Comparison of flavor changes of grass carp between brine injection and brining at 4°C and 20°C

ABSTRACT

Grass carp is the most important freshwater fish in China, and salting is the main processing method. This study investigated the effect of brine injection on flavor of grass carp muscle compared with brining at low temperature (4°C) and room temperature (20°C) respectively. Salt content, free amino acids, nucleotides, equivalent umami concentration (EUC) and texture were evaluated as flavor indicators during salting. The results showed that brine injection groups reached equilibrium in 3 h and 5 h at 20°C and 4°C, respectively. Brining groups reached equilibrium in 5 h and 7 h at 20°C and 4°C, respectively. The equilibrium salt content was about 1.76%, and there was no significant deterioration in terms of the other indicators mentioned above by two different methods. The results of this research indicate the feasibility and the great economic potential of brine injection, and also provide the theoretical basis to promote this method in processing of aquatic products.

RESUMEN

La carpa herbívora es el pez de agua dulce más importante de China. La salazón es el principal método utilizado para su procesamiento. El presente estudio investigó los cambios identificados en el sabor del músculo de la carpa herbívora cuando se la procesó con inyección de salmuera en comparación con su procesamiento en salmuera a baja temperatura (4°C) y a temperatura ambiente (20°C). Los indicadores de sabor evaluados durante la salazón fueron: contenido de sal, de aminoácidos libres y de nucleótidos, concentración equivalente de umami (EUC) y textura. Los resultados dan cuenta de que los grupos inyectados con salmuera alcanzaron el equilibrio en 3 horas y 5 horas a 20°C y 4°C, respectivamente. Los grupos procesados con salmuera alcanzaron el equilibrio en 5 horas y 7 horas a 20°C y 4°C, respectivamente. El contenido de sal de equilibrio fue de alrededor de 1.76%, y, de acuerdo con dos métodos diferentes, se constató que no hubo un deterioro significativo en cuanto a los otros indicadores mencionados anteriormente. Los resultados de esta investigación indican la viabilidad y el gran potencial económico que conlleva la inyección de salmuera, a la vez que proporcionan la base teórica para promover este método en la elaboración de productos acuáticos.

KEYWORDS:

• Brine injection
• brining
• salt content
• free amino acids
• nucleotides

PALABRAS CLAVE:

• inyección de salmuera
• salmuera
• contenido de sal
• aminoácidos libres
• nucleótidos

1. Introduction

Grass carp (Ctenopharyngodon idellus) with fast growth rate, easy cultivation, high feed efficiency ratio, high nutritional value, and relatively low price, has become one of the main freshwater fish species in the world. The yield of grass carp was about 6.06 million tons in 2016 (FAO, 2018), and it was ranked the first place among principal aquaculture species. With the increase of production and consumption, more and more researchers and freshwater fish processing plants have focused on the quality improvement and shelf life extension of grass carp. The effectiveness of many methods has been proved such as vacuum packaging (Pattarapon et al., 2018), freezing (Cheng et al., 2017), preservative application (T. Li et al., 2016), drying (Wu & Mao, 2008) and salting (Jonsdottir et al., 2011).

Salting is a traditional processing method of aquatic products in the world, which is widely used because of low cost, simplicity of process, long shelf life and special relish (Martínez-Alvarez & Gómez-Guillén, 2013). However, excessive intake of salt is increasingly recognized as a serious, worldwide public health concern to give rise to high blood pressure and stomach cancer (Graudal et al., 2017; Tsugane, 2005). Because of this risk, reducing salt content in processed food has received considerable attention. In recent years, lightly salted fish with a salt content approximately of 2% is popular in Southern Europe, especially in Spain, Italy, and Greece (Gudjonsdottir et al., 2010). Previous work indicated that decreasing brine concentration retarded water exudation and salt diffusion in muscle (Van Nguyen et al., 2010), which resulted in an extension of brining time, thus deteriorating the flavor quality. Questions have been raised about the methods of improving the speed of brining, and previous studies have reported several innovative technologies applied, such as ultrasonic (Siró et al., 2009), vacuum (Chiralt et al., 2001) and injection (Sheard & Tali, 2004). In a recent study, vacuum, ultrasonic, brining and injection, were compared, and the results showed that injection was superior to other brining methods (Zhao et al., 2017). Thus, it can be seen that brine injection has great potential to develop in the future.

In China, brine injection as a new kind of food processing technology is characterized by high efficiency, can shorten brining time, and improve yield and even salt distribution in foods. This method has already been widely used in the processing industry of pork (Desmond et al., 2002), beef (Boles & Shand, 2001), and chicken (Yusop et al., 2012). In Iceland and Norway, it is also applied to the processing of cod and other aquatic products. Brine injection did not have any advantage to improve the flavor (Almli & Hersleth, 2013). The advantages were reflected in the salting speed and great applicability for many kinds of brine such as phosphate and antioxidant. Until now, related studies have been focused on the changes in yield, microstructure, and protein aggregation as affected by brine injection (K. A. Thorarinsdottir et al., 2011a, 2011b, 2010), little attention has been paid to the impact of brine injection on flavor of fish meat.

The diffusion coefficients of salt and water during brining not only depend on the concentration of brine, but also the temperature (Corzo & Bracho, 2004). Salting at lower temperatures leads to high weight gain (Birkeland et al., 2005), but it prolongs the brining time and affects the proteolysis process (Arnau et al., 1997). For better quality and economic benefit, different temperatures are commonly used in the industry during salting. Nowadays, the current demand for salted fish is stimulated by enjoying special sensory experience, rather than just preservation (Mujaffar & Sankat, 2005). Therefore, the main purpose of this study is to investigate the differences in flavor of grass carp affected by brine injection and brining at room temperature (represented by 20°C) and low temperature (represented by 4°C).

2. Materials and methods

2.1. Sample preparation and salting

Twenty cultured grass carp (weight 2000 ± 200 g, length 55 ± 2.5 cm, width 8.5 ± 1 cm) were purchased from an aquatic products market in Shanghai, China, in mid-January in 2020, and transported to the laboratory alive within 30 min. These grass carp were stunned by blowing the head, chopped off the head, gutted, and washed. The dorsal muscle was obtained and cut into fillets with a weight of 25 ± 3 g (about 3 cm × 3 cm × 2 cm).

The fillets were selected randomly for each salting condition. For brining groups, the fillets were immersed in 5% NaCl brine (w/v) with a fish to solution ratio of 1:3 (w/v) for different time (0 h, 0.5 h, 1 h, 2 h, 3 h, 5 h, 7 h) at 4°C and 20°C. For injection groups, the fillets were injected with 4% of total brine (same as the brining group) and then subjected to the same salting procedure as the brining groups.

2.2. Determination of salt content

Sodium chloride (NaCl) content was determined by the Volhard method (AOAC., 2000). Minced meat was weighed out 2.00 g accurately into a conical flask, and distilled water (200 mL) was added before the flask was placed on an electric shaker for 45 min. The supernatant (20 mL) was pipetted into Erlenmeyer flasks, and the chloride ions precipitated by adding AgNO₃ (0.1 M, 5–10 mL). The AgNO₃ excess was back-titrated with 0.1 M NH₄SCN solution. A ferric indicator (FeNH₄(SO₄)₂)•12H₂O in diluted HNO₃ was added for the determination of the endpoint.

2.3. Determination of free amino acids

Free amino acids were detected according to the method described by Yu et al. (2018) with slight modification. Minced meat was weighed out 2 g accurately and homogenized with 15 mL of 15% trichloroacetic acid solution for 2 min, and then the homogenate was stood for 2 h. After centrifuging at 9500 × g for 15 min at 4°C, 5 mL supernatant was collected and pH was adjusted to 2.0 with 3 M NaOH solution. Then it was diluted to 10 mL with ultra-pure water, and filtered with 0.22 μm water phase filtration membrane. The entire process was operated at 0–4°C.

Free amino acids were analyzed by an automatic amino acid analyzer (L-8800, Hitachi, Japan). The identity and quantity of the amino acids were assessed by comparison the retention times and peak areas with the standard amino acids.

