Chitosan coatings enriched with active shrimp waste for shrimp preservation

Accepted Manuscript Chitosan coatings enriched with active shrimp waste for shrimp preservation M.Y. Arancibia, M.E. López-Caballero, M.C. Gómez-Guillén, P. Montero PII:

S0956-7135(15)00084-5

DOI:

10.1016/j.foodcont.2015.02.004

Reference:

JFCO 4292

To appear in:

Food Control

Received Date: 10 September 2014 Revised Date:

3 February 2015

Accepted Date: 4 February 2015

Please cite this article as: Arancibia M.Y., López-Caballero M.E., Gómez-Guillén M.C. & Montero P., Chitosan coatings enriched with active shrimp waste for shrimp preservation, Food Control (2015), doi: 10.1016/j.foodcont.2015.02.004. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT 1

Chitosan coatings enriched with active shrimp waste for shrimp preservation

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M.Y. Arancibiaa,b, M.E. López-Caballeroa*, M.C. Gómez-Guilléna, P. Monteroa

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a

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del Frío), C/ José Antonio Nováis, 10, 28040. Madrid, Spain.

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Instituto de Ciencia y Tecnología de Alimentos y Nutrición (ICTAN), CSIC. (formerly Instituto

Technical University of Ambato (UTA). Av. Los Chasquis y Río Payamino. Ambato (Ecuador).

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Tel.: +34 91 5492300; fax: +34 91 5493627.

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ACCEPTED MANUSCRIPT Abstract

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An active coating solution composed of chitosan (Ch) and a shrimp protein-lipid concentrate

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(PCc), both obtained from Litopenaeus vannamei processing wastes, was applied to preserve

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shrimp during chilled storage. The addition of PCc increased the antioxidant capacity of the Ch

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coating, yielding a lower-viscosity mixture which, however, was viscous enough to adhere to

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the shrimp while maintaining its activity. The shrimp storage trial showed that the Ch coatings,

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especially when enriched with PCc (Ch-PCc), delayed microbial growth, mainly by extending

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the lag phase. Pseudomonas spp. appeared to be the predominant bacteria in the microbiota.

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H2S-producing organisms and luminescent colonies were especially sensitive to this active

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coating, with inhibition greater than 4 and 2 log cycle respectively, while the growth of lactic

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acid bacteria was not favoured. The Ch-PCc coating delayed the onset of melanosis and did not

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confer any sensorially detectable colour, taste or odour. It therefore shows promise as a

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means to improve the quality of shrimp during cold storage.

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Keywords: shrimp waste, chitosan coating, protein-lipid concentrate, shrimp storage,

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microorganisms, melanosis.

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ACCEPTED MANUSCRIPT 1. INTRODUCTION

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Shrimp are highly susceptible to deterioration and microbial spoilage during storage. Among

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the methods frequently used for shrimp preservation are freezing, cold storage or a

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combination of brining and chilling. These methods often fail to effectively slow down spoilage

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and can produce unacceptable sensory changes or compromise the quality of the shrimp.

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Furthermore, the spoilage may be aggravated by enzymatic browning or melanosis, which

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occurs as soon as the crustaceans are harvested and stored, either in ice or in cold chambers.

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Melanosis is a natural postmortem mechanism involving an enzymatic complex, polyphenol

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oxidase (PPO), which in the presence of oxygen forms compounds that can polymerise into

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insoluble pigments (McEvily, Iyengar, & Otwell, 1991). Antimelanosic sulphite-based

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formulations are used to mitigate this problem, but in recent years adverse reactions around

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the sulphides (Simon, 1992) have prompted a search for alternative means of reducing

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melanosis, using compounds of natural origin such as chitosan (López-Caballero, Martínez-

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Álvarez, Gómez-Guillén, & Montero, 2006).

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In this context, chitosan, a natural polymer obtained from crustacean exoskeletons, plays an

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important role due to its well-documented antimicrobial (Kong, Chen, Xing, & Park, 2010; No,

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Meyers, Prinyawiwatkul, & Xu, 2007) and antioxidant (Kim & Thomas, 2007) properties.

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Methods for obtaining chitosan generally entail consumption of considerable amounts of

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chemical reagents, with consequent economic and environmental problems posed by waste

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management. However, an eco-friendly method to obtain chitosan with similar properties

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(molecular weight, degree of deacetylation) to commercial chitosan has recently been

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reported (Arancibia et al., 2014).

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There is currently increasing interest in the development of packaging materials for food

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preservation, especially bio-based polymers made from a variety of agricultural commodities

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and/or food waste products (Vásconez, Flores, Campos, Alvarado, & Gerschenson, 2009).

