Enterococcus faecium HKS023 and application thereof

Abstract

The invention discloses a strain of enterococcus faecium HKS023 and application of the enterococcus faecium HKS023. The enterococcus faecium HKS023 is preserved in China General Microbiological Culture Collection Center on February 14, 2023, and the preservation number of the enterococcus faecium HKS023 is CGMCC No.26552. The enterococcus faecium HKS023 is named as enterococcus faecium HKS023. According to the invention, a new strain of enterococcus faecium HKS023 is screened from fresh feces of healthy weaned piglets, corn flour is taken as a raw material, L-lactic acid is produced through fermentation, the acid yield is high, and the enterococcus faecium HKS023 has a very strong inhibition effect on the growth of salmonella and can inhibit the growth of escherichia coli and staphylococcus aureus to a certain extent. The enterococcus faecium HKS023 microbial feed additive is produced by effectively combining and fermenting common raw materials including calcium carbonate, beef extract, yeast extract, peptone, glucose, sodium acetate, dipotassium phosphate, magnesium sulfate, manganese sulfate and the like, the growth performance of livestock and poultry can be effectively improved, the diarrhea rate is reduced, the immunity of the livestock and poultry is improved, and the economic and social benefits are huge.

Description

Technical Field

[0001] The present invention relates to a strain of Enterococcus faecium HKS023 and its application, belonging to the field of microbial technology. Background Art

[0002] Salmonella is a common intestinal pathogen that exists widely in nature. It can cause various diseases such as typhoid fever, paratyphoid fever, and food poisoning, and also lead to problems such as slow growth of livestock and poultry, low feed conversion rate, and extended fattening cycle. At the same time, the feces of carrier animals can also contaminate feed, water sources, and the environment, and are also prone to secondary infections, resulting in a decline in animal immunity and exacerbating the condition. Therefore, Salmonella seriously threatens the economic benefits of the aquaculture industry and public health safety.

[0003] Antibiotics, as a commonly used feed additive, have a long history of research and use in the feed industry, bringing great economic benefits to the aquaculture industry. However, in actual production, it has been found that long-term use will produce many adverse effects. Probiotics are a class of active microorganisms that are beneficial to the health of the host and can improve the balance of the host's intestinal flora. Due to their characteristics such as safety, non-toxicity, and pollution-free, they have become one of the friendly alternatives to antibiotics. Currently, there are many probiotics commonly used in the aquaculture industry, such as Bacillus, Lactobacillus, Saccharomyces cerevisiae, etc. Lactobacillus, etc., are widely present in nature and are important probiotics for animals, with a variety of probiotic functions.

[0004] Lactic acid (LA) is a natural organic acid with wide application value. The lactic acid molecule contains an asymmetric carbon atom, so it is divided into two optical isomers, L-lactic acid and D-lactic acid. After D-lactic acid enters the human body, since the human body (including mammals) does not have an enzyme system for metabolizing D-lactic acid and cannot metabolize it for the human body, excessive intake will cause an increase in blood uric acid and metabolic disorders. Therefore, the World Health Organization (WHO) stipulates that the daily intake of D-lactic acid per kilogram of body weight should not exceed 100 mg, while there is no restriction on L-lactic acid. Currently, L-lactic acid is widely used in food, medicine, feed, and the production of bio-based plastics due to its excellent biodegradability, low toxicity, and high optical purity. In the feed field, L-lactic acid can be used as a feed additive to improve the growth performance and immunity of livestock and poultry.

[0005] Based on the above content, providing a new strain that takes both into account, can effectively inhibit the growth of Salmonella, reduce the prevalence of livestock and poultry, and has good L-lactic acid production performance, can promote the growth of livestock and poultry, and has great social and economic benefits. Summary of the Invention

[0006] In view of the deficiencies of the prior art, the object of the present invention is to provide a new strain of Enterococcus faecium HKS023 and its applications, which can effectively inhibit the growth of Salmonella, and can effectively solve the production of L-lactic acid and Enterococcus faecium microbial feed additives, meeting the actual needs of the market.

[0007] To achieve the above object, on the one hand, the technical solution of the present invention is to provide a strain of Enterococcus faecium, named Enterococcus faecium HKS023, which was deposited at the General Microbiological Center of the China Committee for Culture Collection of Microorganisms on February 14, 2023. The deposit address is: No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit number is: CGMCC No. 26552.

[0008] On the other hand, the technical solution of the present invention is to provide an application of the above-mentioned Enterococcus faecium HKS023 in inhibiting intestinal harmful bacteria and promoting the growth of livestock and poultry.

[0009] Preferably, the intestinal harmful bacteria are Salmonella.

[0010] On the other hand, the technical solution of the present invention is to provide an application of Enterococcus faecium in the production of L-lactic acid, and use the above-mentioned Enterococcus faecium HKS023 to ferment and produce L-lactic acid.

[0011] Furthermore, inoculate Enterococcus faecium HKS023 into a fermentation medium to ferment and obtain an L-lactic acid fermentation broth.

