A microecological preparation for temporary rearing of whiteleg shrimp after capture, and its preparation method and application

Abstract

The invention discloses a probiotic preparation for post-harvest temporary aquaculture of whiteleg shrimp (Penaeus vannamei), a preparation method thereof, and an application thereof. The probiotic preparation is characterized in that the probiotic preparation comprises at least one of a fermented Lactobacillus mucilaginosus RC3 strain and a Lactobacillus plantarum B6 strain, the fermented Lactobacillus mucilaginosus RC3 having a preservation number of CGMCC No. 30602, and the Lactobacillus plantarum B6 having a preservation number of CGMCC No. 30603. The probiotic preparation is prepared by mixing a fermented Lactobacillus mucilaginosus RC3 seed liquid and a Lactobacillus plantarum B6 seed liquid in a volume ratio of (1-2):(1-2), and then inoculating the mixture into an MRS liquid culture medium in a volume ratio of 5-10% to obtain the probiotic preparation for post-harvest temporary aquaculture of whiteleg shrimp. The probiotic preparation has the advantages of reducing the contents of nitrite nitrogen and ammonia nitrogen in aquaculture water, improving the antioxidant and immune properties of shrimp, optimizing intestinal flora, and improving the muscle quality of shrimp.

Description

Technical Field

[0001] The present invention relates to a microecological preparation, in particular to a microecological preparation for temporary rearing of whiteleg shrimp after capture, and a preparation method and application thereof. Background Art

[0002] The whiteleg shrimp (Litopenaeus vannamei) is a member of the Penaeidae family, order Decapoda. Its scientific name is also known as Litopenaeus vannamei. It is known for its excellent traits, including high-temperature and low-salt tolerance, rapid growth, strong disease resistance, suitability for high-density aquaculture, and low feed nutritional requirements. In commercial aquaculture, shrimp undergo a series of processes, including harvesting and transportation to temporary holding ponds. The post-transportation holding period is crucial for determining survival rate and product quality. However, transportation can produce multiple stresses, including mechanical damage, dissolved oxygen fluctuations, and ammonia nitrogen accumulation. These can significantly weaken shrimp immunity, lead to intestinal dysbiosis, and promote the explosive proliferation of opportunistic pathogens such as Vibrio. In recent years, most microecological preparations have focused on long-term aquaculture environments, lacking research on the specific stress conditions after transportation. Furthermore, existing products are relatively limited in their functionality, insufficiently addressing the shrimp's own immune system and the reconstruction of the intestinal microbiome. Summary of the Invention

[0003] The technical problem to be solved by the present invention is to provide a microecological preparation for temporary rearing of whiteleg shrimp after capture, which can reduce the content of nitrite nitrogen and ammonia nitrogen in aquaculture water, improve the antioxidant and immune performance of shrimp, optimize the intestinal flora and improve the muscle quality of shrimp, as well as a preparation method and application thereof.

[0004] The technical solution adopted by the present invention to solve the above technical problems is: a microecological preparation for temporary rearing of white shrimp after capture, the microecological preparation comprising fermented mucus lactobacillus ( Limosilactobacillus fermentum ) RC3 strain and Lactobacillus plantarum ( Lactiplantibacillus plantarum ) at least one of the B6 strains, the preservation number of the fermentative Lactobacillus mucilaginosus RC3 is CGMCC No.30602, and the preservation number of the plant lactobacillus B6 is CGMCC No.30603.

[0005] Preferably, the microecological preparation is fermented by Lactobacillus mucilaginosus ( Limosilactobacillus fermentum ) RC3 seed solution and Lactobacillus plantarum ( Lactiplantibacillus plantarum ) B6 seed liquid is mixed in a volume ratio of (1-2): (1-2), and inoculated into MRS liquid culture medium at a volume ratio of 5-10% to obtain a microecological preparation for temporary rearing of whiteleg shrimp after capture.

[0006] Preferably, the viable count of Lactobacillus mucilaginosus RC3 in the microecological preparation is ≥1.42×109 CFU / mL, the number of viable bacteria of Lactobacillus plantarum B6 ≥1.14×10 9 CFU / mL.

