Enterococcus faecalis and application thereof in aquatic product fermented feed and laying hen premix

Abstract

The invention belongs to the technical field of biology, and provides enterococcus faecalis, the preservation number of which is CGMCC (China General Microbiological Culture Collection Center) No.29087. The enterococcus faecalis is characterized in that the enterococcus faecalis can secrete bacteriocin for inhibiting vibrio parahaemolyticus, staphylococcus aureus and escherichia coli. The invention also provides the enterococcus faecalis and application thereof in an aquatic product fermented feed and a laying hen premix, and the enterococcus faecalis can effectively improve the utilization rate of the feed and the growth and production performance of animals.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology. A strain of Enterococcus faecalis capable of producing bacteriocin that inhibits Vibrio parahaemolyticus, Staphylococcus aureus and Escherichia coli is screened and prepared into aquatic fermented feed and laying hen premix, which is applied to shrimp and laying hen farming to improve the growth performance and production performance of animals. Background Art

[0002] Enterococcus faecalis is a type of lactic acid bacteria that is widely distributed in nature and can be widely used in the food and feed industry for fermented food and yogurt preparation. Enterococcus faecalis grows rapidly and has a strong acid production capacity. During its growth, it can also metabolize and synthesize bacteriocins and other antibacterial active substances, which can inhibit common pathogens such as Vibrio parahaemolyticus, Staphylococcus aureus, and Escherichia coli.

[0003] Enterococcus faecalis is one of the microbial species allowed for use in the food additive catalog and feed additive catalog, and its safety and effectiveness are widely recognized. The use of Enterococcus faecalis has many functions, such as improving inflammatory response, regulating intestinal flora, enhancing immune response, and promoting fat absorption. Therefore, it has a wide range of application potential in food and feed.

[0004] Bacteriocins are a class of bactericidal proteins or peptides synthesized by ribosomes in bacteria. They have a broad bactericidal spectrum and are not prone to drug resistance. Therefore, they are a very important class of antibiotic substitutes. Vibrio is one of the pathogens of aquaculture manufacturers, which seriously threatens the health of shrimp, crabs and other farmed species, and brings huge losses to the aquaculture industry. Common pathogenic Vibrio include Vibrio alginolyticus, Vibrio harveyi and Vibrio parahaemolyticus. The common pathogenic Vibrio in shrimp farming is mainly caused by Vibrio parahaemolyticus, so controlling the number of Vibrio parahaemolyticus in the farming process is crucial to the success rate of farming. Golden glucose and Escherichia coli K88 and K99 are common pathogens that cause diarrhea in laying hens, which bring great harm to the health and production of laying hens. Summary of the invention

[0005] To solve the above problems, the present invention screened a strain of Enterococcus faecalis from the small shed shrimp farming area in Rudong, Jiangsu Province, which can significantly inhibit the growth of Vibrio parahaemolyticus. In the use of fermented shrimp feed, it can significantly improve feed utilization, reduce feed conversion rate, reduce the number of Vibrio in the breeding environment, improve the success rate of shrimp breeding, reduce breeding risks, and increase breeding benefits. Prepared into a premix for laying hens, it can also improve the production performance of laying hens and increase eggshell thickness.

[0006] Firstly, the present invention provides a new strain of Enterococcus faecalis YP-2 which can inhibit Vibrio, Staphylococcus aureus and Escherichia coli.

[0007] The faecal enterococci provided by the present invention are isolated from the white shrimp farming waters in Rudong, Jiangsu Province. Under an optical microscope, they are Gram-stain positive, single or double ovoid cocci, without spores and capsules; and on MRS culture medium, they appear as white, smooth and round colonies with neat edges.

[0008] The Enterococcus faecalis screened by the invention can produce bacteriocin which inhibits Vibrio parahaemolyticus, Staphylococcus aureus and Escherichia coli.

[0009] The screened strains were identified by PCR using universal primers for bacteria: template DNA was extracted according to the instructions of the bacterial DNA extraction kit. The upstream primer 5'-AGAGTTTGATCCTGGCTCAG-3' and the downstream primer 5'-GGTTACCTT GTTACGACT T-3' of the 16S rRNA conservative sequence were used to amplify the bacterial 16S rRNA gene fragment. After sequencing, it was found that the bacteria belonged to Enterococcus faecalis, and the sequencing results of the amplified fragment named YP-2 are shown in SEQ ID No.1.