[0012] Even further, it includes the following steps: (1) Strain activation: Under aseptic operation, pick the strain of Enterococcus faecium HKS023 and inoculate it on an MRS solid medium slant for cultivation; (2) Secondary seed culture: Under aseptic conditions, pick the activated strain and inoculate it into an MRS liquid medium for enlarged enrichment culture to obtain secondary seeds; (3) L-lactic acid production: Inoculate the secondary seeds into a fermentation medium under aseptic conditions and ferment to obtain an L-lactic acid fermentation broth.

[0013] On the other hand, the technical solution of the present invention is to provide an application of Enterococcus faecium in the production of microbial feed additives, and use the above-mentioned Enterococcus faecium HKS023 to culture and produce microbial feed additives.

[0014] Furthermore, inoculate Enterococcus faecium HKS023 into a production medium for cultivation to obtain an Enterococcus faecium microbial feed additive.

[0015] Furthermore, it includes the following steps: (1) Strain activation: Under aseptic operation, pick the Enterococcus faecium HKS023 strain and inoculate it on the inclined plane of MRS solid medium for cultivation; (2) Secondary seed culture: Under aseptic conditions, pick the activated strain and inoculate it into MRS liquid medium for expanded enrichment culture to obtain secondary seeds; (3) Production of microbial feed additive: Inoculate the secondary seeds into the production medium under aseptic conditions for cultivation. After passing the inspection and meeting the enterprise standard, it is the Enterococcus faecium microbial feed additive.

[0016] On the other hand, the technical solution of the present invention is to provide an L-lactic acid fermentation broth produced by the Enterococcus faecium HKS023, an inactivated or non-inactivated microbial feed additive.

[0017] On the other hand, the technical solution of the present invention is to provide an application of the said microbial feed additive in livestock and poultry breeding.

[0018] A new strain of Enterococcus faecium HKS023 was screened from the fresh feces of healthy weaned piglets in the present invention. Using corn flour as the raw material, L-lactic acid was fermented and produced. The content of L-lactic acid in the obtained crude L-lactic acid fermentation broth could reach 133.1 μmoL / L. And Enterococcus faecium HKS023 has a very strong inhibitory effect on the growth of Salmonella, and can also inhibit the growth of Escherichia coli and Staphylococcus aureus to a certain extent. The present invention uses the effective combination of common raw materials such as calcium carbonate, beef extract, yeast extract, peptone, glucose, sodium acetate, dipotassium hydrogen phosphate, magnesium sulfate, manganese sulfate, etc. to ferment and produce Enterococcus faecium HKS023 microbial feed additive, which can effectively improve the growth performance of livestock and poultry, reduce the diarrhea rate, improve the immunity of livestock and poultry, meet the market demand, and have great economic and social benefits. Description of the Drawings

[0019] Figure 1 It is the hydrolysis clear zone for screening strain HKS023.

[0020] Figure 2 It is the colony morphology for screening strain HKS023.

[0021] Figure 3 It is the microscopic morphological characteristics for screening strain HKS023.

[0022] Figure 4 It is the antibacterial effect of screening strain HKS023 against Salmonella (A), Staphylococcus aureus (B), and Escherichia coli (C). Detailed Embodiments

[0023] The following further elaborates the detailed embodiments of the present invention in conjunction with the examples.

[0024] The culture media used in the present invention are as follows:

[0025] MRS solid medium (by mass percentage): beef extract 1%, yeast extract 0.5%, peptone 1%, sodium acetate 0.5%, diammonium citrate 0.2%, glucose 2%, magnesium sulfate 0.05%, manganese sulfate 0.025%, Tween 8%, agar powder 2%.

[0026] MRS liquid medium (by mass percentage): beef extract 1%, yeast extract 0.5%, peptone 1%, sodium acetate 0.5%, diammonium citrate 0.2%, glucose 2%, magnesium sulfate 0.05%, manganese sulfate 0.025%, Tween 8%.

[0027] Fermentation medium: 10 g of corn flour (passed through a 40-mesh sieve), add 100 mL of distilled water, heat up to 90 °C, add 15 - 20 U of α-amylase per g of starch for gelatinization for 10 min, heat up to 100 °C to inactivate the enzyme, cool down to 55 °C, add 150 - 200 U of glucoamylase per g of starch and incubate for saccharification for 4 h, add 2 wt% of calcium carbonate and 0.2 wt% of dipotassium hydrogen phosphate, then it is obtained.

[0028] Production medium (by mass percentage): calcium carbonate 2%, beef extract 1%, yeast extract 0.5%, peptone 1%, glucose 2%, sodium acetate 0.5%, dipotassium hydrogen phosphate 0.2%, magnesium sulfate 0.05%, manganese sulfate 0.025%, Tween 8%.

[0029] LB liquid medium (by mass percentage): sodium chloride 1%, peptone 1%, yeast extract 0.5%.

[0030] Nutrient agar medium (by mass percentage): beef extract 0.3%, sodium chloride 0.5%, peptone 1%, agar powder 2%.

