítulos. Neste Capítulo 1 - ESTRUTURA DA TESE, INTRODUÇÃO E OBJETIVOS -
são apresentados, concisamente, o tema principal do estudo, os objetivos pretendidos e as
etapas envolvidas para sua realização. As atividades propostas e realizadas são apresentadas
na Figura 1.
No Capítulo 2 - REVISÃO DA LITERATURA - uma revisão da literatura foi
realizada abrangendo a caracterização da folha de oliveira, as tecnologias de extração
convencionais ou não para obtenção de extratos e o tema das embalagens ativas e as matérias-
primas para sua produção.
Nos capítulos subsequentes estão apresentados os resultados obtidos nesse trabalho na
forma de quatro artigos. Os mesmos encontram-se conforme os moldes das revistas a que
foram submetidos e, subsequentemente, publicados.
Artigo 1 – Eco-friendly extraction for the recovery of bioactive compounds from
Brazilian olive leaves.
Artigo 2 – Optimization of green extraction for the recovery of bioactive compounds
from Brazilian olive crops and evaluation of its potential as a natural preservative.
Artigo 3 – Bio-Based Active Packaging: Carrageenan Film with Olive Leaf Extract
for Lamb Meat Preservation.
O desenvolvimento experimental da pesquisa foi realizado utilizando a infraestrutura
dos laboratórios de Engenharia Química e Engenharia de Alimentos, da UNIPAMPA
(Universidade Federal do Pampa), Bagé, Rio Grande do Sul, Brasil, do laboratório de
Engenharia Química da UFSM (Universidade Federal de Santa Maria), Santa Maria, Rio
Grande do Sul, Brasil e do Bioresource Engineering Laboratory da McGill University,
Montreal, Quebec, Canadá.
No Capítulo 3 - MÉTODOS DE EXTRAÇÃO PARA OBTENÇÃO DO EXTRATO
DE FOLHA DE OLIVEIRA - foram estudadas as técnicas de extração – maceração, extração
assistida por ultrassom e extração assistida por micro-ondas– a fim de se selecionar a melhor
técnica em termos de recuperação de compostos de interesse bioativo e parâmetros
operacionais.
No Capítulo 4 - OTIMIZAÇÃO DA EXTRAÇÃO DOS COMPOSTOS BIOATIVOS
DA FOLHA DE OLIVEIRA - são apresentados os resultados experimentais da pesquisa
envolvendo a otimização da extração assistida por micro-ondas de compostos bioativos das
21
folhas de oliveira. Foram avaliados os parâmetros de extração tempo, temperatura e pH na
atividade antioxidante e os compostos fenólicos dos extratos produzidos.
No Capítulo 5 - EXTRATO DE FOLHA DE OLIVEIRA PARA OBTENÇÃO DE
EMBALAGEM ATIVA - é apresentada uma alternativa de aplicação do extrato de folha de
oliveira obtido. O extrato foi incorporado na matriz polimérica de carragenana para produção
de filmes que funcionam como embalagem ativa.
No Capítulo 6 - DISCUSSÃO, CONCLUSÃO GERAL E SUGESTÕES PARA
TRABALHOS FUTUROS - são resumidos os principais resultados oriundos do
desenvolvimento da tese. Este capítulo reúne as informações mais relevantes obtidas nos
capítulos 3 a 6, bem como apresenta algumas sugestões de pesquisas futuras.
Figura 1 - Estrutura da Tese
Fonte: Autora (2021).
Artigo 1
Artigo 2
Artigo 3
Maceração
Ultrassom
Micro-ondas
22
1.2 INTRODUÇÃO
A Olea europaea L., conhecida como oliveira, é uma planta nativa da região do
Mediterrâneo, perfazendo áreas da ordem de milhões de hectares. Mas essa cultura vem se
expandindo para outros territórios, principalmente no Brasil na região Sul. O cultivo da
oliveira vem crescendo e mudando a paisagem, antes apenas com vastos campos agrícolas,
agora como olivais a perder de vista. As terras brasileiras se mostraram boas para o cultivo da
oliveira, produzindo azeites premiados mundialmente (CAVALHEIRO et al., 2015;
COUTINHO et al., 2009; FOSCOLOU; CRITSELIS; PANAGIOTAKOS, 2018). Haja vista
essa expansão e que as condições climáticas e locacionais influenciam a composição das
plantas (HODAIFA et al., 2019; TALHAOUI et al., 2014), pesquisas com as lavouras
cultivadas no Brasil são fundamentais.
Do ponto de vista econômico, o interesse pelas oliveiras está ligado à produção de
azeitonas e azeite, porém, as folhas de oliveira são ricas em compostos com potencial
bioativo, os quais exibem propriedades antioxidantes e antimicrobianas, as quais têm sido
relacionadas com suas características conservantes (ALBUQUERQUE et al., 2018;
BENAVENTE-GARCÍA et al., 2000; WICHERS; SOLER-RIVAS; ESPI, 2000). Além disso,
outra vantagem da utilização de folhas de oliveira é que elas são consideradas como um
subproduto da indústria do azeite, podem representar entre 5% e 10% em peso da azeitona
que entra para processamento (BOUDHRIOUA et al., 2008; EL; KARAKAYA, 2009;
HERRERO; CIFUENTES; IBAÑEZ, 2006). Nesse contexto, a incorporação de extrato de
folhas de oliveira em sistemas de embalagem permitiria a substituição de aditivos sintéticos,
como os conservantes alimentícios.
Ainda é possível notar que o uso das folhas de oliveira como extrato valoriza esse
subproduto trazendo uma perspectiva sustentável dentro do contexto produtivo em que são
geradas. Não obstante, destaca-se que a olivicultura, por se tratar de uma cultura incipiente na
região sul do Brasil, não possui muitas pesquisas relacionadas aos seus subprodutos gerados,
como as folhas. Ressaltando-se que a origem geográfica da planta representa mudanças em
sua composição, que consequentemente impacta no seu potencial bioativo.
A diminuição do uso de conservantes químicos é uma tendência atual, devido aos
possíveis riscos à saúde. Na última década, o mercado nutracêutico aumentou a demanda pela
produção e incorporação de conservantes naturais em uma ampla gama de produtos (AHMAD
et al., 2011; GARCÍA-MORENO et al., 2014). No entanto, como qualquer mudança, no
mercado de alimentos, sofre com a adaptação necessária no setor industrial, sendo o processo
23
de extração de aditivos apontado como a principal dificuldade (ALBUQUERQUE et al.,
2018).
Existem diversos métodos relacionados à extração dos compostos bioativos de
materiais vegetais, porém cada um possui vantagens e desvantagens. São requeridos processos
de extração que combinem requisitos de gasto energético razoável com tempos curtos e uso
reduzido de solventes, bem como a escolha de solventes atóxicos, mantendo a eficiência de
extrações sólido-líquido, convencionais, como por exemplo, a maceração
(MEULLEMIESTRE et al., 2016; WANG; WELLER, 2006). Sendo assim, técnicas de
extração não convencionais como extração assistida por micro-ondas, extração assistida por
ultrassom e extração pelo uso de fluido supercrítico estão sendo aplicadas para obter valiosos
compostos bioativos de matrizes naturais (ROSELLÓ-SOTO et al., 2015).
Atualmente, a fim de atender a demanda de embalagens para a indústria alimentícia,
milhares de toneladas de polímeros sintéticos são produzidos, como o policloreto de vinila
(PVC) e o polietileno tereftalato (PET). Apesar de suas inúmeras vantagens, a maioria desses
polímeros é resistente à biodegradação, causando sérios problemas ambientais com seu
descarte (ABDOU; SOROUR, 2014; FANG et al., 2005). Esses problemas enfatizam a
importância de desenvolver alternativas de embalagens que possam substituir o uso de
polímeros convencionais. Dessa forma, a embalagem antimicrobiana à base de biopolímeros,
as chamadas embalagens biodegradáveis ativas, que podem ser na forma de filmes
biodegradáveis, apresenta-se como uma importante alternativa (ANDRADE-MAHECHA;
TAPIA-BLÁCIDO; MENEGALLI, 2012; ROBERTSON, 2013).
As pesquisas de novas fontes para a produção de filmes biodegradáveis cresceram nos
últimos anos. Em particular, fontes não convencionais e pouco exploradas têm sido alvo de
interesse, como as fontes de origem marinha (alginato e quitosana). Porém, ainda as principais
tecnologias empregadas para solucionar o problema são o desenvolvimento de filmes a partir
de amido extraídos das mais diversas matrizes, como batata e milho, por exemplo.
Recentemente, estudos referentes a produção de filmes biodegradáveis com a incorporação de
aditivos antimicrobianos e antioxidantes para aplicação como embalagem foram publicados
(ALBERTOS et al., 2017; BERMÚDEZ-ORIA et al., 2017; NUR FATIN NAZURAH; NUR
HANANI, 2017).
O biopolímero estudado nesse trabalho para a produção de filmes biodegradáveis foi a
carragenana pertencente à família dos polissacarídeos, que possui a capacidade de formar
bons filmes, aqueles que são capazes de serem competitivos com os filmes plásticos
comercializados, apresentando as propriedades físicas requeridas (ABDOU; SOROUR, 2014;
24
PARK, 1996; SHOJAEE-ALIABADI et al., 2014). Ela é um hidrocolóide galactano sulfatado
extraído das algas vermelhas (Rhodophyta) (SMIDSROD; GRASDALEN, 1982). As
carragenanas têm propriedades que lhes garantem aplicação comercial como aditivos na
indústria alimentícia, sendo agentes gelificantes, emulsificantes, estabilizantes e espessantes
(WILLIAMS; PHILLIPS, 2009). Além disso, vêm ganhando gradativamente reconhecimento
como fonte de novos materiais com valiosas aplicações. Por isso, é necessário intensificar a
sua pesquisa, a fim de descobrir novas funcionalidades, aproveitando ao máximo seu
potencial, entre eles a produção de filmes biodegradáveis.
Face ao contexto apresentado, a incorporação de um composto bioativo natural - o
extrato de folhas de oliveira - para a produção de uma embalagem baseada em uma matriz
polimérica biodegradável - as carragenanas - representa uma direção tanto para a substituição
de plásticos não biodegradáveis, como para a extensão do prazo de validade de produtos
alimentícios sem adição de conservantes químicos.
Em linhas gerais, até o momento, não foram encontrados trabalhos na literatura, a
partir das plataformas de buscas científicas, que utilizam as folhas de oliveira originária do sul
do Brasil para a otimização da obtenção de seus extratos, a fim de estudar seu potencial
bioativo e empregá-los como conservante natural em embalagens ativas a base de
carragenana. Procura-se demostrar que os extratos constituem uma excelente fonte de
recuperação de compostos bioativos e que a nova embalagem desenvolvida possui
propriedades desejáveis. Essa hipótese foi testada aplicando a nova embalagem no
armazenamento de carne de cordeiro. Assim, a proposta dessa pesquisa surge como uma
alternativa inovadora, que busca integrar o reaproveitamento de um resíduo na produção de
uma nova embalagem, com vistas à preservação do meio ambiente e proteção dos alimentos e
da saúde humana.
25
1.3 OBJETIVOS
1.3.1 Objetivo geral
Este trabalho teve como objetivo geral obter extrato de folhas de oliveira para
posterior aplicação do extrato na elaboração de filme biodegradável ativo.
1.3.2 Objetivos específicos
Obtenção extratos das folhas de oliveira a partir da extração assistida por micro-ondas e
ultrassom e a extração por maceração;
Otimizar as condições de extração do método avaliado, a partir dos resultados de
compostos fenólicos e atividade antioxidante;
Comparar a extração assistida por micro-ondas com a extração por maceração e a
extração assistida por ultrassom na obtenção de extratos de folha de oliveira;
Determinar o potencial de inibição do extrato na condição otimizada contra Escherichia
coli;
Determinar o conteúdo de oleuropeína e hidroxitirosol dos extratos;
Produzir filmes biodegradáveis de carragenana com incorporação do extrato de folha de
oliveira;
Caracterizar os filmes biodegradáveis obtidos quanto à espessura média, às suas
propriedades mecânicas (tensão de ruptura, elongamento e módulo de elasticidade),
permeabilidade ao vapor d’água e cor.
Avaliar o potencial de aplicação do filme biodegradável como embalagem ativa em
carne de cordeiro.
26
- CAPÍTULO 2 -
REVISÃO DA LITERATURA
27
2.1 A OLIVEIRA
Majoritariamente cultivada na região Mediterrânea a Olea europaea L., popularmente
conhecida como oliveira, é uma planta arbórea e é a única espécie da família Oleaceae que
apresenta fruto comestível, as azeitonas. Além da produção de azeitonas, é usada para fins
ornamentais e produção de azeite (COUTINHO et al., 2009; FLORES et al., 2013).
A planta (Figura 1) é de tamanho médio, de formato arredondado, com tronco
contorcido, casca grossa e folhas perenes. Suas características como porte, densidade da copa,
cor da madeira e folha variam em função das condições de cultivo e do cultivar (RAPOPORT,
1998).
Figura 1 - Apresentação da planta
Fonte: Autora (2021).
Os países europeus apresentam 75% dos olivais do mundo, porém as áreas nesses
países já estão exauridas, havendo uma tendência da ocupação dessa cultura em novas áreas
(RAMOS et al., 2013; WREGE et al., 2015). O Brasil figura entre os principais países
importadores de azeitonas e azeite, sendo o quinto país que mais consome azeite (WREGE et
al., 2015). A olivicultura no Brasil ainda é embrionária, porém com o passar dos anos análises
laboratoriais comprovaram que o azeite brasileiro não perde em qualidade para os produtos
importados, assim impulsionando o plantio, sendo que os estados das regiões Sul e Sudeste se
28
destacam na produção. Mais especificamente, no Rio Grande do Sul as áreas com plantios
comerciais estão localizadas principalmente nas cidades de Bagé, Cachoeira do Sul, Caçapava
do Sul, Dom Pedrito, Encruzilhada do Sul, Rio Grande, Santana do Livramento e Vacaria
(COUTINHO et al., 2009).
A extração do azeite e a produção de azeitonas representam uma atividade econômica
e social altamente relevante. Do ponto de vista nutricional esses produtos são recomendados
por estarem associados a benefícios à saúde (RODRIGUES; PIMENTEL; OLIVEIRA, 2015).
Durante muitos séculos, a folha de oliveira ou os seus extratos foram associados à saúde e
preservação. Na medicina popular estes foram usados para tratar diabetes e hipertensão
(BENAVENTE-GARCÍA et al., 2000; BOCK; ROMBOUTS, 2013).
2.2 AS FOLHAS DE OLIVEIRA E SEUS EXTRATOS
O aumento da demanda pela valorização de resíduos e subprodutos do processo de
produção de azeite levou a indústria de alimentos a explorar novas alternativas. Durante o
processamento do azeite, uma variedade de resíduos e subprodutos é produzida. Os principais
com interesse nutricional e tecnológico, são o bagaço de azeitona, as águas residuais, as folhas
de oliveira e a semente, que podem ser valorizados compondo produtos inovadores (NUNES
et al., 2016; RODRIGUES; PIMENTEL; OLIVEIRA, 2015).
As folhas são consideradas um subproduto gerado em grande quantidade na prática da
olivicultura. Esse subproduto é encontrado principalmente em dois pontos durante a produção
do azeite. Em primeiro lugar, são descartadas durante a poda das oliveiras, cada árvore de
oliveira produz cerca de 25 kg de folhas e ramos e, em segundo lugar, na instalação de
produção de azeite, onde as folhas são separadas das azeitonas por uma máquina sopradora, e
podem representar entre 5% e 10% em peso, da azeitona que entra para o processamento. Só
na Espanha, são geradas 0,2 milhões de toneladas de folhas de oliveira por ano. Sendo assim,
torna-se interessante a valorização e o aproveitamento desse subproduto devido ao seu baixo
custo e grande acúmulo nessa atividade agrícola (BOUDHRIOUA et al., 2008; EL;
KARAKAYA, 2009; GUINDA et al., 2004; ROMERO-GARCÍA et al., 2014; ROMERO et
al., 2018).
As folhas quando adultas são compridas e estreitas, de forma elíptica, elíptica-
lanceolada e lanceolada. Possuem comprimento variando de 5,0 a 7,0 cm e largura de 1,0 a
1,5 cm e coloração verde escura e brilhante na região ventral e verde acinzentada ou
esbranquiçada na região dorsal (RAPOPORT, 1998). A Figura 2 ilustra as folhas de oliveira.
29
Figura 2 - Folhas de oliveira
(A) Região ventral da folha. (B) Região dorsal da folha.
Fonte: Autora (2021).
As folhas de oliveira despertam o interesse da comunidade científica e das indústrias
em todo o mundo, uma vez que os seus benefícios de promoção da saúde são constantemente
mostrados por um número cada vez maior de dados científicos (RODRIGUES; PIMENTEL;
OLIVEIRA, 2015). De acordo com De Castro; Capote, (2010) e Japón-Luján; Luque-
Rodrígues; Castro (2006), as folhas têm o maior poder de extração de compostos com
potencial bioativo. Vários estudos mostraram a grande diversidade de fenóis nas folhas de
oliveira (KIRITSAKIS; GOULA; ADAMOPOULOS, 2017; RODRIGUES; PIMENTEL;
OLIVEIRA, 2015; SAHIN; BILGIN, 2018; WICHERS; SOLER-RIVAS; ESPI, 2000). O
composto fenólico mais abundante é a oleuropeína (GIKAS; BAZOTI; TSARBOPOULOS,
2007), cerca de 60-90 mg/g de folha seca, sendo 73% do total de seus constituintes
(SUDJANA et al., 2009). Muitos estudos têm demonstrado a atividade antimicrobiana de
compostos fenólicos, mostrando assim as potencialidades de aplicação desses como
alternativa aos conservantes químicos (NUNES et al., 2016; SAHIN; BILGIN, 2018).
Liu; Mckeever; Malik (2017) investigaram o efeito antimicrobiano de extrato bruto de
folhas de oliveira e demonstraram que na concentração de 62,5 mg/mL, o extrato inibiu quase
A B
30
completamente o crescimento de Listeria monocytogenes, Escherichia coli e Salmonella
Enteritidis. Concluíram que o extrato poderia ser potencialmente utilizado no controle de
agentes patogênicos em produtos alimentares.
Pereira et al. (2006) relataram que os extratos de azeitonas inibiram o crescimento das
bactérias Bacillus cereus, Bacillus subtilis, Staphylococcus aureus, E. coli e Klebsiella
pneumoniae. Sudjana et al. (2009) estudaram a inibição do extrato comercial de folhas de
oliveira para a atividade inibitória de Salmonella enterica, E. coli e Listeria. monocytogenes.
Lee; Lee (2010) e Albertos et al. (2017) não encontraram atividade inibitória do extrato de
folhas de oliveira contra E. coli, embora tenha havido inibição contra outras espécies
bacterianas. Estes resultados divergentes em relação ao efeito inibitório do extrato podem ser
decorrentes de diferentes metodologias de extração e concentração do extrato, bem como o
uso de diferentes solventes na obtenção dos extratos.
Por fim, figura entre as novas tendências o uso de resíduos e subprodutos da azeitona
como fonte de polifenóis, em especial as folhas de oliveira, que podem ser usadas para
diferentes propósitos, como aditivos alimentares. Nesta linha, foram desenvolvidas várias
patentes e aplicações comerciais baseadas na recuperação de polifenóis, especialmente
hidroxitirosol e oleuropeína, a partir de resíduos e subprodutos da produção do azeite
(ROSELLÓ-SOTO et al., 2015).
Todo conteúdo exposto nessa seção referente ao potencial do reaproveitamento das
folhas de oliveira, tornam seus extratos promissores para a aplicação em filmes
biodegradáveis, como um ingrediente com propriedades bioativas.
2.3 MÉTODOS DE EXTRAÇÃO
A extração de produtos naturais é uma operação de retirada seletiva e eficaz de
compostos ou frações de interesse, que estão dentro de uma matriz vegetal (SIMÕES et al.,
2010). Porém, cada material vegetal possui suas propriedades características em termos da
extração de compostos biologicamente ativos, sendo muito importante desenvolver as
condições ótimas de extração. O sucesso da extração é influenciado por inúmeros fatores, mas
principalmente pela estabilidade térmica dos compostos ativos, tecnologia de extração, pH,
tipo de solvente, temperatura e tempo de extração (PANJA, 2017; PUTNIK et al., 2017a;
ROSELLÓ-SOTO et al., 2015). Por isso, é importante avaliar várias tecnologias que
favorecerão a estabilidade dos compostos bioativos durante as extrações, que sejam
ecologicamente corretas, eficientes e custo-efetivo.