2.4. Determination of nucleotides

ATP-related compounds were extracted according to previous procedure (H. Wang et al., 2018) with slight modification. The sample (5.00 g) was homogenized with 10 mL of 10% cold perchloric acid using FM-200 homogenizer (Shanghai Fokker Equipment Co. Ltd., Shanghai, China) for 30 s and centrifuged at 9500 × g for 15 min at 4°C using H2050R high-speed freezing centrifuge (Changsha Xiangyi Co. Ltd., China). The supernatant was collected, and the precipitate was washed with 5 mL of 5% cold perchloric acid and centrifuged under the same conditions and the procedure was repeated twice. The pH of the combined supernatant was adjusted to 6.5 with KOH solutions (1 M and 10 M), and stood for 30 min. The supernatant was diluted to 50 mL with ultra-pure water and filtered with a 0.45 μm membrane. The entire process was operated at 0–4°C.

ATP-related compounds were analyzed by high-performance liquid chromatography (HPLC) (Waters Co., USA). The HPLC conditions were set as follows: Inertsil COSMOSIL 5C18-PAQ column (4.6 ID × 250 mm) (GL Sciences, Inc., Tokyo, Japan). Flow rate: 1 mL/min; injection volume: 10 μL; UV detector wavelength: 254 nm. Mobile phase A was methanol, while phase B was phosphate buffer solution (0.05 M, pH = 6.5).

2.5. Determination of equivalent umami concentration value (EUC)

The equivalent umami concentration value is an index which can quantify the synergistic effect between the umami taste 5′-nucleotides which are inosincacid (IMP) and adenylate (AMP), and umami taste-free amino acids which are glutamic acid (Glu) and aspartic acid (Asp), which equal to the concentration of L-glutamate (MSG). It can be expressed by an equation raised by Kawai et al. (2002) as follows:

$\mathrm{E}\mathrm{U}\mathrm{C}=\sum {\mathrm{a}}_{\mathrm{i}}{\mathrm{b}}_{\mathrm{i}}+1218\left(\sum {\mathrm{a}}_{\mathrm{i}}{\mathrm{b}}_{\mathrm{i}}\right)\left(\sum {\mathrm{a}}_{\mathrm{j}}{\mathrm{b}}_{\mathrm{j}}\right)$

Where EUC means the equivalent umami concentration (gMSG/100 g), a_i is the concentration of umami taste amino acids (Glu or Asp)(g/100 g); b_i is the relative coefficient of the amino acids (Glu is 1, Asp is 0.77); a_j is the concentration (g/100 g) of 5′-nucleotides (5′-IMP, 5′-AMP); b_j is the relative coefficient of the 5′-nucleotides (5′-IMP is 1, 5′-AMP is 0.18); 1218 is a synergistic constant based on much of sensory analysis.

2.6. Texture measurements

The samples were cut into pieces with a size of 2 cm × 2 cm × 1 cm. Textural properties were determined by a TA-XT Plus (Stable Micro System, U.K.) which was equipped with a P/6 cylindrical probe. The method described by Tang (Tang et al., 2015) was adopted with slight modification. The test was implemented by twice compression (50%) to simulate the bite by human teeth with an interval of 5 s at a constant speed of 1.0 mm/s carried by probe. Trigger value was set to 5 g, and 200 pps for data collection rate. The test was implemented at 15°C. The value of hardness, springiness, and chewiness were measured and eight samples were measured for each condition.

2.7. Sensory analysis

The samples were steamed for 8 min and cooled to about 40°C. As shown in Table 1, the prepared samples were assessed by five indicators (salty, odor, morphology, mouthfeel, and color). Ten trained panelists (four males and six females, average age was 25 years old) participated in the sensory evaluation in the sensory assessment laboratory of Shanghai Ocean University (Gao et al., 2013; Gu et al., 2018). The total score of 100 points was deemed best.

Table 1. Standards of sensory assessment applied for the salted grass carp.

Tabla 1. Estándares de evaluación sensorial aplicados a la carpa herbívora salada.

Quality parameters	Description points
	1–4	5–8	9–12	13–16	17–20
Salty	Unacceptable	Very salty	Slight salty	Moderate salty	Excellent
Odor	Unacceptable	Off-odor	Slightly off-odor	Desirable	Extremely desirable
Appearance	Dull	Slight dull	Slight bright	Bright	Very bright
Morphology	Very loose	Loose	Partial loose	Tight	Very tight
Texture	Very soft or stiff	Soft	Slight elastic	Elastic	Firm

2.8. Statistical analysis

The data of this study were analyzed by SPSS 24.0 (SPSS Inc., Chicago, IL, U.S.A.) with LSD and Duncan methods. The results were expressed as mean value ± standard deviations and significant difference among means were compared at the level of p < .05.

3. Results and discussion

3.1. Changes in NaCl content in fish meat

During the period of salting, the mass transfer of salt and water occurs basically, where salt permeates into fish muscle by free diffusion while water is lost under osmotic pressure (Chaijan, 2011; Oliveira et al., 2012). When the fish muscle is immersed in brine, the outermost cells of the tissue are in contact with the brine. There is a concentration gradient formed between the intracellular fluid of outermost cells and the brine, and the outermost cells shrink due to water loss. At the same time, there is another concentration gradient formed between the intracellular fluid of wizened outermost cells and their adjacent cells, the mass transfer occurs and the adjacent cells shrink (D. Wang et al., 1998). With the development of osmosis, mass transfer and cellular structure shrink occur from the surface to the center of fish muscle. This is the biggest difference between brine injection and brining. For brine injection, the brine is injected into the center of the muscle, so that the mass transfer occurs from the center to outside and the surface to inside in the meantime. This is an efficient method to diffuse salt evenly and quickly to get a pressure balance.

As depicted in Figure 1, salt content increased rapidly during the initial period of 0–2 h due to high osmotic pressure, and equilibrium was reached after 3 h slowly. For temperature, the time of reaching maximum was about 2 h shorter at 20°C than that at 4°C. The maximum salt content reached 1.76% and 1.75% in brining and injection groups at 20°C, respectively. And 1.74% and 1.78% in brining and injection at 4°C, respectively. There were no significant differences in the maximum salt content in all groups (p > .05). Several studies have reported temperature and brine concentration are the most important factors to salt diffusion. Temperature had much influence to distribution coefficients of salt and water in sardine and eel, and higher temperatures led to faster diffusion speed (Corzo & Bracho, 2004; Zhang & Xia, 2006). Another important finding was the method of brine injection made the equilibrium earlier than only brining for about 2 h. That means the effect of brine injection was equal to raise 16°C. In industry, injection is easier than controlling the temperature steady, and this may imply a huge business potential (K. A. Thorarinsdottir et al., 2010; Tressler, 1920).

Figure 1. The changes of salt content in grass carp muscle by different methods during salting at 4°C and 20°C.

Figura 1. Cambios detectados en el contenido de sal del músculo de la carpa herbívora al aplicar diferentes métodos durante la salazón a 4°C y 20°C.

Note: Brand In indicate the brine and injection group respectively, same as shown below. The data (mean ± SD) with different letters in the same salty condition are significantly different (p <0.05).Nota: Bre In indican el grupo de salmuera y de inyección respectivamente, mismo que se muestra a continuación. Los datos (media ± DE) con diferentes letras en la misma condición de salado son significativamente diferentes (p < 0.05).

3.2. Changes in free amino acids content in fish meat

Free amino acids as the most vital non-protein nitrogenous compounds have been widely considered as an indicator to evaluate the flavor of fish and other aquatic products (Hwang et al., 2000). Some amino acids can provide distinct flavor characteristics. For example, aspartic acid, glutamic acid, glycine (Gly), alanine (Ala) and proline (Pro) can be recognized as sweet and umami, and histidine (His) and lysine (Lys) are bitter taste amino acids (Shi et al., 2014). Glycine, which has the simplest structure of amino acids, is widely distributed in nature especially in the aquatic products. It is the main flavoring components of the aquatic products that produce strong sweetness while removing the bitterness and saltness, and it has a synergistic effect with other umami components (He & Yang, 2005). Although histidine is a bitter amino acid, it can provide the flavor of fish and meat (Deng et al., 2010). Some bitter amino acids have a synergistic effect with other sweet and umami amino acids to enhance the taste like phenylalanine (Phe) and tyrosine (Tyr), without reaching the threshold value (Lioe et al., 2005).

