Food Chemistry 159 (2014) 20–28

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Biogenic amine and quality changes in lightly salt- and sugar-salted black carp (Mylopharyngodon piceus) fillets stored at 4 °C Hongbing Fan, Yongkang Luo ⇑, Xiaofei Yin, Yulong Bao, Ligeng Feng College of Food Science and Nutritional Engineering, China Agricultural University, Beijing Higher Institution Engineering Research Center of Animal Product, P.O. Box 112, Beijing 100083, PR China

a r t i c l e

i n f o

Article history: Received 26 September 2013 Received in revised form 21 February 2014 Accepted 27 February 2014 Available online 12 March 2014 Keywords: Black carp Salt Sugar Biogenic amine Quality changes Postmortem changes

a b s t r a c t The effects of low salt and sugar dry-curing on the quality changes of black carp (Mylopharyngodon piceus) fillets stored at 4 °C were evaluated by sensory, physical, chemical, and microbiological methods. Fish samples were left untreated (control), or were dry-cured with 1.5% salt (T1) or 1.5% salt + 1.2% sugar (T2). Curing treatments reduced chemical changes reflected in HxR, Hx, pH, and total volatile base nitrogen (TVB-N); decreased cooking loss; and increased overall sensory quality of fish (p < 0.05) compared to untreated samples. Significantly lower values of cadaverine and putrescine were observed in T1 and T2 compared to the control after the 2nd and 4th day, respectively (p < 0.05). There were significant differences (p < 0.05) between T1 and T2 for pH, TVB-N, total aerobic counts (TAC), and sensory characteristics. Sensory characteristics were significantly correlated with TAC, TVB-N, putrescine, and cadaverine in all samples (p < 0.01). Ó 2014 Elsevier Ltd. All rights reserved.

1. Introduction Black carp (Mylopharyngodon piceus) is one of the main freshwater fish species aquacultured in China and is mostly distributed in the middle and lower reaches of the Yangtze River and its affiliated lakes. The yield of black carp in China in 2012 was 494,908 tonnes. Owing to its rapid growth and relatively high market price, as well as the healthcare potential for biological control of nuisance aquatic mollusks, black carp has a higher economic value than other species of farmed fish. Hitherto, studies on black carp have mainly related to artificial breeding (Rothbard et al., 1997), aquaculture (Sun, Ye, Chen, Wang, & Chen, 2011), and biocontrol of mollusks (Venable, Gaudé, & Klerks, 2000). However, reports are scarce on the postmortem quality changes of black carp muscle. High moisture levels, rich nutrient content, and microbial activity render fish an easily perishable food product. The spoilage of fish, accompanied with physical and chemical changes and microbial growth, is a very complex process. Numerous measures have been taken to improve fish quality and extend shelf life, such as high hydrostatic pressure (Erkan & Üretener, 2010), vacuum packaging (Lyhs et al., 2001), edible films coatings (Song, Liu, Shen, You, & Luo, 2011), and salting (Yanar, Çelik, & Akamca, 2006), ⇑ Corresponding author. Tel./fax: +86 10 62737385. E-mail addresses: [email protected], [email protected] (Y. Luo). http://dx.doi.org/10.1016/j.foodchem.2014.02.158 0308-8146/Ó 2014 Elsevier Ltd. All rights reserved.

among others. Salting is among the most popular one and its preservative effect, which also enhances the flavour, can extend the shelf life of fish by reducing water activity, decreasing opportunity for microbial attack, and enhancing functional properties of fish protein (Yanar et al., 2006). However, at high salt concentration the proteins may denature, resulting in stronger protein-protein bonds, muscle shrinkage, and dehydration (Thorarinsdottir, Arason, Geirsdottir, Bogason, & Kristbergsson, 2002). As consumer preference for prepared foods is increasing, the demand for lightly salted fish fillets is growing. It is thus meaningful to investigate how low levels of salt influence fish quality. Sugar-salted fish products, termed gravad, are very popular dishes traditionally manufactured in Nordic countries (Lyhs et al., 2001). Sugar is also widely used in Chinese dishes, especially those in China’s southeastern coastal cities. Sugar has been used to stabilize frozen surimi as a cryoprotectant (Sultanbawa & Li-Chan, 1998), protects fish myofibrillar proteins (Lee, 1984), and decreases biogenic amine accumulation in slightly fermented sausages (Bover-Cid, Izquierdo-Pulido, & Vidal-Carou, 2001), among others. Hong, Luo, Zhou, and Shen (2012) investigated the effects of brining with low concentration of sucrose combined with salt on the quality parameters of bighead carp fillets stored at 4 °C. However, limited information is available on the effect of sugar on the formation of biogenic amines (BAs) in fish. The aim of this work was to evaluate the effects of low salt and sugar on the quality of black carp fillets by evaluating the sensory

