Shelf-life of shucked oyster in epigallocatechin-3-gallate with slightly acidic electrolyzed water washing under refrigeration temperature

Journal Pre-proof Shelf-life of shucked oyster in epigallocatechin-3-gallate with slightly acidic electrolyzed water washing under refrigeration temperature Sumate Tantratian, Kanchana Kaephen PII:

S0023-6438(19)31075-8

DOI:

https://doi.org/10.1016/j.lwt.2019.108733

Reference:

YFSTL 108733

To appear in:

LWT - Food Science and Technology

Received Date: 12 June 2019 Revised Date:

30 September 2019

Accepted Date: 13 October 2019

Please cite this article as: Tantratian, S., Kaephen, K., Shelf-life of shucked oyster in epigallocatechin-3gallate with slightly acidic electrolyzed water washing under refrigeration temperature, LWT - Food Science and Technology (2019), doi: https://doi.org/10.1016/j.lwt.2019.108733. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Ltd.

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Shelf-life of shucked oyster in epigallocatechin-3-gallate with slightly acidic

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electrolyzed water washing under refrigeration temperature

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Sumate Tantratian* and Kanchana Kaephen

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Department of Food Technology, Faculty of Science, Chulalongkorn University, Bangkok 10330 THAILAND

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*Corresponding author: [email protected]

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Abstract The purpose of this study is to investigate the effect of soaking oyster meat with

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epigallocatechin-3-gallate (EGCG) after washing with slightly acidic electrolyzed water (SAEW) on

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microbiological, physico-chemical properties under cold storage. The results showed that the 60 ppm

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SAEW (pH 6.14) reduced the number of contaminated bacteria with small change of physico-

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chemical properties of oyster. Soaking the oyster meat in 5.0 µg mL-1 EGCG under refrigeration

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temperature for 13 days provided the bacterial total viable count less than 6 log, considered a good

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bacteriological quality, with undetectable of Escherichia coli, Salmonella spp. and Vibrio

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parahaemolyticus. During the storage, oyster meat illustrated pH higher than 6 and with acceptable

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level of total volatile basic nitrogen value. The cutting strength of oyster meat was gradually

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decreased until the storage ended. This study demonstrated a potential application of EGCG in

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controlling pathogenic bacteria in foods processed without a bacterial reduction step.

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Key words; shucked oyster, epigallocatechin-3-gallate, shelf-life, slightly acidic electrolyzed water,

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cold storage

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Introduction Oysters (Saccostrea commercialis) are bivalve mollusc that are highly commercially valued.

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They have short shelf life owing to their high water activity, neutral pH and the excellent source of

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protein (Costa et al., 2014). A wide range of microorganisms was found in oyster meat including

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spoilage and pathogens. Oysters are often consumed raw or lightly cooked, thus contaminated

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pathogenic microorganisms are easily transmitted to the consumers (CDC, 2006). These pathogens

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may cause diseases ranging from mild gastroenteritis to life-threatening syndromes (Rippey, 1994).

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Fishery products and oysters are susceptible to microbial spoilage due to high water content

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and neutral pH (Erkmen and Bozoglu, 2016). To prevent deterioration of oysters, processing steps

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and chemical agents are studied. The application of 150 ppm chlorine water for 20-30 min was

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applied in washing steps was studied by Pace et al. (1988). Dipping in sodium acetate, coating with

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sodium alginate combination with packing under MAP extended the shelf-life of oyster from 57 h to

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160 h (Costa et al., 2014). Efiuvwevwere and Izapka (2000) reported the combination of potassium

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sorbate and cold storage to extend the shelf-life of shucked oyster. Efiuvwevwere and Amadi (2015)

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investigated the quality changes in mangrove oysters when exposed to sodium benzoate, sodium

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chloride and potassium aluminum sulphate stored under ambient temperature in Nigeria. There are two types of electrolyzed oxidized water. The acidic electrolyzed water has pH

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value of less than 3.0, and slightly acidic electrolyzed water (SAEW) has pH value of 5-6.5 (Hao et

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al., 2014). The bacteriocidal effect of SAEW is due to the available chlorine compounds (Zheng et

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al., 2012). At the pH value of 5.0-6.5, the chlorine compound is mainly in the form of hypochlorous

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acid, which has strong antimicrobial activity (Yoshifumi, 2003). Many reports on effectiveness of the

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SAEW antimicrobial activity against foodborne pathogens were found, including Escherichia coli,

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Staphylococcus aureus, Salmonella and Vibrio parahaemolyticus (Al-Holy and Rasco, 2015; Wang et

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al., 2014). Ding et al. (2016) suggested that the treatment with SAEW demonstrated higher

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disinfection efficacy than NaClO and HCl. Application of SAEW on seafood products including

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shrimp (Ratana-Arporn and Jommark, 2014), fish (Al-Holy and Rasco, 2015; Ozer and Demirci,

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2006).

