A strain of Bacillus subtilis isolated from frozen soil and its application

Abstract

The present invention provides a strain of Bacillus subtilis isolated from frozen soil and its application. The Bacillus subtilis, named Bacillus subtilis (Bacillus subtilis) BLCC1-1520, has been deposited in the China Center for Type Culture Collection in Wuhan, China on September 9, 2024, with a deposit number of CCTCC NO: M 20241938. The strain has good probiotic properties and good safety, can enhance antioxidant capacity, improve intestinal barrier function, improve immune function and anti-oxidative stress ability, has antibacterial and anti-inflammatory effects, promotes animal growth, and shows significant advantages in animal husbandry and health fields, and is particularly suitable for maintaining animal health and improving production efficiency under stress conditions.

Description

Technical Field

[0001] The invention relates to the field of microorganisms, and in particular to a strain of Bacillus subtilis separated from frozen soil and application thereof. Background Art

[0002] Any discussion of the prior art throughout the specification should not be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0003] Microorganisms are an important life group in the cryosphere and have many unique biological functions. At present, it is known that psychrophiles can still maintain metabolism and life activities at temperatures of -20℃ or even lower. In order to adapt to the low-temperature environment below freezing point, psychrophiles have evolved a variety of survival strategies and mechanisms from the physiological, biochemical and genetic levels, including the production of antifreeze proteins, low-temperature enzymes, and lipid membranes that maintain membrane fluidity at low temperatures, which give them huge development and application potential. Through the research and application of these strains, feasible solutions can be provided for fields such as environmental protection, agricultural development and biotechnology innovation. The application of antifreeze proteins and psychrophiles in animal production shows that they can improve the cold resistance and production performance of animals, increase the output and quality of livestock products, reduce the incidence and mortality of animal diseases, and improve the economic benefits and sustainable development capabilities of animal breeding. However, there are relatively few related studies at present, mainly focusing on cold-water fish farming and the preparation of silage and fermented materials in cold areas. The effects on livestock and poultry need further in-depth research and application promotion.

[0004] Bacillus subtilis (BS) is one of the most widely used feed probiotics with good tolerance, safety and functionality. Adding Bacillus subtilis to feed can improve intestinal morphology, enhance the body's immunity and antioxidant capacity, maintain the intestinal health of livestock and poultry, and improve feed conversion efficiency and resistance to diseases. This is of great significance for reducing the occurrence and mortality of diseases, improving the health status of livestock and poultry and breeding benefits. At the same time, using Bacillus subtilis to improve the immune level and growth performance of livestock and poultry can reduce dependence on antibiotics, thereby reducing the risk of environmental pollution and antibiotic residues. Summary of the invention

[0005] The present invention provides a strain of Bacillus subtilis isolated from frozen soil and its application. The strain has good probiotic properties and good safety, can enhance antioxidant capacity, improve intestinal barrier function, improve immune function and antioxidant stress resistance, has antibacterial and anti-inflammatory effects, promotes animal growth, shows significant advantages in the field of animal husbandry and health, and is particularly suitable for maintaining animal health and improving production efficiency under stress conditions.

[0006] In a first aspect of the present invention, a strain of Bacillus subtilis is provided, which is named Bacillus subtilis BLCC1-1520, which was deposited in the China Center for Type Culture Collection in Wuhan, China on September 9, 2024, with a deposit number of CCTCC NO: M 20241938.

[0007] The strain has good acid and bile salt resistance. In an environment with a relatively low pH value (pH=2 to pH=2.5), the acid resistance is outstanding, which reflects its stronger adaptability in extreme environments. The strain also exhibits significant tolerance in environments with bile salt concentrations ranging from 0.1% to 0.5%, especially at a high bile salt concentration (0.5%), the survival rate is still close to 50%, which indicates that it has a strong survival advantage in environments with high bile salt content such as the digestive tract.

[0008] The strain has good growth ability at low temperatures and shows obvious superiority in growth at lower temperatures (especially 10°C), indicating that it has strong adaptability to cold environments, giving it broad application prospects in cold regions and is particularly suitable for use in microbial preparations that require low-temperature resistance.

[0009] In a second aspect of the present invention, a fermentation product of Bacillus subtilis according to the first aspect is provided.

[0010] In the present invention, "fermentation product" refers to the general term for all products produced by the fermentation process of Bacillus subtilis BLCC1-1520 under suitable conditions. The fermentation product may include, but is not limited to, the following components: living bacteria, dead bacteria, extracellular metabolites, and culture medium components. In the present invention, the fermentation product may be in liquid, semi-solid, or solid form after treatment.

[0011] In a third aspect of the present invention, a product is provided, which comprises the fermentation product of the Bacillus subtilis described in the first aspect or the Bacillus subtilis described in the second aspect.

[0012] In some embodiments of the present invention, the product comprises a feed additive, an antibacterial agent or a medicine.

[0013] In the fourth aspect of the present invention, there is provided the use of the Bacillus subtilis described in the first aspect or the fermentation product of the Bacillus subtilis described in the second aspect or the product described in the third aspect in the preparation of a product having antioxidant capacity and / or antioxidant stress capacity and / or anti-inflammatory capacity.

[0014] In some embodiments of the present invention, the antioxidant capacity and / or antioxidant stress resistance includes reducing oxidative damage and increasing antioxidant enzyme activity.

[0015] In some embodiments of the present invention, the antioxidant capacity and / or antioxidant stress resistance includes enhancing the total antioxidant capacity (T-AOC) and superoxide dismutase (T-SOD) activity, and reducing the content of malondialdehyde (MDA).

[0016] In some embodiments of the present invention, the anti-inflammatory ability includes increasing the level of anti-inflammatory factors and reducing the level of pro-inflammatory factors, wherein the anti-inflammatory factors include IL-10 and IL-4, and the pro-inflammatory factors include IL-6 and IL-2.

[0017] In the fifth aspect of the present invention, there is provided use of the Bacillus subtilis described in the first aspect or the fermentation product of Bacillus subtilis described in the second aspect or the product described in the third aspect in preparing a product with antibacterial ability.

