Composite biological preservative for penaeus vannamei boone

Abstract

The invention discloses a composite biological fresh-keeping agent for penaeus vannamei boone. The composite biological fresh-keeping agent is prepared from three biological fresh-keeping agents including tremella polysaccharide, Nisin and lysozyme, and water. The composite biological fresh-keeping agent is prepared from the following components in percentage by mass: 0.6% of tremella polysaccharide, 0.6% of vitamin C, 0.6% of vitamin C and the balance of water, wherein the total amount is 100% and the unit is g / ml; the content of the Nisin is 0.03%; the content of the lysozyme is 0.05%; and the balance of water. The composite preservative disclosed by the invention has the capabilities of resisting oxidation, destroying a microbial structure and inhibiting enzyme activity, so that the effects of preserving freshness and inhibiting blackening are achieved, and compared with a single biological preservative, the composite preservative disclosed by the invention is more remarkable in preservation effect.

Description

Technical Field

[0001] The invention relates to the technical field of aquatic product preservation, in particular to a composite biological preservative for white shrimp. Background Art

[0002] In recent years, with the rapid popularization of healthy diet, people pay more and more attention to food nutrition. Shrimp meat has the characteristics of high protein, low fat and low calories, which meets people's definition of healthy food, so the demand for shrimp at home and abroad is on the rise.

[0003] The whiteleg shrimp, scientifically known as Penaeus vannamei, is famous for its delicious taste and high nutritional content. It is edible for both the elderly and children and has won unanimous praise from consumers at home and abroad. It is an important economic species in the world, accounting for 90% of the global aquaculture shrimp production.

[0004] However, after the death of whiteleg shrimp, due to the influence of factors such as storage temperature, storage time and storage method, changes such as lipid oxidation, protein degradation and massive reproduction of microorganisms will occur, causing the shrimp to deteriorate and blacken, etc., which limits the shelf life of the shrimp and affects its sales and circulation.

[0005] At present, the main preservation technologies for whiteleg shrimp include low-temperature preservation technology, controlled atmosphere preservation packaging technology, chemical preservation technology, and biological preservation technology.

[0006] However, the quality of shrimp meat preserved at low temperatures tends to deteriorate after thawing, controlled atmosphere preservation requires high standards of equipment and operation, and excessive use of chemical preservatives can cause potential harm to the human body and the environment. Summary of the invention

[0007] In order to solve the above problem, the present invention provides a composite biological preservative for white shrimp to solve the problem.

[0008] In order to achieve the above objectives, this application provides the following technical solutions:

[0009] A composite biological preservative for white shrimp comprises four components: tremella polysaccharide, Nisin, lysozyme and water, wherein the mass proportions of the tremella polysaccharide, Nisin and lysozyme are respectively tremella polysaccharide: 0.6%; Nisin: 0.03%; lysozyme: 0.05%, and the rest is water.

[0010] Compared with the prior art, the beneficial technical effects of the present invention are:

[0011] 1. The biological fresh-keeping solution prepared by the present invention has an antioxidant effect, can inhibit the oxidation and rancidity of fat and the self-degradation of protein, and slow down the loss of shrimp nutrition;

[0012] 2. The biological fresh-keeping solution prepared by the present invention also has an antibacterial effect, can effectively delay the increase of the total number of bacteria and volatile basic nitrogen, has a good water-locking function, slows down the rate of water loss of shrimps during transportation or storage, and greatly prolongs the fresh-keeping time of shrimps while ensuring the flavor and taste of shrimps. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0014] Figure 1 Schematic diagram of the effects of different concentrations of Tremella polysaccharide solution, Nisin solution and lysozyme solution on the total bacterial count.

[0015] Figure 2 Schematic diagram of the effects of Tremella polysaccharide and sodium sulfite on the blackening of white shrimp.

[0016] Figure 3 Schematic diagram of the effects of Tremella polysaccharide and sodium sulfite on the pH value of white shrimp.

[0017] Figure 4 Schematic diagram of the effects of Tremella polysaccharide and sodium sulfite on the TVB-N value of white shrimp.

[0018] Figure 5 This is a schematic diagram of the effects of Tremella polysaccharides and sodium sulfite on the TBA value of white shrimp.

[0019] Figure 6 Schematic diagram of the effects of Tremella polysaccharide and sodium sulfite on the color difference of white shrimp.

[0020] Figure 7 Schematic diagram of the effects of Tremella polysaccharide and sodium sulfite on the water holding capacity of white shrimp.

[0021] Figure 8 Schematic diagram of the effects of Tremella polysaccharide and sodium sulfite on the total bacterial count of Penaeus vannamei.

[0022] Fig. 9 Schematic diagram of the effects of composite biological preservatives and Tremella polysaccharide on the blackening of white shrimp.

[0023] Fig.10 Schematic diagram of the effects of composite biological preservatives and Tremella polysaccharide on the pH value of white shrimp.

[0024] Fig.11Schematic diagram of the effects of composite biological preservatives and Tremella fuciformis polysaccharides on the TVB-N value of white tremella.

[0025] Fig.12 Schematic diagram of the effects of composite biological preservatives and Tremella polysaccharide on the TBA value of white shrimp.

[0026] Fig.13 Schematic diagram of the effects of composite biological preservatives and Tremella polysaccharide on the color difference of white shrimp.

