Preparation and application of mink bacillus subtilis as well as probiotics and metabiotics of mink bacillus subtilis

Abstract

The invention discloses preparation and application of mink bacillus subtilis as well as probiotics and metabiotics of the mink bacillus subtilis. The invention provides a bacillus subtilis strain which is separated and purified from intestinal contents of healthy minks. The bacillus subtilis strain is preserved in China General Microbiological Culture Collection Center on December 7, 2023, and the preservation number is CGMCC (China General Microbiological Culture Collection Center) No. 29263. The bacillus subtilis provided by the invention is good in stability, and the viable count is gt when the bacillus subtilis is stored at 4 DEG C for 30 days; and 2.3 * 10 < 7 > CFU / mL. The bacillus subtilis can generate active ingredients with the effects of resisting inflammation, inhibiting bacteria, improving intestinal health, regulating immunity, resisting oxidation and the like. The strain can be prepared into bacillus subtilis probiotics to be directly added into a mink feed, and also can be prepared into a postbiotic to be added into the mink feed, so that the growth performance, the oxidation resistance and the immunocompetence of minks are improved.

Description

Technical Field

[0001] The invention belongs to the field of microbial engineering and relates to probiotics, specifically to a mink Bacillus subtilis strain and the preparation and application of probiotics and postbiotics thereof. Background Art

[0002] Bacillus subtilis is a type of Bacillus, without capsule, with flagella, capable of movement, and Gram-positive bacteria. Bacillus subtilis microecological preparations are ideal feed additives to replace antibiotics. They have high biological activity in livestock, poultry, and aquaculture applications, and can improve feed efficiency, enhance animal immunity, and improve animal intestinal health. Bacillus subtilis is also a recognized safe microorganism approved by the Food and Drug Administration because it does not produce any toxic components, which provides safety guarantees for the practical application of Bacillus subtilis.

[0003] Bacillus subtilis can secrete digestive enzymes, thereby improving the animal's ability to digest feed and improving the digestion and utilization rate of feed. Bacillus subtilis can produce antibacterial substances such as bacteriocins, cell wall degrading enzymes, antibacterial active proteins, iturin, fengycin and surfactin, which have inhibitory effects on Clostridium perfringens and Salmonella, and have the potential to replace antibiotics. After Bacillus subtilis spores enter the intestines of animals, they go from a dormant state to a state of rapid growth and reproduction. In this process, a large amount of oxygen is consumed, providing an anaerobic environment for beneficial intestinal bacteria such as lactobacillus and bifidobacteria, and inhibiting the growth of aerobic and harmful bacteria such as Escherichia coli and Salmonella. Summary of the invention

[0004] The present invention provides a strain of Bacillus subtilis, which is a strain isolated and purified from the intestinal contents of a healthy mink. The Bacillus subtilis strain was deposited in the General Microbiological Center of the China Microbiological Culture Collection Administration (No. 3, Yard No. 1, Beichen West Road, Chaoyang District, Beijing) on ​​December 7, 2023, with a deposit number of CGMCC No. 29263. The classification name is Bacillus subtilis, and the Latin name is Bacillus subtilis.

[0005] The present invention also provides the Bacillus subtilis fermentation product and a preparation method thereof, the method comprising the following steps:

[0006] S1. Preparation of seed solution

[0007] The Bacillus subtilis strains are activated and propagated with NB medium to obtain Bacillus subtilis seed liquid;

[0008] S2. Fermentation of bacteria

[0009] The seed liquid is inoculated into a fermentation medium, and fermented for 24 hours to obtain a fermentation liquid;

[0010] Specifically, the steps are as follows:

[0011] S1. Preparation of seed solution

[0012] The Bacillus subtilis isolated and purified from the mink intestine was propagated in NB medium, inoculated into NB medium at a 2% inoculum, and propagated at a constant temperature of 37°C to obtain Bacillus subtilis seed liquid. The average viable count of the seed liquid was measured to be 2.96×10 12 CFU / mL;

[0013] S2. Fermentation of bacteria

[0014] The seed liquid was inoculated into NB medium at a 2% inoculation rate and cultured at 37°C for 24 h. The number of viable bacteria in the fermentation liquid was 4.73×10 12 CFU / mL.

