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NEWS FROM EU RESEARCH

PREPARATION OF ACTIVE PACKAGING WITH ANTIOXIDANT AND ANTIMICROBIAL ACTIVITY BASED ON USING ASTAXANTHIN AND CHITOSAN FROM SHRIMP WASTE

Ana Sanches Silva1; Helena Soares Costa1; Perfecto Paseiro Losada2; Raquel Sendón2; Dalia I. Sánchez-Machado3; Herlinda Soto Valdez4; Inmaculada Angulo Varona5; Jaime López-Cervantes3

1 Departamento de Alimentação e Nutrição, Instituto Nacional de Saúde Doutor Ricardo Jorge, I.P., Av. Padre Cruz, 1649-016, Lisboa, Portugal

2 Department of Analytical Chemistry, Nutrition and Food Science, Faculty of Pharmacy, University of Santiago de Compostela, E-15782 Spain

3 Departamento de Biotecnologia y Ciencias alimentarias, Instituto Tecnológico de Sonora, P.O. Box 541, Cd. Obregón, Sonora, México

4 Centro de Investigación en Alimentación y Desarrollo, A.C., CTAOV, Apdo. Postal 1735 Hermosillo, Sonora 83000, México

5 Gaiker, Technological Center. Parque tecnológico Edificio 202. 48170 Zamudio, Spain
Correspondence: Ana Sanches Silva, Departamento de Alimentação e Nutrição, Instituto Nacional de Saúde Doutor Ricardo Jorge, I.P., Av. Padre Cruz, 1649-016, Lisboa, Portugal.

E-mail: anateress@gmail.com ana.silva@insa.min-saude.pt



Abstract

The project “Preparation of active packaging with antioxidant and antimicrobial activity based on astaxanthin and chitosan” (PAPAAABAC, in Spanish PEACAABAQ) brings together a multi-disciplinary team from Mexico, Spain and Portugal, with expertise in different areas of food and polymer sciences. The working programme includes optimisation of the extraction and characterisation of astaxanthin and chitosan from shrimp waste, and incorporation of these compounds into plastic (polymeric matrices), in order to obtain new packaging with antioxidant and antimicrobial properties (active packaging) and finally control-release studies which are carried out in order to determine the quantity of active compounds that should be included in the packaging. This is a two-year project which started in 2009 and is funded by FONCYCIT (Fund for International Cooperation of Science and Technology EU-Mexico; Fondo de Cooperación Internacional de Ciencia y Tecnologia Unión Europea-Mexico) under the coordination of Professor. Jaime López Cervantes from the Technological Institute of Sonora (ITSON).



Keywords: astaxanthin; chitosan; packaging; antioxidant activity; antimicrobial activity; shrimp waste.

Introduction

Nowadays a lot of attention is focused on the use of industrial waste, namely food industry by-products. The use of biotechnology in the utilisation of shrimp waste in order to eliminate the effects of its accumulation in the environment and the development of active new (active) packaging with antioxidant and antimicrobial activity, incorporating astaxanthin and chitosan extracted from this waste into plastics ( polymeric matrices), are aims of this project. There is an increased interest in the development of active new packaging with these types of natural compounds because of the potential increase in shelf- life of foods and to increase their safety.

Astaxanthin has great scientific and commercial interest, since it is an active molecule of natural origin and has great prospects for its application. For example, in the pharmaceutical industry it is used as a marker of cells and as an antioxidant, in the cosmetics industry it is used as a colouring agent and antioxidant and in the food industry it is used as supplement and to complement of the colour of several products, such as the intensification of the yellow colour of egg yolks.

Chitosan is a natural fibre, ususallyusually coming from crustaceans such as shrimp, lobster and crab. This fibre has several uses in biotechnology, mainly in pharmacology, being widely used in controlled-release drugs (drug-delivery). Moreover, in recent years antimicrobial capacity has also been attributed to chitosan.

In previous studies carried out by the research group of the Technological Institute of Sonora (ITSON), the processing of shrimp waste, by lactic fermentation, produced highly satisfactory results. This work, with the collaboration of entrepreneurs from Sonora, enabled studies to be scaled up to industrial level and the creation of a new company (BIODERPAC, SA de CV).