The changes of free amino acids in processed grass carp muscle are shown in Tables 2–3. The concentrations of glycine, alanine, histidine and proline were dominant, and they were very close to or even beyond the thresholds (130 mg/100 g, 60 mg/100 g, 20 mg/100 g, 300 mg/100 g, respectively) except proline. So, these amino acids were the main components influencing the flavor, while threonine (Thr), serine (Ser) and leucine (Leu) had a minor effect on taste due to the large gap with thresholds (260 mg/100 g, 150 mg/100 g and 190 mg/100 g, respectively). The total free amino acids (TFAA) declined rapidly at initial stage, and then slowed down, similar to the change rate of salt content. This obvious change could be due to the diffusion of soluble amino acids from fish muscle to the brine (Jin et al., 2014). The concentration of amino acids in the brine should increase, and this point has been confirmed (Y. Li et al., 2010). From those two tables, it is easy to find that the concentration of total free amino acids at 20°C was less than those at 4°C significantly (p< .05). This result is consistent with the previous study that the process of osmotic dehydration in pike eel muscle during salting (Zhang & Xia, 2006). The higher temperature could lead to faster and more mass transfer, which means the free amino acids in the muscle immigrated to the brine faster and more (Zhang et al., 2017; Zhang & Xia, 2006). For the sake of nutrition and flavor, low temperature is a better choice. The most interesting finding was that, during the last period of salting, the concentration of total free amino acids rose again non-significantly. This might be because the speed of the proteolysis was faster than the losing speed of amino acids. As is known to all, amino acids are produced by hydrolysis of protein, and enzymes play a key role in this way. During the salting process, the proteinase especially cathepsins B, D, H, and L can hydrolyze the protein into peptides, while the exopeptidases especially peptidases and aminopeptidases can hydrolyze the peptides into amino acids (Toldrá et al., 1997). The enzyme activity of cathepsins B, L, and H has been reported to be promoted in a low salinity environment (Wu et al., 2018, 2016). Several studies have revealed that free amino acids mainly from the hydrolysis of peptides and proteins by aminopeptidases, and the enzyme activity of aminopeptidases could retain more than 70% of its original activity in 15% NaCl solution (Giyatmi & Irianto, 2016; Toldrá et al., 2000; Vo et al., 1983). On the base of the above, at the end of salting, the osmotic pressure closed to balance and the mass transfer was close to finish, where the loss of free amino acid was too little to be detected and increase turned into the main role. The tables also showed that the content of total free amino acids in the injection groups were lower than of the brining groups. It could be ascribed to the yield-increasing effect of injection. Previous researches have reported that injection causes lower degree of protein aggregation and larger inter-cellular space so the water-holding capacity of products is improved (K. A. Thorarinsdottir et al., 2011a, 2011b). It means that the unit mass of muscle treated by injection was increased by water, and the total free amino acids could be less relatively. Taking brining time into consideration, less time was taken to reach the equilibrium in the injection group, so less free amino acids were lost due to diffusion. The content of total free amino acids was 438.02 mg/100 g after brining at 4°C for 5 h, while the value was 427.28 mg/100 g in injection group for 3 h, where the salt content was almost the same in both groups (1.60% and 1.53%, respectively). Similarly, the content of total free amino acids by brining till 3 h at 20°C was 365.04 mg/100 g and by injection till 2 h was 331.92 mg/100 g, both two groups with almost the same salt content (1.74% and 1.73%, respectively). Therefore, the products by the two salting methods did not have significant differences in terms of free amino acids content.

Table 2. The changes of free amino acids content in grass carp muscle by different methods during salting at 4°C.

Tabla 2. Cambios detectados en el contenido de aminoácidos libres del músculo de la carpa herbívora al aplicar diferentes métodos durante la salazón a 4°C y 20°C.