[0015] The present invention also provides the Bacillus subtilis postbiotic and a preparation method of the postbiotic, wherein the postbiotic comprises the Bacillus subtilis and / or its metabolites; the preparation method comprises the following steps:

[0016] S1. Preparation of seed solution

[0017] The Bacillus subtilis strains are activated and propagated with NB medium to obtain Bacillus subtilis seed liquid;

[0018] S2. Fermentation of bacteria

[0019] The seed liquid is inoculated into a fermentation medium, and fermented for 24 hours to obtain a fermentation liquid;

[0020] S3. Inactivation and drying of bacteria

[0021] The fermentation liquid obtained by fermentation and cultivation for 24 hours in S2 is inactivated by high temperature and high pressure, and milk powder containing zeolite powder is added, mixed evenly, and dried at low temperature to obtain Bacillus subtilis postbiotics.

[0022] Furthermore, the present invention also provides a bacterial agent, comprising the Bacillus subtilis and / or the fermentation product of the Bacillus subtilis and / or the postbiotics of the Bacillus subtilis.

[0023] Furthermore, the present invention also provides the use of the Bacillus subtilis or the Bacillus subtilis fermentation product or the Bacillus subtilis postbiotic in the preparation of a probiotic preparation.

[0024] Furthermore, the present invention also provides the use of the Bacillus subtilis and / or the Bacillus subtilis fermentation product and / or the Bacillus subtilis postbiotics in the breeding of minks and / or other non-mink livestock and poultry.

[0025] Furthermore, the present invention also provides a feed additive, which comprises the Bacillus subtilis and / or Bacillus subtilis fermentation product and / or Bacillus subtilis postbiotics. The feed additive is used in mink feed in an amount of 0.15% to 0.45% based on the Bacillus subtilis postbiotic dry powder preparation.

[0026] Beneficial Effects

[0027] Compared with the prior art, the present invention has the following technical advances:

[0028] ① The Bacillus subtilis provided by the present invention is a strain separated and purified from the intestinal contents of healthy minks. The strain is used to produce probiotics or postbiotics specifically for minks, and the application is safer and more reliable. It is a probiotic strain with great potential.

[0029] ② The Bacillus subtilis provided by the present invention has good stability and can be stored at 4°C for 30 days with a viable count of >2.3×10 7 CFU / mL, which can be used to prepare Bacillus subtilis probiotics and postbiotics.

[0030] ③ The preparation method of Bacillus subtilis probiotics and postbiotics for minks provided by the present invention is determined in combination with the characteristics of Bacillus subtilis strains. The bacterial liquid is cultured for 24 hours as the source of Bacillus subtilis probiotics and postbiotics. At this time, the number of viable bacteria is the highest, and the active ingredients of the prepared postbiotics are the highest. The inactivation of Bacillus subtilis is prepared by a high temperature and high pressure method, which fully destroys the cell wall and releases the intracellular effective ingredients. The drying method selects a carrier adsorption and low-temperature drying method. This method uses fermentation liquid to prepare postbiotics, retaining the active substances generated by Bacillus subtilis during the fermentation process; carrier adsorption can reduce water content and reduce drying time; zeolite is added to the carrier to increase the fluidity of the final product; low-temperature drying can effectively retain the active ingredients produced by Bacillus subtilis, and compared with the freeze-drying method, the equipment is simple and the cost is low, which is conducive to industrial production.

[0031] ④ The Bacillus subtilis probiotics and Bacillus subtilis postbiotics prepared by the present invention can meet the different needs in the mink production process. Because mink feed is mainly fresh feed, the addition of Bacillus subtilis is beneficial to the preservation of feed and the colonization of Bacillus subtilis in minks.

[0032] ⑤ After detection and analysis of the metabolic components of Bacillus subtilis, more than 400 up-regulated differential metabolites were obtained, including active ingredients with anti-inflammatory, antibacterial, intestinal health improvement, immune regulation and antioxidant effects. This shows that the Bacillus subtilis and postbiotics of the present invention can play a role in improving the intestinal health of minks, and enhancing the antioxidant and immune abilities of minks.

[0033] In summary, the Bacillus subtilis for minks provided by the present invention has the characteristics of safety and reliability, stable growth performance, good fermentation performance, and rich and diverse active ingredients. The Bacillus subtilis can be used to prepare safe, stable and efficient Bacillus subtilis probiotics and postbiotics for minks, which can be used as functional feed additives in mink production. The preparation method of the probiotics and postbiotics is simple and has high production efficiency, which is conducive to large-scale and industrialized production. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 The colony image (A) and Gram staining microscopic image (B) of the Bacillus subtilis in Example 1 of the present invention are shown.