[0018] The strain exhibits stronger antibacterial ability in inhibiting Escherichia coli, Staphylococcus aureus and Clostridium perfringens in vitro, and has particularly significant inhibitory effects on Staphylococcus aureus and Clostridium perfringens, and has stronger antibacterial properties.

[0019] In the sixth aspect of the present invention, there is provided use of the Bacillus subtilis described in the first aspect or the fermentation product of Bacillus subtilis described in the second aspect or the product described in the third aspect in the preparation of a product for enhancing immune function.

[0020] In the seventh aspect of the present invention, there is provided use of the Bacillus subtilis described in the first aspect, the fermentation product of Bacillus subtilis described in the second aspect, or the product described in the third aspect in the preparation of a product for protecting intestinal health.

[0021] In some embodiments of the present invention, protecting intestinal health includes improving intestinal barrier function and enhancing nutrient absorption.

[0022] In some embodiments of the present invention, improving the intestinal barrier includes improving the physical barrier and the immune barrier.

[0023] Wherein, the improvement of the physical barrier includes increasing the content of Occludin protein and Claudin protein. The improvement of the immune barrier includes increasing the content of secretory IgA (sIgA) and reducing the content of tumor necrosis factor TNF-α.

[0024] Compared with the prior art, the advantages of the present invention include:

[0025] The Bacillus subtilis provided by the present invention has acid and bile salt resistance and can grow well in a low temperature environment, which reflects its stronger adaptability in extreme environments and stronger survival advantage in environments with high bile salt content such as the digestive tract.

[0026] The Bacillus subtilis provided by the present invention can enhance the ability to resist oxidative stress. The strain or its fermented product can increase the activity of antioxidant enzymes (such as SOD), significantly enhance the body's ability to cope with oxidative stress, effectively remove free radicals, and reduce cell damage. This makes the present invention have significant advantages in coping with stress conditions such as high temperature, cold, and transportation.

[0027] The Bacillus subtilis provided by the present invention can improve the intestinal barrier function. The strain or its fermented product can enhance the physical barrier and immune barrier function of the intestine by regulating the content of intestinal tight junction protein and sIgA, which not only promotes nutrient absorption, but also improves the defense ability of the intestine against harmful substances and pathogens. This feature is superior to the existing technology in maintaining intestinal health and reducing the occurrence of diseases.

[0028] The Bacillus subtilis provided by the present invention can promote the improvement of immune function. The strain or its fermented product can reduce inflammation and improve the body's immune function by enhancing antioxidant capacity and regulating immune response, thereby helping animals better resist infection when facing pathogen invasion. This has significant advantages in improving animal immune health.

[0029] The Bacillus subtilis provided by the present invention can improve feed conversion rate and growth performance. The strain or its fermented product can significantly improve feed conversion efficiency, especially promote animal growth under stress conditions, and reduce the decline in production performance caused by environmental stress. This has obvious advantages in improving breeding benefits and reducing feed costs.

[0030] The Bacillus subtilis provided by the present invention has a wide range of antibacterial and anti-inflammatory effects. Compared with the prior art, the strain or its fermented product has a wide range of antibacterial effects, can effectively inhibit the growth of multiple harmful bacteria, and maintain intestinal health by reducing inflammatory reactions. This makes the present invention show greater advantages in reducing the use of antibiotics and improving the health level of animals.

[0031] The Bacillus subtilis provided by the present invention can have multifunctional probiotic properties. The strain or its fermentation product not only has the properties of anti-oxidation, antibacterial and promoting intestinal health, but also can comprehensively regulate the immune function and stress tolerance of animals, thereby surpassing the single functional products of the prior art in improving the overall health and production performance of animals.

[0032] In summary, the main advantages of the present invention lie in the strain's powerful anti-oxidative stress ability, enhanced intestinal barrier function, significant immune enhancement effect, improved feed conversion rate and antibacterial and anti-inflammatory ability, which make it have higher application value and broader application prospects in animal breeding and health management compared with the existing technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The accompanying drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. The embodiments of the present invention are described in detail below in conjunction with the accompanying drawings, wherein:

[0034] Figure 1 The colony morphology of strain BLCC1-1520 is shown.

[0035] Figure 2 The bacterial morphology of strain BLCC1-1520 is shown.

[0036] Figure 3 Shown are changes in broiler immunoglobulin IgG levels.

[0037] Figure 4 Shown are changes in immunoglobulin IgA levels in broiler chickens.

[0038] Figure 5 Shown are changes in broiler immunoglobulin IgM levels.

[0039] Figure 6 The statistical results of broiler growth performance are shown, including the average daily weight gain and feed-to-meat ratio of broilers. Different lowercase letters in different groups under the same comparison item indicate significant differences (P < 0.05). DETAILED DESCRIPTION

[0040] The present invention will be further described below in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present invention and are not intended to limit the scope of the present invention. The experimental methods in the following examples without specifying specific conditions are usually carried out under conventional conditions or under conditions recommended by the manufacturer.

[0041] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. The reagents or raw materials used in the present invention can be purchased through conventional channels. Unless otherwise specified, the reagents or raw materials used in the present invention are used in a conventional manner in the art or in accordance with the product instructions. In addition, any method and material similar to or equivalent to the described content can be applied to the method of the present invention. The preferred implementation methods and materials described in the text are for demonstration purposes only.

[0042] The inventors isolated a strain of Bacillus from frozen soil collected in Xinjiang Uygur Autonomous Region, inoculated it into NA solid plate medium, and incubated it at 37°C for 18-24h to form irregular dirty white colonies with a rough surface, translucent to opaque, flat, and irregular edges. The colony morphology is shown in Figure 1 The bacteria are rod-shaped, arranged in clusters and singly, forming endospores, which are oval, centrally located, and Gram-positive, see Figure 2 . After comparison of 16S rRNA sequence (SEQ ID NO: 1), it was identified as a strain of Bacillus subtilis, named Bacillus subtilis BLCC1-1520, and deposited in the China Center for Type Culture Collection in Wuhan, China on September 9, 2024, with the deposit number CCTCC NO: M 20241938. In the following examples, the strain is also referred to as frozen soil bacteria.