[0031] Calcium carbonate solid medium (by mass percentage): calcium carbonate 2%, beef extract 1%, yeast extract 0.5%, peptone 1%, glucose 2%, diammonium citrate 0.2%, sodium acetate 0.5%, magnesium sulfate 0.05%, Tween 8%, manganese sulfate 0.025%, agar powder 2%.

[0032] Calcium carbonate liquid medium (by mass percentage): calcium carbonate 2%, beef extract 1%, yeast extract 0.5%, peptone 1%, glucose 2%, diammonium citrate 0.2%, sodium acetate 0.5%, magnesium sulfate 0.05%, Tween 8%, manganese sulfate 0.025%.

[0033] The above media are sterilized at 121 °C for 20 min.

[0034] Example 1: Screening and identification of strains

[0035] Collect fresh fecal samples from healthy weaned piglets within 20 days after birth in a farm in Chengliu Town, Jiyuan City. After collecting the feces using a sterile bag, place them in dry ice and transport them to the analysis laboratory within 3 hours. Using these as experimental materials, conduct a preliminary screening by the clear zone method and a directional re-screening by measuring the L-lactic acid (L-LA) production using an L-lactic acid content detection kit (product number: BC2235, specification: 100T / 48S) produced by Beijing Solarbio Science & Technology Co., Ltd., to obtain a strain of Enterococcus faecium with high L-lactic acid production, named Enterococcus faecium HKS023. The specific method is as follows:

[0036] (1) Preliminary screening: Place the collected samples in a laminar flow hood. Weigh 0.2 - 0.3 g of fecal samples respectively and immerse them in 5 mL of sterilized physiological saline. After vortex oscillation, filter using a sterile gauze and remove solid substances. Dilute the filtered fecal suspension, and then take 10 -5 to 10 -7 gradient fecal dilutions. After mixing them evenly, pipette 100 μL of the liquid and spread it on an MRS solid medium plate. Incubate it upside down at 37°C for 24 h. Pick single colonies with different morphologies and streak them again on an MRS solid medium plate for purification culture. After purification, inoculate the single colonies into MRS liquid medium respectively and conduct an enlarged enrichment culture at 37°C and 180 r / min for 12 h. Take a part of the bacterial liquid, mix it evenly with an equal volume of 50% glycerol, number it and store it in a -80°C refrigerator. Take the purified single colonies and spot them onto a calcium carbonate solid medium, incubate it upside down at 37°C until clear zones are produced, observe and select the single colonies with a larger ratio of clear zone diameter (D) to colony diameter (d) for acid production measurement and re-screening. Finally, 6 strains of bacteria producing clear zones are isolated and screened from fresh piglet feces by the clear zone method.

[0037] (2) Re-screening: Inoculate the 6 strains with a larger D / d ratio obtained from the preliminary screening into a calcium carbonate liquid medium, shake culture at 37°C and 180 r / min for 24 h, then centrifuge at 4°C and 8000 r / min for 10 min. Take the supernatant and use an L-lactic acid (L-LA) content detection kit to measure L-lactic acid, compare the L-lactic acid production amounts, select the strain with the highest L-lactic acid production for identification. Finally, 1 strain with relatively high L-lactic acid production is obtained, named HKS023. The clear zone on its calcium carbonate solid medium plate is as Figure 1 (the diameter D / d ratio is 2.59). After detection, the L-lactic acid content in the supernatant can reach 129.1 μmoL / L.

[0038] (3) Morphological observation of L-lactic acid-producing strains

[0039] The strain HKS023 with a large ratio of the diameter of the transparent zone (D) to the colony diameter (d) and the highest L-lactic acid production was isolated by streaking on an MRS solid medium plate. After culturing in an inverted position at 37 °C for 24 h, the morphological characteristics of the strain were observed and recorded; after Gram staining of the bacterial strain, the morphology was observed microscopically through a microscope and the results were recorded. After streaking and inoculating the colonies of strain HKS023 onto an MRS solid medium plate and culturing in an inverted position at 37 °C for 24 h, the colony morphology is shown in Figure 2 , and the microscopic morphology is shown in Figure 3 . As can be seen from Figure 2 , the colony appearance of strain HKS023 can be observed to be white, round, with a moist surface, opaque, and a neat edge. As can be seen from Figure 3 , the Gram staining result is positive, and the microscopic morphology shows that the cells are ellipsoidal, with a size of (0.8 - 1.0) μm × (0.7 - 1.7) μm, and the cells are arranged singly or in pairs.