[0035] Figure 2 This is the growth curve of Bacillus subtilis in Example 1.

[0036] Figure 3 This is a classification diagram of differential metabolites between postbiotics and NB culture medium of Bacillus subtilis in Example 4 of the present invention. Among them, A: Classification of differential metabolites between postbiotics and NB culture medium (HMDB Super Class); B: Classification of differential metabolites between postbiotics and NB culture medium (HMDB Class); C: Classification of differential metabolites between postbiotics and NB culture medium (Ontology).

[0037] Figure 4 The KEGG pathway enrichment bar graph of the Bacillus subtilis postbiotics in Example 4 of the present invention. The KEGG pathway enrichment bar graph of FX-8 (sample) vs. NB (control) (top 30 at most, different colors belong to level 2 classification). Level 1 pathway classification: Metabolism (Metabolism, M), Genetic Information Processing (Genetic Information Processing, G), Environmental Information Processing (Environmental Information Processing, E), Cellular Processes (Cellular Processes, C), Organismal Systems (Organismal Systems, O), Human Diseases (Human Diseases, H), Drug Development (Drug Development, D). DETAILED DESCRIPTION

[0038] The present invention is further described in detail below by specific examples. It should be understood that the described examples are only used to explain the present invention, and are not intended to limit the present invention.

[0039] Unless otherwise specified, the materials and reagents used in the examples of the present invention can be obtained from commercial sources. Experimental methods without specific conditions in the examples are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0040] Example 1 Obtaining and Preserving Bacillus subtilis from Mink

[0041] This embodiment is the acquisition and preservation of Bacillus subtilis for minks. The process of obtaining the bacteria includes the separation and purification of Bacillus subtilis. The separation and purification method is carried out in the following steps:

[0042] (1) Acquisition of strains

[0043] The Bacillus subtilis involved in this embodiment is obtained by the following operation:

[0044] ①Sample collection

[0045] Scrape 0.1 g of the intestinal contents of the mink into a sterilized test tube and dilute it 10 times to obtain 10 -1 ~10 -7 Concentration of dilution.

[0046] ② Cultivation of strains

[0047] Accurately pipette 10 -4 ~10 -7 100 μl of the dilution with the concentration was inoculated into a NA culture dish and cultured at 37°C for 24 h. NA culture medium is shown in Table 1.

[0048] Table 1 NA culture medium ingredients

[0049]

[0050] ③Strain isolation and purification

[0051] Pick up single colonies with rough and opaque surface, dirty white or slightly yellow, continue to culture by plate streak method, and perform single strain purification three times.

[0052] (2) Identification of strains

[0053] DNA was extracted from the isolated and purified strain, and PCR amplification was performed using the extracted DNA as a template and bacterial 16S rDNA universal primers. The successfully amplified PCR product was purified and sequenced. The 16S rDNA gene sequence is shown in SEQ ID NO.1:

[0054]

[0055] The sequencing results were compared with the 16S rDNA sequence of the standard strain in Gen Bank by BLAST at NCBI, and the isolated strain was determined to be Bacillus subtilis, with the Latin name Bacillus subtilis.

[0056] The Bacillus subtilis strain was deposited in the General Microbiology Center of China Microorganism Culture Collection Administration on December 7, 2023, with the deposit number CGMCC No.29263.

[0057] (3) Identification of physical and chemical properties of strains

[0058] ①1①Morphological observation and Gram staining

[0059] Morphological observation: The colonies of Bacillus subtilis are round, with irregular edges, a rough, dry, dull surface, and a layer of membrane on the surface that forms wrinkles with a central bulge. The colonies are opaque grayish white (see Figure 1 A).

