An Enterococcus faecalis and its applications in aquatic fermented feed and laying hen premix

Abstract

The present invention belongs to the field of biotechnology and provides a strain of Enterococcus faecalis with a preservation number of CGMCC No. 29087, which is characterized in that the Enterococcus faecalis can secrete bacteriocins that inhibit Vibrio parahaemolyticus, Staphylococcus aureus and Escherichia coli. The present invention also provides the application of the Enterococcus faecalis and the same in aquatic fermented feed and laying hen premix, which can effectively improve the utilization rate of feed and the growth and production performance of animals.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology. A strain of Enterococcus faecalis that can produce bacteriocin inhibiting Vibrio parahaemolyticus, Staphylococcus aureus and Escherichia coli is screened, and is prepared into an aquatic fermentation feed and a premix for laying hens, and is applied to the cultivation of penaeid shrimps and laying hens, so as to improve the growth performance and production performance of animals. Background Art

[0002] Enterococcus faecalis is a type of lactic acid bacteria, which is widely distributed in nature and can be widely applied to the food and feed industries, such as for fermenting foods and preparing yogurts. Enterococcus faecalis grows rapidly and has a strong acid-producing ability. During the growth process, it can also metabolize and synthesize bacteriostatic active substances such as bacteriocin, and can inhibit common pathogenic bacteria such as Vibrio parahaemolyticus, Staphylococcus aureus and Escherichia coli.

[0003] Enterococcus faecalis is one of the microbial strains allowed to be used in the food additive catalog and the feed additive catalog, and its safety and effectiveness are widely recognized. The use of Enterococcus faecalis has multiple functions such as improving inflammatory response, regulating intestinal flora, enhancing immune response, and promoting fat absorption, so it has broad application potential in foods and feeds.

[0004] Bacteriocin is a type of protein or polypeptide with bactericidal effects synthesized by ribosomes in bacteria, and has the characteristics of a broad bactericidal spectrum and being not easy to produce drug resistance. Therefore, it is a very important alternative product to antibiotics. Vibrio is one of the pathogenic bacteria in the process of aquaculture, which seriously threatens the health of cultured varieties such as penaeid shrimps and crabs, bringing huge losses to the aquaculture industry. Common pathogenic Vibrio include multiple species such as Vibrio alginolyticus, Vibrio harveyi and Vibrio parahaemolyticus. In the process of penaeid shrimp cultivation, the common pathogenic Vibrio is mainly caused by Vibrio parahaemolyticus. Therefore, controlling the number of Vibrio parahaemolyticus in the cultivation process is crucial for the cultivation success rate. Staphylococcus aureus and Escherichia coli K88 and K99 are common pathogenic bacteria causing diarrhea in laying hens, bringing huge harm to the health and production of laying hens. Summary of the Invention

[0005] To solve the above problems, the present invention screens a strain of Enterococcus faecalis from the small shed shrimp cultivation area in Rudong, Jiangsu. This strain can significantly inhibit the growth of Vibrio parahaemolyticus. During the use of the penaeid shrimp fermentation feed, it can significantly improve the feed utilization rate, reduce the bait coefficient, and can reduce the number of Vibrio in the cultivation environment, improve the cultivation success rate of penaeid shrimps, reduce the cultivation risk, and increase the cultivation income. Prepared into a premix for laying hens, it can also improve the production performance of laying hens and increase the eggshell thickness.

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

[0007] The Enterococcus faecalis provided by the present invention was isolated from the culture waters of Litopenaeus vannamei in Rudong area, Jiangsu Province. It is Gram-positive under an optical microscope, with single or paired oval cocci, no endospores, and no capsules; on MRS medium, it presents white, neat-edged, smooth and round colonies.

[0008] The Enterococcus faecalis screened by the present invention can produce bacteriocins that inhibit Vibrio parahaemolyticus, Staphylococcus aureus, and Escherichia coli.

[0009] PCR identification was performed on the screened strains using universal primers for bacteria: Template DNA was extracted according to the operating instructions of the bacterial DNA extraction kit. The upstream primer 5'-AGAGTTTGATCC TGG CTCAG-3' and the downstream primer 5'-GGTTACCTT GTT ACG ACT T-3' of the 16S rRNA conserved sequence were used to amplify the 16S rRNA gene fragment of bacteria. Through sequencing, it was found that this bacterium belongs to Enterococcus faecalis, named YP-2. The sequencing result of the amplified fragment is shown in SEQ ID No.1.

[0010] Using MRS medium: 10 g peptone, 5 g beef powder, 4 g yeast powder, 2 g glucose, 1 ml Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g ammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, 1000 ml distilled water. The strain YP-2 was inoculated and cultured for 48 h, and the inhibitory effects of its fermentation broth on Vibrio parahaemolyticus, Staphylococcus aureus, and Escherichia coli were detected.