[0043] Example 1 In vitro probiotic properties of frozen soil bacteria

[0044] Preparation of fermentation broth: The activated frozen soil bacteria (i.e., Bacillus subtilis BLCC1-1520) and the control bacteria (Bacillus subtilis BL-085 from non-frozen soil, preserved in the strain bank of Shandong Baolai Lilai Biotechnology Co., Ltd.) were inoculated into 250mL Erlenmeyer flasks containing 50mL of LB liquid culture medium, and cultured at 37°C and 180r / min for 24h-30h. After microscopic examination, the culture was stopped when the sporulation rate was about 80%.

[0045] The culture media involved in this example were all purchased from Qingdao Hibo Biotechnology.

[0046] 1.1 Enzyme production performance

[0047] The fermentation broth was centrifuged at 4000 r / min for 10 min, and the supernatant was taken to measure the activities of neutral protease (SB / T10317-1999), cellulase (sodium carboxymethyl cellulose saccharification method) and α-amylase (GB 8275-2009), respectively. The results are shown in Table 1.

[0048] Table 1 Enzyme production performance

[0049]

[0050] 1.2 Tolerance to acid and bile salts

[0051] Take 10 mL of the above fermentation bacterial solution, centrifuge at 4000 r / min for 10 min, discard the supernatant, add 5 mL of physiological saline and mix well to prepare a bacterial suspension as the original bacterial solution.

[0052] Acid resistance test: The original bacterial liquid of the above two strains was added to physiological saline with natural pH (about 6.5), pH=2, pH=2.5, pH=3, and pH=4 at a 5% inoculation rate, respectively, and cultured at 37°C and 180r / min for 2h, counted, and the survival rate was calculated. Among them, natural pH refers to the acidity and alkalinity of the solution formed naturally without artificial adjustment, that is, the original pH value of the solution.

[0053] Bile salt tolerance test: The original bacterial liquid of the above two strains was added into physiological saline with bile salt concentrations of 0, 0.1%, 0.3%, and 0.5% at a 5% inoculation rate, respectively, and cultured at 37°C and 180r / min for 4h. The cells were counted and the survival rate was calculated.

[0054] Table 2 Acid resistance test results

[0055]

[0056] Table 3 Bile salt tolerance test results

[0057]

[0058] As can be seen from the data in Table 2, the survival rate of permafrost bacteria in all acidic environments is better than that of the control bacteria. Even in environments with lower pH values ​​(pH = 2 to pH = 2.5), the acid resistance is outstanding, reflecting its stronger adaptability in extreme environments. This test result shows that permafrost bacteria are much more adaptable to acidic environments than the control bacteria, so they may have greater application potential in dealing with acidic environments in animals.

[0059] As can be seen from the data in Table 3, the frozen soil bacteria showed significant tolerance in environments with bile salt concentrations ranging from 0.1% to 0.5%. Even at high bile salt concentrations (0.5%), the survival rate was still close to 50%, indicating that it has a strong survival advantage in environments with high bile salt content such as the digestive tract. The bile salt tolerance of the control bacteria is weaker, especially when the bile salt concentration reaches 0.3% and 0.5%, its survival rate drops sharply, and it is almost impossible to survive in a high-concentration bile salt environment. This bile salt tolerance of frozen soil bacteria makes it more potential in feed additives, and it can better adapt to the bile salt environment in the animal digestive system, thereby exerting a longer-lasting probiotic effect.

[0060] 1.3 Antibacterial properties

[0061] Preparation of pathogenic bacteria solution: Activated Escherichia coli, Staphylococcus aureus, and Salmonella enteritidis were inoculated into TSB medium and cultured with shaking for 16h-18h. Clostridium perfringens was cultured in FT medium at 42℃ for 6h. The four strains were diluted to 10 6 cfu / mL.

[0062] Antibacterial test: Use the punching method to absorb 1mL (10 6cfu / mL) was added to a sterile culture dish, and then 20 mL of TSA medium was drawn into the sterile culture dish and poured into the dish, and the dish was shaken to make it evenly distributed on the bottom of the dish and solidified. After solidification, holes were punched (the hole diameter of the puncher was 5 mm), and 50 μL of the supernatant of frozen soil bacteria and control bacteria was added to each hole (centrifuged at 4000 rpm for 10 minutes), and cultured overnight. Three parallels were made for each, and the size of the inhibition zone was counted.

[0063] Table 4 In vitro antibacterial performance (mean mm, n = 3) (inhibition zone diameter in mm)

[0064]

[0065] According to the results in Table 4, in comparison, the frozen soil bacteria generally showed stronger antibacterial ability in inhibiting Escherichia coli, Staphylococcus aureus and Clostridium perfringens, especially the inhibitory effect on Staphylococcus aureus and Clostridium perfringens, and had stronger antibacterial properties than the control bacteria. These results show that frozen soil bacteria have great potential when used as animal feed additives or antimicrobial agents, especially in dealing with infections caused by certain pathogens.

[0066] 1.4 Tolerance to culture temperature

[0067] The activated frozen soil bacteria and control bacteria were inoculated into 250 mL Erlenmeyer flasks containing 50 mL of LB liquid culture medium, respectively, placed in different culture temperatures, shaken and cultured, and the absorbance at 600 nm was measured.

[0068] Table 5 OD at different culture temperatures 600 value

[0069]

[0070] Note: - means that spore production reached 80% after 24 hours of culture and culture was not continued to 48 hours.

[0071] According to the results in Table 5, the frozen soil bacteria showed obvious advantages in low temperature (10℃ and 15℃) environment. 600 The value is much higher than that of the control bacteria, indicating that it has strong growth ability and adaptability in cold environments. At 20℃ and 37℃, the growth difference between the frozen soil bacteria and the control bacteria is small, and both show good growth ability. The good growth ability of frozen soil bacteria at low temperatures gives it a broad application prospect in cold regions, and it is particularly suitable for microbial preparations that require low temperature resistance.

[0072] Example 2 Effects on Growth, Immune and Digestive Function of Broilers

[0073] 2.1 Trial Grouping

[0074] Healthy broilers at 0 days old were divided into 3 groups (control group, comparison bacteria group, and frozen soil bacteria group) according to the principle of no difference in weight, with 6 replicates in each group and 10 chickens in each replicate. The test strain was spray-dried after fermentation to prepare bacterial powder, and the number of viable bacteria was about 100 billion / g, which was added through mixing feed.