[0027] Fig.14 Schematic diagram of the effects of composite biological preservatives and Tremella polysaccharide on the water holding capacity of white shrimp.

[0028] Fig.15 Schematic diagram of the effects of composite biological preservatives and Tremella polysaccharide on the total bacterial count of Penaeus vannamei.

[0029] Fig.16 Schematic diagram of the effect of Tremella polysaccharide composite biological preservative on water migration of white shrimp.

[0030] Fig.17 Schematic diagram of the effect of Tremella polysaccharide composite biological preservative on the protein of white shrimp.

[0031] Fig.18 Schematic diagram of the effect of Tremella fuciformis polysaccharide composite biological preservative on the microstructure of muscle tissue of white shrimp.

[0032] Fig.19 Schematic diagram of dilution curve (A) and Shannon-Wiener curve (B) analysis.

[0033] Fig. 20 Schematic diagram of the Alpha diversity index analysis of each group of samples.

[0034] Fig.21 Schematic diagram of the relative abundance of bacterial flora in each group of samples at the order level.

[0035] Fig. 22 Schematic diagram of the relative abundance of bacterial communities in each group of samples at the family level. DETAILED DESCRIPTION

[0036] Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in the field without making any creative work shall fall within the scope of protection of the present invention.

[0037] Embodiment 1:

[0038] The composite biological preservative for white shrimp disclosed in this embodiment comprises 40-80 parts of Tremella polysaccharide solution, 1-3 parts of Nisin solution and 3-5 parts of lysozyme solution, and the preparation method thereof comprises the following steps:

[0039] Step 1: weigh 50% of Tremella polysaccharide, add distilled water to make the volume to 500 mL, and prepare a Tremella polysaccharide solution; weigh Nisin, add distilled water to make the volume to 500 mL, and prepare a Nisin solution; weigh lysozyme, add distilled water to make the volume to 500 mL, and prepare a lysozyme solution;

[0040] Step 2: Evenly mix the solution prepared in step 1 to prepare a composite biological preservative, and soak it for preserving whiteleg shrimp;

[0041] Furthermore, the concentration ratio of Tremella polysaccharide solution, Nisin solution and lysozyme solution is (40-80):(1-3):(3-5).

[0042] Embodiment 2:

[0043] The composite biological preservative for white shrimp disclosed in this embodiment comprises a Tremella polysaccharide solution, a Nisin solution and a lysozyme solution in a concentration ratio of 20:1:1, and a preparation method thereof comprises the following steps:

[0044] Step 1: weigh 50% of Tremella polysaccharide, add distilled water to make the volume to 500 mL, and prepare a Tremella polysaccharide solution; weigh Nisin, add distilled water to make the volume to 500 mL, and prepare a Nisin solution; weigh lysozyme, add distilled water to make the volume to 500 mL, and prepare a lysozyme solution;

[0045] Step 2: Evenly mix the solution prepared in step 1 to prepare a composite biological preservative for soaking and preserving the white shrimp;

[0046] Furthermore, the concentration ratio of the Tremella polysaccharide solution, the Nisin solution and the lysozyme solution is 20:1:1.

[0047] Comparative Example 1:

[0048] Weigh 2g, 4g, 6g, 8g, and 10g of 50% Tremella polysaccharide, add distilled water to 500mL, and make the concentrations of 0.2% (corresponding to Figure 1 TP1 in), 0.4% (corresponding to Figure 1 TP2 in), 0.6% (corresponding to Figure 1 TP3 in), 0.8% (corresponding to Figure 1 TP4 in), 1.0% (corresponding to Figure 1 The Tremella polysaccharide solution of TP5 in the five different concentrations was used to soak and preserve the whiteleg shrimp.

[0049] Comparative Example 2:

[0050] Weigh 0.05g, 0.1g, 0.15g, 0.2g, and 0.25g of Nisin, add distilled water to 500mL, and make the concentrations into 0.01% (corresponding to Figure 1 NI1), 0.02% (corresponding to Figure 1 NI2 in), 0.03% (corresponding to Figure 1 NI3 in), 0.04% (corresponding to Figure 1 NI4), 0.05% (corresponding to Figure 1 Nisin solution of NI5) in the mixture was prepared, and five Nisin solutions of different concentrations were used to soak and preserve whiteleg shrimp.

[0051] Comparative Example 3:

[0052] Weigh 0.05g, 0.1g, 0.15g, 0.2g, and 0.25g of lysozyme, add distilled water to 500mL, and make the concentrations into 0.01% (corresponding to Figure 1 LY1 in), 0.02% (corresponding to Figure 1 LY2 in), 0.03% (corresponding to Figure 1 LY3 in), 0.04% (corresponding to Figure 1 LY4 in), 0.05% (corresponding to Figure 1 The lysozyme solution of LY5) in the five different concentrations was used to soak and preserve the whiteleg shrimp.