[0011] The fermentation broth was added to the Oxford cups containing plates of Vibrio parahaemolyticus, Staphylococcus aureus, and Escherichia coli, and obvious inhibition zones were observed.

[0012] The screened Enterococcus faecalis was named YP-2 and was deposited on November 21, 2023, at the General Microbiology Center of the China Microbial Culture Collection Center, 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 a fermented feed for inhibiting the number of vibrios in the shrimp culture environment, and its fermentation product contains Enterococcus faecalis CGMCC No. 29087. According to the usage method described in this patent, the feed conversion ratio of shrimp can be effectively reduced by 0.15 - 0.3.

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

[0015] The developed fermented shrimp feed and premix for laying hens of the present invention can effectively inhibit the growth of pathogenic bacteria Vibrio parahaemolyticus, Staphylococcus aureus and Escherichia coli, reduce the incidence rate during animal breeding, and improve animal growth and production performance. Detailed implementation manners

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

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

[0018] (1) Culturing method of Vibrio

[0019] 1 ml of TCBS liquid 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 subjected to streak pure culture, and then single colonies were picked and inoculated into TCBS liquid medium, added with 50% glycerol, and stored frozen.

[0020] (2) Preparation of screening plate

[0021] The frozen-stored Vibrio parahaemolyticus was inoculated into TCBS medium for activation, and 0.2 ml of the bacterial cells were aspirated with a sterile pipette and evenly spread on the TCBS solid medium.

[0022] (3) Screening method with Oxford cup

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

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

[0025] (4) Screening Enterococcus faecalis by Oxford cup method

[0026] The sterile Oxford cups were placed on the plates prepared in methods (2) and (3), 200 μL of Enterococcus faecalis YP-2 fermentation broth with different dilution multiples was aspirated with a sterile pipette and added into the Oxford cups, and cultured in an incubator at 28°C for 24 h. Observe whether there is an obvious inhibition zone around the Oxford cup and measure its diameter, so as to judge whether there is an antibacterial substance against Vibrio parahaemolyticus in the fermentation broth and its antibacterial ability.

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

[0028] Sample Number Undiluted Stock Solution Diluted 1-fold Diluted 2-fold Diluted 4-fold Diluted 8-fold Inhibition Zone Diameter (mm) 15.7±0.2 8.4±0.5 5.1±0.4 1.0±0.2 0.3±0.1

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

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

[0031] (1) Culture 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 subjected to streak pure culture, and then a single colony was picked and inoculated into TSB liquid medium, added with 50% glycerol, and stored frozen.

[0033] (2) Culture methods of Escherichia coli K88 and K99

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

[0035] (3) Preparation of screening plates

[0036] The frozen-preserved Staphylococcus aureus was inoculated into TSB medium for activation, and then 0.2 ml of the bacterial cells was aspirated with a sterile pipette and evenly spread on TSB solid medium; the frozen-preserved Escherichia coli K88 and K99 were respectively inoculated into LB medium for activation, and then 0.2 ml of the bacterial cells was aspirated 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 spread evenly and the LB plates inoculated with Escherichia coli K88 and K99 respectively.

[0039] (5) Detection of inhibition diameter by Oxford cup method

[0040] The sterile Oxford cups were placed on the plates prepared in methods (3) and (4), and 200 μL of the fermentation broth of Enterococcus faecalis YP-2 was aspirated with a sterile pipette and added into the Oxford cups, and then cultured in an incubator at 30 °C and 37 °C for 24 h respectively, and the diameter of the inhibition zone around the Oxford cups was observed.

[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 K99 Inhibition Zone Diameter (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 obvious inhibitory effects on Staphylococcus aureus, Escherichia coli K88 and K99.

[0044] Example 3 Detection of Bacteriostatic Substances of Enterococcus faecalis CGMCC No. 29087

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

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

[0047] (3) Protease experiment: After treating the fermentation broth of Enterococcus faecalis with pepsin, trypsin, proteinase K, papain and bromelain for 4 h, its activity was detected, and the results are shown in Table 3.

[0048] Table 3 Experimental Results of Protease Treatment of Enterococcus faecalis Fermentation Broth

[0049]

[0050] Note: The + quantity represents the size of the inhibition zone

[0051] As can be seen from Table 3, after the fermentation supernatant of Enterococcus faecalis CGMCC No. 29087 was treated with protease and trypsin, the bacteriostatic effect decreased significantly, while it had strong resistance to proteinase K, papain and bromelain. The experimental results show that the bacteriostatic substance can be degraded by protease and trypsin, belongs to small peptide antibacterial substances, and belongs to bacteriocin.

