Study of the Quality and Nutritional Value of Alosa sapidissima in the Postmortem Process

Abstract

The American shad (Alosa sapidissima) is an important freshwater food fish, yet little is known about its postmortem quality. We sampled the meat of American shad and tracked the changes in color, pH, shear stress, cooking loss and total volatile basic nitrogen (TVB-N) over 48 h of storage at 24 °C (unrefrigerated) or 4 °C (refrigerated). Thereafter, the essential nutrients, fatty acids, hydrolyzed amino acids, free amino acids, and electronic tongue were evaluated. The results show that the L* and a* values decreased as storage progressed, while the b* value increased. The shear force decreased, but cooking losses in the American shad increased; the TVB-N value continuously increased over the duration of storage. The TVB-N content deviated from the freshness range at 48 h when stored at 24 °C. At 24 °C, the sweet amino acids reached a maximum at 6 h, and the bitter amino acids reached a maximum at 48 h. At 4 °C, there was a significant difference in bitter free amino acids at 48 h (p < 0.05). The nutrient composition showed that the contents of fat and protein decreased, whereas the water content increased. These results showed that American shad should be eaten within 6 h when stored at 24 °C, while American shad stored at 4 °C can maintain freshness for 24 h to obtain better product quality.

1. Introduction

Shad (subfamily Alosinae of the family Clupeidae) are recognized as one of the top food fishes in China owing to their delicious taste. Tenualosa reevesii is widely distributed in the western North Pacific but has been potentially extirpated from many parts of its range, including in the Yangtze River [1]. The American shad Alosa sapidissima is another eurythermal, highly migratory clupeid, with a distribution mainly along the east coast of North America [2]; this species is popularly eaten in China because of its similarity to Reeve’s shad. American shad has a very high economic value and is sold mainly in the form of live fish [3,4]. Although there are significant differences in freshness and spoilage patterns among species of fish, fresh fish are more prone to rapid changes after death than other meats because of their rich nutrients, higher water content, and higher protease activity [5,6].

The postmortem process of fish can be generally divided into four stages: early biochemical changes, followed by rigid dissolution, self-dissolution, and finally decay [7,8]. These postmortem changes in fish muscle are also influenced by several factors prior to harvest (e.g., age, diet, species) as well as handling during capture and storage [9]. The storage temperature has a significant effect on the freshness and quality of fish meat. Meng [10] studied quality changes of Pneumatophorus japonicus stored at 0, 15, and 25 °C. The sensory study showed that the good-quality life of fish stored at 0, 15, and 25 °C was 144, 24, and 12 h, respectively, and the shelf life was 192, 48, and 20 h. Hence, the study of different temperatures to determine the best eating time after death of American shad provided a theoretical basis for processing it as an edible fish.

Numerous changes occur naturally in fish muscle postmortem. Total volatile basic nitrogen (TVB-N) is often used to monitor fish freshness. There are also changes in pH and texture, as well as the amount of free amino acids (FAAs), all of which influence the freshness of the fish. Taste differences between samples can be distinguished by the electronic tongue. This is a robotic system with an array of sensors that combines good reproducibility with low detection limits and high sensitivities and is thus often used for screening taste attributes [11,12]. Bao [13] studied the effect of different cold storage temperatures on the quality change of fresh salmon. The results showed that storage at 0 °C significantly inhibited the deterioration of TVB-N, juice loss, color, and texture properties. Zhang [14] studied the quality changes of grass carp under different storage temperatures (−3, 0, 3, 9, and 15 °C) and found that lower temperatures and micro-freezing storage significantly extended the shelf life of the product. Wang [15] studied changes in the pH, texture, lactic acid, ATP-related compounds, K value, and free amino acids (FAAs) content in grass carp stored at 4 °C for 192 h. The electronic tongue indicated that the differences were distributed in various areas between the first 2 days and the last 6 days. They recommend that grass carp should be consumed within 6 days when stored at 4 °C. To our knowledge, there has been little research on postmortem muscle changes in American shad during storage.

This study aimed to assess changes in the quality of American shad meat during storage. This was achieved by evaluating changes in the color, pH, shear force, cooking loss, and TVB-N of the meat, as well as its nutrient composition (essential nutrients, fatty acids, hydrolyzed amino acids, and free amino acids), and electronic tongue analysis. Thus, the nutritional value and quality indices of American shad under different storage temperatures (refrigerated at 4 °C or unrefrigerated at 24 °C) were determined and then analyzed to effectively characterize quality changes in this aquatic product specifically, and also to enrich the basic theory of what happens to fish after they die; the results will also provide farmers with valuable insights into quality retention.

2. Materials and Methods

2.1. Sample Preparation

Live Alosa sapidissima (average weight 700 ± 110 g, length 42.1 ± 3.5 cm) were purchased from Noah’s Ark Agricultural Science & Technology Company in Changzhou (Changzhou, China). The 2–3 randomly selected American shad were taken for analysis at specific time points. The American shad were gutted and washed, then the fish were packed in polyethylene bags, which were variously stored at 24 ± 1 °C or 4 ± 1 °C, for 0 h, 6 h, 24 h, and 48 h. After the skin was removed, muscle samples were taken in bags and then packed with dry ice and transported to the laboratory. Three replicate samples were taken for all analyses.

2.2. Color Measurement

The sampled muscle was cut into pieces (3 × 1 × 1 cm) and placed in plastic bags (one piece per bag). The color of the muscle was measured using a colorimeter (NR10QC, 3nh, Shenzhen, China). After calibration with a standard color plate, the color of the sample was measured, and the values of brightness (L*), redness (a*), and yellow and blue (b*) of each detection point were measured. L* = 0 represents black, and L* = 100 represents white. A larger value of a* signifies the color is closer to red, and a smaller value signifies the color is closer to green. A larger value of b* denotes that the color tends toward yellow, and a smaller value denotes the color tends toward blue. Each piece of muscle was measured three times, and the results were expressed as mean values [16].

2.3. Shear Stress Measurement

To measure this, muscle of American shad for shearing was cut into pieces (3 × 1 × 1 cm). Each piece was placed in the middle of a digital meat tenderness meter (C-LM3B, Tenovo, Beijing, China), with the cutter perpendicular to the fiber and fat bands for cutting. The maximum force produced in the shearing process of fish meat is denoted as the shear force [17].

2.4. Determination of Cooking Loss

Muscle samples were cut into pieces (3 × 1 × 1 cm), placed in plastic bags (one piece per bag), and then cooked at 80 °C for 20 min in a water bath. The cooked pieces were placed on filter paper for 5 min to drain off the water. The weight of the raw and cooked samples was recorded to calculate cooking loss. Each piece of fish flesh was measured three times and the results were expressed as mean values [13].

The percentage of cooking loss in the American shad was calculated as:

$$\mathrm{Cooking} \mathrm{loss} (\%)=\frac{\mathrm{Weight} \mathrm{of} \mathrm{raw} \mathrm{sample} - \mathrm{Weight} \mathrm{of} \mathrm{cooked} \mathrm{sample}}{\mathrm{Weight} \mathrm{of} \mathrm{raw} \mathrm{sample}} \times 100$$

2.5. Determination of TVB-N and pH

An amount of 10.0 g of each muscle sample was weighed and digested; 1 g of magnesium oxide and 75 mL of distilled water were added and mixed; finally, the mixture was thoroughly distilled and determined by using a Kjeltec Analyzer Unit (VAPODEST 450; Gerhardt, Konigswinter, Germany) [18].

For pH determination, an amount of 2.0 g of each sample was weighed (as described in Section 2.3) and placed in a beaker with 18 mL of distilled water; this mixture was homogenized and centrifugated at 10,000 rpm for 10 min at room temperature. Quickly after filtration, the supernatant was measured for pH (PB-10, Sartoorius, Gottingen, Germany).