[0053] The present invention uses the sensory evaluation, TVB-N value and total bacteria count of white shrimp stored at 4°C for 8 days as evaluation indicators, and conducts a single factor bacterial inhibition effect test on the solutions prepared in Comparative Example 1, Comparative Example 2 and Comparative Example 3 (the experimental results are shown in Figure 1 ); According to the results of the single factor test, the present invention selects three concentrations of Tremella polysaccharide, Nisin and lysozyme, conducts a three-factor, three-level orthogonal test, and screens the composite biological preservative with the best ratio, wherein the sensory evaluation standard of Penaeus vannamei is shown in Table 1, and the orthogonal test design and results of the composite biological preservative are shown in Table 2:

[0054] Table 1: Sensory evaluation criteria for white shrimp:

[0055]

[0056]

[0057] Table 2: Orthogonal test design and results of composite biological preservatives (effects of different concentrations of Tremella polysaccharide, Nisin, and lysozyme on white shrimp)

[0058]

[0059]

[0060] From Table 1 and Table 2, we can see that the indexes of white shrimp vary with the influence of various factors. According to the range value R, we can know the priority order of various factors, and thus determine the optimal ratio of the composite biological preservative. The priority order is: sensory evaluation is A>C>B, TVB-N value is C>A>B, and total bacteria count is B>C>A;

[0061] Among them, the range value is also called the range error or the full range, represented by R, which is used to indicate the amount of variation in statistical data, the difference between the maximum value and the minimum value, that is, the data obtained by subtracting the maximum value from the minimum value. The range = maximum mark value - minimum mark value. The larger the range value R, the more significant the influence of the factor.

[0062] According to the data in the table, the optimal ratio combination is A2, B3, and C3, that is, a composite biological preservative composed of a 0.6% tremella polysaccharide solution, a 0.03% Nisin solution, and a 0.05% lysozyme solution.

[0063] Experimental Example 1:

[0064] Step 1: Clean the whiteleg shrimp and kill them suddenly in crushed ice. Select the shrimps with intact bodies and a mass of (10±2) g and randomly divide them into 7 groups, with 30 shrimps in each group.

[0065] Step 2: soak the 7 groups of shrimps in 0.2% Tremella polysaccharide solution (TP1), 0.4% Tremella polysaccharide solution (TP2), 0.6% Tremella polysaccharide solution (TP3), 0.8% Tremella polysaccharide solution (TP4), 1% Tremella polysaccharide solution (TP5), 1% sodium sulfite solution (CK1) and distilled water (CK2) for 30 min respectively;

[0066] Step 3: After soaking, take out the shrimps, drain them, and pack them into sealed bags. Each group has 5 bags, and each bag has 6 shrimps.

[0067] Step 4: The packaged shrimps were stored at 4°C, and the sensory score of the white shrimps was measured every 2 days (the results are shown in Table 3) and the blackening score was measured (the results are shown in Table 4). Figure 2 ), pH value determination (the determination results are shown in Figure 3 ), TVB-N value determination (the determination results are shown in Figure 4 ), TBA value determination (the determination results are shown in Figure 5 ), color difference / whiteness measurement (the measurement results are shown in Figure 6 ), water holding capacity determination (the determination results are shown in Figure 7 ), total bacterial count (the results are shown in Figure 8 ).

[0068] Table 3: Effects of Tremella polysaccharide and sodium sulfite on sensory quality of Penaeus vannamei

[0069]

[0070] Sensory scores in Table 3 = mean ± deviation 显著性差异 As shown in Table 3, with the extension of storage time, the sensory scores of each group gradually decreased during storage. The sensory scores of TP2-TP5 groups were significantly higher than those of CK1 group, and TP3 group had the highest score. Therefore, Tremella polysaccharide solution can maintain the sensory quality of white shrimp and the effect is better than that of sodium sulfite solution.

[0071] See also Figure 2 With the extension of storage time, the degree of melanization in each group gradually increased. After 8 days of storage, the melanization score of TP group was significantly lower than that of CKI group and CK2 group, and the melanization score of TP3 group was the lowest, indicating that the effect of Tremella polysaccharide solution in inhibiting melanization was better than that of sodium sulfite solution, and the effect of Tremella polysaccharide solution with a concentration of 0.6% was the best.

[0072] Reference Figure 3 As the storage time prolonged, the pH values ​​of each group gradually increased. The increase in pH value caused the shrimp meat to spoil. After 8 days of storage, the pH value of TP3 group was the lowest, which was 7.51, while the pH values ​​of other groups were all higher than 7.60, and the shrimp meat was unacceptable. This shows that the effect of 0.6% Tremella polysaccharide solution in delaying the increase in pH of white shrimp is better than sodium sulfite.

[0073] Reference Figure 4 As the storage time prolonged, the TVB-N values ​​of each group gradually increased. After 8 days of storage, the TVB-N value of the TP3 group was significantly lower than that of the CK1 group, indicating that the effect of the Tremella polysaccharide solution in delaying the increase of the TVB-N value was better than that of the sodium sulfite solution.

[0074] Reference Figure 5 The TBA value can indicate the degree of fat oxidation and is an important indicator for judging the degree of shrimp meat spoilage. With the extension of storage time, the TBA values ​​of each group gradually increased. The TBA value of the CK2 group increased faster, while the TBA value of the Tremella polysaccharide group increased slowly. After 8 days of storage, the TBA value of the TP1 group was the lowest, which was significantly lower than that of the CK1 and CK2 groups.

[0075] Reference Figure 6As the storage time prolonged, the whiteness values ​​of the control groups (CK1 and CK2 groups) first increased and then decreased, while the whiteness values ​​of the treatment groups (TP1-TP5 groups) gradually decreased. After 8 days of storage, the whiteness values ​​of the treatment groups were significantly higher than those of the CK1 group, but there were no significant differences among the treatment groups, among which the TP3 group had the highest whiteness value.