There are three main products obtained in the first stage of the process which aims to extract the bioactive compounds (chitosan and astaxantin) from shrimp waste. There are three phases: the solid phase (from which chitin is obtained), the liquid phase (proteins, minerals and free amino acids) and the lipid phase (lipid and astaxanthin).

. The production of shrimp in Sonora, in 2007 was about 65,000 tonnes, of which 35% is considered to be waste (22,750 tonnes), BIODERPAC is interested in performing the necessary studies to develop a methodology to separate astaxanthin from the lipid and protein phases, after fermentation. In addition, there is great interest in studying the antioxidant effects of astaxanthin, when it is incorporated into an active packaging, thus enabling an increase in added value from this compound, and potentially opening up a new market for its application.

The solid phase obtained from the fermentation produces chitin, equivalent to 15% of the waste generated, resulting in a potential production of 3,400 tonnes of chitin, which when converted into chitosan gives 2900 tonnes,. Chitosan is considered to be an inhibitor of microbial growth, therefore there is interest in developing a new methodology to incorporate it into plastics used in the manufacture of packaging, which would help inhibit microbial growth in foods.

Background to the project

Early studies involving the utilisation of shrimp farming waste began at the ITSON in 1998, with a project funded by CONACYT (Project number 980101020). The main goal of the project was to find viable ways of utilising the shrimp waste that was generated by the processing industry, producing optimal conditions for the waste processing by lactic acid fermentation, at laboratory level. Subsequent projects (supported by the programme of Professor’s Improvement from the Public Education Department, PROMEP) looked at the optimiszation of the process variables and managed to reduced the fermentation time to 24 hours, allowing the possibility of scale- up studies at industrial level by southern Sonora entrepreneurs who have participated in this project (López-Cervantes et al. 2006; López-Cervantes et al. 2007). The infrastructure necessary for industrial production, allowing the extraction of chitin, protein and fat liquor, containing lipids and astaxanthin, has now been developed. The functional and nutritional characteristics of the proteins derived from the fermentation process were studied in the project (Project number: SON-2004-C03-016, FOMIX-CONACYT). Relevant information has been obtained for the applications of the resulting protein liquor, which has been successfully used in agriculture as a plant nutrient on different crops.

The extraction of astaxanthin from blue crab shell waste and shrimp waste has been studied by other researchers (Félix-Valenzuela et al. 2001; Gimeno et al. 2007; Handayani et al. 2008). Following previously established procedures, the aim of the present project is to explore the applications of astaxanthin and chitosan, obtained from chitin, by incorporating them into plastics (polymer matrices). The antioxidant capacity of astaxanthin will be determined and for chitosan, the antimicrobial capacity. Methods will be developed for the separation of astaxanthin with a higher purity from lipids, and for chitosan by a biological method.

There are two fundamental characteristics of carotenoid pigments. First, they are coloured organic compounds so they can act as pigments, and secondly, they have antioxidant capacity , under specific conditions. Thus, while carotenoids are labile to light and in the presence of oxygen, in the absence of these factors, these pigments are stable at elevated temperatures. On the other hand, however, lipid oxidation with the formation of free radicals accelerates the degradation of carotenoids, forming colourless compounds (Torrissen et al. 1989; Niamnuy 2008).

Recently, chitosan has gained importance in the food and pharmaceutical industries, as well as in other applications, due to its antimicrobial activity which has been highlighted as one of its most important characteristics. The degree of deacetylation of chitosan is a key factor in determining its properties, primarily for its solubility and ability to defend against microorganisms. Low molecular weight chitosan (4.6 kDa) has been described as being highly effective against Escherichia coli, Pseudomonas aureofaciens, Enterobacter agglomerans, Bacillus subtilis, Candida kruisei and Fusarium oxysporum, and it also has the ability to suppress the growth of fungal colonies and spore germination at concentrations of 0.01% (Tikhonov et al. 2006). According to Leon and Santiago (2007), chitosan has antimicrobial properties due to the presence of positively charged amine groups that interact with the cell membranes of bacteria, causing the deterioration degredationdegradation degradation of proteins and other membrane components of microorganisms. Moreover it is also biodegradable, non-toxic and biocompatible. Kong et al. (2008) evaluated the antibacterial mechanism through the interfacial contacting inhibition behaviour of chitosan antimicrobials against Escherichia coli. Chitosan microspheres (CMs) were prepared by emulsification cross-linking reaction and oleoyl-CMs (OCMs) were obtained by the introduction of oleoyl groups to the chitosan. These microspheres changed the permeability of membranes and caused cellular leakage that correlated with the hydrophobic interaction between microspheres and cytoplasmic membrane phospholipids of Gram-negative bacteria such as E. coli.

