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Nowadays, there is increasing interest in natural antioxidants from food by-products. Astaxanthin is a potent antioxidant and one of the major carotenoids in crustaceans and salmonids. An ultra-high pressure liquid chromatographic method was developed and validated for the determination of astaxanthin in shrimp by-products, and its migration from new packaging materials to food simulants was also studied. The method uses an UPLCW BEH guard-column (2.1 x 5 mm, 1.7mm particle size) and an UPLCW BEH analytical column (2.1 x 50 mm, 1.7mm particle size). Chromatographic separation was achieved using a programmed gradient mobile phase consisting of (A) acetonitrile–methanol (containing 0.05 M ammonium acetate)–dichloromethane (75:20:5, v/v/v) and (B) ultrapure water. This method was evaluated with respect to validation parameters such as linearity, precision, limit of detection, limit of quantification and recovery. Low-density polyethylene films were prepared with different amounts of the lipid fraction of fermented shrimp waste by extrusion, and migration was evaluated into food simulants (isooctane and ethanol 95%, v/v). Migration was not detected under the tested conditions. This work was funded under Project no. 95935 by FONCICYT C002-2008-1/ALA – 127 249. The authors are grateful for the postdoctoral contract of Ana Sanches Silva in the frame of the Program ‘Science 2007’, funded by Fundação para a Ciência e a Tecnologia, Portugal.

Introduction: Astaxanthin (3,3’-dihydroxy-β-β´-carotene-4-4´-dione), a potent antioxidant, is one of the major carotenoids in crustaceans. In the frame of the project ‘Preparation of active packaging with antioxidant and antimicrobial activity based on astaxanthin and chitosan’, a methodology for the incorporation of compounds obtained from shrimp waste in plastic matrices was developed to produce an active packaging with antioxidant properties. The aim of the present work was to develop and optimize a method to determine astaxanthin by ultra-high pressure liquid chromatography in fermented shrimp waste. Moreover, the method was also applied to determine the migration of astaxanthin from plastic films containing different amounts of shrimp waste to food simulants. Material and Methods: The method was optimized to determine astaxanthin by ultra-high pressure liquid chromatography (UHPLC) with diode array detection (DAD). The chromatographic separation was achieved using a vanguard pre-column (UPLCÒ BEH, 1.7 µm particle size) and a column (UPLCÒ BEH, 2.1 x 50 mm, 1.7 µm particle size) at 20 °C. The mobile phase was a gradient of A (dichloromethane/methanol with ammonium acetate/acetonitrile 5:20:75 (v/v)) and B (ultrapure water) with a flow rate of 0.5 mL/min. The optimized UPLC method allowed an excellent resolution of astaxanthin. The method was also evaluated in what concerns to validation parameters such as linearity, precision, limit of detection, limit of quantification and recovery. Low density polyethylene plastic films produced by extrusion with different amounts of the lipid fraction of shrimp waste were prepared and tested regarding migration into fatty food stimulants (isooctane and ethanol 95%, v/v). Results and conclusion: The proposed method to determine astaxanthin in shrimp waste is simple and has a low detection level (0.054 μg/mL). The concentration of astaxanthin found in the lipid fraction of fermented shrimp waste was 453.8 μg/g. The films produced by extrusion with the lipid fraction of the fermented shrimp waste did not originate astaxanthin migration into the tested fatty food simulants. Further studies could be made in order to evaluate the capacity of these films in protecting packed food from oxidation. This work was funded under the Project no. 95935 from FONCICYT C002-2008-1/ ALA – 127 249. The authors are grateful to the postdoctoral contract of Ana Sanches Silva in the frame of the Program “Science 2007” funded by “Fundação para a Ciência e para a Tecnologia”.