[0076] Reference Figure 7 During the storage of white shrimp, the body water was lost as the shrimp meat tissue degraded. The water holding capacity reflects the ability of muscle tissue to retain water. After 8 days of storage, the water holding capacity of the treated groups was significantly higher than that of the CK1 group, among which the TP2 group had the highest water holding capacity.

[0077] Reference Figure 8 The total bacterial count is an important indicator for detecting the degree of spoilage of white shrimp. After 8 days of storage, the total bacterial count CFU / g values ​​of each treatment group exceeded 6.0. The total bacterial count of the TP4 group was 6.73 CFU / g, which was significantly lower than that of the CK1 and CK2 groups, indicating that the antibacterial effect of the Tremella polysaccharide solution was better than that of the sodium sulfite solution.

[0078] Experimental Example 2:

[0079] Step 1: Weigh 6 g of 50% Tremella polysaccharide, add distilled water to make up to 500 mL, and prepare a Tremella polysaccharide solution with a concentration of 0.6%; weigh 0.15 g of Nisin, add distilled water to make up to 500 mL, and prepare a Nisin solution with a concentration of 0.03%; weigh 0.15 g of lysozyme, add distilled water to make up to 500 mL, and prepare a lysozyme solution with a concentration of 0.03%;

[0080] Step 2: Evenly mix the solution prepared in step 1 to obtain a composite biological preservative (TNL) with an optimal ratio;

[0081] Step 3: Clean the whiteleg shrimp and kill them suddenly in crushed ice, and select the shrimps with intact bodies and a mass of (10±2) g;

[0082] Step 4: The treated whiteleg shrimps were randomly divided into three groups, and the three groups of whiteleg shrimps were immersed in a composite biological preservative (TNL), a 0.6% tremella polysaccharide solution (TP), and distilled water (CK) for 30 min respectively;

[0083] Step 5: After soaking, take out the shrimps, drain them, and pack them into sealed bags;

[0084] Step 6: Store the packaged shrimp at 4°C for 8 days, and take samples regularly for index determination; the determination items include sensory score determination (the determination results are shown in Table 4), blackening score determination (the determination results are shown in Table 4), and Fig. 9 ), pH value determination (the determination results are shown in Fig.10), TVB-N value determination (the determination results are shown in Fig.11 ), TBA value determination (the determination results are shown in Fig.12 ), color difference / whiteness measurement (the measurement results are shown in Fig.13 ), water holding capacity determination (the determination results are shown in Fig.14 ), total bacterial count (the results are shown in Fig.15 ).

[0085] Table 4: Effects of composite biological preservatives and Tremella polysaccharide on sensory quality of Penaeus vannamei

[0086]

[0087]

[0088] As shown in Table 4, with the extension of storage time, the sensory scores of each group gradually decreased. When the score was lower than 6 points, the shrimp was unacceptable. After 8 days of storage, the sensory score of the TNL group was significantly higher than that of the TP group and the CK group. The above results show that the composite biological preservative has a significant effect in delaying the deterioration of the quality of white shrimp. At the same time, the effect of the composite biological preservative in delaying the quality change of white shrimp is better than that of a single Tremella polysaccharide solution.

[0089] Reference Fig. 9 The blackening scores increased gradually with the extension of storage time. The scores of TNL group and TP group were significantly lower than those of CK group, indicating that both groups had an inhibitory effect on the blackening of white shrimp. After 8 days of storage, the blackening score of CK group was 8.83, indicating that the white shrimp in this group had serious blackening. The blackening score of TNL group was the lowest, but the difference with TP group was not significant, indicating that TNL group had the best effect in delaying the blackening of shrimp body.

[0090] Reference Fig.10 After the death of Penaeus vannamei, the pH value of each treatment group showed a trend of first decreasing and then increasing. When the pH value of shrimp meat was 7.6, it was unacceptable. The increase rate of pH value of TNL group and TP group was lower than that of CK group, indicating that TNL group and TP group had an inhibitory effect on pH value. After 8 days of storage, the pH value of CK group was the lowest, and the pH value of TP group was 7.64, which was inedible. The pH value of TNL group was significantly lower than that of CK group and TP group.

[0091] Reference Fig.11 With the extension of storage time, the TVB-N values ​​of each treatment group gradually increased, and the increasing rate of the CK group was the fastest. After 8 days of storage, the freshness grades of the TNL group and the TP group dropped to grade three, and the TVB-N value of the TNL group was significantly lower than that of the TP group. The effect of the composite biological preservative in delaying the increase of the TVB-N value of white shrimp was better than that of the single Tremella polysaccharide solution.

[0092] Reference Fig.12 During the storage of white shrimp, the TBA values ​​of the TNL group and TP group increased more slowly than that of the CK group. After 8 days of storage, due to the free radical scavenging effect of Tremella polysaccharide in the composite biological preservative, the Nisin solution and lysozyme solution inhibited the growth of microorganisms, the esterase content produced by microbial metabolism decreased, the decomposition of fatty acids was delayed, and the increase of TBA value was alleviated. The preservation effect of the composite biological preservative was better than that of single Tremella polysaccharide.

[0093] Reference Fig.13 The body of fresh white shrimp was relatively transparent, with an initial whiteness value of 31.47. As the storage time prolonged, the whiteness value of the CK group showed a trend of first increasing and then decreasing, while that of the TNL and TP groups gradually decreased. After 8 days of storage, the whiteness values ​​of the TNL and TP groups were significantly higher than those of the CK group. The TNL group had the highest whiteness value of 25.82, followed by the TP group with a whiteness value of 24.93.

