Chemical and quality evaluation of Pacific white shrimp Litopenaeus vannamei: Influence of strains on flesh nutrition

Abstract The Pacific white shrimp, Litopenaeus vannamei, is an important fisheries resource in China. To investigate the differences in nutritional quality among strains, we analyzed and compared the basic muscle nutritional components, amino acid (hydrolyzed and free) compositions, and fatty acid compositions among four L. vannamei strains (Universal, KH‐1, Syaqua, and common). The result showed that under an efficiency aquaculture model, all four strains had high protein (21.1%‐22.3%) and low fat (0.8%‐1.1%). The Universal strain was highest in protein and fat as well as essential amino acid score (147.97). The Syaqua strain had the highest levels of polyunsaturated fatty acids (44.88%). The KH‐1 strain had the highest free amino acid content (2.68%), which contributes to the taste. Our findings revealed that strain‐specific variability exists in chemical composition of the shrimp L. vannamei under controlled condition, which may provide buying reference for consumers.

strains under the same cultivated condition can provide novel nutritional information to consumers.
The objective of this study is to evaluate and compare the basic muscle nutritional components, amino acid (hydrolyzed and free) compositions, and fatty acid compositions of four L. vannamei strains (Universal, KH-1, Syaqua, and common) under the efficiency aquaculture model. Our findings will provide essential information for the comprehensive development, utilization, and promotion of L. vannamei strains.

| Experimental design
The 90-day feeding trial was performed from May 2020 to August 2020. The experiment was conducted in a shrimp cultural farm (Binzhou, China). Prior to the experiment, juvenile shrimps (Universal, KH-1, Syaqua, and common) with initial weights of 0.63 ± 0.02 g were temporarily reared for seven days to acclimate. Next, the shrimps were placed into 12 experimental ponds (30 m 3 ) to constitute four groups in triplicate. The stocking density was about 300 individuals per square meter. There were no significant differences in survival rate among the group (78.42% ± 1.29%).
The seawater used in the experiment was filtered using a composite sand filter. The chamfer was shaded to preserve temperature and control light. The culture conditions were maintained at 29℃ ± 1℃, pH 7.5-8.0, dissolved oxygen >5.0 mg/L, salinity 29 -31. Biological preparations, such as photosynthetic bacteria, were added to the aquaria ponds every seven days (Li et al., 2016).
The special formula feed for shrimp (contain 40% crude protein, 8% crude lipid) was purchased from a local commercial feed company. This feed was mainly made of fish meal, fermented soybean meal, vitamin, etc. All shrimp groups were fed daily at a rate of 6%-10% of body weight, divided into two equal (at time 8:30 and 16:30).
The daily feeding dose was recalculated and adjusted every 15 d.
The feeding experiment lasted for 90 days. Diets were hand-fed until apparent satiation. The feeding rates were selected to assure apparent satiation was reached without overfeeding (Zhou et al., 2013).
Sewage was discharged from the central sump 2 hr after feeding, followed by the addition of 10% fresh seawater.

| Sample collection
After the 90-day feeding trial, shrimps in each pond were sampled 24 hr after the last feeding. 30 shrimps from each pond (20.10 ± 2.30 g) were randomly selected to analyze approximate meat composition. All samples were kept on ice until they reached the laboratory. The abdominal meat was obtained from the shell, got rid of the intestinal tract, pooled in triplicate, and homogenized using a grinder. The samples were stored in PE bags in the dark at 20℃ until analysis (within 2 w).

| The determination of proximate composition
Crude protein, crude lipid, moisture, and ash content in the shrimp meat were determined following the procedures of the Association of Official Analytical Chemists (AOAC, 1995). First, the samples were oven-dried until a constant weight was reached at 105℃ to determine the moisture content. Crude protein content was estimated using the Kjeldahl method (Nitrogen Analyzer 8,400, Foss, Denmark), and a conversion factor of 6.25 was used to convert total nitrogen to crude protein. Crude lipid was assayed by ether extraction method using the Soxhlet extraction method (Sotex-8000, Foss, Denmark). The ash content was determined by dry ashing in a muffle oven at 550℃ for 24 hr.