2.6. Proximate and Fatty Acid Composition

The moisture content of each fish sample was determined by oven drying at 105 °C for 24 h until constant weight was achieved, and ash content was determined using a muffle furnace at 550 °C for 24 h, according to standard methods [19]. Total nitrogen content was estimated by the Kjeldahl method. Crude protein was measured by multiplying the total nitrogen content by a factor of 6.25. Crude fat was determined gravimetrically after Soxhlet extraction of dried muscle with hexane in hot extraction mode at 60 °C for 4 h; the extract was then dried in an oven at 100 °C, until a constant weight was achieved [20].

Approximately 2 g of fish extract was weighed out into a methylation glass tube and evaporated under a stream of nitrogen until dry. A mixture containing 100 μL of internal standard solution (C23:0), 200 μL of heptane with BHT, and 100 μL of toluene were added to the dry extract. Samples were methylated in a microwave oven (Multiwave 3000 SOLV; Anton Paar, Ashland, VA, USA) for 5 min at 100 °C and a power level of 500 W. After methylation, heptane with BHT (0.7 mL) and saturated salt water (1 mL) were added. The upper phase (heptane) was transferred into HPLC vials and analyzed using gas chromatography (HP-5890 A; Agilent Technologies, Santa Clara, CA, USA). Fatty acid methyl esters were separated by a capillary gas chromatography column (10 × 100 × 0.1 μm) (Agilent DB-WAX 127–7012; Agilent Technologies, Santa Clara, CA, USA). The Fatty Acid Methyl Esters Standard Mixture (Sigma, St. Louis, MO, USA) was used for fatty acid identification. Fatty acids were quantified as the area (%) of total fatty acids [21].

2.7. Amino Acid Composition

The amino acid content of a freeze-dried powder of the edible body parts of A. sapidissima was analyzed in accordance with the method described by Hu et al. [22], using an Agilent 1100 Series Liquid Chromatograph (Agilent Technologies, Inc., Santa Clara, CA, USA). Briefly, the amino acids were hydrolyzed with 6.0 M hydrochloric acid solution for 22 h at 120 °C, then neutralized with NaOH, and the supernatant was collected for analysis. The essential amino acid score (EAAS) was assessed in reference to the recommendations of the Food and Agriculture Organization of the United Nations (FAO)/World Health Organization (WHO)/United Nations University (UNU) initiative for the AA requirement for adult maintenance (

Table 1. Essential amino acid content in “ideal” protein in amino acid scoring patterns in the 2007 FAO/WHO/UNU and FAO pattern reports.

Essential Amino Acid	WHO/FAO/UNN 2007	FAO Parttern 1984
Ile	30	40
Leu	59	70
Lys	45	55
Thr	23	40
Val	39	50
Met + Cys	22	35
Phe + Tyr	38	60

) [23,24]. EAAI represents the geometric mean of the ratio of the content of essential amino acids in protein to that in egg protein [25]. Free amino acids were adjusted to an appropriate volume with 5% trichloroacetic acid, mixed well, then allowed to stand for 2 h, and then filtered. Finally, the supernatant was collected for analysis.

2.8. Electronic Tongue

The taste traits of A. sapidissima muscles were evaluated by electronic tongue. According to the method described by [26] with a slight modification: to 10 g of cooked muscle sample 20 mL of distilled water was added, followed by homogenization, filtration, and centrifuging at 10,000× g for 20 min; 15 mL of supernatant was used. The sectors of astringency, bitterness, saltiness, umami, and sweetness were activated in the reference solution for 24 h. The sectors were soaked in the reference solution for 30 s, the reference potential Vr was measured, then the sensor was soaked in the sample solution for 30 s, the potential of the sample solution Vs, Vs–Vr was measured, showing the intensity values of bitterness, astringency, umami, richness, and saltiness of the sample; afterwards the sensors of bitterness, astringency, and richness were placed in the reference solution for 30 s, and the electric potential Vr’ was measured. Vr’–Vr were the aftertaste A (bitterness aftertaste) and richness (umami aftertaste) intensity values. Each sample was repeated three times.

2.9. Statistical Analysis

All the experiments were performed in triplicate. The results are reported as mean ± standard error of the mean (SDM). The Shapiro–Wilk test and Levene’s test were used to analyze the data for normal distribution and homogeneity of variance. The differences between groups were compared through an independent-samples t-test, and one-way analysis of variance (ANOVA), with a post hoc Duncan’s multiple range test used to determine the significance level among the groups; p < 0.05 was considered significant. Statistical analyses were conducted using SPSS 22.0 (IBM Corp., Armonk, NY, USA). Principal component analysis (PCA) was used to analyze the significantly different variables.

3. Results

3.1. Color Analysis

As shown in

Table 2. Color of American shad meat during storage at different temperatures (i.e., unrefrigerated and refrigerated).

Temperature	Time (h)	L*	a*	b*
24 °C	0	55.56 ± 0.49 a	5.90 ± 0.36 a	6.44 ± 0.56 d
	6	53.33 ± 0.44 b	4.66 ± 0.06 b	7.38 ± 0.28 bc
	24	51.43 ± 0.56 c	3.07 ± 0.34 c	10.48 ± 0.32 b**
	48	49.60 ± 1.02 d	2.95 ± 0.71 c	11.77 ± 0.29 a**
4 °C	0	55.56 ± 0.49 a	5.90 ± 0.36 a	6.44 ± 0.57 c
	6	54.17 ± 0.23 b	4.93 ± 0.10 a	6.82 ± 0.59 bc
	24	52.50 ± 0.31 c	3.75 ± 0.08 b	7.56 ± 0.32 b**
	48	51.54 ± 0.52 d	3.32 ± 0.10 c	8.59 ± 0.24 a**

Note: Values are given as mean ± SD from triplicates determination. Different lowercase letters indicate significant differences at the different times (p < 0.05). Asterisks indicate significant differences at different storage temperatures (* p < 0.05; ** p < 0.01).

, at 0 h the values of L*, a* and b* were 55.56, 5.90, and 6.44, respectively. At 24 °C anAd 4 °C, the L* and a* values decreased as storage progressed, while the b* value increased (p < 0.05); the b* values of the two storage temperatures were significantly different after 24 h (p < 0.05).

3.2. Change in Shear Stress

The changes in shear stress of American shad meat during storage are shown in Figure 1A. During storage, the shear stress in the unrefrigerated temperature storage group (24 °C) decreased sharply and then tended to stabilize, whereas the shear stress in the refrigerated storage group (4 °C) decreased gradually (p < 0.05). The rate of decline in shear stress slowed after 6 h.

3.3. Change in Cooking Loss

The changes in cooking loss are shown in Figure 1B. Cooking losses in American shad increased; the cooking loss increased rapidly during the period 0–6 h in the unrefrigerated group (24 °C) but changed little in the refrigerated group (4 °C). At 48 h, there was little difference in the cooking loss of the fish meat between the two storage temperature groups.

3.4. Analysis of pH and TVB-N

As shown in Figure 1D, at 0 h the pH value of American shad was 6.35. At 6 h, there was a significant difference between the unrefrigerated group (24 °C) and the refrigerated group (4 °C) (p < 0.05). As shown in Figure 1C, the TVB-N content of the samples increased slowly at first, but in the later stage of storage increased rapidly. At 24 h and 48 h, the TVB-N content differed significantly between the two storage-temperature groups (p < 0.05).

3.5. Proximate Composition Analysis

From the four conventional biochemical components of water, ash, crude fat, and crude protein (

Table 3. Proximate composition of American shad under different storage conditions.