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Epigallocatechin-3-gallate (EGCG) is the most abundant polyphenol in green tea (Mereles

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and Hunstein, 2011). The galloyl moiety of this substance plays roles in lipid lowering effect (Ikeda,

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2008) and possesses biological activities including angiogenesis (Kondo et al., 2002). The

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bioavailability of EGCG remains uncertain (Naumovski et al., 2015). Some inhibitory effects from

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EGCG are including antioxidant, anticancer and anti-inflammatory (Chu et al., 2017). Antimicrobial

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activity of EGCG also receive attention from many researchers, antagonistic effect on bacteria, fungi

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and virus were reported (Daglia, 2012; Hui et al., 2017; Nikoo et al., 2018). The reports on

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antimicrobial activity of EGCG on a variety of foodborne pathogenic bacteria are including E. coli,

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Salmonella, Staphylococcus aureus (Steinmann et al., 2013) and Bacillus anthracis (Falcinelli et al.,

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2017). To incorporate EGCG into food products, it has to be in dissolved form, but the dissolved

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form could affect the stability of the EGCG lead to its degradation (Wang and Zhou, 2004; Istenic et

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al., 2016). The application of EGCG in edible film for controlling the number or bacteria on foods

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was reported by Wu et al. (2010). The degradation of EGCG depends on pH, concentration and

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temperature. Moreover, they tend to associate with protein and other food compounds which reduce

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their activities (Weerawatanakorn et al., 2014).

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Studies of EGCG application on food products are still scarcely found. The increased

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concern about synthetic antimicrobial agents and negative health risk collaborated with their use have

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led to a replacing them with safe or natural products. The objective of this study is to observe the

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bacterial and physico-chemical qualities of shucked oyster when stored in EGCG combination with

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SAEW washing.

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Materials and Methods

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Fresh oysters, 6.0+0.5 cm in length about 50 g each for the total of 10 kg, were brought from

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a farm in Chonburi province, Thailand. They were chilled in ice water in polystyrene containers.

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They were conducted to reach Food Microbiology Laboratory in Chulalongkorn University within 3

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hours. Right at arrival, they were cleaned with a brush and clean water to remove mud and debris

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adhering to the shells and chucked. The oyster meat, weight 11.05+3.86 g each, was separated and

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put into cold water until experiment within 24 h after harvest. The prepared oyster meat was divided

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into a set of 15 pieces to be treated as one set of sample.

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The electrolyzed water was prepared by passing 10% NaCl solution through an electrolyzed

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water generator (ROX-10 WA E, Hoshizaki Electric CO., Ltd., Japan). The slightly acidic

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electrolyzed water (SAEW) was prepared by adjusting the electrolyzed water with sterile water to

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reach the intended available chlorine concentrations. The free chlorine concentration was determined

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using method described in ISO7393-3 (ISO, 1990).

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Antimicrobial activity of epigallocatechin gallate (EGCG) against pathogenic bacteria

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The bacterial strains used in the experiment included Staphylococcus aureus ATTCC25923,

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Bacillus cereus ATCC6633, Vibrio parahaemolyticus ATCC17802, Salmonella Typhimurium

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ATTCC13311 and Escherichia coli ATTCC25922. They were kindly provided from the Food

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research and testing laboratory, Chulalongkorn University. The Lactobacillus plantarum Plan10621

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was the culture isolated in Food Microbiology Laboratory. The cultures were grown on Nutrient

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Agar, except L. plantarum was grown on de Mar Rogosa and Sharpe (MRS) agar and stored under

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refrigerator conditions.

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All bacteria were transferred to sterile Mueller Hinton Broth (MHB) and incubated for 18-24

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h at 35+2oC. They were centrifuged and cell pellets were washed with sterile saline water for 2 times.

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The cell suspensions were adjusted to 0.5 McFarland which contained the cell concentration 8 log

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CFU mL-1.

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The determination of minimal inhibitory concentration (MIC) and minimal bactericidal

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concentration (MBC) was following the macro broth dilution technique described in CLSI (2006).

