Effects of Ascorbic Acid and β-1,3-Glucan on Survival, Physiological Response and Flesh Quality of Cultured Tiger Grouper (Epinephelus fuscoguttatus) during Simulated Transport in Water

Transport in water is the most common method for achieving high survival rates when transporting cultured fish in China; yet, transport success relies on proper water quality and conditions. This research was designed to explore the effects of ascorbic acid and β-1,3-glucan on survival, physiological responses, and flesh quality of farmed tiger grouper (Epinephelus fuscoguttatus) during simulated transport. The transport water temperature for live tiger grouper was 15 °C, which had the highest survival rate, the lowest stress response, and metabolic rate, and this will reduce the susceptibility to diseases. It is stated that β-1,3-glucan influences the changes of cortisol content, heat shock protein 70, IL-1β, and IgM transcription levels during simulated transport. Rather than using ascorbic acid alone (the A-group), β-1,3-glucan (3.2 mg/L) in the presence of ascorbic acid (25 mg/L) can effectively reduce the increase of transport-induced serum cortisol content, heat shock protein 70, and IL-1β, but stimulated IgM. 25 mg/L ascorbic acid and 3.2 mg/L β-1,3-glucan had no obvious effect on the nutritional indexes and flavor of live tiger grouper; however, these can effectively reduce the stress response, improve the innate immune activity, and ensure a higher survival rate.


Introduction
Aquaculture is a rapidly growing industry in the world, providing one of the most sustainable forms of edible protein and nutrient production [1]. According to a report by FAO in 2014, the global aquaculture production has doubled in the past decade and it now accounts for about 50% of fishery products. China produced 41.1 million tons of farmed food fish in 2012, contributing to approximately 61.7% of total world production [2]. High demand for fish is due to an increased consumer awareness of healthy food [3]. These increases have occurred in some commercially important species, such as grouper, seabass, rainbow trout, tilapia, large yellow croaker, and catfish. Live fish species are sold at a higher price than frozen ones. Therefore, research regarding the handling and transport of live fish to improve the viability of commercial fish is essential. Table 1. Experimental design for exposure to anti-stress agents of live tiger grouper during simulated transport in water.

Serum Cortisol Assessment
Serum cortisol was measured by cortisol ELISA kits (Jiancheng Biological Engineering Institute, Nanjing, China), while following the manufacturer's instructions.

Real-Time PCR
Relative expression levels of HSP70, IgM, and IL-1β were determined by RT-PCR, as described by Lee et al. [39]. The total RNA from liver tissues was extracted with RNA rapid extraction kit (TaKaRa Biological Engineering Co., LTD., Dalian, China), quantified, and spectrophotometrically assessed for purity. RNA was then treated with DNase I (TaKaRa Biological Engineering Co., LTD., Dalian, China) to remove gDNA contamination, and complementary DNA (cDNA) was synthesized with M-MuLV reverse transcriptase. RT-PCR analyzed the expression levels of the selected immune-related genes. This was done in a 10 µm total volume while using SYBR Green I chimeric fluorescence, and with 500 nmol primers. PCR cycling conditions for all genes were, as follows: 94 • C for 10 min., 45 cycles at 95 • C for 30 s, 60 • C for 30 s and 72 • C for 30 s, followed by 10 min. at 72 • C. Relative expression levels of the target genes transcript (HSP70, IgM, and IL-1β), with GAPDH as an internal control, Biology 2020, 9, 37 4 of 18 were calculated using a CFX manager software version 2.0 (Bio-Rad). Table 2 shows the primers used. The threshold cycle (Ct) values were obtained from each sample after finishing the program. Ash, fat, moisture content, and total protein were measured according to Ayanda et al. [40].