[0060] Perform Gram staining on the isolated and purified strains. Use a rubber-tipped dropper to draw a drop of saline in the center of a clean glass slide, use a sterilized inoculation needle to pick up a single colony, mix it evenly with the saline on the glass slide, and air-dry it over an alcohol lamp for 3-5 times to fix it. Stain with the crystal violet stain in the Gram staining kit for 120 seconds, and rinse with distilled water until the glass slide is colorless. Then stain with iodine solution for 60 seconds, and rinse with distilled water until it is colorless. Decolorize with anhydrous ethanol for 30 seconds, and continue to rinse with distilled water until it is colorless. Finally, stain with safranin counterstain for 60 seconds, and rinse with distilled water until it is colorless. After the glass slide is air-dried, observe under a microscope (see Figure 1 B). Figure 1 B As can be seen, Bacillus subtilis is purple and the cells are rod-shaped. Bacillus subtilis is arranged as single cells or in clusters, thus forming a chain or grape-like structure. The spores are located in the center of the bacteria and are cylindrical.

[0061] ②Identification of biochemical characteristics of strains

[0062] Using biochemical identification tubes, the obtained Bacillus subtilis strains were subjected to starch hydrolysis test, glucose, maltose, sucrose, lactose fermentation test, D-xylose, L-arabinose, D-mannitol, propionate, citrate utilization test, nitrate reduction test, gelatin liquefaction test, and the results are shown in Table 2.

[0063] Table 2 Biochemical characteristics identification test of Bacillus subtilis

[0064]

[0065]

[0066] *+ represents positive, - represents negative, and (+) in the anaerobic growth column represents facultative anaerobe.

[0067] By testing the biochemical reactions of Bacillus subtilis, its adaptability to different nutrients and growth environments can be identified.

[0068] ③Growth and pH characteristics

[0069] The identified strain was inoculated into NB medium at 2% inoculation volume and cultured in a 37°C air bath constant temperature oscillator (180r / min). The absorbance of the Bacillus subtilis suspension was measured every 2 hours to determine its growth characteristics. The absorbance was measured with the sterile NB medium as the reference and the wavelength was 600nm. 600 Value Figure 2 The ingredients of NB culture medium are shown in Table 3.

[0070] Table 3 NB culture medium ingredients

[0071]

[0072] OD after fermentation 600 As can be seen from the figure, the strain has a short lag phase, entering the exponential growth phase after 2 hours of culture and entering the stable phase after 16 hours.

[0073] ④Drug resistance

[0074] The Bacillus subtilis suspension was spread on NA medium, and a drug sensitivity test paper was placed in the center of the medium. The culture was cultured at 37°C for 24 h, and the diameter of the inhibition zone was measured. The results are shown in Table 4.

[0075] Table 4 Drug sensitivity test results of Bacillus subtilis

[0076]

[0077]

[0078] *S: sensitive, inhibition zone diameter > 1.5 cm; I: moderately sensitive, inhibition zone diameter 1.0 cm ≤ ≤ 1.5 cm; R: resistant, inhibition zone diameter less than 1.0 cm

[0079] As shown in the results in Table 4, the Bacillus subtilis of the present invention is sensitive to multiple drugs.

[0080] ⑤ Antibacterial properties

[0081] Using Escherichia coli and Salmonella as indicator strains, the antibacterial activity of Bacillus subtilis strains was determined by the Oxford cup method. The Bacillus subtilis strain was inoculated into NB medium at an inoculation amount of 2%, and cultured at 37°C and 180 r / min for 24 hours. After that, 2 mL of the bacterial solution was taken and placed in a centrifuge tube, centrifuged at 12,000×g for 20 min, and the supernatant was added to the Oxford cups of the sterile nutrient agar plates containing Escherichia coli and Salmonella respectively. After culturing at 37°C for 48 h, the size of the antibacterial zone was measured, and the experimental results are shown in Table 5. It can be seen from Table 5 that Bacillus subtilis has antibacterial effects on Escherichia coli and Salmonella.

[0082] Table 5 Antibacterial results of Bacillus subtilis

[0083]

[0084] *Insensitive, DIZ = 7.8 mm; Sensitive, 7.8 mm < DIZ ≤ 16.0 mm; Highly sensitive, DIZ > 16.0 mm

[0085] Example 2 Preparation method of Bacillus subtilis probiotics for mink

[0086] This example is a preparation method of Bacillus subtilis probiotics for mink, which specifically includes the following steps carried out in sequence:

[0087] S1. Preparation of seed liquid

[0088] The Bacillus subtilis isolated and purified from mink intestines obtained in Example 1 was propagated in NB medium. It was inoculated into NB medium at an inoculation amount of 2% and incubated at a constant temperature of 37°C for propagation to obtain a seed liquid of Bacillus subtilis for mink. The average viable bacteria count of this seed liquid was measured to be 2.96×10 12 CFU / mL.