[0052] Example 4 Preparation of High-Density Fermentation Broth of Enterococcus faecalis CGMCC No. 29087

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

[0054] (2) Preparation of primary seeds: Transfer the Enterococcus faecalis CGMCC No. 29087 strain cultured in step (1) to an eggplant bottle MRS slant medium and culture at 30 °C for 24 h until it is in the late logarithmic phase to obtain primary seeds.

[0055] (3) Preparation of secondary seeds: Make the primary seeds prepared in step (2) into a bacterial suspension with sterile water and inoculate it into a 100 L seed tank containing 60 L of MRS seed medium. The temperature is 30 °C, the rotation speed is 120 r / min, the tank pressure is 0.05 Mpa, and the ventilation ratio is 1:0.2. Culture for 18 h to obtain a secondary seed liquid.

[0056] (4) Preparation of Enterococcus faecalis CGMCC No. 29087 fermentation broth: Inoculate the secondary seed liquid prepared in step (3) into a 1 m 3 fermentation tank containing 600 L of fermentation medium at an inoculation amount of 1%. The temperature is 30 °C, the rotation speed is 120 r / min, the tank pressure is 0.05 Mpa, and the ventilation ratio is 1:0.2. Culture for 18 h to obtain Enterococcus faecalis CGMCC No. 29087 with a viable count of 3×10 9 CFU / ml.

[0057] The fermentation medium is (g / L): 10 g peptone, 10 g beef extract, 20 g yeast powder, 20 g glucose, 1 ml Tween 80, 2 g dipotassium hydrogen phosphate, 5 g sodium acetate, 2 g ammonium citrate, 0.2 g magnesium sulfate, 0.05 g manganese sulfate, 15 g agar powder, 0.5 g antifoaming agent, 1000 ml 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., and are prepared into pellet feed with a particle size of 0.8 - 1.0 through a shrimp feed granule production line.

[0060] (2) Preparation of fermented feed: Add 2 kg of Enterococcus faecalis CGMCC No. 29087 bacterial powder, 1 kg of Bacillus subtilis bacterial powder, 1 kg of Saccharomyces boulardii, 0.5 kg of mannan oligosaccharide, 4 kg of Astragalus membranaceus, 2 kg of Momordica grosvenori, and 0.5 kg of Dolichos lablab to 1000 kg of pellet feed prepared through the feed production line, mix evenly, put it into a fermentation bag with a breathing membrane, and ferment at 30 °C for 5 - 7 days.

[0061] (3) Detection of viable count of fermented feed: Take 10 g of fermented shrimp feed, grind it evenly and dissolve it in 100 ml of sterile water. After shaking for 30 min, centrifuge at 5000 r / min for 10 minutes and take the supernatant. Bacillus subtilis is counted by plate counting using LB solid medium, Saccharomyces cerevisiae is counted by plate counting using solid Rose Bengal medium, and Enterococcus faecalis CGMCC No. 29087 is counted by plate counting using solid MRS medium. The viable counts of Bacillus subtilis: Enterococcus faecalis CGMCC No. 29087: Saccharomyces boulardii are 2.1×10 9 CFU / g, 1.8×10 8 CFU / g and 4.3×10 8 CFU / g.

[0062] Application of fermented shrimp feed containing Enterococcus faecalis CGMCC No. 29087 in Example 6

[0063] Apply the fermented shrimp feed prepared in Example 5 in the small shed shrimp farming mode for shrimp. The experiment is divided into three groups, two control groups (Control Group 1 and Control Group 2) and one experimental group. Each group has 9 small sheds (with an area of 500 square meters and a water depth of 0.8 meters), and there are 3 parallels in each group. The fry and stocking density of the control group and the experimental group are the same. Control Group 1 is only fed with shrimp feed, and Control Group 2 is fed with fermented feed without adding Enterococcus faecalis CGMCC No. 29087 (the preparation method is to replace 2 kg of Enterococcus faecalis CGMCC No. 29087 powder with commercial Enterococcus faecalis freeze-dried powder on the basis of Example 5). The experimental group is fed with fermented feed prepared according to Example 5 and added with Enterococcus faecalis CGMCC No. 29087. The feeding method of the experimental group is: shrimp feed: fermented feed = 4:1. The control group and the experimental group are fed 3 times a day. The feeding experiment lasts for 80 days, and growth indexes such as total weight of shrimp and feed coefficient and serum immune indexes such as total superoxide dismutase and malondialdehyde are counted.