31
Existem muitos métodos e patentes para extrair compostos fenólicos das folhas de
oliveira, pois as folhas podem conter até 70 g de compostos fenólicos /kg de folhas
(ROMERO et al., 2018). Os métodos de extração mais utilizados são a maceração, a extração
assistida por ultrassom e extração assistida por micro-ondas os quais são descritos em maiores
detalhes nas próximas seções.
2.3.1 Extração por Maceração
A extração por maceração é um método tradicional que resulta em um menor
rendimento de compostos fenólicos, porém ainda é a principal técnica de extração utilizada
(DENG et al., 2017). É um exemplo de extração sólido-líquido, onde emprega-se calor e/ou
agitação com o objetivo de realizar a dissolução dos compostos presentes na amostra sólida
com o solvente extrator, ou seja, consiste no contato de partes sólidas do material com o
solvente. Ocorre o fenômeno de difusão do solvente através do material vegetal, em que um
número de compostos são transferidos da matriz para o solvente, enquanto o contato entre eles
é mantido (ALEIXANDRE-TUDO; TOIT, 2018; DE CASTRO; CAPOTE, 2010).
A matéria-prima deve ser dividida em pequenos fragmentos e, às vezes pulverizada, de
modo a provocar um aumento considerável da área oferecida à ação do solvente extrator que
deve ser deixado em contato com o material vegetal por um determinado tempo. Aliás, o
tempo é um dos principais problemas dessa técnica, pois geralmente são longos, sendo
frequentemente na região entre 24 h e 48 h. Esta técnica pode ser estática, quando o contato
entre o soluto e o solvente é feito por um tempo estabelecido e em repouso, ou dinâmica,
quando a mistura em extração é mantida sob agitação por um tempo determinado (DE
CASTRO; CAPOTE, 2010; VINATORU; MASON; CALINESCU, 2017).
Jimenez et al. (2011) produziram extratos de folhas de oliveira a partir da maceração.
As folhas secas foram submetidas à extração com etanol e água (1:1), por 24 h à temperatura
ambiente. Como resultado obtiveram extratos hidroalcoólicos com cerca de 29% de
oleuropeína.
Ansari, Kazemipour e Fathi (ANSARI; KAZEMIPOUR; FATHI, 2011) também
investigaram extratos de folhas de oliveira obtidos através de maceração com água, dentre
outros solventes, e ainda otimizaram parâmetros como pH, temperatura e tempo de extração.
Seus resultados mostraram que a extração com água deionizada em pH 3, por 4 h na
temperatura de 60°C, resultou na melhor eficiência. Os autores justificaram esse resultado,
com base em que a oleuropeína é um composto fenólico solúvel em água, cuja solubilidade
32
aumenta com a elevação da temperatura. Contudo, fazem a ressalva de que em temperaturas
maiores que 80°C a oleuropeína pode sofrer degradação.
Ahmad-Qasem et al. (2013) estudaram a extração por maceração de compostos
bioativos de folhas de oliveira utilizando uma solução de 80:20 (v/v) etanol-água por 24 h a
25°C. Eles encontraram resultados como 66 mgGAE/g (b.s) de compostos fenólicos totais e 74
mg/g (b.s) de oleuropeína.
2.3.2 Extração assistida por ultrassom
Uma das energias disponíveis para favorecer os processos de extração são as ondas de
ultrassom, que são ondas sonoras com alta frequência além da capacidade auditiva humana,
assim, os dispositivos de ultrassom para extração funcionam na faixa de 20 kHz a 2 MHz
(PANJA, 2017). A extração assistida por ultrassom vale-se da cavitação, ou seja, da formação
de pequenas bolhas sujeitas a rápidas compressões e expansões adiabáticas. Esse fenômeno
favorece a penetração e a transferência de massa, pois ocorre a ruptura das paredes das células
das plantas, levando a uma melhor liberação dos compostos alvo, assim havendo uma maior
solubilidade e difusividade dos compostos de interesse. Além disso, há também a energia
oxidativa dos radicais criados durante a aplicação do ultrassom no solvente, o que resulta em
um alto poder extrativo (JAPÓN-LUJÁN; JANEIRO; DE CASTRO, 2008; JAPÓN-LUJÁN;
LUQUE-RODRÍGUES; CASTRO, 2006a).
A extração assistida por ultrassom para materiais vegetais pode ser descrita em quatro
etapas. Primeiro, as bolhas de cavitação são geradas perto da superfície da matriz da planta
durante a aplicação do ultrassom. Na segunda etapa, ocorre a implosão das bolhas de
cavitação na superfície do produto, gerando micro jatos que resultam em vários efeitos, como
descamação superficial, erosão e quebra de partículas. Na terceira etapa, a superfície da
matriz rompida entra em contato direto com o solvente. Por fim, os componentes ativos são
liberados e transportados para o solvente (CHEMAT et al., 2017; PANJA, 2017).
Essa tecnologia apresenta muitas vantagens para a extração de compostos naturais, são
elas: maior penetração do solvente no material celular, menor tempo de processamento e
residência, maiores rendimentos e reprodutibilidade do produto, menor consumo de solvente,
alta taxa de processamento, economia significativa em manutenção, menos energia necessária
para o processamento, mais barata e menos tóxica (DENG et al., 2017; PANJA, 2017;
ROSELLÓ-SOTO et al., 2015).
33
Japón-Luján; Janeiro; De Castro (2008) enriqueceram óleos comestíveis (oliva,
girassol e soja) com extrato de folhas de oliveira obtidos por extração assistida por ultrassom.
Concluíram que os baixos custos de aquisição e manutenção de uma fonte de ultrassom e sua
aplicação em um sistema dinâmico tornam recomendável a implementação industrial do
método proposto.
Ahmad-Qasem et al. (2013) produziram extratos hidroalcoólicos (80:20 (v/v))de
folhas de oliveira utilizando ultrassom e concluíram que a aplicação de energia de ultrassom
pode ser considerada uma alternativa como meio de intensificar o processo de extração de
compostos fenólicos de folhas de oliveira.
2.3.3 Extração assistida por micro-ondas
Entre as novas técnicas estudadas, com alto potencial industrial, a extração assistida
por micro-ondas tem recebido significativa atenção na extração de bioativos de plantas. O
método está ganhando popularidade porque os equipamentos de micro-ondas estão facilmente
disponíveis a baixo custo (PANJA, 2017; ROSELLÓ-SOTO et al., 2015; SEOANE et al.,
2017).
A extração assistida por micro-ondas é baseada no aquecimento do solvente pela
aplicação de energia de micro-ondas, que é a radiação eletromagnética com comprimentos de
onda variando de 1 mm a 1 m e com frequências de 300 MHz (1 m) a 300 GHz (1 mm)
(PANJA, 2017; SEOANE et al., 2017). No processo, a energia de micro-ondas é usada para
aquecer os solventes em contato com as amostras sólidas e extrair compostos de interesse da
amostra para o solvente, isso se dá pelo aumento da pressão interna dentro da célula que
subsequentemente facilita a ruptura da parede celular e a liberação de compostos ativos
(CHEMAT et al., 2017; DE CASTRO; CAPOTE, 2010; XIE et al., 2015a).
A radiação de micro-ondas provoca o aquecimento direto da parte interna da matriz
sólida e o gradiente de temperatura é invertido em relação ao aquecimento convencional.
Portanto, essa técnica faz uma combinação sinérgica de transferência de massa e de
transferência de calor atuando na mesma direção, de dentro da matriz sólida para o exterior,
evitando as perdas de energia para o meio ambiente. Algumas vantagens desse tipo de
extração são o aquecimento rápido, menor necessidade de solvente e trata-se de um processo
limpo, dependendo do solvente empregado (SEOANE et al., 2017).
As técnicas de maceração e micro-ondas foram comparadas na extração de compostos
fenólicos de folhas de oliveira (RAFIEE et al., 2011). Os pesquisadores observaram que a
34
extração assistida por micro-ondas tinha várias vantagens sobre a maceração em termos de
rendimento e tempo de extração.
Şahin et al. (2017) otimizaram os compostos fenólicos totais e o conteúdo de
oleuropeína em extratos de folhas de oliveira obtidos por extração assistida por micro-ondas.
As condições ótimas de extração foram: potência de irradiação de 250 W, tempo de extração
de 2 min e quantidade de amostra de 5 g. Nestas condições, eles obtiveram o rendimento
máximo de oleuropeína (0,060 ± 0,012 ppm) e a quantidade de compostos fenólicos totais foi
de 2,480 ± 0,060 ppm. Além disso, os extratos de folhas de oliveira obtidos sob condições
ótimas mostraram atividade antibacteriana contra Staphylococcus aureus e Staphylococcus
epidermidis, com uma concentração inibitória mínima (CIM) de 1,25 mg/mL.
2.4 SISTEMAS DE EMBALAGENS
Os sistemas de embalagens ocupam uma posição de destaque no processamento de
alimentos, sendo seu uso indispensável para a distribuição e comercialização de produtos no
mercado. A seleção de materiais e sistemas de embalagem adequados é parte integrante do
processamento de alimentos (BERK, 2018). Robertson (2013) atribui às embalagens quatro
funções: contenção, proteção, conveniência e comunicação. As embalagens para alimentos
sofrem cada vez mais a influência do surgimento de novas tecnologias e novos materiais. Elas
configuram, aproximadamente, metade do total de embalagens produzidas nos países
industrializados (ANDRADE, 2003).
Os alimentos embalados sofrem com dois tipos de problemas. O primeiro é o de
influência direta, como por exemplo, perda do valor nutricional, contaminação por poeiras,
ataque microbiológico e oxidação. O segundo provém da embalagem, como por exemplo,
permeabilidade ao oxigênio, retenção de umidade ou perda de água e compatibilidade do
material com o alimento. Diante disso, é de vital importância que a embalagem seja
constituída de propriedades que atendam às exigências de qualidade do produto (ANDRADE,
2003).
2.4.1 Embalagens ativas
O termo embalagem ativa foi usado pela primeira vez em uma conferência na Islândia,
em 1987, por Theodore Labuza (ROBERTSON, 2013). Embalagem ativa é definida como
35
embalagem que modifica a condição do alimento embalado, de modo a prolongar sua vida útil
e melhorar sua segurança (AHVENAINEN, 2003).
Os sistemas de embalagens ativas são divididos em duas categorias: aquelas em que os
princípios ativos são incluídos na embalagem e aquelas em que são incorporados ao material
da embalagem. Alguns desses princípios são “modificadores da atmosfera”, como
absorvedores de oxigênio, absorvedores ou geradores de dióxido de carbono, absorvedores de
etileno e reguladores de umidade. Estes são normalmente incluídos na embalagem como uma
fase separada (por exemplo, os saquinhos de sílica usados como absorventes de umidade).
Quando o princípio ativo é incorporado na formulação da embalagem, ele pode ser um agente
preservador ou um antioxidante e é liberado lentamente para a atmosfera da embalagem
durante o armazenamento (BERK, 2018; ROBERTSON, 2013; SUPPAKUL et al., 2003).
A perda de alimentos por deterioração microbiana é bastante expressiva, sendo
fundamental o uso de embalagens adequadas à sua conservação. Estudos referentes a
embalagens ativas com a incorporação de antimicrobianos vêm se intensificando. A
incorporação de aditivos antimicrobianos em embalagens aumenta a segurança para o
consumo dos produtos, uma vez que esses compostos não são adicionados diretamente ao
alimento, o que poderia comprometer as características sensoriais do mesmo
(AHVENAINEN, 2003; KECHICHIAN, 2007).
O interesse por esse tipo de embalagem é reflexo da demanda dos consumidores por
alimentos frescos, minimamente processados e isentos de conservantes. Além disso, é sabido
que na maioria dos alimentos sólidos e semissólidos, o desenvolvimento de micro-organismos
ocorre principalmente na superfície, assim há um maior contato entre o produto e o agente
antimicrobiano (AHVENAINEN, 2003; ROBERTSON, 2013).
Uma vantagem importante da utilização de embalagens ativas antimicrobianas é que
apenas pequenas quantidades de conservante entram em contato com os alimentos, em
comparação com a adição direta de conservantes ao alimento. A composição dessas
embalagens tem por objetivo prolongar o período de latência e reduzir a taxa de crescimento
de micro-organismos para prolongar a vida útil e manter a segurança alimentar. Podem ser
aplicadas em: cereais, carnes, peixes, pães, lanches, queijos, frutas e legumes (ROBERTSON,
2013).
Dando ênfase àquela forma de embalagem ativa antimicrobiana quando os agentes
antimicrobianos são adicionados na composição da embalagem, os mesmos ao encontrarem a
superfície do produto são absorvidos e então se difundem conferindo o efeito antimicrobiano
(LÓPEZ-RUBIO et al., 2004). Um exemplo dessa aplicação é a embalagem antimicrobiana
36
para produtos cárneos, produzida com polímero convencional, em que os antimicrobianos
presentes no polímero entram em contato com o produto e migram por difusão
(QUINTAVALLA; VICINI, 2002).
Em uma revisão abrangente Joerger (2007) catalogou e analisou os resultados de 129
estudos publicados sobre filmes antimicrobianos e observou que a bacteriocina nisina foi a
mais comumente incorporada em filmes, seguida por ácidos e sais, quitosana, extratos
vegetais e as enzimas lisozimas e lactoperoxidase.
As aplicações potenciais das embalagens ativas antimicrobianas, para prolongar a vida
útil de carnes e dos produtos derivados foram analisadas por Camo; Beltrán; Roncalés (2008).
Kechichian (2007), em seu trabalho avaliou filmes biodegradáveis a base de fécula de
mandioca incorporando antimicrobianos naturais (cravo em pó, canela em pó, pimenta
vermelha em pó, óleo essencial de laranja, café em pó, mel e extrato de própolis). Kuorwel et
al. (2011) estudou agentes antimicrobianos sintéticos e naturais incorporando-os em filmes
para embalagem, utilizou manjericão, orégano e tomilho e seus óleos essenciais. Ugalde
(2014) produziu filmes biodegradáveis ativos incorporando óleos essenciais de alecrim,
cravo-da-índia, orégano e sálvia e obteve bons resultados para o desempenho dos filmes.
Houve uma melhor inibição frente às bactérias Gram-positivas do que Gram-negativas.
Vale ressaltar que para comprovar o potencial dos compostos antimicrobianos
naturais, bem como um potencial antioxidante para aplicações em embalagens, deve-se testar
as suas propriedades antimicrobianas em sistemas alimentares em condições reais de
armazenamento e distribuição (AHVENAINEN, 2003; SILVA et al., 2017).
2.4.2 Embalagens biodegradáveis
Diferentemente das embalagens produzidas a partir de polímeros convencionais
advindos da indústria petroquímica, as embalagens biodegradáveis são feitas a partir de
matérias-primas renováveis, que podem ser degradadas pela ação de seres vivos, que são
comumente bactérias e fungos (ALVES; TOMÁS, 2003).
Grande parte dos resíduos sólidos municipais é constituída por embalagens, o que
torna interessante e sustentável o desenvolvimento de pesquisas no âmbito dos polímeros
biodegradáveis com o objetivo de produzir embalagens, que vêm com a tarefa de reduzir o
acúmulo de lixo e os impactos ambientais. Assim, um dos desafios das indústrias de
embalagens alimentares é produzir embalagens biodegradáveis que atendam aos requisitos de
resistência e tempo de prateleira do produto. Biopolímeros como carboidratos, proteínas e
37
lipídios têm sido utilizados para a confecção dessas embalagens biodegradáveis (ALVES;
TOMÁS, 2002).
Para mostrar que esse é um tema imprescindível e atual, tramita no Senado Federal
Brasileiro o Projeto de Lei n° 263, de 2018, o qual dispõe:
Art. 2º O Capítulo VI do Título III da Lei nº 12.305, de 2 de agosto de 2010, passa a
vigorar acrescido do seguinte art. 49-A:
“Art. 49-A. São proibidas a fabricação, a importação, a distribuição, ainda que a
título gratuito, e a comercialização de sacolas plásticas para acondicionamento e
transporte de mercadorias, bem como de utensílios plásticos descartáveis para
consumo de alimentos e bebidas.
Parágrafo único. Excetuam-se da proibição estabelecida no caput as sacolas e os
utensílios descartáveis fabricados com material integralmente biodegradável, na
forma do regulamento.” (COMISSÃO DE DIREITOS HUMANOS E
LEGISLAÇÃO PARTICIPATIVA, 2018).
Nesse contexto, os biopolímeros vêm sendo explorados para a fabricação de
embalagens biodegradáveis.
2.5 BIOPOLÍMEROS
Os materiais poliméricos são compostos sólidos, não metálicos, de alto peso
molecular. Eles são compostos por macromoléculas repetidas e têm características variadas
dependendo de sua composição. Os biopolímeros são aqueles polímeros que ocorrem
normalmente na natureza e seu uso é cada vez mais frequente na fabricação de embalagens.
Porém, eles apresentam limitações ao serem processados com as tecnologias tradicionais e
alcançam desempenhos inferiores em termos de propriedades funcionais e estruturais, quando
comparados com os polímeros convencionais (ROCHA; SOUZA; PRENTICE, 2018).
Existem diversos biopolímeros, que ocorrem na natureza e que podem ser utilizados
na fabricação de filmes biodegradáveis. Tharanathan (2003) descreve-os como sendo de
origem animal (colágeno e gelatina), de resíduos do processamento de alimentos de origem
marinha (quitina e quitosana), de fontes microbianas, além dos de origem agrícola (lipídios,
proteínas e polissacarídeos).
Robertson (2013) cita uma gama de matrizes para filmes biodegradáveis comestíveis
que é dominada pelos polissacarídeos quitosana, alginato, κ-carragenana, éteres de celulose,
38
produto de alfa amilose e derivados de amido, filmes à base de proteína feitos com glúten de
trigo, soja, zeína, gelatina, soro e caseína.
Inúmeras são as pesquisas que estudaram as potencialidades da fabricação de filmes
biodegradáveis a partir de variados biopolímeros. No entanto, pouco se sabe sobre a
elaboração de filmes a partir de um biopolímero obtido de algas vermelhas: as carragenanas.
2.5.1 Carragenanas
A diversidade das espécies de algas é algo que se destaca diante da imensidão dos
oceanos que cobrem nosso planeta. O aproveitamento das algas se dá no nosso cotidiano de
diversas maneiras, diretamente na alimentação, extração de ficocolóides, aplicações na
medicina, investigação científica e indústria farmacêutica. Porém, as algas vêm ganhando
gradativo reconhecimento como fonte de materiais valiosos, sendo assim necessário
intensificar a investigação fundamental, de modo a descobrir novos usos, aproveitando todas
as suas potencialidades, sobretudo no que diz respeito aos biopolímeros (PEREIRA et al.,
2003, 2009; VIDOTTI; ROLLEMBERG, 2004; WEBBER et al., 2012).
Dentre as mais variadas espécies algais, as algas vermelhas são do filo Rhodophyta,
que inclui uma única classe, a Rhodophyceae. As espécies mais importantes incluem as
carragenófitas, que como o próprio nome sugere são as algas que nos fornecem as
carragenanas (PEREIRA, 2004). A principal característica dessa alga é a presença do
pigmento ficoeritrina, responsável pela coloração com tonalidades que variam do rosa-claro
ao vermelho-escuro (CARLUCCI et al., 1997; PEREIRA, 2004; VIDOTTI; ROLLEMBERG,
2004).
Carragenana é a denominação dada à família de polissacarídeos extraídos das algas
vermelhas. Assim como o ágar e o alginato as carragenanas são parte de um grupo de
substâncias complexas biopoliméricas chamadas ficocolóides. É o terceiro hidrocolóide
(gomas solúveis em água) mais importante na indústria alimentar, antecedem a gelatina e o
amido (PEREIRA et al., 2003; WEBBER et al., 2012). São polímeros sulfatados, de elevada
massa molecular e com um alto grau de polidispersividade, que possuem duas características
principais: são constituídas a partir de um monômero, a galactose, e contêm uma forte
proporção de ésteres sulfato (O-SO3-). São classificadas de acordo com a presença de ligações
3,6-anidro ligadas ao resíduo de galactose e com a posição e o número de grupos sulfato. Por
isso, são geralmente distribuídas em três categorias denominadas -carragenana, -
carragenana e -carragenana, que podem ser distinguidas por suas estruturas primárias. A
39
Figura 3 mostra as estruturas básicas que se transformam nas várias carragenanas (CAMPO et
al., 2009; PEREIRA et al., 2009; PEREIRA, 2004; WEBBER et al., 2012).