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attributes, physicochemical quality, and microbial changes, as well as by determining biogenic amines of fillets during storage at 4 °C. 2. Materials and methods 2.1. Sampling, preparation and storage conditions Twelve black carps (weight of 6750 ± 225 g, length of 80.08 ± 2.75 cm) were purchased from an aquatic product wholesale market in Beijing, China, in January 2013, and were immediately delivered to the laboratory alive. Fish were euthanized by concussion, scaled, gutted, deheaded, washed, and cut into small pieces (about 7–8 cm in length). Each piece was filleted into two sides, after which the fillets were left to drain on sterile stainless steel wire mesh for 3 min and then prepared for dry-curing. Prior to performing this experiment, different curing concentrations were prepared and tested for consumer acceptability. Two acceptable curing concentrations were obtained (1.5% salt and 1.5% salt + 1.2% sugar) during this testing. Black carp fillets were randomly divided into three groups. The untreated group (control, n = 39) was used as control. The treated groups were dry cured with 1.5% salt (T1, n = 42) and 1.5% salt + 1.2% sugar (T2, n = 42), respectively. Dry salt or salt-sugar mixture was carefully and evenly sprinkled on the surface of the fish fillets. The addition of salt and sugar was expressed as percentage of initial fillet weight. Ordinary commercial food grade salt (CNSIC, Beijing, China) and granulated white sugar (COFCO Tunhe, Xinjiang, China) were used for dry-curing. The treated and untreated samples were packed in polyvinyl chloride bags and stored in refrigerators at 4 °C. Samples of white dorsal muscle from each fish fillet were taken randomly for analysis at selected time intervals (0, 2, 4, 8, 12, 24, 36, and 48 h for postmortem storage and 4, 6, 8, 10, 12, 14, and 16 days for long-term storage). 2.2. Methods 2.2.1. Nucleotide degradation products Preparation of ATP and its degradation products followed the procedure described by Zhu, Luo, Hong, Feng, and Shen (2013). The prepared supernatant sample was filtered through a 0.22 lm membrane filter and analyzed for nucleotide degradation products using reverse phase high-performance liquid chromatography (HPLC) (Shimadzu, LC-10AT series, Kyoto, Japan) equipped with SPD-10A (V) detector, COSMOSIL 5C18-PAQ column (4.6ID  250 mm) (Nacalai Tesque, Inc., Kyoto, Japan). The mobile phase was 0.05 M phosphate buffer (pH 6.8); flow rate was 1 mL/min; injection volume was 20 lL; detection wavelength was 254 nm. ATP and its degradation products were identified and quantified based on the commercially obtained standard ATP, ADP, AMP, IMP, inosine (HxR), and hypoxanthine (Hx). K value was calculated as follows (Saito, Arai, & Matsuyoshi, 1959):

K value % ¼ ½ðHxR þ HxÞ=ðATP þ ADP þ AMP þ IMP þ HxR þ HxÞ  100

2.2.2. pH value, electrical conductivity (EC), and total volatile base nitrogen (TVB-N) A sample (10 g) of fish flesh was dispersed in 100 mL of distilled water and stirred for 30 min by an electric blender, after which the mixture was filtered. The filtrate was collected at room temperature (about 20 °C) for further analysis. The pH value of the filtrate was measured using a digital pH meter (Mettler Toledo FE20/EL20, Shanghai, China). The TVB-N was determined according to the

method of Song et al. (2011). The EC was measured as described by Hong et al. (2012). 2.2.3. Cooking loss A 15  15  10 mm sample of fish flesh was packed in polyethylene bags and then immersed in a water bath at 85 °C for 15 min. Samples were weighed before (Wb) and after (Wa) cooking. Cooking loss was calculated as follows:

Cooking loss % ¼

Wb  Wa  100 Wb

2.2.4. Texture and colour Texture profile analysis (TPA) was determined using the method described by Zhu et al. (2013). The colour of black carp fillets was measured following the method of Hong et al. (2012). 2.2.5. Sensory assessment (SA) SA was performed according to the method described by Ojagh, Rezaei, Razavi, and Hosseini (2010) with some modifications. The attributes of raw fish fillets and cooked fish fillets (steamed for 5 min at 100 °C) were evaluated by a panel of 9 trained members. Each panel member was asked to rate the appearance, odour, texture, and morphology of raw fish muscle, as well as the flavour, taste, and broth turbidity of cooked fish muscle. A rating scale of 1–5 points was used for each attribute, with 5 equivalent to top quality fresh black carp and 1 indicative of borderline freshness. The total scores for each sample were calculated by adding the average score given by each panel member for each of the seven attributes. 2.2.6. Microbiological analysis Total aerobic counts (TAC) indicated as aerobic plate counts were determined following Song et al. (2011). Twenty-five grams of fish flesh was weighed aseptically and then homogenized with 225 mL of sterilized 0.9% physiological saline for 1 min. A series of 1:10 (v/v) dilutions were made; 1 mL of serial dilution was plated onto plate count agar; and the plates were incubated at 30 °C for 72 h. All counts were expressed as log10 CFU/g and performed in duplicate. 2.2.7. Biogenic amines (BAs) Preparation and derivatization of BAs of black carp fillets followed the method of Ikonic´ et al. (2013). Identification and quantification of BAs were performed by using HPLC (Shimadzu, LC-10AT series, Kyoto, Japan) equipped with SPD-10A (V) detector, COSMOSIL 5C18-PAQ column (4.6ID  250 mm) (Nacalai Tesque, Inc., Kyoto, Japan). Chromatographic separation was carried out using a gradient elution of Ammonium acetate (0.1 M, solvent A) and acetonitrile (100%, solvent B) as follows: 0 min, 50% B; 25 min, 90% B; 35 min, 90% B; 45 min, 50% B. Flow rate was 0.8 mL/min; column temperature was 30 °C; injection volume was 20 lL; and peaks were detected at 254 nm. 2.2.8. Statistical analysis All analyses were run in triplicate (except microbiological analyses, which were performed in duplicate). Data were expressed as mean values accompanied by the standard deviation of means. The least significant difference (LSD) procedure was used to test for difference between means using SAS software (2008). The significance level was set at 5%. Pearson’s regression analysis was performed to determine the relationship between sensory, physical, chemical, and microbial quality.