[0010] MRS medium: 10g protein Chen, 5g beef powder, 4g yeast powder, 2g glucose, 1ml Tween 80, 2g potassium dihydrogen phosphate, 5g sodium acetate, 2g ammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate, 1000ml distilled water. Inoculate strain YP-2 and culture for 48h to detect the inhibitory effect of its fermentation broth on Vibrio parahaemolyticus, Staphylococcus aureus and Escherichia coli.

[0011] The fermentation liquid was added into the Oxford cup containing Vibrio parahaemolyticus, Staphylococcus aureus and Escherichia coli plates, and obvious inhibition zones were observed.

[0012] The screened Enterococcus faecalis was named YP-2 and deposited on November 21, 2023 at the General Microbiology Center of China Culture Collection Administration, address: Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, with the deposit number: CGMCC No.29087.

[0013] The present invention also provides a method for preparing fermented feed for inhibiting the number of Vibrio in a shrimp breeding environment, wherein the fermented product contains Enterococcus faecalis CGMCC No. 29087. The use method described in the patent can effectively reduce the feed coefficient of shrimp by 0.15 to 0.3.

[0014] The invention also provides another laying hen premix containing Enterococcus faecalis CGMCC No.29087, which can reduce the incidence of enteritis in laying hens and increase the thickness of egg shells.

[0015] The fermented shrimp feed and laying hen premix developed by the present invention can effectively inhibit the growth of pathogenic bacteria Vibrio parahaemolyticus, Staphylococcus aureus and Escherichia coli, reduce the incidence rate in the animal breeding process, and improve the growth and production performance of animals. DETAILED DESCRIPTION

[0016] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention.

[0017] Example 1 Experimental method for inhibiting Vibrio parahaemolyticus by Enterococcus faecalis CGMCC No. 29087

[0018] (1) Vibrio culture method

[0019] 1 ml of TCBS liquid culture medium was placed in a 10 ml test tube, inoculated with 0.5% Vibrio parahaemolyticus, and cultured overnight at 28° C. The cultured bacteria were streaked for pure culture, and then a single colony was picked and inoculated into TCBS liquid culture medium, 50% glycerol was added, and frozen for storage.

[0020] (2) Screening plate production

[0021] After the frozen Vibrio parahaemolyticus was inoculated into TCBS medium for activation, 0.2 ml of the bacteria was taken up with a sterile pipette and evenly spread on the TCBS solid medium.

[0022] (3) Oxford Cup Screening Method

[0023] Place 3-4 sterile Oxford cups evenly on the TCBS plate inoculated with Vibrio parahaemolyticus and evenly coated.

[0024] (4) Enterococcus faecalis YP-2 was cultured in MRS medium for 24 h and then diluted 1-fold, 2-fold, 4-fold, and 8-fold with sterile water.

[0025] (4) Screening of Enterococcus faecalis by Oxford Cup Method

[0026] Place a sterile Oxford cup on the plate prepared by methods (2) and (3), use a sterile pipette to draw 200 μL of Enterococcus faecalis YP-2 fermentation broth of different dilution multiples and add it to the Oxford cup, place it in an incubator at 28°C and culture it for 24 hours, observe whether there is an obvious antibacterial zone around the Oxford cup and measure its diameter, so as to determine whether the fermentation broth contains antibacterial substances of Vibrio parahaemolyticus and its antibacterial ability.

[0027] Table 1 Evaluation of the ability of Enterococcus faecalis to inhibit Vibrio parahaemolyticus

[0028] Sample No. Do not dilute the stock solution Dilute 1x Dilute 2 times Dilute 4 times Dilute 8 times Inhibition zone diameter (mm) 15.7±0.2 8.4±0.5 5.1±0.4 1.0±0.2 03±0.1

[0029] As can be seen from Table 1, the diameter of the inhibition zone of Enterococcus faecalis CGMCC No. 29087 reached 15.7 mm when it was not diluted, and the diameter of the inhibition zone was still 5.1 mm after being diluted 2 times, but the inhibition zone was not obvious when it was diluted more than 4 times.

[0030] Example 2 Method for inhibiting Staphylococcus aureus and Escherichia coli by Enterococcus faecalis CGMCC No. 29087

[0031] (1) Cultivation method of Staphylococcus aureus

[0032] 1 ml of TSB liquid medium was placed in a 10 ml test tube, inoculated with 0.5% Staphylococcus aureus, and cultured overnight at 30° C. The cultured bacteria were streaked for pure culture, and then a single colony was picked and inoculated into TSB liquid medium, 50% glycerol was added, and frozen for storage.