Esses biopolímeros possuem a capacidade de formar géis termoreversíveis ou soluções
viscosas quando adicionados a soluções salinas. Os géis de carragenanas se estabilizam em
pH de 5,0 a 7,0 e apresentam algumas propriedades de sólido e outras de líquido, o que lhes
conferem a aplicação como aditivos na indústria de alimentos, sendo agentes gelificantes,
emulsionantes, estabilizantes e espessantes (PEREIRA et al., 2009; PEREIRA, 2004;
WEBBER et al., 2012).
Figura 3 - Estruturas básicas das carragenanas
Fonte: Pereira et al. (2009).
Além do uso das carragenanas como um gel, novas pesquisas sugerem sua utilização
como filme biodegradável. Abdou; Sorour (2014) prepararam e caracterizaram filmes
produzidos com carragenanas e amido, em variadas proporções. Obtiveram como resultado
40
filmes em que as propriedades mecânicas e a permeabilidade ao vapor de água aumentaram
com o aumento da concentração de carragenanas. Paula et al. (2015) desenvolveram e
caracterizaram filmes a partir de misturas de ɩ-carragenana, κ-carragenana e alginato.
Concluíram que filmes formados com κ-carragenana, apresentaram melhores propriedades
quando comparados com filmes compostos por ɩ-carragenana. Apesar desses estudos, ainda é
escassa a procura por novos potenciais usos para esse biopolímero.
Alguns estudos também sugerem o aproveitamento das algas vermelhas como um
produto com potenciais antimicrobiano, antioxidante, antiviral, antitumoral e anticoagulante,
bem como no tratamento de algumas doenças. A detecção, caracterização e isolamento de
substâncias ativas presentes em algas tem crescido nos últimos anos (STEIN, 2011).
2.6 FILMES BIODEGRADÁVEIS
Os filmes biodegradáveis caracterizam-se por filmes finos, não tóxicos e não
poluentes, produzidos a partir dos formadores de matriz (lipídios, proteínas e polissacarídeos),
solventes (água e etanol) e agentes plastificantes (glicerol, sorbitol, triacetina e ácidos graxos)
(BATISTA, 2004). A pesquisa envolvendo a produção e caracterização de filmes
biodegradáveis aumentou significativamente, principalmente devido ao interesse em
minimizar o impacto ecológico causado pelo uso de materiais de embalagem sintética. Dadas
as crescentes preocupações ambientais pelo acúmulo excessivo de plástico, as indústrias de
embalagens de alimentos também tem demonstrado interesse nos filmes biodegradáveis nas
duas últimas décadas (MIR et al., 2018).
Além disso, outro grande benefício dos filmes biodegradáveis, é que os mesmos
podem ser utilizados como meios de transmissão de aromas, antioxidantes, agentes
antimicrobianos e corantes, que serão liberados para o sistema alimentar durante o tempo de
estocagem, atuando como embalagem ativa (KROCHTA; MULDER-JOHNSTON, 1997;
MILLER; KROCHTA, 1997). Eles têm sido considerados alternativas atraentes às
embalagens plásticas, devido à sua excelente biodegradabilidade, biocompatibilidade,
comestibilidade e possíveis aplicações, com a incorporação de compostos bioativos (MIR et
al., 2018). Na Tabela 1, podem-se observar exemplos de estudos que incorporaram compostos
antimicrobianos e antioxidantes em filmes biodegradáveis, com intuito de obter embalagem
ativa para alimentos.
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Tabela 1 - Aplicação de filmes biodegradáveis incorporados com antimicrobianos ou
antioxidantes.
Biopolímero Antimicrobiano*/Antioxidante Alimento
testado Referência
Quitosana Óleo de canela* Truta arco-íris Ojagh et al. (2010)
Goma foliar de
Hsian-tsao Extrato de chá verde* carne de porco Chiu; Lai (2010)
κ-carragenana Sorbato de potássio* Peito de
frango Seol et al. (2009)
Proteína de soro Polilisina* Carne fresca
Zinoviadou;
Koutsoumanis;
Biliaderis (2010)
Gelatina/quitosana Extratos de orégano e
alecrim*
Sardinha
defumada
Gómez-Estaca et
al. (2007)
Galactomannan Nisina* Ricota Martins et al.
(2010)
Quitosana/amido Quitosana* Salmão Vásconez et al.
(2009)
Amido de milho Lisozima * In vitro
Fabra; Sánchez-
González; Chiralt
(2013)
Amido de milho Nisina* In vitro Sanchez-Ortega et
al. (2016)
Amido de
mandioca Extrato de alecrim In vitro
Piñeros-
Hernandez et al.
(2017)
Ecoflex®/ácido
polilático
α-tocoferol e extrato de folhas
de oliveira In vitro
Marcos et al.
(2014)
Polietileno/papel Extrato de folhas de oliveira In vitro Moudache et al.
(2016)
Gelatina de peixe Casca de manga In vitro Adilah et al.
(2018)
Celulose Extratos de canela, cravo,
gengibre, chá verde e tomilho Óleo de soja
Phoopuritham et
al. (2012)
Fonte: Autora (2021).
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2.6.1 Técnicas para a preparação de filmes biodegradáveis
A elaboração de filmes biodegradáveis está ancorada na presença de três constituintes
principais: um formador de matriz, um plastificante e um solvente (BARRETO, 2003). Os
formadores de matriz são a estrutura básica do filme biodegradável e podem ser à base de
hidrocolóides (celulose, alginatos, amidos e outros polissacarídeos), de lipídios (ácidos
graxos, ceras e gliceróis) ou de proteínas (colágeno, gelatina e caseína). Já o plastificante é
uma substância não volátil, que quando adicionado em sistemas diversos promove alterações
físicas e/ou mecânicas. O plastificante deve ser miscível no polímero e ambos devem possuir
solubilidade no solvente. O glicerol é um dos agentes plastificantes mais aplicados na
elaboração de filmes biodegradáveis, devido sua compatibilidade e estabilidade com as
cadeias dos biopolímeros. Dependendo do objetivo podem ser utilizados os mais diversos
solventes: água, etanol, metanol, acetona, entre outros, sendo o mais comum a água
(BARRETO, 2003; UGALDE, 2014).
A elaboração de filmes biodegradáveis envolve ligações químicas cruzadas entre
cadeias de polímeros para formar uma matriz tridimensional semi-rígida que interage com o
solvente. A sua estabilidade depende da estrutura do polímero, processo de fabricação,
parâmetros físicos, tais como temperatura, pressão e tipo de solvente, presença de plastificante
e de aditivos (GONTARD; GUILBERT, 1994).
Para a formação de filmes biodegradáveis, frequentemente é preparada uma solução,
chamada de solução filmogênica, e eles se formam pela remoção do solvente, técnica é
conhecida como casting. A solução pode ser aplicada diretamente sobre o produto a ser
recoberto ou ser utilizado um suporte, para posterior remoção e utilização do filme.
Geralmente, designam-se por coberturas as soluções filmogênicas aplicadas e formadas
diretamente na superfície do produto, enquanto os filmes biodegradáveis são formados
separadamente como filmes finos e então aplicados (ROCHA; SOUZA; PRENTICE, 2018).
Dois estudos recentes obtiveram sucesso na produção de filmes biodegradáveis com a
adição de extratos vegetais da oliveira. Albertos et al. (2017) produziram filmes de gelatina de
peixe com a adição de extrato de folhas de oliveira e utilizaram como embalagem para salmão
defumado frio, os filmes com o extrato diminuíram o crescimento de L. monocytogenes. Já
Bermúdez-Oria et al. (2017) produziram filmes biodegradáveis à base de pectina e proteína de
pele de peixe com a adição de extratos de azeitona, o filme preservou os morangos contra
mofo durante o armazenamento. Seol et al. (2009) avaliaram filmes de carragenana
suplementados com sorbato de potássio e EDTA quando usados em embalagens de peito de
43
frango. Eles observaram que os filmes mostraram o efeito da inibição contra bactérias
aeróbias totais e E. coli durante o armazenamento a 5°C. Além disso, alegaram que estudos
adicionais deveriam ser feitos para investigar outras aplicações para produtos cárneos e
diferentes micro-organismos devido à sua eficácia. Outros estudos avaliaram a produção de
filmes antimicrobianos biodegradáveis, mas as dificuldades estão na escolha de um agente
com características antimicrobianas adequadas, assim como na escolha de uma matriz
polimérica que resulte em filmes biodegradáveis com características desejáveis (CHIU; LAI,
2010; GÓMEZ-ESTACA et al., 2007; MARTINS et al., 2010; OJAGH et al., 2010;
VÁSCONEZ et al., 2009; ZINOVIADOU; KOUTSOUMANIS; BILIADERIS, 2010). Ao
contrário de alguns estudos citados que utilizaram um conservante químico para conferir a
atividade antimicrobiana/antioxidante, ou polímero sintético na formulação dos filmes, a
presente pesquisa propõe a utilização do extrato de folhas de oliveira (produto natural) e da
carragenana.
2.6.2 Propriedades de filmes biodegradáveis
A aplicação de filmes e coberturas para embalagens de alimentos requer o prévio
conhecimento de suas características funcionais e estruturais, a saber mecânicas, óticas, de
barreira, solubilidade, sensoriais e de composição. Estas vão depender do composto utilizado
como base do filme, da técnica de fabricação e das condições ambientais (SOBRAL, 2000).
De modo geral, são avaliadas as propriedades mecânicas, sendo elas resistência máxima à
tração e porcentagem de elongação na ruptura, as propriedades físico-químicas, como
solubilidade em água e espessura, a propriedade de barreira, que é a permeabilidade ao vapor
de água (PVA) e a propriedade ótica, cor.
Como já mencionado, os filmes biodegradáveis podem ser aditivados com compostos
antimicrobianos, sendo assim, as propriedades desses materiais também devem ser avaliadas.
Filmes formados pela incorporação de extratos vegetais em polímeros geralmente resultam
em modificações físico-químicas, mecânicas e de barreira, antioxidantes e antimicrobianas em
comparação com filmes feitos de componentes individuais. A maximização dessas
propriedades para a obtenção de filmes biodegradáveis aptos a embalagem tem sido o objetivo
de diversas pesquisas. Para isso, são fabricados filmes biodegradáveis com as mais variadas
composições, fazendo-se blendas de biopolímeros, diferentes proporções de plastificantes,
incorporação de compostos com potencial antimicrobiano e até mesmo a proposição do uso de
novos biopolímeros para o fim de embalagem. A adição de plastificantes exerce efeito sobre
44
as propriedades mecânicas dos filmes biopoliméricos (KECHICHIAN, 2007; MIR et al.,
2018; ROCHA; SOUZA; PRENTICE, 2018).
Veiga-santos et al. (2005) avaliaram a influência da adição dos plastificantes sacarose,
açúcar invertido e fosfato de sódio em filmes biodegradáveis a base de fécula de mandioca.
Foram realizados ensaios de barreira ao vapor de água e ao oxigênio, resistência máxima a
tração e porcentagem de elongação na ruptura, além da determinação de propriedades
térmicas. Os autores concluíram que os ingredientes utilizados afetaram de forma negativa os
resultados de resistência máxima a tração, porém afetaram de forma positiva os resultados de
porcentagem de elongação na ruptura. A permeabilidade ao vapor de água não sofreu
alterações significativas entre as amostras de filmes biodegradáveis elaborados.
Longares et al. (2005) produziram e caracterizaram filmes biodegradáveis com
variadas proporções de proteína do soro do leite e caseinato de sódio, utilizando glicerol como
plastificante. A caracterização foi realizada através de propriedades físicas, de barreira e de
solubilidade. Dentre os filmes elaborados apenas com caseinato de sódio apresentou
resistência máxima a tração e a permeabilidade ao vapor de água semelhante aos filmes
preparados com a combinação de proteína do soro do leite e caseinato de sódio. Filmes
contendo somente proteína e glicerol apresentaram melhores resultados de porcentagem de
elongação na ruptura, quando comparados com filmes preparados apenas com caseinato de
sódio e glicerol.
O aspecto visual está relacionado a aparência do filme biodegradável, que pode
implicar na aceitação dos consumidores do produto por ele embalado, além de determinar a
aplicação ou não do filme biodegradável. A avaliação do aspecto visual é feita de forma
subjetiva através de observações visuais e táteis.
O filme biodegradável deve apresentar uma superfície continua e homogênea, dessa
forma não deve apresentar bolhas, fissuras, partículas insolúveis, poros abertos e diferenças de
coloração (CARVALHO, 1997).
As propriedades óticas dos materiais de embalagem são de importância prática, porque
elas afetam a função de proteção e influenciam na aparência e atratividade. Muitas reações
deteriorantes são catalisadas pela luz, que incluem oxidação, geração de sabor, descoloração,
destruição de componentes de importância nutricional. Embalagens transparentes permitem
que os consumidores vejam o produto através da embalagem e possam julgar a qualidade pela
sua aparência. É possível aliar o compromisso entre a proteção da luz e a transparência
usando-se embalagens coloridas (BERK, 2018).
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A cor e a opacidade destacam-se dentre as propriedades óticas para avaliação de
filmes biodegradáveis. A cor está associada com os ingredientes utilizados na elaboração dos
filmes e pode ser uma boa ferramenta na caracterização dos mesmos. É geralmente avaliada
utilizando-se um colorímetro, por meio dos padrões CIE (Comission Internacionale de
I’Eclairage) Lab, L* variando de 0 (branco) a 100 (preto), a* do verde (-) ao vermelho (+) e
b* do azul (-) ao amarelo (+) (UGALDE, 2014).
Pelissari (2009) produziu filmes de amido de mandioca, quitosana e glicerol. Como
resultado obteve que filmes com maiores concentrações de quitosana originaram filmes mais
opacos e escuros, fato que pode ser devido a sua coloração amarelada característica.
A espessura é definida como sendo a distância entre duas superfícies do material. Esta
propriedade é muito importante, pois através dela é possível caracterizar os filmes
biodegradáveis quanto suas propriedades mecânicas e de barreira (OLIVEIRA; ALVES;
SARANTÓPOULOS, 1996).
O controle do processo de fabricação dos filmes biodegradáveis é muito importante
para que sejam garantidas espessuras uniformes e para que haja repetibilidade das
propriedades estudadas. Quando se utiliza a técnica de casting para produção de filmes
biodegradáveis, deve-se controlar o nível do local, pois desníveis podem provocar diferenças
na espessura (GENNADIOS; WELLER; TESTIN, 1993).
Segundo Park; Chinnan (1995) a permeabilidade ao vapor de água pode variar com a
espessura, pois existem mudanças estruturais na matriz filmogênica, que afetam a estrutura
dos filmes e provocam tensões internas que podem influenciar na permeação. Em sua
pesquisa verificaram que a permeabilidade ao vapor de água aumentava proporcionalmente
com a espessura dos filmes protéicos.
A capacidade de uma embalagem de proteger seu conteúdo contra forças externas
depende de suas propriedades mecânicas. A avaliação dessas propriedades mecânicas é
essencial para filmes biodegradáveis, visto que refletem sua durabilidade e capacidade de
acondicionar alimentos. As propriedades mecânicas estão intimamente ligadas à composição
do filme biodegradável e seu poder de formar estruturas estáveis, bem como às condições
ambientais, como temperatura e umidade (WARD, I. M.; HARDLEY, 1998).
A resistência à tração, porcentagem de elongação na ruptura e o módulo de
elasticidade são propriedades mecânicas avaliadas em filmes biodegradáveis. A resistência à
tração é a máxima tensão suportada pelo filme até o momento de sua ruptura. A elongação é a
capacidade do filme em deformar antes de ocorrer sua ruptura, define sua maleabilidade.
Filmes biodegradáveis quebradiços apresentam baixos valores de elongação (MACLEOD, G.
46
S.; FELL, J. T.; COLLETT, 1997). Já o módulo de elasticidade indica a rigidez do filme, um
valor mais alto corresponde a um material mais rígido (DE CAMPO et al., 2016).
As propriedades de barreira estão ligadas a passagem de gases, especialmente o
oxigênio, e vapor de água para o interior do sistema de embalagem. O oxigênio e o vapor de
água são responsáveis pela perda das características sensoriais, modificações físico-químicas
e deterioração microbiológica de um alimento (BATISTA, 2004).
Três fenômenos podem descrever o transporte de gases e vapor de água através de um
filme polimérico, são eles: a difusão, que é o movimento de uma molécula através de uma
matriz polimérica, a solubilidade, que é o comportamento de partição de uma molécula
permeante entre a superfície do filme polimérico e uma posição vizinha na matriz polimérica,
e a permeabilidade, que é a taxa de transporte de uma molécula através de um filme
polimérico como resultado da combinação dos fenômenos de difusão e solubilidade
(MILLER; KROCHTA, 1997).
A permeabilidade é medida através da exposição dos filmes a um meio com o
permeante, que passa de um lado do filme para o outro, quando há diferentes concentrações
desse permeante em um determinado tempo. Para medição dessa propriedade é importante o
conhecimento da espessura e da área exposta do filme. As propriedades de barreira são
bastante afetadas pela variação na concentração do biopolímero utilizado para a elaboração do
filme biodegradável (BATISTA, 2004).
A solubilidade em água é uma característica importante nos filmes biopoliméricos e
trata da resistência do filme à água (RHIM, 2012). É uma propriedade relevante, sobretudo
para o delineamento da área de aplicação do filme.
47
- CAPÍTULO 3 -
MÉTODOS DE EXTRAÇÃO PARA PRODUÇÃO DO EXTRATO DE FOLHA DE
OLIVEIRA
48
O Capítulo 3 visou a pesquisa dos métodos de extração para a seleção daquele com a
melhor possibilidade de recuperação de compostos de interesse bioativos das folhas de
oliveira. Essa pesquisa está apresentada no artigo 1.
Artigo 1
Eco-friendly extraction for the recovery of bioactive compounds from Brazilian olive
leaves
Gabriela Silveira da Rosa1, 2*, Thamiris Renata Martiny3, Guilherme Luiz Dotto3, Sai Kranthi
Vanga2, Débora Parrine2, Yvan Gariepy2, Mark Lefsrud2, Vijaya Raghavan2
1. Graduate Program in Materials Science and Engineering, Federal University of
Pampa, 1650 Maria Anunciação Gomes Godoy Avenue, Bagé, Rio Grande do Sul,
Brazil, 96413-172
2. Department of Bioresource Engineering, McGill University, 21111 Lakeshore Road,
Ste-Anne-de-Bellevue, Quebec, Canada, H9X 3V9
3. Department of Chemical Engineering, Federal University of Santa Maria, 97105–900, Santa
Maria, Rio Grande do Sul, Brazil
*Corresponding author: Unipampa, 1650, Maria Anunciação Gomes de Godoy Avenue,
Bagé, Rio Grande do Sul, Brazil. E–mail address: [email protected], phone
number (+55) 53 99967-2226
Artigo publicado no formato de short communication no periódico Sustainable Materials and
Technologies
Volume 28, July 2021, e00276
ISSN: 2214-9937. DOI: https://doi.org/10.1016/j.susmat.2021.e00276
49
Graphical Abstract
Abstract
The aim of this study was to recover value-added compounds from Brazilian olive tree leaves
using organic solvent-free green extraction techniques. The key molecules obtained by
microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE) and maceration
were characterized for total phenolic compounds (TP), antioxidant activity (AA), oleuropein
(OP) and hydroxytyrosol (HT) contents. In the study, we also determined the lowest
concentration of extract that causes growth inhibition of Type 1 Escherichia coli. The results
show that green technologies were suitable for the extraction of bioactive compounds from
olive tree leaves. MAE was more efficient than UAE and maceration. OP was the
predominant phenolic compound found in the extracts. At a concentration of 50 mg.mL-1, the
extract produced by MAE inhibited the growth of E. coli by 100%, compared to 80.9% for the
extract produced by UAE. As for the inhibition kinetics of the extracts, no bacterial growth
was observed up to 75 min after inoculation. This study demonstrates that green technologies
can be used successfully to extract compounds with antioxidant and antibacterial properties
from olive culture byproducts, and that Brazilian crops are a viable environmental source.