Temperature	Time (h)	Moisture (%)	Ash (%)	Crude Protein (%)	Crude Fat (%)
24 °C	0	65.74 ± 3.90 b	1.41 ± 0.03 a	15.06 ± 1.41 a	15.43 ± 0.094 a
	6	71.96 ± 4.72 a	1.52 ± 0.07 a	13.62 ± 0.82 a	12.14 ± 2.54 b
	24	73.94 ± 2.29 a	1.57 ± 0.01 a	13.73 ± 1.37 a	9.01 ± 3.42 c
	48	74.15 ± 2.49 a*	1.53 ± 0.26 a	13.62 ± 0.31 a	8.63 ± 1.11 c
4 °C	0	65.74 ± 3.90 a	1.41 ± 0.03 a	15.06 ± 1.41 a	15.43 ± 0.94 a
	6	70.14 ± 4.63 a	1.48 ± 0.26 a	14.69 ± 1.00 a	13.90 ± 5.00 ab
	24	69.68 ± 1.66 a	1.56 ± 0.10 a	13.92 ± 0.43 a	10.49 ± 1.28 b
	48	64.91 ± 1.23 a*	1.53 ± 0.12 a	13.71 ± 0.29 a	10.18 ± 2.10 b

Note: Values are given as mean ± SD from triplicates determination. Different lowercase letters indicate significant differences at different storage times (p < 0.05). Asterisks indicate significant differences at different storage temperatures (* p < 0.05).

), the moisture content of American shad showed an increasing trend under normal temperature (p < 0.05). The moisture content first increased and then decreased during cold storage (p > 0.05); at 48 h, the moisture content was significantly different between the refrigerated and unrefrigerated groups (p < 0.05). There was no difference in the ash and protein contents between the start and end of storage (p > 0.05). The protein and fat contents decreased gradually with the duration of storage.

3.6. Fatty Acid Analysis

Postmortem changes in the fatty acid content of American shad during storage are listed in

Table 4. Fatty acid (FA) content (%) of American shad as storage progressed at 24 °C (unrefrigerated) and 4 °C (refrigerated).

FA (%)	0		6 h		24 h		48 h
	24 °C	4 °C	24 °C	4 °C	24 °C	4 °C	24 °C	4 °C
C12:0	0.05 ± 0.00 b	0.05 ± 0.00 c	0.06 ± 0.00 a	0.06 ± 0.01 a	0.05 ± 0.01 ab	0.05 ± 0.00 bc	0.05 ± 0.00 ab	0.06 ± 0.00 ab
C14:0	2.03 ± 0.14 a	2.03 ± 0.14 b	2.17 ± 0.05 a	2.19 ± 0.03 b	2.14 ± 0.11 a	2.14 ± 0.02 b	2.20 ± 0.07 a*	3.44 ± 0.02 a*
C15:0	0.27 ± 0.01 b	0.27 ± 0.01 b	0.30 ± 0.01 ab	0.28 ± 0.01 b	0.29 ± 0.02 ab	0.28 ± 0.00 b	0.30 ± 0.01 a*	0.36 ± 0.00 a*
C16:0	18.92 ± 0.22 b	18.92 ± 0.22 b	19.94 ± 0.15 a	19.42 ± 0.61 b	20.50 ± 0.76 a	19.09 ± 0.20 b	19.70 ± 0.71 ab*	22.47 ± 0.24 a*
C17:0	0.39 ± 0.03 a	0.39 ± 0.03 ab	0.41 ± 0.02 a	0.38 ± 0.04 ab	0.41 ± 0.02 a	0.38 ± 0.01 a	0.42 ± 0.02 a*	0.29 ± 0.00 b*
C18:0	6.54 ± 0.50 a	6.54 ± 0.50 a	6.36 ± 0.18 a	6.06 ± 0.11 a	6.46 ± 0.31 a	6.08 ± 0.07 a	6.37 ± 0.41 a*	3.32 ± 0.08 b*
C20:0	0.30 ± 0.06 a	0.30 ± 0.06 a	0.25 ± 0.01 a	0.25 ± 0.01 ab	0.30 ± 0.01 a	0.23 ± 0.04 b	0.27 ± 0.04 a	0.16 ± 0.01 c
C22:0	0.18 ± 0.07 a	0.18 ± 0.07 a	0.12 ± 0.00 a	0.12 ± 0.02 a	0.17 ± 0.02 a	0.12 ± 0.02 a	0.13 ± 0.03 a	0.09 ± 0.02 a
C14:1	0.03 ± 0.00 a	0.03 ± 0.00 b	0.03 ± 0.00 b	0.03 ± 0.01 b	0.03 ± 0.00 ab	0.04 ± 0.01 b	0.03 ± 0.00 b*	0.06 ± 0.00 a*
C16:1	2.59 ± 0.14 a	2.59 ± 0.14 bc	2.71 ± 0.02 a	2.67 ± 0.05 b	2.50 ± 0.31 a	2.36 ± 0.24 c	2.63 ± 0.11 a*	6.11 ± 0.04 a
C17:1	0.30 ± 0.01 a	0.30 ± 0.01 b	0.31 ± 0.02 a	0.30 ± 0.01 b	0.29 ± 0.01 a	0.29 ± 0.02 b	0.31 ± 0.00 a*	0.37 ± 0.01 a*
C18:1	30.46 ± 1.57 a	30.46 ± 1.57 ab	28.91 ± 0.37 a	29.29 ± 0.23 a	26.75 ± 2.07 a	29.15 ± 1.18 ab	27.82 ± 0.46 a*	25.02 ± 0.19 b*
C20:1	1.95 ± 0.22 a	1.95 ± 0.22 a	1.64 ± 0.11 ab	1.58 ± 0.11 b	1.46 ± 0.22 b	1.43 ± 0.17 b	1.61 ± 0.14 ab*	0.79 ± 0.06 c*
C18:2	23.17 ± 1.66 a	23.17 ± 1.66 a	24.72 ± 0.51 a	23.98 ± 0.36 a	24.27 ± 1.37 a	24.63 ± 0.20 a	25.09 ± 0.54 a	24.30 ± 0.59 a
C18:3n6	1.69 ± 0.07 a	1.69 ± 0.07 a	1.42 ± 0.05 b	1.98 ± 0.12 a	1.69 ± 0.08 a	1.65 ± 0.26 a	1.33 ± 0.19 b*	0.13 ± 0.03 b
C18:3n3	2.33 ± 0.24 a	2.33 ± 0.24 a	2.57 ± 0.11 a	2.57 ± 0.10 a	2.41 ± 0.17 a	2.56 ± 0.11 a	2.58 ± 0.09 a	2.57 ± 0.09 a
C20:2	0.89 ± 0.09 ab	0.89 ± 0.09 a	0.81 ± 0.03 b	0.82 ± 0.05 a	0.89 ± 0.04 ab	0.81 ± 0.04 a	0.94 ± 0.02 a	0.41 ± 0.04 b*
C20:3n6	0.97 ± 0.05 ab	0.97 ± 0.05 a	0.77 ± 0.03 c	1.01 ± 0.09 a	1.07 ± 0.05 a	0.97 ± 0.15 ab	0.83 ± 0.14 bc*	0.13 ± 0.01 b*
C20:4	0.58 ± 0.04 b	0.58 ± 0.04 a	0.57 ± 0.03 b	0.55 ± 0.02 a	0.70 ± 0.02 a	0.65 ± 0.11 a	0.63 ± 0.03 b*	0.43 ± 0.04 b*
C20:3n3	0.15 ± 0.01 a	0.15 ± 0.01 b	0.14 ± 0.01 a	0.15 ± 0.01 b	0.14 ± 0.01 a	0.14 ± 0.02 b	0.15 ± 0.01 a	0.19 ± 0.00 a
C20:5	0.95 ± 0.14 a	0.95 ± 0.14 ab	1.06 ± 0.03 a	0.99 ± 0.00 b	1.00 ± 0.04 a	0.96 ± 0.15 ab	0.98 ± 0.04 a*	1.40 ± 0.05 a*
C22:6	6.23 ± 0.45 bc	6.23 ± 0.45 b	5.79 ± 0.23 c	6.30 ± 0.31 b	7.48 ± 0.33 a	6.97 ± 1.05 ab	6.61 ± 0.28 b*	9.30 ± 0.11 a*
∑SFA	28.68 ± 0.73 b	28.68 ± 0.73 b	29.60 ± 0.29 ab	28.76 ± 0.75 b	30.32 ± 0.75 a	28.36 ± 0.32 b	30.19 ± 0.35 ab	29.43 ± 0.44 a
∑MUFA	35.32 ± 1.64 a	35.32 ± 1.64 ab	33.61 ± 0.44 a	33.87 ± 0.34 a	31.03 ± 1.98 a	33.27 ± 1.56 ab	32.36 ± 0.24 a	32.40 ± 0.44 b
∑PUFA	36.00 ± 1.71 b	36.00 ± 1.71 b	36.79 ± 0.71 ab	37.37 ± 0.44 ab	38.65 ± 1.58 a	38.36 ± 1.25 a	37.45 ± 0.58 ab	38.17 ± 0.17 ab
∑PUFA/∑SFA	1.26 ± 0.08 a	1.26 ± 0.08 b	1.24 ± 0.04 a	1.30 ± 0.05 ab	1.27 ± 0.05 a	1.35 ± 0.03 a	1.30 ± 0.02 a	1.24 ± 0.03 b