[0075] The control group (CK group) was fed with a basal diet.

[0076] The control group was fed with a basic diet and the conventionally isolated Bacillus subtilis BL-085 (isolated from non-frozen soil) powder was added at a dosage of 5×10 5 cfu / g.

[0077] The frozen soil bacteria group was fed with the basic diet and Bacillus subtilis BLCC1-1520 powder was added at a dosage of 5×10 5 cfu / g.

[0078] 2.2 Test methods

[0079] The experimental broilers were fed and cleaned in groups every day, and the feed was mixed and the feces were cleaned every 2-3 days. The broilers were immunized with Newcastle disease virus vaccine at 7 days of age, and the second immunization was performed at 23 days of age. Serum was collected before immunization at 7 days of age, 12 days of age, 21 days of age, 28 days of age, 35 days of age, and 42 days of age, and the antibody and titer changes before and after the first and second immunizations were measured, and the feed was weighed in groups. At 42 days of age, chickens close to the average weight in each group were selected and killed, and the immune organ index and the relative length of each intestinal segment were measured.

[0080] 2.3 Detection indicators and methods

[0081] 2.3.1 Growth performance: The experimental broilers were weighed and fed weekly, and the average daily weight gain and feed-to-weight ratio of the broilers at each stage and throughout the entire period were calculated.

[0082] 2.3.2 Slaughter performance:

[0083] Slaughter rate: The weight after bleeding, removing feathers, leg cuticles, toe shells and beak shells is the carcass weight. Slaughter rate (%) = carcass weight / live weight × 100%.

[0084] Eviscerated: The weight of the carcass minus the trachea, esophagus, crop, proventriculus, gizzard, abdominal fat, intestines, spleen, pancreas, gallbladder, reproductive organs, head and feet. Eviscerated % = Eviscerated weight / live weight × 100%.

[0085] Liver index % = liver weight / live weight × 100%.

[0086] Chest muscle rate: Cut the skin along the sternal spine and peel it off toward the back. Use a knife to cut off the muscles attached to the sides of the sternal spine and the scapular tendons. Then, the whole piece of skinned chest muscle can be peeled off and weighed. Chest muscle rate (%) = chest muscles on both sides / live weight × 100%.

[0087] 2.3.3 Immune performance:

[0088] Immune organ index: The three immune organs of thymus, spleen and bursa of Fabricius are completely dissected and weighed (g). Immune organ index (g / kg) = immune organ weight (g) / live weight (kg);

[0089] Immunoglobulins: IgG, IgA, and IgM were tested using Elisa test kits;

[0090] Immune factor and inflammatory factor levels: detected using Elisa test kits;

[0091] Newcastle disease antibody titer: measured by hemagglutination inhibition method.

[0092] 2.3.4 Intestinal performance:

[0093] Relative length of intestine (cm / kg) = length of each section of intestine (cm) / live weight (kg)

[0094] The activities of lipase, amylase and trypsin in the duodenum of broiler chickens were determined as follows:

[0095] Lipase: Collect 0.5 g of duodenal contents (mixture of chyme and intestinal mucosa), add cold physiological saline at a ratio of weight (g): volume (mL) = 1:4, shake and mix, centrifuge at 4000 r / min for 8 minutes, take the supernatant (5×) for determination, and determine the lipase determination kit method of Nanjing Jiancheng Bioengineering Institute.

[0096] Amylase: Sample treatment was the same as lipase. The supernatant was diluted to 200 times according to the preliminary test results and determined using the amylase assay kit of Nanjing Jiancheng Bioengineering Institute.

[0097] Trypsin: Collect 0.5g of duodenal contents (mixture of chyme and intestinal mucosa), add homogenate medium in a ratio of weight (g): volume (mL) = 1:4, shake and mix, centrifuge at 4000r / min for 8 minutes, take the supernatant (5×) as the basic sample, package and freeze for testing. Thaw the basic sample (5×) and mix thoroughly, centrifuge at 12000r / min for 4 minutes until the supernatant is transparent, separate the supernatant to detect trypsin activity, and test strictly according to the Nanjing Jiancheng trypsin kit method.

[0098] Note: Before the formal test, a preliminary test is required to determine the dilution multiple of each enzyme activity test sample. At the same time, the total protein content in the supernatant is detected by the Coomassie Brilliant Blue method. According to the preliminary test, the supernatant needs to be diluted 3 times.

[0099] Determination of the content of tight junction proteins (ZO-1, Occludin and Claudin) in chicken intestine:

[0100] 0.5 g of jejunal intestinal mucosa was collected and added to PBS at a ratio of weight (g): volume (mL) = 1:9. The mixture was shaken and mixed, and centrifuged at 4000 r / min for 8 minutes. The supernatant (10×) was taken for determination. The content of chicken intestinal tight junction proteins (ZO-1, Claudin and Occludin) was determined using the Elisa detection kit of Shanghai ELISA Biological Company.

[0101] The sIgA content and TNF-α in the intestinal mucosa were determined using the Elisa detection kit from Shanghai ELISA Biological Company.

[0102] 2.3.5 Serum biochemical indexes

[0103] BS230 biochemical analyzer was used to test the following indicators, including alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), triglyceride (TG), total cholesterol (TC), glucose (GLU), albumin (Alb), globulin (GLB), total protein (TP), albumin-globulin ratio (A / G), creatinine (CREA) and urea nitrogen (UREA), to investigate the safety of the strain.

[0104] 2.4 Test results

[0105] 2.4.1 Effects on the growth performance of experimental broilers

[0106] Table 6 Growth performance of broiler chickens in early and late stages

[0107]

[0108] Note: Data in the same column with different letters on the shoulders indicate significant differences (P<0.05), data with the same letters or no letters on the shoulders indicate no significant differences (P>0.05), the same as in the following table.