Keywords: phenolic compounds; oleuropein; hydroxytyrosol; antioxidant; antimicrobial.
50
3.1 INTRODUCTION
Olive trees (Olea europaea L.) produce many byproducts (leaves, branches, solid and
liquid waste), however olive tree leaves stand out, they are the part of tree containing the
highest levels of biophenols [1,2]. These biophenols have bioactive capacity and can act with
antioxidants, antimicrobials, anti-inflammatories, anti‐atherogenic, in which the antiviral
capacity stands out in the face of the current pandemic needs that we are experiencing [3].
The oleuropein (OP) and the hydroxytyrosol (HT) are the main olive phenols present in the
leaves and they provide a natural defense for the leaves against biological predators [4,5].
There have been many publications on the activity properties of this compound. Researchers
have studied its antioxidant activity [6] and antimicrobial activity [7], thus showing the
potential application of oleuropein as an alternative to chemical preservatives and antibiotics.
The leaves represent an important byproduct considering that at the olive oil extraction
plant, the olive tree leaves represent up to 10% of the mass of the olive arriving from the
fields and need to be removed from the fruits in the early steps of olive cleaning [8].
Currently, this byproduct has no value, and it is even costly to dispose of. The current
practices are either to burn it, or to bury it along with other byproducts in the ground.
Occasionally, the residues are used as animal feed [9]. In terms of sustainability, assessing the
value of biomass based on raw materials has become an urgent need. Therefore, it is
inevitable to evaluate mainly agricultural residues that have no economic value or even
threaten the environment [10].
It is very important to note that there is a variability in olive crops, which are grown
mostly in the Mediterranean region. However, Brazil has increased its cultivated area,
especially in the southern region [11]. Since climatic and location conditions influence the
composition of plants [9,12], research on crops grown in Brazil is fundamental.
The inherent diversity and interest of food industry on natural products to replace
synthetic additives is increasing. Production of bioactive compounds at industrial scale
requires fast and simple extraction processes which are environmental friendly, energy
efficient and simple to implement. Nevertheless, the biophenol contents of the olive leaf
extract varies according to the method and conditions extractions. Therefore, it is important to
evaluate different extraction methods to select a method that maintains the stability of
phenolic compounds during the extraction process, while being effective and economical [3].
Several techniques have been proposed for the recovery of bioactive compounds from olive
tree leaves. Recent development in microwave-assisted extraction (MAE) and in ultrasound
51
assisted extraction (UAE) suggests that these techniques can be successfully used for the
extraction of bioactive compounds from olive leaves [2,13].
Both MAE and UAE are techniques that are related to increasing the bioaccessibility
of phenolics. UAE can be used to accelerate adsorption uptake and kinetics in comparison
with conventional technique (maceration) [14]. The cavitation bubbles generated by the
ultrasound collaborate to rupture the wall of the extraction matrix, thus releasing more
compounds [15]. MAE is known for accelerating the extraction process by heating inside out
caused by microwave energy, minimal degradation of the target components and better
extraction yield, together with less solvent use compared to maceration and other advanced
extraction methods [16].
The scientifically proven sanitary properties of olive phenols and their concentration
levels in olive leaf extracts are promoting the development of new, faster and more efficient
extraction methods for their industrial exploitation. There are few studies that deal with the
extraction and characterization of bioactive compounds from leaves that grow in the southern
region of Brazil. Thus, the aim of this study was to address the lack of knowledge in this area,
investigating the potential benefits of using MAE and UAE for the extraction of value added
compounds, specifically OP and HT, bringing an important assessment of their antimicrobial
capacity, using green extraction techniques without organic solvent.
3.2 MATERIALS AND METHODS
3.2.1 Reagents
Analytical grade 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin Ciocalteu’s reagent,
anhydrous sodium carbonate and gallic acid were obtained from Sigma Aldrich (St. Louis,
USA). HPLC grade water, acetonitrile, acetic acid, methanol, oleuropein and hydroxytyrosol
from Sigma Aldrich (St. Louis, USA) were also utilized.
For the antimicrobial analysis, Nutrient and Müller-Hinton broth and Kanamycin were
purchased from Sigma Aldrich (St. Louis, USA). Escherichia coli K12 (ATCC 10798) was
obtained from Cederlane (Burlington, USA).
52
3.2.2 Sample Preparation
The olive leaves (variety Arbequina) were collected at the Rio Grande do Sul state in
Brazil (31º30'04.0"S, 53º30'42.0"W). The leaves were prepared according to Martiny [17],
briefly, the leaves were signaled with sterile distilled water and a 2% sodium hypochlorite
solution. The initial moisture of the leaves was at 51.7% (w.b.). The leaves were dried for 24
h in a forced air oven operated at 40°C to a final moisture content of 3.1% (w.b.). Prior to
analysis, the fraction retained at 60-mesh sieve was used to ensure an uniform particle size.
3.2.3 Extraction Procedure
The MAE was performed in a Mini-WAVE microwave unit (SCP Science, Canada).
The microwave unit operated at a frequency of 2.45 GHz with a maximum power of 1000 W.
A MAE extract was produced at a 86°C temperature, with a duration of 3 min.
The UAE was performed in an ultrasound system with a working frequency of 20 kHz
and a power output of 450 W (Branson Sonifier 450, USA). The sample was exposed to the
acoustic waves while the ultrasonic probe was immersed 1 cm into the solution for 29 min.
The amplitude of the ultrasonic power has been adjusted to a desired level of 55% (percentage
of output power) of the maximum power. The temperature of the sample was maintained by a
cold-water bath at 27°C and the duty cycle was set up at 50%.
For the maceration extraction, the samples were blended with water and stirred
(Cimarec2, Thermolyne, USA) for 24 h at 25oC with agitation.
The extraction conditions chosen for this research for all techniques used (MAE, UAE
and maceration) are optimized conditions obtained by preliminary studies [18]. In which for
MAE different temperatures and extraction times were evaluated, for UAE different times and
ultrasound power output were evaluated and for maceration different solvents (water and
ethanol in different proportions) were evaluated. All extractions were carried out with an
amount of 0.5 g of ground material in 25 mL of distilled water used as solvent.
3.2.4 Total Phenolics (TP)
Singleton and Rossi [19] method was used to quantify total phenolics content with
Folin-Ciocalteu reagent. TP were quantified using a standard curve of absorbance obtained
53
with concentrations of gallic acid ranging from 70 to 1800 mg.L-1. The concentration was
expressed as milligrams of gallic acid equivalent per gram of dry matter. All measurements
were made in triplicate.
3.2.5 Antioxidant Activity (AA)
The AA was measured by the Brand-Williams et al. [20] method. The analysis was
conducted in triplicate. The percentage of free radicals scavenged by DPPH radical was
calculated using Equation 1.
𝐴𝐴 (%) = (𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙−𝐴𝑠𝑎𝑚𝑝𝑙𝑒
𝐴𝑐𝑜𝑛𝑡𝑟𝑜𝑙) ∙ 100 (1)
where, Acontrol is the absorbance of the blank solution (with water), and Asample is the
absorbance of the extract solution. The absorbance was measured at 517 nm.
3.2.6 Oleuropein (OP) and Hydroxytyrosol (HT)
An Agilent 1100 series instrument (Santa Clara, USA), high performance liquid
chromatograph (HPLC) equipped with a variable wavelength detector (VWD) was used to
identify and quantify the amount of OP and HT in the extract. The separation was conducted
at 25ºC using a reversed phase C18 discovery column (5 μm, 25 cm×4.6 mm) (Supelco,
USA), fitted with a Supelguard C18 cartridge (5 μm, 2 cm×4 mm) (Discovery, USA). The
mobile phase was composed by a water/acetonitrile/acetic acid solution (80/19/1 v/v/v) [21].
Extracts obtained by the MAE, UAE and maceration methods were filtered through a 0.45
mm syringe filter and directly injected in the HPLC. The concentrations of OP and HT in the
extracts were quantified using standard curves. The extraction yields of OP and HT were
expressed in miligrams per gram of olive tree leaves (mg.g-1 ) (d.b.).
3.2.7 Antimicrobial Inhibition (AI)
The extract AI analysis was performed with the gram-negative bacteria Escherichia
coli K12 (ATCC 10798), following the micro-dilution method of the Clinical and Laboratory
Standards Institute [22]. Lyophilized extracts were used to determine the minimum inhibitory
54
concentration (MIC). The lyophilized extracts were obtained using a freeze dryer (Gamma 1-
16 LSC, Christ, Germany) for 48 h and the yield for extract produced was 22.9% (w/w).
Samples were tested at concentrations ranging from 5 to 75 mg.mL-1. The E. coli was cultured
in a nutrient broth at 37°C for 24 h in shaken flasks until a 0.5 optical density (600 nm) was
reached. A mixture of extract (135 μL), sterile Muller-Hinton broth (145 μL) and E. coli
culture (20 μL) was added to a 96-well microtiter plates. A positive control containing only
the inoculum (with no extract) and a negative control of inoculum and kanamycin antibiotic
(50 ug.mL-1) were added to the microplate. The assay was carried out in a microplate
incubated in a spectrophotometer (PowerWave XS, Biotek, USA) for 16 h, with the
temperature kept at 37°C. Absorbances (620 nm) were taken at different times during the
incubation period to establish the inhibition kinetics. The analysis were conducted in
biological triplicates. AI was calculated using Equation 2.
𝐴𝐼 = [1 − (𝑂𝐷𝑒𝑥𝑡2−𝑂𝐷𝑒𝑥𝑡1
𝑂𝐷𝑐𝑜𝑛𝑡𝑟𝑜𝑙2−𝑂𝐷𝑐𝑜𝑛𝑡𝑟𝑜𝑙1) 𝑥 100 ] (2)
where, ODext2 is the optical density of the sample after the incubation period, ODext1 is the
optical density of the sample before the incubation period, ODcontrol2 is the optical density of
the control after the incubation period, and ODcontrol1 is the optical density of the control
before the incubation period.
3.2.8 Statistical analysis
Experimental data were expressed as average values ± mean deviation. Significant
differences among the means were determined by Tukey test at p < 0.05 by Statistica software
(StatSoft Inc., 10, Tulsa, OK, USA).
3.3 RESULTS AND DISCUSSION
The values of total phenolic compounds (TP), antioxidant activity (AA), oleuropein
content (OP) and hydroxytyrosol content (HT) obtained with MAE, UAE and maceration are
presented in Table 1.
55
Table 1. Total phenolic (TP), antioxidant activity (AA), oleuropein (OP) and hydroxytyrosol
(HT).
Extraction
technique
TP
(mgGAE.g-1 d.b.)
AA
(%)
OP
(mgOP.g-1 d.b.)
HT
(mgHT.g-1 d.b.)
MAE 104.22±0.61a 90.03±0.04a 14.468±0.405a 0.590±0.001a
UAE 80.51±1.52b 91.45±1.29a 6.914±0.276b 0.547±0.001b
Maceration 57.28±0.95c 67.25±0.03b 0.051±0.001c 0.027±0.008c
Data reported are average values and ± mean deviation. Means with different superscript alphabets in the
columns of each analysis are significantly different at p<0.05.
The different extraction methods had a significant impact on the results of TP, AA, OP
and HT, with the exception of AA for MAE and UAE in which there was no significant
difference. Generally, the MAE increased significantly (p≤0.05) all the results. A previous
study reported a better performance of MAE attributed primarily to the greater cell wall
disruption under microwave processing, which resulted in faster release of the cell compounds
into the solvent [23]. The temperature applied in MAE improved the permeation and the
solubilization processes to wash the intracellular compounds out of the matrix [24]. Many
plant compounds are sensitive to high temperature, therefore, the use of microwave energy
during the extraction may yield poor results due to the degradation of these compounds. In
general, the extraction yield increases with the rising temperature in a shorter extraction time,
as it peaks at a maximum before flatlining or declining [25]. Thus, extraction of thermal
sensitive compounds, as oleuropein, has been a challenge. The results in this study prove that
a high extraction temperature (86oC) does not cause the degradation of antioxidant
compounds from olive tree leaves. The use of a temperature of 80°C is recommended to MAE
of olive tree leaves to minimize compound degradation [26].
An increase of the TP yield using MAE with similar short extraction times (2.5 to 4
min) has already been observed [13,27]. As expected, the AA values measured from the
extracts were higher and the values observed for the UAE (91.45%) and MAE (90.03%)
methods were similar to those found in the literature (95.4% - 95.5610%) [28,29]. Although
the extracts obtained by maceration resulted in a much lower yield (TP 57.28 mgGAE.g-1) than
the ones obtained by UAE or MAE, they were twice as high as those reported in the literature.
Ahmad-Qasem et al.[30] and Cavalheiro et al. [31] reported an extraction rate of 29 mgGAE.g-1
for olive leaves under maceration extraction in ethanol, at room temperature condition with
durations ranging from 5 to 24 h.
56
Acoustic cavitation was observed in the solvent by ultrasonic waves. Interestingly,
MAE using water as solvent was found to be faster and more efficient than the two other
extraction methods tested. The use of water, compared to standard methods that utilize organic
solvent, has the advantage of water being safer, less expansive and environment-friendly.
Bilgin and Sahin [32] also studied the extraction of phenolic compounds from olive leaves,
but using methanol as a solvent, in techniques such as homogenization and ultrasound
extraction. The authors found for the yield of phenolic extracts with the homogenization
technique values between 10.11 - 61.66 mgGAE/g dry leaf, while for ultrasound, the content
varied between 7.35 - 38.66 mgGAE/g of dry leaf. It is worth mentioning that, although some
studies have indicated that methanol is a good solvent to extract oleuropein, it has high
toxicity and may compromise the use of extracts in foods. Martín-García et al. [3] studied
pressurized liquid extraction to recover phenolic compounds from olive leaves, found better
results using ethanol as a solvent. Although ethanol is a safe solvent, extraction with it implies
more expenses with the process. In a recent study Kırbaşlar and Şahin [10] In a recent study
Kırbaşlar and Şahin [10] evaluated the extraction of phenolic compounds from olive leaves
using MAE, and obtained better results (10.45 mgGAE/g dry leaf) using acetonitrile as a
solvent, which is a harmful solvent. Therefore, the use of water as a solvent reduces the cost
of the process, makes it safer and cleaner, and expands the possibilities for future applications.
The OP was the predominant phenolic compound found in the extracts, followed by
HT. Oleuropein has been shown to be a powerful inhibitor of viruses, blocking the synthesis
of enzymes with roles in virus replication [33]. In a recent study, analyzing solutions to deal
with the COVID-19 pandemic, the oleuropein compound of olive leaves was mentioned for
its antiviral potential [33]. The suitability of each technique changes based on the properties
of the target class of compound for extraction and MAE has been shown to be more efficient
for extracting more polar compounds such as oleuropein [26]. The OP content reported in this
study for the MAE was 14.468 mg.g-1 (d.b.), a two-fold increase compared to the UAE. In
many cases, the UAE is used as a supplementary technique to increase the yield, as
demonstrated bt Xie et al. [34] when they obtained higher yields for oleuropein when they
used the UAE as a combined extraction technique. Yateem et al. [35] reported a high yield of
oleuropein, due to an increase in solubility of this compound, which was obtained by using a
higher temperature (60°C). In the literature, the measured concentrations of OP in olive leaves
are in the range of 6 to 20 mg.g-1 (d.b.) [12,36,37]. However, the extraction conditions
reported by the studies apply toxic solvents, such as acetonitrile and methanol. Şahin et al.
[27] also explored solvent-free MAE methods for the extraction of polyphenolic compounds,
57
and oleuropein from olive tree leaves. Yet, the yield observed by them was considerably
lower than the one reported in this work. They obtained, in the optimum extraction conditions
(250 W and 2 min), a oleuropein yield of 0.00006 mg.g-1 (d.b.), and a TP of 0.00248 mgGAE.g-
1 (d.b.).
The AI of olive leaf aqueous extracts against Escherichia coli was investigated in this
study. Though most of the strains of E. coli are harmless and thrive in the lower intestine of
human body, few strains can lead to food poisoning and gastroenteritis. E. coli contamination
in food products is one of the major causes of economic loss due to product recalls in North
America and Europe [38]. The antimicrobial analysis results for the olive extract obtained by
MAE and UAE are reported in Table 2.
Table 2. Antimicrobial Inhibition (AI) effect of extracts obtained from MAE and UAE against
E. coli.
Olive leaf extract
(mg.mL-1)
AI (%)
MAE UAE
75 100 91.8
50 100 80.9
25 85.4 0
10 38.6 0
5 23.9 0
The extract produced by MAE showed an inhibition of E. coli growth of 100% at a
concentration of 50 mg.mL-1, while at the same concentration, the UAE extract inhibition was
of 80.9%. The inhibition effect reached almost 92% for the UAE extract at 75 mg.mL-1. Liu et
al. [39] has reported that at a concentration of 62.5 mg.mL-1 the extract from olive leaves
inhibited 95% of the growth of E. coli. Similar to our results, however, utilizing a commercial
extract of olive leaves, Sudjana et al. [7] also observed a 100% of inhibitory activity for E.
coli. These differences are due to the use of different extraction methodologies, the origin of
the leaves, and the concentrations of synthesized bioactive compounds. The impact of the
geographic origin of the leaves on the concentration of the phenolic compounds has already
been proven. The concentration of phenolic compounds, obtained by UAE, ranging from 7.35
to 38.66 mgGAE/g-dried leaf have been obtained in metanoic extracts of olive leaves from
different regions of Anatolia [40]. Certainly, the chemical composition of the extracts
conditioned the observed antimicrobial effects [41], suggesting that Brazilian olive cultivars
58
have an enormous potential for recovery of bioactive compounds. The literature shows that
plant extracts with high phenolic content have a strong antibacterial activity [42]. Therefore,
our findings revealed that the high antibacterial activity of the MAE extract could be
attributed to the high OP and TP contents, compared to the one obtained by other extraction
methods.
OP and HT have been proven to inhibit or delay the growth rate of several pathogens [43].
Additionally, the use of extracts as antibacterial agents might be more beneficial and
synergistic than isolated constituents. Bioactive individual compounds can change their
properties in the presence of other compounds that are naturally present in the leaf extracts
[44]. This enhanced property, due to synergistic effect, is important in the bio-active
enrichment and makes the use of leaf extracts in the food and biopharma industries extremely
interesting [45].
Time-kill studies are important because the comprehensive information on
pharmacodynamics of an antimicrobial agent may not be obtained simply through endpoints
such as MIC [46]. Turbidimetry was used as the method to determine the growth of the
microorganism; the gradual cellular growth was inferred by absorbance measurements (Fig.
1).
Fig. 1. Kinetic growth profiles of E. coli against olive leaf extracts. MAE=microwave-assisted
extraction; UAE=ultrasound-assisted extraction; NC=negative control; PC=positive control.
59
Fig. 1 shows the kinetics of antimicrobial inhibition of olive leaves extracts at a
concentration of 50 mg.mL-1 against E. coli during the incubation period. The positive control
showed a typical expected growth curve including the exponential and stationary phases.
According to the findings, no bacterial growth was observed on extracts during the 75 min
after inoculation. Differences in antimicrobial activity between the MAE and UAE were
observed. After 75 min, the extract obtained from MAE presented a slight growth. The results
from the samples containing the UAE extract showed a less pronounced cell inhibition. In this
case, there was a point where the bacterial cells obtained large sizes and deposited in the
bottom, causing the backscattering effect, in which there is an increase of the spread of light
in the suspension, causing the false conclusion of a decrease in bacterial growth. However,
there was still a substantial significant difference in bacterial viability between the control and
the bacterial sample exposed to olive leaf extracts 16 hours after the inoculation. Thus, we
report on the antibacterial effect of the extract of Brazilian olive leaves against E. coli, as it
interferes with microbial growth, which can lead to bacteria death.
3.4 CONCLUSION
Different extraction techniques were used and compared to extract bioactive
compounds from Brazilian olive leaves using water as solvent. The results obtained in this
study demonstrate that MAE is more efficient than UAE and maceration. Under these
conditions to MAE, the OP and HT extractions were 14.468 and 0.590 mg.g-1 (d.b.).