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ACCEPTED MANUSCRIPT Edible coatings can improve the quality of food products by retarding lipid oxidation,

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preventing the loss of protein functionality and the formation of off odours, and reducing

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discolouring and moisture losses (Lu, Liu, Ye, Wei, & Liu, 2009; Nussinovitch, 2009; Sathivel,

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2005; Wu et al., 2000). One possible approach is the development of antimicrobial films or

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coatings using antimicrobial based-polymers for application to the surface of food products

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(López-Caballero, Gómez-Guillén, Pérez-Mateos, & Montero, 2005a; Ojagh, Sahari, Rezaei, &

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Hosseini, 2011; Salgado, López-Caballero, Gómez-Guillén, Mauri, & Montero, 2013) or the

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incorporation of bioactive compounds in the films or coatings (Cagri, Ustunol, & Ryser, 2004;

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Lu, Liu, Ye, Wei, & Liu, 2009; Nussinovitch, 2009).

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Various potentially functional materials including polysaccharides, proteins, or lipids, and their

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derivatives, alone or blended have been widely investigated as edible coatings or carrier

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materials (Nussinovitch, 2009). In the last few years chitosan-based antimicrobial coatings

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have been developed incorporating natural active materials (Guo et al., 2013; Li, Li, Hu, & Li,

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2013). Very recently, the addition of a carotenoprotein-lipid concentrate to a chitosan coating

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has shown promise of conferring active properties (Arancibia et al., 2014). However, no

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information is available about the application of an edible chitosan coating blended with a

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protein-lipid concentrate for food preservation. The objective of this work was to develop an

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antimicrobial coating formulation from new active composite materials, namely chitosan and

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shrimp protein-lipid concentrate, and to consider its application to prolong the shelf life of raw

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shrimp under chilled storage.

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2. MATERIAL AND METHODS

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2.1 Recovery of coating forming material

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The protein-lipid concentrate was prepared from frozen shrimp (Litopenaeus vannamei), kindly

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provided by Angulas Aguinaga Burgos (Burgos, Spain), as described in a previous work

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(Arancibia et al., 2014). Briefly, shrimp waste (cephalothorax and exoskeleton) was suspended

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ACCEPTED MANUSCRIPT in water (1:1 ratio w/v) and subjected to autolysis by incubation at 40⁰C for 4h with constant

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stirring. After thermal inactivation at 80 °C for 20 min, the suspension with the autolysed

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residues was kept at 2 °C overnight. An organic extraction was carried out using an

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acetone:ethanol mixture (1:1 v/v) at 40 °C for 2 h with constant stirring, with a waste

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suspension:solvent ratio of 1:3 (w/v). After 3 h decanting, three well-defined phases were

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formed. The intermediate phase, with an intense orange color, opaque and viscous

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appearance, was filtered through cheesecloth to separate the solid residue, consisting of

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chitinous material. The liquid phase was further subjected to organic extraction under the

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same conditions as described above. The resulting aqueous phase was centrifuged (10000xg -

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30 min – 5 °C), the supernatant was discarded and the pellet was lyophilized (VirTis BenchTop

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Pro Freeze Dryer, mod. GKBTEL, Gardiner, U.S.A.) at -85 ºC and constituted the protein-lipid

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concentrate, PCc.

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Mild-processed chitosan was obtained from chitinous material, following a demineralization

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using lactic acid (75.6 g/L), ratio 1:3 (w/v) at 21 °C for 36 h. The residue was then collected and

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the protein was removed through an enzymatic hydrolysis with Viscozyme® L (Sigma-Aldrich

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Quimica, S.L. Spain) (pH 4.5, 50 °C) followed by hydrolysis with Alcalase® (Sigma-Aldrich

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Quimica, S.L. Spain) 2.4L (pH 8.5, 50 °C), using the pH-stat (TIM 856, Radiometer Analytical,

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Villeurbanne Cedex). The enzyme inactivation was carried out at 90 °C for 10 min. Removal of

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acetyl groups from the chitin was achieved by using 10% NaOH solution, ratio 1:7 (w/v) at 100

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°C for 72h with constant stirring. The resulting chitosan was washed to neutrality with distilled

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water.

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2.2 Chitosan characterization

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2.2.1 Molecular weight

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ACCEPTED MANUSCRIPT The viscosity average molecular weight (Mwv) of chitosan was calculated from experimental

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intrinsic viscosity [η] (mL/g) data by utilizing the Mark-Houwink-Sakurada-Staudinger equation

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(Roberts and Domszy, 1982).