Nucleotides
The nucleotide extracts were prepared based on the method of Fang et al. [42]. ATP-related compounds, including inosine monophosphate (IMP), inosine (HxR), and hypoxanthine (Hx), were analyzed while using HPLC (Waters 2695, Milford, MA, USA), equipped with a VP-CDS C18 column (150 × 46 mm). 0.05 M phosphate buffer solution (pH 6.7) was used as the mobile phase. The flow rate was 1 mL/min., and the injection volume was 10 µL. The peak was detected at 254 nm.
The taste activity value (TAV) was calculated as the following equation: in which C corresponds to the absolute concentration of taste substances, mg/100 g, and T reflects the taste threshold, mg/100 g, (IMP: 25 mg/100 g, AMP: 50 mg/100 g).
2.8. Free Amino Acids (FAAs) Assessment 5 g mashed tiger grouper muscle tissue sample and 15 mL of 15% cold trichloroacetic acid were mixed and homogenized at 10,000 rpm, for 5 min. After standing at 4 • C for 2 h, the homogenate was centrifuged at 5980× g for 15 min, at 4 • C. Next, 5 mL supernatant was immediately neutralized to pH 2.00 and then diluted to 10 mL with ultrapure water [43]. The mixture was then filtered through a 0.22 µm, and an amino acid analyzer determined the contents of FAAs (Hitachi L-8800, Tokyo, Japan).

Statistical Analysis
All of the assumptions were met prior to data analysis. Data were expressed as the mean ± SD and the one-way analysis of variance (ANOVA) procedure followed by Duncan's multiple range tests was adopted to determine the significant difference (p < 0.05) between treatments.

Effect of Temperature on Stress Responses of Tiger Grouper during Simulated Transport
HSP is a protein that will respond to external stressful conditions [44]. It protects cells from extreme physiological, pathological, and environmental conditions, and plays a role in protein misfolding correction, preserving immature polypeptides from aggregation under stress [45,46]. Cortisol is the main glucocorticoid hormone in teleosts that are involved in the regulation of metabolic adjustments. Under stress conditions, the increase of plasma cortisol promotes protein, glucose, and lipids mobilization in the skeletal muscle, which provide energy to overcome the stress [47]. The transcriptional level of HSP70 in the liver of all tiger grouper samples, at different transport temperatures, increased to the maximum value at 10 h, and then decreased, as shown in Figure 1a. Besides, higher transport temperatures seem to correlate with a higher level of HSP70 during simulated transport. After 17 h transport, the HSP70 values gradually recovered, but did not return to the initial values, except at 15 • C. Cortisol showed a similar trend as HSP70, with significantly higher values at 27 • C than at other temperatures. At the end of transport, the cortisol concentration at 15, 18, and 21 • C recovered to the initial levels. HSP70 and cortisol increased with the transport temperature increase, which could illustrate that the tiger grouper had transport stress responses during simulated transport. A higher transport temperature could lead to unrecoverable stress response, resulting in death. Additionally, stress response was not significant, and fish could maintain the body balance through self-regulation [48]. Cortisol showed a similar trend as HSP70, with significantly higher values at 27 °C than at other temperatures. At the end of transport, the cortisol concentration at 15, 18, and 21 °C recovered to the initial levels. HSP70 and cortisol increased with the transport temperature increase, which could illustrate that the tiger grouper had transport stress responses during simulated transport. A higher transport temperature could lead to unrecoverable stress response, resulting in death. Additionally, stress response was not significant, and fish could maintain the body balance through self-regulation [48].

Effect of Temperature on Antioxidant Enzyme of Tiger Grouper during Simulated Transport
Fish exposition to anoxia and hypoxia may result in oxidative changes, because oxygen consumption determines the levels of ROS generated, and also the antioxidant status [49]. Some clues could be given by an increase in activities of antioxidant enzymes under anoxic Figure 1. Effects of temperature on HSP70 (a) and serum cortisol (b) of tiger grouper during simulated transport. Among different temperature transport groups, different small and capital letters indicate the results of Duncan's test at different transport time. The same letters mean no significant difference (p > 0.05), while different letters mean significant difference (p < 0.05). Different letters without * = p-value < 0.05, different letters with ** = p-value < 0.01.