[0089] S2. Fermentation of bacterial strains

[0090] The seed liquid was inoculated into NB medium at an inoculation amount of 2% and incubated at a constant temperature of 37°C for 24 h. The viable bacteria count in the fermented liquid was actually measured to be 4.73×10 12 CFU / mL. When this bacterial liquid was stored at 4°C for 30 days, the viable bacteria count > 2.3×10 7 CFU / mL. This bacterial liquid can be directly used as a source of Bacillus subtilis probiotics and can be used as a probiotic within 30 days of storage at 4°C.

[0091] Example 3 Preparation method of Bacillus subtilis postbiotics for mink

[0092] This example is a preparation method of Bacillus subtilis postbiotics for mink, which specifically includes the following steps carried out in sequence:

[0093] S1. Preparation of seed solution

[0094] The Bacillus subtilis isolated and purified from the mink intestine obtained in Example 1 was propagated in NB medium, inoculated into NB medium at a 2% inoculum, and placed at 37°C for constant temperature propagation to obtain a Bacillus subtilis seed solution for minks. The average viable count of the seed solution was 2.96×10 12 CFU / mL.

[0095] S2. Fermentation of bacteria

[0096] The seed solution was inoculated with 2% of the inoculum into NB medium and cultured at 37°C for 24 h. The actual number of viable bacteria was 4.73×10 12 CFU / mL.

[0097] S3. Inactivation and drying of bacteria

[0098] The fermentation liquid obtained by fermentation culture in S2 for 24 hours was treated at 131° C. for 30 minutes to obtain the Bacillus subtilis postbiotic preparation liquid.

[0099] 100 mL of the prepared solution was mixed evenly with 300 g of milk powder containing 1% zeolite powder, dried at 65° C., crushed, and passed through an 80-mesh sieve to obtain a Bacillus subtilis postbiotic dry powder preparation. The mass of the postbiotic preparation was about 310 g.

[0100] Example 4 Determination and analysis of metabolites of Bacillus subtilis postbiotics for minks

[0101] The above-mentioned high-temperature treated Bacillus subtilis fermentation broth was subjected to metabolite determination and differential metabolites classification between Bacillus subtilis postbiotics and NB medium. The results are shown in Figure 3 .

[0102] The results showed that there were 427 upregulated metabolites in the postbiotics of the present invention. According to the HMDB Class of the human metabolome database, the upregulated metabolites included 8 azole compounds, 26 benzene and substituted derivatives, 80 carboxylic acids and their derivatives, 7 diazonaphthalene compounds, 21 fatty acyl compounds, 9 flavonoid compounds, 8 imidazopyrimidine compounds, 25 indoles and their derivatives, 8 organic nitrogen-containing compounds, 25 organic oxygen-containing compounds, 10 peptidomimetic compounds, 37 isoprenoid lipids, 12 pyridines and pyridine derivatives, and 16 steroids and steroid derivatives.

[0103] Among the above metabolites, azole compounds, such as nizatidine, are H2 receptor antagonists used to treat gastrointestinal diseases such as gastric ulcer, duodenal ulcer, and gastroesophageal reflux disease. Benzene and its derivatives have medicinal value, such as acetaminophen (containing a benzene ring structure), chloramphenicol, niflumic acid, and benzoic acid, which have antibacterial and analgesic effects. Carboxylic acids and their derivatives, such as aspirin (acetylsalicylic acid), pantothenic acid, apiramycin, and γ-aminobutyric acid, have anti-inflammatory, antiparasitic, and metabolic activities. Organic oxygen-containing compounds, such as pseudoneurotrophin A, have specific antioxidant activity, streptomycin, and glutamine have antifungal activity. Indole and its derivatives, such as alkaloids and tryptophan, have antibacterial and antiviral effects, and melatonin, tryptamine, and tryptamine-3-ethanol have anti-inflammatory and antioxidant effects. Fatty acyl compounds, such as thermomycin, have antifungal activity. Flavonoids are natural antioxidants, such as apigenin, galangin, baicalein-7-methyl ether, catechins, etc. Imidazolopyrimidine compounds participate in some metabolic processes or signal transduction pathways in the body. Organic nitrogen-containing compounds, such as penicillin and cephalosporin, have antibacterial effects, and agmatine has anti-inflammatory activity. Peptidomimetic compounds, such as surfactin A, surfactin B, surfactin C13, anserine or anserine, have antioxidant and metabolic regulating effects. Isoprene lipids, such as Deltaline, aconitine, soybean saponin I, paclitaxel, etc., have anti-inflammatory activity. Pyridine and pyridine derivatives, such as vitamin B6, are related to material metabolism. Steroids and steroid derivatives have anti-inflammatory and antioxidant effects.

