Lactobacillus brevis and application thereof

Abstract

The invention discloses a lactobacillus brevis MyhYC-01, which is preserved in the China General Microbiological Culture Collection Center (CGMCC) on January 16, 2025, and the preservation number is CGMCC No.33441. The lactobacillus brevis MyhYC-01 is a lactobacillus brevis strain. The strain is screened from healthy Guayi lake ecological breeding river crabs, has better oxidation resistance, pathogenic bacterium inhibition and oxygen resistance, and can be used for effectively fermenting feed, pre-decomposing nutritional ingredients in the feed and improving the utilization efficiency of the feed. Through breeding experiments, the inventor finds that the strain has a relatively good effect on improving liver and intestine health and immunity of the river crabs. The lactobacillus brevis Myh YC-01 disclosed by the invention has the potential of serving as aquatic probiotics, and is very wide in application prospect.

Description

Technical Field

[0001] The invention relates to a lactobacillus brevis and application thereof in preparing fermented crab feed, belonging to the technical field of microorganisms. Background Art

[0002] Chinese mitten crab (Eriocheirsinensis), also known as river crab, is widely distributed in freshwater lakes across my country. It is favored by Chinese consumers for its unique flavor and rich in various nutrients. Therefore, in recent years, the farming of river crabs has developed rapidly and has become an important economic species of farming in my country. At present, many animal protein feeds for river crabs on the market are mostly fresh or frozen young fish. However, feeding a large amount of chilled small fish can easily cause frequent crab diseases and pollute the water quality. In 2020, the Ministry of Agriculture and Rural Affairs issued a document proposing five major actions to promote green and healthy aquaculture. Among them, "compound feed replacing young fish" requires the use of compound feed throughout the process of river crab farming.

[0003] In recent years, with the rapid development of river crab farming, the output of river crab feed has continued to increase. However, due to the shortage of feed resources, the price of raw materials remains high, and the cheap miscellaneous meal cannot be digested, absorbed and utilized by animals, and is discarded in large quantities. Faced with such problems, bio-fermented feed has gradually become popular in the industry. Bio-fermented feed is a general term for a new type of feed that uses feed or feed raw materials as the processing object and uses biotechnology such as fermentation engineering and protein engineering as the processing method. It mainly includes: microbial fermented feed, fermented and enzymatic feed, and feed enzyme preparations. There are many types of probiotics used in fermented feed, generally Bacillus, lactic acid bacteria, yeast, etc. Through single or composite strains, the feed protein is decomposed and converted into microbial bacterial protein, bioactive small peptide amino acids and active probiotics, which can remove anti-nutritional factors in the feed while also facilitating the absorption and utilization of nutrients by the animal body.

[0004] Most of the strains used in the current fermented crab feed are not isolated from farmed animals, resulting in the inability of the microorganisms to efficiently colonize in the intestines and the inability to maintain the continued function of the probiotic strains. Summary of the invention

[0005] The purpose of the present invention is to solve the above-mentioned shortcomings and provide a Lactobacillus brevis strain, which is screened from the healthy ecologically cultured river crabs of Gaoyou Lake, has good antioxidant, pathogen inhibition and oxygen tolerance capabilities, can be efficiently colonized in the intestines of river crabs, and is used to prepare fermented river crab feed, which can effectively improve the liver and intestinal health and immune ability of river crabs.

[0006] To achieve the above object, the present invention adopts the following technical solution:

[0007] A Lactobacillus brevis Myh YC-01 was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on January 16, 2025, with the deposit number CGMCC No. 33441.

[0008] The Lactobacillus brevis Myh YC-01 of the present invention was screened from healthy Eriocheir sinensis cultured in the ecological environment of Gaoyou Lake, and has good antioxidant, pathogen inhibitory and oxygen tolerance abilities; it can improve the quality of fermented feed, significantly improve the liver and intestine health of Eriocheir sinensis, and enhance the immunity of Eriocheir sinensis.

[0009] Use of the above-mentioned Lactobacillus brevis in antioxidant aspect.

[0010] Use of the above-mentioned Lactobacillus brevis in the preparation of fermented feed for Eriocheir sinensis to improve the liver and intestine health of Eriocheir sinensis and enhance the immunity of Eriocheir sinensis.