[0040] (4) Physiological and biochemical analysis of L-lactic acid-producing strains

[0041] For the screened target strain HKS023, the biochemical characteristics were identified using the VITEK GP Gram-positive bacterial identification card (Shanghai Jingxin Industrial Development Co., Ltd.). The results of the physiological and biochemical analysis are shown in Table 1. As can be seen from Table 1, strain HKS023 can use D-ribose, D-maltose, D-mannitol, D-trehalose, D-mannose, salicin, D-galactose, N-acetyl-D-glucosamine, sucrose, etc. as carbon sources for growth, and cannot use D-amygdalin, D-sorbitol, D-raffinose, D-xylose, lactose, methyl-β-D-glucopyranoside, cyclodextrin, amylopectin, etc. Strain HKS023 grows in enzymes such as L-pyrrolidone arylamidase, arginine dihydrolase, and tyrosine arylamidase, but does not grow in β-galactosidase, L-aspartate arylamidase, leucine arylamidase, phosphatidylphospholipase C, β-galactopyranosidase, L-proline arylamidase, urease, alanine-phenylalanine-proline arylamidase, α-mannosidase, alanine arylamidase, α-galactosidase. Strain HKS023 is also tolerant to 6.5% NaCl, optochin, novobiocin, polymyxin B, bacitracin, O / 129, but is not tolerant to L-lactic acid salinization. The above physiological and biochemical identification results were input into the API 50CHB identification system of bioMérieux, and HKS023 was preliminarily identified as a species in the genus Enterococcus.

[0042] Table 1 Results of physiological and biochemical analysis of strain HKS023

[0043]

[0044] Note: "+" indicates positive, "-" indicates negative

[0045] (5) Molecular biological identification of L-lactic acid producing strains

[0046] The target strain HKS023 obtained by screening was inoculated into MRS liquid culture medium and cultured in a shaking incubator at 37°C and 180 r / min for 24 h. The genomic DNA of the screened strain was obtained using the bacterial genomic deoxyribonucleic acid (DNA) extraction kit (D1600-100) produced by Beijing Solebow Technology Co., Ltd. The extracted genomic DNA was used as a template, and bacterial universal primers 27F (5'-AGAGTTTGATCCTGGCTAG-3') and 1492R were used.

[0047] PCR amplification of 16S rDNA gene sequence fragment (5'-GGTTACCTTGTTACGACTT-3') was performed. PCR amplification system (50 μL): 2× Super PCR Mix 25 μL, template DNA 1 μL (50 μg / mL), forward primer 27F 1 μL, reverse primer 1492R 1 μL, dd H 2 O 22 μL (the universal primers 27F, 1492R, and 2×Super PCR Mix for PCR amplification of the 16S rDNA gene of the strain were provided by Beijing Liuhe BGI Gene Technology Co., Ltd.), and the amplification conditions were as follows: pre-denaturation at 94°C for 4 min, denaturation at 94°C for 30 s, annealing at 60°C for 30 s, extension at 72°C for 1 min for 30 cycles, and final extension at 72°C for 5 min.

[0048] The amplified PCR products were verified by 1% agarose gel electrophoresis and then sent to Beijing Liuhe BGI Genomics Co., Ltd. for sequencing. The sequencing results were submitted to the GenBan database in the National Center for Biotechnology Information (NCBI) of the United States. The Basic Local Alignment Search Tool (BLAST) was used for homology search and alignment, and the phylogenetic tree was constructed using the proximity joining method using the software MEGA 7.0.

[0049] The 16S rDNA amplification product of strain HKS023 was sent to BGI for sequencing. The sequencing results showed that the gene sequence length was 1437 bp, which was consistent with the bacterial 16S rDNA fragment. The sequence alignment results are shown in Table 2. A BLAST homology search was performed in the GenBan database of NCBI, and the similarity with Enterococcus faecium LMG 11423 (AJ301830) was 100%.

[0050] Based on the comprehensive results of the observation of the strain's appearance and morphology, physiological and biochemical analysis, and molecular biological identification, this strain HKS023 was Enterococcus faecium. It was deposited in the General Microbiology Center of the China Committee for Culture Collection of Microorganisms on February 14, 2023. The deposit address is No. 3, Building 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences. The deposit number is CGMCC No. 26552.

[0051] Table 2 16S rDNA sequence of strain HKS023

[0052]

[0053]

[0054] Example 2: Application of Enterococcus faecium HKS023 in the production of L-lactic acid

[0055] (1) Strain activation: Under aseptic conditions, pick 1-2 loops of Enterococcus faecium HKS023 strain and inoculate it on the slant of MRS solid medium, and culture it at 37°C for 24 h;

[0056] (2) Secondary seed culture: Under aseptic conditions, pick 3-5 loops of the activated strain and inoculate it into MRS liquid medium. The loading volume is 150 mL / 250 mL Erlenmeyer flask, and perform an enlarged enrichment culture at 37°C and 100 r / min for 18 h to obtain the secondary seed;

[0057] (3) Production of L-lactic acid: Under aseptic conditions, inoculate the secondary seed into the fermentation medium, and the inoculation amount is 3-5% (mass ratio). Culture it at 33°C and 100 r / min for 3 d to obtain the crude L-lactic acid fermentation broth.

[0058] The crude L-lactic acid fermentation broth was centrifuged at 4°C and 8000 r / min for 10 min. The supernatant was taken and the L-lactic acid content was measured using an L-lactic acid (L-LA) content detection kit. After detection, the L-lactic acid content in the crude L-lactic acid fermentation broth could reach 133.1 μmoL / L.