[0094] Reference Fig.14 During the storage of whiteleg shrimp, the muscle tissue was oxidized, the filamentous spaces contracted, water was precipitated from the shrimp meat as exudate, and nutrients such as amino acids and peptides were decomposed by microorganisms, which hindered the hydration and led to a decrease in water holding capacity. At the beginning of storage of whiteleg shrimp, the water holding capacity was 90.44%, and then the water holding capacity of the three groups showed a downward trend. After 8 days of storage, the water holding capacity of the TNL group was significantly higher than that of the TP group and the CK group, and the TNL group showed better water holding capacity.

[0095] Reference Fig.15 The total bacterial counts in the three groups gradually increased with the extension of storage time. At the beginning of storage, the total bacterial count was 3.56 CFU / g, and the white shrimp was at the first-level freshness. After 8 days of storage, the total bacterial count in the TNL group was 5.78 CFU / g, which was significantly lower than that in the TP group. The TP group was no longer acceptable, indicating that the antibacterial effect of the composite biological preservative was better than that of a single Tremella polysaccharide solution.

[0096] Table 5: Correlation analysis of various indicators in Experimental Example 2

[0097]

[0098] As shown in Table 5, the sensory score of white shrimp was significantly positively correlated with color difference and water holding capacity, and significantly negatively correlated with blackening score, pH value, TVB-N value, TBA value and total bacteria count. The correlation coefficients between each index and the sensory score were all greater than 0.8, indicating that the sensory quality had a strong correlation with blackening score, pH value, TVB-N value, TBA value and other indicators.

[0099] Muscle tissue oxidation leads to an increase in TBA value, a decrease in water holding capacity, and an increase in color difference. As the storage time of whiteleg shrimp increases, microorganisms grow and multiply and produce volatile amines, which increase the TVB-N value and pH value. Under the action of polyphenol oxidase, the color of the shrimp body becomes darker. Changes in the above indicators can affect the sensory quality of whiteleg shrimp.

[0100] Table 6: Linear regression equation of TVB-N value, total bacteria count and sensory score

[0101]

[0102] Table 7: TVB-N value and shelf life critical value of total bacterial count

[0103]

[0104] As shown in Tables 6 and 7, the regression equations of TVB-N value, total bacteria count and sensory score were established through linear regression analysis. A sensory score lower than 6 points in the sensory evaluation indicated that the shrimp was unacceptable, and 6 points was the end point of the shelf life. By substituting it into the regression equation, the critical values ​​of TVB-N value and total bacteria count corresponding to the sensory quality shelf life could be obtained.

[0105] Table 8: Shelf life of Penaeus vannamei stored in different treatment groups

[0106]

[0107] As shown in Table 8, the sensory evaluation showed that the shelf life of white shrimp was the shortest and the TVB-N value was the longest. Tremella polysaccharide can extend the shelf life of white shrimp by about 1-3 days, and the composite biological preservative is better than Tremella polysaccharide in extending the shelf life of white shrimp by 2-4 days.

[0108] Experimental Example 3:

[0109] Step 1: weigh 12 g of Tremella polysaccharide, 0.3 g of Nisin and 0.5 g of lysozyme, add distilled water to a 500 mL beaker and dissolve, then use a 1 L volumetric flask to make a Tremella polysaccharide composite biological preservative (TNL) with a concentration ratio of 0.6% Tremella polysaccharide, 0.03% Nisin and 0.05% lysozyme;

[0110] Step 2: Clean the white shrimp and kill them suddenly in crushed ice. Select the shrimps with intact limbs and a mass of (10±2) g and randomly divide them into 3 groups, 36 shrimps in each group, and soak them in a composite biological preservative (TNL), a 0.6% tremella polysaccharide solution (TP), and distilled water (CK) for 30 min respectively;

[0111] Step 3: After soaking, take out the shrimps, drain them, and pack them into sealed bags, 6 bags per group, 6 shrimps per bag, and store them at 4℃;

[0112] Step 4: Select a sample of whiteleg shrimp every 2 days, cut the second abdominal segment muscle of whiteleg shrimp into a cylinder with a diameter of 0.8 mm and a length of 1.5 cm, wipe off the surface moisture, wrap it with plastic wrap, and put it in a nuclear magnetic detection tube with a diameter of 1 cm. Use spin echo (Carr-purcell-meiboom-gill sequence, CPMG) to measure the NMR transverse relaxation time T2, and perform batch inversion of the exponential decay graph obtained by software to obtain the transverse relaxation time T2 spectrum of whiteleg shrimp.

[0113] Reference Fig.16 In this experiment, LF-NMR was used to measure the distribution and migration changes of water in white shrimp. The relaxation time (T2) of the continuous distribution was obtained through CPMG data. The peak diagram obtained by inversion can reflect the physical and chemical state of water.

[0114] The larger the T2 value, the looser the bond between water molecules and macromolecules, and the freer the water. The change in T2 value can reflect the flow characteristics of water molecules, and the different peaks in the peak graph represent different states of water molecules, and the peak area represents the relative content of water in this state: at the beginning of storage, the non-flowable water content was 94.82% and the free water content was 0.53%. With the extension of storage time, the non-flowable water ratio of each group gradually decreased, and the free water ratio gradually increased. With the decrease of the water holding capacity of muscle tissue, the water in the muscle migrated, and the non-flowable water gradually converted into free water (Christian et al. 2015).