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3. Results and discussion 3.1. Nucleotide degradation products Variations of nucleotide degradation products (IMP, HxR, and Hx) and K value in the muscle of black carp fillets during the 16-day storage are shown in Fig. 1. Nucleotide degradation products (ATP ? ADP ? AMP ? IMP ? HxR ? Hx), especially IMP, HxR, and Hx, have been commonly used as indicators of fish freshness before bacterial spoilage. The breakdown of IMP to Hx is slow and is caused by both autolytic and microbial enzymes (Alasalvar, Taylor, Öksüz, Shahidi, & Alexis, 2002). IMP is a flavour enhancer and is strongly associated with acceptable levels of fish freshness. Conversely, Hx conduces to the gradual loss of desirable flavour and the development of bitter off-flavours. Its accumulation in fish tissue reflects the initial stage of autolytic degradation as well as subsequent bacterial spoilage (Liu et al., 2010). The initial level of IMP in the muscle of black carp was 5.409 lmol/g. As shown in Fig. 1(a), the concentration of IMP had no significant changes within the first 2 days but afterwards decreased sharply. No significant differences (p > 0.05) in the level of IMP were

observed between control, T1, and T2 samples during the storage period. The initial levels of HxR and Hx of raw black carp fillets were 0.343 and 0 lmol/g, respectively. Changes in concentrations of HxR and Hx of all samples showed the same trend in which the values of HxR increased initially and then decreased while those of Hx consistently increased throughout storage. Treated samples (T1 and T2) showed higher concentrations of HxR and lower concentrations of Hx than control samples after being stored for 6 and 4 days (p < 0.05), respectively, from which it can be concluded that 1.5% salt and 1.5% salt + 1.2% sugar treatment can delay the rate of breakdown from HxR to Hx. This delay was possibly due to the inhibitory effect of salt on autolytic degradation or on the growth of spoilage bacteria, which may secrete related enzymes that contribute to the breakdown of HxR to Hx. There seemed to be no significant differences (p > 0.05) in the levels of HxR and Hx in T1 and T2 samples throughout storage. K value is commonly used to evaluate fish freshness and presents a good correlation with the storage time of fish. The initial K value of raw black carp fillets was 5.63 and increased steadily until the end of storage. K values of control, T1, and T2 samples

Fig. 1. Changes in IMP, HxR, Hx, and K value of black carp fillets treated with low salt and sugar during storage at 4 °C for 16 days (Control: untreated; T1: treated with 1.5% salt; T2: treated with 1.5% salt + 1.2% sugar).

H. Fan et al. / Food Chemistry 159 (2014) 20–28

increased to the maximum levels of 83.89, 91.93, and 87.67 on day 12, 16, and 16, respectively. There were no significant differences (p > 0.05) in K value between all three samples over the storage period. Saito et al. (1959) described fish products with K values lower than 20% as very fresh, less than 50% as moderately fresh, and higher than 70% as not fresh. Based on these K value categories, the raw black carp fillets under the experimental conditions of this study could be considered very fresh within 2 days, moderately fresh within 4 days and not fresh around the 8th day. However, it is important to note that the classification previously described depends on the species. 3.2. pH value Changes in pH value of black carp fillets during 16 days of storage are shown in Fig. 2. The initial pH value of the fish sample was 6.86, similar to the value (6.78) of fresh ray fish reported by Ocaño-Higuera et al. (2011). Variations among the initial pH values may be due to the species, season, diet, level of activity, and other factors. Changes in pH values of different treatments showed the same trend in which the values decreased initially and then increased until the end of storage. The initial decrease of pH value may be attributed to the decomposition of glycogen, ATP, and creatine phosphate in fish muscle, while the subsequent increase is due to the production of alkaline substances caused by degradation of protein by either endogenous or microbial enzymes (Ruiz-Capillas & Moral, 2001). There were no significant differences (p > 0.05) among all fish samples during 6 days of storage, but thereafter pH increased at different rates until the end of storage. T1 and T2 samples showed a lower pH than control samples; T2 samples reached the lowest pH (6.37) on the 10th day, which may be due to glycolysis of sugar. Other researchers reported that this lower pH may be attributed to the growth of lactic acid bacteria (LAB). These bacteria may contribute to a lowering of muscle pH by inhibiting the growth of other kinds of bacteria, as well as by buffering the basic metabolites produced (Calo-Mata et al., 2008). It can be concluded that the lower pH value of T1 and T2 might restrain microbial growth and inhibit the activity of the endogenous proteases to varying degrees, thus leading to the extension of preservation time of black carp fillets.

Fig. 2. Changes in pH of black carp fillets treated with low salt and sugar during storage at 4 °C for 16; (a) Enlarged figure of pH changes (0–0.5 day) (Control: untreated; T1: treated with 1.5% salt; T2: treated with 1.5% salt + 1.2% sugar).