[0109] Early stage (0-21 days old): There was no significant difference in feed-to-meat ratio or average daily weight gain between the groups supplemented with frozen soil bacteria and control bacteria and the control group (CK group), indicating that in the early growth stage, the effects of these two bacteria on broilers were not much different.

[0110] Late stage (21-42 days old): The frozen soil bacteria group showed significant advantages in the late stage, with the lowest feed-to-meat ratio (P<0.05) and the highest average daily weight gain (P<0.05), which significantly improved the growth performance and feed utilization of broilers. In contrast, although the control bacteria group showed some improvement in the late stage, the difference was not significant compared with the control group.

[0111] Overall, the effect of permafrost bacteria is more significant in the later stage, and can effectively improve the growth performance and feed utilization rate of broilers. Therefore, it has great potential for application in broiler farming.

[0112] 2.4.2 Effects on broiler slaughter performance

[0113] Table 7 Slaughter performance

[0114]

[0115] As shown in Table 7, the slaughter rate of the frozen soil bacteria group was significantly higher than that of the control group (CK group) by 2.40%. The rate of evisceration and breast muscle in the control bacteria group was higher, but the difference was not significant compared with the control group.

[0116] 2.4.3 Effects on intestinal function of broiler chickens

[0117] (1) Relative length of intestine

[0118] Table 8 Relative length of intestinal tract of experimental broiler chickens at 42 days of age (cm / kg)

[0119]

[0120] As can be seen from the data in Table 8, both strains of bacteria used in the experiment can increase the relative length of the intestine of broilers. The frozen soil bacteria group performed better in the length of the jejunum, ileum and total intestine, and the relative length of the intestine was significantly higher than that of the control group, indicating that the frozen soil bacteria has a stronger promoting effect on the intestine. Overall, the addition of frozen soil bacteria can significantly increase the relative length of the intestine of broilers, especially in the length of the jejunum and ileum, which may help improve the absorption efficiency of nutrients and thus improve the growth performance of broilers.

[0121] (2) Determination of duodenal digestive enzyme activity

[0122] Table 9 Activities of three digestive enzymes in duodenum

[0123]

[0124] The duodenum is an important digestive tract in broilers. The activity of digestive enzymes reflects the level of digestion and absorption of nutrients by broilers, which has a direct impact on the growth performance of broilers.

[0125] It can be seen from the data in Table 9 that compared with the control group, the activities of trypsin, lipase and amylase in the group adding frozen soil bacteria were significantly increased, with trypsin activity significantly increased by 152.42%, lipase activity increased by 232.78%, and amylase activity significantly increased by 73.06%.

[0126] (3) Tight junction protein content in chicken intestines - physical barrier

[0127] ZO-1, Occludin and Claudin are three important proteins associated with tight junctions between cells. They play a key role in maintaining cell barrier function and regulating the permeability of substances passing through the intercellular space. In the intestine, these tight junction proteins can help limit the entry of harmful substances and pathogens from the intestinal cavity into the body, and also affect the absorption of nutrients by regulating the permeability of tight junctions. The content of these proteins is often used to assess the integrity and health of the intestinal barrier function. Increased or decreased expression of ZO-1, Occludin and Claudin will affect the intestinal barrier function and the efficiency of intercellular material exchange.

[0128] Table 10 Results of determination of tight junction protein content in jejunum

[0129]

[0130] As can be seen from the data in Table 10, in terms of ZO-1 content, the ZO-1 content of the frozen soil bacteria group was significantly lower than that of the control bacteria group (P<0.05), and the difference was significant compared with the CK group (P<0.05). This indicates that the addition of frozen soil bacteria may make the intestinal barrier more open and promote the passage and absorption of nutrients. The ZO-1 content of the control bacteria group was slightly higher than that of the CK group, but the difference was not significant. In terms of Occludin content, the Occludin content of the frozen soil bacteria group was significantly higher than that of the control bacteria group (P<0.05), and also higher than that of the control group, indicating that the tightness of the intestinal barrier was enhanced after the addition of frozen soil bacteria. This may help to better limit the penetration of harmful substances and pathogens and enhance the defense ability of the intestine. The Claudin content of the frozen soil bacteria group was also the highest, significantly higher than that of the control group and the control bacteria group, although the difference was not statistically significant (P>0.05). The high expression of Claudin indicates that the intestinal tight junctions of the frozen soil bacteria group are more solid, which helps to regulate the permeability of the paracellular channels and enhance the barrier function of the intestine.

[0131] (4) Chicken intestinal mucosal immunoglobulin sIgA content (immune barrier)

[0132] Table 11 Results of determination of jejunal sIgA and TNF-α contents

[0133]

[0134] Secretory IgA (sIgA) is an important immune defense component in the intestine, which can neutralize pathogens and prevent them from entering the body. As shown in Table 11, the sIgA content of the frozen soil bacteria group and the control bacteria group was significantly higher than that of the CK group (P<0.05), indicating that the addition of these two bacteria can enhance the local immune function of the intestine and improve the ability of the intestine to resist foreign pathogens. TNF-α is a pro-inflammatory cytokine, and high levels of TNF-α are usually associated with inflammation and tissue damage. The TNF-α content of the frozen soil bacteria group was significantly lower than that of the control group (P<0.05), and was also lower than that of the control bacteria group, indicating that the addition of frozen soil bacteria can effectively inhibit the inflammatory response and reduce the expression of inflammatory factors in the intestine, which may help reduce the occurrence of intestinal inflammation and related diseases. Overall, frozen soil bacteria are particularly outstanding in improving intestinal immune function (by increasing sIgA content) and reducing inflammatory response (by reducing TNF-α content), which helps to enhance the intestinal health and overall immunity of broilers.