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The 0.5 mL of two fold dilution of EGCG (Cayman Chemical, Michigan, USA) from 2560 to 1.25 µg

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mL-1 were combined with 0.5 mL of double strength MHB in each tube. One mL of each cell

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suspension was added to each tubes. The 1 mL of double strength MHB added with 1 mL sterile

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water or 1 mL cell suspension were designated as negative control or positive control. The inhibitory

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concentration was detected by the lowest concentration that did not show the sign of microbial growth

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(turbidity). The concentration that was able to kill bacteria could be described by one with no

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bacterial colony being detected on Muller Hinton Agar (MHA), except MRS agar for L. plantarum,

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after incubation at 37oC for 24 h. The MIC and MBC values of each bacteria reported by the

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statistical mode (highest frequencies) of minimal concentration that inhibit the growth or kill that

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bacterial, respectively.

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Effect of slightly acidic electrolyzed water on microbial quality, and physicochemical properties of

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shucked oyster

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Each set oyster meat was transferred from cold water to slightly acidic electrolyzed water

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(SAEW) at the concentration of available chlorine concentration 0, 20, 40, 60, 80 and 100 ppm. The

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pH of SAEW were 6.48, 6.35, 6.20, 6.14, 5.95 and 5.82, respectively. The portion of oyster meat to

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electrolyzed water was 1:10 (w:v). They were treated for 30 min under 4oC. Then they were soaked

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in sterile water for another 10 min to get rid of SAEW. All samples were subjected to microbiological

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and physico-chemical quality determination. The microbiological determinations were the total

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viable count (TVC) (AOAC, 2012), the number of V. parahaemolyticus (BAM, 2004), E. coli

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(AOAC, 2012), S. aureus (AOAC, 2012), and lactic acid bacteria (LAB) followed ISO 15214 (2015).

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The Salmonella spp. was determined using 3M™ Petrifilm™ (AOAC, 2012).

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Physicochemical qualities were determined including pH, color, total volatile base-nitrogen

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and cutting force. The pH of oyster meat was followed Lekjing et al. (2017). The 10 g of oyster meat

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was homogenized with 20 ml of distilled water using hand held homogenizer. The pH homogenate

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oyster meat was measured using pH meter (Mettler Toledo, Switzerland). The color of oyster was

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determined using a Chroma meter (Model CR-400 series, Minolta, Japan).

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Total volatile base – nitrogen (TVB-N) was measured by micro-diffusion analysis using a

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Conway’s unit according to Conway and O’Malley (1942). The 4 g of blended oyster meat was

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added with 16 mL of 4%TCA. They were mixed and left under ambient condition for 30 min. The

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mixture was filtered through a Whatman filter No. 41 the supernatant was collected. The clean

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Conway unit was applied the Vaseline on the outer edge. The 1 mL of 1% boric acid was pipetted

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into the inner ring. The outer ring was filled with 1 mL of the sample extract and 1 mL of saturated

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K2CO3. The unit was covered and closed with clip. Gentle rotating was applied, to prevent any

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solution mixing from one ring to other. After then the unit was placed in an incubator of 37oC for 60

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min. After the cover was removed, the solution of inner ring was titrated with 0.01N HCl with

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bromocresol green was indicator. A blank test was carried out with 1 ml of 1%TCA. The cutting force of belly and adductor muscle was followed Cruz-Romeo et al. (2008) using

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texture analyzer (TA-XT2i, Stable Micro Systems, Goldaming, UK) and a cutting blade (HDP/BS).

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The operation was cutting speed of 2.00 mm s-1, cutting distance of 5 mm. Cutting force was reported

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as the mean of four measurements on different oysters of similar size oysters per treatment and

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expressed as the force (gf) required for cutting 1 mm into oyster tissue. The SAEW with concentration of available chlorine that was able to reduce the number of

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bacteria without or less deteriorate the physico-chemical quality was selected for further experiments.

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Microbial and physico-chemical qualities of shucked oyster soaked in EGCG solution under cold

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storage

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The oyster meat was soaked for 30 min in the SAEW at the selected available chlorine

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concentration. The oyster meat was transferred to sterile water for 10 min and let dry for 5 min. Then

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they were transferred into a polypropylene container filling with a solution of 5 µg mL-1 EGCG. The

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proportion of oyster meat to EGCG solution was 1:2 (w/v) and kept under 4+2oC. The oyster meat

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soaked in cold distilled water for 30 min and kept in 0.1% potassium sorbate under 4+2oC was set as

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control. The microbiological qualities were determined for the total viable count (TVC), the number

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of V. parahaemolyticus, E. coli, S. aureus, Salmonella spp. and lactic acid bacteria (LAB) as

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described. The physico-chemical qualities were determined for pH, TVB-N, color and cutting

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strength. They were performed as described. The differences in color of the oyster meat, from day 0

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and another day, in both belly and adducted muscle during storage were calculated.

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The difference in color

E*ab was calculated using the equation 1.

E*ab = √ (L*1-L*2)2+(a*1-a*2)2+(b*1-b*2)2 ………………………. (1)

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Statistical analysis The experiments were designed using a completely randomized designed with 3 replications.

