Cat-source bacillus subtilis and application thereof

Abstract

The invention belongs to the technical field of microorganism application, and particularly relates to cat-source bacillus subtilis and application thereof. The strain number of the cat source bacillus subtilis is 11-6Q1, the cat source bacillus subtilis is preserved in the China Center for Type Culture Collection, and the preservation number of the cat source bacillus subtilis is CCTCC NO: M 20241474. The bacillus subtilis strain 11-6Q1 disclosed by the invention is separated from healthy pet cat excrement and has acid-resistant and high-temperature-resistant effects. The bacterium has a strong hydrolysis effect on protein, and can relieve digestion and absorption of protein in intestinal tracts, maintain animal intestinal flora balance, improve animal health and improve animal immunity.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial application, and particularly relates to a cat-derived Bacillus subtilis strain and application thereof. Background Art

[0002] Animal intestinal microorganisms participate in the physiological activities of the animal body and play an important role in maintaining the health of the host. Under normal circumstances, intestinal microorganisms maintain a dynamic balance and protect the host animal from the occurrence of diseases; when the microbiome is disturbed, the balance of the intestinal microecology will be broken, affecting the health of the animal and even causing various diseases.

[0003] Intestinal diseases are one of the most common diseases in dogs and cats. Diarrhea is the most common symptom, which has an adverse effect on the health of dogs and cats. At present, the main means of treating intestinal diseases in clinical practice is the use of antibiotics, but irrational or high-dose use of antibiotics can easily cause pathogens to develop drug resistance and destroy normal intestinal microbiota. Many studies have shown that probiotics have a certain effect on the prevention and treatment of intestinal diseases in dogs and cats, and can maintain host health through antagonism, rejection, and immune regulation.

[0004] Bacillus is a type of Gram-positive bacteria that can produce resistant endospores and is widely present in organisms and the environment. Cao Shuwei et al. showed that Bacillus has antibacterial and antiviral activities; it also has the functions of promoting growth and digestion, maintaining the balance of intestinal flora, inhibiting pathogens and improving the body's immunity. For example, adding Bacillus subtilis to the feed can reduce the diarrhea rate of piglets, while reducing the body's inflammatory response and improving intestinal absorption function. Studies have shown that adding Bacillus subtilis to the feed can regulate the intestinal flora of laying hens, improve the antioxidant status and barrier integrity of the duodenum, reduce the expression of proinflammatory cytokines, and reduce intestinal pathogens. Bacillus has a strong survival ability, and the antibacterial substances it produces generally have broad-spectrum bactericidal activity. The antibacterial mechanism is mainly to resist the adverse intestinal environment through biological oxygen deprivation, flora adjustment, biological antagonism, improving immune function and enhancing enzyme activity. For example, Bacillus subtilis can produce antibacterial substances, antimicrobial peptides, during metabolism, which can inhibit the growth of harmful bacteria such as streptococci, Escherichia coli, and Salmonella. Summary of the invention

[0005] In order to overcome the shortcomings of the prior art, the present invention first provides a cat-origin Bacillus subtilis, the cat-origin Bacillus subtilis (Bacillus subtilis) strain number is 11-6Q1, the preservation date is July 3, 2024, and it is preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: M 20241474.

[0006] The present invention also provides a bacterial agent, which comprises a substance obtained by culturing the above-mentioned cat-origin Bacillus subtilis 11-6Q1 in a microbial culture medium.

[0007] The present invention also provides a composition, which comprises the above-mentioned cat-origin Bacillus subtilis 11-6Q1 and / or the bacterial agent.

[0008] Application of the above composition in the preparation of animal food additives.

[0009] Furthermore, the animal is a feline or a canine.

[0010] Furthermore, the feline is a cat.