Methods	Amino acid species	Taste characteristics	Threshold (mg/100 g)	Contents (mg/100 g)
				0 h	0.5 h	1 h	2 h	3 h	5 h	7 h
Brining	Asp^★	fresh/sweet (+)	100	0.67 ± 0.12^a	1.01 ± 0.01^b	0.92 ± 0^b	0.77 ± 0.04^a	0.75 ± 0.08^a	0.77 ± 0.05^a	0.75 ± 0.01^a
	Thr^▲	bitter (-)	260	18.97 ± 2.94 ^c	15.61 ± 1.38^bc	14.75 ± 1.61^b	9.88 ± 0.18^a	8.51 ± 1.42^a	9.69 ± 0.68^a	16.76 ± 0.43^bc
	Ser^★	sweet (+)	150	6.7 ± 1.05^b	6.6 ± 1.11^b	6.41 ± 1.25^b	3.34 ± 0.12^a	2.61 ± 0.48^a	3.18 ± 0.24^a	7.23 ± 0.07^b
	Glu^★	fresh (+)	30	1.62 ± 0.34^a	1.71 ± 0.37^a	1.83 ± 0.39^ab	1.2 ± 0.1^a	1.36 ± 0.36^a	1.46 ± 0.05^a	2.43 ± 0.15^b
	Gly^★	sweet (+)	130	109.32 ± 15.25^b	106.32 ± 11.33^b	89.67 ± 12.05^ab	71.06 ± 3.96^a	69.6 ± 11.12^a	75.09 ± 6.98^a	104.97 ± 2.86^b
	Ala^★	sweet (+)	60	30.76 ± 4.32^bcd	35.83 ± 0.72^d	33.48 ± 0.07 ^cd	24.57 ± 0.43^ab	25.74 ± 3.99^ab	28.63 ± 3.02^bc	21.16 ± 0.18^a
	Cys		ND	1.77 ± 0.33^c	1.49 ± 0.13^bc	1.49 ± 0.23^bc	1.15 ± 0.02^ab	0.93 ± 0.15^a	1.09 ± 0.09^ab	1.72 ± 0.14 ^c
	Val^▲	sweet/bitter (-)	40	11 ± 1.66 ^cd	12.07 ± 0.32^d	9.41 ± 0.32^abc	7.86 ± 0.34^ab	7.31 ± 1.19^a	7.94 ± 0.65^ab	9.56 ± 0.09^bc
	Met	bitter/sweet (-)	30	2.41 ± 0.38^b	2.2 ± 0.39^b	2.11 ± 0.22^b	1.25 ± 0.12^a	1.11 ± 0.17^a	1.22 ± 0.07^a	2.46 ± 0.09^b
	Ile^▲	bitter (-)	90	7.68 ± 0.16^d	6.51 ± 0.12^c	5.85 ± 0.1^b	3.78 ± 0.21^a	3.73 ± 0.62^a	3.55 ± 0.08^a	5.62 ± 0.02^b
	Leu^▲	bitter (-)	190	12.36 ± 0.63^d	10.57 ± 0.47 ^c	9.65 ± 0.12^bc	6.77 ± 0.52^a	6.53 ± 1.13^a	6.33 ± 0.1^a	9.15 ± 0.22^b
	Tyr	bitter (-)	ND	10.08 ± 0.86^b	8.65 ± 0.26^ab	6.4 ± 1.06^a	4.31 ± 1.49^a	4.08 ± 2.82^a	3.16 ± 0.56^a	4.73 ± 0.38^a
	Phe^▲	bitter (-)	90	5.1 ± 3.79^a	4.19 ± 3.09^b	3.36 ± 2.62^a	1.23 ± 0.28^a	2.92 ± 2.83^a	0.95 ± 0.14^a	3.24 ± 0.25^a
	Lys^▲	sweet/bitter (-)	50	15.27 ± 0.64^d	14.52 ± 1.61^bc	11.4 ± 0.7^bc	9.03 ± 1.38^ab	7.76 ± 1.58^a	7.51 ± 0.47^a	12.6 ± 0.97 ^c
	His^▲	bitter (-)	20	298.94 ± 44.19^bc	305.97 ± 13.02^c	261.12 ± 6.53^abc	246.41 ± 5.75^ab	222.36 ± 31.44^a	248.65 ± 20.69^abc	219.95 ± 12.19^a
	Arg^▲	sweet/bitter (-)	50	4.02 ± 0.64^bc	3.78 ± 0.74^b	3.6 ± 0.91^b	1.78 ± 0.17^a	1.48 ± 0.25^a	1.73 ± 0.12^a	5.11 ± 0.02 ^c
	Pro^★	sweet/bitter (+)	300	32.76 ± 4.69^abc	22.2 ± 7.11^ab	27.16 ± 3.85^abc	33.51 ± 1.65^bc	35.32 ± 5.98 ^c	37.08 ± 3.62 ^c	21.3 ± 3.55^a
	BFAA			366.86 ± 43.65^a	370.24 ± 10.46^a	312.9 ± 8.27^b	282.42 ± 2.52^ab	257.29 ± 24.06^a	281.04 ± 20.54^ab	272.42 ± 13.99^ab
	FAA			181.84 ± 25.77^b	173.66 ± 19.18^ab	159.47 ± 14.19^ab	134.46 ± 6.22^a	135.38 ± 22.01^a	146.2 ± 13.95^ab	157.84 ± 6.15^ab
	TFAA			569.45 ± 72.68^b	561 ± 31.16^b	488.61 ± 20.61^ab	427.91 ± 8.89^a	402.11 ± 47.65^a	438.02 ± 35.27^a	448.74 ± 19.57^a
	FAA/TFAA			31.9 ± 0.45^a	30.91 ± 1.7^a	32.61 ± 1.53^ab	31.41 ± 0.8^a	33.58 ± 1.5^ab	33.36 ± 0.5^b	35.18 ± 0.16^b
Injection	Asp^★	fresh/sweet (+)	100	0.67 ± 0.12^ab	0.77 ± 0.17^ab	0.66 ± 0.02^ab	0.51 ± 0.17^a	0.64 ± 0.09^ab	0.67 ± 0.08^ab	0.84 ± 0.07^b
	Thr^▲	bitter (-)	260	18.97 ± 2.94 ^c	14.64 ± 2.44^bc	10.5 ± 1.79^ab	7.91 ± 1.84^a	10.09 ± 0.7^ab	12.65 ± 1.92^ab	14.51 ± 0.61^bc
	Ser^★	sweet (+)	150	6.7 ± 1.05^b	6.68 ± 1.19^b	4.72 ± 1.28^ab	3.52 ± 0.82^a	4.13 ± 0.2^a	5.15 ± 0.78^ab	6.37 ± 0.26^b
	Glu^★	fresh (+)	30	1.62 ± 0.34^ab	1.76 ± 0.17^ab	1.55 ± 0.37^ab	1.28 ± 0.3^a	1.12 ± 0.68^a	2.14 ± 0.32^ab	2.77 ± 0.17^b
	Gly^★	sweet (+)	130	109.32 ± 15.25^b	96.18 ± 5.76^ab	89.65 ± 11.57^ab	72.08 ± 16.63^a	87.9 ± 2.12^ab	77.93 ± 10.68^a	88.64 ± 4.38^ab
	Ala^★	sweet (+)	60	30.76 ± 4.32^bc	24.88 ± 5.63^bc	18.03 ± 1.97^ab	15.43 ± 1.11^a	18.67 ± 2.73^ab	18.95 ± 2.33^ab	18.03 ± 0.8^ab
	Cys		ND	1.77 ± 0.33 ^c	1.46 ± 0.31^bc	2.48 ± 0.36^d	0.74 ± 0.13^ab	1.03 ± 0.23^ab	1.24 ± 0.14^ac	1.59 ± 0.04^bc
	Val^▲	sweet/bitter (-)	40	11 ± 1.66^bc	8.83 ± 1.67^ab	12.39 ± 1.47 ^c	5.95 ± 1.15^a	7.27 ± 0.36^a	7.6 ± 1.02^a	8.09 ± 0.35^ab
	Met	bitter/sweet (-)	30	2.41 ± 0.38 ^c	1.95 ± 0.29^bc	3.65 ± 0.37^d	1.05 ± 0.36^a	1.23 ± 0.01^ab	1.84 ± 0.36^bc	2.18 ± 0.05 ^c
	Ile^▲	bitter (-)	90	7.68 ± 0.16^d	5.19 ± 0.12 ^c	4.83 ± 0.14^bc	4.41 ± 0.32^ab	3.73 ± 0.21^a	4.33 ± 0.6^ab	5.03 ± 0.06^bc
	Leu^▲	bitter (-)	190	12.36 ± 0.63^d	8.77 ± 0.23 ^c	8.25 ± 0.08^bc	7.64 ± 0.46^abc	6.58 ± 0.24^a	7.1 ± 0.99^ab	8.27 ± 0.08^bc
	Tyr	bitter (-)	ND	10.08 ± 0.86^d	4.7 ± 0.93^ab	7.59 ± 0.96 ^c	5.85 ± 1.71^bc	3.07 ± 0.26^a	3.28 ± 0.55^a	4.41 ± 0.16^ab
	Phe^▲	bitter (-)	90	5.1 ± 3.79^a	1.36 ± 0.17^a	1.68 ± 0.23^a	3.71 ± 3.46^a	0.84 ± 0.13^a	1.68 ± 0.77^a	4.31 ± 0.23^a
	Lys^▲	sweet/bitter (-)	50	15.27 ± 0.64 ^c	11.45 ± 0.76^b	9.74 ± 0.19^a	9.21 ± 0.5^a	8.53 ± 0.69^a	9.28 ± 1.17^a	11.44 ± 0.12^b
	His^▲	bitter (-)	20	298.94 ± 44.19^c	266.92 ± 31.95^bc	247.79 ± 27.5^abc	212.66 ± 9.6^ab	240.49 ± 20.45^abc	211.1 ± 21.4^ab	192.36 ± 8.76^a
	Arg^▲	sweet/bitter (-)	50	4.02 ± 0.64 ^cd	3.9 ± 0.88^bcd	3.03 ± 0.93^abc	1.83 ± 0.53^a	2.33 ± 0.02^ab	3.76 ± 0.66^bcd	4.79 ± 0.13^d
	Pro^★	sweet/bitter (+)	300	32.76 ± 4.69^d	24.62 ± 3.87^bc	17.34 ± 1.71^ab	27.36 ± 4.95 ^cd	29.65 ± 2.07 ^cd	18.02 ± 1.19^ab	14.83 ± 0.68^a
	BFAA			366.86 ± 43.65 ^c	313.08 ± 34.8^bc	298.95 ± 31.32^ab	252.3 ± 5.19^ab	274.06 ± 20.65^ab	249.98 ± 25.99^ab	240.88 ± 9.17^a
	FAA			181.84 ± 25.77^b	155.15 ± 5.64^ab	131.97 ± 16.89^a	120.18 ± 23.98^a	142.11 ± 1.81^ab	122.86 ± 15.39^a	131.49 ± 6.37^a
	TFAA			569.45 ± 72.68^b	484.34 ± 43.18^ab	443.9 ± 50.36^a	381.13 ± 31.14^a	427.28 ± 23.38^a	386.73 ± 43.43^a	388.48 ± 16.18^a
	FAA/TFAA			31.9 ± 0.45^a	32.11 ± 1.7^a	29.7 ± 0.44^a	31.38 ± 3.73^a	33.3 ± 1.4^a	31.75 ± 0.41^a	33.84 ± 0.23^a

All data were presented as means ± standard error. ^★ represents flavor amino acids; ^▲represents bitter amino acids; ND represents the threshold was not detected; TFAA means total free amino acids; BFAA means bitter amino acids; FAA means flavor amino acids;

Means of content with different letters within each row were significantly different (p < 0.05) at different time points. The same below.

Todos los datos se presentaron como media ± error estándar. ^★ representa los aminoácidos de sabor; ^▲representa los aminoácidos amargos; ND expresa que el umbral no fue detectado; TFAA significa aminoácidos libres totales; BFAA significa aminoácidos amargos; FAA significa aminoácidos de sabor.

Las medias de contenido indicadas con diferentes letras dentro de cada fila son significativamente diferentes (p < 0.05) en diferentes puntos de tiempo. Lo mismo abajo.

Table 3. The changes of free amino acids content in grass carp muscle by different methods during salting at 20°C.

Tabla 3. Cambios detectados en el contenido de aminoácidos libres del músculo de la carpa herbívora al aplicar diferentes métodos durante la salazón a 20°C.

