Green mussel (Perna viridis L.) is a common ma- rine animal in the Asia-Pacific region [1]. This is a large bivalve with smooth, elongated  shell  typical  of  seve- ral mytilids. Flesh  of  the  green  mussel  is  con- sidered  an  excellent   source   of   selenium,   cal- cium,       iron,       magnesium,       phosphorous       and

vitamins   (A,    B1,    B2,   B6,    B12   and    C)   [2,    3]. In addition, mussel fat is rich in polyunsaturated fat- ty acids (PUFA,  37–48% of total  fatty acids) mainly

ω–3 PUFA [4], which is biologically important and can decrease the risk of cardiovascular disease [5]. Howe- ver, this filter feeder animal has a high risk of microbio- logical contamination that lead to a short shelf-life, poor quality, and  economic  losses.  In  addition,  only  well– cooked green mussels are microbiologically safe.

Marinating is a seasoning process mainly using or- ganic acid containing liquids [6]. Liquid marinades ge-

nerally include sugar, spices, oil, and acid such as vine- gar, fruit juice or wine. Marinating improves tenderness, juiciness, taste and aroma, as well as improves the shelf- life of red meat, poultry, seafood, and vegetables [7–10].

Vietnamese tamarind fish sauce is popular in its country of origin. The main ingredients are tamarind pulp, fish sauce and sugar, while some fresh garlic and chili are used to enhance taste. The Vietnamese con- sume this sauce with various foods, such as seafood, fried food, dried food. It is even used as a condiment in various kinds of food products. Ready-to-cook pro- ducts are expected to gain popularity in South-East Asia as well. Mineral deficiencies, including iron and zinc, remain problematic in developing countries. Thus, the enrichment of food products with minerals can be an effective way to solve this problem. However,  accor- ding to a market survey, green mussels marinated with enriched tamarind fish sauce are not commercially avai-

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lable, especially when packed in a modified atmosphere. Modified atmosphere packaging (MAP) is a technology that prolongs the shelf-life of food products by retard- ing microbial growth and biochemical reactions, often with an elevated carbon dioxide level [11]. Moreover, in MAP oxygen may inhibit the growth of anaerobic bacte- ria and accumulate toxins from Clostridium botulinum, type E [12]. However, botulinum toxins can form at be- low 2% oxygen [13, 14].

Therefore, the aim of this study was to determine the shelf-life of green mussels marinated with tamarind fish sauce enriched with iron and zinc. The mussels were packed in modified atmosphere and stored at 4°C.

STUDY OBJECTS AND METHODS

(ZnSO ·7(H O)) were taken from Sigma–Aldrich Com-

foam agent were added. 5 ml from 50 ml of the distillate were mixed with 5 ml of 0.02 M 2-thiobarbituric acid in 90% glacial acetic acid (TBA reagent). The mixture was placed in a vial that was capped and heated in a water bath for 35 min. Afterwards, the vial was cooled down and the sample was measured at 532 nm using a spec- trophotometer (UV/VIS T180, PG Instrument Ltd., UK). The results are expressed in mg malondialdehyde/kg sample.

Glycogen content. Glycogen content was deter- mined based on the method of [16]. 50 mg of chopped marinated green mussels were boiled for 20 min with 400 µl of 33% KOH, cooled down, and 700 µl of 96% ethanol was added. The mixture was placed in an ice bath for 2 hours to reach complete precipitation. There- after, the mixture was centrifuged at 7,500 g for 20 min using a microlitre centrifuge (Z-233-M2, Hermle– Germany) and the supernatant was collected. The su- pernatant was mixed with 1 ml of distilled water and sonicated for 5 min using Crest Ultrasonic (575 DAE, Malaysia). Then 100 µl of the solution and 2 ml of an- throne reagent were kept at 90°C for 20 min in dark- ness. The reagent consisted of 38 ml of concentrated

sulphuric acid with 15 ml of distilled water and 0.075 g

4            2

pany (Steinheim, Germany).