[0011] A method for preparing fermented feed for Eriocheir sinensis: First, mix sterilized water and basal feed evenly, then add the above-mentioned Lactobacillus brevis bacterial liquid, and ferment anaerobically at 37°C for 7 days to obtain fermented feed for Eriocheir sinensis;

[0012] The basal feed is composed of the following components in mass percentage: fish meal 8 - 12%, chicken meal 6 - 10%, blood globulin meal 2 - 3%, soybean meal 18 - 22%, peanut meal 14 - 16%, rapeseed meal 15 - 17%, yeast protein 1 - 3%, flour 11 - 13%, lysine 0.3 - 1%, methionine 0.1 - 0.3%, calcium dihydrogen phosphate 1 - 2.5%, soybean oil 2 - 4%, shrimp paste 1 - 3%, multi-vitamin and multi-mineral premix 0.5 - 1.5%, antioxidant 0.1 - 0.2%, sodium humate 1 - 3%, molt-inhibiting hormone 0.1 - 0.2%, bentonite 4 - 4.5%.

[0013] Furthermore, the mass ratio of the basal feed to the sterilized water is 1:0.4.

[0014] Furthermore, the addition amount of the Lactobacillus brevis is 10 6 CFU / g.

[0015] Furthermore, the method for preparing the Lactobacillus brevis bacterial liquid: Pick single colonies of Lactobacillus brevis from the isolated and purified solid medium and inoculate them into MRS liquid medium, anaerobically culture statically at 37°C for 24 h, inoculate them into MRS medium at an inoculation amount of 1% (volume percentage), and anaerobically culture statically at 37°C for 6 - 8 h to obtain Lactobacillus brevis bacterial liquid.

[0016] Advantages of the present invention: The present invention provides a Lactobacillus brevis Myh YC-01, which is a strain first isolated and screened from the intestine of Eriocheir sinensis. This strain has strong anti-inflammatory and antibacterial properties. Using this strain can effectively ferment feed, pre-digest the nutrients in the feed, and improve the utilization efficiency of the feed. Through breeding experiments, it is found that this strain has a good effect on improving the liver-intestine health and immune capacity of Eriocheir sinensis. The Lactobacillus brevis Myh YC-01 of the present invention has the potential to be used as an aquatic probiotic, and its application prospect is very broad. Brief Description of the Drawings

[0017] Figure 1 Phylogenetic tree of Lactobacillus brevis MyhYC-01 based on 16S rDNA sequence;

[0018] Figure 2 Growth curve of Lactobacillus brevis Myh YC-01;

[0019] Figure 3 Morphological feature diagram of Lactobacillus brevis MyhYC-01;

[0020] Figure 4 Test results of the antibacterial ability of Lactobacillus brevis MyhYC-01 against three pathogenic bacteria;

[0021] Figure 5 Effect of Lactobacillus brevis-fermented feed on antioxidant enzymes in the hepatopancreas of Eriocheir sinensis;

[0022] Figure 6 Effect of Lactobacillus brevis-fermented feed on the immune enzyme activity in the blood of Eriocheir sinensis;

[0023] Figure 7 Effect of Lactobacillus brevis-fermented feed on the expression of immune-related genes in the hepatopancreas of Eriocheir sinensis;

[0024] Figure 8 Effect of Lactobacillus brevis-fermented feed on the expression of genes related to intestinal health in Eriocheir sinensis. Detailed Embodiments

[0025] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and specific embodiments.

[0026] Example 1

[0027] 1. Isolation and identification of Lactobacillus brevis

[0028] The inventors collected the intestines of wild Eriocheir sinensis from Gaoyou Lake for the isolation and screening of Lactobacillus brevis. Specific method: Use sterile forceps to pick the intact intestines of Eriocheir sinensis, take the anterior and middle intestines and place them in 1 mL of pre-cooled sterile PBS buffer, and use a high-throughput tissue homogenizer to crush and homogenize thoroughly. After gradient dilution of the homogenate, take 0.1 mL and spread it on MRS solid medium, and incubate it statically at 37 °C for 48 h. Pick colonies with white color, smooth edges, and raised middle and inoculate them in MRS liquid medium for expanded culture and then streak culture. Repeat more than 3 times until the colony morphology is completely consistent. Transfer the picked single colonies to liquid medium for enrichment, and then store them in -80 °C refrigerator with 20% glycerol.