[0011] The beneficial effects of the present invention are as follows: the present invention obtains a Bacillus subtilis strain 11-6Q1 with the strongest enzyme production ability through screening, which has the effects of acid and high temperature resistance. The bacteria can ease the digestion and absorption of protein in the intestine, maintain the balance of animal intestinal flora, improve animal health, improve animal inflammatory response, and enhance animal immunity, and has a good application prospect as an animal food additive. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 is the morphological diagram of strain 11-6Q1; Figure 2 is the Gram staining result of strain 11-6Q1; Figure 3 is the result of agarose gel electrophoresis (M stands for Marker, from top to bottom are 5000, 3000, 2000, 1000, 750, 500, 250, 100 bp); Figure 4 is the growth curve of strain 11-6Q1; Figure 5 This is a graph showing the results of a hemolytic test of strain 11-6Q1; Figure 6 is the antibacterial ability of strain 11-6Q1; Figure 7 Comparison of the weight growth rate of mice under different dosage conditions; Figure 8 Comparison of the weight of mouse viscera under different dosage conditions; Fig. 9 It is the Venn diagram under different dosage conditions; Fig.10 For comparison of components at the gate level; Fig.11 For the comparison of composition at the genus level; Fig.12 Comparison photos of feces before and after the test in the medium-dose and high-dose groups; Fig.13 Changes in IL-4 levels; Fig.14 is the change of IL-1β level; Fig.15 is the change of TNF-α level; Fig.16 For SOD level detection. DETAILED DESCRIPTION

[0013] Below, in conjunction with the accompanying drawings and specific embodiments, the present invention is further described. It should be noted that, under the premise of no conflict, the embodiments described below or the technical features can be arbitrarily combined to form new embodiments. The specific conditions not specified in the embodiments are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used, unless otherwise specified, are conventional products obtained through commercial channels.

[0014] Example 1 Isolation, screening and identification of Bacillus subtilis: (1) Isolation of Bacillus subtilis strains Collect feces from pet cats and prepare bacterial suspension. Spread the diluted liquids in different proportions on enzyme-producing solid culture plates, neutral protease-producing strain screening culture plates, and lipase-producing strain screening culture plates. Culture at 37°C for 2 days, and observe the growth status of the colonies every 12 hours. Observe the growth status and colony morphology of the coated plates, select the appropriate dilution (about 10 to 200 colonies), select single colonies of different morphologies, number them, and then streak them, and culture them at 28°C for 20 hours. Observe whether the morphological characteristics of the streaked culture colonies are single. For plates with non-single colony morphological structures, continue to pick single colonies for streaking culture until a pure cultured single bacterium with consistent colony morphological characteristics is obtained. Pick the separated and purified pure cultured single colonies and inoculate them into the liquid culture medium, and culture them at 200 rpm and 37°C at a constant temperature until the OD 600 value of the bacterial solution reaches about 1.0 to obtain a purified isolated bacterial strain.

[0015] (2) Screening of Bacillus subtilis strains Place the purified bacterial solution obtained in step (1) into a beaker, add 10 times the amount of sterile saline, treat at 90°C for 10 min, and then perform 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5After dilution, 3 plates were coated for each dilution, and then coated on the corresponding plates and cultured at 37°C for 48 h to screen various enzyme-producing strains. The first batch of 5 strains with the largest ratio of transparent circle to colony diameter were screened by observing whether colonies grew on the screening plates for amylase, lipase, and protease production.

[0016] Table 1 Results of initial screening of enzyme production Note: - : indicates that it cannot be measured; + : indicates that it can be measured.

[0017] The results are shown in Table 1. By observing whether colonies grew on the screening plates for amylase, lipase and protease production, the first five strains with the largest ratio of transparent zone to colony diameter were screened out, namely 7-6D, 7-6E, 11-302, 11-303 and 11-6Q1.

[0018] In order to further confirm the enzyme activity of the five strains 7-6D, 7-6E, 11-302, 11-303, and 11-6Q, the size of the transparent zone was preliminarily determined and the enzyme activity was tested.

[0019] Table 2 Enzyme production of candidate strains The results are shown in Table 2. Finally, the strain 11-6Q1 with the strongest protease production ability was screened out, with a protease activity of 2.33U and an amylase activity of 1.67U.