From the antimicrobial inhibition results, the use of olive leaves as nutraceuticals may
lower the risk of E. coli contamination, mainly due to the protective action provided by its
bioactive compounds, mainly attributable to the presence of OP and HT. The extract produced
by MAE showed a MIC at concentration of 50 mg.mL-1. This study also reports on the
inhibition kinetics, which shows a quick effect (after 75 min).
Based on the results presented, antioxidant and antimicrobial properties were verified
and the potential for valorization of byproducts from olive cultivation, especially in emerging
plantations in southern Brazil. In the food industry, olive leaf extracts have shown to have a
broad application prospect in food industries to improve the nutritional profile of foods. Also
important to the food industry and others, the antibacterial effect of the extract should be
explored. Although future studies aiming at characterizing the antibacterial activity of the
extract, along with the biomolecular mechanism of the inhibition will be important to
60
elucidate this finding, this study is the first in reporting the important, and environmental-
friendly, potential of olive leaves extract as antibacterial agents.
Thus, in future work, olive leaf extracts can be used for the preparation of food
coatings in order to increase the shelf life of products. Furthermore, regarding the Covid-19
pandemic, that brought fast and substantial changes in the most diverse systems and
organizations, the academic-scientific environment are encouraged to find advances. We
intent in our future studies to produce nanofibers masks added with olive leaf extracts, which
may have the potential to provide extra biological protection through antimicrobial or
antiviral action.
Acknowledgements
The authors would like to thank the Coordination for the Improvement of Higher
Education Personnel and the Natural Sciences (CAPES), process number:
88881.119571/2016-01, and Natural Sciences and Engineering Research Council of Canada
for their financial support. This research was also supported by the Brazilian National
Counsel of Technological and Scientific Development (CNPq), process number:
249096/2013-7.
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- CAPÍTULO 4 -
OTIMIZAÇÃO DA EXTRAÇÃO DE COMPOSTOS BIOATIVOS DA FOLHA DE
OLIVEIRA
66
O Capítulo 3 revelou que o melhor método de extração para recuperação de compostos
com potencial bioativo é a extração assistida por micro-ondas, diante disso o Capítulo 4
explorou essa técnica de extração visando a determinação das condições ótimas de extração
desses compostos de folhas de oliveira a partir do estudo de otimização. Essa pesquisa está
apresentada no artigo 2.
Artigo 2
Optimization of green extraction for the recovery of bioactive compounds from
Brazilian olive crops and evaluation of its potential as a natural preservative
Thamiris Renata Martiny1,2, Vijaya Raghavan3, Caroline Costa de Moraes4, Gabriela Silveira
da Rosa2,4, Guilherme Luiz Dotto1*
1Department of Chemical Engineering, Federal University of Santa Maria, 97105–900, Santa
Maria, Rio Grande do Sul, Brazil.
2Engineering Graduate Program, Federal University of Pampa, 1650, Maria Anunciação
Gomes de Godoy Avenue, Bagé, Rio Grande do Sul, Brazil.
3Department of Bioresource Engineering, McGill University, 21111 Lakeshore Road, Ste–
Anne–de–Bellevue, Quebec, Canada, H9X 3V9.
4Materials Science and Engineering Graduate Program, Federal University of Pampa, 1650,
Maria Anunciação Gomes de Godoy Avenue, Bagé, Rio Grande do Sul, Brazil.
Thamiris Renata Martiny Email: [email protected]
Vijaya Raghavan Email: [email protected]
Caroline Costa de Moraes Email: [email protected]
Gabriela Silveira da Rosa Email: [email protected]
Guilherme Luiz Dotto Email: [email protected]
*Corresponding author: UFSM, 1000, Roraima Avenue, 97105–900, Santa Maria, RS, Brazil.
Email address: [email protected], phone number (+55) 55 32208448
Artigo publicado no periódico Journal of Environmental Chemical Engineering
Volume 9, Issue 2, 2021, 105130
ISSN: 2213-3437. DOI: https://doi.org/10.1016/j.jece.2021.105130
67
Graphical Abstract
Abstract
Olive leaves are a great resource to obtain bioactive compounds, contributing to sustainability
in agriculture. This work aimed to optimize the extraction conditions of bioactive compounds
from Brazilian olive leaves using a green technique and evaluate the potential of the extract as
a food additive with antioxidant and antibacterial activities. The microwave-assisted
extraction (MAE) was used to obtain the olive leaves extracts (O.L.Extracts). A central
composite rotational design was applied to optimize the MAE as a function of pH,
temperature and, irradiation time. The response variables were total phenolic (T.phenolic) and
antioxidant activity (Antioxidant.A). The optimal conditions for MAE were: 100 °C, 2 min,
and pH 6. Under these conditions, it is possible to obtain Antioxidant. A equal to 92.87% and
T.phenolic equal to 103.87 mgGAE.g– 1(d.b), according to the desirability function.
O.L.Extracts obtained under optimal conditions of MAE extraction showed antibacterial
activity against Escherichia coli, with a minimum inhibitory concentration value of 50 mg
mL-1 and the oleuropein content was 11.59 ± 0.004 mg g-1 (d.b). In addition to being able to
be recycled, olive leaves represent a viable source of bioactive compounds from green
extraction. Hence, their extracts have enormous potential for application in various areas,
especially natural additives, in the food area.
Keywords: olive leaves; microwave-assisted extraction; phenolic compounds; antioxidant
activity; oleuropein.
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4.1 INTRODUCTION
The olive tree is a fruit plant, scientific name Olea europaea L., family Oleaceae.
Olive tree crops exist throughout the world, making up areas of the order of millions of
hectares, of which 90% are located on the coast of the Mediterranean Sea [1]. But this culture
has been expanding into other territories, especially in Brazil. The cultivation of olive trees
has been growing and changing the landscape, before with only vast agriculture fields, now as
olive groves as far as the eye can see [2,3]. The Brazilian lands proved to be good for
cultivating the olive tree, producing olive oils awarded worldwide. This scenario demonstrates
the economic and social importance of this crop and the emerging opportunity of using any of
its by-products of olive crops. From the economic viewpoint, the interest in olive trees is
linked to olives and olive oil production. In this production, many residues are generated,
such as pomace, mill wastewater, and leaves. Many studies have been published about other
olive-based residues in the production of value-added products, such as the use of olive
pomace for the production of cosmetics and supplementation of diets and the recovery of
bioactive compounds from mill wastewater have been appreciated in food and pharmaceutical
industry [4,5]. Nevertheless, the recycling of olive leaves must be considered, as well as the
most effective methods to extract all its bioactive properties.
This plant's leaves are considered by-products generated in large quantities, about 25
kg of leaves and branches, per tree, from the pruning activities [6–8]. Despite this,
historically, olive leaves are used for medicinal purposes, such as fighting fevers. Besides,
they are known to possess bioactive potential, which exhibits antioxidant and antimicrobial
properties, which have been linked to their strong preservative characteristics [9–11].
Therefore, it is interesting to appreciate and use this by-product due to its low cost and great
accumulation in this agricultural activity, representing an environmental, technical, and
economical solution to manage these by-products [6,8,12,13].
Olive leaves are of interest to science and industry worldwide [14]. According to De
Castro and Capote [15] and Japón-Luján [16], the leaves have the greatest power to extract
compounds with bioactive potential. Several studies have shown the great diversity of phenols
in olive leaves [10,14,17–19]. The most abundant phenolic compound is oleuropein [20],
about 60-90 mg/g of dry leaf, 73% of the total of its constituents [21]. Oleuropein has strong
antimicrobial activity. The main mechanisms proposed include damage to the cell membrane
and interference with the production of certain amino acids essential for these microorganisms
[22]. Furthermore, such compounds play important roles as antioxidant agents due to their
69
ability to scavenge free radicals, thus saving the cell from different diseases [23]. Larrosa et
al. [24] already highlighted vegetables as important sources of exogenous antioxidants like
phenolic compounds.
Given the exposition, the investigation of olive leaves extracts in the most diverse
industrial sectors is relevant, especially concerning the application as a natural additive in the
food area. With the benefit of the chemical industry's advancement, the food industry started
to use many chemical additives to improve the storage conditions. However, there is no
concern about the risks to health or the environment of these synthetic additives [25]. For this
reason, it is important to search for potential natural food additives, such as olive leaf extract,
which, due to its characteristics already mentioned, can act as a preservative and antioxidant
additive. Some studies point in this direction. The results obtained by Lee and Lee [26] show
that the combination of olive leaf extract phenolics possessed antimicrobial and antioxidant
activities. Also, Şahin et al. [27] obtained extracts from olive leaves that showed excellent
antibacterial and antioxidant activities. However, antioxidant activity and phenolics in olive
leaves were reported [6,28,29], but few studies are available on optimum extraction
conditions and quantification of individual phenolic compounds. When it comes to the olive
tree of Brazilian geographical origin, studies are non-existent.
The extraction of natural products is an operation of selective and effective removal of
compounds or fractions of interest within a plant matrix [30]. However, each plant has its
specific properties in terms of the extraction of biologically active compounds. It is then
important to obtain the optimal extraction conditions. Extraction success is influenced by
several factors, but mainly by the active compounds' thermal stability, extraction technology,
pH, temperature, solvent, and extraction time [31–33]. Therefore, it is fundamental to evaluate
several technologies that favor bioactive compounds' stability during extractions, which are
ecologically correct, efficient, and cost-effective. The quantity and quality of bioactive
compounds present in a plant depend on numerous conditions [34]. These include factors such
as drying, extraction, separation, and purification. Among these, extraction is a basic and
foremost important step in extracting the optimum amount of bioactive compounds. Some
alternative extraction techniques such as microwave-assisted extraction (MAE), ultrasonic-
assisted extraction, pressurized liquid extraction, and supercritical fluid extraction are
available and proven effective for phytochemicals [34]. The MAE is known to speed-up the
extraction process, low-energy requirement, minimum degradation of target components, and
better extraction yield along with lesser solvent use compared with conventional and other
advanced extraction techniques [35].
70
This work is of great value and will make a difference for the wider public. It provides
data on the extraction of bioactive compounds from olive leaves from plantations in southern
Brazil, whose information is still limited, given that the cultivation of olive trees in this area
region started recently. Coupled to this, the research seeks to solve a waste problem generated
in the cultivation of the olive tree, which is the leaves, making use of this waste that has
enormous potential as a source of bioactive compounds. Besides, it explores aspects of a
fundamental unit operation in chemical engineering, which is extraction. The research seeks
alternative forms of extraction that do not harm the environment, using water as a solvent,
providing valuable information within this research line.
The main goal of this work is to study the green extraction of olive tree leaves using
the MAE approach to optimize the extraction conditions and to prove that the extracts
produced under the optimum conditions are viable for future applications like natural additive
for the food industry, investigating their oleuropein content and their antimicrobial action.
4.2 MATERIALS AND METHODS
4.2.1. Olive leaves
The leaves, when grown, are long and narrow, elliptical, elliptical-lanceolate, and
lanceolate. They have a length ranging from 5.0 to 7.0 cm and a width of 1.0 to 1.5 cm and a
dark green and bright color in the ventral region and greyish or whitish green in the dorsal
region [36]. Fig. 1 represents the olive leaves of cultivar Arbequina collected in the region of
the Rio Grande do Sul campaign in southern Brazil (31º30'04.0 "S, 53º30'42.0" W). After
collection, the leaves were oven-dried (ETHIK, Brazil) (40°C-24 h). Then, the leaves were
milled (IKA® A11BS32, China) and sieved (metal mesh 60, Bertel, Brazil). Leaf powder
(particles less than 0.272 mm in diameter) was used in the extraction processes.
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Fig. 1. Olive leaves.
4.2.2 Chemicals
The reagents used were: Folin Ciocalteu’s, anhydrous sodium carbonate, 2,2-diphenyl-
1-picrylhydrazyl (DPPH), methanol (HPLC grade), gallic acid, oleuropein, acetonitrile
(HPLC grade), and acetic acid (HPLC grade). All reagents were obtained from Sigma Aldrich
(St. Louis, USA).
4.2.3 Apparatus and extraction
Olive leaves extracts (O.L.Extracts) were obtained by microwave-assisted extraction
(MAE). MAE was conducted using a multimode (closed) microwave unit (MiniWAVE SCP
Science, Canada) with the frequency and irradiation power of 2.45 GHz and 1000 W. 0.5 g of
powdered olive leaves was added to 25 mL of distilled water [37]. The vessels were placed in
equipment, and the MAE was performed. After extractions were complete, the solids were
separated from the mixture by vacuum filtration (Sigma Aldrich, St. Louis, USA).
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4.2.4 Experimental design
The type of solvent, extraction time, temperature, solvent concentration,
sample/solvent ratio, and microwave power are fundamental factors for the recovery of
polyphenols using microwave-assisted extraction [33]. Therefore, different extraction
conditions were tested. In this study, a central composite rotational design (CCRD) was used
to determine the main affecting factors and to verify the existence of quadratic terms in the
regression model. To evaluate the pure error, the design consisted of fourteen randomized
runs with three replicates at the central values. Independent variables used in the experimental
design were pH, irradiation time, and temperature (see Fig. 2). The independent variables
(Table 1) were coded in levels, and the answers are presented. The obtained parameters were
replaced in the second-order polynomial model shown in Equation 1.
Υ = 𝛽0 + ∑ 𝛽𝑖𝜅𝑖 = 1 Χ𝑖 + ∑ 𝛽𝑖𝑖
𝜅𝑖=1 Χ𝑖𝑖
2 + ∑ 𝛽𝑖𝑗𝜅−1𝑖 Χ𝑖 Χ𝑗 + (1)
where Υ is the predicted response (Antioxidant.A and T.phenolics), 𝛽0 is the constant
coefficient, Χ𝑖 are non-coded values of independent variables (pH, temperature, and
irradiation time), 𝛽𝑖, 𝛽𝑖𝑖, and 𝛽𝑖𝑗 are the linear, quadratic, and cross-product coefficients, and
is error.
Fig. 2. Central rotational composite design for microwave-assisted extraction (MAE) with
experimental values and coded levels of independent variables.
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4.2.5 Estimation of total phenolic compounds and antioxidant activity
To obtain total phenolic compounds (T.phenolics) spectrometric method was used
[38]. For this, an aliquot (500 µL) of O.L.Extracts is mixed with 1000 µL of distilled water
and 100 µL of Folin-Ciocalteu. After 5 min, 8000 µL of 7.5% (w / v) aqueous sodium
carbonate solution is added. The mixture is stored in the dark for two hours. Then, the
mixture's absorbance is measured at a wavelength of 765 nm with a spectrophotometer
(Ultraspec 2100 pro, Biochrom Ltd., Cambridge, United Kingdom).
2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging method [39] was used
to determine the antioxidant activity (Antioxiant.A). Briefly, the O.L.Extracts (200 µL) is
mixed with 7800 µL of DPPH solution and kept for 30 min in the dark at room temperature.
The same procedure is carried out with an aliquot of water to obtain the control. The
absorbance of the control and the extracts samples are measured using a spectrophotometer
(Ultraspec 2100 pro, Biochrom Ltd., Cambridge, United Kingdom) at 517 nm.
4.2.6 Potential of the leaf extract as a food preservative
The O.L.Extract's potential as a food preservative was determined using two
indicators: the oleuropein content and the antimicrobial action.
Quantitative analyzes of oleuropein in O.L.Extracts were performed by high-
performance liquid chromatography - HPLC (Agilent 1100 series instrument, USA).
Oleuropein was separated on a Discovery column (Supelco, USA) RP C18 (5 μm, 25 cm ×
4.6 mm), equipped with a Supelguard cartridge (Discovery, USA), C18 (5 μm, 2 cm × 4 mm)
and then analyzed using a variable wavelength detector (VWD) that is set to 280 nm. The
mobile phase is a mixture of water, acetonitrile, and acetic acid (80/19/1 v/v/v). The extract
was filtered through a 0.45 mm syringe filter and injected directly into the HPLC. The column
temperature is maintained at 25 °C, and the mobile flow was fixed at 1.0 mL.min-1.
Oleuropein was identified and quantified using external standards and a calibration curve. The
oleuropein content is expressed in mg.g-1 of olive leaves on a dry basis.
The Minimum Inhibitory Concentration (MIC) was evaluated against Escherichia coli
(E. coli) (ATCC 11229). E. coli was chosen because it is an important bacterium that
indicates food contamination, thus being able to limit or make dangerous its consumption
[40]. The MIC of the extract was defined as the lowest concentration of the extract necessary
for complete inhibition of visible growth [27]; concentrations ranged from 5 to 150 mg.mL-1
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of extract. The antimicrobial action of the extracts was determined by the Elisa plate culture
adapted method [41,42]. In a 96-well microplate, 145 μL of sterile Mueller-Hinton broth, 135
μL of extract (in each of the tested concentrations), and 20 μL of the culture containing the E.
coli were pipetted into well. The antimicrobial action was quantified by the absorbance
(wavelength of 630 nm) difference between the two readings, one in the initial time and the
other after 16 h of incubation, of the samples containing each extract in different
concentrations concerning the mean absorbance of the control (with water) samples.
4.2.7 Statistical Analysis
All the experimental analyses were performed in triplicate, and the results are the
means with the mean deviation. All results were analyzed statistically with the aid of the
statistical software Statistica®, version 10.0. The adequacy of ANOVA for the models for all
independent variables without interaction was assessed using the Fisher's F-test and the lack
of fit test (at a 5% level of statistical significance) with an adjusted R2 value. The optimal
conditions of extraction were calculated using the procedure of desirability (in optimal values)
to maximize the response variables, optimizing processes with multiple responses.
4.3 RESULTS AND DISCUSSION
4.3.1 Optimization of microwave-assisted extraction conditions
Table 1 shows the values of phenolic compounds and antioxidant activity of the
O.L.Extracts obtained by microwave-assisted extraction according to the CCRD conditions.
Fig. 3 shows the O.L.Extracts obtained in each of the 17 extraction conditions; the visual
analysis assumes that the results of the extractions were different. The extraction process's
good reproducibility was verified through the results obtained by experiments 15 to 17, which
correspond to the central points for both total phenolic compounds and antioxidant activity.
From Table 1, it was found that for the experimental conditions, the range obtained for the
total phenolic compounds was from 67.84 to 119.16 mgGAE.g–1 (d.b). For antioxidant activity,
values between 77.56 and 93.58% were obtained.
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Fig. 3. Extracts obtained from microwave extraction. The numbers correspond to each extract
obtained in the respective extraction condition (1-17) of Table 1.
Table 1. Total phenolic compounds (T.phenolics) and antioxidant activity (Antioxidant.A) of
extracts obtained by microwave.
Extration
condition
Irradiation
time (min) pH
Temperature
(ºC)
T.phenolics
(mgGAE.g–1
d.b)*
Antioxidant.A
(%)*
1 2 (-1) 3 (-1) 60 (-1) 79.85 ± 0.89 92.71 ± 0.003
2 6 (1) 3 (-1) 60 (-1) 88.79 ± 1.78 82.17 ± 0.1015
3 2 (-1) 9 (1) 60 (-1) 80.09 ± 0.84 79.70 ± 0.0041
4 6 (1) 9 (1) 60 (-1) 89.08 ± 1.18 84.49 ± 0.0038
5 2 (-1) 3 (-1) 100 (1) 103.24 ± 0.28 93.04 ± 0.0008
6 6 (1) 3 (-1) 100 (1) 110.02 ± 0.86 92.19 ± 0.0007
7 2 (-1) 9 (1) 100 (1) 105.91 ± 0.53 86.96 ± 0.0045
8 6 (1) 9 (1) 100 (1) 106.16± 0.86 83.81 ± 0.0069
9 4 (0) 6 (0) 46 (-1.68) 67.84 ± 0.62 90.49 ± 0.0058
10 4 (0) 6 (0) 114 (+1.68) 115.80 ± 0.49 92.12 ± 0.0054
11 4 (0) 1 (-1.68) 80 (0) 119.16 ± 1.61 93.58 ± 0.0071
12 4 (0) 11(+1.68) 80 (0) 111.32 ± 0.36 77.57 ± 0.0081
13 0.64 (-1.68) 6 (0) 80 (0) 89.32 ± 1.81 92.05 ± 0.0014
14 7.36 (+1.68) 6 (0) 80(0) 99.64 ± 1.15 91.14 ± 0.0016
15 4 (0) 6 (0) 80 (0) 100.46 ± 0.05 92,06 ± 0.0043
16 4 (0) 6 (0) 80 (0) 100.33 ± 0.33 92.59 ± 0.003
17 4 (0) 6 (0) 80 (0) 100.18 ± 1.57 92.38 ± 0.0023
* mean ± standard deviation (n = 3).