[0029] To further identify the strain, the genomic DNA of the strain was extracted, amplified using the universal primers of 16s rDNA, and the gene sequence of 16s rDNA was obtained by sequencing. Through BLAST analysis on the NCBI website and constructing a phylogenetic evolution tree, as Figure 1 . The results showed that the strain belongs to Lactobacillus brevis in Lactobacillus, named Lactobacillus brevis MyhYC-01, and was deposited in the General Microbiology Center of the China Microbial Culture Collection Center on January 16, 2025, with the deposit number CGMCC No. 33441.

[0030] 2. Growth performance and morphological biology of Lactobacillus brevis MyhYC-01

[0031] Take 3 anaerobic culture tubes filled with sterilized MRS liquid medium, inoculate 1% of the activated Lactobacillus brevis culture solution, place them in a constant temperature incubator at 37 °C, and take a small amount of liquid at 0, 1, 2, 3, 4, 6, 8, 10, 12, 14, and 16 h to measure the OD value.

[0032] Figure 2 is the growth curve of Lactobacillus brevis MyhYC-01. It can be seen from Figure 2 that when inoculated for 1 - 8 h, the strain enters the logarithmic growth phase, and the fastest growth rate is around 5 h.

[0033] Figure 3 is the morphological characteristic diagram of Lactobacillus brevis MyhYC-01. It can be seen that the colonies of Lactobacillus brevis are raised, slightly transparent milky white, round and smooth, with neat edges.

[0034] 3. Drug sensitivity test and antibacterial test of Lactobacillus brevis MyhYC-01

[0035] (1) The drug sensitivity test was performed using the K-B disc diffusion method. Specifically: The drug sensitivity discs were evenly patched on the plate coated with Lactobacillus brevis. After culturing at 37 °C for 24 h, the diameter of the inhibition zone was measured. R represents drug resistance, with an inhibition zone less than 10 mm; I represents moderate sensitivity, with an inhibition zone of 10 - 20 mm; S represents sensitivity, with an inhibition zone greater than 20 mm. The results are shown in Table 1:

[0036] Table 1 Results of the drug sensitivity test

[0037]

[0038]

[0039] As can be seen from Table 1, this Lactobacillus brevis is resistant to 14 drugs such as penicillin, oxacillin, cephalexin, cefazolin, cefuroxime sodium, ceftazidime, ceftriaxone, streptomycin, norfloxacin, ciprofloxacin, vancomycin, co-trimoxazole, clindamycin, and levofloxacin; moderately sensitive to 6 drugs such as ampicillin, cefoperazone, amikacin, kanamycin, erythromycin, and lincomycin; and sensitive to 10 drugs such as piperacillin, gentamicin, tetracycline, minocycline, azithromycin, polymyxin b, chloramphenicol, imipenem, doxycycline, and florfenicol.

[0040] (2) The antibacterial experiment was performed using the punching method. Specifically: Sterilized medical cotton swabs were used to dip into the bacterial suspensions of 3 common aquatic pathogenic bacteria, Staphylococcus aureus, Aeromonas veronii, and Aeromonas hydrophila, and evenly spread them on the nutrient agar solid medium. A 1 mL pipette tip (outer diameter: 9 mm) was used to punch holes in the nutrient agar medium. The fermentation broth of Lactobacillus brevis was aspirated and added to the corresponding holes, making the bacterial suspension in the holes full but not overflowing. The petri dish with the added sample was placed in a 4 °C refrigerator and left standing for 3 h, then transferred to a 37 °C incubator for anaerobic culture for 12 h. The diameter of the inhibition zone in the petri dish was measured and recorded to judge the antibacterial effect of Lactobacillus brevis. The test results are shown in Table 2 and Figure 4 :

[0041] Table 2 Diameters of the inhibition zones of three pathogenic bacteria

[0042]

[0043] As can be seen from Table 2 and Figure 4 it can be known that the diameters of the inhibition zones of Lactobacillus brevis against Aeromonas hydrophila and Aeromonas veronii are both > 15 mm. Therefore, Lactobacillus brevis has a high antibacterial effect on these two pathogenic bacteria. 15 mm > the inhibition zone of Staphylococcus aureus > 10 mm. Therefore, Lactobacillus brevis has a moderate antibacterial effect on Staphylococcus aureus.

[0044] 4. Oxygen tolerance experiment, physiological and biochemical reaction experiment, and DPPH free radical scavenging rate experiment of Lactobacillus brevis Myh YC-01.