[0115] After 8 days of storage, the free water ratio of TNL and TP groups was lower than that of CK group, and the composite biological preservative had a better ability to delay the loss of immobile water and retain free water. This result was consistent with the result of water holding capacity.

[0116] Experimental Example 4:

[0117] Step 1: weigh 12 g of Tremella polysaccharide, 0.3 g of Nisin and 0.5 g of lysozyme, add distilled water to a 500 mL beaker and dissolve, then use a 1 L volumetric flask to make a Tremella polysaccharide composite biological preservative (TNL) with a concentration ratio of 0.6% Tremella polysaccharide, 0.03% Nisin and 0.05% lysozyme;

[0118] Step 2: Clean the white shrimp and kill them suddenly in crushed ice. Select the shrimps with intact limbs and a mass of (10±2) g and randomly divide them into 3 groups, 36 shrimps in each group, and soak them in a composite biological preservative (TNL), a 0.6% tremella polysaccharide solution (TP), and distilled water (CK) for 30 min respectively;

[0119] Step 3: After soaking, take out the shrimp and drain it. Weigh 3g of fresh Penaeus vannamei, 3g of fresh Penaeus vannamei soaked in a composite biological preservative (TNL), 3g of fresh Penaeus vannamei soaked in a Tremella polysaccharide solution (TP), and 3g of fresh Penaeus vannamei soaked in distilled water (CK). Perform the following operations on each group of fresh Penaeus vannamei:

[0120] Add 6 mL of physiological saline, homogenize for 1 min, place in a 60°C water bath for 1 h, centrifuge at 10,000 r / min for 10 min, take the precipitate and repeat the above steps twice, take 3 mL of supernatant, add 1 mL of loading buffer, boil in boiling water for 10 min, centrifuge at 10,000 r / min for 10 min, and the supernatant is the sample to be electrophoresed;

[0121] Step 4: Prepare 5% stacking gel and 12% separation gel, and load 6 μL of protein standard (Maker) and 8 μL of sample to be electrophoresed in electrophoresis buffer at pH 6.8, wherein the voltage of the stacking gel is 80V and the voltage of the separation gel is 120V.

[0122] The film was stained with Coomassie Brilliant Blue staining solution. After staining for 2 h, it was placed in ethanol decolorizing solution for decolorization until the protein bands and film background were clear, and the gel was photographed.

[0123] In this experimental example, the above operation was repeated separately for each group of samples to be electrophoresed to obtain four groups of gels.

[0124] Reference Fig.17 The molecular weight of the protein of Penaeus vannamei is mainly distributed in the range of 15-100kDa, among which the paramyosin with a molecular weight of about 100kDa and the actin with a molecular weight of about 40kDa are more obvious. The tropomyosin with a molecular weight of about 38kDa and the myosin light chain (MLC) with a molecular weight of about 20kDa are lighter in color.

[0125] After 8 days of storage, the paramyosin, actin, tropomyosin and MLC bands of the three groups were significantly different. Compared with the bands of fresh white shrimp, the paramyosin bands and actin bands of TP group and CK group were significantly narrowed, and the tropomyosin bands and MLC bands disappeared, indicating that the proteins in TP group and CK group underwent a certain degree of oxidation and the protein content decreased. The protein bands of TNL group were not significantly different from those of fresh white shrimp, indicating that the Tremella polysaccharide composite biological preservative can slow down the decomposition of polypeptides by microorganisms, delay the decrease of protein content, and better maintain the integrity of protein bands.

[0126] Experimental Example 5:

[0127] Step 1: weigh 12 g of Tremella polysaccharide, 0.3 g of Nisin and 0.5 g of lysozyme, add distilled water to a 500 mL beaker and dissolve, then use a 1 L volumetric flask to make a Tremella polysaccharide composite biological preservative (TNL) with a concentration ratio of 0.6% Tremella polysaccharide, 0.03% Nisin and 0.05% lysozyme;

[0128] Step 2: Clean the white shrimp and kill them suddenly in crushed ice. Select the shrimps with intact limbs and a mass of (10±2) g and randomly divide them into 3 groups, 36 shrimps in each group, and soak them in a composite biological preservative (TNL), a 0.6% tremella polysaccharide solution (TP), and distilled water (CK) for 30 min respectively;

[0129] Step 3: After soaking, take out the shrimp and drain it. Perform the following operations on the fresh whiteleg shrimp, the fresh whiteleg shrimp soaked in the composite biological preservative (TNL), the fresh whiteleg shrimp soaked in the Tremella polysaccharide solution (TP), and the fresh whiteleg shrimp soaked in distilled water (CK):

[0130] The shrimp samples were cut into small pieces of 3 mm × 5 mm × 5 mm, and dehydrated step by step for 40 min in ethanol solutions with volume fractions of 75%, 85%, 95%, 100%, and 100%. The samples were immersed in three xylenes in turn, each for 1 h, and then immersed in three paraffin cylinders in turn, the first cylinder for 1 h, the second cylinder for 1.5 h, and the third cylinder for 2 h, for paraffin embedding. The pre-cooled wax blocks were cut into slices with a thickness of 3 μm and stained with hematoxylin. After staining, the slices were eluted with ethanol and xylene, sealed with neutral gum, and observed under a microscope and photographed for preservation.