From the CCRD response matrix (Table 1), Pareto charts were generated to verify the
significance of temperature, time, pH, and their interactions in the considered responses.
Pareto plots for total phenolic compound responses and antioxidant activity are shown in Fig.
4 (a) and 4 (b), respectively. Fig. 4 shows that all the main effects (temperature, time, and
76
pH), both linear and quadratic, and also the interaction effect, significantly influenced
(p≤0.05) the response to total phenolic compounds (Fig. 4 (a)). Şahin et al. [27] also found
that the variable extraction time was significant when they studied the extraction of phenolic
compounds from olive leaves. For the antioxidant activity response (Fig. 4 (b)), only the
interaction between linear temperature and time was not significant.
Fig. 4. Pareto chart of the responses (a) total phenolic compounds and (b) antioxidant activity
77
Second-order Equations 2 and 3 are derived for total phenolic compounds
(T.phenolics), and antioxidant activity (Antioxidant.A), respectively, as a function of
temperature (T), time (t), and pH.
T. phenolics = 100,66 − 4,06T2 − 3,25t2 + 4,15pH2 + 12,27T + 3,10t − 1,01pH −
+0,22TpH − 1,36Tt − 0,81pHt (2)
Antioxidant. A = 92,50 − 0,87T2 − 0,80t2 − 2,99pH2 + 1,43T − 3,82pH − 0,47TpH +
+1,62pHt (3)
To verify whether the statistical models (Equations 2 and 3) were predictive and
significant, the analysis of variance and Fisher's F-test (Tables 2 and 3) were used. The model
that represents the total phenolic compounds as a function of the independent variables
(Equation 2) was statistically significant (R2 = 0.92) and predictive since the F calculated
value (Fvalue= 15.62) was about four times higher than the standard F (Ftable = 3.68). The
model that represents the antioxidant activity (Equation 3) was also statistically significant
(R2 = 0.82) and not predictive since the F calculated value was approximately equal to the F
table (Fvalue= 3.79 and Ftable = 3.68). Regardless, the results can be included in the analysis by
the recommendations from other authors who also found similar behavior in plant matrices
[43,44]. Thus, the models can adequately adjust to the experimental data, indicating the
relationship between the dependent and independent parameters. The experimental designs
were found to be adequate to describe and predict the extraction process of phenolic
compounds and antioxidant activity from olive leaves, providing useful models that
sophisticated the research process towards better research parameters, allowing to guide
decision making.
Table 2. Analysis of variance for phenolic compounds.
Sum of
squares
Degrees of
freedom
Quadratic
mean F value F table
Regression 2915.77 9 323.97 15.62 3.68
Residual 145.17 7 20.74
Lack of fit 145.13 5 29.03
Pure error 0.04 2 0.02
Total 3060.94 16
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Table 3. Analysis of variance for antioxidant activity.
Sum of
squares
Degrees of
freedom
Quadratic
mean F value F table
Regression 381.66 9 42.41 3.79 3.68
Residual 78.34 7 11.19
Lack of fit 79.19 5 0.02
Pure error 0.14 2
Total 460.01 16
The estimation of the optimal conditions for the response variables defined in the
experimental design was performed based on the proposed statistical models and "Function
Desirability." Table 4 presents the specifications of the numerical values for the lower limit,
average value, and upper limit, in addition to the exponents s and τ and the desirability
required by the Statistica software as input for the optimization of the extraction. The values
in parentheses are the desirability (Table 4). For antioxidant activity, responses above 85.57%
were considered acceptable, whereas total phenolic compounds are considered unsatisfactory
below 67.84 mgGAE.g– 1 (d.b) acceptable close to the midpoint and satisfactory close to the
upper limit. The aim was to maximize these responses; the upper limits were considered
desirable. The high value chosen against the low value of τ causes the desirability to decrease
rapidly, becoming very low unless the predicted response is very close to the target. In this
case, it is more acceptable for the response value to be above the target than below it. Fig. 5
shows the results of the optimization algorithm.
Table 4. Parameters used in the simultaneous optimization of microwave extraction
responses.
Response Inferior limit Midpoint Superior limit s τ
Antioxidant activity 77.57 (0) 85.57 (1) 93.58 (1) 10 1
Total phenolic compounds 67.84 (0) 93.50 (0.5) 119.1 (1) 10 1
79
Fig. 5. Profile of predicted values and desirability for antioxidant activity and total phenolic
compounds for microwave extraction.
The curves show how the responses vary with each factor, keeping the levels of the
other factors fixed at the specified values. In the second graph of the first column, for
example, it can be seen that the antioxidant activity is little affected by the temperature
variation. Time has little influence on the responses since temperature and pH are the
determining factors for obtaining the optimum point. The desirability graphs as a function of
temperature and pH (fourth line) show that any change in temperature or time will cause a
sharp drop in desirability. The vertical dashed lines indicate maximum global desirability
conditions, which for this set of tests reached 0.84 (blue dashed line). It was obtained at a
temperature of 100 °C in 2 min and pH 6 (red dashed line). Under these conditions, it will be
possible to obtain the antioxidant activity equal to 92.87% and the total phenolic compounds
equal to 103.87 mgGAE.g– 1 (d.b), as shown by the values marked on the respective axes (solid
blue line).
80
The condition optimized by the "Desirability function" was tested experimentally for
validation. The result for total phenolic compounds was 115.96 ± 0.56 mgGAE.g–1(d.b), and the
result for antioxidant activity was 89.52 ± 0.013 %. The result of total phenolic compounds
exceeded the model's expectation, whereas the antioxidant activity resulted below the
expected result. The results indicated the suitability of the model. The Desirability was
successfully applied to optimize the extraction process in a shorter extraction time and at a pH
without the need for correction, since pH 6 was that of the distilled water used.
The optimal extraction conditions are following the data reported in the literature.
Şahin et al. [27] also reported very short extraction times. They also find a better time in 2
min during the extraction of phenolic compounds from olive leaves using microwaves. The
short time is one of MAE's main related advantages because conventional techniques require
a long extraction time, causing the degradation of bioactive compounds [37,45]. As
previously reported, MAE presents efficient extraction yields in a shorter time and without the
degradation of the extracted bioactive compounds [46]. In addition, because it does not
require a long extraction time and has a good yield, the MAE justifies its use despite its
energy expenditure.
About the temperature effect, in extraction with microwaves, the elevated temperature
favored the removal of the bioactive compounds present in the leaf matrix. The increase in the
temperature decreases solvent viscosity, causing an increase in the intermolecular interaction
resulting in increased compounds solubility, increasing the mass transfer coefficients. Higher
temperatures can also cause cell disruption due to the increase in intracellular pressure [47].
All these facts corroborate the result of a higher temperature for the optimal extraction
condition.
Finally, for the variable pH, extraction proceeds better at more moderate pHs.
Regarding the recovery of polyphenols, pH can impact different mechanisms, such as
increased solute solubility and alternation of antioxidant interactions with other plant material
[48]. According to Japón-Luján et al. [49], the largest amount of phenolic compound
oleuropein extracted from olive leaves was found at pH 7. In contrast, the authors used the
ultrasound-assisted extraction technique.
Regarding the yield of phenolic compounds and antioxidant activity, the results found
were excellent, indicating a good prospect for future applications of the produced
O.L.Extracts. In recent contribuitions from our research group [50] was reported that
Arbequina olive leaves collected from the same location showed lower (41.40 mgGAE.g–1)
phenolic content when maceration process with water was applied. Compared to other
81
researches, similar antioxidant activity results of up to 94% were obtained for olive leaf
extracts [4,18,23]. In line with the results obtained in the present study, Rosa et al. [37] also
produced olive leaf extract by MAE but did not explore the pH variable. They found 104.22
mgGAE.g–1 (d.b.) for total phenolic compounds and 90.03% for antioxidant activity, at a
temperature of 86 °C and extraction time of 3 min. Abaza et al. [28] also produced aqueous
extracts of olive leaves, but using room temperature and maceration as an extraction
technique, and found 16.52 mgGAE.g–1 (d.b). Ahmad Qasem et al. [51] studied the phenolic
extraction of olive leaves by maceration and found the value of 66 mgGAE.g–1 (d.b.) for the
total phenolic compounds. Rafiee et al. [52] reported that the phenolics compounds yield of
54.08 mgGAE.g−1 (d.b.) for olive leaf extract was obtained using water as a solvent in MAE
with 4 min of extraction time.
4.3.2 Oleuropein content and antimicrobial action of the O.L.Extracts
The oleuropein content quantified under optimized microwave-assisted extraction
conditions was 11.59 ± 0.004 mg.g-1 (d.b.). In a study by Japón-Luján et al. [53], 23 mg.g-1
(d.b.) of oleuropein was extracted using the microwave technique in a mixture of ethanol and
water (80:20 v/v). In contrast, Khemakhem et al. [54] used the maceration technique and
produced aqueous extracts of olive leaves, obtaining 2.65 mg.g-1 (d.b.) of oleuropein. Jemai et
al. [55] extracted 0.0432 mg.g-1 (d.b.) of oleuropein from olive leaves using a mixture of
methanol and water (200 mL, 4: 1 v/v) by maceration. Ansari et al. [56] extracted the
oleuropein with water at 60 °C and obtained approximately 15 mg.g-1 (d.b.). Coppa et al. [57]
obtained a high oleuropein value of 65.9 mg.g-1 (d.b.) but used methanol as a solvent, and the
olive leaves came from Spain. In another study published by Stamatopoulos et al. [19], the
authors extracted oleuropein from dried olive leaves using the bleaching method steam in
multiple stages, for 48 h at 40 °C, and ethanol and water ratio 70:30. These conditions
resulted in an oleuropein content of 4.6 mg.g-1 (d.b.). The value obtained in this research was
comparable to the results reported in the cited literature. It is worth mentioning that important
differences are observed, such as the type of solvent, extraction technique, and origin of the
plant matrix. Another relevant fact was that water was used as a solvent (safe and non-toxic)
in this research, which showed a positive result in the oleuropein content obtained, thus
making its use competitive in substitution to methanol and ethanol, which are commonly used
in extractions.
82
The O.L.Extracts obtained under the optimal microwave extraction conditions showed
antibacterial activity against Escherichia coli, with a MIC value of 50 mg mL-1. This result is
a half of MIC reported in previous study [50] with same samples, but using maceration as an
extraction method. Therefore, it is noteworthy that the methodology and optimized conditions
achieved in the present study were efficient in increasing the antibacterial activity of the
O.LExtract. The result also is in close agreement with the data previously reported by Pereira
et al. [22]. They also found antimicrobial activity dependent on the concentration of olive leaf
extracts against bacteria and fungi. They attributed the antimicrobial activity to oleuropein
and the total phenolic compounds identified in the extract. Şahin et al. [27] similarly produced
olive leaves extracts using the microwave technique but evaluated the variables irradiation
power, irradiation time, and leaf mass. Their study revealed that olive leaf extract obtained in
optimal conditions showed antibacterial activity against Staphylococcus aureus and
Staphylococcus epidermidis, with a MIC value of 1.250 mg mL-1. Dominciano [58] evaluated
oleuropein's efficiency as antimicrobial power against the bacteria Listeria monocytogenes,
Staphylococcus aureus, and Escherichia coli. They found that the oleuropein also had an
antimicrobial effect (reduction: 91.49%).
The results of oleuropein content and antimicrobial action of the O.L.Extracts obtained
at optimal MAE conditions have revealed that olive leaf extract is a potentially cheap,
renewable, and abundant bioactive compound source. These valuable compounds in olive
leaves are responsible for many health benefits, and their use represents benefits for the
environment. However, the O.L.Extracts characteristics, as demonstrated in this work, are
affected by technological parameters, type of solvent, temperature, extraction time, pH, and
geographical origin. Therefore, there is a growing interest in using O.L.Extracts in various
industrial applications, such as natural food additives. Some works already point in this
direction, especially in film production. For example, the research developed by Carvalho et
al. [59] produced chitosan films with anthocyanins, which also have antioxidant power. More
specifically, Rosa et al. [60] demonstrated that when the olive leaf extract obtained by MAE is
incorporated in biodegradable carrageenan films, the films presented antioxidant
characteristics appear to be a potential strategy to add natural food additives. Martiny et al.
[50] also incorporated olive leaves extract obtained by maceration into carrageenan matrix
film, and they discovered that the film could be used as antimicrobial food packaging. Marcos
et al. [61] developed polyester films with olive leaf extract. They found that extract has
antioxidant activity in vitro. Finally, Botsoglou et al. [62] proved that olive leaves extract
could also be used as an antioxidant in meat stored.
83
The proposed MAE method allows the extraction of these compounds in a shorter time
(2 min) with higher efficiency without using any toxic solvent. Therefore, MAE proved to be
an attractive alternative to conventional extraction methods to extract bioactive compounds
from olive leaves. It is noteworthy that this research has found superior results especially
when comparing the results of phenolic compounds and antioxidant activity using the MAE
for this purpose with viable and competitive parameters. The literature cited makes use of
other extraction methods, such as maceration and ultrasound-assisted extraction. When
comparing the use of MAE in the cited literature, it appears that all parameters that were
explored here were not explored in a combined manner. It is important to note that all this was
achieved using water as an extractor solvent, which is an important fact, since most other
research to have good extraction yields uses organic solvents (methanol and ethanol), which
can limit the application of the extracts later. In comparision with the conventional extraction
[50] it was possible to increase the phenolic compounds in almost 3-fold and to ensure the
antibacterial activity against Escherichia coli with a half of MIC. Combining all obtained
results, MAE showed advantages and could be successfully used at the optimal conditions
proposed for the recovery of olive leaves bioactive compounds in order to produce a natural
additive with antioxidant and antimicrobial capacities. The O.L.Extracts produced in this
work can be applied in several industry sectors, but special attention can be given to its use as
a natural preservative in food packaging. This fact was confirmed in a recent work published
using the extract obtained from this research. Martiny et al. [63] produced carrageenan-based
active packaging film using the same O.L.Extracts from this research as a bioactive agent, and
the study revealed that the film with the O.L.Extracts was shown to have an antimicrobial
capacity during the storage of lamb meat with the potential to increase the shelf life of it.
4.4 CONCLUSIONS
This work studied the effects of temperature, irradiation time, and pH during the
microwave-assisted extraction (MAE) of bioactive compounds from southern Brazil's olive
leaves. The regression models can accurately predict the phenolic compounds and antioxidant
activity of the MAE. The ideal extraction conditions for the examined responses were as
follows: pH = 6, irradiation time = 2 min and temperature = 100 °C. Under these ideal
conditions, the oleuropein concentration was 11.59 mg.g-1 (d.b.), and the olive leaf extract
showed antimicrobial action against the bacterium Escherichia coli with a MIC of 50 mg mL-
1. The data of this research demonstrate that the olive leaves can supply compounds with
84
bioactive potential, which can be used as a resource and should not be discarded as residue.
This fact reinforces the questions about sustainability in olive growing. The extract produced
represents an alternative to chemical additives in the food industry, exploiting their
antioxidant and antimicrobial activities.
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90
- CAPÍTULO 5 -
EXTRATO DE FOLHA DE OLIVEIRA PARA OBTENÇÃO DE EMBALAGEM
ATIVA
91
O Capítulo 5 explorou a aplicação do extrato de folha de oliveira obtido nas condições
ótimas de extração, determinadas no Capítulo 4, na efetiva utilização como potencial aditivo
conservante em embalagem biodegradável ativa. O artigo 3 reuniu os resultados dessa
pesquisa.
Artigo 3
Bio-Based Active Packaging: Carrageenan Film with Olive Leaf Extract for Lamb Meat
Preservation
Thamiris Renata Martiny1,2, Vijaya Raghavan3, Caroline Costa de Moraes4, Gabriela
Silveira da Rosa1,4,* and Guilherme Luiz Dotto2
1 Engineering Graduate Program, Federal University of Pampa, 1650, Maria Anunciação
Gomes de Godoy Avenue, Bagé, 96413-172 Rio Grande do Sul, Brazil;
2 Chemical Engineering Department, Federal University of Santa Maria, Santa Maria,
97105-900, Rio Grande do Sul, Brazil; [email protected]
3 Department of Bioresource Engineering, McGill University, 21111 Lakeshore Road, Ste-
Anne-de-Bellevue, H9X 3V9 Montreal, QC, Canada; [email protected]
4 Graduate Program in Materials Science and Engineering, Federal University of Pampa,
1650, Maria Anunciação Gomes de Godoy Avenue, Bagé, 96413-172 Rio Grande do Sul,
Brazil; [email protected]
* Correspondence: [email protected]; Tel.: +55 53-9996-722-26.
Artigo publicado no periódico Foods
Volume 9, Issue 12, 2020, 1759
ISSN: 2304-8158. DOI: https://doi.org/10.3390/foods9121759
92
Graphical Abstract
Abstract: Carrageenan-based active packaging film was prepared by adding olive leaf
extract (OLE) as a bioactive agent to the lamb meat packaging. The OLE was characterized
in terms of its phenolic compounds (T.ph), antioxidant activity (AA), oleuropein, and
minimum inhibitory concentration (MIC) against Escherichia coli. The film’s formulation
consisted of carrageenan, glycerol as a plasticizer, water as a solvent, and OLE. The effects
of the OLE on the thickness, water vapor permeability (WVP), tensile strength (TS),
elongation at break (EB), elastic modulus (EM), color, solubility, and antimicrobial capacity
of the carrageenan film were determined. The OLE had the following excellent
characteristics: the T.ph value was 115.96 mgGAE∙g−1 (d.b), the AA was 89.52%, the
oleuropein value was 11.59 mg∙g−1, and the MIC was 50 mg∙mL−1. The results showed that
the addition of OLE increased the thickness, EB, and WVP, and decreased the TS and EM of
the film. The solubility was not significantly affected by the OLE. The color difference with
the addition of OLE was 64.72%, which had the benefit of being a barrier to oxidative
processes related to light. The film with the OLE was shown to have an antimicrobial
capacity during the storage of lamb meat, reducing the count of psychrophiles five-fold when
compared to the samples packed by the control and commercial films; therefore, this novel
film has the potential to increase the shelf life of lamb meat, and as such, is suitable for use
as active packaging.
Keywords: active packaging film; microwave-assisted extraction; Olea europaea;
antimicrobial capacity; antioxidant activity; lamb meat
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5.1 INTRODUCTION
Packaging systems occupy a prominent position in food processing and their use is
indispensable for the distribution and commercialization of products on the market. Food
packaging is increasingly influenced by the emergence of new technologies and new
materials, such as active packaging [1]. This is a reflection of consumer demand for food that
is original, underprocessed, and without chemical additives, and for more sustainable
packaging materials [1,2]. Active packaging refers to packaging that modifies the condition of
packaged food. Therefore, active packaging materials are used to extend a food’s shelf life
and improve food safety [2,3]. Although extensive research is being carried out on active
packaging, many have not yet been successfully tested on real systems. Thus, antimicrobial
packaging in the form of biopolymeric films represents a viable alternative to active
packaging [4,5].
From this perspective, new and unprecedented materials for bio-based packaging have
been and continue to be developed, especially when it comes to biodegradable films [6], for
example, biopolymers that are made from marine sources (chitosan, alginate) or raw materials
from agriculture (corn and potato starches, zein) [7,8]. However, little is known about making
films from a biopolymer obtained from red algae, namely, carrageenans, which is a group of
complex biopolymeric substances called phycocolloids and represents a great prospect for the
future [9,10]. The advantage of using carrageenan is that its gels have properties that alternate
between liquids and solids, which allows for applications as additives in food manufacturing
and the potential to form good biodegradable films due to their gelling power with excellent
mechanical properties [11,12]. In addition, when making biodegradable films, a plasticizer is
required. The plasticizer is a non-volatile substance that, when added to different systems,
promotes physical and mechanical changes, allowing the films to become more malleable and
resistant in terms of tension and elongation. Glycerol is a commonly used plasticizing agent in
the production of films due to its compatibility and stability with biopolymer chains [13].