[0033] (2) Cultivation method of Escherichia coli K88 and K99

[0034] 1 ml of LB liquid medium was placed in a 10 ml test tube, and 0.5% of E. coli K88 and K99 were inoculated respectively, and cultured overnight at 37° C. The cultured bacteria were streaked for pure culture, and then a single colony was picked and inoculated into LB liquid medium, 50% glycerol was added, and frozen for storage.

[0035] (3) Screening plate production

[0036] After the frozen Staphylococcus aureus was inoculated into TSB medium for activation, 0.2 ml of the bacteria was taken up with a sterile pipette and evenly spread on TSB solid medium; after the frozen Escherichia coli K88 and K99 were inoculated into LB medium for activation, 0.2 ml of the bacteria was taken up with a sterile pipette and evenly spread on LB solid medium.

[0037] (4) Oxford Cup Screening Method

[0038] 3-4 sterile Oxford cups were evenly placed on the TSB plate inoculated with Staphylococcus aureus and evenly coated, and the LB plate inoculated with Escherichia coli K88 and K99.

[0039] (5) Oxford cup method for detecting antibacterial diameter

[0040] Place a sterile Oxford cup on the plate prepared by methods (3) and (4), use a sterile pipette to draw 200 μL of Enterococcus faecalis YP-2 fermentation liquid and add it to the Oxford cup, place it in an incubator at 30°C and 37°C for 24 h, and observe the diameter of the inhibition zone around the Oxford cup.

[0041] Table 2 Evaluation of the ability of Enterococcus faecalis to inhibit Staphylococcus aureus and Escherichia coli

[0042] Indicator bacteria Staphylococcus aureus Escherichia coli K88 Escherichia coli K88 Diameter of inhibition zone (mm) 13.5±0.5 19.3±0.5 16.4±0.4

[0043] As can be seen from Table 2, strain YP-2 has a significant inhibitory effect on Staphylococcus aureus, Escherichia coli K88 and K99.

[0044] Example 3 Detection of antibacterial substances of Enterococcus faecalis CGMCC No.29087

[0045] (1) Elimination of the antibacterial effect of acid: When the pH value of the supernatant of the fermentation culture of Enterococcus faecalis CGMCC No. 29087 was adjusted to 7.0, it was found that it still had an inhibitory effect on Vibrio parahaemolyticus, Staphylococcus aureus and Escherichia coli, indicating that the antibacterial activity was not produced by acid but by active substances.

[0046] (2) Catalase experiment: Catalase was added to the supernatant of the fermentation culture of Enterococcus faecalis CGMCC No. 29087 to decompose the possible hydrogen peroxide. The experiment found that the fermentation supernatant of Enterococcus faecalis CGMCC No. 29087 still had antibacterial activity, indicating that the antibacterial effect was not produced by hydrogen peroxide.

[0047] (3) Protease experiment: The fermentation broth of Enterococcus faecalis was treated with pepsin, trypsin, proteinase K, papain and bromelain for 4 h, and then their activities were detected. The results are shown in Table 3.

[0048] Table 3 Results of the protease treatment experiment of Enterococcus faecalis fermentation broth

[0049]

[0050] Note: + number indicates the size of the inhibition zone

[0051] As can be seen from Table 3, the antibacterial effect of the fermentation supernatant of Enterococcus faecalis CGMCC No. 29087 was significantly reduced after being treated with protease and trypsin, while it was more resistant to proteinase K, papain and bromelain. The experimental results show that the antibacterial substances can be degraded by protease and trypsin, belong to small peptide antibacterial substances, and belong to bacteriocins.

[0052] Example 4 Preparation of high-density fermentation broth of Enterococcus faecalis CGMCC No.29087

[0053] (1) Plate culture rejuvenation: Enterococcus faecalis CGMCC No. 29087 was inoculated on MRS plate culture medium and cultured at 30°C for 24 h to allow Enterococcus faecalis CGMCC No. 29087 to be rejuvenated and form single colonies. Single colonies were picked and placed on inoculation medium and cultured at 30°C for 24 h.

[0054] (2) Preparation of primary seeds: The Enterococcus faecalis CGMCC No. 29087 cultured in step 1) was transferred to the MRS slant medium in the eggplant bottle and cultured at 30° C. for 24 h until the culture was in the late logarithmic phase, thereby obtaining primary seeds.