[0059] Example 3: Application of Enterococcus faecium HKS023 in the production of microbial feed additives

[0060] (1) Strain activation: Under aseptic operation, pick 1-2 loops of Enterococcus faecium HKS023 strain and inoculate it on the slant of MRS solid medium, and culture it at 37°C for 24 h.

[0061] (2) Secondary seed culture: Under aseptic conditions, pick 3-5 loops of the activated strain and inoculate it into MRS liquid medium. The loading amount is 150 mL / 250 mL Erlenmeyer flask, and perform enlarged enrichment culture at 37°C and 100 r / min for 18 h to obtain secondary seeds.

[0062] (3) Production of microbial feed additive: Under aseptic conditions, inoculate the secondary seeds into the production medium, and the inoculation amount is 3-5% (mass ratio). Culture it at 33°C and 100 r / min for 22-25 h, and conduct detection in accordance with the industry standard NY / T1444-2007 "General Rules for Microbial Feed Additives" of the Ministry of Agriculture and Rural Affairs of the People's Republic of China. After passing the inspection and meeting the enterprise standard, it is the Enterococcus faecium microbial feed additive.

[0063] After inspection, Enterococcus faecium ≥ 10 9 CFU / mL, meeting the enterprise standard of Institute of Biology, Henan Academy of Sciences Co., Ltd.

[0064] Example 4: Application of Enterococcus faecium HKS023 in inhibiting intestinal harmful bacteria

[0065] (1) Inoculate three harmful bacteria, namely Escherichia coli (ATCC25922), Staphylococcus aureus (ATCC25923), and Salmonella (ATCC13076), into LB liquid medium respectively, and culture them at 37°C and 180 r / min for 24 h. Then evenly coat the bacterial liquid on the nutrient agar medium plate.

[0066] (2) After the screened strain HKS023 grows in calcium carbonate liquid medium at 37°C and 180 r / min on a shaker for 24 h, centrifuge it at 4°C and 8000 r / min for 10 min, and take the supernatant. Place the sterilized round filter paper (diameter about 0.6 cm) on the nutrient agar medium plate, suck 20 μL of the above supernatant and drop it onto the center of the filter paper. After culturing at 37°C for 24 h, check and record the size of the inhibition zone.

[0067] The antibacterial experimental results of strain HKS023 against Salmonella, Staphylococcus aureus, and Escherichia coli are shown in Figure 4 . By Figure 4It can be seen that strain HKS023 has a certain inhibitory effect on Staphylococcus aureus and Escherichia coli. The diameter of the inhibition zone against Staphylococcus aureus is 13.6 mm, and the diameter of the inhibition zone against Escherichia coli is 8.6 mm. It has a very strong inhibitory effect on Salmonella, and the diameter of the inhibition zone can reach 55.6 mm, far exceeding the antibacterial performance of the publicly available Enterococcus faecium against Salmonella (Zhang Xiaoyong, Lan Wei, Wang Ya, et al. Isolation, identification and antibacterial characteristics of Enterococcus faecium FDT2 [J]. China Animal Husbandry & Veterinary Medicine, 2024, 51(7): 3020-3029. The diameter of the inhibition zone of Enterococcus faecium FDT2 screened from yak feces against Salmonella is 15.68 mm; Wan Ying. Analysis of the biological characteristics of Enterococcus faecium F11.1G and exploration of its metabolites [D]. Tarim University, 2024. The diameter of the inhibition zone of Enterococcus faecium F11.1G isolated and screened by Wan Ying et al. against Salmonella is 11.28 mm).

[0068] Example 5: Application of the microbial feed additive produced by Enterococcus faecium HKS023 in the breeding of "Liangfenghua" broilers

[0069] (1) Experimental animals

[0070] The experimental animals were 50-day-old healthy "Liangfenghua" broilers with good mental state and consistent body weight, provided by Weishi County Wugu Nongjia Farm.

[0071] (2) Feeding diet

[0072] The basic diet formula and nutritional level are shown in Table 3.

[0073] Table 3 Basic diet ratio and main nutritional levels

[0074]

[0075] The additive contains (per kilogram): copper sulfate 0.211 g, ferrous sulfate 0.466 g, zinc sulfate 0.36 g, manganese sulfate 0.227 g, potassium iodide 0.0008 g, sodium selenite 0.0011 g, cobalt chloride 0.00042 g, limestone powder 9.8 g, calcium bicarbonate 11.6 g, chicken vitamins 0.15 g, choline chloride 0.35 g, methionine 1.6 g, flavomycin 0.005, mycocide 2.5 g.

[0076] (3) Experimental design

[0077] A total of 100 50-day-old healthy "Liangfenghua" broilers with good mental state and consistent body weight were selected for the experiment. They were randomly divided into 2 groups, with 5 replicates in each group and 10 chickens in each replicate. The control group was fed the basic diet, and the experimental group was fed the basic diet supplemented with 0.1% of the microbial feed additive produced by Enterococcus faecium HKS023 based on the quality of the basic diet. The preliminary trial period was 7 days, and the formal trial period was 49 days.