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3.3. Cooking loss, texture and colour Measurements of cooking loss, texture and colour parameters of black carp fillets are shown in Table 1. Cooking loss generally includes a combination of liquid and soluble substances lost from meat during cooking; the principle component is water. T1 and T2 samples showed a lower (p < 0.05) cooking loss than control samples during the storage period, which was in agreement with what has been reported with regard to the addition of small amounts of salt to low-sodium ground meat patties (Ruusunen et al., 2005). No significant differences (p > 0.05) were observed between T1 and T2. Mendiratta, Kondaiah, Anjaneyulu, and Sharma (2008) reported that change in cooking loss may be related to pH and WHC (water holding capacity) of muscle. The curing process brings about changes in muscle proteins, in salt-soluble proteins in particular, which are strongly associated with WHC of muscle. The hardness of raw fish samples increased significantly (p < 0.05) during the first 4 h and then decreased with time to the end of storage, which indicated that the maximum rigidity state of black carp was reached at 4 h postmortem. Gumminess and chewiness followed the same trend; whereas springiness showed a reverse trend. Significantly higher values of springiness (p < 0.05) were detected after the curing treatments. T1 and T2 showed a higher value of chewiness than the control after storage for 2 days (p < 0.05), which indicated that low salt (1.5%) treatment could offer a better mouth feel to black carp fillets. Surface colour of muscle food is an important parameter in acceptability of muscle foods and influences consumers’ acceptance to products. T1 and T2 samples showed a significantly (p < 0.05) lower L value during the first 8 days of storage, and higher b value after 4 days of storage, than the control samples, respectively. These data indicated that curing had significant effects on the colour changes of black carp fillets. There were no significant differences (p > 0.05) between T1 and T2 samples in L, a, and b values during the entire storage period. T1 and T2 had higher L and b values than the control at the end of storage. 3.4. Changes in SA, EC, TAC, TVB-N, and BAs Fig. 3(a) shows changes in sensory assessment (SA) of black carp fillets stored at 4 °C for 16 days. Curing treatment lowered the scores of appearance of black carp fillets while improved the taste to some extent. However, treatment with 1.5% salt or 1.5% salt + 1.2% sugar did not have a significant effect on the initial overall sensory scores. The average overall sensory scores of all samples decreased continuously with the increase of time. After being stored for 8 days, the sensory scores for the control, T1 and T2 samples decreased from initial values of 34.0, 32.3, and 31.7 to 16.3, 21.5, and 22.3, respectively. The black carp fillets were considered acceptable for human consumption until the sensory score reached 15. Therefore, after 8 days of storage, control samples had almost reached this unacceptable limit score, whereas the treated samples had not. Compared to the control, treatments T1 and T2 significantly (p < 0.05) delayed decreases in sensory quality. The slower speed of decline in sensory quality of treated samples was possibly due to higher osmotic pressure inhibiting enzymatic activity and microbial growth in fish muscle. T2 samples showed a lower score (p < 0.05) than T1 samples after being stored for 12 days. This reduction was attributed to the slightly sour odour produced by fermentation of sugar by acid-forming bacteria; this odour was not acceptable to panelists. According to the above-mentioned limit, T1 and T2 samples maintained relatively good quality up to 12 and 14 days, respectively. As an index of the concentration of electrolytes in the muscle tissues, electrical conductivity (EC) can be used to characterize the texture of fish tissue. Fig. 3(b) shows changes in EC value of

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Table 1 Changes in cooking loss, texture and surface colour analyses of black carp fillets treated with low salt and sugar during storage at 4 °C. Parameters

Cooking Loss (%)

Gumminess (g) Chewiness (mJ) Springiness (mm) L

a

b

Control T1 T2 Control T1 T2 Control T1 T2 Control T1 T2 Control T1 T2 Control T1 T2 Control T1 T2 Control T1 T2

Storage time (h)

Storage time (days)

0

2

4

8

12

24

36

48

4

8

12

23.55 ± 0.43

23.29 ± 0.72a 16.06 ± 0.25b 15.24 ± 0.28b 901 ± 108a 253 ± 28b 231 ± 17b 317 ± 17a 202 ± 19b 152 ± 26b 9.4 ± 0.6a 6.2 ± 0.2b 7.0 ± 1.3b 2.90 ± 0.04a 3.93 ± 0.08b 4.04 ± 0.03b 56.64 ± 0.20a 54.45 ± 0.52b 51.99 ± 0.56c 5.30 ± 0.28a 5.01 ± 0.23a 5.63 ± 0.36a 9.54 ± 0.30a 8.76 ± 0.12b 7.54 ± 0.31c

23.60 ± 1.34a 15.16 ± 0.34b 14.39 ± 0.32b 1645 ± 138a 288 ± 28b 223 ± 28b 655 ± 33a 175 ± 10b 132 ± 13c 19.1 ± 1.8a 6.0 ± 0.2b 7.2 ± 0.4c 2.81 ± 0.09a 4.22 ± 0.03b 4.17 ± 0.26b 55.63 ± 0.27a 52.15 ± 0.92b 51.38 ± 0.34b 5.50 ± 0.10a 6.31 ± 0.41b 6.48 ± 0.15b 8.96 ± 0.24a 8.05 ± 0.41b 7.33 ± 0.31b

23.40 ± 0.52a 13.69 ± 1.18b 14.29 ± 0.15b 1409 ± 76a 278 ± 35b 209 ± 15b 468 ± 28a 172 ± 7b 159 ± 9b 11.7 ± 0.6a 6.7 ± 0.9b 7.2 ± 0.6b 2.51 ± 0.03a 4.10 ± 0.04b 4.49 ± 0.13c 54.47 ± 0.39a 51.99 ± 0.93b 51.37 ± 0.86b 6.45 ± 0.08a 6.99 ± 0.77ab 7.26 ± 0.10b 8.70 ± 0.32a 7.84 ± 0.55ab 7.40 ± 0.30b

22.99 ± 0.66a 12.95 ± 1.09b 14.42 ± 0.60b 1029 ± 21a 269 ± 39b 254 ± 11b 389 ± 35a 181 ± 12b 184 ± 11b 11.1 ± 0.5a 6.4 ± 0.2b 7.4 ± 1.0b 2.59 ± 0.05a 4.32 ± 0.24b 4.82 ± 0.11c 54.32 ± 0.58a 52.25 ± 1.01b 51.99 ± 0.28b 6.12 ± 0.21a 6.91 ± 0.24b 6.82 ± 0.13b 8.73 ± 0.54a 7.86 ± 0.27ab 7.34 ± 0.52b

19.20 ± 0.40a 11.29 ± 0.52b 10.74 ± 0.18b 600 ± 29a 343 ± 30b 267 ± 18c 305 ± 8a 208 ± 18b 175 ± 16b 10.3 ± 0.8a 8.7 ± 0.6b 7.3 ± 0.9b 2.73 ± 0.10a 4.65 ± 0.14b 4.72 ± 0.05b 54.10 ± 0.34a 51.92 ± 0.25b 52.14 ± 0.23b 5.81 ± 0.11a 6.01 ± 0.22a 5.92 ± 0.39a 9.29 ± 0.14a 8.35 ± 0.22b 8.45 ± 0.22b