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Analysis of variance and Duncan's new multiple comparison range test were utilized with the

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application of IBM SPSS Statistics, version 22.0 (IBM Co., Armonk, NY, U.S.A.) at a confidence

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level of 95%.

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Results and Discussion

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Antimicrobial activity of epigallocatechin gallate (EGCG) against tested pathogenic bacteria The MIC and MBC of EGCG against 6 tested bacteria were shown in Table 1. The MIC and

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MBC of tested gram negative bacteria were 2.5 and 5.0 µg mL-1. The MIC of EGCG on E. coli was

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3.3 µg mL-1 reported by Noormandi and Dabaghzadeh (2015). The three gram positive bacteria

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demonstrated more sensitivity to EGCG than tested gram negative bacteria. This result agreed with

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reports (Akinyemi et al., 2006; Nakayama et al., 2011; Cui et al., 2012). There are contradictory

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reports related to sensitivity of gram negative bacteria on EGCG (Taylor et al., 2005; Yoda et al.,

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2004).

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The mechanism in antimicrobial activity of EGCG was not cleared. The aggregation of

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membrane proteins which resulted in inhibition of their functions was reported by Nakayama et al.

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(2015). The inhibition of EGCG on the gram negative bacteria, E. coli, was mainly by hydrogen

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peroxide production (Cui et al., 2012). Gradisar et al. (2007) reported that EGCG can lead to the

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death of bacteria by inhibiting bacterial DNA gyrase.

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Effect of SAEW on microbial quality, and physico-chemical properties of shucked oyster

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The shucked oyster was washed in available chlorine 0-100 ppm SAEW for 30 min. Washing

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with 0 ppm SAEW showed insignificant (p>0.05) reduction of bacterial number on oyster meat. The

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significant (p<0.05) reduction in the number of TVC, S. aureus and lactic acid bacteria were found on

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those treated with concentration of 40 ppm and higher (Table 2). The 60 ppm SAEW was able to

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reduce the number of TVC, S. aureus and V. parahaemolyticus to 1.36+0.11 log10 CFU g-1, 1.02+0.08

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log10 CFU g-1 and 3.00+1.73 MPN g-1, respectively. Ratana-Arporn and Jommark (2014) reported the

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elimination of 4.16 log10 CFU g-1 of V. parahaemolyticus on shrimp after treatment with 50 ppm

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neutral electrolyzed water for 15 min. The antimicrobial mechanism of SAEW is still unknown.

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Ding et al. (2016) suggested the disinfection mechanism of SAEW was disrupting the permeability of

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cell membrane and agglutinating the cellular inclusion. Ye et al. (2017) reported the morphology

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changes on E. coli when treated with SAEW, including cell expansion, cell elongation and increased

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membrane permeability.

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The color and TVB-N value of oyster meat were not significantly (p>0.05) changed after the

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SAEW treatments for 30 min. The color before and after SAEW treatment in CIE Lab, the value of

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L*, a* and b*, of oyster belly meat, were at 68.16+1.15, -0.58+0.17, 7.08+0.32, respectively and

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oyster adductor muscle were 69.48+0.47, 0.70+0.05 and 7.54+0.97, respectively. The average TVB-

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N of the samples was 3.26+1.29 mg N100g-1. The volatile basic nitrogen compounds found in oyster

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are the results of degradation of nitrogenous compounds caused by microbial activity (Ruiz-Capillas

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and Moral, 2005).

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The fresh oyster meat illustrated pH value at 6.55+0.20. The data conformed to the

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International Commission on Microbial Specification for Foods (ICMSF: 2005) that the fresh oysters

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have pH values about 6.2 to 6.5. Cook (1991) suggested the oyster in liquor with pH higher than 6.0

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was in good quality, while those with pH lower than 5.0 was decomposed. The pH of 0-100 ppm

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SAEW were 6.48 to 5.82. Oyster treated with 80 and 100 ppm SAEW demonstrated pH lower than

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6.0 after treatments.

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Texture of shucked oyster after washing in 60 ppm SAEW, pH 6.20+0.10, was losing their

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firmness (Table 3). The cutting force was changed from 552.32+5.47 g force in untreated sample to

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521.01+7.12 g force. The low pH in SAEW caused denaturation of protein in shucked oyster that

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resulted in less water holding capability and losing their firmness (Huang et al,. 2008).

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Treating oyster meat at 60 ppm SAEW significantly (p<0.05) reduced the number of TVC, V.

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parahaemolyticus and S. aureus while the pH of oysters was remained over 6.0. This concentration of

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SAEW was chosen for the further experiments.