Introduction: Chitosan has multiple applications and as inhibitor of microbial growth, there is interest in developing new methodologies to its incorporation into plastics, which would avoid microbial growth in foods. In the frame of the project “Preparation of active packaging with antioxidant and antimicrobial activity based on astaxanthin and chitosan” (PAPAAABAC), chitosan was extracted from shrimp by-products and used to prepare plastic films by solvent evaporation (casting) and extrusion. Material and Methods: Chitosan films were prepared by extrusion of polyamide and by casting. By casting, films were prepared at different concentrations (1%, 2%, and 3%) by dissolving chitosan in acetic acid aqueous solution 1% (w/v), with and without plasticizer (1% glycerol). By extrusion, chitosan pellets were made of polyamide 6 at 2%, 5%, 6%, 8%, and 10% with two different particle sizes (180 and 300 µm). Then, they were rolled in an extruder using a specific screw for polyamide. In order to determine chitosan in food simulants (ultrapure water and ethanol 95% (v/v)), it was degraded into the glucosamine units by hydrolysis and quantified by ultra high pressure liquid chromatography coupled with diode array detection after its derivatization with 9-fluorenylmethyl chloroformate. Results and conclusion: From the plastic films prepared by casting and by extrusion, only those prepared by casting without plasticizer presented chitosan migration that increases with the amount of chitosan added. These films could not be used to pack aqueous foodstuffs. However, the addition of a plasticizer (glycerol) has avoided the migration of chitosan. Therefore, the use of casting films with chitosan shall include a plasticizer in the formulation. Films prepared by extrusion presented no migration into both simulants indicating suitability to pack both aqueous and fatty foodstuffs. This work was funded under the Project no. 95935 from FONCICYT C002-2008-1/ ALA – 127 249. The authors are grateful to the postdoctoral contract of Ana Sanches Silva in the frame of the Program “Science 2007” funded by “Fundação para a Ciência e para a Tecnologia”.

Accumulation of organic wastes in intensive crustaceans culture ponds and nearby coastal waters has become a serious environmental and economical problem. For this reason, new ecofriendly and economically feasible products from agricultural wastes or byproducts for shrimp farms have been developed. This biowaste could be used as an important source of the useful biopolymer chitin and others components such as proteins or carotenoids like asthaxanthin [1]. Chitin is the most abundant polysaccharide after cellulose and the main source is the shell of crustaceans. Chitosan, derived from chitin, has proven useful for a wide range of applications due to its biodegradability, biocompatibility, antimicrobial activity, non-toxicity and versatile physicochemical properties. These properties make the chitosan an excellent candidate to use in food packaging [2]. The development of active packaging with antimicrobial and antioxidant activity based on chitosan and asthaxanthin obtained from shrimp waste is the main goal of the project: “Preparation of active packaging with antioxidant and antimicrobial activity based on asthaxanthin and chitosan” funded by FONCYCIT. The characterization of chitosan in the development of active materials is a key issue since their properties play an important role in its effectiveness as an antimicrobial agent. These properties are mainly molecular weight (Mw), acetylation degree (DA) and polymerization degree (PA). In addition, in mediums of low pH, the antimicrobial activity of chitosan increases [3]. The objective of the present study was characterized three different samples of chitosan obtained from shrimp waste by using two microscopy techniques, Scanning Electron Microscopy (SEM) and Transmission Electron Microscopy (TEM). Additionally, the films developed after the chitosan incorporation in the polyamide matrix were also characterized. Shrimp waste (heads and cephalotorax) samples were collected from local shrimp processing factories in South Sonora, Mexico. The waste was minced, fermented and centrifuged. After the treatment, three fractions were obtained: chitin-rich fraction, protein rich liquor and lipid fraction. Scanning Electron Microscopy was used to investigate the structure properties relationships of chitosan. Samples were spread on a carbon conducting adhesive tape pasted on a metallic stub, subjected to gold covering and observed. The samples for TEM observation were embedded in EPON resin and polymerized at 60 ºC; then were cut at (-120 ºC) using a Leica Ultracut crio-ultramicrotome. The images obtained showed the particle of chitosan embedded into the polyamide matrix. FONCYCIT (Fund for International Cooperation of Science and Technology EU-Mexico)

