Research on the quality changes of grass carp during brine salting

Abstract The research on the quality changes of grass carp during brine salting with 6%, 8%, and 10% salt additions was evaluated by chemical and physical indicators, as well as a sensory assessment and microbiological analysis. The NaCl content was proportional to salt addition and salting time. The increase of salt addition could lead to the increase of hardness and chewiness in which change trends were contrary to the pH within 24 hr. All K values were less than 10% during brine salting. The effect of 8% salt additions on free amino acids was relatively smaller. Higher levels of salt additions could inhibit bacterial growth. Combined with sensory assessment, equivalent umami concentration (EUC), and taste activity value (TAV) to analysis comprehensively, it was suggested that grass carp meat should be eaten at 4–8 hr of brine salting with 8% salt additions or processed for the next step, in which the grass carp meat had a better taste and quality.

some chronic diseases, like hypertension and cardiovascular diseases (Gallart-Jornet, Rustad, Barat, Fito, & Escriche, 2007). Light salting fish has been used to increase the flavor of food, to reduce drip loss, and to counteract the negative effects of freezing (Gudjónsdóttir et al., 2011). The uptake and distribution of salt largely depend on the method used, salt concentration, and salting time (Birkeland, Akse, Joensen, Tobiassen, & Skara, 2007).
There have been many studies on physicochemical properties and quality changes of aquatic products during salting. Chaijan (2011) investigated the effect of wet and dry salting on the physicochemical changes of tilapia muscle and found that dry salting resulted in a higher oxidation of tilapia muscle lipid throughout the salting period. Fan, Luo, and Yin (2014) believed that salting treatments could reduce chemical changes reflected in HxR, Hx, pH, and total volatile base nitrogen, decrease cooking loss, and increase overall sensory quality of black carp fillets stored at 4°C. Qin et al. (2017) studied the influence of lightly salting (dry salting) and sugaring on the quality and water distribution of grass carp during super-chilled storage, and the results showed that salting treatments predominately accelerated IMP formation, inhibited bacterial growth, retarded cooking loss, and improved the overall sensory quality. There also have been many reports focused on the grass carp, including volatile flavor substances of grass carp and studies of evaluating the quality changes of grass carp majorly involving the different season (Chen, Fang, Shi, & Chen, 2017) and storage process (Wang, Zhu, Zhang, Wang, & Shi, 2018) and so on. However, the report of the effect of brine salting on the quality changes in grass carp muscle was scarce. Therefore, the research mainly focused on the quality changes of grass carp meat during brine salting with different salt additions will be of certain significance.
The products of salted grass carp can be steamed or fried, which are traditional Chinese dishes and in which fish fillets are salted in brine first generally. Salt concentration and salting time have great influences on the quality change of fish and the flavor of the final product during the brine salting process. Therefore, the main objective of this work was to study the quality changes of grass carp meat during brine salting with different salt additions (6%, 8%, and 10% (w/w)). This was achieved by evaluating the meat moisture content, sodium chloride content, texture properties, color changes and pH, ATP-related compounds, K value, and levels of free amino acids, as well as a sensory assessment, microbiological analysis, and electronic tongue evaluation. In addition, the indicators of equivalent umami concentration and taste activity value were analyzed. It was desired to establish a firm theoretical foundation for the quality control of grass carp during salting. These will also promote the study of the mechanism of flavor changes of aquatic products in the process of salting.

| Sample preparation and Salting
Twenty fresh grass carp (weight of 2.3 ± 0.3 kg, length of 58.5 ± 3.2 cm, and width of 9.2 ± 0.7 cm) were from captured an aquaculture farm in Shanghai, China, in January 2019 and then transported alive to the laboratory within half an hour. They were stunned by a physical blow to the head, gutted, beheaded, scaled, washed, drained, and cut into skin-on fillets with an average weight of 3.5 ± 3 g (about 3.5 × 3.5 × 2.5 cm).
The fillets were collected randomly and salted by immersing them in saline with three different salt concentrations (6%, 8%, and 10%) at a fish-to-brine ratio of 1:1.5. Noniodized food grade salt (Shanghai, China) and distilled water were used to make the saline.
The salting process was performed at 4°C for 48 hr. Samples from each sampling point (0, 4, 8, 16, 24, 36, and 48 hr) were brined in one closed plastic container, and ten fillets were chosen randomly at each sampling point for analyses. The brined fillets were drained for 2-3 min on grid before determining.

| Determination of muscle pH
The determination of muscle pH was determined using the method described by Fan et al. (2014) and modified slightly. A sample (2.00 g) of salting fillets was dispersed in 18 ml of distilled water and centrifuged at 10,000 g for 10 min, and then filtered. The pH value of the filtrate was determined by a digital pH meter (Mettler Toledo FE20/ EL20) and performed in triplicate.