Effect of Temperature on Antioxidant Enzyme of Tiger Grouper during Simulated Transport
Fish exposition to anoxia and hypoxia may result in oxidative changes, because oxygen consumption determines the levels of ROS generated, and also the antioxidant status [49]. Some clues could be given by an increase in activities of antioxidant enzymes under anoxic conditions [50]. Oxidative reactions are essential in normal metabolism of aerobic organisms, but ROS are produced during the oxidative metabolism, generating free radicals [51]. In a situation of oxidative stress, fish might show a typical reaction for ROS, involving lipoperoxidation (LPO), which can be quantified by an increase in TBARS levels. On the other hand, the deleterious effect of ROS can be balanced by the production of antioxidant defenses [52], such as CAT.
GR is an important indicator for evaluating the degree of oxidative stress [53]. Figure 2 showed that the GR levels increased first, and then decreased following transport time, reaching a peak level at 10 h, which was similar to cortisol and HSP70. It indicated that all tiger groupers were under different degrees of oxidative stress during early simulated transport. GR activity of tiger grouper transported at 27 • C was significantly higher than those at low temperatures, and the initial levels could not be recovered at the end of transport. In the 27 • C transport group, all of the tiger grouper suffered severe oxidation reaction, which caused irreversible damage and further affected the survival. However, GR activity recovered to the initial levels that were transported at 18 and 21 • C. Yan et al. [54] showed that fugu also had an oxidative stress reaction, due to the stressor of temperature. It led to HSP70 and GR activity increase. Therefore, a low temperature is suitable for tiger grouper transport, because of the decreased stress response and increased survival.

Effect of Temperature on Metabolic and Immune Enzyme Activity of Tiger Grouper during Simulated Transport
Changes in the transport temperature not only induced a stress response in tiger grouper, but also lead to immune suppression. The main evaluation indexes of humoral immunity in fish include LZM [55,56]. AKP and ACP play important roles in phosphate hydrolysis in metabolic process and are key compounds in lysosomal digestion of invading organisms in the immune system [57]. AKP values significantly increased and reached a peak level at 10 h, and then decreased, as shown in Figure 3 (Figure 3a). The final AKP values did not recover to the initial values at 24 °C, which meant that high temperature simulated transport could affect fish metabolism. However, the final AKP values could recover to the initial values at 15, 18, and 21 °C. ACP activities had similar trends to AKP; however, it reached a peak at 3 h ( Figure  3b). The results suggested that the low temperature induced immune suppression response at the early stage of transport. As the first barrier of immunity, fish skin contains a large number of innate immune factors, as LZM in skin mucus, which mediates the protection against exogenous pathogen infection [58]. When the body is attacked by pathogens, LZM is secreted in the blood and mucus to eliminate these by activating blood cells and complement, and Effect of temperature on glutathione reductase (GR) of tiger grouper during simulated transport. Among different temperature transport groups, different small and capital letters indicate the results of Duncan's test at different transport time. The same letters mean no significant difference (p > 0.05), while different letters mean significant difference (p < 0.05). Different letters without * = p-value < 0.05, different letters with ** = p-value < 0.01.

Effect of Temperature on Metabolic and Immune Enzyme Activity of Tiger Grouper during Simulated Transport
Changes in the transport temperature not only induced a stress response in tiger grouper, but also lead to immune suppression. The main evaluation indexes of humoral immunity in fish include LZM [55,56]. AKP and ACP play important roles in phosphate hydrolysis in metabolic process and are key compounds in lysosomal digestion of invading organisms in the immune system [57]. AKP values significantly increased and reached a peak level at 10 h, and then decreased, as shown in Figure 3a. The final AKP values did not recover to the initial values at 24 • C, which meant that high temperature simulated transport could affect fish metabolism. However, the final AKP values could recover to the initial values at 15, 18, and 21 • C. ACP activities had similar trends to AKP; however, it reached a peak at Biology 2020, 9, 37 7 of 18 3 h (Figure 3b). The results suggested that the low temperature induced immune suppression response at the early stage of transport. As the first barrier of immunity, fish skin contains a large number of innate immune factors, as LZM in skin mucus, which mediates the protection against exogenous pathogen infection [58]. When the body is attacked by pathogens, LZM is secreted in the blood and mucus to eliminate these by activating blood cells and complement, and phagocytes in the liver and pancreas [59]. LZM values in the skin mucus of all tiger grouper firstly increased, and then decreased during simulated transport, as shown in Figure 3c. The highest value of LZM activity reached at 3 h, and then LZM activity plummeted to lower levels than the initial ones, indicating that the response of the innate immune system to temperature was at least partially suppressed. LZM activity of tiger grouper transported at 15 and 18 • C for 24 h could recover to the initial values, which indicate that fish damage can be decreased to the minimum at these temperatures.