[0078] (4) Feeding and management

[0079] The chicken house is semi - open. The experimental chickens are raised in three - layer cages, with 5 chickens in each cage. Natural ventilation combined with negative pressure ventilation is adopted in the house. During the experimental period, the chickens are fed with the basal diet according to the feeding standard of broilers in China. They are fed once at 6:00 every day. 5 chickens in each cage are fed quantitatively with free access to food and water. The disinfection of the chicken house and the routine immunization of laying hens are carried out according to the routine feeding management of the chicken farm. The feed intake and the number of dead and culled chickens are recorded every day, and the chickens are weighed on an empty stomach once a week. The average daily feed intake, average daily weight gain and feed - to - meat ratio are calculated respectively. At the end of the experiment, serum is collected for the detection of serological immune indexes.

[0080] (5) Experimental results

[0081] Table 4 Experimental results

[0082]

[0083] According to the experimental results, 5 chickens died and were culled in the control group, and there was no death or culling in the experimental group. As can be seen from Table 4, on the basis of feeding the same basal diet, the average daily weight gain of the experimental group was 16.8% higher than that of the control group (P < 0.05), and the feed - to - meat ratio of the experimental group was 14.4% lower than that of the control group (P < 0.05), significantly improving the production performance.

[0084] Table 5 Detection results of serological immune indexes

[0085]

[0086] As can be seen from Table 5, the immunoglobulin M in the serum of the experimental group was 24.45% higher than that of the control group (P < 0.05), the immunoglobulin G in the experimental group was 20.39% higher than that of the control group (P < 0.05), the serum immunoglobulin A in the experimental group was 24.48% higher than that of the control group (P < 0.05), the interleukin - 6 in the experimental group was 20.66% higher than that of the control group (P < 0.05), the interleukin - 10 in the experimental group was 10.67% higher than that of the control group (P < 0.05), and the total lipocalin - 2 in the experimental group was 26.17% higher than that of the control group (P < 0.05), all reaching a significant difference level, greatly improving the immunity of "Liangfenghua" broilers.

[0087] Staphylococcus aureus was not detected in the chicken manure of the control group, Salmonella was 10 CFU / g, and Escherichia coli was 10 4 CFU / g. Staphylococcus aureus, Salmonella and Escherichia coli were not detected in the experimental group.

[0088] Example 6: Application of the microbial feed additive produced by Enterococcus faecium HKS023 in piglets

[0089] (1) Experimental animals

[0090] 7-day-old piglets (breed: Duroc×Landrace×Yorkshire).

[0091] (2) Feeding diet

[0092] Basic formula of piglet compound feed (by mass percentage): extruded corn 25%, corn 15%, rice 10%, soybean meal 22%, fermented soybean meal 7%, fish meal 3%, whey powder 8%, plasma protein powder 5%, milk powder 1%, premix 4% (each kilogram of premix contains 110 mg of copper, 1250 mg of iron, 1500 mg of zinc, 250 mg of manganese, 56000 IU of VA 3 6500 IU, 1500 IU of VE, 50 mg of VK 3 50 mg, 125 mg of VB 2 125 mg). Nutritional levels: digestible energy (MC / kg) 3.81, metabolic energy (MC / kg) 2.29, crude protein 20.1%, calcium 0.56%, available phosphorus 0.38%, lysine 1.46%.

[0093] (3) Experimental design

[0094] Fifty 7-day-old piglets (breed: Duroc×Landrace×Yorkshire) were selected for the experiment and randomly divided into two treatments: a control group and an experimental group. Each treatment had 5 replicates, with 5 piglets in each replicate. The control group was fed the basic formula of piglet compound feed, and the experimental group was supplemented with a microbial feed additive produced by Enterococcus faecium HKS023 at 0.1% of the mass of the basic formula diet. Experimental period: 25 days.

[0095] (4) Feeding management

[0096] Feeding was carried out according to the conventional feeding management of the pig farm. The feed intake and diarrhea situation were recorded every day, and the pigs were weighed on an empty stomach once a week. The average daily feed intake, average daily weight gain, feed-to-gain ratio, and diarrhea rate were calculated respectively. Statistical analysis was performed using SPSS statistical software 25.0, and the results are shown in Table 6.

[0097] Table 6 Effects on the production performance of piglets

[0098]

[0099] In the table, different letters represent significant differences.

[0100] Test results: In the experimental group compared with the control group, the average daily feed intake in the experimental group increased slightly by 2.84% (P > 0.05) compared with the control group, and the difference was not significant; the average daily weight gain increased by 22.98% (P < 0.05), reaching a significant level; the feed-to-meat ratio in the experimental group decreased by 16.57% (P < 0.05) compared with the control group, reaching a significant level. There was no diarrhea in the pigs in the experimental group, while the diarrhea rate in the control group was 10.16% (P < 0.05), reaching a significant level.