Marinated green mussel preparation. The green mussels were washed during 2 hours with tap water, then blanched in boiling water at 100°C for 30 s to fa- cilitate meat removal. A ratio of sample to water was 1:2  w/w.  Afterwards,  the  meat  of   green   mussels was separated from the shells. We prepared tam- arind fish sauce contained  42%  of  tamarind  slur- ry, 37% of sugar, and 21% of fish sauce.   236 mg of

FeNaEDTA·3(H O)   and   88.5 mg   of   ZnSO ·7(H O)

of anthrone. The solution was measured at 620 nm using

a spectrophotometer. Glycogen concentration was calcu- lated from a standard curve for glycogen from Sigma– Aldrich Company (Steinheim, Germany).

Total volatile base nitrogen (TVB – N). Total vola- tile base (TVB – N) contents in the green mussels were determined using Conway micro-diffusion method de- scribed in [17]. 2 g of a sample were mixed with 8 ml of 4% tricloroacetic acid (TCA), then homogenized with

a high-speed homogenizer for 1 min. The supernatant

2                                                                                      4            2

were added into 30 g of the sauce, which was 36 mg of Fe and 20 mg of Zn, respectively. The green mus- sel meat was soaked in the enriched sauce at the ra- tio of 1:1.  The  marinated  meat  was  packaged  in 15 х 30 cm Havel Vacuum bags, b.v. (Europac Co., Ltd)

was filtered by Whatman No. 41 filter paper (Whatman

transmission  rate  46.6  cm3m2   day–1    at  38°C,

ple to initiate the reaction after mixing. Thereafter, the

1 atm. We tested five samples of green mussels: meat without marinade solution packed under normal air (C1), marinated meat without gases (C2), and meat packed in the modified atmosphere (M1, M2, and M3). A percent-

mixture was incubated at room temperature for 3 hours.

Afterwards, the inner ring solution was titrated using 0.02N HCl until the green color turned to pink similar to a blank sample. TVB-N content was calculated as fol-

age of O :CO :N

was 5:50:45 for M1, 5:70:25 for M2,

lows, mg/100 g:

2           2      2

and 5:90:5 for M3. All samples were stored at 4°C for

30 days. Microbiological, chemical and sensory analyses were performed every 3 days.

pH. The marinated green mussels were blended with sterilized distilled water at the ratio of 1:5 and allowed to stand for 2 min. Then pH was measured by using a pH meter (Mettler 350, Singapore).

Thiobarbituric acid reactive substances (TBARS). The thiobarbituric acid reactive substances (TBARS) were determined by the method described in [15]. 10 g of chopped samples were homogenized with 50 ml of distilled water for 2 min and then transferred to distil- lation tubes. 47.5 ml of distilled water was additional- ly infused into the distillation tube to avoid losing the

sample. 2.5 ml of 4 N HCl and a few drops of an anti-

(mg/100 g) ,

where: N is normality of HCl in titrant; A is titration vo- lume for actual sample; B is titration volume for blank; V is total initial volume of sample with TCA.

Iron and zinc content. Samples of 0.5 g in mass were digested in 5 ml of concentrated nitric acid with 2 ml of 30% hydrogen peroxide under pressure in a closed vessel before heating in a microwave oven un- til the samples were digested. Afterwards,  the  sam- ples were cooled down and filtered by Whatman filter paper No. 1 with a pore size of 110 nm (Whatman In-

ternational, Ltd., Maidstone, UK)). Each sample solu-

tion was then transferred into a 25 ml volumetric flask and the volume was made up with distilled water [18]. The extract samples were used to determine iron and zinc by Inductively Couple Plasma Mass Spectrometry (ICP-MS) (Perkin-Elmer SCIEX, Shelton, CT, USA). The RF power was 1,350 W, the plasma gas flow rate was 15 l/min, the carrier gas flow was 0.94 l/min, and make-up gas flow was 0.15 l/min. The sample take rate was 100 µl/min and the sample depth was 6.0 mm.

Microbiological quality. 25 g of the marinated green mussels were blended with 225 ml of 0.1% ste- rilized peptone water. From the 10–1 dilution, dilutions to 10–2 and 10–3  were prepared with 0.1% sterilized peptone water. Mesophilic and psychrophilic bacteria, Esche- richia coli, Salmonella spp, Vibrio cholera, and Staphy- lococcus aureus were  determined  using  the  method in [19]. Lactic acid bacteria were determined using de Man Rogosa and Shape agar by pour plate method, with incubation in anaerobic conditions at 35°C for 48 hours before colony count [20].