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2.2.2 Deacetylation degree

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The deacetylation degree (DD) was determined by two methods: nitrogen content (Niola,

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Basora, Chornet &Vidal, 1993) and infrared spectroscopy (FTIR) (Khan et al., 2002).

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2.3 Coatings preparations

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A starter chitosan solution and a protein-lipid concentrate solution were prepared separately.

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Chitosan solution (2% w/w) was made using a 75.5% deacetylated chitosan synthesized in our

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lab and dissolved in 0.15 M lactic acid solution (pH 2.3). Separately, the protein-lipid

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concentrate (11.48% lipids and 42.27% proteins) (1% w/w) recovered, was dispersed in 0.15 M

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lactic acid solution and sonicated (Q700, Qsonica, CT, USA) for 1 min at 100% amplitude.

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The chitosan coating solution (Ch) was prepared by blending the chitosan solution with 0.15 M

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lactic acid solution (1:1 v/v). The blended chitosan-protein-lipid concentrate coating (Ch-PCc)

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was also prepared with a relation 1:1 (v/v) of each starter solution. The final pH of the coating

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solutions was 3.2.

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2.4 Viscosity of coatings

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Viscosity of the coatings was determined at 25 ± 0.1 °C using a Bohlin CVO-100 rheometer

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(Bohlin Instruments Ltd., Gloucestershire, UK) with cone-plate geometry (cone angle 4, gap

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0.15 mm), at a constant shear rate of 0.5 s-1. Results were averages of five determinations and

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were expressed as mPa.s.

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2.5 Antioxidant properties

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ACCEPTED MANUSCRIPT ABTS radical [2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid)] scavenging capacity and

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FRAP (ferric reducing ability of plasma) were used to measure the antioxidant activity of the

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coatings (Ch, Ch-PCc). Both coatings were dissolved in distilled water and shaken until they

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were totally homogeneous. The coating solutions were filtered through Whatman No. 1 paper.

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The method used for the FRAP and ABTS assays was previously described by Alemán, Giménez,

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Montero, & Gómez-Guillén (2011). Results were expressed as µmol Fe2+ equivalents/L for FRAP

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assay and mg Vitamin C Equivalent Antioxidant Capacity (VCEAC)/L) for ABTS assay, based on

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standard curves of FeSO4·7H2O and vitamin C, respectively. All determinations were performed

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at least in triplicate.

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Total Folin-reactive substances content was determined spectrophotometrically in triplicate

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using gallic acid as a standard according to a modified method of Slinkard & Singleton (1977),

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with the Folin Ciocalteau’s reagent. An aliquot of 10 µl of sample was mixed with 750 µl of

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distilled water and oxidized with 50 µl of Folin Ciocalteau’s reagent sample. The reaction was

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neutralized with 150 µl of sodium carbonate solution (75 g/L) and incubated for 2 h at room

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temperature. The absorbance of the resulting blue color was measured at 765 nm (UV-1601,

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model CPS-240, Shimadzu, Kyoto, Japan). Results were expressed as mg gallic acid (GA)

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equivalent/L of sample. All determinations were performed at least in triplicate.

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2.6 Storage trial

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Frozen shrimp (Penaeus vannamei) acquired at a local market was divided randomly to

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prepare the following batches (approximately 100 g each):shrimp coated with a chitosan

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coating (S-Ch), shrimp coated in a chitosan-protein-lipid concentrate coating (S-Ch-PCc),

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shrimp dipped in a lactic acid solution (S-LA) and shrimp un-coated used as control (S-C). In all

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cases, shrimp were immersed for 1 min in each coating solution without further drying. All

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batches were then packed in polyethylene bags (Cofresco®, Minden, Germany) stored at 5 °C

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for 17 days and periodically sampled.

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ACCEPTED MANUSCRIPT 2.6.1 pH

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Approximately 5 g of peeled shrimp were homogenized with distilled water (1:2, w/v) at room

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temperature for 5 min, the pH was determined with a pHm93 pH-meter and a combined pH

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electrode (Radiometer, Copenhagen, Denmark). The experiments were performed at least in

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triplicate.