Effect of Temperature on the Relative Expression of Immune Indexes of Tiger Grouper during Simulated Transport
IgM and IL-1β are two important immune factors in fish immunity. IL-1β is one of the most important pro-inflammatory cytokines, which has a variety of immune response functions in viral infection, including the activation of innate immunity and regulation of adaptive immune response [60]. When affected by temperature stress, it can produce an acute-phase protein to activate innate immune regulation function [61]. IgM is one of the most important anti-pathogen antibodies, and the main immunoglobulin mediating humoral adaptive immunity of fish [62]. The expression levels of IgM and IL-1β significantly increased and came to a peak level at 3h and 10h, and then decreased, as shown in Figure 4. It was found that the expression of IgM in rainbow trout and Nile tilapia increased in a high-temperature environment, as compared to a low-temperature environment [63]. IgM expression in serum of tiger grouper transported at 27 °C was significantly higher than at other temperatures. The expression level at the end of transport was lower than the initial levels, which indicated that temperature stress made tiger grouper reach the threshold of innate immune ability, and unable to maintain the normal immune level through self-regulation. However, the tiger grouper transported at 15 °C could activate the innate immune system to maintain the immune balance and ensure the survival rate. This might be due to the dormancy induced in Figure 3. Effect of temperature on alkaline phosphatase (AKP) (a), acid phosphatase (ACP) (b), and lysozyme (LZM) (c) of tiger grouper during simulated transport. Among different temperature transport groups, different small and capital letters indicate the results of Duncan's test at different transport time. The same letters mean no significant difference (p > 0.05), while different letters mean significant difference (p < 0.05). Different letters without * = p-value < 0.05, different letters with ** = p-value < 0.01.

Effect of Temperature on the Relative Expression of Immune Indexes of Tiger Grouper during Simulated Transport
IgM and IL-1β are two important immune factors in fish immunity. IL-1β is one of the most important pro-inflammatory cytokines, which has a variety of immune response functions in viral infection, including the activation of innate immunity and regulation of adaptive immune response [60]. When affected by temperature stress, it can produce an acute-phase protein to activate innate immune regulation function [61]. IgM is one of the most important anti-pathogen antibodies, and the main immunoglobulin mediating humoral adaptive immunity of fish [62]. The expression levels of IgM and IL-1β significantly increased and came to a peak level at 3h and 10h, and then decreased, as shown in Figure 4. It was found that the expression of IgM in rainbow trout and Nile tilapia increased in a high-temperature environment, as compared to a low-temperature environment [63]. IgM expression Biology 2020, 9, 37 8 of 18 in serum of tiger grouper transported at 27 • C was significantly higher than at other temperatures. The expression level at the end of transport was lower than the initial levels, which indicated that temperature stress made tiger grouper reach the threshold of innate immune ability, and unable to maintain the normal immune level through self-regulation. However, the tiger grouper transported at 15 • C could activate the innate immune system to maintain the immune balance and ensure the survival rate. This might be due to the dormancy induced in fish by this low-temperature.

Effect of Ascorbic Acid and β-1,3-Glucan Addition on Stress Responses of Tiger Grouper during Simulated Transport
HSP70 and serum cortisol are indicators of transport stress response. The expression of HSP70 and the content of serum cortisol increased, and the treated samples were lower than that of CK, as recorded in Figure 5. The content of HSP70 and cortisol in A-G2 could recover to the initial level, which confirms the conclusion of Henrique et al. [64] that ascorbic acid addition can adjust the level of HSP70 and cortisol and, thus, regulate stress responses during fish transport. Therefore, ascorbic acid and β-1,3-glucan can be used to decrease transport stress responses for tiger grouper.

Effect of Ascorbic Acid and β-1,3-Glucan Addition on Stress Responses of Tiger Grouper during Simulated Transport
HSP70 and serum cortisol are indicators of transport stress response. The expression of HSP70 and the content of serum cortisol increased, and the treated samples were lower than that of CK, as recorded in Figure 5. The content of HSP70 and cortisol in A-G2 could recover to the initial level, which confirms the conclusion of Henrique et al. [64] that ascorbic acid addition can adjust the level of HSP70 and cortisol and, thus, regulate stress responses during fish transport. Therefore, ascorbic acid and β-1,3-glucan can be used to decrease transport stress responses for tiger grouper.