[0045] (1) Dilute and spread the fermentation supernatant of Lactobacillus brevis Myh YC-01 after 24 h of cultivation. After exposing it to air for 0 min, 15 min, and 30 min respectively, place it in an anaerobic incubator and cultivate anaerobically at 37 °C for 24 h. Take it out and observe and count the growth status of the strains on each plate. Each sample is repeated three times to ensure the accuracy of the experimental results. Calculate the survival rate of Lactobacillus brevis with the viable count of the 0-min exposure as 100%. The test results are shown in Table 3:

[0046] Table 3 Test results of oxygen tolerance experiment

[0047]

[0048] As can be seen from Table 3, Lactobacillus brevis has a relatively high tolerance to oxygen.

[0049] (2) Conduct physiological and biochemical experiments on Lactobacillus brevis Myh YC-01 using a lactobacillus biochemical reaction tube (Qingdao Haibo Biotechnology Co., Ltd.).

[0050] Test results: Lactobacillus brevis can utilize cellobiose, maltose, mannitol, salicin, sorbitol, sucrose, raffinose, inulin, and lactose, but cannot utilize esculin.

[0051] (3) The DPPH free radical scavenging rate represents the antioxidant capacity of the strain. Take 1 mL of the fermentation supernatant of Lactobacillus brevis Myh YC-01, mix it evenly with 1 mL of 1.50 mmol / L DPPH anhydrous ethanol solution, react in the dark at room temperature for 30 min, and measure the absorbance at 517 nm. In the blank group, an equal volume of ethanol is used instead of the DPPH solution, in the control group, an equal volume of sterile water is used instead of the Lactobacillus brevis fermentation supernatant, and 0.125 g / L vitamin C is used as a positive control. The scavenging rate calculation formula:

[0052] Scavenging rate / % = [1 - (A 1 - A) / A 0 × 100%

[0053] In the formula, A is the absorbance value of the blank group, A 0 is the absorbance value of the control group, and A 1 is the absorbance value of the sample group.

[0054] Measured by the above experimental steps, Lactobacillus brevis Myh YC-01 has a good DPPH free radical scavenging rate, and the scavenging rate is 62.53%.

[0055] Example 2

[0056] Effect of Lactobacillus brevis on the growth performance, immune ability and hepatopancreas-intestine health of Chinese mitten crabs:

[0057] 120 Chinese mitten crabs were selected for the experiment and evenly divided into 3 groups, namely the control group, the blank fermented feed group, and the Lactobacillus brevis fermented feed group. Each group had 4 replicates, with 10 crabs in each replicate. They were cultured for 10 weeks and fed ad libitum twice a day (08:00 and 17:00). Among them, the control group was fed with the basal feed, the blank fermented feed group was fed with the blank fermented feed, and the Lactobacillus brevis fermented feed group was fed with the Lactobacillus brevis fermented feed. After 1 h of feeding, the residual bait and feces were removed by suction.

[0058] Preparation method of Lactobacillus brevis fermented feed: Pick a single colony of Lactobacillus brevis Myh YC-01 from the isolated and purified solid medium and inoculate it into MRS liquid medium. Anaerobic culture was carried out statically at 37 °C for 24 h, and then inoculated into MRS medium at an inoculation amount of 1% (volume percentage). Anaerobic culture was carried out statically at 37 °C until the end of the logarithmic phase to obtain Lactobacillus brevis bacterial liquid; Mix sterilized water and basal feed (basal feed formula: fish meal 10%, chicken meal 8%, blood globulin meal 2.5%, soybean meal 20%, peanut meal 15%, rapeseed meal 16%, yeast protein 2%, flour 12%, lysine 0.5%, methionine 0.1%, calcium dihydrogen phosphate 1.5%, soybean oil 3%, shrimp paste 2%, multi-vitamin and multi-mineral premix 1%, antioxidant 0.1%, sodium humate 2%, molting hormone 0.1%, bentonite 4.2%.) evenly at a mass ratio of 1:0.4, and then add the Lactobacillus brevis bacterial liquid to the sterilized MRS liquid medium and mix it into the feed (the initial addition amount of Lactobacillus brevis in the feed is 10 6 CFU / g), immediately vacuum-seal it, and put it in an incubator at 37 °C for fermentation for 7 d to obtain Lactobacillus brevis fermented feed, and store it at 4 °C after taking it out.