[0104] KEGG pathway analysis was performed based on its metabolites, see Figure 4The differential metabolites of FX-8 (sample) vs. NB (control) involved a total of 182 pathways, among which the 10 most affected pathways were ATP transporters (ABC transporters), protein digestion and absorption, metabolic pathways, arginine and proline metabolism, purine metabolism, D-amino acid metabolism, biosynthesis of amino acids, biosynthesis of cofactors, microbial metabolism in diverse environments, and biosynthesis of secondary metabolites.

[0105] More than 400 upregulated differential metabolites were obtained, including active ingredients with anti-inflammatory, antibacterial, intestinal health improvement, immune regulation and anti-oxidation effects. The above results show that the Bacillus subtilis and postbiotics of the present invention can play a role in improving the intestinal health of minks, and enhancing the antioxidant and immune abilities of minks.

[0106] Example 5 Application of Bacillus subtilis postbiotics for minks on male minks

[0107] This example investigates the effects of Bacillus subtilis postbiotics on growth performance and serum immune indexes of male minks in the growing period, and the method is as follows:

[0108] The experiment selected 120 12-week-old white male minks and randomly divided them into 4 groups, with 15 replicates in each group and 2 minks in each replicate. The addition amounts of Bacillus subtilis postbiotic preparation liquid (the inactivated fermentation liquid obtained in preparation step S3) in the 4 groups of diets were 0, 0.05%, 0.1%, and 0.15%, respectively (or the addition amounts of the postbiotic dry powder preparation obtained in preparation step S3 were 0, 0.15%, 0.3%, and 0.45%, respectively. Note: The addition amount refers to the addition according to this ratio, for example, the addition amount of 0.05% is 100g×0.05%=0.05g in 100g diet, 1000g×0.05%=0.5g in 1000g diet, and 1000kg×0.05%=0.5kg in 1 ton diet). The pre-test period is 1 week and the formal test is 8 weeks.

[0109] At the beginning and end of the experiment, each mink was weighed on an empty stomach, and the initial and final weights were recorded. Three days were randomly selected each week to accurately record the amount of feed and the amount of leftover feed. The average daily weight gain, average daily feed intake, and feed-to-weight ratio of the minks were calculated.

[0110] After the feeding experiment, 10 mL of blood was collected from the hearts of 8 minks randomly selected from each group, placed in a centrifuge tube, and centrifuged at 3000×g for 10 min to prepare serum for the determination of serum immunoglobulin indicators. The serum immunoglobulins were determined by enzyme-linked immunosorbent assay for immunoglobulin A, immunoglobulin G, and immunoglobulin M. Malondialdehyde, superoxide dismutase, glutathione peroxidase, and total antioxidant capacity were detected by thiobarbituric acid method, hydroxylamine method, and colorimetric method, respectively.

[0111] The results of the effect of Bacillus subtilis postbiotics on the production performance of male minks in the growing period are shown in Table 6. As shown in Table 6, Bacillus subtilis postbiotics can significantly affect the average daily weight gain and feed-to-weight ratio in the first 0-4 weeks, 5-8 weeks and 0-8 weeks (P<0.05), and significantly affect the mid-term weight and final weight of minks (P<0.05). Compared with the control group, the body weight of the Bacillus subtilis postbiotics group increased significantly at 4 weeks and 8 weeks (P<0.05), and the average daily weight gain of 0-4 weeks, 5-8 weeks and 0-8 weeks increased significantly (P<0.05), and the feed-to-weight ratio decreased significantly (P<0.05).