[0131] Reference Fig.18 The myofibrils of fresh white shrimp were densely arranged, the muscle tissues were tightly connected, the sarcomeres were plump, there were almost no gaps, and the endomysium was intact. After 8 days of storage, the muscle tissue gaps in the TNL group were fewer, and the muscle fibers were relatively tight. The muscle tissue distribution in the TP group was more regular, but compared with the TNL group, the muscle tissue gaps increased and the gaps were larger. The muscle structure of the CK group was severely broken and the tissue structure was loose, which was significantly different from the TNL group, indicating that the TNL group had the best effect in delaying the rupture of the myofibril structure of shrimp meat.

[0132] Experimental Example 6:

[0133] Step 1: Weigh 12g of Tremella polysaccharide, 0.3g of Nisin and 0.5g of lysozyme into a 500mL beaker, add distilled water to dissolve, and then use a 1L volumetric flask to make a Tremella polysaccharide composite biological preservative with a ratio of 0.6% Tremella polysaccharide, 0.03% Nisin and 0.05% lysozyme.

[0134] Step 2: Clean the whiteleg shrimp and kill them suddenly in crushed ice. Select the shrimps with intact limbs and a mass of (10±2) g and randomly divide them into 3 groups, with 36 shrimps in each group. They are placed in a composite biological preservative (TNL), a 0.6% tremella polysaccharide solution (TP), and distilled water (CK) for 30 minutes respectively; FP (fresh whiteleg shrimp).

[0135] Step 3: After soaking, take out the shrimps, drain them, and pack them into sealed bags, 6 bags per group, 6 shrimps per bag, and store them at 4°C. Select samples for index determination on the 0th, 2nd, 4th, 6th, 8th, and 20th days.

[0136] Step 4: Take fresh white shrimp samples and samples soaked in a composite biological preservative (TNL), a 0.6% tremella polysaccharide solution (TP), and distilled water (CK) and stored for 20 days, and perform the following operations on them respectively:

[0137] Under sterile environmental conditions, the shrimp intestines were extracted using sterilized dissecting tools, immersed in sterile physiological saline, and sealed and stored at -80°C;

[0138] Among them, S and I represent the surface and intestine of white shrimp, respectively.

[0139] Step 5: Use a genomic DNA extraction kit to extract bacterial DNA from the sample and evaluate the DNA quality and quantity by the ratios of 260nm / 280nm and 260nm / 230nm.

[0140] Step 6: Amplify the V3-V4 region of the 16S rRNA gene using primers 338F (5'-ACTCCTACGGGAGGCAGCA-3') and 806R (5'-GGACTACHVGGGTWTCTAAT-3').

[0141] By Quant-iT TM All PCR products were quantified by dsDNA HS reagent and pooled together, and then the purified pooled samples were subjected to high-throughput sequencing analysis of bacterial rRNA genes using the Illumina HiSeq 2500 platform (PE250).

[0142] The software Trimmomatic was used to filter the raw data obtained by sequencing; then the software Cutadapt was used to identify and remove the primer sequences to obtain high-quality reads that did not contain primer sequences. The software FLASH was then used to splice the high-quality reads of each sample based on the overlapping relationship between the reads, and the spliced ​​sequence obtained was the high-quality data. The software UCHIME was then used to identify and remove the chimera sequences to obtain the final valid data (see Table 9 and Figure 19-22 ).

[0143] Table 9: Statistics of valid sequences and OTU numbers of each group of samples

[0144]

[0145] As shown in Table 9, the types of microorganisms on the surface of white shrimp are more than those in the intestine. After 20 days of storage, the number of OTUs in each group decreased compared with fresh white shrimp. The number of OTUs on the surface and intestine of white shrimp treated with the composite biological preservative was the lowest, indicating that it had the least number of microbial species, indicating that the composite biological preservative inhibited the growth of some microorganisms to a certain extent.

[0146] Reference Fig.19 The rarefaction curve refers to a curve constructed by randomly extracting a certain number of sequences from a sample and the corresponding number of OTUs. It is used to verify whether the amount of sequencing data is reasonable and can reflect the richness of microbial species in the sample.

[0147] When the number of sample sequencing increases to 10,000, the dilution curve rises sharply, indicating that a large number of microbial species are found in the sample. Subsequently, the dilution curves of each group gradually flatten out, indicating that the species in this environment will not increase with the increase in sequencing numbers, indicating that the sample sequence is sufficient and can reflect most of the microbial species in the sample.

[0148] The Shannon index is used to measure the diversity of microbial species. The larger the Shannon index, the richer the species. When the curve gradually flattens, it indicates that the number of sequencing is reasonable and can cover the vast majority of microbial species information in the sample.

[0149] Both the rarefaction curve and the Shannon-wiener curve showed that the number of sequencing samples in each group was sufficient and the sequencing results were scientifically credible.

[0150] Reference Fig. 20,Single sample (Alpha) diversity can reflect the abundance and species diversity of microbial species, including ACE index, Chaol index, Shannon index and Simpson index. ACE and Chaol index measure the abundance of microbial species, while Shannon and Simpson index measure the diversity of species.