[0059] Preparation method of blank fermented feed: Without adding Lactobacillus brevis bacterial liquid, only add sterilized water and sterilized MRS liquid medium for fermentation (the other steps are the same as above) to obtain blank fermented feed, and store it at 4 °C after taking it out.

[0060] At the end of the breeding experiment, each crab was weighed and counted one by one to calculate its growth performance. Samples of muscle, hepatopancreas and intestine of Chinese mitten crabs in each group were collected to measure the enzyme activity of hepatopancreas and the gene expression of hepatopancreas and intestine. Take the hemolymph at the base joint membrane of the third walking leg of the crab, add anticoagulant, centrifuge at 12000 r / min at 4 °C for 10 min, and collect the supernatant for the determination of immune enzyme activity.

[0061] Enzyme activity assay method:

[0062] The intestinal and hepatopancreas samples were prepared into homogenates using a high-throughput grinding tissue homogenizer, and the determination of intestinal digestive enzymes and hepatopancreas antioxidant enzymes was carried out according to the kits of Nanjing Jiancheng Bioengineering Institute. The determination of alkaline phosphatase (AKP), acid phosphatase (ACP), lysozyme (LZM), and peroxidase (POD) in blood samples was carried out according to the instructions of the kits of Nanjing Jiancheng Bioengineering Institute.

[0063] Gene expression level assay method:

[0064] The cDNA was synthesized using TransScript All-in-One First-strand cDNA Synthesis SuperMix for qPCR reagent (Transgen Biotech, Beijing, China). The gene primers were synthesized by Tsingke Biotechnology Co., Ltd. (Beijing, China). The qRT-PCR reaction was carried out on a real-time fluorescence quantitative PCR instrument using ChamQ SYBR qPCR Master Mix (Vazyme, Nanjing, China). The reaction system and conditions were set according to the instructions of the detection kit. The total reaction volume was 20 μL, with β-actin as the internal reference gene, and the relative expression level of the gene mRNA was calculated using the 2 -△△Ct method.

[0065] Experimental results:

[0066] The test results of the effects of Lactobacillus brevis-fermented feed on the survival rate, growth performance, and feed conversion ratio of Chinese mitten crabs are shown in Table 4:

[0067] Table 4 Effects of Lactobacillus brevis-fermented feed on the survival rate, growth performance, and feed conversion ratio of Chinese mitten crabs

[0068]

[0069] It can be seen from Table 4 that the weight gain rate of the Lactobacillus brevis-fermented feed group was significantly higher than that of the other two groups (P < 0.05), and the feed conversion ratio was significantly reduced (P < 0.05), indicating that the utilization efficiency of Lactobacillus brevis-fermented feed was better.

[0070] The effects of Lactobacillus brevis-fermented feed on the antioxidant enzymes in the hepatopancreas of Chinese mitten crabs are shown in Figure 5 , in which, Figure 5 A is the effect on T-AOC, Figure 5 B is the effect on GSH-PX, Figure 5C is the impact on MDA. Both GSH-PX and T-AOC are important indicators representing the antioxidant capacity of Chinese mitten crabs, while the level of MDA indicates the degree of damage to hepatopancreas cells. The results show that feeding the fermented feed of Lactobacillus brevis significantly promoted the activities of GSH-PX and T-AOC (P < 0.05), enhancing the antioxidant capacity of the hepatopancreas of Chinese mitten crabs. At the same time, the level of MDA decreased significantly (P < 0.05), and the hepatocytes were healthy.

[0071] Figure 6 For the impact of the fermented feed of Lactobacillus brevis on the immune enzyme activities in the blood of Chinese mitten crabs, among which, Figure 6 A is the impact on lysozyme LZM, Figure 6 B is the impact on peroxidase POD, Figure 6 C is the impact on acid phosphatase ACP, Figure 6 D is the impact on alkaline phosphatase AKP. The results show that the activities of POD, LZM, and AKP in the blood of the group fed with the fermented feed of Lactobacillus brevis were all significantly increased (P < 0.05), indicating that feeding enhanced the non-specific immune capacity of Chinese mitten crabs.