Methods	Amino acid species	Taste characteristics	Threshold (mg/100 g)	Contents (mg/100 g)
				0 h	0.5 h	1 h	2 h	3 h	5 h	7 h
Brining	Asp^★	fresh/sweet (+)	100	0.67 ± 0.12 ^cd	0.72 ± 0.02^d	0.59 ± 0.06^bcd	0.54 ± 0.05^abc	0.49 ± 0.06^ab	0.42 ± 0.07^a	0.39 ± 0.03^a
	Thr^▲	bitter (-)	260	18.97 ± 2.94^b	9.84 ± 0.05^a	8.31 ± 0.15^a	8.77 ± 0.25^a	8.83 ± 0.33^a	8.48 ± 0.66^a	8.4 ± 0.44^a
	Ser^★	sweet (+)	150	6.7 ± 1.05 ^c	4.56 ± 0.03^b	3.64 ± 0.22^ab	3.79 ± 0.2^ab	3.65 ± 0.25^ab	2.84 ± 0.23^a	3.51 ± 0.16^ab
	Glu^★	fresh (+)	30	1.62 ± 0.34 ^c	1.21 ± 0.09^abc	1.17 ± 0.22^abc	1.42 ± 0.1^bc	1.32 ± 0.13^abc	0.88 ± 0.08^a	1.07 ± 0.14^ab
	Gly^★	sweet (+)	130	109.32 ± 15.25^d	95.53 ± 1.97 ^cd	77.12 ± 0.78^ab	79.25 ± 1.99^ab	82.92 ± 2.16^bc	66.05 ± 5.4^a	77.01 ± 1.19^ab
	Ala^★	sweet (+)	60	30.76 ± 4.32^b	20.93 ± 0.09^a	19.2 ± 0.02^a	20.3 ± 0^a	19.19 ± 0.54^a	17.72 ± 1.29^a	19.35 ± 0.52^a
	Cys		ND	1.77 ± 0.33^b	1.45 ± 0.02^ab	1.35 ± 0.01^a	1.45 ± 0.03^ab	1.44 ± 0^ab	1.23 ± 0.11^a	1.41 ± 0.03^a
	Val^▲	sweet/bitter (-)	40	11 ± 1.66^b	5.39 ± 0.1^a	4.47 ± 0.04^a	4.49 ± 0.16^a	4.74 ± 0.16^a	4.2 ± 0.33^a	4.36 ± 0.2^a
	Met	bitter/sweet (-)	30	2.41 ± 0.38^b	1.11 ± 0.1^a	1.09 ± 0.09^a	1.33 ± 0.13^a	1.33 ± 0.09^a	1.16 ± 0.1^a	1.34 ± 0.08^a
	Ile^▲	bitter (-)	90	7.68 ± 0.16 ^c	4 ± 0.09^ab	3.53 ± 0^a	3.89 ± 0.01^ab	4.07 ± 0.08^ab	3.67 ± 0.33^a	4.71 ± 0.89^b
	Leu^▲	bitter (-)	190	12.36 ± 0.63^d	6.9 ± 0.16^bc	6.1 ± 0.06^ab	6.37 ± 0.03^abc	6.92 ± 0.13^bc	5.93 ± 0.53^a	7.08 ± 0.4^c
	Tyr	bitter (-)	ND	10.08 ± 0.86 ^c	2.28 ± 0.19^ab	1.86 ± 0.05^a	2.04 ± 0.08^ab	2.61 ± 0.12^ab	2.11 ± 0.54^ab	2.91 ± 0.13^b
	Phe^▲	bitter (-)	90	5.1 ± 3.79^a	2.56 ± 0.15^a	2.19 ± 0.03^a	2.31 ± 0^a	2.7 ± 0.08^a	2.21 ± 0.33^a	2.78 ± 0^a
	Lys^▲	sweet/bitter (-)	50	15.27 ± 0.64^d	6.17 ± 0.02 ^c	4.87 ± 0.05^a	5.03 ± 0.45^ab	6.09 ± 0.09 ^c	5.7 ± 0.59^abc	5.94 ± 0.18^bc
	His^▲	bitter (-)	20	298.94 ± 44.19 ^c	223.04 ± 5.21^b	194.8 ± 2.02^ab	184.72 ± 2.8^ab	202.97 ± 2.56^ab	179.96 ± 11.75^ab	166.21 ± 6.7^a
	Arg^▲	sweet/bitter (-)	50	4.02 ± 0.64 ^c	2.21 ± 0.02^b	1.56 ± 0.05^a	1.76 ± 0.09^ab	2.04 ± 0.12^ab	1.66 ± 0.09^ab	1.76 ± 0.05^ab
	Pro^★	sweet/bitter (+)	300	32.76 ± 4.69^b	17.53 ± 0.39^a	15.24 ± 0^a	14.6 ± 0.01^a	13.75 ± 0.35^a	13.75 ± 0.44^a	13.1 ± 0.44^a
	BFAA			366.86 ± 43.65 ^c	253.67 ± 6.04^b	220.47 ± 2.3^ab	211.94 ± 3.74^ab	233.45 ± 2.27^ab	206.6 ± 14.59^a	197.1 ± 8.63^a
	FAA			181.84 ± 25.77 ^c	140.47 ± 2.36^b	116.96 ± 0.3^ab	119.9 ± 2.32^ab	121.32 ± 3.49^ab	101.66 ± 7.52^a	114.43 ± 2.48^ab
	TFAA			569.45 ± 72.68 ^c	405.43 ± 8.46^b	347.09 ± 2.45^ab	342.05 ± 6.34^ab	365.04 ± 1.55^ab	317.97 ± 22.87^a	321.35 ± 11.58^a
	FAA/TFAA			31.9 ± 0.45^a	34.65 ± 0.14 ^cd	33.7 ± 0.15^bc	35.05 ± 0.03^d	33.23 ± 0.81^b	31.97 ± 0.06^a	35.62 ± 0.51^d
Injection	Asp^★	fresh/sweet (+)	100	0.67 ± 0.12 ^c	0.79 ± 0.01^d	0.49 ± 0^b	0.38 ± 0.01^a	0.46 ± 0.01^ab	0.39 ± 0^ab	0.4 ± 0^ab
	Thr^▲	bitter (-)	260	18.97 ± 2.94^b	8.81 ± 0.4^a	8.93 ± 0.82^a	7.4 ± 0.04^a	7.71 ± 0.2^a	8.21 ± 0.08^a	8.17 ± 0.03^a
	Ser^★	sweet (+)	150	6.7 ± 1.05^b	3.89 ± 0.14^ab	3.47 ± 0.22^ab	2.81 ± 0.01^ab	3.19 ± 0.04^a	3.45 ± 0.29^a	3.1 ± 0.01^a
	Glu^★	fresh (+)	30	1.62 ± 0.34^d	1.15 ± 0.03^ac	1.33 ± 0.19 ^cd	0.97 ± 0^ac	1.48 ± 0.19^d	0.82 ± 0.01^a	0.91 ± 0.02^a
	Gly^★	sweet (+)	130	109.32 ± 15.25^d	97.54 ± 1.99 ^cd	87.02 ± 0.81^bc	77.68 ± 0.74^ab	73.12 ± 0.12^ab	80.01 ± 11.03^abc	63.12 ± 1.4^a
	Ala^★	sweet (+)	60	30.76 ± 4.32 ^c	20.6 ± 0.56^b	18.53 ± 1.14^ab	16.81 ± 0.01^ab	17.47 ± 0.45^ab	15.54 ± 1.18^a	16.96 ± 0.15^ab
	Cys		ND	1.77 ± 0.33^b	1.27 ± 0.01^a	1.17 ± 0.05^a	1.14 ± 0.02^a	1.23 ± 0.06^a	1.32 ± 0.04^a	1.29 ± 0.04^a
	Val^▲	sweet/bitter (-)	40	11 ± 1.66^b	5.15 ± 0.2^a	4.85 ± 0.17^a	4.22 ± 0.01^a	4.27 ± 0.15^a	4.34 ± 0.05^a	4.04 ± 0.05^a
	Met	bitter/sweet (-)	30	2.41 ± 0.38^b	1.26 ± 0.23^a	1.33 ± 0.01^a	1.02 ± 0.25^a	1.13 ± 0.02^a	1.25 ± 0.03^a	1.24 ± 0.04^a
	Ile^▲	bitter (-)	90	7.68 ± 0.16 ^c	4 ± 0.3^b	3.94 ± 0.08^b	3.48 ± 0.08^a	3.74 ± 0.07^ab	3.98 ± 0.05^b	3.84 ± 0.23^ab
	Leu^▲	bitter (-)	190	12.36 ± 0.63^c	6.82 ± 0.42^b	6.52 ± 0.32^ab	5.81 ± 0.1^a	6.23 ± 0.04^ab	6.55 ± 0.03^ab	6.35 ± 0.34^ab
	Tyr	bitter (-)	ND	10.08 ± 0.86^b	2.25 ± 0.28^a	2.06 ± 0.25^a	2.04 ± 0.17^a	2.19 ± 0.27^a	2.93 ± 0.07^a	2.49 ± 0.6^a
	Phe^▲	bitter (-)	90	5.1 ± 3.79^a	2.47 ± 0.28^a	2.36 ± 0.14^a	2.25 ± 0.14^a	2.3 ± 0.18^a	2.85 ± 0.09^a	2.54 ± 0.4^a
	Lys^▲	sweet/bitter (-)	50	15.27 ± 0.64 ^c	5.6 ± 0.1^ab	5.37 ± 0.1^ab	5.08 ± 0.03^a	5.28 ± 0.1^ab	5.88 ± 0.21^b	5.61 ± 0.36^ab
	His^▲	bitter (-)	20	298.94 ± 44.19^b	212.25 ± 4.52^a	217.42 ± 3.28^a	184.24 ± 3.59^a	167.81 ± 6.48^a	196.65 ± 27.59^a	173.73 ± 4.4^a
	Arg^▲	sweet/bitter (-)	50	4.02 ± 0.64^b	1.97 ± 0.04^a	1.78 ± 0.07^a	1.61 ± 0.07^a	1.55 ± 0.11^a	1.79 ± 0.13^a	1.62 ± 0.04^a
	Pro^★	sweet/bitter (+)	300	32.76 ± 4.69 ^c	19.01 ± 1.05^b	19.57 ± 0.04^b	14.98 ± 0.58^ab	15.21 ± 0.41^ab	15.41 ± 1.18^ab	12.61 ± 0.09^a
	BFAA			366.86 ± 43.65^b	241.76 ± 6.37^a	245.63 ± 2.3^a	209.76 ± 4.37^a	194.5 ± 6.31^a	226.23 ± 27.84^a	201.45 ± 6.45^a
	FAA			181.84 ± 25.77^d	142.98 ± 3.78 ^c	130.4 ± 0.71^bc	113.63 ± 1.31^ab	110.92 ± 0.74^ab	115.62 ± 13.68^abc	97.1 ± 1.65^a
	TFAA			569.45 ± 72.68 ^c	394.81 ± 10.54^b	386.13 ± 3.89^ab	331.92 ± 3.08^ab	314.37 ± 7.31^ab	351.39 ± 41.64^ab	308.02 ± 8.16^a
	FAA/TFAA			31.9 ± 0.45^a	36.22 ± 0.01^d	33.77 ± 0.16^bc	34.24 ± 0.71^c	35.29 ± 0.59^d	32.9 ± 0.01^b	31.53 ± 0.3^a