[0101] Staphylococcus aureus was not detected in the feces of pigs in the control group, Salmonella was not detected, and Escherichia coli was 10 2 CFU / g. Staphylococcus aureus, Salmonella, and Escherichia coli were not detected in the experimental group.

[0102] Example 7: Application of inactivated Enterococcus faecium HKS023 bacterial liquid in the breeding of "Liangfenghua" broilers

[0103] This experiment studied the application of the microbial agent (feed additive) produced by Enterococcus faecium HKS023 after being kept at 80 °C for 1 h, that is, the inactivated Enterococcus faecium HKS023 bacterial liquid in the breeding of "Liangfenghua" broilers.

[0104] (1) Experimental animals

[0105] The experimental animals were 50-day-old healthy "Liangfenghua" broilers with good mental state and consistent body weight, provided by Wushi County Wugu Nongjia Breeding Farm.

[0106] (2) Fed diets

[0107] The basic diet formula and nutritional levels are shown in Table 7.

[0108] Table 7 Basic diet ratio and main nutritional levels

[0109]

[0110] The additives contained (per kilogram): copper sulfate 0.211 g, ferrous sulfate 0.466 g, zinc sulfate 0.36 g, manganese sulfate 0.227 g, potassium iodide 0.0008 g, sodium selenite 0.0011 g, cobalt chloride 0.00042 g, limestone powder 9.8 g, calcium hydrogen carbonate 11.6 g, chicken vitamins 0.15 g, choline chloride 0.35 g, methionine 1.6 g, flavomycin 0.005, and mold inhibitor 2.5 g.

[0111] (3) Experimental design

[0112] A total of 100 healthy "Liangfenghua" broilers at 50 days of age with good mental state and consistent body weight were randomly divided into 2 groups, with 5 replicates in each group and 10 chickens in each replicate. The control group was fed a basal diet, and the experimental group was fed a basal diet supplemented with 0.1% of the inactivated Enterococcus faecium HKS023 bacterial solution based on the quality of the basal diet. The preliminary trial period was 7 days, and the formal trial period was 49 days.

[0113] (4) Feeding and management

[0114] The chicken house was semi-open, and the experimental chickens were raised in three-tier cages, with 5 chickens in each cage. Natural ventilation was combined with negative pressure ventilation in the house. During the experimental period, the chickens were fed according to the national broiler feeding standard of the basal diet, and were fed once at 6:00 every day. The 5 chickens in each cage were fed quantitatively with free access to food and water. The disinfection of the chicken house and the routine immunization of laying hens were carried out according to the routine feeding management of the chicken farm. The feed intake and the number of dead and culled chickens were recorded every day, and the chickens were weighed on an empty stomach once a week. The average daily feed intake, average daily weight gain, and feed-to-meat ratio were calculated respectively. At the end of the experiment, serum was collected for the detection of serological immune indexes.

[0115] (5) Experimental results

[0116] Table 8 Experimental results of average daily feed intake, average daily weight gain, and feed-to-meat ratio

[0117]

[0118] According to the experimental results, 5 chickens died and were culled in the control group, and there was no death and culling in the experimental group. As can be seen from Table 8, on the basis of feeding the same basal diet, the average daily weight gain of the experimental group was 17.3% higher than that of the control group (P<0.05), and the feed-to-meat ratio of the experimental group was 14.5% lower than that of the control group (P<0.05), significantly improving the production performance.

[0119] Table 9 Detection results of serological immune indexes

[0120]

[0121]

[0122] As can be seen from Table 9, the immunoglobulin M in the serum of the experimental group was 23.25% higher than that of the control group (P<0.05), the immunoglobulin G in the experimental group was 18.60% higher than that of the control group (P<0.05), the serum immunoglobulin A in the experimental group was 22.83% higher than that of the control group (P<0.05), the interleukin-6 in the experimental group was 17.71% higher than that of the control group (P<0.05), the interleukin-10 in the experimental group was 9.30% higher than that of the control group (P<0.05), and the total lipocalin-2 in the experimental group was 24.57% higher than that of the control group

[0123] (P<0.05), all reaching a significant difference level, greatly improving the immunity of "Liangfenghua" broilers.

[0124] Example 8: Application of Inactivated Enterococcus faecium HKS023 Bacterial Solution in Suckling Pigs

[0125] This experiment studied the application of the microbial inoculant (feed additive) produced by Enterococcus faecium HKS023, namely the inactivated Enterococcus faecium HKS023 bacterial solution obtained after incubating at 80 °C for 1 h, in 7-day-old suckling pigs (breed: Duroc×Landrace×Yorkshire).

[0126] (1) Experimental Animals

[0127] 7-day-old suckling pigs (breed: Duroc×Landrace×Yorkshire).

[0128] (2) Feeding Diet

[0129] Basic formula of the suckling pig compound feed: extruded corn 25%, corn 15%, rice 10%, soybean meal 22%, fermented soybean meal 7%, fish meal 3%, whey powder 8%, plasma protein powder 5%, milk powder 1%, premix 4% (each kilogram of premix contains 110 mg of copper, 1250 mg of iron, 1500 mg of zinc, 250 mg of manganese, 56000 IU of VA 3 6500 IU, 1500 IU of VE, VK 3 50 mg, VB 2 125 mg), nutritional level: digestible energy (MC / kg) 3.81, metabolizable energy (MC / kg) 2.29, crude protein 20.1%, calcium 0.56%, available phosphorus 0.38%, lysine 1.46%.