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2.6.2 Total Volatile Basic Nitrogen (TVB-N)

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Total volatile basic nitrogen (TVB-N) determinations were carried out over the storage period

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using the method of Antonacopoulos and Vyncke (1989). Samples of peeled shrimp (10 g) were

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homogenized with 90 mL of perchloric acid (6%) in an Osterizer (at 5000 rpm for 1 min) to

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precipitate proteins. The mixture obtained was filtered through Whatman Nº 1 paper, washed

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with 5 mL of perchloric acid, and adjusted to 100 mL. The filtrate was distilled in a Tecator AB

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device (model 1002, Kjeltec Systems, Sweden). The distillate was collected on boric acid (0.3%

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w/v) and was titrated with 0.05 M HCl. Analyses were performed at least in triplicate, and the

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results were expressed as mg TVB-N/100 g muscle.

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2.6.3 Microbiological analyses

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To determine the antimicrobial effect of the coating during storage, shrimp were aseptically

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peeled and a total amount of 10 g was weighed and transferred into sterile bags (Sterilin,

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Stone, Staffordshire, UK), combined with 90 ml of buffered 0.1% peptone water (Oxoid,

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Basingstoke, UK) and shaken vigorously for 1 min in a Stomacher blender (model Colworth

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400, Seward, London, UK), appropriate dilutions were prepared for the following

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microorganism determinations: (i) total bacterial counts (TBC) on spread plates of Iron Agar

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(Scharlab, Barcelona, Spain) 1% NaCl, incubated at 15 °C for 3 days; (ii) H2S-producers

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organisms, as black colonies, on spread plates of Iron Agar incubated at 15 °C for 3 days; (iii)

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luminescent bacteria on spread plates of Iron Agar 1% NaCl incubated at 15 °C for 5 days; (iv)

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ACCEPTED MANUSCRIPT total aerobic mesophiles on pour plates of Plate Count Agar, PCA (Oxoid) incubated at 30 °C for

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72 h; (v) Pseudomonas on spread plates of Pseudomonas Agar Base (Oxoid) with added CFC

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(Cetrimide, Fucidine, Cephalosporine) supplement for Pseudomonas spp. (Oxoid) incubated at

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25 ºC for 48 h; (vi) Enterobacteriaceae on double-layered plates of Violet Red Bile Glucose Agar

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(VRBG, Oxoid) incubated at 30 °C for 48 h; and (vii) lactic acid bacteria on double-layered

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plates of MRS Agar (Oxoid) incubated at 30 °C for 72 h. All analyses were perfomed at 0, 3, 5,

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7, 10 and 14 days by duplicate. The day when the shrimp were coated was taken as day 0.

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Microbiological counts are expressed as the log of the colony-forming units per gram (log

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CFU/g) of sample.

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2.6.4 Colour parameters

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The colour parameters, lightness (L*), redness (a*), and yellowness (b*) were measured using

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a Konica Minolta CM-3500d colorimeter (Osaka, Japan). Measurements were taken at different

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locations of the shrimp's caparace and each point is the mean of at least nine measurements.

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∆L* (lightness) was calculated by ∆L* = L f * - L i *, where i and f represent the beginning and

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the end of the storage trial respectively.

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2.6.5 Sensory analysis

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Throughout the storage period, a trained panelists evaluated the appearance of shrimp every

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2-3 days (eight individuals per treatment evaluation) as described by López-Caballero,

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Martínez-Álvarez, Gómez-Guillén and Montero, (2007) with some modifications. The panelist

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evaluated the odor and taste on a hedonic scale from 0 to 5, as follows: 5 = typical

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characteristic in fresh crustacean and 0 = off-odour. The acceptability limit was considered

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below 2.5. Similarly, the melanosis was evaluated according to a scale from 0 to 5, where 5 =

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no melanosis and 0 = substantial black spots in the whole shrimp.

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2.7 Statistical analysis

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ACCEPTED MANUSCRIPT Results were expressed as mean ± standard deviation. Results were analyzed by two-way

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ANOVA (two factors: batch and storage time). Means were tested with the Tukey's test for

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paired comparison, with a significance level α= 0.05, using the Graph Pad Prism v5.03

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(GraphPad Software, California, USA).

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3. RESULTS AND DISCUSSION

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The Ch (320 kDa, 74% DD) solution was more viscous (5.86 Pa.s) than the Ch-PCc blend

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solution (2.68 Pa.s). Thus, PCc could have disrupted the chitosan network, breaking the

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hydrogen bonding and thus reducing the viscosity. Moreover, the lipids in PCc (11.48%) could

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act as a surfactant, and that should cause a drop in the viscosity of the system as suggested by

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Nyström, Kjøniksen, & Iversen (1999). On the other hand, Jeon et al., (2002) asserted that the

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viscosity and the preservative efficacy of chitosan were inter-related (the higher the viscosity,

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the greater the efficacy). It has been suggested that the high viscosity of chitosan could be

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related to stronger surface adhesion capacity (Yamada et al., 2000). In the present work, Ch-

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PCc was less viscous and hence less adhesive, even although it inhibited microbial growth

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more efficiently, as detailed further below.