[0007] The present invention also provides a method for preparing the above-mentioned microecological preparation for temporary rearing of whiteleg shrimp after capture, comprising the following steps:

[0008] Step 1, taking a loop of fermented Lactobacillus mucilaginosus RC3 and Lactobacillus plantarum B6 respectively with an inoculating loop, activating them by a three-zone line method, placing them in a 36-38° C. constant temperature incubator and inverting them for 20-28 hours to obtain single colonies of fermented Lactobacillus mucilaginosus RC3 and Lactobacillus plantarum B6, respectively; picking single colonies of fermented Lactobacillus mucilaginosus RC3 and Lactobacillus plantarum B6 respectively and inoculating them into MRS liquid culture medium, culturing them at 28-38° C. for 12-24 hours for activation, and continuously activating them for two generations to obtain fermented Lactobacillus mucilaginosus RC3 seed liquid and Lactobacillus plantarum B6 seed liquid;

[0009] Step 2: After mixing the fermented Lactobacillus mucilaginosus RC3 seed liquid and the Lactobacillus plantarum B6 seed liquid in a volume ratio of (1-2): (1-2), the mixed bacterial liquid is inoculated into MRS liquid culture medium in a volume ratio of 5-10%, and cultured at 28-38° C. for 12-24 hours to obtain a microecological preparation for temporary rearing of whiteleg shrimp after capture.

[0010] Furthermore, in step 2, the fermented Lactobacillus mucilaginosus RC3 seed liquid and the Lactobacillus plantarum B6 seed liquid are mixed in a volume ratio of 2:1, and the mixed bacterial liquid is inoculated into MRS liquid culture medium at a volume ratio of 5%, and cultured at 28-38° C. for 12-24 hours to obtain a microecological preparation for temporary rearing of whiteleg shrimp after capture.

[0011] Furthermore, the viable count of Lactobacillus mucilaginosus RC3 in the microecological preparation is ≥1.42×10 9 CFU / mL, the number of viable bacteria of Lactobacillus plantarum B6 ≥1.14×10 9 CFU / mL.

[0012] The present invention also provides the use of the above-mentioned microecological preparation for temporary rearing of whiteleg shrimp after capture as a nitrite nitrogen degrader and / or ammonia nitrogen degrader in aquaculture seawater.

[0013] The present invention also provides the use of the microecological preparation for temporary rearing of whiteleg shrimp after capture in the preparation of a shrimp antioxidant enhancement preparation and / or a shrimp immune enhancer.

[0014] The present invention also provides the use of the microecological preparation for temporary rearing of whiteleg shrimp after capture in the preparation of a shrimp intestinal probiotic preparation.

[0015] The present invention also provides the use of the above-mentioned microecological preparation for temporary rearing of whiteleg shrimp after capture in the preparation of a Vibrio harveyi inhibitor.

[0016] Compared with the prior art, the advantages of the present invention are: the present invention provides a microecological preparation for temporary rearing of whiteleg shrimp after capture, and its preparation method and application, which effectively reduces the levels of nitrite nitrogen and ammonia nitrogen in aquaculture water through the microecological preparation. At the same time, mechanical damage, dissolved oxygen fluctuations, ammonia nitrogen accumulation, etc. will occur to shrimp during transportation, which will induce excessive reactive oxygen and cause oxidative stress in shrimp. The organism relies on antioxidant enzymes to maintain redox homeostasis. Studies have shown that the microecological preparation can significantly increase the activity of SOD and T-AOC, reduce the MDA content, enhance the ability of shrimp to resist lipid peroxidation, and effectively reduce the accumulation of MDA in muscle. In addition, the ACP and ALP immune enzyme activities are significantly higher than those of the blank group shrimp; at the same time, it can also increase the diversity of beneficial bacteria in the shrimp intestine and effectively inhibit the growth of potential pathogenic bacteria in the intestine, thereby significantly improving the survival rate of whiteleg shrimp after transportation.