[0112] Table 6 Effects of Bacillus subtilis postbiotics on the production performance of male minks in the growing period

[0113]

[0114] The results of the effect of Bacillus subtilis postbiotics on serum immunity of male minks in the rearing period are shown in Table 7. As shown in Table 7, Bacillus subtilis postbiotics significantly affected serum immunoglobulin G (P<0.05). Compared with the control group, the serum immunoglobulin G content of the 0.05% Bacillus subtilis postbiotics supplementation group increased significantly (P<0.05).

[0115] Table 7 Effects of Bacillus subtilis postbiotics on serum immunity of male minks during the rearing period

[0116]

[0117] The results of the effects of Bacillus subtilis postbiotics on the antioxidant indexes of male minks in the growing period are shown in Table 8. As shown in Table 8, Bacillus subtilis postbiotics had no significant effect on the malondialdehyde content, superoxide dismutase and glutathione peroxidase activities in the serum of male minks (P>0.05), but affected the total antioxidant activity (P<0.05). Compared with the control group, the total antioxidant capacity of the 0.15% Bacillus subtilis postbiotic group increased significantly (P<0.05).

[0118] Table 8 Effects of Bacillus subtilis postbiotics on serum antioxidant indexes of male minks during the breeding period

[0119]

[0120] It can be seen from this that adding 0.05% Bacillus subtilis postbiotic preparation liquid (or 0.15% Bacillus subtilis postbiotic dry powder preparation) to mink feed is most effective for the growth performance, antioxidant capacity and immune capacity of male minks.

[0121] Example 6 Application of Bacillus subtilis postbiotics for minks on female minks

[0122] This example investigates the effects of Bacillus subtilis postbiotics on growth performance, serum immune indexes and antioxidant indexes of female minks during the growing period, and the specific methods are as follows:

[0123] The experiment selected 120 12-week-old white female minks and randomly divided them into 4 groups, with 15 replicates in each group and 2 minks in each replicate. The addition amount of Bacillus subtilis postbiotic preparation liquid (the inactivated fermentation liquid obtained in preparation step S3) in the 4 groups of diets was 0, 0.05%, 0.1%, and 0.15%, respectively (or the postbiotic dry powder preparation obtained in preparation step S3, then the addition amount is 0, 0.15%, 0.3%, and 0.45%, respectively; Note: The addition amount refers to the addition according to this ratio, for example, the addition amount of 0.05% is 100g × 0.05% = 0.05g in 100g diet, 1000g × 0.05% = 0.5g in 1000g diet, and 1000kg × 0.05% = 0.5kg in 1 ton diet). The pre-test period is 1 week, and the formal test is 8 weeks.

[0124] At the beginning and end of the experiment, each mink was weighed on an empty stomach, and the initial and final weights were recorded. Three days were randomly selected each week to accurately record the amount of feed and the amount of leftover feed. The average daily weight gain, average daily feed intake, and feed-to-weight ratio of the minks were calculated.

[0125] After the feeding experiment, 10 mL of blood was collected from the hearts of 8 minks randomly selected from each group, placed in a centrifuge tube, and centrifuged at 3000×g for 10 min to prepare serum for the determination of serum immunoglobulins and antioxidant indicators. The serum immunoglobulins were determined by enzyme-linked immunosorbent assay for immunoglobulin A, immunoglobulin G, and immunoglobulin M. Malondialdehyde, superoxide dismutase, glutathione peroxidase, and total antioxidant capacity were detected by thiobarbituric acid method, hydroxylamine method, and colorimetric method, respectively.

[0126] The results of the effect of Bacillus subtilis postbiotics on the production performance of female minks in the rearing period are shown in Table 9. As shown in Table 9, Bacillus subtilis postbiotics can significantly affect the average daily weight gain, final weight and feed-to-weight ratio (P<0.05), but have no significant effect on the average daily feed intake (P>0.05). Compared with the control group, the feed-to-weight ratio of the 0.05%, 0.1% and 0.15% Bacillus subtilis postbiotics supplementation groups was significantly reduced (P<0.05), and the average daily weight gain and final weight of the 0.15% Bacillus subtilis postbiotics supplementation group were significantly increased (P<0.05).

[0127] Table 9 Effects of Bacillus subtilis postbiotics on the production performance of female minks during the rearing period

[0128]

[0129] The results of the effect of Bacillus subtilis postbiotics on serum immunity of female minks during the rearing period are shown in Table 10. As shown in Table 10, Bacillus subtilis postbiotics significantly affected the serum immunoglobulin A content (P<0.05). Compared with the control group, the serum immunoglobulin A content of the 0.1% and 0.15% Bacillus subtilis postbiotics supplementation groups increased significantly (P<0.05).