19.40 ± 0.74a 11.74 ± 0.74b 11.28 ± 0.55b 716 ± 47a 325 ± 40b 225 ± 18c 294 ± 20a 204 ± 40b 158 ± 15b 9.1 ± 0.4a 8.3 ± 0.6ab 7.5 ± 0.5b 2.98 ± 0.05a 4.75 ± 0.04b 4.67 ± 0.16b 53.71 ± 0.27a 51.84 ± 0.26b 51.62 ± 0.95b 7.30 ± 0.20a 6.73 ± 0.14b 7.29 ± 0.30a 9.25 ± 0.21a 8.82 ± 0.53ab 8.52 ± 0.13b

22.23 ± 0.43a 12.72 ± 0.94b 12.06 ± 0.81b 512 ± 53a 334 ± 19b 308 ± 11b 298 ± 13a 227 ± 26b 183 ± 24b 9.7 ± 0.4a 8.4 ± 0.1b 8.4 ± 0.8ab 3.03 ± 0.11a 4.37 ± 0.11b 4.79 ± 0.11c 54.63 ± 0.07a 51.08 ± 0.17b 51.54 ± 0.37b 6.57 ± 0.38a 6.72 ± 0.32a 6.34 ± 0.49a 9.14 ± 0.10a 8.53 ± 0.52ab 8.36 ± 0.49b

21.89 ± 0.92a 12.69 ± 0.64b 10.45 ± 0.63b 474 ± 14a 397 ± 38b 305 ± 37b 248 ± 24a 242 ± 36a 199 ± 18a 6.1 ± 0.4a 8.6 ± 0.4b 8.4 ± 1.0b 3.07 ± 0.14a 4.07 ± 0.04b 4.24 ± 0.16b 55.17 ± 0.09a 52.23 ± 0.31b 51.13 ± 0.14c 5.20 ± 0.14a 6.08 ± 0.45b 5.80 ± 0.03b 9.66 ± 0.21a 10.82 ± 0.32b 10.09 ± 0.24a

18.82 ± 0.32a 9.85 ± 0.37b 8.57 ± 0.29c 389 ± 89a 367 ± 28a 239 ± 24b 161 ± 30a 191 ± 37a 141 ± 24a 4.4 ± 0.8a 8.5 ± 1.6b 7.0 ± 0.4b 2.64 ± 0.04a 4.43 ± 0.11b 4.30 ± 0.28b 51.51 ± 0.31a 49.61 ± 0.11b 48.96 ± 0.84b 6.29 ± 0.39a 7.25 ± 0.27b 7.36 ± 0.37b 9.65 ± 0.76a 11.25 ± 0.51b 11.83 ± 0.19b

21.51 ± 0.45a 10.45 ± 0.39b 10.19 ± 1.57b 258 ± 42a 360 ± 41b 193 ± 31a 113 ± 18a 177 ± 26b 131 ± 13ab 3.3 ± 0.4a 8.6 ± 1.2b 6.6 ± 0.9b 2.41 ± 0.10a 4.22 ± 0.04b 3.66 ± 0.18c 54.57 ± 0.69a 57.13 ± 1.18b 56.00 ± 0.16b 5.65 ± 0.22a 6.21 ± 0.32a 6.35 ± 0.98a 9.07 ± 0.18a 12.51 ± 0.72b 12.95 ± 0.60b

710 ± 53

262 ± 13

6.9 ± 0.6

2.88 ± 0.06

58.00 ± 0.08

4.93 ± 0.29

10.24 ± 0.36

Results are presented as mean ± standard deviation. Same superscript lowercase letters in a column indicate no significant differences (p > 0.05). Control: untreated; T1, treated with 1.5% salt; T2: treated with 1.5% salt + 1.2% sugar.

H. Fan et al. / Food Chemistry 159 (2014) 20–28

Hardness (g)

Treatment

H. Fan et al. / Food Chemistry 159 (2014) 20–28

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Fig. 3. Changes in sensory scores, EC, TAC, and TVB-N of black carp fillets treated with low salt and sugar during storage at 4 °C for 16 days (Control: untreated; T1: treated with 1.5% salt; T2: treated with 1.5% salt + 1.2% sugar).

minced black carp muscle. The initial EC values of T1 and T2 treated samples were 2835 lS cm1 and 2657 lS cm1, respectively, both of which were significantly (p < 0.05) higher than those of control samples (1037 lS cm1) because of the high ionic strength of sodium chloride solution. The EC value of control samples increased with time particularly from the 8th day to the 12th day of storage; this increase indicated that drastic spoilage occurred after the 8th day in the control samples. An evident increase in EC values of T1 (530 lS cm1) and T2 (683 lS cm1) samples during the first 2 days was observed. This dramatic increase may be due to the higher initial osmotic pressure, which led to leaky membrane structures and then an increase in extracellular volume. Afterwards the EC values of these two samples gradually increased with time, although a slight fluctuation in the value was observed from the 6th to the 16th day. The EC increase rate of control samples was 34.0 lS cm1 day1 from the 2nd day to the end of storage, which was significantly higher than that of T1 (23.2 lS cm1 day1) and T2 (20.14 lS cm1 day1), from which it can be concluded that treatment of both T1 and T2 could delay the increase of EC in fish samples. Gibson (1985) reported that