In order to transfer the additional antioxidant and antimicrobial properties to the films,
the use of plant extracts is increasing [2] and olive leaf extract has characteristics that are
suitable for this application. Olive leaves have substantial amounts of bioactive compounds
(oleuropein, verbascoside, luteolin-7-O-glucoside, apigenin-7-O-glucoside, hydroxytyrosol,
and tyrosol) in their composition, resulting in antioxidant and antimicrobial properties, which
have been associated with their strong characteristics as preservatives [14–16]. The presence
of phenolics and antioxidant activity in olive leaf extract has been reported [17,18]. Liu et al.
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[19] studied the antimicrobial activity of crude olive leaf extract and found that the extract
nearly completely inhibited the growth of Escherichia coli and Salmonella enteritidis. They
concluded that the extract could potentially be used to control pathogens in food products.
Another advantage is that the leaves are a waste product of olive oil manufacture, where they
make up an average of 10% by weight in the processing of olives [20,21].
The supplementation of olive leaf extract in biodegradable films has already been
tested in some research [22,23]. Albertos et al. [22] produced fish gelatin films with the
addition of olive leaf extract and used them as packaging for cold-smoked salmon; the films
with the extract decreased the growth of Listeria monocytogenes. Bermúdez-Oria et al. [24]
produced biodegradable films based on pectin and fish skin protein with the addition of olive
extracts; the film preserved strawberries against mold during storage. It is also worth
mentioning that two recent BR (Brazil) patents related to this research have shown the
effectiveness of films produced with carrageenan and an incorporated olive leaf extract
[25,26]. All information regarding the potential of reusing olive leaves shows their extracts as
being promising as an ingredient with bioactive properties for applications in active
packaging.
The effort to use less plastic and new packaging technologies is being mobilized
around the world and is being felt even in the meat industry. Despite this, the development of
meat packaging is a challenge because meat products often require materials with low
transmission rates to extend and guarantee their useful life [7,27]. In this context, lamb meat
is a suitable candidate for investment in protective packaging, as there are few studies on the
changes in the quality of lamb meat during its storage. Lamb meat preservation methods
include freezing, cooling, a vacuum, and a modified atmosphere [28], although the shelf life is
limited by microorganism growth and lipid oxidation; however, these troubles can be solved
by adding extracts of plants to the formulation of the packaging, making it active.
The potential applications for active antimicrobial packaging to extend the shelf life of
meat and meat products were analyzed by Camo et al. [29]. Kuorwel et al. [30] studied
synthetic and natural antimicrobial agents by incorporating them in films for packaging used
basil, oregano, and thyme, as well as their essential oils. Given the above, the implementation
of active packaging in the storage of lamb meat can be an innovative technology for the
longer preservation of this meat and has the advantage that the extracts are not applied
directly to the surface of the meat but are incorporated into the internal part of the packaging
material [6,28,31].
95
Thus, the aims of this unprecedented research were to produce and evaluate the
properties of an active carrageenan film containing olive leaf extract and to investigate its
influences during the refrigerated storage of fresh lamb meat to produce an active packaging
with the potential to replace films produced with synthetic polymers.
5.2 MATERIALS AND METHODS
5.2.1 Materials
5.2.1.1 Olive Leaves
The Olea europaea L. type Arbequina was grown in southern Brazil (31°30'04.0"S,
53°30'42.0"W). After the collection, the leaves were oven-dried (ETHIK, Vargem Grande
Paulista, Brazil) (40 °C, 24 h). Then, the leaves were ground (IKA® A11BS32, Shanghai,
China) and sieved (metal mesh 60, Metalúrgica Indústria Bertel, Caieiras, Brazil). Particles
with a diameter of less than 0.272 mm were used.
5.2.1.2 Chemicals
The carrageenan was purchased from Sigma-Aldrich (St. Louis, MO, USA), where the
type was κ-carrageenan. The plasticizer used was glycerol, which was purchased from
Mistura da Terra (Bagé, Brazil). The following reagents were bought from Sigma-Aldrich (St.
Louis, MO, USA) and were of an analytical standard: Folin Ciocalteu’s phenol reagent, 2,2-
diphenyl-1-picrylhydrazyl (DPPH), methanol, anhydrous sodium carbonate, gallic acid, and
oleuropein. For microbiological analyses: nutrient broth, Müller–Hinton broth, PCA agar, and
peptone (Himedia, Bengaluru, India), as well as sterilized distilled water, were used.
5.2.1.3 Bacterial Isolates
Escherichia coli ATCC 11229 was obtained from Oswaldo Cruz Foundation, Rio de
Janeiro, Brazil. Before the tests, the bacterial culture was grown for 24 h in a nutrient broth at
35 °C.
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5.2.2 Preparation of the Plant Extract
In order to obtain olive leaf extracts (OLEs), microwave-assisted extraction was
performed in a multimode (closed) microwave unit (SCP Science, Baie-D'Urfe, QC, Canada)
by applying the methodology of Rosa et al. [32]. The extraction was performed with 0.5 g of
olive leaf powder in 25 mL of distilled water. Subsequently, the extracts were vacuum-filtered
using Whatman® Grade 4 filter paper (Sigma-Aldrich, St. Louis, MI, USA). Finally, they
were lyophilized (Gamma 1–16 LSC, Christ, Osterode, Germany). Using the experimental
optimization methodology (preliminary tests), the best extraction conditions were the
following: 100 °C for 2 min at pH 6.
5.2.3 Extract Characterization
The total phenolics (T.ph) were determined using a method adapted from Singleton
and Rossi [33]. The procedure was analogous to that of Martiny et al. [34]. The T.ph results
were expressed in milligrams of gallic acid equivalent (GAE) per gram of dry matter. The
analysis was performed in triplicate.
The method of Brand-Williams et al. [35] was used for the measurement of the
antioxidant activity. A total of 0.2 mL of OLE was blended with 7.8 mL of DPPH (6 × 10−5
M) solution and stood still for thirty minutes at room temperature without light. The same
procedure was performed with an aliquot of water in order to obtain control samples. The
absorbance of the control and extract samples were measured using a spectrophotometer
(Ultraspec1000, Amersham Pharmacia Biotech, Cambridge, England) at 517 nm. The free
radicals captured by the DPPH were calculated using Equation (1) in triplicate:
A. activity = (𝐴C−𝐴OLE
𝐴C) × 100%, (1)
where A. activity is the antioxidant activity expressed as a percentage (%), 𝐴C is the
absorbance of the control, which was water, and 𝐴OLE is the absorbance of the OLE samples.
Quantitative analyses of the oleuropein in the OLE were performed using HPLC
(Agilent 1100 series instrument, Santa Clara, CA, USA). Oleuropein was separated on a
Discovery RP C18 column (Supelco, PA, USA; 5 μm, 25 cm × 4.6 mm) equipped with a
Supelguard C18 cartridge (Discovery, St. Louis, MO, USA; 5 μm, 2 cm × 4 mm); it was then
97
analyzed using a variable wavelength detector (VWD) that was set to 280 nm. The mobile
phase was a mixture of water, acetonitrile, and acetic acid (80/19/1 v/v/v). The oleuropein was
identified and quantified (mg∙g−1 of olive leaves (d.b.) (dry base) using an external standard
and a calibration curve.
The minimum inhibitory concentration (MIC) was evaluated against E. coli (ATCC
11229). E. coli was chosen because this bacterium is a potential causative agent of food-
related diseases since meat is an environment that is favorable for its development. The MIC
is the lowest concentration of the extract necessary for the complete inhibition of visible
growth, where concentrations ranged from 5 to 150 mg∙mL−1 of the lyophilized extract. The
antimicrobial activity was determined via inhibition analysis of the extracts obtained and was
performed using the ELISA plate culture method adapted from [36,37]. In an ELISA
microplate, 145 μL of sterile Mueller–Hinton broth, 135 μL of extract, and 20 μL of the
culture containing the microorganism were pipetted into wells in triplicate. The antimicrobial
activity was quantified using the absorbance (wavelength of 630 nm) difference between the
two readings of the samples containing the extract in relation to the mean absorbance of the
control samples in a spectrophotometer (PowerWave XS, Biotek, Winooski, VT, USA).
Equation (2) was used to calculate the inhibition:
microbial. A = [ 1 − (𝐴2OLE−𝐴1OLE
𝐴2C−𝐴1C)] × 100%,
(2)
where microbial. A is the inhibition (%) and A is the absorbance, where the subindex "1"
refers to the readings at 0 h, the subindex "2" refers to the readings at 16 h, the subindex
"OLE" refers to samples containing extracts, and the subindex "C" refers to the mean
absorbances for the control samples, which were the inoculants with water.
5.2.4 Carrageenan Biodegradable Films
5.2.4.1 Film Preparation
The biodegradable films were formed according to the casting technique by Rosa et al.
[23]. The proportion used for the film-forming solution was 1% (w/v) of carrageenan, 37.5%
(w/w) glycerol (based on the carrageenan mass), and 62.5% (w/w) of OLE (based on
98
carrageenan mass), which were dissolved in 50 mL of distilled water under agitation (1100
rpm) on a magnetic stirrer with heating (QUIMIS-Q261M23, Diadema, Brazil) at a
temperature of 70 °C for 15 min. These conditions were established in preliminary tests.
Initial studies were carried out to find the most suitable plasticizer and OLE concentrations.
The results showed that films without a plasticizer were brittle, while those with high amounts
of glycerol were stringy and difficult to remove from the plates. Thus, the concentration of
37.5% (w/w) glycerol was chosen for this research. It was determined that films made with
OLE at concentrations below 62.5% (w/w) showed low or no antimicrobial effect; therefore,
we chose to use a concentration of 62.5% (w/w) OLE for the formulation of the films. Films
without the extract were produced as control films and called CAR-C, while films with the
extract were called CAR-OLE. The filmogenic solutions were poured into 150 mm diameter
acrylic plates and subjected to dehydration in an oven at 40 °C for 24 h.
5.2.4.2 Film Properties
With the support of a digital micrometer (Insize-IP65, São Paulo, Brazil), the
measurements were taken at ten different positions of the film, thus detecting the mean
thickness of the different samples of the biodegradable films produced.
The water vapor permeability (WVP) was ascertained using the ASTM standard
E96/E96M methodology [38]. The mass gain, measured via anhydrous calcium chloride
absorption, was monitored over ten days and the WVP was calculated using Equation (3):
WVP = 𝑊
𝑡
𝑒
𝑎𝑃, (3)
where WVP is the water vapor permeability (g∙m−1∙Pa−1∙s−1), 𝑊 is the absorbed moisture (g), t
is the time (s), e is the thickness (m), a is the exposed film surface (m²), and ΔP the partial
pressure difference (1176.17 Pa at 294.31 K).
The tensile strength (TS), elongation at break (EB) point, and elastic modulus (EM) of
the films were measured using the ASTM standard D882-09 methodology [39]. The apparatus
used was a Texturometer Analyzer (Stable Micro System TA.XTplus, Richmond, U.K.).
The film color was determined using the methodology proposed by Martiny et. al.
[34]. The color difference was calculated using Equation (4):
99
∆𝐸∗ = √(𝐿𝐶∗ − 𝐿𝑂𝐿𝐸
∗ )2 + (𝑎𝐶∗ − 𝑎𝑂𝐿𝐸
∗ )2 + (𝑏𝐶∗ − 𝑏𝑂𝐿𝐸
∗ )2, (4)
where ∆𝐸∗ is the color difference (%); 𝐿𝐶∗ , 𝑎𝐶
∗ , and 𝑏𝐶∗ are the color parameters of the CAR-C
films; 𝐿𝑂𝐿𝐸∗ , 𝑎𝑂𝐿𝐸
∗ , and 𝑏𝑂𝐿𝐸∗ are the color parameters of the CAR-OLE films. The L*
parameter ranges from 0 (black) to 100 (white). The a* parameter measures the degree of red
(+a) or green (−a) color and the b* parameter measures the degree of yellow (+b) or blue (−b)
color.
The water solubility of carrageenan-based films was determined using the method
proposed by Gontard and Guilbert [40]. The film samples were initially dried to find the
initial mass dry of the film samples. The samples were cut into 2 cm diameter discs, then
immersed in 50 mL of water and the system received gentle agitation (100 rpm) at 20 °C for
24 h using a shaker incubator (SOLAB-SL 223, Piracicaba, Brazil). To determine the final
amount of dry matter, the sample was dried (105 °C for 24 h). The solubility in water was
calculated from the triplicate results using Equation (5):
WS = 𝑚𝑖−𝑚𝑓
𝑚𝑖× 100%, (5)
where WS is the water solubility (%), 𝑚𝑖 is the initial dry mass (g), and 𝑚𝑓 is the final dry
mass (g).
5.2.5 Storage Study: Inhibition of Psychrophiles in Lamb Meat
To evaluate whether the biodegradable films of carrageenan with the olive leaf extract
(CAR-OLE) had an influence on the housing of lamb meat, analyses of the growth of
psychrophiles were made. Chilled lamb samples were purchased from the local market in
Bagé, a city in southern Brazil. The tested meat was fresh and raw. The initial psychrophiles
count was determined. Then, the samples were packed separately for each different film in
duplicate, namely, CAR-C, CAR-OLE, and commercial polyvinyl chloride (PVC) film (Royal
Pack, Alto Aririu, Brazil) (Figure 1). The packages were sealed using a heat-sealing machine
(Lenor CP1-TH, ShenZhen, China) at 150 °C for 10 s. The packages were stored at 7 °C for 2
days. After the storage period, the final psychrophiles count was performed. The methodology
used was the standard plate count [41] according to the American Public Health Association
(APHA) [42]. The plate counts were made using a colony counter microprocessor
(Electronics India, Parwanoo, India). The results were presented as the colony-forming units
per gram of lamb meat (CFU∙g−1).
100
Figure 1. Samples of lamb meat that were packed using different films: (A) polyvinyl
chloride (PVC) film, (B) carrageenan control film (CAR-C), and (C) carrageenan film with
olive leaf extract (CAR-OLE).
5.2.6 Statistical Analysis
The means of the experimental data and their respective deviations were calculated,
where three replicates were performed. Significant differences between the means were
determined via Tukey’s test at p < 0.05 using Statistica software (Stat Soft Inc., version 10,
Tulsa, OK, USA).
5.3 RESULTS AND DISCUSSION
5.3.1 Olive Leaf Extract
Olive leaf is an excellent resource of phenolic compounds with an elevated antioxidant
activity [32]. The results found in the current research also demonstrated these attributes. The
total phenolic compounds (T.ph) were 115.96 ± 0.56 mgGAE∙g−1 (d.b.) and the result for the
antioxidant activity was 89.52 ± 0.013%. Similar results for the antioxidant activity of up to
94% were found for olive leaf extracts obtained using a microwave [43,44]. The oleuropein
content was 11.59 ± 0.004 mg∙g-1 (d.b.). In a study by Japón-Luján et al. [45], 23 mg∙g−1 (d.b.)
oleuropein was extracted via the microwave technique using ethanol and water (80:20 v/v). In
contrast, Khemakhem et al. [46] used the maceration technique and produced aqueous
extracts of olive leaves, obtaining 2.65 mg∙g−1 (d.b.) of oleuropein. Jemai et al. [47] extracted
0.0432 mg∙g−1 (d.b.) of oleuropein from olive leaves using methanol with water (4:1 v/v) via
maceration. Ansari et al. [48] extracted oleuropein with water at 60 °C and obtained 15
101
mg∙g−1 (d.b.). The values obtained in this research were comparable to the results reported in
the literature cited. It is noteworthy that important differences were observed, such as the
solvent type, extraction technique, and plant matrix origin. Another relevant fact was that in
this research, water was used as a solvent (safe and non-toxic), which had a positive result in
the oleuropein content obtained; as such, its use becomes competitive as a substitution for
methanol and ethanol, which are commonly used in extractions.
Extracts of olive leaves produced via microwave-assisted extraction demonstrated
antibacterial activity against E. coli, with an MIC value of 50 mg∙mL−1. This result is in
accordance with the data previously reported by Pereira et al. [49], where they also observed
an antimicrobial effect that was related to the concentration of OLE against bacteria and
fungi. They found that the antimicrobial effect was attributed to oleuropein and the T.ph
found in the extract, corroborating the results found in this research. Şahin et al. [43] similarly
produced olive leaf extracts using the microwave technique but evaluated variables such as
irradiation potency, irradiation time, and leaf mass. Their study revealed that the extract
obtained under optimal conditions demonstrated an antibacterial effect against Staphylococcus
aureus, with an MIC value of 1.25 mg∙mL−1.
5.3.2 Film Evaluation
Figure 2 illustrates the visual appearance of the biodegradable carrageenan films. The
carrageenan-based biodegradable films were homogeneous, uniform, and could be easily
removed from the plate. Films with no extract addition were clear and transparent and films
with the extract addition were greenish-brown.
Figure 2. Visual appearance of the carrageenan films: (A) CAR-C and (B) CAR-OLE. CAR–
C: carrageenan film, CAR–OLE: carrageenan film with olive leaf extract.
102
Table 1 provides the thickness, WVP, mechanical properties, solubility, and optical
properties data of the formulated films. These results show that the incorporation of the OLE
into the films caused an increase in the thickness. The thickness of the films increased linearly
with the increasing mass; the addition of the extract in the film formulation caused an increase
in the mass, which consequently caused an increase in thickness. Rhim [50] produced
agar/carrageenan composite films (50:50) with incorporated clay nanocomposites; he obtained
thicknesses of 0.0582 mm and 0.0643 mm as a result. The differences in the thicknesses
found in the literature are due to the different methodologies used in the experimental
preparation procedures, composition, and formulation of the film solutions.
There were significant differences in the WVPs of the films CAR-OLE and CAR-C,
which was an indication that the water vapor permeability gradient was not the same for both
films tested. The difference in the % relative humidity at the interfaces of both films was the
driving force behind the diffusion of the water [51]. The WVP of a film depends on several
variables, especially the thickness [52]. As there were differences in the permeability values
between films with an OLE concentration, the increase in WVP may be due to the difference
in the thickness. The increase in thickness in CAR-OLE was already expected due to the
increase in the mass of the film-forming solution due to the incorporation of the extract. Films
with OLE had a significantly higher thickness than those without OLE (Table 1). This
observed effect may have been due to the interaction of the polysaccharides and OLE, which
may have changed the saturation point. These interaction effects of the polymeric matrix and
the different additives were also observed by Turco et al. [53], where they produced poly
(lactic acid)/thermoplastic starch films with cardoon seed epoxidized oil and observed that the
higher the tension at the interface (less compatible mixtures), the greater the spaces between
the phases, which favored the diffusion of water. Therefore, the OLE probably decreased the
interfacial adhesion between the polymeric matrix and the dispersed phase, decreasing both
the surface tension of the carrageenan and the intermediate space between the
macromolecular chains, thus causing the acceleration of the diffusion of water molecules.
Ideally, a film for the storage of lamb meat requires a low WVP; this result was found in this
research and was corroborated by other studies that produced carrageenan films or films with
the addition of OLE [22,23].
Comparing the data found in Table 1 with the properties of the commercial PVC film,
which had a thickness of 0.004 mm, a WVP of 2.47 g∙m−1∙Pa−1∙s−1, elongation at break of
108.79%, and a tensile strength of 21.55 MPa based on data reported by Martiny et al. [34], it
was concluded that the produced carrageenan films presented a superior thickness and WVP
103
and inferior mechanical properties. These results suggest that the films can be improved in
terms of their physical properties, where the investigation of biopolymeric blends for this
purpose may be an option.
Table 1. Thickness, water vapor permeability (WVP), mechanical properties, solubility, and
optical properties of carrageenan films.
Physical Properties CAR-C CAR-OLE
Thickness (mm) 0.032 ± 0.004a 0.048 ± 0.004b
WVP (g∙m−1∙s−1∙Pa−1) 6.61×10−11 ± 1.6×10−12a 7.43×10−11 ± 9.1×10−13b
Elongation at break (%) 29.21 ± 0.12a 36.58 ± 1.70b
Tensile strength (MPa) 11.83 ± 0.23a 8.51 ± 0.09b
Elastic modulus (MPa) 40.50 ± 0.97a 23.34 ± 1.33b
Solubility (%) 82.60 ± 3.47a 76.60 ± 0.33a
Optical Properties CAR-C CAR-OLE
L* 94.49 ± 0.21a 71.41 ± 0.92b
a* −0.145 ± 0.03a 7.78 ± 0.43b
b* 3.32 ± 0.31a 63.26 ± 1.59b
ΔE - 64.72
Data reported as the mean values ± mean deviation. CAR–C: carrageenan film, CAR–OLE: carrageenan
film with OLE. ∆𝐸∗ is the color difference (%); L*, a*and b* are the color parameters Different letters in
the same line indicate significant differences between samples (p < 0.05).