[0055] (3) Preparation of secondary seeds: The primary seeds prepared in step 2) were made into a bacterial suspension with sterile water, and inoculated into a 100 L seed tank containing 60 L MRS seed culture medium at a temperature of 30°C, a rotation speed of 120 r / min, a tank pressure of 0.05 MPa, and a ventilation ratio of 1:0.2 for 18 h to obtain a secondary seed solution.

[0056] (4) Preparation of Enterococcus faecalis CGMCC No. 29087 fermentation broth: The secondary seed solution prepared in step 3) was inoculated into 1 mL of 600 L fermentation medium at a rate of 1%. 3 In the fermentation tank, the temperature was 30℃, the speed was 120r / min, the tank pressure was 0.05Mpa, the ventilation ratio was 1:0.2, the culture time was 18h, and the number of viable bacteria was 3×10 9 CFU / ml of Enterococcus faecalis CGMCC No.29087.

[0057] The fermentation medium is (g / L): 10g protein Chen, 10g beef extract, 20g yeast powder, 20g glucose, 1ml Tween 80, 2g dipotassium hydrogen phosphate, 5g sodium acetate, 2g triammonium citrate, 0.2g magnesium sulfate, 0.05g manganese sulfate, 15g agar powder, 0.5g defoamer, 1000ml distilled water, and the pH is adjusted to 7.0.

[0058] Example 5 Preparation of fermented feed

[0059] (1) Preparation of shrimp feed: The main raw materials of shrimp feed include imported fish meal, soybean meal, phospholipid oil, lysine, calcium dihydrogen phosphate, etc., which are prepared into pellet feed with a particle size of 0.8-1.0 through the shrimp feed pellet production line.

[0060] (2) Preparation of fermented feed: 1000 kg of pelleted feed prepared by the feed production line was added with 2 kg of Enterococcus faecalis CGMCC No.29087 powder, 1 kg of Bacillus subtilis powder, 1 kg of Saccharomyces boulardii, 0.5 kg of mannan peptide, 4 kg of Astragalus, 2 kg of Momordica grosvenori, and 0.5 kg of white lentil. The mixture was evenly mixed, put into a fermentation bag with a breathing membrane, and sealed and fermented at 30°C for 5N7 days.

[0061] (3) Detection of the number of live bacteria in fermented feed: Take 10g of shrimp fermented feed, grind it evenly, and dissolve it in 100ml of sterile water. After shaking for 30min, centrifuge it at 5000r / min for 10min, and take the supernatant. Bacillus subtilis was counted on LB solid medium, yeast was counted on solid Bengal red medium, and Enterococcus faecalis CGMCC No.29087 was counted on solid MRS medium. The number of live bacteria of Bacillus subtilis, Enterococcus faecalis CGMCC No.29087 and Saccharomyces boulardii were 2.1×10 9 CFU / g, 1.8×10 8 CFU / g and 4.3×10 8 CFU / g.

[0062] Example 7 Application of fermented shrimp feed containing Enterococcus faecalis CGMCC No.29087

[0063] The fermented shrimp feed prepared in Implementation Case 5 was applied in the shrimp farming model of small sheds. The experiment was divided into three groups, two control groups (control group 1 and control group 2) and one experimental group. Each group had 9 small sheds (area of ​​500 square meters, water depth of 0.8 meters), and each group had 3 parallels. The seedlings and seeding density of the control group and the experimental group were the same. The control group 1 was fed only with shrimp feed, the control group 2 was fed with fermented feed without adding Enterococcus faecalis CGMCC No.29087 (the preparation method was to replace 2 kg of Enterococcus faecalis CGMCC No.29087 powder with commercial freeze-dried Enterococcus faecalis powder on the basis of Implementation Case 5), and the experimental group was fed with fermented feed with Enterococcus faecalis CGMCC No.29087 prepared according to Implementation Case 5. The feeding method of the experimental group feed was: shrimp feed: fermented feed = 4:1, and the control group and the experimental group were fed 3 times a day. The feeding experiment lasted for 80 days, and the growth indicators such as total weight of shrimp, feed conversion ratio and serum immune indicators such as total superoxide dismutase and malondialdehyde were counted.