[0017] The fermented Lactobacillus mucilaginosus ( Limosilactobacillus fermentum ) RC3 strain, with the deposit number CGMCC No.30602, was deposited in the General Microbiology Center of China Culture Collection Administration on May 13, 2024, and the deposit address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0018] The above-mentioned Lactobacillus plantarum ( Lactiplantibacillus plantarum ) B6 strain, deposited with CGMCC No. 30603, was deposited on May 13, 2024 at the General Microbiology Center of China Culture Collection Administration, with the deposit address being No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Effects of four post-transportation treatments on the SOD levels in the hepatopancreas of Penaeus vannamei;

[0020] Figure 2 Effects of four post-transportation handling methods on the hepatopancreas T-AOC level of Penaeus vannamei;

[0021] Figure 3 Effects of four post-transportation treatments on MDA levels in the hepatopancreas of Penaeus vannamei;

[0022] Figure 4 Effects of four post-transportation handling methods on the ACP levels in the hepatopancreas of Penaeus vannamei;

[0023] Figure 5 Effects of four post-transportation handling methods on the ALP level in the hepatopancreas of Penaeus vannamei;

[0024] Figure 6 Effects of low-temperature transportation on the hepatopancreas tissue structure and morphology of untreated whiteleg shrimp;

[0025] Figure 7 Effects of four treatments on the hepatopancreas structure and morphology of white shrimp during post-transportation and temporary rearing, where a is the experimental group, b is the Vc group, c is the combined group, and d is the control group;

[0026] Figure 8 Effects of four post-transportation handling methods on the muscle texture of whiteleg shrimp.

[0027] Figure 9 Effects of low-temperature transportation on the intestinal tissue structure and morphology of untreated whiteleg shrimp;

[0028] Figure 10 Effects of four treatments on the intestinal tissue structure and morphology of whiteleg shrimp during post-transportation and temporary rearing, where a is the experimental group, b is the Vc group, c is the combined group, and d is the control group;

[0029] Figure 11 Analysis of β-diversity of intestinal microbiota of whiteleg shrimp in four post-transportation rearing treatments;

[0030] Figure 12 Analysis of the differences in LEfSe of intestinal flora of white shrimp in four treatments during temporary rearing after transportation. DETAILED DESCRIPTION

[0031] The present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

[0032] Specific Example 1: Preparation of a microecological preparation for temporary rearing of whiteleg shrimp after capture.

[0033] Example 1: A method for preparing a microecological preparation for temporary rearing of whiteleg shrimp after capture, comprising the following steps:

[0034] Step 1, strain selection: fermentative Lactobacillus mucilaginosus RC3 and Lactobacillus plantarum B6 with good antibacterial properties, nitrite nitrogen and ammonia nitrogen reduction capabilities, and good salt tolerance and acid production characteristics were screened from the strain collection center of the Animal Products Processing Laboratory of Ningbo University;

[0035] Step 2, culturing lactic acid bacteria seed liquid: preparing MRS solid culture medium and MRS liquid culture medium, sterilizing with high-pressure steam, pouring the solid culture medium into a plate, and setting it aside for use after solidification; taking out fermentation mucus Lactobacillus RC3 and plant lactobacillus B6 stored in a -80°C glycerol tube, and then taking one loop each with an inoculating loop, using a three-zone line method to activate, placing in a 37°C constant temperature incubator and inverting and culturing for 24 hours to obtain single colonies of fermentation mucus Lactobacillus RC3 and plant lactobacillus B6, respectively; picking single colonies of fermentation mucus Lactobacillus RC3 and plant lactobacillus B6, respectively, inoculating them into MRS liquid culture medium, culturing at 37°C for 12 hours for activation, and continuously activating for two generations to obtain seed liquid;

[0036] Step 3, fermentation of the probiotic preparation: fermentation Lactobacillus mucilaginosus RC3 seed liquid and Lactobacillus plantarum B6 seed liquid were mixed at a volume ratio of 2:1, and then inoculated into MRS liquid medium at a volume ratio of 5% and cultured at 37°C for 12 hours to obtain a probiotic preparation for temporary rearing of whiteleg shrimp after capture, wherein the number of viable bacteria of fermentation Lactobacillus mucilaginosus RC3 in the probiotic preparation was ≥1.42×10 9 CFU / mL, the number of viable bacteria of Lactobacillus plantarum B6 ≥1.14×10 9 CFU / mL.