One of the main causes of food spoilage is the development of microorganisms. In order to inhibit or retard the growth of microorganisms and consequently, improve food security and extend the shelf life of food products, in the past years, active packaging and particularly films with antimicrobial properties have attracted the attention of the scientists. One of the approaches used is to add chitosan to the film. Due to their excellent properties, non-toxic, biodegradable, biofunctional and biocompatible with others antimicrobials, chitosan is one of the antimicrobial agents most appropriate for the development of active materials [1]. Chitosan (CAS nº 9012-76-4) is a polysaccharide, with the structure of a linear polymer of (1-4)-linked 2-amino-deoxy-β-D-glucan, obtained by the partial deacetylation of chitin, one of the most abundant polysaccharides in nature, found in shells of crustaceans [2]. Shrimp waste (heads and cephalothorax) samples were collected from local shrimp processing factories in South Sonora, Mexico. The waste was minced, fermented and centrifuged. After the treatment, three fractions were obtained: chitin rich fraction, protein rich liquor and lipid fraction. In this work, Fourier Transformed Infrared Spectroscopy (FTIR) and Nuclear Magnetic Resonance (NMR) were used to characterize three different samples of chitosan. Moreover, 100% chitosan-based films and films of polyamide with chitosan incorporated were also evaluated. The degree of acetylation (DA) values obtained may change, depending on the nature and level of impurities, source and polymer morphologies [3]. To evaluate the DA, FTIR and NMR were used. The samples were prepared as a thin pellet made from a mixture of KBr and the chitosan powder. To evaluate the DA in films, Fourier Transform Total Reflection infrared Spectroscopy (FTIR-ATR) was used. All spectra were recorded in the range of 400-4000 cm-1. Nuclear Magnetic Resonance (NMR) was also employed to evaluate the DA. Two experiments were tentatively carried out: 1H NMR and 13C NMR. In both cases approximately 5 mg of each sample were diluted in 1 % (v/v) CD3COOD in D2O. All data were compared with three commercially available standards submitted to the same experiments as samples. The sample 1 corresponding to chitosan obtained from shrimp waste and with high viscosity presented the lowest DA, therefore had higher antimicrobial activity. Both techniques; FTIR and NMR, led to the same conclusion. FONCICYT (Fund for International Cooperation of Science and Technology EU-Mexico)

This work was funded under Project no. 95935 by FONCICYT C002-2008-1/ALA – 127 249. The authors are grateful for the postdoctoral contract of Ana Sanches Silva in the frame of the Program ‘Science 2007’, funded by Fundação para a Ciência e a Tecnologia, Portugal.

Glucosamine is one of the most abundant monosacharides and is part of the structure of the chitosan and chitin, which compose the exoskeletons of crustaceans, other arthropods and the fungi cell walls. Shrimp by-products are a good source of glucosamine because about 45% of the animal is composed of inedible cephalothorax and exoskeleton. The project ‘Preparation of active packaging with antioxidant and antimicrobial activity based on astaxanthin and chitosan’ aims to develop a methodology for the incorporation of compounds obtained from shrimp waste in plastic matrices for the development of an active packaging with antimicrobial and antioxidant properties. In the frame of this project, shrimp by-products were fermented and insoluble chitin was purified by a sequence of steps: depigmentation, deproteinization, demineralization and blanching. The aim of the present work was to optimize a method to determine glucosamine by Ultra-high Pressure Liquid chromatography (Ultra Performance Liquid Chromatography, UPLC) with diode array detection (DAD) from shrimp by-products. First, an acid hydrolysis of the chitin of shrimp by-products is carried out. Afterwards, a derivatization is done because glucosamine does not contain a chromophore. The selected derivatization reagent was 9-fluorenylmethyl-chloroformate (Fmoc-Cl). The chromatographic separation is achieved using a vanguard pre-column (Acquity UPLCÒ HSS T3, 1.8 µm particle size) and a column (Acquity UPLCÒ HSS T3, 2.1 x 50 mm, 1.7 µm particle size) at 38 °C. Quantification of glucosamine was carried out at 265 nm. The mobile phase is a gradient of phase A [water (pH 6.5)/ methanol, 85:15 (v/v)] and phase B (acetonitrile) with a flow rate of 0.3 mL/min. The optimized UPLC method allows an excellent separation in just 10 min of the two anomers of glucosamine, of the Fmoc-Cl and of its corresponding alcohol, which is the hydrolysis product of the spontaneous reaction between Fmoc-Cl with water. Several samples have been analysed, some are commercial samples (chitosan with low molecular weight; chitosan with medium molecular weight; chitosan from shrimp shells; chitosan from crab shells), and three other samples produced from shrimp by-products in the frame of the project. Commercial samples presented a higher amount of glucosamine content than in-house produced samples. This work was funded under Project ‘Preparation of active packaging with antioxidant and antimicrobial activity based on astaxanthin and chitosan’ (PAPAAABAC, in Spanish PEACAABAQ) no. 95935 from FONCICYT C002-2008-1/ALA – 127 249.