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Microbial quality of oyster in EGCG solution under cold storage

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The TVC of oyster meat was 5.74+0.10 log10 CFU g-1. After washed with 60 ppm SAEW the

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TVC was 4.90+0.11 log10 CFUg-1. The washing of oyster meat with 60 ppm SAEW caused in the

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reduction of total bacterial number, E. coli, Salmonella spp., S. aureus and lactic acid bacteria at

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0.70+0.08, 0.36+0.13, 0.41+0.15, 1.50+0.23 and 0.52+0.08 log10 CFU g-1, respectively.

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The bacterial count on the control sample reached 6 log10 CFU g-1 after day 5 (Table 4),

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whilst the sample treated with EW and EGCG reached the same level after day 13 storage. The

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concentration of 5 µg mL-1 EGCG was illustrated to be the MIC for tested bacteria. The combination

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with refrigeration temperature hold the number of TVC steady for 5 days (Table 5), then the TVC

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number was increased gradually. This may be due to the degradation of EGCG, since EGCG is also a

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potent antioxidant and tend to be oxidized easily (Zhou et al., 2003).

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The experiment on control samples was terminated after day 5 because the microbial quality

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was unacceptable related to ICMSF suggestion. The ICMSF (2005) suggested criteria for fresh

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seafood products are of good quality when standard plate count less than 6 log10 CFU g-1, whilst

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products with the count exceeded 7 log10 CFU g-1 are unacceptable. Cook (1991) suggested the oyster

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meat quality is of substandard when bacterial population of oyster meat exceeds 6 log10 CFU g-1.

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Efiuvwevwere and Izakpa (2000) reported a shelf-life of 4 days for shucked oyster treated in 0.2%

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potassium sorbate and kept under 5oC. Savaminee et al. (2014) reported the microbiological quality

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of shucked oysters soaked in 0.3% potassium sorbate and kept in 4oC was less than 6 log10 CFU g-1

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until the day 8 storage. Cao et al. (2009) reported the aerobic plate count of pacific oysters was 3

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log10 CFU g-1 and increased to higher than 6 log10 CFU g-1 after 8 day storage under 5oC.

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The bacterial number on oyster was declined in the 1st day of storage. It could be the transferring of some bacteria on oyster meat to the aqueous solution and combining with the

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antimicrobial effect of EGCG and sorbate (Table 4 and Table 5). The number of S. aureus was less

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than 5 log10 CFU g-1 until the day 3 storage in sorbate. The threshold level for enterotoxin level

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expressed as the number of S. aureus bacteria of 5 to 8 log10 CFU g-1 had been suggested (Lindqvist et

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al., 2002). Samples in EGCG solution illustrated the number of S. aureus below 5 log10 CFU g-1

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throughout the storage period. It was found that the samples stored in EGCG solution demonstrated

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undetectable of E. coli, Salmonella spp. and V. parahaemolyticus after day 1. The reduction of the

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number of E. coli and Salmonella spp. were also found in the first day treatment in 0.1% potassium

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sorbate. The EGCG solution prolonged the microbial quality of oyster longer than sorbate.

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Stability of EGCG in the solution is influenced by several factors including temperature, pH

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and ionic strength. The soluble EGCG in pH less than 5 under 4oC is quite stable. It was found that

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the rate of degradation was at most in neutral (pH 7). The metal ion also increase the degradation

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(Proniuk et al., 2002). The EGCG in the solution in our study, pH closed to 7, could be rapidly

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degraded. There is no antioxidant and chelating agent added to the oyster soaking solution.

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Somehow, oyster is also rich in minerals that can rapidly degrade EGCG. It was found that the

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number of total and some pathogenic bacteria were increased at the end of storage.

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The lactic acid bacteria and S. aureus were undetectable at the early of storage. After that

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they were rapidly increased to the end of storage. This indicated the reduction of effectiveness of

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EGCG by the degradation of EGCG in aqueous solution. The degradation was rapidly found in the

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pH value higher than 6 (Krook and Hagerman, 2012).

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Physico-chemical changes of oyster meat in EGCG solution under cold storage

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The initial average pH of fresh oyster meat was 6.55. The pH of control samples reached the

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level of 6 after day 7 (Table 6). The oyster meat washed with SAEW had the pH value of 6.20+0.15

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and fall below pH 6 after 13 days in EGCG (Table 7). Bunruk et al. (2013) reported the pH of

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marinated oyster meat before chilled storage by was 6.6+0.10 and decreased to lower than 6.0 after

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stored for 5 day. Cao et al. (2009) stored shucked and individually wrapped pacific oyster under 5oC

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and found the pH of gradually decreased from 6.30+0.12 to 5.89+0.13 in 18 days. The pH of oyster

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higher than 6.0 considered good in quality (Ward and Hackney, 1991; Gacutan, 1974), while ICMSF

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(2005) suggested the pH of oyster decreases to 5.8 or below during spoilage.