the increase of EC values in fish muscle is mainly due to the metabolism of the constituents of the culture medium by microorganisms, in particular an increase in the number of ion carriers, because of the catabolism of high molecular weight materials, e.g., proteins, to low molecular weight compounds, e.g., ammonia and amines. The EC values of T1 and T2 were significantly higher than those of control (p < 0.05), and no significant differences (p > 0.05) were detected between T1 and T2 throughout the storage period. Changes in total aerobic counts (TAC) of black carp fillets during 16-day storage at 4 °C are presented in Fig. 3(c). The initial quality of fish used in this study was high, as indicated by a low initial number of bacteria (3.81 log10 CFU/g) before fish were subjected to different treatments. A similar initial microbial load (3.86 log10 CFU/g) was reported by Ojagh et al. (2010) for rainbow trout stored at 4 °C. TAC of control samples continuously increased with storage time. TAC of control samples increased faster than that of T1 and T2 samples during the first 8 days (p < 0.05), whereas no significant differences (p > 0.05) were observed between T1 and T2. This slow speed of increase for T1 and T2 may

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be attributed to the inhibitory effect of salt on spoilage bacteria. TAC of T2 samples was significantly (p < 0.05) higher than that of T1 samples after 8 days of storage. Noticing the previous discussion of SA for T1 and T2 samples, this variance may be attributed to the fact that sugar itself is a nutrient and was likely utilized by some specific bacteria, e.g., acid-forming bacteria, which then promoted their reproduction. According to the microbiological tolerable limit of 7 log10 CFU/g proposed for freshwater and marine fish by ICMSF. (1986), control, T1, and T2 samples maintained relatively good quality up to 4, 6, and 6 days, respectively. It can be concluded from other discussions on fish quality that TAC may not be suitable for determining the shelf life of black carp fillets. Therefore, in order to evaluate the shelf life of black carp fillets comprehensively, other indicators (K value, SA and TVB-N) should also be considered. Fig. 3(d) shows the effects of different treatments on TVB-N production in black carp fillet samples stored at 4 °C. The initial TVB-N level in raw black carp fillets was 7.18 mg/100 g. From the 1st day to the 6th day of storage, there were no significant differences (p > 0.05) among all fish samples; however, TVB-N thereafter began to increase with storage time. Many researchers have reported that the increase of TVB-N with storage time is related to the activity of endogenous enzymes and spoilage bacteria (Song et al., 2011). Compared to the control, treatment T1 and T2 delayed the increase speed of TVB-N (p < 0.05) after being stored for 6 days; this delay may be attributed to the preservative effect of salt. The TVB-N content of T2 was significantly lower (p < 0.05) than that of T1 after 10 days of storage. This reduction was possibly owing to the reproduction of acid-forming bacteria, which can produce organic acid to neutralize volatile basic nitrogenous compounds. A comparison with the proposed highest acceptable level of TVBN (25 mg/100 g) by Ojagh et al. (2010) showed that fillets maintained good quality up to 8, 10, and 12 days for control, T1, and T2, respectively. It can be concluded that fillets treated by 1.5% salt and 1.5% salt + 1.2% sugar can reduce chemical changes in fish muscle and extend the shelf life of black carp fillets. Changes in the concentrations of biogenic amines (BAs) in black carp fillets are shown in Table 2. That the initial concentrations of eight BAs were very low in all samples indicates the high quality of raw fish materials used in this study. Putrescine, cadaverine, and tyramine were the main BAs found to form in black carp muscle. Zhang, Lin, and Nie (2013) likewise reported putrescine, cadaverine, and tyramine were the main BAs formed in silver carp sausages during the entire processing period. Treatments T1 and T2 significantly (p < 0.05) reduced the accumulation of putrescine and tyramine compared to the control after being stored for 4 and 8 days, respectively. Cadaverine in treated samples showed no significant difference (p > 0.05) compared to the control samples during the first 2 days of storage but was then observed to be significantly lower (p < 0.05) than that of the control except for the 10th and 12th day for T2 samples. Noticing cadaverine was not detected at the 4th day for treated samples, this variance may be because the values of cadaverine were very low and meanwhile had a certain fluctuation during the early stages of storage period. Similar phenomenon was also observed during the ripening of dry-fermented sausage (Ikonic´ et al., 2013). The initial concentration of tryptamine was 9.47 mg/kg; values of all samples decreased over time, especially for treated samples, whose values approached 0 after the 4th day. There seemed to be no significant differences (p > 0.05) between T1 and T2 in BAs accumulation, indicating that 1.2% sugar had no significant effect on the accumulation of BAs in black carp muscle in this study. The rise in concentrations of cadaverine and putrescine can be observed during the spoilage of various foods. Continuous increases in putrescine and cadaverine concentrations were observed in black carp muscle throughout the storage. Bover-Cid et al. (2001)