| The determination of hydrolyzed amino acid (HAA) and free amino acid (FAA)
HAA content was measured according to the GB/T 5,009.124-2016in China (2016. The sample was hydrolyzed with 6 mol L mol/L HCl at 110℃ for 24 hr in sealed glass tubes filled with nitrogen; then, 1 ml of hydrolysate was evaporated to dryness at 45℃ to remove the HCl. The hydrolysate was dissolved in 5 ml of 0.02 mol/L HCl, centrifuged (5,000 rpm, 10 min, 4℃), and then filtered. 1 µl aliquot of the supernatant was used for the amino acid analysis. Cysteine and methionine were determined as cysteic acid and methionine sulfone, respectively, by performic acid oxidation prior to their digestion in 6 N HCl. The content of tryptophan was determined after alkaline hydrolysis (5 mol/L NaOH) of each sample. All determinations were performed in triplicate.
Free amino acid (FAA) was carried out using the method of Li et al., (2014) with some modification. The sample was mixed with 0.01 mol/L HCl, supersonic extracted for 30 min, and then filtered.
1 µl aliquot of the supernatant was used for the amino acid analysis.
The amino acid composition was analyzed using an automatic amino acid analyzer (LA-8080, Hitachi, Tokyo, Japan) equipped with a 4.6 mm ×60 mm Hitachi 2,622 column. The identity and quantity of the amino acids were assessed by comparison with the retention times and peak areas of amino acid standards. Each sample was measured thrice and presented as the average of three replicates.
The essential amino acid score was calculated with respect to the Amino acid score =sample amino acid/reference amino acid ×100.

| The determination of fatty acid
Fatty acids were extracted from the shrimp samples and analyzed according to the ISO 5509 method (2000) involving Soxhlet extraction, saponification, esterification, and finally fatty acid methyl ester (FAME) extraction in hexane. Gas chromatography was performed using an Agilent Technologies 6,890 gas chromatograph equipped with an HP-5 cross-linked methyl silicone-fused-silica capillary column (30 m × 0.32 mm I.D., 0.5 µm film thickness). Individual components were identified by comparing the mass spectral data and retention time data with those obtained for authentic and laboratory standards. Each sample value represents the mean of three measurements.

| Statistical analysis
All results were expressed as mean ± SD, and one-way analysis of variance (ANOVA) was performed using statistical analysis software (SPSS Version 16.0). Differences in the concentration of nutritional elements among shrimp strains were tested with ANOVA followed by a multiple comparison test (Tukey's HSD). Differences were considered to be significant at p <.05.

| Ethical statement
The Pacific white shrimp is an aquaculture species. All experiments in this study were approved by the Animal Care and Use guideline of the Marine Science Research Institute of Shandong Province.

| Proximate composition of four shrimp strains
The proximate composition of each strain is shown in Table 1. Crude protein content ranged from 21.1% to 22.3%, crude fat content ranged from 0.8% to 1.1%, and the crude ash did not differ among strains. These results are consistent with Ma et al. (2018) who found that crude protein and fat content were relatively uniform among shrimp strains. Among the four strains examined in the present study, crude protein content was the highest in Universal (22.3%).
Crude fat content was the lowest in common shrimp, and crude ash content was the lowest in KH-1 and common shrimp. The samples came from the same culture area, but results were found to be comparable. A similar crude protein content was found in Aristaeomorpha foliacea (Bonoet al., 2012) and Penaeus monodon (Sriket et al., 2007) (87.7 g 100g -1 dry matter), while Fenneropenaeus chinensis had a low crude protein content of 20.60% . Shrimp cultured in pond had higher values of crude fat, as reported by Sriket et al. (2007). In general, the chemical composition analyses confirmed that the four strains were to be excellent food resources, with good balance of nutrients and good level of protein.

| HAA profiles of four shrimp strains
The hydrolyzed amino acid profiles (HAA) of four shrimp strains are presented in Table 2. In all strains, glutamic acid was the most abundant amino acid, followed by lysine, aspartic acid, and leucine. These four amino acids constituted 41.34%-42.64% of the total amino acids (TAA). The KH-1 had the highest TAA amounts at 82.07 g 100g -1 (DW). Additionally, the essential amino acid (EAA) was high.
The nutritional value of food depends on amino acid composition and content. For humans, food with higher amino acid content can provide better nutritional value (Shi et al., 2013). TAA and EAA are also important indicators of the nutritional value of aquatic products (Wu et al., 2014). The four shrimp strains evaluated in this study had glutamic acid, content ranging from 172.8 mg g -1 (KH-1) to 176.6 mg g -1 (Syaqua) crude protein. These values are significantly higher than those reported for fish: 114 mg/g crude protein in Pampus punctatissimus (Zhao et al., 2010), 117 mg/g crude protein in Paralichthys olivaceus (Ning et al., 2018); crabs: 147 mg/g crude protein in Carcinus maenas (Naczk et al., 2004), 151 mg/g crude protein in Eriocheir sinensis (Chen et al., 2007); and other shrimps: 135 mg/g crude protein in Euphausia superba . Glutamate contributes to biochemical metabolism in brain tissue, synthesizing active substances and playing an important role in synaptic transmissions between nervous systems (Morris et al., 1987). Furthermore, the efflux of glutamic acid from meat in response to critical illness can serve as an important carrier of ammonia (nitrogen) to the splanchnic area and the immune system (Deutz et al., 1992). The shrimp protein was also rich in TA B L E 1 Proximate composition of four shrimp strains (mean±SD) (g 100g -1 wet weight) lysine, which is the limiting amino acid in the cereal-based diets of children in developing countries (Kim et al., 2000).
The EAA scores of each shrimp strain are presented in Table 3.
When compared to the reference amino acid pattern of preschool children (2-5 years old), all amino acid scores were >100, except for tryptophan. Tryptophan had the lowest scores (especially in Syaqua), which would be considered the limiting amino acid in shrimp. However, in Chinese recipes, shrimp is often cooked with pork, mutton, and millet, which will supply the limiting amino acid (Jilin and Peck, 1995). Lysine and sulfur-containing amino acids (cysteine and methionine) had the highest scores in all strains. In general, the shrimp muscle protein was of high quality and exhibited wellbalanced EAA compositions.