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The cutting force of oyster meat, belly and adductor muscle, continuously declined (p<0.05)

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during storage (Table 5). The EGCG soaked oyster demonstrated the slower reduction than those

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soaked in potassium sorbate solution. It was found that the belly demonstrated the reduction of

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cutting strength faster than adductor muscle. The decline in cutting strength related to pH value and

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the number of bacteria. The autolysis enzymes in oyster and from spoilage bacteria caused the

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degradation of the muscles (Mc Millin, 2008). The declining in pH is a key in influencing the ability

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of muscle cells to retain water (Huff-Lonergan and Lonergan, 2005).

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The degradation of protein and non-protein nitrogenous compounds by microbial activity

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causes change in the total volatile basic nitrogen (TVB-N) (Ruiz-capillas and Moral, 2005). The

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quality limit of acceptability for fish products at 20-25 mg N 100g-1 was suggested by Ababouch et al.

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(1996). The level of >30 mg N 100g-1 TVB-N is considered unfit for human consumption (Harpaz et

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al., 2003). The average TVB-N value of fresh oysters was 4.07+1.10 and raised to 18.97+1.10 mg N

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100g-1 at the end of storage. The oyster soaked in potassium sorbate, control sample, reached

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27.76+0.36 mg N 100g-1 within 7 days (Table 5). The TVB-N of fresh eastern oyster was 4.02±1.29

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mg N 100g-1 (Zhang et al., 2016). Cao et al. (2009) reported the TVB-N of pacific oyster was initially

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at 4.25+0.34 and increased to 53.33+0.26 mg N 100g-1 after 18 day storage at 4oC. The TVB-N value

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of 15 day storage oyster meat in EGCG solution still in the acceptable limit, but the control sample’s

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was over the acceptability limit of fish product

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The color of oyster meat was changed during storage (Table 5 and Table 6). The decreasing

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(p<0.05) in L* and a* values of belly section indicated loosing of brightness and increasing of

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greenness, respectively. In adductor muscle, the L* value was decreased (p<0.05) while a* and b*

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values were increased (p<0.05) which indicated increasing in redness and yellowness. The increase in

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difference of color was related to the change of TVB-N which indicated the degradation of oyster

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protein. The change in color was likely due to denaturation of protein (Ludikhuyze and Hendrickx,

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2002). The adductor muscle, which are mainly muscular system, illustrated faster change in color

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than belly section. In the CIELAB color space, the value of “delta E” larger than 2.3 is suggested as

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just noticeable difference (Gaurav, 2003). The noticeable change in color of adductor muscle, was

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found on the first and third day of storage in potassium sorbate and EGCG solution, respectively

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(Table 5 and Table 6). The belly section were noticed after 11 and 5 day of storage, in EGCG and

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potassium sorbate, respectively. The increasing of total color differences during storage, in our data,

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is in agreement with the untreated oyster in the report of Cruz-Romero et al. (2008). In perception,

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oyster meat was darker when stored longer. The oyster meat stored in potassium sorbate had color

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alteration greater than those in EGCG.

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Conclusion

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The application of 60 ppm SAEW in washing the oyster meat reduced the number of total

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bacteria about 1 log CFU g-1. The oyster meat soaking in 5.0 µg mL-1 (1 MBC) EGCG at 4oC could

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maintain the total viable count under 6 log CFU g-1 for 13 days. During the storage, E. coli,

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Salmonella spp. and V. parahaemolyticus were not detectable, while the number of S. aureus was

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below the threshold of enterotoxin risk. The pH of the oyster meat demonstrated the values higher

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than 6.0 and TVB-N values were less than 30 mg N 100g-1, during 13 day storage.

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EGCG, a tea extract, at low concentration provided a marvelous inhibitory effect on

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pathogenic bacteria and extended the shelf-life of oyster meat. The oyster meat soaking in EGCG

298

could be stored for 13 days with good quality both microbiological and physico-chemical properties.

299

The combination of SAEW and EGCG has a potential to be applied in foods which processed without

300

a bacterial reduction step and preferred consumption in raw.

301

Acknowledgement

302

This research was funded by the 90th Anniversary of Chulalongkorn University Fund

303

(Ratchadaphiseksomphot Endowment Fund) No.GCUGR1125614050M under the Graduate school,

304

Chulalongkorn University. The electrolyzed water supported by Dr. Warapa Mahakarnchanakul, the

305

Department of Agro-industry, Kasetsart Univeristy, Bangkok, is appreciated.