Carotenoids have antioxidant properties allowing protection of tissues from oxidative damages and they are also beneficial in cardiovascular, immune, inflammatory and neurodegenerative diseases. Astaxanthin (3,3’-dihydroxy-β-β´-carotene-4-4´-dione) is a carotenoid classified as xanthophyl and it is one of the major carotenoids in crustaceans. The project ‘Preparation of active packaging with antioxidant and antimicrobial activity based on astaxanthin and chitosan’ aims to develop a methodology for the incorporation of compounds obtained from shrimp waste in plastic matrices for the development of an active packaging with antimicrobial and antioxidant properties. In the frame of this project, shrimp by-products were fermented and the silage was centrifuged. Three fractions were obtained and the upper phase, corresponding to the lipid fraction, was further analysed to determine astaxanthin content. The aim of the present work was to optimize a method to determine astaxanthin as well as seven other carotenoids and two vitamins (A and E) by ultra-high pressure liquid chromatography (Ultra Performance Liquid Chromatography, UPLC) with diode array detection (DAD) method in shrimp by-products. The chromatographic separation is achieved using a vanguard pre-column (UPLCÒ BEH, 1.7 µm particle size) and a column (UPLCÒ BEH, 2.1 x 50 mm, 1.7 µm particle size) at 20 °C. The mobile phase is a gradient of A (dichoromethane/methanol with ammonium acetate/acetonitrile 5:20:75 (v/v)) and B (ultrapure water) with a flow rate of 0.5 mL/min. The optimized UPLC method allows excellent peaks separation. Shrimp by-products (lipid fraction of shrimp waste, head and shell of cooked and raw shrimp) have been analysed. Moreover, low density polyethylene plastic films produced by extrusion with different amount of the lipid fraction of shrimp waste were prepared and tested regarding migration into fatty food simulants. Migration tests were carried out with isooctane and ethanol 95% (v/v), both alternative fatty food simulants. No migration was detected at the conditions tested, which are conventionally considered the most severe when the food contact material is intended for use at room temperature. This work was funded under Project ‘Preparation of active packaging with antioxidant and antimicrobial activity based on astaxanthin and chitosan’ (PAPAAABAC, in Spanish PEACAABAQ) no. 95935 from FONCICYT C002-2008-1/ALA – 127 249.

Chitosan is a hydrophilic polysaccharide which derives from chitin by deacetylation. It has several applications, namely as a film that can be applied to preserve the quality and increase the shelf-life of food. Chitosan is insoluble in most solvents but it is soluble in dilute organic acids such as formic acid and acetic acid[1]. The properties of chitosan depend on the degree of deacetylation (DA) and molecular weight (MW). A broad antimicrobial activity has been attributed to chitosan, either for gram-negative, gram-positive bacteria and fungi. The aim of the present study is to evaluate the antimicrobial activity of a chitosan film prepared by casting. The chitosan was obtained from shrimp waste collected from shrimp processing factories of South Sonora (Mexico). Four bacteria (Bacillus cereus; Escherichia coli; Staphylococcus aureus and Listeria monocytogenes) and one fungus (Botrytis cinerea) were evaluated. Although L. monocytogenes and B. cinerea growth was not inhibited by the chitosan film, results showed a clear growth-inhibitory effect, at the two bacteria concentration levels tested, for B. Cereus, E. coli and S. aureus. Different antibacterial mechanisms have been proposed to explain chitosan antimicrobial activity[2-3]: i) chitosan may form an external barrier which inhibits essential nutrients adsorption; ii) chitosan can also penetrate the microbial cell, disturbing the metabolism of the cell by inhibiting the mRNA and protein synthesis; iii) chitosan may have an ionic surface interaction with the bacteria originating wall cell leakage. Although these mechanisms may take place simultaneously, the antimicrobial activity may also depend on the properties of chitosan (DA and MW). This work was carried out under the Project no. 95935 from FONCICYT C002-2008-1/ ALA – 127 249, with the financial support of the European Union and CONACYT. The authors are grateful to the postdoctoral contract of Ana Sanches Silva in the frame of the Program ‘‘Science 2007’’ funded by “Fundação para a Ciência e a Tecnologia’’, Portugal.