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Both the ABTS (45.25 mg Vit C eq/g) and FRAP (85.39 µmol Fe/g) analyses revealed that the Ch

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solution showed a noticeable antioxidant capacity. These values were slightly higher than

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reported by Arancibia et al. (2014) for chitosan with a higher degree of deacetylation.

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According to Kim & Thomas (2007), the basis of chitosan’s effectiveness as a free radical

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scavenger is mainly the degree of amino group exposure as a function of its molecular weight

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(320 kDa). The addition of PCc to the formulation significantly increased (p≤ 0.05) the ABTS

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and FRAP values (Table 1). Carotenoprotein-rich extracts from Pacific white shrimp (L.

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vannamei) wastes with notable radical scavenging capacity and ferric ion reducing power have

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been reported (Senphan, Benjakul, & Kishimura, 2014; Binsan et al., 2008). Furthermore,

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ACCEPTED MANUSCRIPT Arancibia et al. (2014) reported that the antioxidant activity of a chitosan solution was

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improved by the addition of a shrimp waste protein-lipid concentrate.

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The addition of PCc increased significantly the total phenol content of the chitosan solution

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(Table 1), which was consistent with previous results (Arancibia et al., 2014) and could be due

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to the presence of a number of free radical reactive sites from phenols in PCc. Casettari,

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Gennari, Angelino, Ninfali, & Castagnino (2012) also observed very low total phenol values in

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native chitosan. Natural antioxidant compounds present in shrimp shell waste, mainly

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phenolic, were characterized by Seymour, Li, & Morrissey, (1996). The presence of antioxidant

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peptides in the protein concentrate (He, Chen, Sun, Zhang, & Gao, 2006; Mendis, Rajapakse,

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Byun, & Kim, 2005), could also contribute to the positive reaction to the Folin-reagent.

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Initially the pH of shrimp muscle was 7.25, which is in agreement with the values reported by

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other authors for chilled shrimp (Parapenaeus longirostris) (Gonçalves, López-Caballero, &

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Nunes, 2003; López-Caballero, Martínez-Alvarez, Gómez-Guillén, & Montero, 2007). The pH of

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the shrimp muscle increased during storage; the lowest values, approximately 7.6 after 15

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days (p≤0.05), were recorded in batch S-Ch-PCc. To evaluate the effect of the acidic media

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where the chitosan was dissolved, shrimp was dipped in a lactic acid solution 0.15M. The

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results were similar to the control batch (Fig. 1a). In this connection, Martínez-Álvarez, López-

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Caballero, Montero, & Gómez-Guillén, (2005), reported that the presence of acid in the

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antimelanosic solutions did not reduce the pH in muscle of prawns (Marsupenaeus japonicus)

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because of its buffering capacity. The increase in pH reflected the production of alkaline

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bacterial metabolites in spoiling shrimp, which is presumably related to an increase in total

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volatile basic nitrogen (TVBN) (Fig. 1b).

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TVB-N increased in shrimp samples during storage, to different extents depending on the

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treatment (p≤0.05). The initial value was 7.1 mg TVB-N/100 g, which agrees with values

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reported by Huang, Chen, Qiu, & Li, (2012) and Wang et al., (2014) in White leg shrimp

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ACCEPTED MANUSCRIPT (Litopenaeus vannamei). The chitosan coating delayed TVB-N formation compared to the

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control and lactic acid solution batches. This effect was enhanced when PCc was incorporated

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in the formulation (Fig. 1b). Other authors have reported a delay in the production of TVB-N

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compounds in shrimp coated with 1-1.5% O-carboxymethyl chitosan and 1-1.5% chitosan

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(Huang, Chen, Qiu, & Li, 2012), and 2% chitosan (Simpson, Gagne, Ashie, & Noroozi, 1997) . In

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the present work, S-Ch-PCc batch registered approximately half the value raised by S-C batch

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at the end of the storage trial (Fig. 1b), which led in the absence unpleasant odors in the S-Ch-

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PCc as will be described below.

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The initial total bacteria count was around 2.5 log CFU/g (Fig. 2a). Similarly, López-Caballero et

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al. (2002) reported counts on Iron Agar of around 2-3 log CFU/g in good quality pink shrimp

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(Parapenaeus longirostris). All the batches evolved in a similar way except S-Ch-PCc, where

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counts were 1.5 log cycles lower than the rest between day 5-7 of storage. From that day

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onwards, the counts increased, reaching 8-9 log cfu/g at the end of the storage trial (p>0.05).