306

14

307

The authors declare no conflict.

308

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Table 4. The changes in the number of bacteria on the oyster meat after distilled water washed and keeping in 0.1% potassium sorbate. 0 day 1 day 3 day 5 day TVC 5.66dc+0.17 5.21bc+0.42 4.05a+0.03 4.94b+0.01 LAB 4.47d+0.17 2.72a+0.69 2.76a+0.05 3.95b+0.04 b a b S. aureus 4.09 +0.22 3.08 +0.50 4.01 +0.02 4.94c+0.07 b E. coli 2.74 +0.38 V. parahaemolyticus (MPN g-1) 7.86+0.01 <2 <2 <2 Note a,b,c, …. means in the same row with different letter are significantly different (p<0.05); n=3. -

means not detectable

7 day 5.75d+0.26 4.23c+0.35 5.23d+0,11 2.40a+0.15 <2

9 day 7.57e+0.21 4.87e+0.11 5.50e+0.09 2.83b+0.20 <2

Table 1. Antimicrobial activities of epigallocatechin gallate (EGCG) against pathogenic bacteria in suspension.

S. aureus B. cereus L. plantarum S. Typhimurium E. coli V. parahaemolyticum

EGCG (µg mL-1) MIC MBC 1.25 2.5 1.25 2.5 1.25 2.5 2.5 5.0 2.5 5.0 2.5 5.0

Table 2. Reduction of microbial numbers (log10 CFU g-1) on shucked oyster after soaked in various concentration of available chlorine in slightly acidic electrolyzed water for 30 min. 0 ppm

20 ppm

40 ppm

60 ppm

80 ppm

100 ppm

TVC

0.07a+0.01

0.11ab+0.02

0.18b+0.03

1.35c+0.01

1.41d+0.01

1.56e+0.03

LAB

0.05a+0.07

0.09a+0.07

0.19b+0.05

0.16b+0.05

0.26c+0.09

0.56d+0.08

S. aureus

0.01a+0.01

0.07a+0.01

0.12b+0.01

1.01c+0.02

1.07c+0.06

1.15d+0.07

V. parahaemolyticus (MPN g-1)

-

0.33a+0.57

2.33b+2.08

3.00bc+1.73

2.33b+1.52

3.00bc+1.00

Note a,b,c, …. means in the same row with different letter are significantly different (p<0.05); n=3.

Table 3. The pH and cutting force (gf) of oyster after soaked in various available chlorine SAEW for 30 min. 0 ppm

20 ppm

40 ppm

60 ppm

pH 6.50f+0.04 6.34e+0.02 6.25d+0.03 6.15c+0.01 d c b Cutting force 585.07 +9.82 565.52 +14.25 549.11 +5.47 550.44b+3.90 Note a,b,c, …. means in the same row with different letter are significantly different (p<0.05); n=3.

80 ppm

100 ppm

5.92b+0.03 542.29ab+3.34

5.85a+0.03 535.01a+3.78

Table 5. The changes in the number of bacteria on the oyster meat after electrolyte water soaked and keeping in EGCG 0 day 1 day 3 day 5 day 7 day 9 day TVC 5.06d+0.42 2.62a+0.04 2.75a+0.07 2.65a+0.32 3.28b+0.19 3.68c+0.12 LAB 3.93e+0.10 2.22a+0.62 2.80ab+0.05 3.08c+0.02 bc a S. aureus 3.10 +0.16 2.43 +0.50 2.72ab+0.52 3.29c+0.16 3.81d+0.10 E. coli 2.37+0.16 V. parahaemolyticus 7.20+0.12 <2 <2 <2 <2 <2 (MPN g-1) Note a,b,c, …. means in the same row with different letter are significantly different (p<0.05); n=3. -

Not detectable

11 day 5.35d+0.06 3.51d+0.08 3.89d+0.06 <2

13 day 6.17e+0.04 3.97ef+0.24 4.24e+0.04 <2

15 day 7.48f+0.18 4.04f+0.05 4.50f+0.07 <2

Table 6. The changes in physico-chemical properties of oyster meat after washed in distilled water and keeping in 0.1% potassium sorbate, as a control