[0151] The ACE and Chaol indexes of the FP-S group were higher than those of the CK-S, TNL-S and TP-S groups, and the ACE and Chaol indexes of the FP-I group were higher than those of the CK-I, TNL-I and TP-I groups, indicating that the abundance of microorganisms on the surface and in the intestine of white shrimp stored for 20 days decreased with the extension of storage time.

[0152] The ACE and Chaol indexes on the surface and intestine of white shrimp treated with different preservatives were CK group > TP group > TNL group, indicating that the composite biological preservative had the best effect in inhibiting microbial growth, indicating that the addition of Nisin and lysozyme enhanced the antibacterial effect.

[0153] Reference Fig.21 In the relative abundance of the bacterial flora of each group of samples at the order level, the microorganisms on the surface of fresh white shrimp were mainly Lactobacillales, Bacillales, Pseudomonadales and Enterobacteriales, and their relative abundance accounted for 71% of the total bacterial flora, among which the relative abundance of Lactobacillales accounted for 41% of the total bacterial flora, and it was the dominant bacterial species on the surface of fresh white shrimp.

[0154] The intestinal microorganisms of fresh whiteleg shrimp are mainly Lactobacillales and Pseudomonasales, with a relative abundance of 88% of the total bacterial flora, among which the relative abundance of Pseudomonasales accounts for 54% of the total bacterial flora, making it the dominant bacterial species in the intestine of fresh whiteleg shrimp.

[0155] Compared with TP-S and CK-S, the relative abundance of Pseudomonadales in the TNL-S group was lower, at 12%, indicating that the Tremella polysaccharide composite biological preservative had a certain inhibitory effect on the Pseudomonadales on the surface of white shrimp.

[0156] Reference Fig. 22In the relative abundance of the flora of each group of samples at the family level, the surface microorganisms of fresh whiteleg shrimp were mainly Lactobacillaceae, Carnobacteriaceae and Enterobacteriaceae, with relative abundances of 19%, 17% and 12%, respectively, and the relative abundance of other species was 27%, indicating that fresh whiteleg shrimp showed relatively rich microbial species, which was consistent with the analysis results of the microbial diversity index.

[0157] Moraxellaceae and Carnobacteriaceae are the main microorganisms in the intestine of fresh white shrimp, with relative abundances of 54% and 22%, respectively. A large proportion of Moraxellaceae is present in the intestine of white shrimp.

[0158] After 20 days of storage, the relative abundance of Moraxellaceae on the surface of each group increased, among which the relative abundance of TNL-S group was the lowest, at 11%, followed by CK-S group, at 39%, and TP-S group was the highest, at 44%. The relative abundance of Moraxellaceae in the intestine of white shrimp decreased, with the highest relative abundance of TNL-I group at 34%, followed by TP-I and CK-I groups, both at 26%.

[0159] In addition, compared with fresh white shrimp, the relative abundance of Listeriaceae in TNL-S and TNL-I groups was reduced, indicating that the composite biological preservative can inhibit the growth of Moraxellaceae and Listeriaceae, and the effect of the preservative on the surface of white shrimp is more obvious. Therefore, during storage, the Tremella polysaccharide composite biological preservative can delay the water migration of white shrimp, have a certain protective effect on muscle tissue, change the microbial flora of white shrimp, and delay the quality deterioration of white shrimp.

Claims

1. A composite biological preservative for Penaeus vannamei, characterized in that: It consists of four ingredients: Tremella polysaccharide, Nisin, lysozyme and water.

2. The composite biological preservative for Penaeus vannamei according to claim 1, characterized in that: The concentration ratio of the Tremella polysaccharide solution, Nisin solution and lysozyme solution is (40-80): (1-3): (3-5).

3. The composite biological preservative for Penaeus vannamei according to claim 1, characterized in that: The concentration ratio of the Tremella polysaccharide solution, Nisin solution and lysozyme solution is 20:1:

1.

4. The composite biological preservative for Penaeus vannamei according to claim 1, characterized in that: The mass ratios of Tremella polysaccharide, Nisin and lysozyme are respectively Tremella polysaccharide: 0.6%; Nisin: 0.03%; Lysozyme: 0.05%, the rest is water.

5. The composite biological preservative for Penaeus vannamei according to claim 4, characterized in that: The specific preparation steps are as follows: Step 1: Preparation of Tremella polysaccharide solution; Step 2: Nisin solution preparation; Step 3: Preparation of lysozyme solution; Step 4: Evenly mix the solutions prepared in steps 1-3 to obtain the composite biological preservative.

6. The composite biological preservative for Penaeus vannamei according to claim 5, characterized in that: The preparation method in step 1 is: weigh 6 g of 50% Tremella polysaccharide, add distilled water to make the volume to 500 mL, and prepare a Tremella polysaccharide solution with a concentration of 0.6%.

7. The composite biological preservative for Penaeus vannamei according to claim 5, characterized in that: The preparation method in step 2 is: weigh 0.15 g of Nisin, add distilled water to make the volume to 500 mL, and prepare a Nisin solution with a concentration of 0.03%.

8. The composite biological preservative for Penaeus vannamei according to claim 5, characterized in that: The preparation method in step 3 is: weigh 0.15 g of lysozyme, add distilled water to make the volume to 500 mL, and prepare a lysozyme solution with a concentration of 0.03%.

9. An application method of a composite biological preservative for Penaeus vannamei, applied to any one of claims 1 to 8, characterized in that: The whiteleg shrimp was soaked in the composite biological preservative for 30 minutes.

Images