[0037] The preparation method of MRS solid culture medium is as follows: mix 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 2 g of diammonium citrate, 5 g of sodium acetate, 20 g of glucose, 80 ml of Tween, 0.5 g of magnesium sulfate, 0.25 g of manganese sulfate and 15 g of agar powder, dissolve it in 1 L of distilled water, and sterilize it at 121°C for 15 min.

[0038] The preparation method of MRS liquid culture medium is as follows: mix 10 g of peptone, 10 g of beef extract, 5 g of yeast extract, 2 g of diammonium citrate, 5 g of sodium acetate, 20 g of glucose, 80 ml of Tween, 0.5 g of magnesium sulfate and 0.25 g of manganese sulfate, dissolve in 1 L of distilled water, and sterilize at 121°C for 15 min.

[0039] Example 2 is the same as Example 1, except that in step 3, the seed liquid of fermented Lactobacillus mucilaginosus RC3 and the seed liquid of Lactobacillus plantarum B6 are mixed in a volume ratio of 1:1.

[0040] Example 3 is the same as Example 1, except that in step 3, the seed liquid of fermented Lactobacillus mucilaginosus RC3 and the seed liquid of Lactobacillus plantarum B6 are mixed in a volume ratio of 1:2.

[0041] Example 4 is the same as Example 1, except that in step 3, the seed liquid of fermentation Lactobacillus mucilaginosus RC3 is inoculated into MRS liquid culture medium at a volume ratio of 5% and cultured at 37° C. for 12 h.

[0042] Example 5 is the same as Example 1, except that the seed liquid of Lactobacillus plantarum B6 is inoculated into MRS liquid culture medium at a volume ratio of 5% and cultured at 37° C. for 12 h.

[0043] Specific Example 2: Effect test of the probiotic preparation prepared in Specific Example 1.

[0044] 1. Degradation of nitrite nitrogen and ammonia nitrogen in aquaculture water by microecological preparations.

[0045] The probiotics of Examples 1-5 were added to aquaculture wastewater at a volume percentage of 3%. The nitrite nitrogen and ammonia nitrogen contents in the water were measured three days before and after addition. The results were compared with those of a blank control (MRS broth culture medium was added to aquaculture wastewater at a volume percentage of 3%), and the inhibition efficiency was calculated.

[0046] Determination of nitrite nitrogen reduction ability: The naphthylethylenediamine spectrophotometric method (national standard GB17378.4-2007) is used for measurement. Nitrite and sulfonamide are subjected to diazotization reaction under acidic conditions. The product is coupled with naphthylethylenediamine hydrochloride. The absorbance value of the generated red azo dye is measured at a wavelength of 543nm. The nitrite nitrogen concentration is calculated based on the linear relationship between the absorbance value and the nitrite nitrogen concentration (mg / L).

[0047] Determination of ammonia nitrogen reduction ability: The hypobromite oxidation method (national standard GB17378.4-2007) is used for measurement. Ammonia is oxidized to nitrite by hypobromite in an alkaline medium. The total nitrite nitrogen content is then measured by diazo-azo spectrophotometry. The ammonia nitrogen concentration is obtained by subtracting the original nitrite concentration.

[0048] Inhibition efficiency = ΔB - ΔA / ΔB × 100%; ΔA represents the difference between the initial and post-treatment concentrations in the experimental group; ΔB represents the difference between the initial and post-treatment concentrations in the blank group. The results are shown in Table 1.

[0049] Table 1 Degradation effect of microecological preparations on nitrite nitrogen and ammonia nitrogen in aquaculture water

[0050]

[0051] As shown in Table 1, the lactic acid bacteria probiotic preparation of the present invention can be quickly distributed in water bodies, effectively reducing the overall levels of nitrite nitrogen and ammonia nitrogen by decomposing organic matter and metabolic waste in the water, thereby improving the water environment. Compared with Examples 2-5, Example 1 has the strongest comprehensive degradation ability, and the combination of the two lactic acid bacteria synergistically improves the degradation rate of nitrite nitrogen.

[0052] 2. The inhibitory effect of proecological preparations on Vibrio harveyi strains.

[0053] (1) Strain activation

[0054] Lactobacillus strains were inoculated into MRS liquid medium, and Vibrio harveyi was inoculated into 2216E liquid medium. Both were activated and cultured in a 37°C constant temperature incubator for 12 h.