pH

0 day

1 day

3 day

5 day

7 day

6.55e+0.01 528.91e+15.40

6.45d+0.01 443.33dc+29.87

6.32c+0.02 430.62c+1.29

6.24b+0.01 332.32b+4.61

6.00a+0.04 246.01a+31.52

Cutting Force (gf) TVB

belly Add. muscle

548.16e+4.55 4.37a+0.58

529.18d+14.59 15.53b+1.10

466.54c+22.17 20.20c+0.55

349.05b+35.48 24.67d+0.40

290.71a+13.82 27.77e+0.36

Belly color

L*

72.19c+0.05

71.60b+0.36

70.61a+0.21

70.54a+0.23

70.51a+0.43

a*

-0.52a+0.02

-0.59ab+0.04

-0.65b+0.05

-0.66b+0.03

-0.82c+0.01

b* E*ab L*

9.43a+0.57 63.76d+0.08

9.58a+0.86 0.67a+0.02 62.32c+0.26

9.65ab+0.87 1.63b+0.70 60.62b+0.44

10.25b+0.59 1.85b+0.59 60.55b+0.17

11.17c+0.50 2.50c+0.65 59.82a+0.04

a*

3.21a+0.45

3.34a+0.31

3.58ab+0.13

3.97bc+0.56

4.24c+0.47

Add. muscle color

b* 12.25a+0.05 14.21b+0.70 14.79bc+0.68 16.25c+0.57 17.42d+0.61 E*ab 2.45a+0.05 4.07b+0.85 5.18c+0.57 6.60d+0.56 Note a,b,c, …. means in the same row with different letter are significantly different (p<0.05); n=3, except cutting force n=4.

Table 7. The changes in physico-chemical properties of oyster meat after SAEW treated and keeping in EGCG solution pH Cutting force (gf)

0 day 6.22d+0.05 528.91h+15.97 551.37+39.45

1 day 6.26d+0.06 472.29g+1.43 545.69 e+30.00

3 day 6.25d+0.06 456.22f+47.73 536.11e+12.63

L*

4.06a+1.09 72.66c+3.38

5.30b+0.52 72.36c+0.29

a*

-0.36a+0.04

-0.37a+0.04

belly Add. muscle

TVB Belly color

7 day 6.22d+0.06 370.81e+22.52 455.32d+17.98

9 day 6.19d+0.06 335.21d+15.61 444.78d+28.36

11 day 6.16c+0.02 278.96c+8.47 346.22c+7.31

13 day 6.09b+0.06 255.38b+18.67 270.67b+17.09

15 day 5.97a+0.06 242.99a+18.76 221.02a+23.99

7.47c+1.61 72.18bc+1.05

5 day 6.23d+0.06 468.78fg+17.57 475.06de+16.7 7 10.27d+0.12 71.52b+0.49

11.33e+0.14 71.44ab+0.21

12.42f+2.14 71.33ab+1.20

16.20g+0.52 70.21a+0.25

17.40gh+0.52 70.45a+0.44

18.97h+1.09 69.55a+2.55

-0.51b+0.01

-0.54b+0.06

-0.64c+0.04

-0.70d+0.03

-0.82e+0.03

-0.89f+0.07

-0.92g+0.01

11.87cd+1.51 3.09d+0.19 67.68b+0.60 4.21d+0.11 16.57e+0.88 8.34g+0.27

12.55d+0.56 4.20e+0.27 65.53a+0.04 4.78e+0.07 16.65e+0.03 9.39h+0.67

b* 9.72a+1.38 10.17ab+0.22 10.46ab+1.56 11.78c+0.94 11.03b+0.50 11.29bc+0.75 11.44bc+0.42 a a b bc c E*ab 0.51 +0.83 0.86 +0.20 1.53 +0.11 1.77 +0.24 2.05 +0.19 2.27cd+0.16 Add. L* 72.72+0.26 72.68f+0.31 72.51f +0.07 71.33e+0.18 71.21e+0.25 70.34d+0.47 69.38c+0.49 a a ab b bc c muscle a* 2.39 +0.54 2.58 +0.29 2.99 +0.71 3.41 +0.12 3.56 +0.19 3.74 +0.21 3.98cd+0.76 a a b c cd d color b* 11.97 +0.78 12.18 +0.04 14.68 +0.83 16.10 +0.23 16.35 +0.64 16.42 +0.03 16.49de+0.67 E*ab 1.15a+0.45 3.24b+0.31 4.96c+0.07 5.71d+0.44 6.52e+0.15 7.17f+0.76 Note a,b,c, …. means in the same row with different letter are significantly different (p<0.05); n=3, except cutting force n=4.

Highlights; -

Washing with 60 ppm SAEW was able to reduce some bacteria with small texture change. 5.0 µg mL-1 EGCG kept the TVC of oyster under 6 log for 13 days No E. coli, Salmonella sp. and V. parahaemolyticus were found during storage. The oyster meat stored in EGCG still had good physical properties.

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The authors have no affiliation with any organization with a direct or indirect financial interest in the subject matter discussed in the manuscript

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The following authors have affiliations with organizations with direct or indirect financial interest in the subject matter discussed in the manuscript:

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