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Similar behaviour was observed in mesophilic microorganisms, pseudomonas and lactic acid

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bacteria (Fig. 2 d, e, f respectively). Batch S-Ch-PCc presented an increase in the lag phase of

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up to 5 days in mesophilic and lactic acid bacteria counts, which were also the lowest counts.

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The differences in total mesophilics (p≤0.05) reached as high as 3 log units at 5 day. On

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chitosan coated cod patties (López-Caballero, Gómez-Guillén, Pérez-Mateos, & Montero,

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2005a) and culture prawns (Marsupeanaeus tiger) treated with chitosan as an antimelanosic

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formula (López-Caballero, Martínez-Álvarez, Gómez-Guillén, & Montero, 2006), the presence

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of the chitosan coating seemed to stimulate slight growth of the lactic acid bacteria, probably

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because the pH on the patty surface pH was reduced by the acid (acetic acid) solution in which

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the chitosan was dissolved. In this respect, chitosan oligosaccharides have been reported to

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have a bifidogenic effect at concentrations between 0.1 and 0.5% and to stimulate growth of

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Lactobacillus casei and Lactobacillus brevis at 0.1% (Lee, Park, Jung, & Shin, 2002). In the

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ACCEPTED MANUSCRIPT present study, the increase in lactic acid bacteria counts with the chitosan coating solution (in

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lactic acid) was not significant (p>0.05).

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Exponential growth of H2S producing organisms was observed in the control batches (S-C and

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S-LA) from the early stages of the storage trial (p≤0.05). Formulations containing chitosan and

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chitosan-protein-lipid concentrate increased the lag phase of these organisms, presumptive S.

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putrefaciens (López-Caballero, Sánchez-Fernández, & Moral, 2001), by 5 and 7 days

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respectively (Fig. 2b). H2S producers or S. putrefaciens are known to be sensitive to chitosan

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(Arancibia et al., 2014; López-Caballero, Gómez-Guillén, Pérez-Mateos, & Montero, 2005a,

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2005b; López-Caballero, Martínez-Álvarez, Gómez-Guillén, & Montero, 2006). When chitosan

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acetate films were included in packaging treatments (air and vacuum) of sole and hake fillets,

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the lag phase was increased and the final concentration of H2S-producing bacteria reduced

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(Fernández-Saiz, Sánchez, Soler, Lagaron, & Ocio, 2013). Similarly, luminescent colonies

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(presumptive P. phosphoreum) (López-Caballero, Gonçalves, & Nunes, 2002) remained

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constant for 7 days in batch Ch-PCc (p>0.05), growing exponentially thereafter up to 6 log

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CFU/g (Fig. 2c). Inhibition of luminescent colonies (counts < detection limit) in cultured prawns

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treated with chitosan (0.27% w/w) has been reported (López-Caballero, Martínez-Álvarez,

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Gómez-Guillén, & Montero, 2006). In the present work, pseudomonads seemed to dominate

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the microbiota during cold storage of shrimp. And again, chitosan has been reported to inhibit

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the Pseudomonas genus less effectively than it does other Gram-negative and Gram-positive

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microorganisms. López-Caballero, Martínez-Álvarez, Gómez-Guillén, & Montero, (2006) and

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Simpson, Gagne, Ashie, & Noroozi, (1997) attributed the fact that pseudomonas are more

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resistant than the Gram-positives to the protection afforded by the outer membrane of Gram-

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negative cells.

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Previous papers have reported on the antimicrobial activity of chitosan coatings applied to fish

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products and how they help to maintain product stability (Fernández-Saiz, Sánchez, Soler,

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Lagaron, & Ocio, 2013; Jeon, Kamil, & Shahidi, 2002; López-Caballero, Gómez-Guillén, Pérez-

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ACCEPTED MANUSCRIPT Mateos, & Montero, 2005a). However, to our knowledge no information is available on the

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utility of a chitosan coating solution containing a protein-lipid concentrate for seafood

296

preservation. The antimicrobial property of this protein-lipid concentrate was tested, and it

297

showed antimicrobial activity against Gram-negative and Gram-positive microorganisms such

298

as Aeromonas hydrophila (data not shown). The antimicrobial activity of the Ch-PCc coating

299

could be explained by the positive charge on the C-2 of the glucosamine monomer of chitosan