reported that the occurrence of putrescine and cadaverine in meat products was related to ornithine- and lysine-decarboxylase activity, respectively, of Enterobacteriaceae. Treatments T1 and T2 significantly reduced (p < 0.05) the putrescine and cadaverine formation, probably owing to the inhibitory effect of salt on spoilage bacteria such as Enterobacteriaceae; however, no significant difference (p > 0.05) was observed between T1 and T2 in this study. Zhang et al. (2011) reported that the addition of sucrose could significantly (p < 0.05) reduce the production of these two BAs in drycured grass carp (Ctenopharyngodo idellus) during processing, whereas Bover-Cid et al. (2001) found the presence of sugar had no such effect in slightly fermented sausages. The sources of putrescine and cadaverine in meat products are complex and affected by numerous variables, and the impact of sugar on putrescine and cadaverine formation in black carp muscle was unclear. Further studies are required to determine how sugar affects the formation of these two BAs. Much attention has been given to histamine and tyramine, which may cause adverse health effects. The accumulation of tyramine is normally associated with several species of LAB such as Enterococci (Zhang et al., 2013). Some reports have found that a low pH facilitates tyramine production by LAB (Pircher, Bauer, & Paulsen, 2007); however, in this study, no significant difference (p > 0.05) was observed between T1 (without sugar) and T2 (with sugar). This result may be because the pH of T2 was approximately 0.1 lower than that of T1; thus we can presume that sugar did not substantially promote the growth of some species of LAB in black carp muscle. The upper limit of tyramine in foods is 100– 800 mg/kg (Kim, Mah, & Hwang, 2009). Tyramine concentrations in black carp muscle, regardless of the influence of salt and sugar, were much lower than that value. The low level of histamine in black carp muscle throughout the storage period should be noted since histamine is the most important biogenic amine from a toxicological point of view. Histamine concentration varied from 0.41 to 0.87 mg/kg, which is drastically lower than its allowable limit in foods (100 mg/kg) (Kim et al., 2009). This phenomenon may be due to the lack of decarboxylation of amino acids to form histamine in freshwater species or the inhibition of specific strains of some Enterobacteriaceae species capable of decarboxylating histidine (Ikonic´ et al., 2013). Subtle changes were detected in phenylethylamine, spermidine, and spermine concentrations during storage of black carp fillets. Phenylethylamine content varied from 0.43 to 11.39 mg/kg in all samples, which is much lower than the proposed upper limit (30 mg/kg) (Kim et al., 2009). The lack of increase (even a slight reduction during storage) in spermidine and spermine contents may be attributed to two possible speculations: (i) these polyamines naturally exist in black carp muscle and are rarely or not at all derived from the microbial decarboxylation of amino acids, or (ii) deamination reactions as well as microbial consumption contribute to this reduction in polyamines content. Treatments T1 and T2 had no significant effects on phenylethylamine, spermidine, and spermine content during storage (p > 0.05). 3.5. Relationship between SA and EC, TAC, TVB-N, putrescine, and cadaverine of black carp fillets stored at 4 °C The relationships between SA and EC, TAC, TVB-N, putrescine, and cadaverine of black carp fillets stored at 4 °C are shown in Table 3. There were high correlations between SA and TAC, TVB-N, putrescine, and cadaverine with high Pearson’s correlations (p < 0.01) in all samples. EC was highly correlated with SA in control (p < 0.01) and T1 (p < 0.05) samples but not in T2 (0.656) samples. Zhu et al. (2013) reported that high correlations between SA, TAC, TVB-N, and Q value (an index of electrical characteristics) were detected in bighead carp stored at 0 °C and 3 °C. Krˇízˇek,

Table 2 Biogenic amines (BAs) concentration (mg/kg) in black carp fillets treated with low salt and sugar during storage at 4 °C. Treatment

Tryptamine

Control T1 T2 Control T1 T2 Control T1 T2 Control T1 T2 Control T1 T2 Control T1 T2 Control T1 T2 Control T1 T2

Phenylethylamine

Putrescine

Cadaverine

Histamine

Tyramine

Spermidine

Spermine

Storage time (days) 0

2

4

6

8

10

12

9.47 ± 2.67

9.63 ± 0.86a 8.78 ± 3.99a 8.48 ± 2.91a 8.75 ± 0.46a 4.05 ± 2.02b 4.14 ± 0.71b 1.04 ± 0.46a 1.37 ± 1.27a 1.00 ± 0.26a 0.75 ± 0.54a 0.49 ± 0.41a 0.34 ± 0.26a 0.79 ± 0.04a 0.79 ± 0.02a 0.77 ± 0.04a 48.09 ± 3.17a 44.84 ± 3.42a 43.41 ± 0.67a 2.13 ± 0.17a 2.46 ± 0.43a 2.51 ± 0.31a 2.90 ± 0.31a 2.73 ± 0.09a 2.86 ± 0.35a

8.36 ± 1.90a 6.32 ± 1.55a 5.76 ± 0.83a 9.07 ± 2.20a 7.99 ± 1.14a 8.52 ± 0.95a 1.25 ± 0.90a 0.57 ± 0.14a 1.01 ± 0.29a 1.28 ± 0.61a ND ND 0.82 ± 0.14a 0.75 ± 0.04a 0.66 ± 0.10a 51.91 ± 10.55a 42.87 ± 11.42a 40.75 ± 5.04a 2.03 ± 0.53a 2.30 ± 0.57a 1.78 ± 0.14a 3.47 ± 0.44a 3.30 ± 1.39a 2.58 ± 0.41a

4.94 ± 2.88a ND ND 8.20 ± 2.74a 10.71 ± 1.98a 7.97 ± 2.31a 7.57 ± 2.27a 0.53 ± 0.37b 1.37 ± 0.42b 6.75 ± 1.78a ND 2.32 ± 0.81b 0.54 ± 0.04a 0.49 ± 0.03ab 0.46 ± 0.02b 43.14 ± 5.40a 43.85 ± 7.84a 46.78 ± 5.68a 1.88 ± 0.74a 2.34 ± 0.41a 2.70 ± 0.22a 2.52 ± 0.42a 2.80 ± 0.54a 2.95 ± 0.44a

3.03 ± 1.74a ND ND 7.04 ± 1.31a 10.15 ± 8.35a 11.39 ± 2.36a 13.85 ± 5.98a 1.68 ± 1.21b 4.87 ± 3.09b 16.62 ± 5.13a 1.57 ± 0.44b 7.42 ± 4.34b 0.51 ± 0.05a 0.49 ± 0.03a 0.58 ± 0.08a 66.40 ± 18.78a 66.64 ± 14.79a 57.09 ± 7.27a 1.40 ± 0.66a 2.07 ± 1.24a 2.94 ± 0.24a 2.26 ± 0.23a 2.67 ± 1.16a 3.43 ± 0.42a