[0020] (3) Identification of Bacillus subtilis strains Inoculate Bacillus subtilis 11-6Q bacterial solution, streak on the surface of LB solid medium, and invert in a 37°C incubator for 24 h. Record the shape, color, and size of the colonies growing on the plate. In addition, take 20 μL of bacterial solution for smear, stain with crystal violet dye for about 1 min after fixation, rinse with distilled water, and observe the morphology under an optical microscope (100×) after drying and take photos and record.

[0021] The results are as follows Figure 1 As shown, strain 11-6Q1 was milky white on the solid plate, with round colonies, smooth and moist surfaces, neat edges, and raised colonies. Figure 2 As shown, the bacterial morphology of 11-6Q1 under microscopic examination is round or oval, Gram-staining positive, and the bacteria are mostly single, opposite, and rarely in chains.

[0022] (4) Molecular identification results of Bacillus subtilis According to the general method of microbial identification, the 16S rRNA gene of the isolated strain was amplified using universal primers 27 (5′-AGAGTTTGATCCTGGCTCAG-3′) and 1492R (5′-GGYTACCTTGTTACGACTT-3′), and the bands were detected under 1.5% agarose gel electrophoresis. The PCR reaction system and PCR reaction conditions are shown in Tables 3 and 4.

[0023] Table 3 PCR reaction system Table 4 PCR reaction conditions The results are as follows Figure 3 As shown, the PCR product was detected by agarose gel electrophoresis to obtain a target fragment of about 1500 bp, and the sequencing results were submitted to NCBI for Blast analysis. The obtained sequence was compared with the NCBI database, and the results identified that 11-6Q1 belongs to: Bacteria, Firmicutes, Bacilli, Bacillales, Bacillaceae, Bacillus, named Bacillus subtilis, and identified as Bacillus subtilis. The Bacillus subtilis strain was named 11-6Q1 and deposited in the General Microbiology Center of the China Microbiological Culture Collection Administration Committee. The deposit date is July 3, 2024, and the deposit number is CCTCC NO: M 20241474. The sequencing results of Bacillus subtilis 11-6Q1 are shown in Table 5.

[0024] Table 5 Sequencing results of strain 11-6Q1 (5) Routine biochemical identification of Bacillus subtilis Unless otherwise specified, the general morphological, physiological and biochemical properties tests were conducted in accordance with Bergey's Manual of Bacterial Identification (9th Edition) and Microbiology Laboratory Manual. The biochemical reaction of the bacteria showed that the 11-6Q1 strain fully met the characteristics of Bacillus, and the results are shown in Table 6.

[0025] Table 6 Biochemical reaction results of screened strains Note: +: positive; -: negative; (6) Growth curve determination of Bacillus subtilis Bacillus subtilis was inoculated into LB liquid medium and cultured at 37 °C and 150 r / min for 24 h. The fermentation liquid was taken every 2 h. The blank liquid LB medium was used as a control. The absorbance value of OD600 was measured. There were 3 replicates in each group. The growth curve of Bacillus subtilis strain was drawn. The results are shown in Figure 4 shown.

[0026] Example 2 Tolerance and drug sensitivity experiments of Bacillus subtilis 11-6Q1: (1) Tolerance test results In order to understand the resistance of Bacillus subtilis 11-6Q1 screened in this project to digestive effects such as gastric acid and bile salts, an in vitro digestion test simulating the gastrointestinal environment was conducted to compare the bacterial count of Bacillus subtilis 11-6Q1 at different bile salt concentrations and the bacterial survival rate under different pH conditions.

[0027] Table 7 Results of bile salt tolerance survival rate Table 8 Acid resistance survival rate results The results are shown in Tables 7 and 8, which are the bile salt resistance survival rate results and acid resistance survival rate results. It was found that Bacillus subtilis 11-6Q1 still maintained a certain survival rate in an environment of 0.5% bile salt concentration and pH 3-4. Therefore, the strain is able to resist the digestion of substances such as gastric acid and bile salts in the intestine.

[0028] At the same time, considering that there will be high temperature links in the later stage of probiotic preparation, the bacterial count of Bacillus subtilis 11-6Q1 at different temperatures was also measured, and the results are as follows.