[0055] (2) Antibacterial experiment operation

[0056] ① Placement of Oxford cups: Using sterile operation in a clean bench, arrange three Oxford cups in an equilateral triangle on the surface of a flat plate;

[0057] ② Prepare bacterial plates: Take the activated Vibrio harveyi bacterial solution and add 1% (v / v) of 2216E agar medium (45-50°C), shake to mix, and then pour into the plate;

[0058] ③ Sample addition: After the plate solidifies, remove the Oxford cup, take out the activated lactic acid bacteria liquid, mix thoroughly by vortexing, and use a pipette to accurately draw 200 μL into the hole;

[0059] ④ Culture and observation: After the plates were placed in a 37°C constant temperature incubator for 12 h, the diameter of the inhibition zone was measured using an automatic colony counter. The results are shown in Table 2.

[0060] Table 2 Antibacterial effect of probiotics on Vibrio harveyi strains

[0061]

[0062] In the table a, b: different letters indicate significant differences among different treatment groups ( p <0.05).

[0063] As can be seen from Table 2 above, the antibacterial effect is measured by the diameter of the inhibition zone. The antibacterial diameter of the microecological preparation in which RC3 and B6 are compounded in a ratio of 2:1 is 23.695 mm, which is significantly better than the other ratio compounding groups and has no significant difference with B6. The blank group is a group in which 200 μL of water or MRS liquid culture medium is added to the hole, and no inhibition zone appears.

[0064] 3. The effect of microecological preparations on the survival rate of whiteleg shrimp after temporary rearing.

[0065] The whiteleg shrimps after low-temperature transportation were randomly distributed into 12 50L temporary water barrels, with 80 shrimps placed in each barrel. The 12 temporary water barrels were evenly divided into 4 groups. The probiotic preparation of Example 1 was added to the temporary water of the shrimps, and the low (1×10 5CFU / mL), medium (1×10 6 CFU / mL), high (1×10 7 The three concentration gradients of the test samples were used, and no probiotics were added to the control group.

[0066] The number of dead shrimps was observed and recorded every 24 h, and they were removed from the experimental tanks. The survival rate was calculated and shown in Table 3 below.

[0067] Table 3 Survival rate of whiteleg shrimp in temporary culture after being treated with probiotics

[0068]

[0069] The results in Table 3 above show that during the 3-day temporary storage period after low temperature stress, there was no significant difference in the cumulative survival rate between the low-dose probiotic preparation group and the control group; while the medium and high-dose groups showed significant improvement, with their 24-hour survival rates increased by 17.09% and 14.59% respectively compared with the control group, their 48-hour survival rates increased by 27.08% and 24.58% respectively compared with the control group, and their 72-hour survival rates increased by 30.83% and 25.31% respectively compared with the control group, indicating that the appropriate concentration of probiotic preparations can significantly enhance the stress resistance of white shrimp after low-temperature transportation by regulating physiological homeostasis.

[0070] Specific Example 3: Application test of the probiotic preparation prepared in Example 1.

[0071] The whiteleg shrimp that had been transported at low temperatures were randomly distributed into 12 50L temporary holding buckets, with 80 shrimp placed in each bucket. The 12 temporary holding buckets were evenly divided into 4 groups.

[0072] Experimental Group 1: The probiotic preparation in Example 1 was applied to the temporary rearing stage after low-temperature transportation in the supply chain of whiteleg shrimp. The concentration of the probiotic preparation was set at 1×10 6 CFU / mL, recorded as the experimental group.

[0073] Experimental group 2: During the temporary holding stage after low-temperature transportation in the supply chain of whiteleg shrimp, VC was added to the temporary holding water to make the VC concentration in the temporary holding water reach 100 mg / L, recorded as the Vc group.

[0074] Experimental group 3: During the temporary holding stage after low-temperature transportation in the supply chain of whiteleg shrimp, a concentration of 1×10 6 The microecological preparation with a concentration of 100 CFU / mL was combined with Vc with a concentration of 100 mg / L and recorded as the composite group.

[0075] Experimental Group 4: No preservatives were added to the temporary holding water during the temporary holding stage after low-temperature transportation in the whiteleg shrimp supply chain, and was recorded as the control group.