Sensory evaluation. Marinated samples were baked in a casserole pan (House worth HW-1707S, China) at 180°C for 10 min until the core temperature of the meat sample reached 80 ± 2°C. Then the samples were served to fifty untrained panelists of post-graduate students and technicians from the Department of Food Technology, Prince of Songkla University. The panelists were asked to evaluate the likability scores for appearance, aroma, texture, taste and overall on a nine-point hedonic scale.

Statistical analyses.  All experiments  were  run in triplicates. The data were subjected to Analysis of Vari- ance (ANOVA) and the differences between means were assessed for significance by Duncan’s Multiple Range Test [21]. Data analysis was performed with the SPSS package (SPSS 6.0 for Windows, SPSS Inc., Chicago, IL, USA).

RESULTS AND DISCUSSION

We found that the sauce had pH 3.31, total titratable acidity 32.13%, and water activity 0.87. The viscosity of this enriched sauce was 840 cP, with non-Newtonian be- havior. The colour coordinates L*, a*, and b* were 26.44,

15.8 and 33.7, respectively.

pH. pH levels of un-treated and treated green mus- sels are shown in Fig. 1. In general, pH with all treat- ments decreased with storage time. The initial pH of un-treated green mussels (C1) was 6.77 and decreased to

6.21 at 15 days of storage (p < 0.05). The marinated sam- ples had significantly lower pH than the control group due to acidity of the sauce (pH 3.31) (p < 0.05). The pH of marinated green mussels without gases (C2) was

4.48 on day 1 and declined to 4.07 by day 21 of storage (p < 0.05). The initial pH of marinated green mussel (M1, M2, and M3) was about 4.51–4.64 and dropped to 4.24, 4.2 and 3.95, respectively, at 30 days of storage (p < 0.05). The decrease in pH of meat during chilled storage is normally caused by conversion of glycogen to lactic acid and other volatile acids, as a result of mus- cle degradation [22]. Cao et al. also reported that the spoilage of mollusk shellfish was partly by fermentation

indicated by decreasing pH [23]. Moreover, pH was low-

Fig. 1. Dependence of pH values of green mussels on storage time. C1 = control; C2 = marinated with no gases; M1, M2, and M3 = marinated at the O :СO :N  percentage of 5:50:45;

2          2      2

5:70:25; and 5:90:5, respectively.

,

MDA/kg, respectively. It should be noted that the mari- nated green mussels had lower TBARS values than the control samples, apparently due to osmosis during ma- rinating conveying TBARS out from the mussels. Also the bacterial count was higher for C1 than for the other samples, which could also play a role in producing TBARS [28]. The lower TBARS values of the marina-

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Fig. 2. Dependence of TBARS values of green mussel

on storage time. C1 = control; C2 = marinated with no gases; M1, M2, and M3 = marinated at the O :СO :N  percentage

2          2      2

er with MAP using gases than with other treatments,

of 5:50:45; 5:70:25; and 5:90:5, respectively.

2,400   1,800   1,200   600   0

,

Fig. 3. Dependence of TVB – N values of green mussels on storage time. C1 = control; C2 = marinated with no gases; M1, M2, and M3 = marinated at the O :СO :N  percentage

Fig. 4. Dependence of glycogen content of green mussels

on storage time. C1 = control; C2 = marinated with no gases; M1, M2, and M3 = marinated at the O :СO :N  percentage of

2          2      2

2          2      2

of 5:50:45; 5:70:25; and 5:90:5, respectively.