Note: Values are given as mean ± SD from triplicate determinations. Different lowercase letters indicate significant differences at different storage times (p < 0.05). Asterisks indicate significant differences at different storage temperatures (* p < 0.05). ∑SFA: total saturated FAs; ∑MUFA: total monounsaturated FAs; ∑PUFA: total polyunsaturated FAs.

. A total of 22 fatty acids (FA), 8 saturated fatty acids (SFA), 5 monounsaturated fatty acids (MUFA), and 9 polyunsaturated fatty acids (PUFA) were detected in the unrefrigerated shad. Saturated fatty acids reached a maximum level at 24 h; as single unsaturated fatty acids declined, PUFA increased. SFA showed no basic change in the period 0 to 24 h; single unsaturated fatty acids declined, but the change was slower in the refrigerated group, and PUFA increased. The FA content differed significantly between the two storage temperatures at 48 h.

3.7. Hydrolyzed Amino Acid Analysis

A total of 17 amino acids were detected in American shad (

Table 5. Hydrolyzed amino acids content of American shad as storage progressed at 24 °C (unrefrigerated) and 4 °C (refrigerated).

Amino Acids (g/100 g)	0 h		6 h		24 h		48 h
	24 °C	4 °C	24 °C	4 °C	24 °C	4 °C	24 °C	4 °C
Asp	7.96 ± 0.38 b	7.96 ± 0.38 a	7.07 ± 0.59 a	8.45 ± 0.38 a	6.68 ± 0.30 a*	8.59 ± 0.10 a*	7.10 ± 0.91 a	7.14 ± 0.87 a
Glu	12.03 ± 0.53 b	12.03 ± 0.53 a	10.73 ± 0.96 a	12.9 ± 0.75 a	10.19 ± 0.46 a*	13.30 ± 0.12 a*	10.96 ± 1.41 a	11.10 ± 1.27 a
Ser	2.59 ± 0.13 b	2.59 ± 0.13 bc	2.28 ± 0.18 ab	2.81 ± 0.12 ab	2.17 ± 0.10 b	2.92 ± 0.04 a	2.51 ± 0.20 a	2.49 ± 0.25 c
His	2.37 ± 0.12 a	2.37 ± 0.12 a	2.04 ± 0.04 b	1.60 ± 0.40 b	1.90 ± 0.13 b	1.88 ± 0.04 b	1.99 ± 0.23 b	1.82 ± 0.10 b
Gly	3.94 ± 0.16 b	3.94 ± 0.16 ab	3.49 ± 0.33 a	4.02 ± 0.22 ab	3.37 ± 0.15 a*	4.30 ± 0.13 a*	3.45 ± 0.39 a	3.66 ± 0.43 b
Thr	3.09 ± 0.15 b	3.09 ± 0.15 ab	2.72 ± 0.23 ab	3.23 ± 0.27 ab	2.59 ± 0.12 ab*	3.43 ± 0.03 a*	2.89 ± 0.32 b	2.90 ± 0.30 b
Arg	4.24 ± 0.17 b	4.24 ± 0.17 ab	3.71 ± 0.29 ab	4.41 ± 0.37 ab	3.52 ± 0.17 b*	4.70 ± 0.07 b*	3.91 ± 0.39 ab	3.98 ± 0.42 a
Ala	4.48 ± 0.19 b	4.48 ± 0.19 a	3.99 ± 0.34 ab	4.72 ± 0.24 a	3.79 ± 0.15 b*	4.91 ± 0.06 a*	4.03 ± 0.46 ab	4.15 ± 0.46 a
Tyr	2.40 ± 0.11 a	2.40 ± 0.11 a	1.97 ± 0.12 bc	2.34 ± 0.26 ab	1.83 ± 0.14 c	2.33 ± 0.16 ab	2.09 ± 0.01 b	1.98 ± 0.22 b
Cys-s	0.24 ± 0.01 a	0.24 ± 0.01 a	0.18 ± 0.02 ab	0.13 ± 0.08 ab	0.17 ± 0.02 b	0.19 ± 0.01 b	0.16 ± 0.05 b	0.15 ± 0.04 ab
Val	4.22 ± 0.19 b	4.22 ± 0.19 ab	3.69 ± 0.37 ab	4.26 ± 0.31 ab	3.55 ± 0.15 b*	4.41 ± 0.06 a*	3.82 ± 0.31 ab	3.93 ± 0.27 b
Met	2.37 ± 0.09 a	2.37 ± 0.09 a	1.89 ± 0.07 b	2.29 ± 0.25 ab	1.81 ± 0.15 b	2.30 ± 0.02 ab	1.99 ± 0.07 b	1.92 ± 0.36 b
Phe	3.29 ± 0.18 b	3.29 ± 0.18 a	2.88 ± 0.24 ab	3.37 ± 0.17 a	2.73 ± 0.12 b*	3.44 ± 0.02 a*	2.87 ± 0.33 ab	2.91 ± 0.32 a
Ile	3.69 ± 0.16 b	3.69 ± 0.16 ab	3.29 ± 0.29 ab	3.87 ± 0.26 a	3.13 ± 0.12 b*	3.99 ± 0.04 a*	3.35 ± 0.38 ab	3.40 ± 0.35 b
Leu	5.92 ± 0.27 b	5.92 ± 0.27 a	5.26 ± 0.46 a	6.37 ± 0.33 a	5.01 ± 0.21 a*	6.50 ± 0.05 a*	5.43 ± 0.63 a	5.49 ± 0.59 a
Lys	7.41 ± 0.38 a	7.41 ± 0.38 a	6.60 ± 0.62 a	6.26 ± 2.81 a	6.26 ± 0.25 a*	8.00 ± 0.11 a*	6.38 ± 1.00 a	6.68 ± 0.74 a
Pro	1.99 ± 0.27 a	1.99 ± 0.27 a	1.69 ± 0.18 a	1.92 ± 0.38 a	1.96 ± 0.58 a	2.00 ± 0.14 a	2.16 ± 0.33 a	2.14 ± 0.56 a
TAA	72.22 ± 3.30 a	72.22 ± 3.30 ab	63.47 ± 5.04 b	72.94 ± 6.48 ab	60.68 ± 2.23 b*	77.19 ± 0.96 a*	65.09 ± 5.99 ab	65.85 ± 7.31 b
EAA	32.35 ± 1.51 a	32.35 ± 1.51 a	28.38 ± 2.25 b	31.24 ± 4.39 a	26.99 ± 1.15 b*	33.95 ± 0.30 a*	28.72 ± 2.59 ab	29.05 ± 2.83 a
NEAA	39.87 ± 1.80 a	39.87 ± 1.80 ab	35.10 ± 2.82 ab	41.69 ± 2.17 a	33.69 ± 1.08 b*	43.24 ± 0.77 a*	36.36 ± 3.42 ab	36.80 ± 4.49 b
E/T	0.45 ± 0.00 a	0.45 ± 0.00 a	0.45 ± 0.00 a	0.43 ± 0.02 a	0.44 ± 0.00 a	0.44 ± 0.00 a	0.44 ± 0.00 a	0.44 ± 0.00 a
N/T	0.55 ± 0.00 a	0.55 ± 0.00 a	0.55 ± 0.00 a	0.57 ± 0.02 a	0.56 ± 0.00 a	0.56 ± 0.00 a	0.56 ± 0.00 a	0.56 ± 0.00 a
E/N	0.81 ± 0.01 a	0.81 ± 0.01 a	0.81 ± 0.01 a	0.75 ± 0.07 a	0.80 ± 0.01 a	0.79 ± 0.01 a	0.79 ± 0.01 a	0.79 ± 0.02 a