[0072] Figure 7 For the impact of the fermented feed of Lactobacillus brevis on the expression of immune-related genes in the hepatopancreas of Chinese mitten crabs, among which, Figure 7 A is the impact on alf, Figure 7 B is the impact on crustin1, Figure 7 C is the impact on hsp70, Figure 7 D is the impact on hsp90, Figure 7 E is the impact on toll1, Figure 7 F is the impact on pelle, Figure 7 G is the impact on tube, Figure 7 H is the impact on myd88. The toll1, pelle, myd88, and tube genes are important components in the Toll immune pathway. Myd88 activates the production of inflammatory factors, and the antimicrobial peptides alf and crustin1 are key genes for immune defense. Hsp70 and hsp90 both belong to heat shock proteins, which can enable animals to quickly adapt to environmental changes, protect cells from damage, and can induce and activate a variety of immune factors to participate in immune responses. The results show that feeding the fermented feed of Lactobacillus brevis significantly up-regulated the gene expression levels of toll1, toll2, pelle, tube, alf1, alf2, crustin, hsp70, and hsp90 (P < 0.05), and significantly down-regulated the gene expression level of myd88 (P < 0.05). It shows that the immune capacity of the hepatopancreas of Chinese mitten crabs has been enhanced.

[0073] Figure 8 For the impact of the fermented feed of Lactobacillus brevis on the expression of genes related to intestinal health in Chinese mitten crabs, among which, Figure 8A is the impact on ilf2, Figure 8 B is the impact on litaf, Figure 8 C is the impact on p38MAPK, Figure 8 D is the impact on relish, Figure 8 E is the impact on PM, Figure 8 F is the impact on PT, Figure 8 G is the impact on ZO-1. The expression levels of PT and PM can represent the health of the peritrophic membrane barrier in the intestine of Chinese mitten crabs, and ZO-1 is related to intestinal permeability. Ilf2 and litaf are two important pro-inflammatory factors in Eriocheir sinensis and are regarded as marker cytokines for the occurrence of inflammatory responses. P38MAPK initiates the release of litaf. The nuclear transcription factor relish is a member of the NF-κBs superfamily and is an important immune signal transduction effector. The results showed that the expression levels of PT, PM, and ZO-1 in the group fed with Lactobacillus brevis-fermented feed were significantly up-regulated (P < 0.05), and intestinal permeability increased; the pro-inflammatory factors were significantly down-regulated (P < 0.05), indicating that Lactobacillus brevis has a good anti-inflammatory effect.

[0074] The above are only the preferred embodiments of the present invention and are not used to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A Lactobacillus brevis Myh YC-01, deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on January 16, 2025, with the deposit number CGMCC No.33441.

2. Use of the Lactobacillus brevis described in claim 1 in the preparation of fermented crab feed for improving the liver and intestine health of crabs and enhancing the immune ability of crabs.

3. A method for preparing fermented crab feed, characterized in that: First, the sterilized water and the basic feed are mixed evenly, and then the Lactobacillus brevis liquid according to claim 1 is added, and anaerobically fermented at 37° C. for 7 days to obtain the fermented feed for river crabs; The basic feed is composed of the following components in percentage by mass: 8-12% fish meal, 6-10% chicken meal, 2-3% hemoglobin powder, 18-22% soybean meal, 14-16% peanut meal, 15-17% rapeseed meal, 1-3% yeast protein, 11-13% flour, 0.3-1% lysine, 0.1-0.3% methionine, 1-2.5% monocalcium phosphate, 2-4% soybean oil, 1-3% shrimp paste, 0.5-1.5% multivitamin premix, 0.1-0.2% antioxidant, 1-3% sodium humate, 0.1-0.2% dehulling agent, and 4-4.5% bentonite.

4. The method for preparing the fermented crab feed according to claim 3, characterized in that: The mass ratio of the basic feed to sterilized water is 1:0.

4.

5. The method for preparing the fermented crab feed according to claim 3, characterized in that: The amount of Lactobacillus brevis added is 10 6 CFU / g.

6. The method for preparing fermented crab feed according to claim 3, characterized in that: The preparation method of the Lactobacillus brevis bacterial liquid comprises the following steps: picking a single Lactobacillus brevis colony from a separated and purified solid culture medium and placing it in an MRS liquid culture medium for anaerobic culture at 37° C. for 24 hours, inoculating it in an MRS culture medium at a 1% inoculation amount, placing it in an anaerobic culture at 37° C. for 6-8 hours, and obtaining the Lactobacillus brevis bacterial liquid.

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