300

at pH 3.2 (Feng et al., 2006; No, Young Park, Ho Lee, & Meyers, 2002) and the effect of the

301

protein-lipid concentrate, which will be also mainly positively charged at low pH (Devlieghere,

302

Vermeulen, & Debevere, 2004), thus enhancing the antimicrobial activity. According to the

303

agar plate diffusion assay, incorporation of the protein-lipid concentrate that was isolated

304

during waste recovery enhanced the antibacterial activity of the chitosan coating (Arancibia et

305

al., 2014). It is possible that various peptide fractions with antimicrobial activity (produced in

306

the recovery of the protein-lipid concentrate) could be active in this way. Rosa and Barracco

307

(2010) reported the presence of antimicrobial peptides, penaeidins, in shrimp (Penaeus sp.).

308

Antimicrobial peptides have been described as cationic and amphipathic in nature, with low

309

molecular weight (usually less than 10 kDa) and a large proportion of hydrophobic residues

310

(Azmi et al., 2013; Dürr, Sudheendra, & Ramamoorthy, 2006). In the present work, the protein-

311

lipid concentrate that was added to the chitosan coating contained a significant amount of

312

hydrophobic amino acid residues, as well as arginine, proline, glycine, tryptophan and others

313

(Arancibia et al., 2014). Antimicrobial activity of peptides with such an amino acidic profile was

314

previously reported (Rosa & Barracco, 2010).

315

These microbiological results reflect the production of total volatile nitrogen compounds and

316

pH values. Generally, the TVB-N values and the concomitant increase in pH are an indicator of

317

spoilage and may be attributed essentially to ammonia produced from bacterial catabolism of

318

nitrogen-containing compounds (LeBlanc & Gill, 1984). In fact, Nirmal & Benjakul (2009),

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reported that shrimp were not acceptable when the pH was greater than 7.6. In the present

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ACCEPTED MANUSCRIPT work, pH values very close to 7.6 were registered in batch S-Ch-PCc practically up to the end of

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storage, whereas in the other lots this value had been exceeded after 7 days. As reported

322

further below, the coating treatment prevented the production of off-flavours throughout

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storage.

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Changes in shrimp colour during storage are shown in Fig. 3. L* values decreased in all batches

325

over the full storage period (Fig. 3a). In the control batch, it was 12 points below the initial

326

value (L*= 29) at the end of the experiment. This can be attributed to the appearance of black

327

spots (melanosis), as previously found in shrimp treated with several antimelanosic

328

formulations (López-Caballero, Martínez-Alvarez, Gómez-Guillén, & Montero, 2007). The

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incorporation of PCc in the chitosan coating slightly contributed to the maintenance of the L*

330

values through the storage time, since the drop in batch Ch-PCc was the lowest (Fig. 3a). If the

331

increase in the L* parameter (∆L*) was calculated at the end of the storage period, the ∆L*

332

values were considerably higher in the S-C batch (-14.63) followed by the S-LA and S-Ch

333

batches (-11.20 and -7.07, respectively). On the contrary, the presence of the Ch-PCc prevents

334

this drop (-2.11). Shrimp coated with Ch-PCc showed a marked tendency towards red (a*) (Fig.

335

3b). There were significant differences (p≤0.05) between the Ch-PCc coating and the other

336

coatings throughout storage, consistent with a noticeable delay in the onset of melanosis in

337

this batch (Fig. 3b) probably due to the antioxidant activity of the PCc as discussed below. The

338

b∗ values increased during storage; differences between batches were observed (p≤0.05),

339

following a similar trend to a* values (Fig. 3c).

340

All batches received similar sensory scores during the first days of storage (Figure 4). Initially,

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all groups scored between 5 and 4 (typical and neutral). However, after 5 days the scores for

342

odour dropped sharply, evolving to “ammonia” or even “off-odour” and “off—taste”. At this

343

point of the conservation trial, the TVB-N in the S-C and S-LA batch was approximately 15

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mg/100 g (Fig. 1). Han, Lederer, McDaniel, & Zhao (2005), found that chitosan solutions

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chitosan did not cause sensory rejection in terms of flavour and taste, although the presence

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of PCc increased the score. The S-Ch-PCc batch was the only one maintaining scores above 3

348

after one week of storage and this batch was rejected after 10 days of storage (Fig. 4).