Effect of Ascorbic Acid and β-1,3-Glucan Addition on Stress Responses of Tiger Grouper during Simulated Transport
HSP70 and serum cortisol are indicators of transport stress response. The expression of HSP70 and the content of serum cortisol increased, and the treated samples were lower than that of CK, as recorded in Figure 5. The content of HSP70 and cortisol in A-G2 could recover to the initial level, which confirms the conclusion of Henrique et al. [64] that ascorbic acid addition can adjust the level of HSP70 and cortisol and, thus, regulate stress responses during fish transport. Therefore, ascorbic acid and β-1,3-glucan can be used to decrease transport stress responses for tiger grouper.

Effect of Ascorbic Acid and β-1,3-Glucan Addition on Relative Expression of Non-Specific Immune Indexes of Tiger Grouper during Simulated Transport
Simulated transport usually induces stress responses that could lead to increase susceptibility to diseases. Immunostimulants can reduce the outbreak of diseases by facilitating the function of phagocytic cells, improving resistance to bacterial challenges [65]. The expression levels of IL-1β significantly increased, and then decreased, as shown in Figure 6a. IL-1β expression of CK was significantly higher than that of other simulated transport groups. From Figure 6b, the expression of IgM had a similar trend as that of IL-1β, and the expression of IgM in the A-G2 group was significantly higher than that of the other groups, which indicated that ascorbic acid and β-1,3-glucan addition could stimulate non-specific immune factors. Moreover, A-G2 had the highest relative expression of IgM, thus signifying that this concentration of β-1,3-glucan was most effective in this study, and it is considered suitable for transport. Biology 2020, 9, x FOR PEER REVIEW 10 of 20

Effect of Ascorbic Acid and β-1,3-Glucan Addition on Relative Expression of Non-Specific Immune Indexes of Tiger Grouper during Simulated Transport
Simulated transport usually induces stress responses that could lead to increase susceptibility to diseases. Immunostimulants can reduce the outbreak of diseases by facilitating the function of phagocytic cells, improving resistance to bacterial challenges [65]. The expression levels of IL-1β significantly increased, and then decreased, as shown in Figure  6a. IL-1β expression of CK was significantly higher than that of other simulated transport groups. From Figure 6b, the expression of IgM had a similar trend as that of IL-1β, and the expression of IgM in the A-G2 group was significantly higher than that of the other groups, which indicated that ascorbic acid and β-1,3-glucan addition could stimulate non-specific immune factors. Moreover, A-G2 had the highest relative expression of IgM, thus signifying that this concentration of β-1,3-glucan was most effective in this study, and it is considered suitable for transport. Figure 6. Effect of ascorbic acid and β-1,3-glucan addition on relative expression of 1L-1β (a) and IgM (b) of tiger grouper during simulated transport. Among different treatments transport groups, different small letters indicate the results of Duncan's test at different transport time. The same letters mean no significant difference (p > 0.05), while different letters mean significant difference (p < 0.05). Different letters without * = p-value < 0.05, different letters with ** = p-value < 0.01.

Effect of Ascorbic Acid and β-1,3-Glucan Addition on Serum Biochemical Parameters of Tiger Grouper during Simulated Transport
Creatine kinase activity can act as an indicator of live fish metabolism. Increased creatine kinase suggests that muscle and kidney of fish have been damaged [66]. Creatine kinase activity of serum increased in all samples during simulated transport, and then returned to the initial level after recovery, as shown in Table 4. However, there was no significant difference in creatine kinase activity of A-G2 throughout the simulated transport, which indicated that ascorbic acid and β-1,3-glucan addition can effectively reduce the damage to kidneys. Figure 6. Effect of ascorbic acid and β-1,3-glucan addition on relative expression of 1L-1β (a) and IgM (b) of tiger grouper during simulated transport. Among different treatments transport groups, different small letters indicate the results of Duncan's test at different transport time. The same letters mean no significant difference (p > 0.05), while different letters mean significant difference (p < 0.05). Different letters without * = p-value < 0.05, different letters with ** = p-value < 0.01.