[0130] Table 10 Effects of Bacillus subtilis postbiotics on serum immunity of female minks during the rearing period

[0131]

[0132] The results of the effects of Bacillus subtilis postbiotics on the serum antioxidant indexes of female minks during the rearing period are shown in Table 11. As shown in Table 11, Bacillus subtilis postbiotics had no significant effect on the activities of serum superoxide dismutase and glutathione peroxidase (P>0.05), but affected the total antioxidant activity and malondialdehyde content (P<0.05). Compared with the control group, the total antioxidant capacity of the 0.1% and 0.15% Bacillus subtilis postbiotic groups increased significantly (P<0.05), and the malondialdehyde content decreased significantly (P<0.05).

[0133] Table 11 Effects of Bacillus subtilis postbiotics on serum antioxidant indexes of female minks during the breeding period

[0134]

[0135]

[0136] It can be seen from this that adding 0.10% and 0.15% Bacillus subtilis postbiotic preparation liquid (or 0.3% and 0.45% Bacillus subtilis postbiotic dry powder preparation) to mink feed can improve the growth performance, antioxidant capacity and immune capacity of female minks.

[0137] Example 7 A mink feed additive

[0138] A mink feed additive, wherein the main component of the feed additive is the Bacillus subtilis postbiotic dry powder preparation obtained in Example 3, and the postbiotic dry powder preparation is added in an amount of 0.15% to 0.45% in the mink feed.

Claims

1. A strain of Bacillus subtilis, characterized in that: The Bacillus subtilis is deposited in the General Microbiological Center of China Microorganism Culture Collection Administration (CGMCC), and the deposit number is CGMCC No29263.

2. The Bacillus subtilis fermentation product according to claim 1.

3. The method for preparing the Bacillus subtilis fermentation product according to claim 1, characterized in that: The following steps are involved: S1. Preparation of seed solution The Bacillus subtilis strain is activated and propagated with NB medium to obtain Bacillus subtilis seed liquid; S2. Fermentation of bacteria The seed liquid was inoculated into the fermentation medium and fermented for 24 hours to obtain a fermentation liquid; the number of viable bacteria in the fermentation liquid was 4.73×10 12 CFU / mL.

4. The Bacillus subtilis postbiotic according to claim 1, characterized in that Containing the Bacillus subtilis and / or its metabolites according to claim 1.

5. The method for preparing the Bacillus subtilis postbiotic according to claim 4, characterized in that: The following steps are involved: S1. Preparation of seed solution The Bacillus subtilis strains are activated and propagated with NB medium to obtain Bacillus subtilis seed liquid; S2. Fermentation of bacteria The seed liquid is inoculated into a fermentation medium, and fermented for 24 hours to obtain a fermentation liquid; S3. Inactivation and drying of bacteria The fermentation liquid obtained in S2 is inactivated at high temperature and high pressure, and milk powder containing zeolite powder is added, mixed evenly, and dried at low temperature to obtain Bacillus subtilis postbiotics.

6. A bacterial agent, characterized in that: Comprising the Bacillus subtilis according to claim 1 and / or the fermentation product of Bacillus subtilis according to claim 2 and / or the postbiotics of Bacillus subtilis according to claim 3.

7. Use of the Bacillus subtilis according to claim 1, the Bacillus subtilis fermentation product according to claim 2, or the Bacillus subtilis postbiotic according to claim 3 in the preparation of a probiotic preparation.

8. Use of the Bacillus subtilis according to claim 1 and / or the Bacillus subtilis fermentation product according to claim 2 and / or the Bacillus subtilis postbiotics according to claim 3 in mink and / or other non-mink livestock and poultry farming.

9. A feed additive, characterized in that: The feed additive comprises the Bacillus subtilis according to claim 1 and / or the Bacillus subtilis fermentation product according to claim 2 and / or the Bacillus subtilis postbiotics according to claim 3.

10. A mink feed additive, characterized in that: The feed additive comprises the Bacillus subtilis postbiotic according to claim 3; the Bacillus subtilis postbiotic is added in an amount of 0.15% to 0.45% based on dry powder preparation.

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