[0076] Five sampling points were set up: before transportation (abbreviated as BT), after transportation (abbreviated as AT), temporary storage for 12 hours (abbreviated as TC12), temporary storage for 24 hours (abbreviated as TC24), and temporary storage for 36 hours (abbreviated as TC36).

[0077] 1. Effects of probiotics on the activities of various enzymes in shrimp hepatopancreas.

[0078] An appropriate amount of hepatopancreas sample was weighed and homogenized with sterile physiological saline at a mass ratio of 1:9 in an ice bath. The contents were centrifuged at 10,000 rpm and 4°C for 10 min. Superoxide dismutase (SOD), total antioxidant capacity (T-AOC), malondialdehyde (MDA), acid phosphatase (ACP), and alkaline phosphatase (ALP) in the hepatopancreas of shrimp were determined using a kit, and the hepatopancreas tissue of shrimp was sectioned and observed.

[0079] Superoxide dismutase (SOD) is a key defense system for crustaceans against free radicals and other oxidative damage. When an organism is in a state of oxidative stress, cells are exposed to excessive ROS, which activates the antioxidant system and increases SOD enzyme activity. Figure 1 It can be seen that after shrimp were transported in low-temperature water, their SOD activity increased. During the 36-hour temporary culture period, the SOD activity of shrimp in the experimental, vitamin C, and combined groups was higher than that of shrimp in the control group, indicating that the probiotics and vitamin C have a certain promoting effect on SOD enzyme activity. The SOD activity of shrimp in the control group continued to decrease during the temporary culture period, indicating that the shrimp are more susceptible to adverse environmental factors and may die.

[0080] Total antioxidant capacity (T-AOC) is an important indicator to measure the strength and health of the body's defense system. After being transported in low-temperature water, shrimps, in order to resist adverse environmental factors, will experience increased oxidative stress, produce more free radicals, and consume antioxidant enzymes. Figure 2 The results show that the T-AOC of the experimental shrimp was significantly better than that of the normal state after 36 hours of temporary culture. The short-term effect of the Vc group was the most obvious, with a significant increase in T-AOC after 12 hours. The T-AOC of the shrimp in the control group was lower than that of the normal state.

[0081] Malondialdehyde (MDA) activity can be used as a marker of oxidative stress. Higher MDA levels usually indicate that the cells of the shrimp hepatopancreas are oxidatively damaged. This may have a negative impact on the normal function of the hepatopancreas, reduce the activity of digestive enzymes, inhibit protein synthesis, and even lead to apoptosis or necrosis of hepatopancreas cells. Figure 3 It can be seen that after transportation, the MDA content increased significantly. During the 36-hour temporary storage process, the MDA content of the experimental group, Vc group, compound group and control group showed a trend of first increasing and then decreasing. The MDA content of the experimental group, Vc group and compound group were lower than that of the control group within 36 hours, and were significantly lower than the control group at TC12 and TC24.

[0082] ACP and ALP of shrimp are usually related to the health of liver, intestine and immune system. Figure 4 and Figure 5 As shown in the data, after shrimp were transported in low-temperature water, ACP increased significantly and abnormally, and during the 36-hour temporary rearing, the ACP values ​​of the experimental group, Vc group, and compound group were significantly better than those before transportation (BT), while the ACP value of the control group was no different from that before transportation (BT), indicating that both the experimental group and the Vc group could promote ACP enzyme activity; ALP increased slightly, but not significantly. However, during the 36-hour temporary rearing, ALP of the control group continued to increase. It is speculated that the lack of trace elements during the fasting temporary rearing led to damage to the health of the shrimp.

[0083] The hepatopancreas is an important immune and digestive organ of shrimp, which has the functions of secreting digestive enzymes, storing nutrients and detoxifying. Figure 6 As shown in Figure 2, after low-temperature transportation, whiteleg shrimp are subjected to multiple stresses, which lead to the expansion of the lumen of the hepatopancreas of the shrimp, and some of them have vacuoles and rupture. Figure 7 As shown, after 36 hours of culture, the hepatic ducts, lumen, and cellular structure of the experimental and combined groups returned to normal. However, some vacuoles remained in the Vc group, indicating incomplete recovery. Furthermore, after 36 hours of culture, the basement membrane of the control group remained damaged, and B and R cells had not returned to normal. This suggests that probiotics can effectively repair hepatopancreatic tissue and improve its function by inhibiting inflammation and promoting the production of antioxidants.