Effect of Ascorbic Acid and β-1,3-Glucan Addition on Serum Biochemical Parameters of Tiger Grouper during Simulated Transport
Creatine kinase activity can act as an indicator of live fish metabolism. Increased creatine kinase suggests that muscle and kidney of fish have been damaged [66]. Creatine kinase activity of serum increased in all samples during simulated transport, and then returned to the initial level after recovery, as shown in Table 4. However, there was no significant difference in creatine kinase activity of A-G2 throughout the simulated transport, which indicated that ascorbic acid and β-1,3-glucan addition can effectively reduce the damage to kidneys.
Total protein (TP) and albumin (ALB) reflect the liver function. Albumin is synthesized by the liver, and plays a role as a carrier in the blood [67]. TP and ALB in the serum can accurately reflect the absorption and metabolism of the protein. The contents of TP and ALB in serum of CK were significantly higher than in other groups during simulated transport, as shown in Table 5. It suggests that the addition of ascorbic acid and β-1,3-glucan can improve the function of tiger grouper liver. The contents of TB and ALB in treated groups showed no significant difference throughout simulated transport. Moreover, TB and ALB of tiger grouper gradually returned to the initial level after transport and recovery.  Table 5. Effect of ascorbic acid and β-1,3-glucan addition on the free amino acids of tiger grouper during simulated transport (mg/100 g). Urea, creatinine, and uric acid (UA) reflect the renal function. Urea is the product of metabolism of nitrogen compounds, and also an important component in maintaining blood osmotic pressure [68]. At the end of transport, urea, creatinine and UA levels of treated samples decreased (Table 4), and these could not return to the initial level after recovery, which indicates damage that may have contributed to the fish majority during long-term transport.

Transport
3.9. Effect of Ascorbic Acid and β-1,3-Glucan Addition on Nutritional Indexes of Tiger Grouper during Simulated Transport Figure 7 shows the changes in ash, moisture content, crude fat, and crude protein of tiger grouper during simulated transport. The contents of moisture and ash in all samples did not show obvious changes. Protein decreased, possibly because of stress related to transport and temperature change. Among all of the samples, the nutritional components of A-G2 showed no significant changes during simulated transport, which indicates that ascorbic acid and the G2 β-1,3-glucan concentration could effectively reduce the negative impact of transport and temperature changes on the nutritional indexes of tiger group.
serum of CK were significantly higher than in other groups during simulated transport, as shown in Table 5. It suggests that the addition of ascorbic acid and β-1,3-glucan can improve the function of tiger grouper liver. The contents of TB and ALB in treated groups showed no significant difference throughout simulated transport. Moreover, TB and ALB of tiger grouper gradually returned to the initial level after transport and recovery.
Urea, creatinine, and uric acid (UA) reflect the renal function. Urea is the product of metabolism of nitrogen compounds, and also an important component in maintaining blood osmotic pressure [68]. At the end of transport, urea, creatinine and UA levels of treated samples decreased (Table 4), and these could not return to the initial level after recovery, which indicates damage that may have contributed to the fish majority during long-term transport. Figure 7 shows the changes in ash, moisture content, crude fat, and crude protein of tiger grouper during simulated transport. The contents of moisture and ash in all samples did not show obvious changes. Protein decreased, possibly because of stress related to transport and temperature change. Among all of the samples, the nutritional components of A-G2 showed no significant changes during simulated transport, which indicates that ascorbic acid and the G2 β-1,3-glucan concentration could effectively reduce the negative impact of transport and temperature changes on the nutritional indexes of tiger group. Figure 7. Effect of ascorbic acid and β-1,3-glucan addition on ash (a), moisture (b), fat (c) and protein (d) of muscle of tiger grouper during simulated transport. Among different treatments transport groups, different small letters indicate the results of Duncan's test at different transport time. The same letters mean no significant difference (p > 0.05), while different letters mean significant difference (p < 0.05). Different letters without * = p-value < 0.05, different letters with ** = p-value < 0.01.