‘Active packaging’ is defined as a system in which the product, packaging and environment interact in a positive way, in order to extend the shelf-life of foods, and provide further characteristics that cannot be obtained in traditional packaging. The main objective is to extend shelf-life, through the release of antioxidants/antimicrobial substances, such as phytochemicals, vitamins, prebiotics or nanofibers (Mistry 2006). Active packaginge can be based on materials such as non-biodegradable polymers, thermoplastic biomass, nanobiocompounds, proteins, polysaccharides and biopolymers from microorganisms, and these materials can release one or more substances (called migrants) into food (Coma 2008).



Aims of the project

The main aim of the project is to develop a methodology for the incorporation of compounds, obtained from shrimp waste (astaxanthin and chitosan), in plastic matrices of polyethylene (PE), ethylene vinyl acetate (EVA) and polyamide (PA) for the development of ‘active packaging’ with independent antimicrobial and antioxidant properties.

The specific objectives are: (1) to determine optimal conditions for the extraction of astaxanthin present in the fat, which results from the fermentation of shrimp waste by supercritical fluid technology; (2) to obtain the best conditions for the conversion of chitin to chitosan by enzymatic techniques; (3) to develop methodology for the incorporation of astaxanthin in PE and PA for the generation of active packaging with antioxidant properties; (4) to develop methodology for the incorporation of chitosan in PE and PA, for the generation of active packaging with antimicrobial properties; (5) to study the time-dependent release of natural extracts added to the plastic film into food simulants and (6) to verify the antioxidant and antimicrobial capacities of the packaging material developed.

Consortium description

Dr. Jaime Lopez Cervantes is the coordinator of the project and the consortium consists of the following institutions: (1) The Technological Institute of Sonora (ITSON) (Cd. Obregón, Sonora, México). (2) BIODERPAC, SA de CV (México); (3) University of Santiago de Compostela (USC) (Spain); (4) GAIKER Technological Centre (Zamudio, Spain); (5) The Research Centre for Food and Development, AC (CIAD) (México); (6) The National Institute of Health Dr. Ricardo Jorge (INSA) (Portugal).



Ongoing work

During the first six months of the project, astaxanthin and chitosan, extracted by lactic acid fermentation from shrimp waste, have been identified and quantified in natural extracts by techniques such as Gas Chromatography coupled with mass Spectrometry (GC-MS), High Performance Liquid Chromatography coupled with Mass Spectrometry (HPLC-MS), Nuclear Magnetic Resonance(NMR), Infrared radiation (IR), etc. The purification of the identified compounds was carried out using the supercritical fluids technique and potential contaminants, that could alter the food, were removed.

During the next stage of the project, the antimicrobial and antioxidant capacities of astaxanthin and chitosan will be verified. A scale-up of the extraction process will be performed in order to obtain the necessary quantities of the various natural extracts for the subsequent development of several active packaging systems.

During the third stage of the project, a system that allows the use of natural extracts in different plastics will be developed. Also, the study of the release of the natural extracts from plastic to foods or food simulants with time will be performed. The packaging materials will be identified and the extracts incorporated into the packaging. Moreover, the particular formation conditions of the container which can incorporate astaxanthin and chitosan in the plastic matrix, separately, will be established.

At the fourth stage, the antioxidant and antimicrobial capacities of the packaging materials developed will be verified and the degree of oxidation and microbial contamination of the new packaging system will be evaluated.