The addition of the extract in the composition of the films significantly changed the
mechanical properties. There was an increase in the EB and a decrease in the TS. The fact that
the biodegradable films of carrageenan prepared with the extract had a higher EB percentage
may have been due to the fact that the extract had essential oils in its constitution since some
studies show that lipid components can act as plasticizers in films. The addition of essential
oils to the composition of biofilms caused a decrease in the rupture tension and an increase in
the elongation of the films [54]. The EB is due to the alteration of the morphological structure
of the film, which changes to a plastic flow regime up to the breaking limit. In this whole
104
process, the chain alignment in the amorphous regions and the destruction of micro- and
macro-crystalline structures are involved, with the consequent formation of fibrillar structures
[55]. A polymeric material can undergo transformations in its original structure, as indicated
by the EB test, and can limit the possible applications of the packaging [56].
Table 1 presents the values of the EMs for carrageenan films. The addition of OLE in
the carrageenan films had an effect on the elastic modulus. There was a significant difference
(p < 0.05) between both film formulations. The CAR-C film showed the highest EM value
(40.50 MPa), which was in accordance with the result of the TS in this study. Films with
higher EM data have turned out to be less flexible and more rigid than those with lower EM
data [57,58]. The results found in this study showed that the OLE contributed significantly to
the EM, producing more flexible films; this may have been due to the presence of lipid
compounds in the extract that were able to form a continuous and cohesive structure. An EM
result very similar to ours for a carrageenan film that was also plasticized with glycerol was
found by Paula et al. [59], where the authors reported a value of 52.45 MPa. The results of
this study were also in agreement with the study by Pereda et al. [60], which reported that the
addition of olive oil into chitosan films enhanced the EM; according to the authors, this was
because organic compounds, such as fatty acids and lipids, can contribute to the plasticization
of the films. Therefore, the incorporation of the OLE had an effect on the elastic modulus of
the carrageenan films produced in this study.
The addition of the extract to the film formulation did not cause a significant
difference in solubility (Table 1). This high solubility is a feature of films formed from
hydrocolloids, as they are highly hydrophilic, especially those made of polysaccharides and
proteins [61]. In contrast, Rhim [50] only produced glycerol-plasticized carrageenan films,
where the films completely disintegrated in water in just a 30 min test.
The results for the color parameters and color differences are shown in Table 1.
Regarding the display of the carrageenan films, the addition of the extract decreased their
moderate L* value, which is an occurrence that was expected since the extract had a dark
green color. The parameters a* and b* increased with the incorporation of the OLE, with both
being positive, i.e., having mostly red and yellow components, respectively. The increase in
b* may have been linked to the phenolic compounds present in the OLE, which can absorb
light of low wavelengths; similar behavior was obtained by Shojaee-Aliabadi et al. [62] for
carrageenan films with incorporated essential oils. The films with oils differed from the
control at 64.72%; these authors used glycerol plasticized carrageenan (1%) films of the same
description as the CAR-C films, where L* = 88.41, a* = −0.27, and b* = 0.86. Another
105
relevant aspect is color, given that it interferes with the consumption profile. For the
commercial PVC film, the optical parameters were L* = 95.81, b* = 1.088, and a* = −0.14.
As can be seen from Table 1, these data were close to the carrageenan films without the OLE,
whereas for the films with the addition of the extract, the color parameters were significantly
different. Despite the disadvantages attributed to the decrease in transparency, the addition of
the extract has the advantage of decreasing oxidative processes caused by light on the food,
though a more thorough investigation should be done regarding this feature.
5.3.3 Storage Study
The effects of the carrageenan film with or without the OLE on the development of
psychrophiles microorganisms in the lamb meat packaging are shown in Table 2. The lamb
meat was measured for initial psychrophiles and was determined to contain 1.10 × 105
CFU∙g−1. The final concentration of psychrophile microorganisms in the lamb meat increased
substantially during the 2 days of storage in all the samples. This trend was in line with the
findings of Al Sheddy et al. and the EFSA BIOHAZ (European Food Safety Authority
Biological Hazards) Panel [63,64], who demonstrated the ability of these microorganisms to
develop in cool temperatures. It can be observed that during the storage period, the growth of
psychrophile microorganisms was lower in lamb meat packed with the CAR-OLE film; this
was approximately 5 times lower compared to the samples packed with CAR-C or the PVC
film. CAR-OLE was able to slow the growth of psychrophiles compared to the control
sample. The CAR-OLE efficiency can be attributed to its position on the surface of the lamb
meat, where there was a higher microbial concentration.
Table 2. The psychrophiles population measured initially and after 2 days of storage.
Samples packed with different films Initial (CFU∙g−1) 2 Days of Storage (CFU∙g−1)
Fresh lamb meat (1.10 ± 5.66) × 105 -
CAR-C - (27.6 ± 8.66a) × 105
CAR-OLE - (5.51 ± 7.62b) × 105
PVC film - (23.9 ± 7.78c) × 105
Data reported as the average values ± standard deviation. CAR–C: carrageenan film, CAR–OLE: carrageenan
film with OLE, PVC film: polyvinyl chloride commercial film. Tukey’s multiple range tests were executed.
Different letters in the same column indicate significant differences between the samples (p < 0.05).
106
Some studies have also achieved successful results when designing active packaging
for food applications; however, despite the advantages of this type of packaging, there are not
many studies on lamb meat. In one study, Karabagias et al. [65] obtained a significant
reduction in bacterial growth in lamb chops packed in a modified atmosphere packaging
containing thyme oil. This active packaging extended its useful life by 2 to 3 days compared
to that achieved with conventional modified atmosphere packaging. As opposed to using
biodegradable polymers, Camo et al. [29] incorporated oregano extract into polystyrene
(synthetic polymer) polymer matrix films and evaluated the application of this active
packaging to the storage of lamb, successfully increasing the shelf life of the samples.
Jancikova et al. [66] obtained edible carrageenan films with the incorporation of a lapacho
extract; the results they obtained show that the films produced have the potential as a wrapped
food commodity. However, they did not test the application in any food matrix. Saleh et al.
[66] evaluated the effect of the direct use of olive leaf extract (not under a polymeric matrix
like the present study) on the microbial growth of raw poultry meat; the results found by them
revealed that the incorporation of olive leaf extract successfully reduced the microbial growth
and the extract extended the shelf life of the poultry meat.
Other research had already shown that OLE can inhibit the growth of microorganisms
in in vitro assays. However, in this study, it was demonstrated for the first time that the
carrageenan film with OLE could reduce the growth of psychrophile microorganisms in food,
specifically in lamb meat. The application of the active carrageenan film with OLE provided
an additional obstacle to the growth of unwanted microorganisms. The results reported in the
literature prove that the union of a biopolymer, such as carrageenan, and olive leaf extract
represents enormous potential regarding the development and real application as an active
packaging for meat, which was corroborated by the promising results found in the present
study.
5.4 CONCLUSIONS
From the aqueous OLE extraction process using the microwave-assisted extraction
technique, an extract rich in total polyphenols was obtained, with high antioxidant activity
and excellent oleuropein content, which was highly effective against E. coli. In addition, it has
other benefits, such as low cost due to its source being a byproduct stream. When the OLE
was incorporated into the biopolymeric matrix of carrageenan for the production of films, it
decreased the growth rate of psychrophiles in packaged lamb meat and presented competitive
107
physical characteristics relative to conventional polymeric matrices. The characterization of
the biodegradable carrageenan films demonstrated that they were flexible and manageable,
and that the addition of OLE significantly changed the thickness, the color parameters, the
mechanical properties, and the barrier property. However, there was no significant change in
the solubility. The carrageenan films with OLE were found to act as effective packaging that
inhibited microbial growth in lamb. In order to avoid a significant effect on sensory
properties, additional studies are recommended, including sensory tests on the product due to
the application of the film.
Patents
There are two patents that resulted from the work reported in this manuscript.
Patent 1: Registry number: BR 102018013380-2 A2; publication date: 14/01/2020;
title: Filme bioativo antimicrobiano à base de carragenana e extrato de folhas de oliveira.
Patent 2: Registry number: BR 102017013381-8 A2; publication date: 15/01/2019;
title: Biofilmes antimicrobianos para proteção de alimentos.
Author Contributions: Conceptualization, methodology, formal analysis, investigation,
writing—original draft, T.R.M.; supervision, resources, investigation, writing—review and
editing, V.R.; conceptualization, resources, investigation, writing—review and editing,
C.C.M.; conceptualization, supervision, resources, investigation, writing—review and editing,
G.S.R.; supervision, resources, investigation, writing—review and editing, G.L.D.
Funding: The authors would like to thank the Research Support Foundation of the
Coordination for the Improvement of Higher Education Personnel (CAPES) and the Natural
Sciences and Engineering Research Council of Canada (NSERC) for their financial support.
Conflicts of Interest: The authors declare no conflict of interest.
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- CAPÍTULO 6 -
DISCUSSÃO, CONCLUSÃO GERAL E SUGESTÕES PARA TRABALHOS
FUTUROS
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6.1 DISCUSSÃO
O presente trabalho envolveu a produção de três artigos com enfoque a obtenção de
extrato de folha de oliveira utilizando três diferentes métodos de extração (maceração,
extração assistida por ultrassom e micro-ondas), e posteriormente, a sua incorporação em
embalagem ativa a base de carragenana. Os principais resultados foram a obtenção de um
extrato de folha de oliveira por meio da extração assistida por micro-ondas em condições
otimizadas de temperatura, tempo de extração e pH, e a formulação de uma nova embalagem
ativa com comprovada ação antimicrobiana, possibilitando a manutenção da qualidade do
alimento embalado, nesse caso a carne de cordeiro.
O primeiro artigo testou métodos de extração, a maceração e a extração assistida por
ultrassom, além da extração assistida por micro-ondas, e comprovou que esta última foi a
mais eficiente em relação aos resultados de atividade antioxidante, compostos fenólicos,
oleuropeína e hidroxitirosol. Esses melhores resultados foram devido à energia do micro-
ondas que rompe a parede celular, aliada a transferência de massa e calor na mesma direção,
permitindo assim uma maior liberação dos compostos de interesse no solvente. Na extração
assistida por micro-ondas os conteúdos de oleuropeína e hidroxitirosol foram de 14,468 e
0,590 mg.g-1 (b.s.).
O segundo artigo avaliou a extração de compostos bioativos de folhas de oliveira de
plantações do sul do Brasil, especificamente do tipo Arbequina. As informações sobre esta
cultivar ainda são limitadas nesta região e também há poucos estudos, visto que o cultivo na
região iniciou recentemente. Sendo assim, o estudo torna-se importante, pois como já é
conhecido o tipo de cultivar e a origem geográfica onde a planta é cultivada afetam suas
características, como a composição, influenciando as suas propriedades bioativas. Lembrando
que existem diversas variedades desta planta, e na lista das mais citadas estão as cultivares
Picual e Ascolana, para avaliar as propriedades das folhas esta informação deve ser levada em
consideração. Obteve-se extrato de folhas de oliveira através da extração assistida por micro-
ondas, com comprovada atividade antimicrobiana. As melhores condições para a obtenção do
extrato com potencial antimicrobiano foram temperatura 100°C, tempo de extração 2 min e
pH 6. Nessas condições, foi possível obter a atividade antioxidante de 92,87% e os compostos
fenólicos totais de 103,87 mgGAE.g-1 (b.s), de acordo com a função de desejabilidade. Os
extratos obtidos nestas condições apresentaram atividade antibacteriana contra Escherichia
coli, com valor de concentração inibitória mínima de 50 mg. mL-1 e teor de oleuropeína de
11,59 ± 0,004 mg.g-1 (b.s).
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O método de extração assistida por micro-ondas de compostos bioativos das folhas,
não relatado anteriormente na forma como a pesquisa foi realizada, foi avaliado e otimizado.
A combinação de parâmetros nunca antes avaliados para este método, especialmente no que
diz respeito à influência do pH, foi investigada. Modelos estatísticos para atividade
antioxidante e compostos fenólicos totais relacionando três parâmetros importantes,
temperatura, tempo e pH, foram obtidos. A pesquisa buscou uma forma alternativa de
extração em que impacto negativo no meio ambiente fosse reduzido, sendo possível obter um
extrato à base de água com excelentes propriedades bioativas. Destaca-se o seu resultado do
teor de oleuropeína, que foi superior ao encontrado na literatura sob condições análogas.
Apesar da dificuldade, foi possível obter excelentes resultados sem a utilização de solventes
extratores orgânicos e sob condições de extração viáveis e competitivas. Deixando claro que
inúmeros outros trabalhos relacionados com a produção de extratos de folha de oliveira não
alcançariam um bom aproveitamento se não utilizassem solventes orgânicos.
No terceiro artigo, o extrato obtido sob as condições otimizadas foi utilizado com
sucesso na produção de filmes biodegradáveis. Com a incorporação do extrato na matriz
biopolimérica de carragenana observou-se uma redução do crescimento de psicrófilos no
filme utilizado como embalagem para carne de cordeiro. Além disso, essa nova alternativa de
embalagem mostrou-se competitiva frente às embalagens convencionais, apresentando
excelentes propriedades.
A caracterização dos filmes biodegradáveis de carragenana demonstrou que estes
foram flexíveis e manuseáveis e que a adição de extrato de folhas de oliveira na matriz de
carragenana provocou mudanças nas propriedades dos filmes, como o aumento na espessura,
na elongação e na permeabilidade ao vapor d’água, e diminuição na tensão de ruptura e no
módulo de elasticidade dos filmes. A solubilidade não foi significativamente afetada (p >
0,05) pelo extrato. A diferença de cor com a adição do extrato foi de 64,72%. As espessuras
dos filmes controle e preparados com a adição do extrato foram de 0,032 e 0,048 mm,
respectivamente. A permeabilidade ao vapor d’água obteve-se 6,61×10−11 e 7,43×10−11 g. m-1
.s-1.Pa-1, respectivamente para os filmes controle e com extrato. Os resultados de resistência à
tração, elongação e módulo de elasticidade foram 11,83 Mpa, 29,21 % e 40,50 MPa, e 8,51
MPa, 36,58 % e 23,34 MPa para filmes controle e com extrato, respectivamente. Por fim, a
solubilidade foi de 82,60% e 76,60% para filmes controle e com extrato, respectivamente.
Os filmes biodegradáveis de carragenana quando aplicados na embalagem de amostras
de carne de cordeiro apresentaram efeito inibitório na contagem de psicrófilos. Pode-se
observar que durante o período de armazenamento, o crescimento de microrganismos
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psicrófilos foi menor na carne de cordeiro embalada com o filme com adição de extrato; foi
aproximadamente 5 vezes menor em comparação com as amostras embaladas com o filme
controle ou filme de PVC.
Aqui cabe uma importante discussão acerca de testes preliminares não mencionados
ou não publicados. Foi feita a tentativa de extração supercrítica para obtenção do extrato de
folha de oliveira. A técnica de extração por fluido supercrítico também é uma técnica não
convencional de extração emergente em pesquisas, e possui vantagens, sobretudo por
apresentar uma boa recuperação de compostos bioativos sem a necessidade do uso de
solventes orgânicos. Não se logrou sucesso na tentativa do uso dessa técnica para a extração
de compostos bioativos de folhas de oliveira. As extrações foram realizadas com o fluido CO2
na unidade extratora disponível no laboratório de Engenharia Química da UFSM, porém as
dimensões do extrator supercrítico e as características do material a ser extraído, nesse caso as
folhas de oliveira em pó, dificultaram a extração e não permitiram a obtenção de uma
quantidade suficiente de extrato para os testes posteriores necessários, tais como a
quantificação dos compostos fenólicos e a determinação da atividade antioxidante.
Com base na pesquisa realizada, também é possível apontar algumas realidades que
foram observadas tanto no estudo dos extratos e métodos de extração como na produção do
filme, e entre elas estão:
• A folha de oliveira brasileira é uma boa fonte de compostos fenólicos com
atividade antioxidante;
• A extração assistida por micro-ondas mostrou-se um método rápido e eficiente
para a produção de extrato de folhas de oliveira;
• O extrato de folhas de oliveira em condições ideais mostrou atividade
antibacteriana contra Escherichia coli;
• O extrato de folha de oliveira tem um grande potencial de uso como conservante
natural para alimentos;
• Os extratos de folha de oliveira demonstraram ter uma ampla perspectiva de
aplicação na indústria alimentícia;
• As propriedades físicas mostraram que os filmes têm potencial para uso como
embalagem de alimentos;
• Os filmes foram eficazes em retardar o crescimento microbiano na carne de
cordeiro;
• Os filmes têm um uso promissor como embalagens ativas de alimentos.
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6.2 CONCLUSÃO GERAL
O desenvolvimento dessa tese é de grande importância e pode inspirar novas pesquisas
e fornecendo informações valiosas, entre elas, dados da extração de compostos bioativos de
folhas de oliveira de plantações do sul do Brasil, cujas informações estas que ainda são
limitadas, devido ao recente cultivo de oliveiras nessa região. Aliado a isso, a pesquisa busca
resolver um problema de resíduo que é gerado no cultivo da oliveira, que são as folhas, dando
um aproveitamento para esse resíduo que possui um enorme potencial como fonte de
compostos bioativos. O estudo também explora aspectos de uma operação unitária
fundamental na engenharia química, que é a extração, buscando formas alternativas de
extração que não tenham um impacto negativo no meio ambiente, utilizando a água como
solvente. A pesquisa ainda permitiu a elaboração e a caracterização de filmes biodegradáveis
ativos, bem como sua aplicação no acondicionamento de carne de cordeiro. Os resultados
mostraram que a adição de extrato de folhas de oliveira em filmes biodegradáveis a base de
carragenana promove alterações nas propriedades dos filmes e confere atividade
antimicrobiana aos mesmos. Assim, o extrato de folhas de oliveira apresenta-se como uma
alternativa para a formulação de filmes biodegradáveis de carragenana, com grande
prospecção de aplicação no setor de embalagens ativas para alimentos.
6.3 SUGESTÕES PARA TRABALHOS FUTUROS
Da experiência adquirida ao longo da execução dessa tese e com base nos resultados
alcançados, uma lista de sugestões é exposta abaixo com o intuito de estimular pesquisas
futuras que potencializem esse estudo para uma possível aplicação industrial da extração, bem
como o uso do filme obtido. Salienta-se que algumas pesquisas não foram possíveis de ser
executas devidos às limitações impostas pela atual situação de pandemia, e por isso também
se estimula a continuidade a partir dos trabalhos futuros. O Grupo de Pesquisa Engenharia
Processos e Sistemas Particulados (GPEPSP) que colaborou em partes com o
desenvolvimento dessa tese, possui outros trabalhos que envolvem a olivicultura e já vem
desempenhando papel importante na continuidade das pesquisas.
i. Avaliar a estrutura dos filmes biodegradáveis através de técnicas de
microscopia;
119
ii. Avaliar os filmes por Calorimetria Exploratória Diferencial (DSC) e realizar
Análise Termogravimétrica (TGA).
iii. Estudar a biodegradabilidade dos filmes;
iv. Estudar a difusão do agente antimicrobiano;
v. Avaliar a inibição frente à bactéria Listeria monocytogenes;
vi. Estudar a reticulação dos filmes de carragenana;
vii. Avaliar a técnica de extração por fluído supercrítico;
viii. Estudar outros cultivares de oliveira e comparar com o cultivar Arbequina já
estudado;
ix. Avaliar a extração de compostos bioativos de outros resíduos da olivicultura, tal
como o bagaço de azeitona;
x. Estudar outras técnicas de armazenamento dos extratos;
xi. Estudar a influência da liofilização do extrato nos compostos bioativos;
xii. Estudar a desintegração dos filmes em diferentes condições de umidade;
xiii. Quantificar o conteúdo de compostos fenólicos totais nos filmes biodegradáveis
incorporados com extrato de folhas de oliveira;
xiv. Estudar a aplicação dos filmes biodegradáveis ativos em outros alimentos;
xv. Estudar a purificação dos extratos.
120
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