[0135] 2.4.4 Immune performance

[0136] (1) Immune organ index

[0137] Table 12 Immune organ index of experimental broiler chickens at 42 days of age (g / kg)

[0138]

[0139] The test results in Table 12 show that whether adding frozen soil bacteria or control bacteria, it has little effect on the development and function of the thymus and spleen of broilers, and there is no significant difference compared with the control group (P>0.05). The bursa of Fabricius is an organ related to the immune system unique to poultry, and its main function is to generate B lymphocytes, thereby affecting the humoral immune response. The bursa index of the frozen soil bacteria group was significantly higher than that of the control group and the control bacteria group (P<0.05), indicating that the addition of frozen soil bacteria can promote the development of the bursa of Fabricius. Although the bursa index of the control bacteria group was slightly higher than that of the control group, the difference was not significant (P>0.05), and its promoting effect on the bursa of Fabricius was not obvious. Overall, frozen soil bacteria performed outstandingly in enhancing the development and function of broiler immune organs (especially the bursa of Fabricius). At 42 days of age, the thymus and bursa indexes of the frozen soil bacteria group were the highest, and the bursa index was significantly higher than that of the control group and the control bacteria group by 20.37% and 18.18%, respectively, indicating that it has certain advantages in improving the humoral immune response and overall immunity of broilers.

[0140] (2) Newcastle disease antibody titer

[0141] Table 13 Changes in average titer of Newcastle disease antibody in broiler chickens after two immunizations (log2)

[0142]

[0143] As can be seen from the data in Table 13, at 7 days of age, broilers have not yet received immunization, and the antibody titer mainly comes from maternal antibodies. The antibody titers of the three groups are relatively high, and the antibody titer of the frozen soil bacteria group is slightly higher than that of the control group and the comparison bacteria group, indicating that the frozen soil bacteria can better maintain the level of maternal antibodies. Five days after the first immunization (12 days of age), the antibody titer of the frozen soil bacteria group was significantly higher than that of the control group and the comparison bacteria group (P<0.05), indicating that the frozen soil bacteria has a significant advantage in enhancing the immune response ability of broilers. At 21 days of age, the antibody titers of the CK group and the comparison bacteria group were basically the same, and the antibody titer of the frozen soil bacteria group was significantly higher than that of the other two groups (P<0.05), indicating that the frozen soil bacteria can maintain a high level of antibody titer and improve the immune effect of broilers. Five days after the second immunization (28 days of age), the antibody titers of the three groups increased, and the titers of the frozen soil bacteria group were slightly higher than the control bacteria group, and there was little difference from the control group. At 12 days after the second immunization (35 days of age), the antibody titer of the frozen soil bacteria group was significantly higher than that of the control group and the control bacteria group (P<0.05), reaching 7.00log2. This shows that frozen soil bacteria are effective in enhancing the long-term immune response of broilers, helping to increase the antibody level of broilers and maintain it for a long time. Overall, the frozen soil bacteria group showed the best immune enhancement effect, significantly improving the antibody titer level of broilers against Newcastle disease, especially in maintaining the long-term immune effect, which helps to improve the immunity and disease resistance of broilers.

[0144] (3) Changes in immunoglobulin levels in broiler chickens

[0145] The changes in the levels of IgG in broiler chickens are shown in Figure 3 .

[0146] Frozen soil bacteria can significantly increase the level of immunoglobulin IgG after 28 days of age. The IgG content of the CK group was higher in the early stage, but as time went on, the IgG content decreased significantly. The IgG content of the control bacteria group has been relatively stable, without significant changes, and the immune enhancement effect is not as good as that of the frozen soil bacteria group. This shows that frozen soil bacteria can play a better role in improving the immunoglobulin level of broilers, especially in the later stage, significantly increasing the IgG content, which helps to enhance the immunity of broilers.

[0147] Changes in the level of immunoglobulin IgA in broiler chickens Figure 4 .

[0148] The IgA content of the frozen soil bacteria group was higher than that of the other two groups at all time points, especially at 35 days of age, when IgA reached the highest value. The IgA content of the control bacteria group was relatively stable, and although there was no significant change, it was also higher than the control group. The IgA content of the CK group began to decrease significantly from 21 days of age, especially at 35 and 42 days of age, when it dropped to the lowest level, indicating that in the absence of strain addition, the level of immunoglobulin A decreased more significantly and the immune defense ability was weakened. Overall, the addition of frozen soil bacteria is particularly outstanding in increasing the IgA content, which helps to improve the immunity and resistance of broilers.

[0149] Changes in the levels of immunoglobulin IgM in broiler chickens Figure 5 .

[0150] The IgM content of the frozen soil bacteria group recovered to a higher level at 42 days of age, indicating that frozen soil bacteria help maintain a strong immune response. Even after the IgM content decreased, the immune response recovery ability of the frozen soil bacteria group was significantly stronger than that of the other two groups. The IgM content of the control bacteria group has been relatively low at all stages, especially at 35 days of age, when the decline was the largest and the recovery speed was the slowest, indicating that the control bacteria are not as effective as the frozen soil bacteria in enhancing immunity. The IgM content of the CK group remained high before 21 days of age, but dropped sharply at 35 days of age. It recovered at 42 days of age, but the degree of recovery was not as good as that of the frozen soil bacteria group. Overall, the frozen soil bacteria group performed well in enhancing and maintaining IgM content and enhancing immune response, especially in the later stage (42 days of age), showing obvious advantages, which helps to improve the immunity of broilers.

[0151] (4) Inflammatory factors

[0152] Table 14 Inflammatory factors (pg / mL)

[0153]

[0154] As can be seen from the data in Table 14, compared with the control group, the level of anti-inflammatory factors (IL-10 and IL-4) in the frozen soil bacteria group was significantly higher than that in the control group and the comparison bacteria group, indicating that frozen soil bacteria can effectively enhance the anti-inflammatory response and help the body resist inflammation. At the same time, the IL-2 content of the frozen soil bacteria group was significantly lower than that of other groups, further indicating that it performed outstandingly in inhibiting pro-inflammatory responses and reducing inflammatory damage. The level of anti-inflammatory factors in the comparison bacteria group was slightly lower, and the content of pro-inflammatory factors IL-6 and IL-2 was also lower, indicating that it had a certain effect in anti-inflammatory, but overall it was not as significant as the frozen soil bacteria group. Overall, frozen soil bacteria are effective in enhancing anti-inflammatory responses, reducing pro-inflammatory responses, and regulating the balance of the immune system, which helps to improve the body's immune status and reduce inflammatory responses.