| FAA profiles of four shrimp strains
The free amino acid (FAA) profiles of the four shrimp strains are presented in Table 2. The total FAA contents were 7.99 g 100g -1 (DW), 10.89 g 100g -1 (DW), 9.32 g 100g -1 (DW), and 5.60 g 100g -1 (DW), respectively. The most abundant FAA was glutamic acid, glycine, alanine, and lysine, which differed from the HAA profiles. These amino acids constituted 63.05%, 57.83%, 60.61%, and 75.10% of the total FAA (TFAA), respectively. The least abundant FAA in the meat of all four shrimp strains was tryptophan.
The taste activity value (TAV) of the FAA identified in the four shrimp strains is presented in Table 4. FAAs are important components of the meat that can affect the flavor of aquatic animals . Each amino acid has a unique taste, the flavor contribution of which is dependent on the TAV, the ratio between the taste substance contained in the sample and its threshold value (Chen et al., 2012;Li et al., 2014). A TAV >1 indicates an important contribution to the taste of food . However, TAV does not consider the interaction between the components, which may have a synergistic effect on food taste. Maybe sensory evaluation should be used in our future research. Among the four shrimp strains, glutamic acid, glycine, valine, methionine, lysine, and histidine had TAVs greater than one. Amino acids, especially glutamic acid, glycine, alanine, and aspartic acid greatly influence the shrimp flavor (Liu et al.,l., 2017). Figure 1 shows the comparative content model charts and TAV model charts of the major FAA in the four shrimps, respectively.
All strains had the similar FAA compositions, except the common shrimp, which had higher glycine levels (Figure 1a). The FAA content model among the other three strains was similar, but the model area was highest for KH-1, followed by Syaqua, and then Universal, which indicates that the FAA content of KH-1 was higher.
The FAA content models were similar among strains, except for the common shrimp (Figure 1b). KH-1 had the largest model area.
The top contributor to taste was glutamine, with a TAV of 14.33. The TAVs of alanine and glycine were 7.83 and 3.69, respectively, which also significantly influence the taste of shrimp meat. The models indicate that KH-1 would taste fresher and sweeter than the other strains.

| Fatty acid profiles of four shrimp strains
The fatty acid profiles and levels, which varied among the four strains, are shown in Table 5 Lipids store and provide energy to organisms. PUFAs are essential for the reproduction and growth of organisms; they can also increase the flavor of food during heating, reduce blood viscosity, and enhance human immunity (Cui et al., 2018). Among the most valuable FAs, DHA and EPA play important roles in the prevention of inflammatory and cardiovascular diseases due to their serum triglycerides-lowering effects (Bono et al., 2012).
The EPA and DHA contents were moderately high in all the shrimp samples, especially KH-1. However, the EPA content measured in the present research was lower than that reported by Huang TA B L E 4 The taste attribute and TAV of free amino acid in four shrimp strains (mg g -1 )  et al., (2004) for L. vannamei (11.5 to 13.7 g100 g −1 FA), while the DHA content, which ranged from 8.9 to 10.4 g 100 g −1 FA, was lower than that of our data.

| CON CLUS ION
The results from this study revealed that strain-specific variability exists in chemical composition of the shrimp L. vannamei under controlled condition. The Universal strain had the highest protein and fat content, and the highest overall EAA score. The Syaqua shrimps showed the highest levels of PUFA. And the KH-1 strain was highest in FAA taste-active components.

This study was funded by Modern Agricultural Industry Technology
System of Shandong Province (SDAIT-13-07).

CO N FLI C T S O F I NTE R E S T
The authors declare that they have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
The authors confirm that the data supporting the findings of this study are available within the article.