Note: Values are given as mean ± SD from triplicate determinations. Different lowercase letters indicate significant differences at different storage times (p < 0.05). Asterisks indicate significant differences at different storage temperatures (* p < 0.05). EAA: Essential amino acids, N-EAA: Non-essential amino acids. TAA: total AA. E/N = ratio of total essential amino acids to total non-essential amino acids; E/T = ratio of essential amino acids to total amino acids; N/T = ratio of total non-essential amino acids to total amino acids.

), including 7 essential amino acids and 10 non-essential amino acids. During storage at 24 °C, from 0 to 24 h the content of hydrolyzed amino acids, essential amino acids and non-essential amino acids decreased gradually (p > 0.05). During refrigeration (4 °C) the content of hydrolyzed amino acids changed little from 0 to 6 h but increased in the period 6–24 h. There was a significant difference between groups in the total amount of essential and non-essential amino acids at 24 h (p < 0.05), but thereafter the difference was not significant.

The AA content per gram of protein is shown in

Table 6. Amino acids (AAs) content per gram of protein of American shad as storage progressed at 24 °C (unrefrigerated) and 4 °C (refrigerated).

AAs	0 h		6 h		24 h		48 h
	24 °C	4 °C	24 °C	4 °C	24 °C	4 °C	24 °C	4 °C
Ile	84.05 ± 8.96 a	84.05 ± 8.96 a	67.44 ± 9.69 a	78.86 ± 13.38 a	59.46 ± 2.79 a**	86.92 ± 2.87 a**	64.23 ± 14.66 a	87.16 ± 11.8 a
Leu	134.91 ± 14.46 a	134.91 ± 14.46 a	107.95 ± 15.95 a	129.62 ± 21.42 a	95.28 ± 4.75 a**	141.53 ± 4.4 a**	104.08 ± 24.19 a	140.97 ± 19.85 a
Lys	168.82 ± 19.48 a	168.82 ± 19.48 a	135.29 ± 19.16 a	125.96 ± 57.97 a	118.97 ± 5.6 a**	174.06 ± 3.89 a**	122.46 ± 32.15 a	171.51 ± 24.36 a
Thr	70.28 ± 7.68 a	70.28 ± 7.68 a	55.97 ± 9.02 a	65.73 ± 11.86 a	49.24 ± 2.57 a**	74.61 ± 2.89 a**	55.38 ± 12.52 a	74.33 ± 10.02 a
Val	96.04 ± 10.25 a	96.04 ± 10.25 a	75.41 ± 8.94 b	86.76 ± 15.44 a	67.37 ± 3.45 b**	95.95 ± 2.17 a**	73.06 ± 14.33 b	100.75 ± 10.3 a
Met + Cys	108.44 ± 8.23 a	108.44 ± 8.23 a	42.79 ± 9.13 b	48.79 ± 4.95 b	37.77 ± 3.75 b**	54.2 ± 1.82 b**	40.88 ± 3.46 b	53.17 ± 11.52 b
Phe + Tyr	129.56 ± 14.79 a	129.56 ± 14.79 a	100.07 ± 18.65 b	116.07 ± 18.19 a	86.86 ± 8.73 b**	125.72 ± 6.36 a**	94.65 ± 16.89 b	125.27 ± 17.15 a
Total content	792.11 ± 83.63 a	792.11 ± 83.63 a	584.93 ± 90.01 b	651.8 ± 119.16 a	514.96 ± 28.43 b**	753 ± 23.83 a**	554.74 ± 117.31 b	753.17 ± 104.39 a

Note: Values are given as mean ± SD from triplicate determinations. Different lowercase letters indicate significant differences at different storage times (p < 0.05). Asterisks indicate significant differences at different storage temperatures (** p < 0.01).

. The EAAS and EAAI of American shad as storage progressed at 24 °C (unrefrigerated) and 4 °C (refrigerated) is shown in

Table 7. Essential amino acids score (EAAS) and essential amino acids index (EAAI) content of American shad as storage progressed at 24 °C (unrefrigerated) and 4 °C (refrigerated).

AAs (mg/g pro)	EAAS
	0 h		6 h		24 h		48 h
	24 °C	4 °C	24 °C	4 °C	24 °C	4 °C	24 °C	4 °C
Ile	280.18 ± 29.85 a	280.18 ± 29.85 a	224.8 ± 32.31 a	262.87 ± 44.6 a	198.2 ± 9.29 a**	289.73 ± 9.57 a**	198.2 ± 9.29 a*	289.73 ± 9.57 a*
Leu	228.67 ± 24.52 a	228.67 ± 24.52 a	182.96 ± 27.03 a	219.69 ± 36.3 a	161.5 ± 8.05 a**	239.88 ± 7.46 a**	161.5 ± 8.05 a	239.88 ± 7.46 a
Lys	375.15 ± 43.29 a	375.15 ± 43.29 a	300.65 ± 42.57 a	279.92 ± 128.83 a	264.38 ± 12.43 a**	386.81 ± 8.63 a**	264.38 ± 12.43 a	386.81 ± 8.63 a
Thr	305.57 ± 33.37 a	305.57 ± 33.37 a	243.36 ± 39.23 a	285.8 ± 51.55 a	214.09 ± 11.19 a**	324.39 ± 12.58 a**	214.09 ± 11.19 a*	324.39 ± 12.58 a*
Val	246.26 ± 26.27 a	246.26 ± 26.27 a	179.55 ± 21.29 b	222.45 ± 39.59 a	172.75 ± 8.84 b**	246.03 ± 5.56 a**	172.75 ± 8.84 b**	246.03 ± 5.56 a**
Met + Cys	492.9 ± 37.42 a	492.9 ± 37.42 a	194.49 ± 41.51 b	221.78 ± 22.52 b	171.7 ± 17.04 b**	246.37 ± 8.27 b**	171.7 ± 17.04 b	246.37 ± 8.27 b
Phe + Tyr	340.95 ± 38.91 a	340.95 ± 38.91 a	263.35 ± 49.08 b	305.46 ± 47.87 a	228.59 ± 22.96 b**	330.85 ± 16.73 a**	228.59 ± 22.96 b*	330.85 ± 16.73 a*
EAAI	223.73 ± 23.42 a	223.73 ± 23.42 a	160.5 ± 25.31 b	179.53 ± 31.87 a	141.4 ± 7.79 b**	196.11 ± 3.57 a**	153.04 ± 30.58 b	206.84 ± 29.55 a

Note: Values are given as mean ± SD from triplicate determinations. Different lowercase letters indicate significant differences at different storage times (p < 0.05). Asterisks indicate significant differences at different storage temperatures (* p < 0.05, ** p < 0.01).