[0155] 2.4.5 Effects on broiler serum physiological and biochemical parameters

[0156] Table 15 Physiological and biochemical parameters of broiler serum

[0157]

[0158] From the data in Table 15, it can be seen that compared with the control group, the control bacteria and frozen soil bacteria had no significant effect on various blood biochemical indicators at the test dose, indicating that both strains of bacteria can be used safely.

[0159] It should be noted that, except for urea nitrogen (UREA), all blood biochemical indicators in the control bacteria group were elevated, aspartate aminotransferase (AST) and alanine aminotransferase (ALT) in liver function were elevated, and creatinine (CREA) and urea nitrogen (UREA) in kidney function were elevated, all indicating that liver and kidney function may be damaged, and a decrease has no pathological significance. Secondly, all serum protein indicators in the control bacteria group were also elevated, indicating that it may have a certain impact on the body's protein metabolism.

[0160] Example 3 Effect on Antioxidant Stress Function of Broilers

[0161] 3.1 Effects on chicks’ resistance to cold stress

[0162] The cold environment causes stress response in the body, and the cold stress response is accompanied by excessive generation of free radicals. Broiler chicks require a room temperature of 34-35°C during the brooding process. Therefore, it is easy to cause stress due to the low temperature in the house during brooding in the cold winter. When livestock and poultry are under stress, the antioxidant capacity in the body is significantly reduced. Improving their antioxidant capacity can reduce the damage caused by stress. This batch of experiments was carried out in Shandong in December. Electric heaters were used to heat the chicken house, and plastic sheets were added to keep warm. The house temperature was 22-29°C, which did not meet the requirements for brooding, and the chicks were prone to huddling.

[0163] 3.1.1 Test methods

[0164] The experimental period was 1-7 days. One-day-old healthy broilers were selected and divided into 16 groups according to the principle of no difference in weight, with 15 chickens in each group. Among them, 8 groups added 1‰ compound multivitamin to drinking water (called multivitamin group), and 8 groups added 1×10 9 cfu / g frozen soil bacteria powder preparation (filled with glucose as auxiliary material) (called frozen soil bacteria group).

[0165] The growth status of the chicks was observed every day, and the feed amount and mortality rate of each group were recorded. On the 7th day, one chicken with a body weight close to the average was selected from each group, and blood was collected from the heart. The serum was separated and used to determine serum antioxidant indices, including total antioxidant capacity (T-AOC), superoxide dismutase (T-SOD) and malondialdehyde (MDA). The determination kits were purchased from Nanjing Jiancheng Bioengineering Research Institute and the determination was carried out strictly according to the kit method.

[0166] 3.1.2 Test results

[0167] Table 16 Growth performance

[0168]

[0169] The results showed that in the 1-7d test, the average daily weight gain of the multi-dimensional group was slightly higher than that of the permafrost bacteria group (a difference of 0.36g), and the feed-to-meat ratio of the permafrost bacteria group was slightly higher than that of the multi-dimensional group, indicating that the feed utilization efficiency of the permafrost bacteria group was slightly lower than that of the multi-dimensional group, but the difference between the two groups was small, indicating that the effect of adding multi-dimensional or permafrost bacteria on the early growth of broilers was not much different. Feed-to-meat ratio is an important indicator for measuring feed utilization efficiency. Although the feed-to-meat ratio of the permafrost bacteria group was slightly higher, it was still within a reasonable range. The survival rate was the most significant difference between the two groups. The survival rate of the permafrost bacteria group was significantly higher than that of the multi-dimensional group, close to 98%, while the survival rate of the multi-dimensional group was about 86%. This difference shows that the addition of permafrost bacteria can effectively improve the survival rate of broilers, which may be related to the fact that permafrost bacteria enhance the immunity of broilers and reduce diseases or stress. In general, the frozen soil bacteria group had better comprehensive performance, especially in improving the survival rate of broilers. The addition of frozen soil bacteria can greatly improve the survival rate of broiler chicks, and the chicks are in good growth condition. It is suitable as a feed additive to improve the health and growth performance of broiler chicks.

[0170] Table 17 Antioxidant indexes in serum of 7-day-old broilers

[0171]

[0172]

[0173] From the data in Table 17, it can be seen that there are differences in the serum antioxidant indicators of 7-day-old broilers between the frozen soil bacteria group and the multidimensional group, especially in terms of total antioxidant capacity (T-AOC) and superoxide dismutase (T-SOD) activity. The frozen soil bacteria group showed better antioxidant effects. The total antioxidant capacity of the frozen soil bacteria group was significantly higher than that of the multidimensional group, increasing by 26.47%. T-AOC represents the comprehensive antioxidant capacity of the body. The higher the value, the stronger the body's ability to cope with oxidative stress. The T-SOD activity of the frozen soil bacteria group was higher than that of the multidimensional group, increasing by 13.31%. SOD is an important antioxidant enzyme responsible for removing superoxide anion free radicals in the body and protecting cells from oxidative damage. The enhanced activity of T-SOD in the frozen soil bacteria group indicates that it plays a positive role in the antioxidant enzyme system and further improves the resistance of broilers to oxidative stress. MDA is a product of lipid peroxidation. The higher the value, the more serious the oxidative damage in the body. The MDA content of the two groups was close, and the frozen soil bacteria group was slightly lower than the multidimensional group, decreasing by 2.38%. Although the difference is not significant, the MDA level in the permafrost group is slightly lower, indicating that it has a certain effect on reducing lipid peroxidation damage and helps protect cell membranes from oxidative damage. Overall, permafrost can effectively enhance the antioxidant capacity of broilers, help the body resist oxidative stress, reduce oxidative damage, and perform more prominently in enhancing the antioxidant defense system of broilers, helping to improve the health status, antioxidant performance and stress resistance of broilers.

[0174] 3.2 Effects on heat stress resistance of broilers around 30 days old

[0175] The test method and grouping were the same as in Example 2. The breeding time was from mid-April to the end of May. Around 30 days of age, broilers grew fast and were easily affected by stress factors, especially poor heat resistance. At this time, the temperature rose sharply and the chicken house was poorly ventilated, causing heat stress.