[0084] 2. The effect of microecological preparations on the texture of shrimp.

[0085] The muscle texture characteristics of whiteleg shrimp (Penaeus vannamei) were measured using a QTS-25 texture analyzer and a 4 mm diameter flat-bottom probe. After removing the cephalothorax and shell, the second abdominal segment muscle was dissected and subjected to a Texture Profile Analysis (TPA) using the texture analyzer. The measured parameters included hardness, adhesion, cohesion, elasticity, stickiness, chewiness, and shear strength. The measurement parameters were: p / 50 probe, pre-test speed of 1 mm / s, test speed of 2 mm / s, post-test speed of 2 mm / s, and a compression ratio of 40%.

[0086] After being transported in low-temperature water, the texture properties of shrimps all decreased to varying degrees. There was no significant effect on hardness, adhesion, cohesion, and elasticity, but the most significant effect was on cohesion and chewiness, indicating that low-temperature water transportation would have an adverse effect on texture quality. Figure 8 As shown in the figure, the texture characteristics were restored during the 36-h storage period, and the chewiness of the experimental group and the composite group was still better than that of the other groups during the 36-h storage period.

[0087] 3. The effects of microecological preparations on the intestines of shrimp.

[0088] The intestinal tissue of Penaeus vannamei was sectioned and observed, and the intestinal flora of Penaeus vannamei was sequenced by 16s rRNA, and microbial diversity analysis was performed to explore the effects of lactic acid bacteria proecological preparations on the intestinal tract of Penaeus vannamei.

[0089] The intestine is the key place for shrimp to digest and absorb nutrients, and it is also the main way for pathogens to invade. The height and density of intestinal microvilli are closely related to the absorption area. Increasing the height and density of microvilli can increase the contact between digestive enzymes and chyme, expand the transportation area of ​​nutrients, and improve the absorption and utilization rate of nutrients in the intestine, thereby promoting the healthy growth of shrimp. Figure 9 As shown in the figure, after the white shrimp was transported at low temperature, the epithelium on the intestinal basement membrane was peeled off and the connection was loose; the intestinal villi were shortened and the density was reduced; some epithelial cells were necrotic and the vacuoles ruptured. Figure 10 Results showed that after 36 hours of temporary storage, the intestinal villi in the experimental and combined groups recovered better than those in the vitamin C and control groups. Furthermore, epithelial cells in the vitamin C and control groups separated from the basement membrane, and numerous vacuoles were present. This suggests that probiotics can enhance intestinal barrier function and accelerate intestinal repair and regeneration by maintaining intestinal flora.

[0090] like Figure 11 As shown in the figure, PCoA analysis showed that the experimental group, Vc group and compound group were significantly separated from the control group, indicating that the use of milk microecological preparations would have a significant effect on the intestinal flora of white shrimp.

[0091] LEfSe analysis was used to identify species that had significant differences in sample partitioning, such as Figure 12 As shown in the figure, after 36 hours of temporary culture, multi-level species analysis of each group revealed that in the experimental group, f_Lactobacillaceae, g_Pediococcus, g_Xanthomarina, and g_Marivita were enriched. The first two belong to the lactic acid bacteria group and are clearly beneficial to white shrimp. In the control group, p_Bacteroidota, o_Flavobacteriales, c_Bacteroidia, f_Alteromonadaceae, o_Corynebacteriales, o_Rhizobiales, g_Meridianimaribacter, g_Ahrensia, and g_Winogradskyella were enriched. Among them, o_Flavobacteriales and o_Corynebacteriales are potential pathogens. This suggests that the use of milk microecological preparations increases the production of beneficial bacteria in the intestinal tract of white shrimp.

[0092] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by persons of ordinary skill in the art within the spirit and scope of the present invention shall also fall within the scope of protection of the present invention.