3.3. Changes in nucleotides content in fish meat

The ATP and its related compounds (adenosine (ADP), adenylate, inosincacid, inosine (HxR) and hypoxanthine (Hx)) are the most important indicators of freshness and flavor quality. IMP and AMP have strong relevance to the umami taste of fish and other aquatic products and their degradation products Hx and HxR represent putrefaction (Howgate, 2006). Hx and HxR have a bitter taste so a high content of them can destroy the whole flavor of fish (Hu et al., 2017). The IMP and AMP have an interesting property of flavor-enhancing, which means they can enhance the taste of other flavor components like flavor amino acids, peptides, organic acids and so on (Hu et al., 2017; Jones, 1969). Previous researches by Yamaguchi and Ninomiya (2000) have reported a hypothesis that IMP itself may not provide the umami taste, but it can commonly strengthen the umami taste of glutamate in the mouth. But the initial concentration of IMP after death is various among species, which leads to different shelf life and best taste period (Bremner et al., 1988).

As shown in Table 4, in view of the overall situation, ATP and AMP showed a decreasing trend, Hx and HxR presented a downtrend at first and then rose up, and IMP was increased firstly and then declined. It can be found that IMP was the dominating component. This could be attributed to the speed of ATP dephosphorylation and AMP deamination was faster significantly than the speed of IMP dephosphorylation comparatively (Spinellj, 1967). This phenomenon resulted in the accumulation of IMP and the decline of ATP and AMP initially. With the reactions proceeding, the ATP and AMP were almost completely degraded, and the IMP started to decrease. This process could be accelerated by high temperatures. The results showed that the decrease of ATP and AMP and the change (increase then decrease) of IMP were more intense at 20°C. The same phenomenon also was observed in Hx and HxR, where they increased due to the degradation of IMP, and both compounds slowly increased comparatively at 4°C. It was indicated that keeping a low temperature could retain the freshness of fish, and was beneficial to the flavor (Hong et al., 2017; Rodríguez et al., 2006). It was also found that the nucleotides content of injection groups showed a lower level than that in the brining groups from the present study. The unit weight of the muscle could contain more water because of injection, and the nucleotides content showed a lower level compared with the brining. Similarly, salting time should be considered, and it was easy to find out that the contents of IMP were 145.28 mg/100 g and 144.74 mg/100 g by injection and brining at the same salt content at 20°C, respectively (3 h for injection and 5 h for brining, salt content were 1.73% and 1.74% respectively). At 4°C, the contents of IMP were 161.14 mg/100 g and 175.99 mg/100 g by injection and brining respectively at the same salt content (1.79% for 5 h injection and 1.74% for 7 h brining). AMP was in a similar way. There was no significant difference in terms of flavor nucleotides between the two methods, but injection could save salting time.

Table 4. The changes of nucleotides content in grass carp muscle by different methods during salting at 4°C and 20°C.

Tabla 4. Cambios detectados en el contenido de nucleótidos del músculo de la carpa herbívora al aplicar diferentes métodos durante la salazón a 4°C y 20°C.