| Microbiological analysis
Samples were weighed 5.00 g aseptically and homogenized (Shanghai Huxi Industrial Co. Ltd., Shanghai, China) with 45 ml of sterilized 0.85% (w/v) physiological saline (Sinopharm Chemical Reagent, Shanghai, China) for 2 min. A series of 1:10 (v/v) dilutions were made by serially diluted with sterile saline for bacteriological analyses. One mL of serial dilution was spread onto plate count agar, and the indicator of total viable counts (TVC) was measured after incubating at 30°C for 72 hr. All counts were performed in duplicate and expressed as log10 CFU/g. The total viable count was calculated according to the Chinese standard method of GB-4789.2 (2016).

| Sensory assessment
The fillets of salted grass carp meat were immersed in pure water for 15 min and steamed for 8 min. When the steamed samples were cooled to about 40°C, sensory assessment was conducted from five aspects (salty, odor, morphology, mouthfeel, and color) by ten trained sensory assessment personnel (4 males and 6 females, average age 25 years old) using standard sensory assessment (SA) methods with slight modifications (Table 1; Gao, Zhu, Wu, Li, & Zhang, 2013;Gu et al., 2018

| Texture measurements
The transverse slices of salted fish fillet were taken and were cut into 2 × 2 × 1 cm pieces. Textural properties were measured by a TA-XT Plus (Stable Micro System) equipped with a P/6 cylindrical probe.
The test was performed by twice compression (50%) with an interval of 5 s at a constant speed of 1.0 mm/s carried by probe (Tang, Xie, Xu, Zhang, & Gao, 2015). The trigger value was 5 g, and the data collection rate was 200 PPS. The values of hardness, springiness, cohesiveness, chewiness, and resilience were calculated as described by Bourne (1978) and measured with eight replications.

| Color measurements
The colorimetric values of the fish sample were obtained by using an automatic chromaticity instrument (CR-400, Konica Minolta, Japan), which was calibrated with standard white board before sample determination. The sample was cut into slices of uniform size and 1 cm thickness, and each group of samples was measured 6 times. The results were expressed in L* (lightness), a* (redness/ greenness), and b* (yellowness/blueness) (Gerdes & Valdez, 1991).

| ATP-related compounds and K value
The extraction method of ATP-related compounds was improved slightly according to the previous methods Yokoyama, Sakaguchi, Kawai, & Kanamori, 1992). The sample (5.00 g) was homogenized with 10 ml of cold perchloric acid (10% (v/v)) by using FM-200 homogenizer (Shanghai Fokker Equipment Co. Ltd.) for 30 s and centrifuged by using H2050R high-speed freezing centrifuge (Changsha Xiangyi Co. Ltd.) at 10,000 g for 15 min at 4°C. The precipitate was washed with 5 ml cold perchloric acid (5% (v/v)) and centrifuged under the same conditions, which was repeated twice. The pH of combined supernatant was adjusted to 6.5 with potassium hydroxide solutions (1 and 10 M) and stand for 30 min. The supernatant was diluted to 50 ml with ultrapure water and filtered with a 0.45-μm membrane. All above operations were carried out below 4°C.
ATP-related compounds were analyzed by high-performance liquid chromatography (HPLC) (Waters Co.) equipped with COSMOSIL 5C18-PAQ column (4.6 ID × 250 mm) (GL Sciences, Inc., Tokyo, Japan) and SPD-10A (V) detector. The 0.05 M phosphate buffer (pH 6.5) and methanol were used for mobile phase to elute with a flow rate of 1 ml/min for 20 min.

| Free amino acids
Free amino acids (FAA) were extracted according to the procedure described by Yu et al. (2018) and slightly modified. The sample (2.00 g) was homogenized with 15 ml trichloroacetic acid solution (15% (v/v)) for 2 min and stand for 2 hr, and then centrifuged at 10,000 g for 15 min below 4°C. The supernatant (5 ml) was adjusted TA B L E 1 Standards of sensory assessment applied for the salted grass carp pH to 2.0 with sodium hydroxide solution (3 M), diluted to 10 ml with ultrapure water, and filtered with a 0.22-μm membrane. All above operations were carried out below 4°C.
Free amino acids were determined and analyzed by automatic amino acid analyzer (L-8800, Hitachi, Japan). The mobile phase was a mixed buffer solution (pH 3.2, 3.3, 4.0, and 4.9) of sodium citrate and citric acid and a ninhydrin buffer (4% (w/v)). Flow rates were 0.4 ml/ min and 0.35 ml/min, respectively.