This project intends to continue with the development of ideas for the exploitation of shrimp waste in order to obtain added-value compounds. Products need to reach the standards of purity required by the market which would give them added value, making the process economically viable.

The project also aims to carry out the relevant studies to assess the degree of fixation of astaxanthin and chitosan to plastic materials, namely PE and PA, since these are two materials that are used widely in flexible packaging, allowing the development of active packaging with antioxidant and antimicrobial properties, respectively.

This project also proposes a natural source of astaxanthin for use in active packaging, which is an innovative key element, because commercial astaxanthin is mainly obtained from chemical synthesis and there is a growing concern regarding the food safety of synthetic pigments.

Finally, the project also intends to develop an entirely biological production process for chitosan, extracted from chitin, using a clean, natural and economically feasible method at industrial scale.

Conclusions

This project has ecological interest because it intends to develops methodologies which are, both technically and financially feasible, for the use of products and / or food by-products that have a direct impact on environmental preservation, through the development of cleaner production processes. Also, the raw materials used are part of a chain of environmental contamination and this has added value components of interest to both Mexico and Europe.

This project also contributes to the training of highly qualified scientists through supporting graduate students (Master thesis and PhD) in both Mexican and European Universities, who are the link with the productive sector of the countries involved.

Finally, this project will lead to the establishment of international networks, allowing the development of new multidisciplinary working groups focused on the interaction between academia and industry.



Acknowledgements

This work was funded under Project no. 95935 from FONCICYT C002-2008-1/ ALA – 127 249.



References

Coma V (2008) Bioactive packaging technologies for extended shelf life of meat-based products. Meat Science 78:90-103.

Félix-Valenzuela L, Higuera-Ciapara I, Goycoolea-Valencia F (2001) Supercritical CO2/ethanol extraction of astaxanthin from blue crab (callinectes sapidus) shell waste. Journal of Food Process Engineering 24: 101-112.

Gimeno M, Ramírez-Hernández JY, Martinez-Ibarra C et al. (2007) One-solvent extraction of astaxanthin from lactic acid fermented shrimp wastes. Journal of Agricultural and Food Chemistry 55: 10345-10350.

Handayani AD, Sutrisno, Indraswati N et al. (2008) Extraction of astaxanthin from giant tiger (Panaeus monodon) shrimp waste using palm oil: studies of extraction kinetics and thermodynamic. Bioresource Technology 99: 4414-4419.

Kong M, Chen X, Liu C, Meng X, et al. (2008) Antibacterial mechanism of chitosan microspheres in a solid dispersing system against E. Coli. Colloids and Surfaces. B, Biointerfaces 65: 197–202.

León K & Santiago J (2007) Propiedades Antimicrobianas de Películas De Quitosano-Alcohol Polivinílico Embebidas en Extracto De Sangre De Grado. Revista de la Sociedad Química del Perú 73: 158-165.

López-Cervantes J, Sánchez-Machado DI, Gutiérrez-Coronado MA et al. (2006) Quantification of astaxanthin in shrimp waste hydrolysate by HPLC. Biomedical Chromatography 20: 981-984.

López-Cervantes J, Sánchez-Machado DI, Delgado-Rosas KE (2007) Quantification of glucosamine from Shrimp waste using HPLC. Journal of Chromatographic Science 45: 195-199.

Mistry Y (2006) Development of LDPE-based antimicrobial films for food packaging. Doctoral Thesis. Victoria University, Australia.

Niamnuy C, Devahastin S, Soponronnarit S et al. (2008) Kinetics of astaxanthin degradation and color changes of dried shrimp during storage. Journal of Food Engineering 87: 591-600.

Tikhonov V, Stepnova E, Babak V, et al. (2006) Bactericidal and antifungal activities of a low molecular weight chitosan and its N-/2(3)-(dodec-2-enyl)succinoyl/-derivatives. Carbohydrate Polymers 64: 66-72.



Torrissen, O (1989) Pigmentation of salmonids: Interactions of astaxanthin and cantaxanthin on pigment depositation in raibou trout. Aquaculture 79: 363-374.

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