[0130] (3) Experimental Design

[0131] Fifty 7-day-old suckling pigs (breed: Duroc×Landrace×Yorkshire) were selected for the experiment and randomly divided into 2 groups: a control group and an experimental group, with 5 replicates in each group and 5 piglets in each replicate. The control group was fed the basic formula of the suckling pig compound feed, and the experimental group was fed the basic formula diet supplemented with 0.1% of the inactivated Enterococcus faecium HKS023 bacterial solution based on the quality of the basic formula diet. Experimental period: 25 days.

[0132] (4) Feeding Management

[0133] Feeding was carried out according to the conventional feeding management of the pig farm. The feed intake and diarrhea situation were recorded daily, and the pigs were weighed on an empty stomach once a week. The average daily feed intake, average daily weight gain, feed-to-gain ratio, and diarrhea rate were calculated respectively. Statistical analysis was performed using SPSS statistical software 25.0, and the results are shown in Table 10.

[0134] Table 10 Effects on the Production Performance of Suckling Pigs

[0135]

[0136]

[0137] In the table, different letters represent significant differences.

[0138] From the results in Table 10, it can be seen that compared with the control group, the average daily feed intake of the experimental group increased slightly by 2.31% (P>0.05), and the difference was not significant; the average daily weight gain increased by 19.88% (P<0.05), reaching a significant level; the feed-to-meat ratio of the experimental group decreased by 16.00% (P<0.05), reaching a significant level. There was no diarrhea in the pigs of the experimental group, while the diarrhea rate in the control group was 10.22% (P<0.05), reaching a significant level.

[0139] As can be seen from the above, the present invention uses corn flour as a raw material to ferment and produce L-lactic acid, with high yield, rich raw material sources, low price, meeting market needs, and having great economic and social benefits. At the same time, the present invention uses commonly used raw materials such as calcium carbonate, beef extract, yeast extract, peptone, glucose, sodium acetate, dipotassium hydrogen phosphate, magnesium sulfate, manganese sulfate, etc. to effectively combine and ferment to produce Enterococcus faecium microbial feed additive. The Enterococcus faecium strain used as a probiotic preparation can not only relieve the pathological diarrhea changes caused by Salmonella, Staphylococcus aureus, and Escherichia coli infections, but also promote the absorption and utilization of feed by animals by changing the intestinal flora structure of animals. At the same time, it can protect animals from pathogen infections, improve the body's immune ability to diseases, achieve the effects of preventing diseases and promoting growth, effectively promote animal growth, improve the texture characteristics and muscle fiber characteristics of animal muscles, and improve meat quality.

Claims

1. A strain of Enterococcus faecium, characterized in that: Named Enterococcus faecium HKS023, it was deposited in the General Microbiology Center of China Culture Collection Administration on February 14, 2023. The deposit address is: No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, Institute of Microbiology, Chinese Academy of Sciences, and the deposit number is: CGMCC No.26552.

2. A use of the Enterococcus faecium HKS023 as claimed in claim 1 in inhibiting harmful intestinal bacteria and promoting the growth of livestock and poultry.

3. The use according to claim 2, characterized in that: Preferably, the intestinal harmful bacteria is Salmonella.

4. An application of Enterococcus faecium in producing L-lactic acid, characterized in that: L-lactic acid is produced by fermentation using the Enterococcus faecium HKS023 described in claim 1.

5. The use according to claim 4, characterized in that: Enterococcus faecium HKS023 was inoculated into a fermentation medium and fermented to obtain L-lactic acid fermentation liquid.

6. The use according to claim 5, characterized in that: The following steps are involved: (1) Activation of bacterial strains: Select the Enterococcus faecium HKS023 strain under sterile operation, inoculate it on the slant of MRS solid culture medium, and culture it; (2) Secondary seed culture: Select the activated bacterial strain under sterile conditions and inoculate it into MRS liquid culture medium to expand the bacterial culture and obtain secondary seeds; (3) L-lactic acid production: Inoculate the secondary seeds into the fermentation medium under sterile conditions and ferment them to obtain L-lactic acid fermentation liquid.

7. Use of Enterococcus faecium in producing a microbial feed additive, characterized in that: The Enterococcus faecium HKS023 described in claim 1 is used to culture and produce a microbial feed additive.

8. The use according to claim 7, characterized in that: Enterococcus faecium HKS023 is inoculated into a production medium and cultured to obtain an Enterococcus faecium microbial feed additive.

9. An L-lactic acid fermentation broth produced by the Enterococcus faecium HKS023 as claimed in claim 1, or an inactivated or non-inactivated microbial feed additive.

10. Use of the microbial feed additive according to claim 9 in livestock and poultry breeding.

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