| Taste active value and equivalent umami concentration
Taste active value (TAV) is the ratio of the concentration of flavor substance in sample to the corresponding threshold, which is widely used in various food flavor studies (Scharbert & Hofmann, 2005).
The taste active substance has a significant contribution to the overall taste of the sample when TAV is greater than 1, and its value is proportional to the contribution. On the contrary, the taste active substance has little contribution to the overall taste of the sample when TAV is less than 1 (Shi, Fang, Wu, Pan, & Hou, 2017).
Equivalent umami concentration (EUC) means that the umami intensity produced by the synergistic effect of umami amino acids (Glu and Asp) and flavor nucleosides (IMP, AMP, etc.) equivalent to the concentration of a single monosodium glutamate (MSG) . The value of EUC was calculated following the formula (Yamaguchi, Yoshikawa, Ikeda, & Ninomiya, 1971): In the formula, EUC is equivalent umami concentration (gMSG/100 g), ai is the concentration of umami amino acid (Asp or Glu) (g/100 g), bi is the umami coefficient of umami amino acid relative to MSG (Glu: 1, Asp: 0.077), aj is the concentration of flavor nucleotide (5'-IMP, 5'-AMP) (g/100 g), bj is the umami coefficient of flavor nucleotide relative to IMP (5'-IMP: 1, 5'-AMP: 0.18), and 1,218 is the synergy constant.

| Electronic tongue
The principal component analysis (PCA) of electronic tongue (Alpha M.O.S., ASTREE) was generally used to analyze the taste of sample. The determination was according to the method described by Zhang, Gu, Ding, Wang, and Jiang (2015) and slightly modified. The sample (2.00 g) was homogenized with 25 ml deionized water for 2 min and stand for 30 min, and then centrifuged at 10,000 g for 10 min at 4°C. The supernatant was diluted to 100 ml with deionized water. The diluted solution (5 ml) was diluted to 80 ml with deionized water and placed it into a special sampling cup of electronic tongue for testing. In order to ensure the stability of the sensors, the data of each sample were collected once every second for 120 s. The sensors were rinsed with deionized water for 10 s between measurements to reach steady readings in water. Seven parallel analyses were performed on the each sample to keep the reliability of the results, and the data of the last three were used as the original data of PCA.

| Statistical analysis
The F I G U R E 1 Changes in moisture and NaCl content (A) and muscle pH (B) of salting grass carp meat during brine salting with salt additions of 6%, 8%, and 10%. Data (mean ± SD) with different letters in the same salt addition are significantly different (p < .05)

| Changes in moisture content and NaCl content and muscle pH
Salting is a process of mass transfer between water and salt.
Salt permeates into grass carp muscle through osmotic mechanism, while water diffuses from muscle through osmotic pressure (Horner, 1997). The content of salt addition is the main factor affecting the diffusion of salt and water. The higher the salt concentration is, the higher the external osmotic pressure and the higher the salt absorption rate are (Fu, 2010;Horner, 1997). From the results (Figure 1a), it can be found that the NaCl content in fish muscle increased with the extension of salting time and with the increase of salt addition in the process of salting. In addition, the NaCl content during brine salting with the salt additions of 6%, 8%, and 10% reached the maximum at 48 hr, which was 5.30%, 7.44%, and 8.31%, respectively. Chaijan (2011) immersed tilapia fillets in 25% brine for 180 min and found that the salt content of salted fish muscle gradually increased and reached the maximum value at 180 min (> 5%). Kosak and Toledo (1981)  The pH changes of grass carp muscle during the salting period of 48 hr were shown in Figure 1b. The initial pH value of the fish sample was 6.72, which was close to the value of fresh ray fish (6.82) reported by Ocaño-Higuera et al. (2011), but lower than the initial value of 6.87 when the black fish was immersed in light salt of 1.5% (Fan et al., 2014). Differences in initial pH values may be due to species, season, diet, activity levels, and other factors (Fan et al., 2014).
In the process of salting with 6%, 8%, and 10% salt additions, the pH values changed in a similar trend. The pH values all decreased at different rates at first and reached the minimum values at 24 hr, which were 6.55, 6.41, and 6.43, respectively. After significant increases at 36 hr, there were downward trends. The initial decrease in pH may be due to the decomposition of glycogen, ATP, and phosphocreatine in fish muscle, while the subsequent increase may be due to endogenous or microbial enzymes (Ruiz-capillas & Moral, 2001).
It can be seen that the pH values were relatively low during brine salting with salt additions of 8% and 10%, which may be due to the growth of lactic acid bacteria. These bacteria may reduce the muscle pH by inhibiting the growth of other bacteria and buffering the basic metabolites produced (Calo-mata et al., 2008).