[0064] Table 4 Experimental results of fermented feed shrimp farming

[0065] project Control group 1 Control group 2 Experimental Group Stocking density (tail / mouth) <![CDATA[50000±500 a ]]> <![CDATA[50000±520 a ]]> <![CDATA[50000±600 a ]]> Total weight of shrimp (kg / mouth) <![CDATA[610.5±21.3 a ]]> <![CDATA[653.3±32.6 b ]]> <![CDATA[783.5±38.8 c ]]> Shrimp feed coefficient <![CDATA[1.33±0.05 c ]]> <![CDATA[1.18±0.05 b ]]> <![CDATA[1.00±0.05 a ]]> Total superoxide dismutase (U / ml) <![CDATA[87.6±3.8 a ]]> <![CDATA[98.9±4.2 b ]]> <![CDATA[106.6±5.6 c ]]> Malondialdehyde (mmol / ml) <![CDATA[17.4±0.8 c ]]> <![CDATA[12.4±1.2 b ]]> <![CDATA[6.6±1.6 a ]]>

[0066] Note: The numbers with different letters have statistically significant differences (p < 0.05).

[0067] As can be seen from Table 4, under the same seedlings and stocking density, the shrimp weight of the experimental group reached 783.5 kg, which was 28.3% higher than that of the control group 1. Similarly, the feed conversion rate (feed coefficient) of the control group 1 was only 1.00, significantly lower than the 1.33 of the control group. Compared with the control group 2, the total weight of the shrimp also increased significantly, with an increase of 19.9%; the feed coefficient decreased by 0.18.

[0068] After the breeding, 10 shrimps were randomly selected from the three experimental groups to measure the serum antioxidant index. The results showed that the total superoxide dismutase index of the experimental group fed with fermented feed was significantly higher than that of the two control groups, reaching 106.6U / ml, which shows that fermented feed improves the antioxidant capacity in the serum of shrimps, can more effectively remove free radicals, and improve the body's resistance to environmental stress; and the content of malondialdehyde is significantly lower than that of the control group 1, only 6.6mmol / ml, which shows that fermented feed can effectively improve the integrity of immune organ cells such as the liver and reduce cell loss.

[0069] The fermented feed breeding experiment shows that adding Enterococcus faecalis CGMCC No.29087 in the production and preparation of fermented feed can significantly increase the functional effects of compound Chinese herbal medicine and other probiotics. In the breeding process of shrimp, adding 20% ​​of it in combination with feed can not only improve feed utilization and weight gain efficiency, but also improve the immunity of shrimp.

[0070] In addition, during the screening process of Enterococcus faecalis CGMCC No. 29087, it was found that the strain could effectively inhibit the growth of Vibrio parahaemolyticus. Therefore, during the experiment, we also detected the changes in the number of Vibrio parahaemolyticus in the aquaculture water. The relevant results are shown in Table 5.

[0071] Table 5 Changes in the number of Vibrio parahaemolyticus during the use of fermented feed

[0072] <![CDATA[Vibrio parahaemolyticus (10 3 CFU / L)]]> Control group 1 Control group 2 Experimental Group Day 0 <![CDATA[4.8±0.5 a ]]> <![CDATA[4.8±0.5 a ]]> <![CDATA[4.8±0.4 a ]]> Day 5 <![CDATA[4.7±0.6 c ]]> <![CDATA[4.3±0.6 b ]]> <![CDATA[0.9±0.5 a ]]> Day 10 <![CDATA[5.7±0.4 c ]]> <![CDATA[3.1±0.4 b ]]> <![CDATA[0.4±0.5 a ]]> Day 15 <![CDATA[6.8±0.5 c ]]> <![CDATA[1.8±0.5 b ]]> <![CDATA[0.2±0.5 a <!-- 5 -->]]> Day 20 <![CDATA[10.7±0.3 c ]]> <![CDATA[1.1±0.3 b ]]> <![CDATA[0.05±0.5 a ]]>

[0073] Note: The numbers with different letters have statistically significant differences (p < 0.05).

[0074] As can be seen from Table 5, the number of Vibrio in the control group 1 showed an increasing trend during the breeding process, which indicates that the number of Vibrio in the breeding water has been in a process of proliferation. After the experimental group used fermented feed, the number of Vibrio in the breeding water was significantly reduced, two orders of magnitude lower than that of the control group; and the continuous use of fermented feed can always control Vibrio parahaemolyticus at a low level, which will greatly reduce the occurrence of diseases during the breeding process. Although the number of Vibrio in the control group 2 decreased, its concentration was still at a high level. This also shows that during the use of fermented feed, the key metabolites that inhibit Vibrio parahaemolyticus are produced by Enterococcus faecalis CGMCC No. 29087, so only the number of Vibrio in the experimental group is maintained at a low level.