[0029] Table 9 High temperature resistance survival rate results The results are shown in Table 9, and it was found that Bacillus subtilis 11-6Q1 still had a certain survival rate at 50-60°C. Therefore, Bacillus subtilis 11-6Q1 also has high heat resistance, which laid the foundation for our subsequent addition to cat food for processing.

[0030] (2) Hemolytic test By culturing Bacillus subtilis 11-6Q1 on medium containing animal or human blood, physical changes caused by hemolytic activity were observed.

[0031] The results are as follows Figure 5 As shown, the cat-derived probiotics screened in this experiment did not undergo hemolysis around the colonies, and there was no color change on the periphery of the colonies, indicating that they were not hemolytic.

[0032] (3) Determination of antibacterial properties of strains The Oxford cup method was used to determine the inhibitory effect of cat-derived probiotics on pathogenic Escherichia coli, Salmonella, Staphylococcus aureus and Shigella, with ampicillin as the positive control and sterile distilled water as the negative control.

[0033] The results are as follows Figure 6 As shown, no obvious inhibition zone was observed, and none of the strains had obvious antibacterial properties.

[0034] (4) Drug sensitivity test of Bacillus subtilis 11-6Q1 Bacillus subtilis 11-6Q1 was transferred and cultured, and samples were taken every 1 h to determine the concentration of the bacterial solution (3.0×10 8 ~8.0×10 8 ) CFU / mL. Pour 10 mL of agar medium into 9 cm culture dishes, take 200 μL of activated bacterial solution and mix with 5 mL of 45℃ 0.6% corresponding soft agar (the concentration of bacterial solution is maintained at 1.5×10 7 CFU / mL~3.0×10 7 CFU / mL), the soft agar was evenly spread on the pre-prepared solid culture medium of the same type, and placed at room temperature for about 15 min. After the upper agar was cooled and solidified, the antibiotic sensitive paper disc containing the antibiotic was aseptically pasted on the agar surface. The 13 drug sensitive paper discs containing experimental antibiotics were tetracycline (TET), penicillin (PEN), gentamicin (GEN), ampicillin (AMP), enrofloxacin (NOR), sulfisoxazole (SIZ), erythromycin (ERY), and florfenicol (FFC). The diameter of the paper disc was 6 mm, which met the antibiotic paper disc standard. Three parallels of each drug sensitive paper disc were diffused at 4℃ for 3 h and then cultured at 37℃ for 24 h. The diameter of the inhibition zone was measured with a vernier caliper, and the average of the three measurements was taken as the result. The results are shown in Table 10.

[0035] Table 10 Results of drug sensitivity test (5) Safety experiment in mice According to the relevant provisions of GB14922.1 and GB14922.2 of Laboratory Animal Microbiology, Parasitology and Monitoring, 5-week-old Kunming mice with a body weight of 24 g were selected as experimental animals. The experimental animals were randomly divided into 4 groups, with 6 females in each group. After Bacillus subtilis strain 11-6Q1 was activated three times continuously, the colonies were scraped and dissolved in physiological saline to make bacterial concentrations of low dose 1×10 7 CFU / mL, medium dose 1×10 8 CFU / mL, high dose 1×10 9 CFU / mL of bacterial suspension. Before the experiment, the mice were kept in the experimental animal room for 5 days for environmental adaptation and quarantine observation. After the adaptation period, the mice in the control group were given oral saline, and the mice in the experimental group were given oral low-dose, medium-dose, and high-dose bacterial suspensions, all at a dose of 0.05 mL, for a total of 1 time. The grouping of mice is shown in Table 11. The weight of the mice was weighed on the 0th, 7th, and 14th days of the gavage experiment ( Figure 7 After the 14-day experiment, the mice were weighed and killed, and their organs were dissected to calculate the organ indexes of the heart, liver, spleen, lung and kidney ( Figure 8 ). And the feces of the mice were taken for high-throughput sequencing to analyze the changes in the bacterial flora ( Fig. 9 ).