| Microbial and sensory assessment analysis
The changes in TVC of grass carp meat during the salting period of 48 hr were presented by Figure 2a. The initial TVC of grass carp was 3.70 log10 CFU/g. The black carp had an initial TVC of 3.81 log10 CFU/g when salted with 1.5% salt addition (Fan et al., 2014). Chytiri, Chouliara, Savvaidis, and Kontominas (2004) showed that the number of bacteria in freshwater fish varied between 2 and 6 log10 CFU/g with the changes of water environment and temperature. The low initial TVC in this study indicated that the quality of grass carp was better. It can be seen from Figure 2a that the TVC of grass carp meat increased with salting time and reached the maximum values at 48 hr during the brine salting with 6%, 8%, and 10% F I G U R E 2 Changes in TVC (A) and sensory score (B) of salting grass carp meat during brine salting with salt additions of 6%, 8%, and 10%. Data (mean ± SD) with different letters in the same salt addition are significantly different (p < .05) salt additions, which were 5.49 log10 CFU/g, 5.15 log10 CFU/g, and 4.79 log10 CFU/g, respectively. Ojagh, Rezaei, Razavi, and Hosseini (2010) showed that the maximum acceptable TVC value of freshwater fish was 7 log10 CFU/g, indicating that the grass carp maintained a relatively good quality during the whole salting period. In addition, it also can be found that the higher the salt addition, the slower the growth rate of TVC, indicating that salt can inhibit the growth of bacteria to a certain extent. Qin et al. (2017) also reached this conclusion when studying lightly salted grass carp meat.
It was generally believed that the four major elements of food quality are nutrition, appearance, texture, and flavor. In addition to nutritional quality, other characteristics were perceived by the senses and had an important impact on the acceptability of products (Liu, 2011). process, was lower than those of 6% and 8%. This indicated that the salted grass carp meat with salt additions of 6% and 8% were more acceptable due to their better taste than those of 10%. In addition, it can be seen from Figure 2b that the sensory scores in 6% and 8% salt additions decreased slowly at 4-16 hr. Combined with the microbial analysis, it may be because the higher osmotic pressure inhibited the enzyme activity and microbial growth in the muscle of grass carp. At 48 hr of salting, the sensory scores were all lower than 60, which were unacceptable to the sensory evaluators. In terms of sensory score, the qualities of grass carp meat salted with 6% and 8% salt additions were better than 10% and within 16 hr comparatively good. In order to comprehensively evaluate the quality of salted grass carp, other indicators (K value, EUC, TAV, etc.) should be considered.

| Texture properties and color analysis
The texture changes of fish meat are mainly related to the destruction of extracellular matrix structure (collagen) and the resulting changes of intracellular myofibrillar protein (Martinez et al., 2011). the hardness gradually decreased, which was contrary to the trends of pH in this study. Hultmann and Rustad (2004) reported that the texture properties of fish, such as hardness and springiness, were affected by some factors including myofibrils decomposition and the rate of pH decline. For fish products, texture, especially hardness, was an important quality factor affecting the acceptability of fish. In addition, during the same salting time, the greater the salt addition was, the greater the hardness was. The increase in hardness may be related to and calcium and magnesium ions in salt and the low pH of muscles (Lauritzsen, Akse, Gundersen, & Olsen, 2004). The changes of chewiness of meat were consistent with the hardness. They also reached the peak values at 24 hr of salting with 6%, 8%, and 10% salt additions, which were 248.95, 250.83, and 266.50 g, respectively. In the process of salting, proteins of fish will be denatured due to the action of salt, resulting in the reduction of their own gel properties and the increase of the toughness of tissue structure, which is manifested as the decrease of fish springiness (Hwang et al., 2012).