3.29 ± 2.86a 3.12 ± 2.24a ND 8.26 ± 3.19a 5.05 ± 0.11a 11.26 ± 4.25a 15.84 ± 2.31a 7.02 ± 2.75b 7.01 ± 2.09b 35.61 ± 15.37a 9.00 ± 1.46b 23.34 ± 6.40a 0.49 ± 0.09a 0.56 ± 0.06a 0.54 ± 0.02a 81.62 ± 5.56a 40.67 ± 11.60b 55.31 ± 4.55b 1.45 ± 0.33a 1.49 ± 0.48a 2.22 ± 0.78a 2.10 ± 0.16a 2.27 ± 0.65a 3.12 ± 1.09a

3.33 ± 2.96a ND ND 4.65 ± 3.59a 10.36 ± 3.67a 9.89 ± 2.56a 25.39 ± 6.99a 12.92 ± 5.84b 9.38 ± 3.47b 45.87 ± 16.08a 14.30 ± 5.99b 27.41 ± 7.71ab 0.47 ± 0.08a 0.53 ± 0.11ab 0.65 ± 0.03b 74.75 ± 8.63a 39.18 ± 7.74b 50.54 ± 3.37b 1.30 ± 0.22a 1.32 ± 0.45a 1.31 ± 0.24a 1.95 ± 0.30a 2.37 ± 0.70a 2.15 ± 0.32a

0.43 ± 0.29

0.60 ± 0.12

1.04 ± 0.33

0.87 ± 0.02

39.12 ± 6.33

2.37 ± 0.84

2.71 ± 0.34

14

16

ND ND

0.06 ± 0.06a ND

5.74 ± 3.61a 7.76 ± 4.15a

6.13 ± 1.12a 3.19 ± 2.04a

16.16 ± 7.73b 14.82 ± 2.44b

10.94 ± 0.75b 10.77 ± 1.69b

23.01 ± 10.69b 31.54 ± 9.23b

21.87 ± 7.84b 26.67 ± 6.62b

0.45 ± 0.04b 0.41 ± 0.00b

0.55 ± 0.15b 0.47 ± 0.11b

46.44 ± 5.41b 49.19 ± 2.33b

41.06 ± 4.67b 45.50 ± 10.77b

0.81 ± 0.48a 1.75 ± 0.26b

0.77 ± 0.21a 0.69 ± 0.09a

1.59 ± 0.59a 2.87 ± 0.37b

1.68 ± 0.14a 1.84 ± 0.46a

H. Fan et al. / Food Chemistry 159 (2014) 20–28

BA

Results are presented as mean ± standard deviation. Same superscript lowercase letters in a column indicate no significant differences (p > 0.05). Control: untreated; T1, treated with 1.5% salt; T2: treated with 1.5% salt + 1.2% sugar. ND, not detected.

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Table 3 Pearson’s correlations between SA and EC, TAC, TVB-N, putrescine and cadaverine of black carp fillets during storage at 4 °C. Parameters

EC TAC TVB-N Putrescine Cadaverine

Sensory assessment (SA) Control

T1

T2

0.921** 0.928** 0.945** 0.917** 0.887**

0.784* 0.950** 0.933** 0.813** 0.841**

0.656 0.891** 0.989** 0.934** 0.875**

Control: untreated; T1, treated with 1.5% salt; T2: treated with 1.5% salt + 1.2% sugar. ND, not detected. * p < 0.05; ** p < 0.01.

Pavlícˇek, and Vácha (2002) investigated the relationship between sensory quality and putrescine and cadaverine content in carp meat and then proposed a critical putrescine content for fish quality (20 mg kg1 for good, acceptable, and poor quality, respectively), based on the sensory scores of carp meat. 4. Conclusion The results of the present study revealed that lightly salted black carp fillets with 1.5% salt or 1.5% salt + 1.2% sugar could delay physical, chemical, and microbial changes and improved sensory attributes; these changes led to retention of high quality characteristics and extension of shelf life. Sugar treatment can significantly reduce pH value and decrease TVB-N accumulation (p < 0.05), though it promoted bacterial growth to some extent and decreased the sensory quality of fish at later stages of storage. It can be concluded that treatment with low salt and sugar is a safe, healthy, and convenient method of preservation for black carp fillets and that this method is suitable not only for household use but also for fish processors. Acknowledgement This study was supported by the earmarked fund for China Agriculture Research System (CARS-46). Appendix A. Supplementary material Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.foodchem.2014. 02.158. References Alasalvar, C., Taylor, K. D. A., Öksüz, A., Shahidi, F., & Alexis, M. (2002). Comparison of freshness quality of cultured and wild sea bass (Dicentrarchus labrax). Journal of Food Science, 67, 3220–3226. Bover-Cid, S., Izquierdo-Pulido, M., & Vidal-Carou, M. C. (2001). Changes in biogenic amine and polyamine contents in slightly fermented sausages manufactured with and without sugar. Meat Science, 57, 215–221. Calo-Mata, P., Arlindo, S., Boehme, K., de Miguel, T., Pascoal, A., & Barros-Velazquez, J. (2008). Current applications and future trends of lactic acid bacteria and their bacteriocins for the biopreservation of aquatic food products. Food and Bioprocess Technology, 1, 43–63. Erkan, N., & Üretener, G. (2010). The effect of high hydrostatic pressure on the microbiological, chemical and sensory quality of fresh gilthead sea bream (Sparus aurata). European Food Research and Technology, 230, 533–542. Gibson, D. M. (1985). Predicting the shelf life of packaged fish from conductance measurements. Journal of Applied Bacteriology, 58, 465–469.

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