[0064] Table 4 Results of the shrimp farming experiment with fermented feed

[0065] Item Control Group 1 Control Group 2 Experimental Group Stocking Density (tails / pond) <![CDATA[50000±500 a > <![CDATA[50000±520 a > <![CDATA[50000±600 a > Total Weight of Prawns (kg / pond) <![CDATA[610.5±21.3 a > <![CDATA[653.3±32.6 b > <![CDATA[783.5±38.8 c > Feed Conversion Ratio of Prawns <![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: Different letters in digital superscripts indicate statistically significant differences (p<0.05)

[0067] It can be seen from Table 4 that under the same fry and stocking density, the weight of shrimp in the experimental group reached 783.5 kg, and the yield increased by 28.3% compared with Control Group 1. The feed conversion rate (feed coefficient) of Control Group 1 was only 1.00, which was significantly lower than 1.33 of the control group. Compared with Control Group 2, the total weight of shrimp also increased significantly, with a 19.9% increase in production; the feed coefficient decreased by 0.18.

[0068] After the breeding ended, 10 shrimps were randomly selected from the three experimental groups, and the serum antioxidant indexes were measured. It was found 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 106.6 U / ml, indicating that the fermented feed improved the antioxidant capacity in the serum of shrimps, could more effectively scavenge free radicals, and enhanced the resistance of the body to environmental stress; while the content of malondialdehyde was significantly lower than that of control group 1, only 6.6 mmol / ml, indicating that the fermented feed could effectively improve the integrity of cells in immune organs such as the liver and reduce cell loss.

[0069] The breeding experiment with fermented feed showed that adding Enterococcus faecalis CGMCC No.29087 during the production and preparation of fermented feed could significantly increase the functional effects of compound Chinese herbal medicines and other probiotics. During the breeding process of shrimps, when used in combination with feed at an addition ratio of 20%, it could not only improve the utilization and weight gain efficiency of feed, but also enhance the immunity of shrimps.

[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 change in the number of Vibrio parahaemolyticus in the breeding water body, and 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: Different letter superscripts of numbers indicate statistically significant differences (p < 0.05)

[0074] As can be seen from Table 5, the number of vibrios in control group 1 showed an increasing trend during the breeding process, indicating that the number of vibrios in the breeding water body was always in the process of proliferation. After the experimental group used the fermented feed, the number of vibrios in the breeding water body decreased significantly, by two orders of magnitude compared with the control group; moreover, the continuous use of the fermented feed could always control the Vibrio parahaemolyticus at a low level, which would greatly reduce the occurrence of diseases during the breeding process. Although the number of vibrios in control group 2 decreased to some extent, its concentration was still at a relatively high level. This also indicated that during the use of the fermented feed, the key metabolite that inhibited Vibrio parahaemolyticus was produced by Enterococcus faecalis CGMCC No.29087, so only the number of vibrios in the experimental group was maintained at a low level.

[0075] Example 7 Application of the core premix containing Enterococcus faecalis CGMCC No.29087 in laying hen breeding

[0076] Add the core premix containing Enterococcus faecalis CGMCC No. 29087 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: a control group, experimental group 1, and experimental group 2. The core premix was not added to the control group. The addition level of Enterococcus faecalis CGMCC No. 29087 in experimental group 1 was 10 3 CFU / g, and the addition level of Enterococcus faecalis CGMCC No. 29087 in experimental group 2 was 10 6 CFU / g. The experiment was carried out for 45 days, and the egg production, feed-to-egg ratio, and the number of Escherichia coli in the intestine were counted.

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

[0078] Item Control Group Experimental Group 1 Experimental Group 2 Enterococcus faecalis Addition Level (CFU / g) 0 <![CDATA[10 3 > <![CDATA[10 6 > Egg Production (g / (bird·d)) <![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 > Haugh 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)]]> <![CDATA[9.97±0.15 c > <![CDATA[9.11±0.18 b > <![CDATA[1.74±0.17 a >

[0079] Note: Different letters in the digital superscripts indicate statistically significant differences (p < 0.05).

[0080] As can be seen from Table 6, adding the core premix containing Enterococcus faecalis CGMCC No. 29087 to the 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) addition amount is better than that of the low-level (experimental group 1) addition. By detecting the thickness of the eggshell, it was also found that the experimental group with the core premix containing Enterococcus faecalis CGMCC No. 29087 was higher than the control group, indicating that Enterococcus faecalis can promote the absorption and conversion of calcium. By detecting the number of Escherichia coli in the intestinal flora, 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-to-egg ratio and 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 It can produce 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 mixture with shrimp feed in a ratio of 10 to 20%, and then sealing and fermenting it for 4 hours to feed the shrimp; (2) adding 10 kg of fermented carbon source and 90 catties of water to 1 kg of fermented feed, and then sealing and fermenting it for 48 hours and then spraying it throughout the pond, with 5 to 10 kg of fermented liquid used per mu of pond; mixing the feed and spraying it throughout the pond can reduce the shrimp feed coefficient by 0.15-0.3, and the method of use is not for therapeutic purposes.

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 CFU / g of Enterococcus faecalis CGMCC No.2908710-15kg, polyglutamic acid 3-5kg, polylysine 0.5-1kg, and mannan peptide 1-1.5kg.

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