| ATP-related compounds and K value analysis
ATP-related compounds are important flavor substances in aquatic products, and K value is often used to monitor fish freshness. Adenosine triphosphate (ATP) is mainly found in fresh fish, but ATP will be degraded by endogenous enzymes into adenosine diphosphate (ADP), adenosine phosphate (AMP), and inosine monophosphate (IMP) after fish die. With the extension of death time, IMP degrades into inosine (HxR) and hypoxanthine (Hx) (Howgate, 2005).
The changes in the contents of ATP and its related compounds of grass carp meat during brine salting with 6%, 8%, and 10% salt additions for 48 hr were shown in Table 3. ATP-related compounds are important flavor substances in fish, among which IMP is one of the important factors for the umami taste of fish, while Hx is closely related to the putrefaction of fish (Howgate, 2005). As can be seen from  (Tersaki, Kajikawa, & Fujita, 1965). Therefore, low-salt brine salting can delay the putrefying of grass carp meat to some extent.
K value is widely used to evaluate the freshness of fish. The smaller the K value is, the better the freshness is. Generally, K value less than 20% is considered as the first order freshness, indicating that the fish is very fresh (Aubourg, Quitral, & Larrain, 2007). As can be seen from As is known to all, IMP is an important umami nucleotide in fish, which has a synergistic effect with AMP to enhance umami. IMP is also an ideal flavor enhancer with a threshold of 25 mg/100 g, while AMP can inhibit bitter taste and make food produce an ideal sweet and salty taste with a threshold of 50 mg/100 g (Takashi, Maya, & Hideyuki, 2007). TAV is the ratio between the content of flavor substances in the sample and its threshold, and TAV greater than 1 indicates that the substance contributes significantly to the overall taste of the sample. As can be seen from Table 3, the TAVs of IMP in all samples were far greater than those of AMP, and the TAVs of IMP reached minimum values of 6.30, 6.71, and 7.82, respectively at 48 hr, all of which were greater than 1, indicating that IMP contributed significantly to the overall taste of grass carp meat during brine salting. Tables 4-6 showed the changes in free amino acid contents of grass carp meat during brine salting with 6%, 8%, and 10% salt additions. It can be seen that the contents of Gly, Ala, His, and Pro were relatively high during brine salting, while threonine (Thr), serine (Ser), and leucine (Leu) had little effect on the taste of grass carp meat in the whole salting process due to their contents lower than their thresholds (260 mg/100 g, 150 mg/100 g, and 190 mg/100 g, respectively), which was consistent with the study on changes in free amino acid contents of grass carp in the process of postmortem . As can be seen from

| EUC and TAV analysis
EUC is used to reflect the synergistic effect of umami amino acids and flavor nucleotides, indicating the intensity of umami. The main umami amino acids in grass carp are Asp and Glu, which can synergize with IMP and AMP to improve the overall umami of grass carp meat . Figure 3 showed the changes of EUC and

| Electronic tongue analysis
The electronic tongue uses a sensor array technology that allows both qualitative and quantitative taste recognition (Beullens et al., 2006). Figure , 1999). There is no overlapping area in PCA, indicating that the electronic tongue can clearly identify the differences between different samples (Beullens et al., 2006). As can be seen from indicating that the taste of grass carp meat was significantly different in different salting times and salting processes. Further analysis showed that there was a significant difference in the taste profile distribution of grass carp meat with different salt additions, which further indicated that the tastes of grass carp meat salted with 6%, 8%, and 10% salt additions were different.

| CON CLUS IONS
The purpose of this study was to research the quality changes of grass carp meat during brine salting with different salt additions.
NaCl contents were proportional to salting time and salt addition, while brine salting had little effect on moisture content of grass carp meat. TVC of grass carp meat increased with the extension of salting time, and the higher salt addition could inhibit the growth of bacteria to some extent. The change trends of hardness of grass carp meat were opposite to pH within 24 hr of salting, and higher salt addition had adverse effects on the hardness and chewiness. Short salting period of low-salt additions was more beneficial to enhance lightness. In the three salting processes, the grass carp meat was very fresh (K values < 10%) and K values were significantly reduced to the minimum values at 4 hr. Among the three salting processes, the contents of TUSAA, TBAA, and TFAA decreased the most slowly with 8% salt additions. PCA showed that the tastes of grass carp meat were significantly different in all salting samples. Sensory assessment showed that the qualities of grass carp meat salted with 6% and 8% salt additions were better than 10%, and the quality of grass carp meat salted within 16 hr was better. Combined EUC and TAV analysis, grass carp meat salted with 8% salt additions for 4-8 hr had a better taste and should not exceed 16 hr. Therefore, this study suggested that grass carp meat should be brine salting with 8% salt additions and eaten at 4-8 hr. At this point, it can also be processed for the next step, such as steaming or frying, in which the grass carp meat had a better taste and quality. These results would provide useful information for quality control of grass carp during salting and theoretical reference for improving the nutritional value of grass carp. In addition, further exploration of volatile components and endogenous protease of grass carp during brine salting will contribute to the improvement of flavor change mechanism and the optimization of salting process of grass carp.

ACK N OWLED G M ENT
This study was supported by the National Key R&D Program of China (Grant No. 2018YFD0901003) and the National Natural Science Foundation of China (Grant No. 31471685).

CO N FLI C T O F I NTE R E S T
We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.