Effect of Ascorbic Acid and β-1,3-Glucan Addition on Free Amino Acids of Tiger Grouper during Simulated Transport
The total content of free amino acids of tiger grouper increased during simulated transport, and recovered to the initial level, as shown in Table 5. Transport stress could promote protein degradation, resulting in higher total free amino acids contents. It should be noted that there was no food for tiger grouper during simulated transport; therefore, transport stress could accelerate protein degradation and lead to nutrient content loss. However, the addition of ascorbic acid and β-1,3-glucan resulted in a reduction of free amino acid and thus probably slowed down the rate of protein degradation during simulated transport. This indicates moderation of the stress response. Free amino acids in the muscle tissue are usually related with different tastes, such as umami, sweetness, bitterness, and sourness. Umami amino acids include Asp and Glu, sweetness amino acids include Thr, Ser, Gly, Ala, and Pro [69]. Figure 8 shows the effect of ascorbic acid and β-1,3-glucan addition on umami and sweet taste amino acids of tiger grouper during simulated transport. In all samples, the amount of umami and sweet taste amino acids increased during simulated transport, due to transport stress response accelerated protein degradation. There was no significant difference in taste amino acids of A-G2 during simulated transport and recovery. The results indicate that ascorbic acid and the G2 β-1,3-glucan concentration can effectively reduce the changes of free amino acids during simulated transport.
Biology 2020, 9, x FOR PEER REVIEW 15 of 20 Figure 8. Effect of ascorbic acid and β-1,3-glucan addition on umami (a) and sweet (b) taste amino acids of tiger grouper during simulated transport. Among different treatments transport groups, different small letters indicate the results of Duncan's test at different transport time. The same letters mean no significant difference (p > 0.05), while different letters mean significant difference (p < 0.05). Different letters without * = p-value < 0.05, different letters with ** = p-value < 0.01.
Nucleotides in aquatic products are of great significance for flavors [70]. IMP and AMP are the two main taste nucleotides in tiger grouper during simulated transport. IMP has a strong umami taste [71]. Table 6 shows the changes in IMP and AMP of tiger grouper during simulated transport. The results demonstrated that value of IMP was higher than 10. Therefore, IMP contributed most to the sweet and meaty flavor of tiger grouper. However, the content of AMP was obviously lower than IMP, and the TAV value of AMP was less than 1. IMP and AMP concentrations values were decreased during simulated transport and were able to recover to the initial levels. Different from the CK samples, the IMP and AMP concentrations in A-G2 samples were higher after recovery, and TAV value of IMP in A-G2 was 16.14, proving ascorbic acid and β-1,3-glucan addition could relieve flavor nucleotides degradation during simulated transport and recovery. Table 6. Effect of ascorbic acid and β-1,3-glucan addition on nucleotides of tiger grouper during simulated transport in water.

Transport
Samples IMP (mg/100 g) TAV AMP (mg/100 g) TAV  Nucleotides in aquatic products are of great significance for flavors [70]. IMP and AMP are the two main taste nucleotides in tiger grouper during simulated transport. IMP has a strong umami taste [71]. Table 6 shows the changes in IMP and AMP of tiger grouper during simulated transport. The results demonstrated that value of IMP was higher than 10. Therefore, IMP contributed most to the sweet and meaty flavor of tiger grouper. However, the content of AMP was obviously lower than IMP, and the TAV value of AMP was less than 1. IMP and AMP concentrations values were decreased during simulated transport and were able to recover to the initial levels. Different from the CK samples, the IMP and AMP concentrations in A-G2 samples were higher after recovery, and TAV value of IMP in A-G2 was 16.14, proving ascorbic acid and β-1,3-glucan addition could relieve flavor nucleotides degradation during simulated transport and recovery. Table 6. Effect of ascorbic acid and β-1,3-glucan addition on nucleotides of tiger grouper during simulated transport in water.

Conclusions
The minimum tolerable temperature of tiger grouper transported by water is 15 • C, and could induce dormancy, thus resulting in reducing life activities. The activities of metabolic enzymes, cortisol, HSP70 transcription level, GR enzyme activity, IL-1β, and IgM transcription levels in tiger grouper serum at 15 • C were significantly lower than in groups transported at other temperatures. Anti-stress agents, including ascorbic acid and β-1,3-glucan was added in the transport water for tiger grouper, during simulated transport and recovery. Cortisol content, HSP70 transcription level, and immune index of tiger grouper serum in A-G2 were lower than in other groups. However, there was no significant difference in nutritional content, taste amino acids, and nucleotides of muscle tissue in A-G2 before and after transport. The addition of ascorbic acid and β-1,3-glucan could effectively reduce the stress response of tiger grouper and improve their immunity and survival. Besides, it did not lead to loss of nutritional valued and flavor.