[0176] Table 18 Growth performance of experimental broilers at 28-35 days of age

[0177]

[0178] From the data in Table 18, it can be seen that there are significant differences in the growth performance of 28-35-day-old broilers between different treatment groups (CK group, control bacteria group and frozen soil bacteria group), especially in feed-to-meat ratio, average daily weight gain and number of deaths. The frozen soil bacteria group showed obvious advantages. The frozen soil bacteria group had the best growth performance, which was manifested by the lowest feed-to-meat ratio (highest feed utilization), the highest average daily weight gain and zero mortality rate, indicating that frozen soil bacteria can significantly improve the resistance of broilers to heat stress and effectively improve the survival rate while ensuring the growth rate. The average daily weight gain and feed-to-meat ratio of the control bacteria group were poor, and the mortality rate was high, indicating that its effect on heat stress resistance was not as good as that of the frozen soil bacteria group. On the other hand, since the deaths of the control bacteria group were all fast-growing and heavy-weight chickens, the final growth performance and average daily weight gain were even slightly inferior to those of the CK group. Overall, frozen soil bacteria have the ability to significantly improve the ability of broilers to resist heat stress, can improve feed utilization and survival rate without affecting growth, and are very suitable as feed additives for broiler breeding in high temperature seasons.

[0179] Table 19 Antioxidant indexes in serum of experimental broiler chickens at 35 days of age

[0180]

[0181] From the data in Table 19, it can be seen that the T-AOC value of the frozen soil bacteria group was the highest, reaching 15.83U / mL, which was higher than that of the CK group and the control bacteria group, indicating that frozen soil bacteria can effectively enhance the overall antioxidant capacity of broilers. This enhanced antioxidant capacity may help broilers better cope with oxidative stress and maintain the health of the body. The T-SOD activity of the frozen soil bacteria group was the highest, reaching 149.92U / mL. This shows that frozen soil bacteria can significantly improve the antioxidant enzyme activity of broilers, further enhance the body's free radical scavenging ability, and reduce the damage caused by oxidative stress.

[0182] Example 4 Application of the composite microecological preparation to improve the body's immune level

[0183] 4.1 Test methods

[0184] Test strains: The frozen soil bacteria powder was compounded with interferon-γ recombinant lactic acid bacteria to form a compound preparation, so that the number of live frozen soil bacteria was 1×10 9 cfu / g, the number of viable lactic acid bacteria recombinant with interferon-γ was 2×10 10 cfu / g, the compound preparation was diluted 100 times with sterile saline before use.

[0185] Experimental animals: 1-day-old AA white-feathered broiler chickens, fed with a basic diet without antibiotics.

[0186] 4.2 Experimental design

[0187] Sixty one-day-old AA white-feathered broilers with uniform body weight and good health were randomly divided into two groups, with three replicates in each group and 10 chickens in each replicate. One group was the blank control group and the other was the experimental group. The experimental group was orally fed with 200 μL / chicken / day of the compound preparation diluent at 7-9 days, 14-16 days, and 21-23 days of age, and the blank control group was orally fed with the same amount of sterile saline.

[0188] 4.3 Detection indicators

[0189] At 28 days of age, the weight of the chicks and the feed intake during the experimental period were counted, and the average daily weight gain and feed-to-meat ratio were calculated. Blood was collected from the wing vein, serum was separated, and serum IL-4 and IL-10 levels were determined using ELISA kits.

[0190] 4.4 Test results

[0191] Results Figure 6 . On the 28th day of the experiment, the average daily weight gain of the experimental group was significantly higher than that of the blank control group (P < 0.05), and the feed-to-meat ratio was significantly lower than that of the blank control group (P < 0.05), indicating that the compound preparation has a significant promoting effect on the growth performance of broilers.

[0192] Table 20 Cytokine levels

[0193]

[0194] As shown in Table 20, the levels of IL-4 and IL-10 in the serum of the experimental group were significantly higher than those of the blank control group (P < 0.05), indicating that the compound preparation can significantly enhance the level of cellular immune response and improve the body's immunity.

[0195] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Although the present invention is described in detail with reference to the aforementioned embodiments, those skilled in the art can still modify the technical solutions described in the aforementioned embodiments or replace some of the technical features therein by equivalents. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A strain of Bacillus subtilis, named Bacillus subtilis ( Bacillus subtilis )BLCC1-1520, which was deposited in the China Center for Type Culture Collection in Wuhan, China on September 9, 2024, with the deposit number CCTCCNO: M 20241938.

2. A product comprising the Bacillus subtilis according to claim 1.

3. The product according to claim 2, characterized in that The product is a feed additive, an antibacterial agent or a medicine.

4. Use of the Bacillus subtilis according to claim 1 in preparing a product having antioxidant capacity and / or antioxidant stress capacity and / or anti-inflammatory capacity; in, The antioxidant capacity and / or antioxidant stress resistance capacity includes reducing oxidative damage and increasing antioxidant enzyme activity; the anti-inflammatory capacity includes increasing the level of anti-inflammatory factors and reducing the level of pro-inflammatory factors.

5. The use according to claim 4, characterized in that: The antioxidant capacity and / or antioxidant stress resistance includes improving the total antioxidant capacity and superoxide dismutase activity, and reducing the content of malondialdehyde.

6. The use according to claim 4, characterized in that: The anti-inflammatory factors include IL-10 and IL-4, and the pro-inflammatory factors include IL-6 and IL-2.

7. Use of the Bacillus subtilis according to claim 1 in preparing a product with antibacterial ability; in, The antibacterial ability includes the ability to inhibit Escherichia coli, Staphylococcus aureus, Salmonella enteritidis and Clostridium perfringens.

8. Use of the Bacillus subtilis according to claim 1 in preparing a product for enhancing immune function.

9. Use of the Bacillus subtilis according to claim 1 in preparing a product for protecting intestinal health; in, The protection of intestinal health includes improving intestinal barrier function and enhancing nutrient absorption.

10. The use according to claim 9, characterized in that: Improving the intestinal barrier includes improving the physical barrier and the immune barrier; Wherein, the improvement of the physical barrier includes increasing the content of Occludin protein and Claudin protein; Wherein, the improvement of immune barrier includes increasing the content of secretory immunoglobulin A and reducing the content of tumor necrosis factor TNF-α.

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