. The first limiting AA in all groups was leucine. The average EAAS of each essential AA in American shad during storage exceeded the requirements of the combined WHO, FAO and UNU standard model (>100%), indicating that the AA content in American shad can meet the nutritional requirements of the human body, and has a high absorption and utilization rate. EAAI decreased with time at 24 °C (unrefrigerated) (p < 0.05) at normal temperature. There was no significant difference in the refrigerated group within 48 h (p > 0.05), but there was a downward trend.

3.8. Free Amino Acid Analysis

Postmortem changes in the free amino acid (FAA) content of American shad during storage are listed in

Table 8. Free amino acid content of American shad as storage progressed at 24 °C (unrefrigerated) and 4 °C (refrigerated).

Amino Acids (g/kg)	Flavor Contribution	T0		T6		T24		T48
		24 °C	4 °C	24 °C	4 °C	24 °C	4 °C	24 °C	4 °C
Asp	Fresh/Acid (+)	0.15 ± 0.01 a	0.15 ± 0.01 a	0.16 ± 0.01 a**	0.13 ± 0.01 b**	0.15 ± 0.01 a	0.14 ± 0.00 bc	0.16 ± 0.01 a*	0.14 ± 0.00 c*
Glu	Fresh/Acid (+)	0.28 ± 0.03 b	0.28 ± 0.03 b	0.78 ± 0.10 a**	0.31 ± 0.04 a**	0.52 ± 0.14 ab	0.32 ± 0.04 a	0.34 ± 0.01 ab**	0.22 ± 0.03 a**
Ser	Sweet (+)	0.08 ± 0.02 a	0.08 ± 0.02 a	0.07 ± 0.03 a	0.04 ± 0.00 b*	0.07 ± 0.01 a	0.04 ± 0.01 b	0.08 ± 0.00 a*	0.04 ± 0.02 b*
His	Bitter (-)	5.48 ± 0.58 a	5.48 ± 0.58 a	5.49 ± 1.37 a	6.23 ± 1.31 b	6.13 ± 1.28 a	6.63 ± 1.25 b	7.68 ± 0.35 a	8.94 ± 0.70 a
Gly	Sweet (+)	0.42 ± 0.06 b	0.42 ± 0.06 b	0.68 ± 0.15 a	0.46 ± 0.15 a	0.51 ± 0.14 ab	0.42 ± 0.09 a	0.61 ± 0.03 ab**	0.45 ± 0.03 a**
Thr	Sweet (+)	0.27 ± 0.05 a	0.27 ± 0.05 a	0.57 ± 0.13 ab*	0.26 ± 0.04 b*	0.32 ± 0.04 a	0.29 ± 0.04 ab	0.48 ± 0.03 b**	0.34 ± 0.02 a**
Arg	Sweet/Bitter (+)	0.18 ± 0.02 a	0.18 ± 0.02 a	0.19 ± 0.04 ab	0.16 ± 0.03 a	0.16 ± 0.05 ab	0.16 ± 0.00 a	0.26 ± 0.01 b	0.19 ± 0.07 a
Ala	Sweet (+)	0.84 ± 0.15 ab	0.84 ± 0.15 ab	1.75 ± 0.40 ab*	0.98 ± 0.21 a*	0.98 ± 0.09 b	1.05 ± 0.08 a	1.51 ± 0.05 a*	1.38 ± 0.02 a*
Tyr	Bitter (-)	0.06 ± 0.01 b	0.06 ± 0.01 b	0.22 ± 0.05 a**	0.04 ± 0.01 b**	0.06 ± 0.04 b	0.06 ± 0.01 b	0.12 ± 0.01	0.09 ± 0.01 a
Cys-s		0.03 ± 0.04 a	0.03 ± 0.04 a	0.10 ± 0.03 a	0.14 ± 0.03 b	0.07 ± 0.07 a	0.15 ± 0.03 b	0.03 ± 0.03 a**	0.21 ± 0.01 b**
Val	Sweet/Bitter (-)	0.13 ± 0.02 a	0.13 ± 0.02 a	0.54 ± 0.13 ab**	0.18 ± 0.03 c**	0.7 ± 0.04 ab	0.23 ± 0.03 bc	0.31 ± 0.03 b	0.30 ± 0.01 a
Met	Bitter/Sweet/Sulfur (-)	0.08 ± 0.02 c	0.08 ± 0.02 c	0.27 ± 0.06 a*	0.11 ± 0.03 bc*	0.13 ± 0.03 bc	0.13 ± 0.01 b	0.16 ± 0.03 b	0.19 ± 0.01 a
Phe	Bitter (-)	0.06 ± 0.02 a	0.06 ± 0.02 a	0.27 ± 0.08 a**	0.04 ± 0.01 b**	0.11 ± 0.02 a*	0.05 ± 0.00 bc*	0.12 ± 0.01 a**	0.08 ± 0.01 a**
Ile	Bitter (-)	0.07 ± 0.01 a	0.07 ± 0.01 a	0.23 ± 0.06 ab*	0.07 ± 0.01 a*	0.10 ± 0.0 a	0.08 ± 0.01 a	0.16 ± 0.00 b**	0.12 ± 0.00 b**
Leu	Bitter (-)	0.12 ± 0.02 a	0.12 ± 0.02 a	0.50 ± 0.13 ac**	0.12 ± 0.02 a**	0.21 ± 0.01 c*	0.14 ± 0.01 a*	0.28 ± 0.00 ab*	0.08 ± 0.12 a*
Lys	Sweet/Bitter (-)	1.43 ± 0.16 b	1.43 ± 0.16 b	2.03 ± 0.72 ab	1.61 ± 0.14 a*	1.41 ± 0.68 ab	1.77 ± 0.31 a	2.43 ± 0.04 a*	1.58 ± 0.62 a*
Pro	Sweet/Bitter (+)	0.66 ± 0.15 ab	0.66 ± 0.15 ab	0.39 ± 0.07 c	0.36 ± 0.08 b	0.43 ± 0.13 bc	0.36 ± 0.01 b	0.72 ± 0.15 a	0.51 ± 0.18 ab
Fresh, sweet free amino acids		2.70 ± 0.43 d	2.70 ± 0.43 d	4.40 ± 0.80 a*	2.54 ± 0.08 a*	2.99 ± 0.41 cd	2.62 ± 0.25 a	3.90 ± 0.15 abc**	3.08 ± 0.20 a**
Bitter free amino acids		7.60 ± 0.83 b	7.60 ± 0.83 b	9.75 ± 2.28 ab*	8.56 ± 1.30 b*	8.58 ± 1.16 ab	9.25 ± 1.63 b	11.52 ± 0.43 a	11.57 ± 0.27 a
TAA		10.33 ± 1.22 c	10.33 ± 1.22 a	14.25 ± 3.11 ab	11.24 ± 1.72 b	11.64 ± 2.04 bc	12.02 ± 1.90 b	15.45 ± 0.32 a*	14.85 ± 0.19 a*

Note: Values are given as mean ± SD from triplicate determinations. Different lowercase letters indicate significant differences at different storage times (p < 0. 05). Asterisks indicate significant differences at different storage temperatures (* p < 0.05, ** p < 0.01). TAA: total AAs.

; 17 different free amino acids were detected. The content of sweet amino acids was higher in the unrefrigerated group. At 6 h, Ser, Thr, Arg, Tyr, Phe, Ile, Leu, and Lys differed significantly between the storage groups (p < 0.05); the total amounts of sweet amino acids, bitter amino acids, and free amino acids in the shad increased during unrefrigerated storage compared with the levels at 0 h.