Temperature	Methods	Time (h)	Contents (mg/100 g)
			IMP	ATP	ADP	AMP	Hx	HXR	Total
20°C	Brining	0	203.68 ± 12.26^a	3.45 ± 0.8^a	9.16 ± 2.08^a	1.5 ± 0.29^a	1.44 ± 0.14^bc	11.84 ± 0.9^c	231.06 ± 14.3^a
		0.5	205.69 ± 17.48^a	2.6 ± 0.08^ab	9.94 ± 1.15^a	1.01 ± 0.06^b	1.37 ± 0.17 ^c	11.84 ± 1.18^c	232.46 ± 19.89^a
		1	184.47 ± 10.11^ab	1.77 ± 0.37^b	10.29 ± 0.38^a	0.67 ± 0.15^b	1.35 ± 0.11 ^c	4.05 ± 5.89^d	202.62 ± 9.69^b
		2	145.57 ± 6.87 ^c	1.66 ± 0.49^b	8.51 ± 0.15^a	0.66 ± 0.2^b	1.57 ± 0.12^bc	16.24 ± 1.2^bc	174.21 ± 8.9^b
		3	162.64 ± 8.71^bc	1.83 ± 0.65^b	8.98 ± 2.49^a	0.86 ± 0.34^b	2.09 ± 0.15^a	22.05 ± 1.11^a	198.43 ± 8.76^b
		5	144.47 ± 17.95 ^c	2.07 ± 0.57^b	7.94 ± 2.5^a	0.98 ± 0.14^b	1.71 ± 0.19^b	19.49 ± 1.64^ab	176.65 ± 21.42^b
		7	143.42 ± 16.86 ^c	2.06 ± 0.68^b	9.71 ± 1.16^a	0.84 ± 0.3^b	2.32 ± 0.27^a	21.92 ± 2.5^a	180.27 ± 21.2^b
	Injection	0	203.68 ± 12.26^a	3.45 ± 0.8^a	9.16 ± 2.08^a	1.5 ± 0.29^a	1.44 ± 0.14^bc	11.84 ± 0.9^b	231.06 ± 14.3^a
		0.5	151.7 ± 4.54^bc	1.49 ± 0.5^b	8.19 ± 1.4^a	0.6 ± 0.25^ab	1.42 ± 0.06^bc	12.85 ± 0.32^b	176.24 ± 5.75^b
		1	161.41 ± 8.21^b	2.36 ± 1.45^ab	6.27 ± 1.76^a	1.07 ± 0.61^b	1.07 ± 0.19 ^c	9.76 ± 1.42^b	181.93 ± 9.52^b
		2	144.54 ± 2.36^bc	1.45 ± 0.27^b	7.5 ± 1.97^a	0.57 ± 0.13^b	1.61 ± 0.44^b	17.35 ± 4.46^a	173.02 ± 8.05^b
		3	145.28 ± 18.49^bc	1.33 ± 0.23^b	9.33 ± 1.77^a	0.52 ± 0.14^b	2.22 ± 0.37^a	21.12 ± 2.79^a	179.8 ± 23.26^b
		5	139.25 ± 2.17 ^cd	2.2 ± 0.54^ab	9 ± 0.89^a	0.94 ± 0.26^b	1.89 ± 0.35^ab	20.27 ± 2.88^a	173.56 ± 5.74^b
		7	126.36 ± 2.71^d	1.31 ± 0.3^b	9.39 ± 1.13^a	0.59 ± 0.1^b	2.19 ± 0.09^a	20.5 ± 1.54^a	160.34 ± 3.84^b
4°C	Brining	0	203.68 ± 12.26^ab	3.45 ± 0.8^a	9.16 ± 2.08^ab	1.5 ± 0.29^a	1.44 ± 0.14 ^c	11.84 ± 0.9^bc	231.06 ± 14.3^a
		0.5	206.43 ± 10.28^ab	2.84 ± 0.79^ab	8.59 ± 0.51^ab	1.31 ± 0.21^ab	1.41 ± 0.06 ^c	9.77 ± 2.57 ^c	230.36 ± 7.19^a
		1	215.86 ± 8.63^a	2.2 ± 0.32^bc	6.97 ± 0.45^b	1.29 ± 0.23^ab	1.35 ± 0.04 ^c	12.83 ± 1.51^bc	240.51 ± 10^a
		2	204.85 ± 9.36^ab	1.9 ± 0.63^bc	7.1 ± 0.86^b	1.33 ± 0.27^ab	1.48 ± 0.1^c	13.86 ± 3.03^b	230.53 ± 6.73^a
		3	189.49 ± 16.18 ^c	1.75 ± 0.31 ^c	9.92 ± 1.5^a	0.98 ± 0.23^bc	1.77 ± 0.1^b	19.2 ± 1.65^a	223.11 ± 15.95^ab
		5	175.99 ± 14.33 ^cd	1.77 ± 0.24 ^c	7.12 ± 1.14^b	0.8 ± 0.06 ^c	1.71 ± 0.04^b	17.4 ± 0.61^a	204.79 ± 13.8^bc
		7	163.13 ± 18.7^d	2.02 ± 0.4^bc	9.17 ± 0.24^ab	0.71 ± 0.06 ^c	2.07 ± 0.08^a	19.4 ± 0.71^a	196.51 ± 18.54 ^c
	Injection	0	203.68 ± 12.26^a	3.45 ± 0.8^a	9.16 ± 2.08^a	1.5 ± 0.29^a	1.44 ± 0.14 ^c	11.84 ± 0.9^e	231.06 ± 14.3^a
		0.5	165.92 ± 2.2^bc	1.74 ± 0.11^b	6.98 ± 0.16^bc	0.83 ± 0.06 ^c	1.04 ± 0.03^d	8.16 ± 0.62 ^f	184.68 ± 2.58^bc
		1	178.51 ± 7.19^b	1.77 ± 0.23^b	6.8 ± 1.05^bc	1.07 ± 0.07^b	0.85 ± 0.04^e	7.49 ± 0.26 ^f	196.49 ± 8.42^b
		2	170.82 ± 8.11^bc	1.68 ± 0.07^b	5.57 ± 0.17 ^c	0.83 ± 0.07 ^c	1.18 ± 0.1^d	13.73 ± 0.46^d	193.8 ± 7.66^bc
		3	171.78 ± 7.83^bc	1.62 ± 0.08^b	7.42 ± 1.75^abc	0.73 ± 0.03 ^c	1.77 ± 0.1^b	16.39 ± 0.43 ^c	199.71 ± 8.56^b
		5	161.14 ± 1.18 ^cd	1.64 ± 0.11^b	9.21 ± 0.09^a	0.69 ± 0.05 ^c	1.68 ± 0.02^b	17.92 ± 0.35^b	192.29 ± 0.88^bc
		7	148.57 ± 6.88^d	1.49 ± 0.06^b	7.92 ± 0.76^ab	0.68 ± 0.13 ^c	2.04 ± 0.05^a	19.56 ± 0.55^a	180.26 ± 7.39 ^c

Different superscript letters in the same column represent a significant difference (p < 0.05) at different time points.

Las distintas letras en superíndice indicadas en la misma columna representan una diferencia significativa (p < 0.05) en diferentes puntos de tiempo.

3.4. Changes in equivalent umami concentration value (EUC) in fish meat

Like the statement above, free amino acids and nucleotides play a key role in the taste. But the flavor of grass carp muscle is not constituted of the single components accumulating, it also has the synergistic effect between umami free amino acids and umami 5′-nucleotides to enhance the overall umami taste (Liu et al., 2009). Umami taste can be well characterized by EUC value, and the value is the umami taste produced by the synergistic effect between free amino acids and 5′-nucleotides which are equivalent to the concentration of monosodium glutamate (g MSG/100 g). It has been revealed by Yamaguchi et al. (1971) first based on much sensory analysis.

As shown in Figure 2, all the TAV of EUC value was over 1 in all groups, indicating the synergistic effect had a great influence on the flavor of fish muscle. It showed a drop from 0.42 g/100 g to 0.19 g/100 g and 0.15 g/100 g (brining and injection respectively) at 20°C, while it showed a tendency to decrease first and then rose back at 4°C. That might be because of the higher speed of degradation of IMP at a higher temperature which could not be compensated by increase of free amino acids. It was also found that the EUC value was higher at 20°C, which could be attributed to the low speed of diffusion of Glu and the degradation of IMP. Therefore, the low temperature was helpful to retain the umami taste. The EUC value of the injection group was lower than the brining group, because of the mass issue stated above and similar comparison methods were adopted. It could be seen that the EUC value was 0.5 g/100 g and 0.43 g/100 g at 4°C with the same salt content (7 h for brining and 5 h for injection respectively), while the EUC value at 20°C was 0.16 g/100 g and 0.27 g/100 g (5 h for brining and 3 h for injection respectively). The injection did not show a lower quality compared with brining, and it has a better economic beneficial.

Figure 2. The changes of EUC and TAV in grass carp muscle by different methods during salting at 4°C and 20°C.

Figura 2. Cambios detectados en el contenido de aminoácidos libres del músculo de la carpa herbívora al aplicar diferentes métodos durante la salazón a 4°C y 20°C.

Note: Different superscript letters indicated in the same column represent a significant difference (p <0.05) at different time points.Nota: Las distintas letras en superíndice indicadas en la misma columna representan una diferencia significativa (p < 0.05) en diferentes puntos de tiempo.

3.5. Changes in texture properties of fish meat

Texture plays a key role in the sensory properties of fish muscle and it is related to the purchasing desire of consumers. The changes in fish muscle texture are mainly due to the extracellular matrix structure and myofibrillar protein properties (Martinez et al., 2011). In terms of fish muscle, hardness and springiness are the main indicators of freshness and quality, and this has been revealed in detail (Sato et al., 1991). As shown in Table 5, the initial hardness of grass carp muscle was 560.86 g, and it increased during salting and reach the maximum value (627.53 g, 649.19 g, 652.06 g, 666.45 g at 20°C and 4°C by brining and injection respectively) in the end. The reason could be attributed to the protein denaturation and the loss of water due to mass transfer (Benito et al., 2003; Rahman & Al-Farsi, 2005). Springiness was increased initially, which might be because the dissolution of protein strengthens the bonding between tissues. And then decreased rapidly, which might be caused by the disintegration of myofibril (Hultmann & Rustad, 2004). The chewiness showed an increase, which is consistent with the previous study. It has reported that chewiness showed a significant positive correlation to the hardness (Rahman & Al-Farsi, 2005). It could be found that various indexes of 20°C were lower than those at 4°C. This could be attributed to the increase in enzyme activity. The enzyme had an important influence on the microstructure and structure protein (Martinez et al., 2011). This result was consistent with the study by Bahuaud et al. (2010) where the relation between cathepsin activity and texture for Atlantic salmon was revealed. The results also indicated that brine injection could enhance the hardness and chewiness significantly (p< .05). That was because the lower degree of protein aggregation leads to the enhancement of water-holding capacity and protein crosslinking (Gallart-Jornet et al., 2007; Kin et al., 2010). The chewiness was increased with hardness. At the end of salting, different salting methods did not have differences in texture analysis. Hardness and springiness were 616.81 g and 0.85 for 5 h and 630.19 g and 0.86 for 3 h by brining and injection respectively (the same salt content) at 20°C, while 652.06 g and 0.85 for 7 h and 648.17 g and 0.85 for 5 h by brining and injection respectively (the same salt content) at 4°C. The low temperature could retain the texture properties better.