[0036] Table 11 Mouse experimental groups The results are as follows Figure 7 As shown in the figure, the mice were weighed on the 0th, 7th and 14th days of the gavage experiment. The weight gain rate of the mice after taking probiotics was higher than that of the control group, indicating that probiotics are beneficial to animal health. Figure 8 As shown in the figure, it was found that the changes in organ weight did not reach the level of significant difference. According to the results of high-throughput sequencing, it was found that the control group and the probiotic group had 291 common OTU units, and in terms of the unique OTU units in each group, the low-dose group, the medium-dose group, and the high-dose group were far higher than the control group. Fig. 9 As shown, the diversity and richness of the bacterial flora increased after mice took probiotics. In terms of feeding concentration, by comparing the three doses of high, medium and low, it can be found that the medium concentration dose is the most ideal, so when feeding cats later, consider the medium dose of 1×10 8 CFU / mL is used as the concentration, but the cat's weight should be fully considered.

[0037] (6) Diversity analysis At the same time, through diversity index analysis, the species richness, diversity, uniformity and coverage of the microbial community can be reflected. Among them, the ACE index and Chao index are often used to reflect species richness, and the Shannon index and Simpson index are often used to reflect community diversity. The larger the Shannon index value, the higher the community diversity; and the larger the Simpson index value, the lower the community diversity. Using the inter-group difference test, the inter-group diversity index value can be tested for significant differences. The diversity statistical results are shown in Table 12: Table 12 Diversity analysis table (7) Analysis and comparison of bacterial species composition Statistical analysis was performed at the phylum level. Those with a relative abundance of less than 1% were classified as others, and the rest were analyzed and counted according to their proportions. The results showed that the phyla with the largest relative abundance at the phylum level were: Bacteroidota, Firmicutes, and Proteobacteria. The results are as follows Fig.10 shown.

[0038] Statistical analysis was performed at the genus level, and those with a relative abundance of less than 1% were classified as others. The rest were analyzed and counted according to their proportions. The results showed that the genera with relatively high relative abundance at the genus level were: norank_Muribaculaceae; Ligilactobacillus; Lactobacillus; Bacteroides; Alloprevotella. Fig.11 shown.

[0039] After completing the data statistics, we conducted a differential analysis of the four groups of data to discover bacterial phyla or genera with significant changes. At the phylum level, the proportion of Cyanobacteria decreased significantly after taking probiotics, and this phylum is directly related to IBD. Some representative species of cyanobacteria, such as Anabaena sp and Microcystis sp, may produce toxins and pose a threat to human or animal health. The proportion of this phylum showed a downward trend after taking probiotics. In terms of intestinal flora, the proportion of Cyanobacteria decreased after taking probiotics, and the intestinal inflammation of animals was alleviated. In the future, it can be considered to continue to develop for the treatment of IBD enteritis.

[0040] Test Example 1: Application of cat-derived probiotics The main ingredients of the compound bacterial powder are: cat-derived probiotics Bacillus subtilis 11-6Q1, Bacillus licheniformis, Saccharomyces boulardii, etc. When the bacterial liquid is freeze-dried to prepare the bacterial powder, skim milk powder is added as a freeze-drying protective agent. The number of live bacteria in the bacterial powder is ≥3*10 8 CFU / g, packaged into 2g bags.

[0041] The experimental animals were based on cats with diarrhea or soft stools in the cattery. Twelve healthy adult cats with good development and an average weight of (2.0±0.2) kg, aged 10 months, were selected and randomly divided into 4 groups, with 3 cats in each group. No antibiotics or any other drugs or preparations known to affect the intestinal flora were used in the 3 months before the experiment. The pre-feeding period was 7 days and the formal test period was 7 days.

[0042] Table 13 Experimental groups According to the nutritional needs of adult cats, the control group was fed with basic food, and the experimental group was fed with compound probiotics on the basic food (the expected number of live bacteria in the bacterial powder at the factory was ≥3*10 8 CFU / g, the dosage was 2g per day). The control group was not treated, the low-dose group was fed half a bag of probiotics per day; the medium-dose group was fed 1 bag of probiotics per day; and the high-dose group was fed 1.5 bags of probiotics per day. During the experiment, the normal diet of the cats was not affected, and they followed their previous diet structure, except that the probiotics were fed once more.