Claims

1. A microecological preparation for temporary rearing of whiteleg shrimp after capture, characterized in that: The microecological preparation includes fermented Lactobacillus mucilaginosus ( Limosilactobacillus fermentum ) RC3 strain and Lactobacillus plantarum ( Lactiplantibacillus plantarum ) B6 strain, the preservation number of the fermentative Lactobacillus mucilaginosus RC3 is CGMCC No.30602, and the preservation number of the plant lactobacillus B6 is CGMCC No.30603.

2. A microecological preparation for temporary rearing of whiteleg shrimp after capture according to claim 1, characterized in that: The microecological preparation is fermented by Lactobacillus mucilaginosus ( Limosilactobacillus fermentum ) RC3 seed solution and Lactobacillus plantarum ( Lactiplantibacillus plantarum ) B6 seed liquid is mixed in a volume ratio of (1-2): (1-2), and inoculated into MRS liquid culture medium at a volume ratio of 5-10% to obtain a microecological preparation for temporary rearing of whiteleg shrimp after capture.

3. The microecological preparation for temporary rearing of whiteleg shrimp after capture according to claim 1, characterized in that: The number of viable bacteria of fermented Lactobacillus mucilaginosus RC3 in the microecological preparation is ≥1.42×10 9 CFU / mL, the number of viable bacteria of Lactobacillus plantarum B6 ≥1.14×10 9 CFU / mL.

4. A method for preparing a microecological preparation for temporary rearing of whiteleg shrimp after capture according to any one of claims 1 to 3, characterized in that The following steps are involved: Step 1, respectively taking fermentation Lactobacillus mucilaginosus RC3 and Lactobacillus plantarum B6 and placing them in a 36-38° C. constant temperature incubator for inversion culture for 20-28 hours to obtain fermentation Lactobacillus mucilaginosus RC3 and Lactobacillus plantarum B6 single colonies; picking single colonies of fermentation Lactobacillus mucilaginosus RC3 and Lactobacillus plantarum B6 and inoculating them into MRS liquid culture medium, culturing them at 28-38° C. for 12-24 hours for activation, and continuously activating for two generations to obtain fermentation Lactobacillus mucilaginosus RC3 seed liquid and Lactobacillus plantarum B6 seed liquid; Step 2: After mixing the fermented Lactobacillus mucilaginosus RC3 seed liquid and the Lactobacillus plantarum B6 seed liquid in a volume ratio of (1-2): (1-2), the mixed bacterial liquid is inoculated into MRS liquid culture medium in a volume ratio of 5-10%, and cultured at 28-38° C. for 12-24 hours to obtain a microecological preparation for temporary rearing of whiteleg shrimp after capture.

5. The method for preparing a probiotic preparation for temporarily rearing whiteleg shrimp after capture according to claim 4, characterized in that: In step 2, the fermented Lactobacillus mucilaginosus RC3 seed liquid and the Lactobacillus plantarum B6 seed liquid are mixed at a volume ratio of 2:1, and the mixed bacterial liquid is inoculated into an MRS liquid culture medium at a volume ratio of 5%, and cultured at 28-38° C. for 12-24 hours to obtain a microecological preparation for temporary rearing of whiteleg shrimp after capture.

6. The method for preparing a probiotic preparation for temporary rearing of whiteleg shrimp after capture according to claim 4, characterized in that: The number of viable bacteria of fermented Lactobacillus mucilaginosus RC3 in the microecological preparation is ≥1.42×10 9 CFU / mL, the number of viable bacteria of Lactobacillus plantarum B6 ≥1.14×10 9 CFU / mL.

7. Use of the probiotic preparation for temporary rearing of whiteleg shrimp after capture according to any one of claims 1 to 3 as a nitrite nitrogen degrader and / or ammonia nitrogen degrader in aquaculture seawater.

8. Use of the probiotic preparation for temporary rearing of whiteleg shrimp after capture according to any one of claims 1 to 3 in the preparation of a shrimp antioxidant enhancement preparation and / or a shrimp immune enhancer.

9. Use of a microecological preparation for temporary rearing of whiteleg shrimp after capture according to any one of claims 1 to 3 in the preparation of a shrimp intestinal probiotic preparation.

10. Use of the probiotic preparation for temporary rearing of whiteleg shrimp after capture according to any one of claims 1 to 3 in the preparation of a Vibrio harveyi inhibitor.

Images