Table 5. The changes of texture properties in grass carp muscle by different methods during salting at 4°C and 20°C.

Tabla 5. Cambios detectados en las propiedades de textura del músculo de la carpa herbívora al aplicar diferentes métodos durante la salazón a 4°C y 20°C.

Temperature	Methods	Time (h)	Hardness (g)	Springiness	Chewiness (g)
20°C	Brining	0	560.86 ± 38.38^a	0.9 ± 0.02^d	192.68 ± 7.19^ab
		0.5	571.51 ± 7.52^a	0.91 ± 0.01^d	190.46 ± 5.45^a
		1	581.7 ± 7.69^ab	0.91 ± 0.01^d	192.55 ± 1.34^ab
		2	586.39 ± 12.87^abc	0.89 ± 0.01^cd	194.53 ± 3.3^abc
		3	606.44 ± 6.49^bcd	0.87 ± 0.02^bc	201.11 ± 11.34^abc
		5	616.81 ± 10.02 ^cd	0.85 ± 0.03^b	204.4 ± 7.97^bc
		7	627.53 ± 7.46^d	0.81 ± 0.02^a	206.04 ± 6.41^c
	Injection	0	560.86 ± 38.38^a	0.9 ± 0.02^b	192.68 ± 7.19^a
		0.5	591.93 ± 8.92^b	0.92 ± 0.01^b	196.91 ± 5.72^ab
		1	596.37 ± 13.19^b	0.92 ± 0.02^b	199.77 ± 5.5^ab
		2	609.36 ± 7.79^bc	0.92 ± 0.02^b	200.15 ± 5.94^ab
		3	630.19 ± 15.95 ^cd	0.86 ± 0.01^a	205.16 ± 3.37^bc
		5	647.31 ± 7.29^d	0.86 ± 0.02^a	213.05 ± 5.28^bc
		7	649.19 ± 7.47^d	0.84 ± 0.02^a	214.79 ± 6.14 ^c
4°C	Brining	0	560.86 ± 38.38^a	0.9 ± 0.02 ^cd	192.68 ± 7.19^a
		0.5	581.57 ± 12.08^ab	0.92 ± 0.01^de	196.36 ± 4.91^a
		1	590.74 ± 3.77^ab	0.93 ± 0.01^e	195.8 ± 3.98^a
		2	597.28 ± 8.48^b	0.92 ± 0.01 ^cde	198.81 ± 4.22^ab
		3	613.11 ± 16.8^bc	0.89 ± 0.02^bc	202.91 ± 7.29^abc
		5	633.47 ± 11.63 ^cd	0.87 ± 0.01^ab	209.79 ± 8.57^bc
		7	652.06 ± 7.5^d	0.85 ± 0.02^a	213.37 ± 6.97 ^c
	Injection	0	560.86 ± 38.38^a	0.9 ± 0.02 ^c	192.68 ± 7.19^a
		0.5	601.55 ± 11.63^b	0.93 ± 0.02 ^cd	202.9 ± 8.52^ab
		1	606.38 ± 14.61^b	0.94 ± 0.01^d	205.3 ± 4.04^b
		2	619.05 ± 9.23^bc	0.9 ± 0.02 ^c	205.23 ± 5.01^b
		3	641.84 ± 9.66 ^cd	0.87 ± 0.02^b	206 ± 4.47^b
		5	648.17 ± 6.27 ^cd	0.85 ± 0.01^a	213.72 ± 3.65^bc
		7	666.45 ± 10.35^d	0.83 ± 0.01^a	218.5 ± 6.56 ^c

Different superscript letters in the same column represent a significant difference (p < 0.05) at different time points.

Las distintas letras en superíndice indicadas en la misma columna representan una diferencia significativa (p < 0.05) en diferentes puntos de tiempo.

3.6. Changes in sensory properties of fish meat

As shown in Table 6, the maximum sensory score (92.5 ± 2.12) was obtained in brining group at 4°C. The sensory scores increased initially and then decreased in all groups with the extension of brining. Salting destroyed the fillets appearance, but removed the fish smell and improved the taste. The result was similar to Fan et al. (2014). The increasing trend was due to the better odor and moderate salt content, and the decrease was due to high salt content and inferior appearance. Salting method and temperature had little effect on the sensory scores except time of reaching maximum scores and injection groups could reach the best taste earlier.

Table 6. The changes of sensory scores in grass carp muscle by different methods during salting at 4°C and 20°C.

Tabla 6. Cambios de las puntuaciones sensoriales del músculo de la carpa herbívora al aplicar diferentes métodos durante la salazón a 4°C y 20°C.

	Scores
Groups	4°C Brining	4°C Injection	20°C Brining	20°C Injection
0 h	76.5 ± 2.12^a	76.5 ± 2.12^a	76.5 ± 2.12^a	76.5 ± 2.12^a
0.5 h	80.5 ± 2.12^ab	81.5 ± 2.12^b	83.5 ± 2.12^b	80.5 ± 2.12^ab
1 h	83.5 ± 2.12^bcd	85 ± 1.41^b	87.5 ± 0.71 ^cd	85.5 ± 2.12^bcd
2 h	87.5 ± 0.71 ^cde	89 ± 1.41 ^cd	89.5 ± 0.71 ^cd	90 ± 2.83^d
3 h	89 ± 1.41^de	91.5 ± 0.71^d	91 ± 1.41^d	88.5 ± 2.12^d
5 h	92.5 ± 2.12^e	85.5 ± 0.71^bc	86 ± 1.41^bc	87 ± 1.41 ^cd
7 h	83 ± 4.24^bc	83.5 ± 2.12^b	78.5 ± 2.12^a	82 ± 2.83^abc

Different superscript letters in the same column represent a significant difference (p < 0.05) at different time points.

Las distintas letras en superíndice indicadas en la misma columna representan una diferencia significativa (p < 0.05) en diferentes puntos de tiempo.

4. Conclusions

The main aim of this research was to investigate the influence of brine injection on the flavor of grass carp muscle compared with conventional brining at low temperature (4°C) and room temperature (20°C). Brine injection showed a better performance than brining in salting speed that reached 40.00% (from 5 h to 3 h) and 28.57% (from 7 h to 5 h) time savings at 20°C and 4°C respectively. The diffusion speed was faster at 20°C, and in this study, the diffusion speed of injection group at 4°C was almost equal to the speed of brining group at 20°C. The equilibrium salt content were 1.76% and 1.75% by brine injection and brining at 20°C, and 1.74% and 1.78% by brine injection and brining at 4°C, respectively. The equilibrium salt content was not significantly different in all groups. As for free amino acids, nucleotides, EUC value and texture, there were no significant differences at the end of salting and the lower temperature was beneficial to retain the flavor quality. Above all, brining could not balance the speed and flavor, but injection could get the same flavor quality in a shorter time at both temperatures. Therefore, this study suggests brine injection is feasible and it has great economic potential. Moreover, further research on volatile components, enzyme activity and protein properties in grass carp muscle may help us to clarify the mechanism of injection impact on flavor.

Disclosure statement

We declare that we do not have any commercial or associative interest that represents a conflict of interest in connection with the work submitted.

Additional information

Funding

This study was supported by the National Key Research and Development Program of China (Grant No. 2019YFD0902003 and 2018YFD0901003) and the National Natural Science Foundation of China (Grant No. 31471685).