3.9. Electronic Tongue Analysis

As shown in Figure 2, the PCA results are displayed as two-dimensional scatter plots. Each plot consists of two axes, PC1 and PC2. The PC1 explains 89.42% of the sample variance and PC2 explains 6.42% in the unrefrigerated group (Figure 2A). The PC1 explains 78.75% of the sample variance and PC2 explains 19.72% during refrigeration (Figure 2B). Postmortem changes in the radar chart of volatile compounds in American shad during storage are listed in Figure 3 and

Table 9. Taste profiles of American shad muscle at 24 °C (unrefrigerated) and 4 °C (refrigerated).

Temperature	Time	Saltiness	Bitterness	Aftertaste-A	Umami	Richness
24 °C	0 h	−1.02 ± 0.08 d	5.02 ± 0.07 ab	−0.85 ± 0.01 b	15.72 ± 0.21 ab	3.18 ± 0.08 a
	6 h	0.70 ± 0.04 a	4.94 ± 0.08 b	−0.93 ± 0.06 ab	16.55 ± 0.06 a	4.29 ± 0.40 a
	24 h	−0.27 ± 0.08 c	5.01 ± 0.05 ab	−0.91 ± 0.04 b	16.12 ± 0.04 b	3.36 ± 0.10 a
	48 h	0.53 ± 0.03 b	5.13 ± 0.02 a	−1.00 ± 0.06 a	16.46 ± 0.13 ab	3.9 ± 0.56 a
4 °C	0 h	−1.02 ± 0.08 c	5.02 ± 0.07 b	−0.85 ± 0.01 a	15.72 ± 0.21 c	3.18 ± 0.08 a
	6 h	−0.01 ± 0.08 a	4.87 ± 0.09 b	−0.98 ± 0.08 b	16.42 ± 0.07 b	3.77 ± 0.71 a
	24 h	−0.39 ± 0.04 b	5.00 ± 0.08 b	−0.93 ± 0.06 ab	16.10 ± 0.09 b	3.88 ± 0.54 a
	48 h	−1.06 ± 0.01 d	5.33 ± 0.11 a	−0.96 ± 0.04 b	16.89 ± 0.20 a	−0.68 ± 0.11 b

Note: Values are given as mean ± SD from triplicate determinations. Different lowercase letters indicate significant differences at different storage times (p < 0.05).

. Saltiness and bitterness differed significantly in the unrefrigerated group (p < 0.05). Saltiness, bitterness, aftertaste-A, umami, and richness differed significantly during refrigeration (4 °C) (p < 0.05).

4. Discussion

4.1. Color

Color is an important index to evaluate the quality of fish, as this aspect of its appearance directly affects consumers’ purchasing desire and the market value of the product. Generally, lipid oxidation during storage changes the color of fish meat [27]. The initial brightness (L*) value of the meat was 55.56; at 48 h, the L* values had decreased to 49.60 (24 °C) and 51.54 (4 °C). This downward trend may be attributable to a decrease in water content in the tissue, resulting in less light reflectance on the fish surface and a gradually darkened color [28]. The initial a* value of the meat was 5.90; at 48 h, the a* values were 2.95 (24 °C) and 3.32 (4 °C), showing a downward trend. Fish muscle browns as hemoglobin and myoglobin combine with oxygen to form oxymyoglobin and methemoglobin, making the fish appear red–orange. With the extension of storage time, oxymyoglobin is gradually oxidized to ferrimyoglobin, gradually turning the color of the fish meat to brownish–yellow, resulting in a lower a* value. A decrease in the a* value may also be caused by lipid oxidation, which produces large amounts of free radicals, H₂O₂, and some oxidation products in the fish muscle, promoting the oxidation of astaxanthin and Fe²⁺ in myoglobin and hemoglobin, and accelerating the browning of the flesh [16]. In addition, severe brown discoloration occurs when these substances react with ammonia, amines, heme, and other components in the fish pieces [29]. It has been reported that the value of b* correlates with lipid oxidation. The initial b* value of the meat was 6.44; at 48 h, the value had risen to 11.77 (24 °C) or 8.59 (4 °C). Free radicals and carbonyl compounds produced by unsaturated fatty acid oxidation in fish can react with free amino acids in proteins to synthesize brown pigments [30]. The decreasing trend of a* and b* values in late storage may correlate with blackening.

4.2. Shear Stress and Cooking Loss

The texture of fish pieces is a characteristic index to determine the monetary value of fish and affects the attractiveness of the product to consumers. The change of shear stress is correlated with the texture of fish, which indirectly reflects the change of the fish tissue structure [31,32]. The initial shear stress of a block of American shad meat was 14.02 N, and the shear force values were 5.33 N (24 °C) and 7.85 N (4 °C) at 6 h. The value of shear stress decreased rapidly at first, and then slowed, possibly because of the loss of water in the cells of tissues, which destroys the cytoskeleton structure and reduces the shear stress. The texture of fish is also closely related to the connective tissue; the collagen fibers in the connective tissue can maintain the toughness and integrity of the texture of fish [33,34]. Yang et al. [35] found that degeneration of low molecular weight myofibrillar structural proteins, such as desmin and troponin-T, resulted in Z-disk weakening and actin loosening. With the prolongation of storage time, more macromolecular myofibrillar structural proteins (such as myponectin and concomitant actin) were degraded, and the Z-disk and M-band disintegrated, leading to a sharp decrease in shear force.

Heat denaturation of muscle protein is the main mechanism leading to water loss in muscle. Most water in muscles is found in the myofibrils—narrow channels between thick and thin filaments. Heat causes myosin degeneration and contraction of the myofibrils, followed by the expulsion of water. Most of the loss during cooking is water; other components lost are lipids and solids, including collagen or gelatin, muscle fragments and congealed sarcoplasmin [34]. The changes in the rate of cooking loss are illustrated in Figure 1B. In shad that had been stored for 6 h, the cooking loss rates were 10.68% (24 °C) and 6.16% (4 °C), compared with 6.29% initially, probably due to less water binding to the myofibrin. After 6 h, the rate gradually increased, and was 12.22% (24 °C) and 11.96% (4 °C) at 48 h. The cell wall of fish muscle tissue is considered relatively elastic, but when the meat becomes soft it loses its ability to bind water [13].

4.3. TVB-N and pH

TVB-N is one of the most important indicators of freshness, and refers to the decomposition of protein and non-protein nitrogen compounds by microorganisms and enzymes to produce volatile ammonia and basic nitrogen compounds, such as dimethylamine and trimethylamine [36]. As shown in Figure 1C, from the start of storage the TVB-N value increased slowly; in the samples stored at 24 °C, the TVB-N content reached 27.00 mg/100 g after 48 h (p < 0.05), thus deviating from the freshness range. In the shad stored at 4 °C, the TVB-N content remained steady and the values were far below the limit up to the end of the 48-h storage period [37]. With rapid propagation of microorganisms, decay accelerates, resulting in a large amount of trimethylamine, sulfide, organic acids, aldehydes, ketones, and a series of small molecular metabolites with a rotten smell. The pH of meat has a different impact on the freshness and quality of fish. As can be seen from Figure 1D, fresh fish is acidic. With the extension of storage time, the pH value of the fish pieces decreased slightly and then increased gradually, showing an overall upward trend [38]. The unrefrigerated fish (24 °C) had a higher pH than refrigerated fish (4 °C) during the storage period. This is likely attributable to the role of microorganisms and endogenous enzymes under the conditions of adequate oxygen and nutrition, as protein decomposition produces alkaline substances, which destroys the original redox balance, changes the concentration of hydrogen ions and free radical ions, and then changes the pH value of the fish flesh. Likewise, refrigeration of fish could well reduce the activity of endogenous enzymes, inhibit the growth of microorganisms, and also delay the color change, shear force decline, cooking loss rate, TVB-N content, and rise in pH rate. Refrigeration may also reduce the decarboxylation and dehydrogenation ability of non-protein nitrogen-containing substances, thereby reducing the degree of damage to the protein [39].