[0043] (1) Fecal measurement During the trial period, the weight of the cats was recorded, and the weight changes of the cats in the experimental group and the control group were compared, and the growth rate was calculated. An objective third party consisting of 6 people scored the feces of each cat. The scoring criteria (feces water content, feces volume) were used to grade the feces quality according to the method of Nery J et al. Before and after the experiment, we used the rectal sampling method to obtain the intestinal excrement of the pet cats and conducted fecal testing. The results are shown in Table 14.

[0044] Table 14 Fecal test results At the same time, we also scored the stool morphology based on a 6-person evaluation panel, and the final results are shown in Table 15.

[0045] Table 15 Comparison of fecal scoring results The results are shown in Table 15. Through scoring and visual observation, it was found that feeding probiotics had a certain degree of improvement in the feces of some cats. The comparison photos of feces before and after the test of the medium-dose and high-dose groups are shown in Table 15. Fig.12 .

[0046] (2) Serum immune factor detection At 9 am on the first and seventh days of the experiment, 3 mL of blood was collected from the left forelimb vein of the cat on an empty stomach. The collected blood was centrifuged at 4000 r / min for 5 min, and the upper serum was separated and collected. The serum was separated by centrifugation at 6000 r / min for 5 min at 4°C. The levels of TNF-α, IL-4 and IL-1β in cat serum were detected based on fluorescence quantitative PCR. TNF-α is an inflammatory cytokine mainly produced by macrophages, and its overexpression is associated with the development of a variety of inflammatory and autoimmune diseases. IL-4 is a pleiotropic cytokine that plays a key role in regulating immune responses and promoting antibody production. IL-1β is an important proinflammatory cytokine that participates in the body's inflammatory response and immune regulation. The changes in the levels of TNF-α, IL-4 and IL-1β in serum were observed to reflect the effects of the probiotic on the immunity of pet cats.

[0047] The results are as follows Fig.13 , Fig.14 , Fig.15 As shown in the figure, after using probiotics, the IL-4 in the cat's serum increased significantly, which helps to relieve inflammation; while the levels of TNF-α and IL-1β decreased significantly, indicating that inflammation was suppressed. Therefore, the detection of cytokines can show that the use of probiotics can significantly improve the immunity of cats.

[0048] (3) Detection of SOD This experiment was based on the serum samples of pet cats and carried out the detection based on superoxide dismutase (SOD). The level of SOD activity indirectly reflects the body's ability to remove gas free radicals.

[0049] The results are as follows Fig.16 As shown in the results, the addition of probiotics to cat food leads to an increase in superoxide dismutase (SOD) in cat serum, which is mainly related to the antioxidant defense system in the body and is essential for resisting oxidative stress, preventing cell damage, and maintaining the stability of physiological functions. Probiotics indirectly enhance the body's antioxidant capacity by improving the balance of intestinal microecology. A good antioxidant state helps protect the intestinal mucosa, improve the absorption efficiency of nutrients, and enhance the body's health and physiological functions.

[0050] The above-mentioned embodiments are only preferred embodiments of the present invention and cannot be used to limit the scope of protection of the present invention. Any non-substantial changes and substitutions made by technicians in this field on the basis of the present invention shall fall within the scope of protection required by the present invention.

Claims

1. A strain of Bacillus subtilis from cat origin, characterized in that: The cat-origin Bacillus subtilis strain number is 11-6Q1, which is preserved in the China Center for Type Culture Collection with a preservation number of CCTCC NO: M 20241474.

2. A bacterial agent, characterized in that The bacterial agent comprises a substance obtained by culturing the cat-derived Bacillus subtilis 11-6Q1 as claimed in claim 1 in a microbial culture medium.

3. A composition, characterized in that The composition comprises the cat-derived Bacillus subtilis 11-6Q1 described in claim 1 and / or the bacterial agent described in claim 2.

4. Use of the composition according to claim 3 in the preparation of animal food additives.

5. Use of the composition according to claim 4 in the preparation of animal food additives, characterized in that: The animal is a feline.

6. Use of the composition according to claim 5 in the preparation of animal food additives, characterized in that: The feline is a cat.

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