5:50:45; 5:70:25; and 5:90:5, respectively.

ted green mussels were consistent throughout the sto- rage, and sulfur compounds from garlic in the marinade may have played a role as antioxidants [29]. However, TBARS in all cases increased during storage because of high content of polyunsaturated fatty acids in mussel fat, as mentioned in the introduction. Moreover, MAP packaging reduced TBARS may be due to O2-elimina-

and M1 samples were 27, 24, and 25 days, respectively. In addition, [36] noticed that for seafood TVB-N indi- cated the freshness of raw materials and acceptability for human consumption. They pointed out that TVB-N increased with storage time as a result of quality dete- rioration. However, the initial TVB-N of the marinated products may not indicate the quality, considering that

proportion [22]. Masniyom et al.

fish sauce and garlic used as ingredients cause erroneous

reported that seabass flesh in MAP did not suffer from

increased lipid oxidation [30]. The TBARS  level  of 5–8 mg/kg malonaldehyde is a standard criterion of the poor quality of oily food [31, 32]. TBARS in the control (C1) reached 8.03 mg MDA/kg on day 12 of storage, while C2 had 7.85 mg MDA/kg on day 18. M1, M2, and M3 had TBARS levels below the standard on day 27, namely 7.59, 6.89, and 6.82 mg MDA/kg, respectively. The marinated green mussels packed at the percentage

of O2:CO2:N2 equaled 5:90:5 gave the least TBARS level at the end of storage (p > 0.05).

Total volatile base nitrogen (TVB-N). Changes in the TVB-N value of the treated and un-treated green mussels during storage are shown in Fig. 3. The TVB-N increased with storage time. The initial TVB-N of green mussels (C1) was 6.44 mg/100 g and increased gradually to 10.22 mg/100 g during storage. The treated samples C2, M1, M2, and M3 had initial TVB-N values 20.6,

results.

Glycogen content. During storage glycogen content decreased gradually in all samples, but declined rapidly in C2 samples (p < 0.05), as shown in Fig. 4. The ini- tial glycogen content in the control sample (C1) was sig- nificantly lower than that in the marinated samples (C2, M1, M2, and M3). Glycogen is a polysaccharide includ- ing glucose units in a branched structure. Extraction and hydrolysis were used to obtain simple sugars in the gly- cogen content determination. As mentioned earlier, the marinade solution had 37% of sugar, so it positively con- tributed to the glycogen content. Jermyn [37] mentioned

Table 1. Iron and zinc content in green mussel samples during storage

Minerals                   Samples  Initially                 End of storage

18.97, 19.53 and 18.94 mg/100 g, respectively. By the end of the storage they reached 30.1, 33.33, 33.67, and

29.69 mg/100 g, respectively (p < 0.05). TVB-N is com- posed of  volatile amines,  typically  dimethylamine, trimethylamine and ammonia [22], that commonly de- termine the spoilage of seafood [33]. The marinated samples had higher TVB-N values than the control even on day 1, due to volatile amines in the fish sauce [34]. In addition, the marinade contained garlic with sulfur compounds that could also react with boric acid, con- tributing to TVB-N content [29]. The increasing trend

of TVB-N with time in this study agrees with the find-

Iron content,

mg/kg

Zinc content, mg/kg

C1            71.44 ± 0.77a           67.61 ± 0.67b

C2            599.62 ± 12.60a      371.47 ± 2.33b M1               389.67 ± 1.14a            381.34 ± 1.99b M2 399.2 ± 1.98a               390.00 ± 1.41b M3                481.93 ± 1.36a        392.36 ± 0.44b C1  11.09 ± 0.40a               8.95 ± 0.13b C2      382.19 ± 8.22a            249.00 ± 1.41b M1 267.20 ± 1.56a            257.74 ± 2.51b M2             280.35 ± 0.64a            266.17 ± 1.17b M3 331.00 ± 1.98a            271.11 ± 2.40b

ings of [35].  Fishery products are acceptable for human

Note: a–b   Means within rows with different superscripts are signifi-

cantly different (p < 0.05). C1 = control; C2 = marinated with no

consumption when TVB-N does not exceed 30 mg/100 g

gases; M1, M2, and M3 = marinated at the O :СO :N

percentage of

,

of sample [34]. Based on this, the shelf-lives of M3, M2,

5:50:45; 5:70:25; and 5:90:5, respectively

2         2      2

Table 2. Microbiological levels in marinated and non-marinated green mussel kept under different conditions during the storage at 4°C for 30 days.

Mesophile, CFU/g

Bac-

Treat-

Storage time, days