4.4. Proximate and Fatty Acids

Based on the usual biochemical markers, the water content of American shad varied from 65.74% to 74.15% during storage at 24 °C for 48 h. Changes in water content during storage may be caused by the loss of proteins and other soluble compounds. American shad refrigerated for 48 h had a lower moisture content than unrefrigerated fish. Moreover, refrigeration slows the damage of tissues and cells. The decrease in protein content may be caused by leaching of soluble components, especially water-soluble protein and urea during storage [40,41]. The decrease in lipid content may be linked with the oxidation of PUFA found in fish tissues, through other products such as aldehydes, free fatty acids, ketones, and peroxides [42].

The composition and proportion of fatty acids are important indicators of the nutritional value of a food. Palmitic (C16:0), oleic (C18:1), and linolenic (C18:2) acids are abundant in American shad (Table 4). Similar findings have been reported for golden mullet fillet [43]. The content of oleic acid (C18:1) is an important index of food quality [44,45]. Reportedly, diets with high concentrations of PUFA significantly reduce the incidence of cardiovascular diseases by lowering blood lipids, inhibiting platelet aggregation, lowering blood pressure, improving biofilm fluidity, anti-tumor capacity, immune regulation, and other effects [46]. In the present study, oleic acid accounted for the highest proportion of FAs in the muscle of A. sapidissima, which shows the benefits of eating A. sapidissima to human health. The PUFA/SFA ratio is commonly used to assess the nutritional quality of meat, as a higher PUFA/SFA value indicates healthier meat [47,48]. In this study, analysis of the ΣSFA, ΣPUFA, and ∑PUFA/∑SFA values of the four groups showed that the meat had better nutritional value at 6–24 h during storage.

4.5. Amino Acid

The amino acid content in American shad is another important indicator of its nutritional value. The greatest component in the muscle was glutamic acid, followed by aspartic acid as well as lysine. Glutamic acid not only provides a better taste, but is also an important amino acid in brain tissue biochemical metabolism, participating in the synthesis of a variety of physiologically active substances. Lysine content was also high; lysine is the first limiting amino acid in human milk, making this fish species a good galactagogue food. In addition, American shad also contains abundant aspartic acid branch-chain amino acids which protect the liver, inhibit cancer cells, and reduce cholesterol, among other effects [49]. Amino acid content is also an important index used to evaluate the nutritional value of fish, among which the ratio of essential amino acids to total amino acids (E/T) is an important index to evaluate the nutritional value of amino acids. It was found that the E/T ratio in good-quality proteins is approximately 0.4. The ratio of total essential amino acids to total non-essential amino acids (E/N) was above 0.6 [50]. Table 5 shows that the E/T ratio of both the unrefrigerated and refrigerated shad was about 0.4 at 48 h, indicating that both storage groups retained sufficient muscle amino acids. The E/T ratio was higher in fish stored at 24 °C compared with 4 °C, but the E/N ratio was lower in the former group. The E/T ratio in the unrefrigerated fish was basically unchanged from 0 to 6 h, and thereafter decreased gradually. The E/T ratio in the refrigerated fish showed little change during storage.

From the perspective of nutrition, it can be seen that the nutritional value of food proteins largely depends on the content and composition of essential amino acids [51]. To compare and evaluate the quality of AAs of each sample, the AA content of each sample was converted into milligrams of AAs per gram of protein, and then the nutritional value was evaluated according to the FAO/WHO optimal ratio model [25]. EAAI is one of the most commonly used indexes to evaluate the nutritional value of proteins. The higher the value, the higher the protein utilization. In this study, the total amount of AAs and the EAI index in the normal temperature group and the refrigerated group showed a downward trend, and the protein utilization rate decreased with the extension of storage time. The decrease of the EAAI index at 24 °C was slower than that at 4 °C. These results indicate that cold storage has a positive effect on the nutritional quality of American shad.

FAAs are important flavor components, providing tastes such as sweet, sour, bitter, and umami, but their content also directly affects the freshness of food. There are seven fresh, sweet amino acids, namely: Asp, Thr, Ser, Glu, Gly, Ala, and Pro. Bitter amino acids include Val, Met, Ile, Leu, Tyr, Phe, Lys, His, and Arg [52]. Thr, Gly, Ala and His were identified as the majority FAAs in American shad (Table 8). Under storage at 24 °C for 6 h, the contents of Glu, Gly, and Thr were 0.78, 0.68, and 0.57 g/kg, respectively, and then were decreased. At 24 °C, the sweet amino acid reached a maximum at 6 h, and the bitter amino acid reached a maximum at 48 h. This suggests that American shad will taste better if eaten within 6 h of harvest. During refrigerated storage, the total amount of FAA in refrigerated fish increased gradually, and there was a significant difference in bitter free amino acids between the groups at 6 h (p < 0.05). The FAA content rose with the duration of storage, although slowly; this is probably because the protein under the action of microbes and protease generates FAAs, increasing the content of FAA; in fish refrigerated at 4 °C, there will be less microbial enzyme activity than if stored at 24 °C, therefore the FAAs increased more slowly. However, differences in the FAA profiles could also be related to other aspects, such as diet, water temperature, or storage time [53].

4.6. Electronic Tongue

A principal component analysis was also used to reflect the results of the E-tongue analysis (Figure 2). In this study, the total contribution rate was 95.84% at 24 °C and 95.47% at 4 °C during storage. PCA more accurately reflects the variations in the taste of the American shad at different storage times. In general, the total contribution rate was more than 85% [54,55]. The scores of American shad at different storage times were not overlapped but scattered; the results showed that there were differences in storage at different temperatures. As shown in Figure 3 and Table 9, during unrefrigerated storage (24 °C), the saltiness of American shad increased and then decreased with time (p < 0.05), bitterness was significantly increased (p < 0.05), and the umami increased first and then decreased, so the American shad had higher umami taste, lower bitterness and higher flavor substance at 6 h. The saltiness of American shad decreased gradually, and the bitter substances were significantly different at 48 h (p < 0.05). The umami increased gradually. The saltiness and sourness of American shad decreased [56]. Time had a certain effect on the saltiness, bitterness and richness of American shad. The chemical indexes combined with the taste substances suggested that American shad kept at 4 °C should be eaten within 24 h.

5. Conclusions

This study investigated changes of meat quality in American shad after harvest when stored at 24 °C and 4 °C. The nutrient composition showed that the contents of fat and protein decreased while the content of water increased during storage. We found that the pH was closely related to the firmness of the fish across the 48-h storage period. In terms of the total quality of fresh amino acids, American shad is acceptable to consume within 6 h of harvest if stored unrefrigerated. The TVB-N content increased gradually with the duration of storage. The color differences were consistent with the TVB-N value and other chemical parameters. At 24 °C, the sweet amino acid reached a maximum at 6 h, and the bitter amino acid reached a maximum at 48 h. At 4 °C, there was a significant difference in bitter free amino acids at 48 h (p < 0.05). Compared with the unrefrigerated fish stored at 24 °C, the fatty acid content, shear stress, rate of cooking loss, and TVB-N content were greater in the refrigerated group (4 °C). Based on these results, combined with the electronic tongue index, it is recommended that American shad kept at 24 °C should be eaten within 6 h, but if kept at 4 °C the fish will retain freshness for 24 h, and such cold storage obtains better product quality. These results provide useful information for producers and product developers trying to improve the quality of American shad products. Further studies of the enzyme activity in shad postmortem will improve our understanding of this process, and thereby allow us to develop specific methods that help keep shad fresh.