349

As reported previously (Martínez-Álvarez, López-Caballero, Montero, & Gómez-Guillén, 2005),

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in the present work melanosis evolved similarly in shrimp head and body (Fig. 4), progressively

351

increasing during storage. From scores of 5 or similar in the early days, the control (S-C) and S-

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LA batches received scores ≤ 3 (denoting severe melanosis) after 5 days, whereas the S-Ch and

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S-Ch-PCc batches still retained melanosis scores of 4-5 (absence). The mechanism of melanosis

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development starts with the enzymatic action of polyphenol oxidase, which oxidizes naturally

355

occurring phenols from amino acids into quinones. Subsequent non-enzymatic polymerization

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of the colourless quinones causes a build-up of high-molecular-weight black pigments

357

(Ramirez, Whitaker, Virador, Voragen, & Wong, 2003), and the appearance of black spots

358

devalues the shrimp. After 7 days, S-Ch-PCc received melanosis scores of 3.5 in head and 3 in

359

body, while the rest of the batches scored less than 2.5. The literature reports that chitosan

360

delays the appearance of black spots in crustaceans, attributing this effect to its chelating

361

action and coating-induced oxygen exclusion, which prevents PPO enzyme activity (Simpson,

362

Gagne, Ashie, & Noroozi, 1997). Similarly, in the present work this effect was observable until

363

day 5 of storage. In contrast, López-Caballero et al. (2006) reported that chitosan did not

364

significantly inhibit PPO activity in shrimp. In the present work, the antimelanosic effect of

365

chitosan was enhanced by incorporation of the shrimp protein-lipid concentrate in the blend

366

coating solution.

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In summary, the chitosan coating solution incorporating an active shrimp protein-lipid

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concentrate increased the lag phase and inhibited the growth of microorganisms involved in

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shrimp spoilage. These results were reflected in the biochemical indices determined in shrimp

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ACCEPTED MANUSCRIPT muscle. The composite chitosan coating was imperceptible from a sensorial point of view and

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prevented black spot development. Then, this coating could therefore be useful for preserving

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shrimp quality during chilled storage with a shelf-life extension in shrimps of about 5 days.

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4. ACKNOWLEDGMENT

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This research was financed by the Spanish Ministry of Economy and Competitiveness through

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project AGL2011-27607. Author M. Arancibia is funded by a SENESCYT Scholarship provided by

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the Ecuadorian government.

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5. REFERENCES

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FIGURE CAPTIONS

534 Figure 1. a) Changes in the pH values and b) Total volatile base content in coated shrimp at 4˚C

536

during a 14-day storage trial. C: control, LA: lactic acid, Ch: chitosan and Ch-PCc:

537

chitosan-protein-lipid concentrate coatings. Different letters in the same sample (A,

538

B…) indicate significant differences with time. Different letters in the same time (a, b…)

539

indicate significant differences between samples.

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Figure 2. Microbial counts (log CFU/g) in shrimp stored at 4°C. a) total viable bacteria, b) H2S-

541

producing microorganisms c) luminescent bacteria, d) mesophilic viable count, e)

542

Pseudomonas sp., f) lactic acid bacteria. S-C: control, S-LA: lactic acid coating, S-Ch:

543

chitosan coating and S-Ch-PCc: chitosan-protein-lipid concentrate coating.

SC

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Figure 3. Colour parameters (L*, a*, and b*) of shrimp stored at 4°C for 14 day. S-C: control, S-

545

LA: lactic acid coating, S-Ch: chitosan coating and S-Ch-PCc: chitosan-protein-lipid

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concentrate coating.

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Figure 4. Sensory evaluation of shrimp samples S-C: control, S-LA: lactic acid coating, S-Ch:

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chitosan coating and S-Ch-PCc: chitosan-protein-lipid concentrate coating, during

549

storage at 4 °C.

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ACCEPTED MANUSCRIPT Table 1. Antioxidant activity and Folin reactive substances of Ch and Ch-PCc coatings

FRAP

Folin reactive

(mg Vit C eq/g)

(µmol Fe/g)

substances (mg/g)

Ch

45.25 ± 0.36a

85.39 ± 0.40a

0.07±0.02a

Ch-PC

118.2 ± 7.74b

315.2 ± 2.14b

222.9 ± 14.63b

Sample

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ABTS

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Results are the mean ± standard deviation. One-way ANOVA: Different letters (a, b) in the same column indicate significant differences between the different films (P≤0.05).

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Figure 1

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Figure 2

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Figure 3

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Figure 4

ACCEPTED MANUSCRIPT Chitosan and protein-lipid concentrate were obtained from shrimp waste An active chitosan coating enriched protein-lipid concentrate was designed Protein-lipid concentrate increased antioxidant capacity of chitosan coating

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Coating prevents microbial growth and extended the shelf-life in shrimp for 5 days