[0075] Example 8 Application of core premix containing Enterococcus faecalis CGMCC No.29087 in laying hen breeding

[0076] The core premix containing Enterococcus faecalis CGMCC No.29087 was added to the laying hen feed to evaluate the effect of the core premix on the production performance of laying hens. The experiment was divided into three groups, the control group, experimental group 1 and experimental group 2. The control group did not add the core premix, the level of Enterococcus faecalis CGMCC No.29087 added to experimental group 1 was 103CFU / g, and the level of Enterococcus faecalis CGMCC No.29087 added to experimental group 2 was 10bCFU / g. The experiment lasted for 45 days, and the egg production, feed-egg ratio and the number of Escherichia coli in the intestine were counted.

[0077] Table 6 Effect of core premix on laying hen production performance

[0078] project Control group Experimental Group 1 Experimental Group 2 Enterococcus faecalis supplementation level (CFU / g) 0 103 106 Egg production (g / (egg·day)) <![CDATA[50.3±1.3 a ]]> <![CDATA[51.2±0.9 b ]]> <![CDATA[56.7±1.2 c ]]> Feed to Egg Ratio 2.23±0.09 2.26±0.11 2.30±0.15 Eggshell thickness (mm) <![CDATA[0.35±0.02 a ]]> <![CDATA[0.36±0.03 a ]]> <![CDATA[0.41±0.02 b ]]> Haff Unit 88.8±1.2 89.2±1.7 88.9±1.3 <![CDATA[Number of Escherichia coli in the intestine (10 5 CFU / g)]]> 9.97±0.15c <![CDATA[9.11±0.18 b ]]> <![CDATA[1.74±0.17 a ]]>

[0079] Note: The numbers with different letters have statistically significant differences (p < 0.05).

[0080] As can be seen from Table 6, adding a core premix containing Enterococcus faecalis CGMCC No.29087 to laying hen feed can significantly increase the egg production of laying hens compared with the control group, and the effect of the high level (experimental group 2) of addition is better than the low level (experimental group 1) of addition. The thickness of the eggshell was also found to be higher in the experimental group containing the core premix of Enterococcus faecalis CGMCC No.29087 than in the control group, indicating that Enterococcus faecalis can promote the absorption and conversion of calcium. The number of Escherichia coli in the intestinal flora was detected, and it was found that the experimental group was lower than the control group, indicating that the colonization of Enterococcus faecalis can inhibit the growth of Escherichia coli. The addition of the core premix had no significant effect on the feed-egg ratio and the Haugh unit.

Claims

1. A new strain of Enterococcus faecalis, whose deposit number is CGMCC No.29087, whose 16S rRNA gene sequence is shown in SEQ ID No.1, characterized in that Produces bacteriocins that inhibit Vibrio parahaemolyticus, Staphylococcus aureus and Escherichia coli.

2. A fermented feed suitable for the cultivation of Penaeus vannamei in small sheds and outdoor ponds, characterized in that: The formula ratio of the fermented feed is: 800-1000kg of shrimp feed, 0.5-2kg of Enterococcus faecalis CGMCC No.29087 powder, 0.5-1kg of Bacillus subtilis powder, 0.5-1kg of Saccharomyces cerevisiaeboulardii powder, 0.5-1kg of mannan peptide, 3-5kg of astragalus, 1-2kg of monk fruit and 0.5-1kg of white hyacinth bean.

3. A method for using a fermented feed containing Enterococcus faecalis CGMCC No. 29087 in aquaculture of white shrimp in small sheds and outdoor ponds: (1) mixing the feed with shrimp feed in a ratio of 10-20%, and then sealing and fermenting for 4 hours to feed the shrimp; (2) adding 10 kg of fermentation carbon source and 90 catties of water to 1 kg of fermented feed, and then sealing and fermenting for 48 hours and then spraying the whole pond, using 5-10 kg of fermented liquid per mu of pond; mixing the feed and spraying the whole pond can reduce the shrimp feed coefficient by 0.15-0.

3.

4. A premix for laying hens that reduces the number of intestinal Escherichia coli and increases eggshell thickness, characterized in that Each ton of premix contains: 1.0~6.0×10 10 10-15 kg of CFU / g of Enterococcus faecalis CGMCC No.29087, 3-5 kg ​​of polyglutamic acid, 0.5-1 kg of polylysine, and 1-1.5 kg of mannan peptide.

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