Bacillus subtilis and application thereof

Abstract

The invention provides bacillus subtilis and application thereof, and relates to the field of microorganisms. The bacillus subtilis disclosed by the invention is named as bacillus subtilis BLDT-9, and is preserved in China Center for Type Culture Collection in Wuhan University in Wuhan City in China on February 17, 2025, and the preservation number of the bacillus subtilis is CCTCC NO: M2025228. The strain can significantly improve multiple performance indexes of poultry: the unqualified egg rate is reduced by more than 50%, the laying rate is improved, and the feed-egg ratio is improved; the shelf life of the eggs can be prolonged, and the rate of AA-grade eggs can be kept above 90% after the eggs are refrigerated for 5 days; the breeding poultry reproductive performance, including the fertilization rate and the hatching rate, can be improved; good production performance of laying hens at the age of 380 days or above and breeding hens at the age of 39 weeks or above can still be maintained; the anti-stress capability of the organism is obviously enhanced, the anti-oxidation index is improved, and the death and culling rate in high-temperature seasons is lower than 0.1%; meanwhile, the utilization efficiency of the feed is improved by improving the intestinal flora structure, and the feed has a wide application prospect.

Description

Technical Field

[0001] This application relates to the fields of microorganisms and fermentation engineering, and specifically relates to a strain of Bacillus subtilis and its applications. Background Art

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

[0003] With the improvement of people's living standards and the enhancement of health awareness, the demand for high-quality, safe and healthy livestock and poultry products is increasing day by day. Laying hens and breeding chickens, as important livestock and poultry breeds, their production performance and health status directly affect the quality of eggs and chicken meat. However, in the modern intensive farming mode, laying hens and breeding chickens face various stress factors, such as high-density breeding, environmental changes, pathogenic microorganism invasion, etc. These factors will lead to an increase in the level of oxidative stress in the body, a large accumulation of free radicals, which will damage cells and tissues, accelerate the aging of the body, reduce production performance and immunity, and ultimately affect the breeding efficiency.

[0004] In recent years, probiotics, as a kind of green and safe feed additive, have been more and more widely used in livestock and poultry breeding. Bacillus subtilis, as a common probiotic, has characteristics such as high temperature resistance, acid resistance, and bile salt resistance, and can colonize in the intestinal tract of livestock and poultry and play a probiotic role. Research shows that Bacillus subtilis can improve the health of livestock and poultry and enhance production performance through various ways such as regulating the balance of intestinal flora, enhancing the body's immunity, and improving antioxidant capacity. However, different strains of Bacillus subtilis have different biological characteristics, and their probiotic effects also vary. Summary of the Invention

[0005] The present invention relates to a strain of Bacillus subtilis and its application in improving the production performance of poultry, especially for improving the production performance, reproductive performance and feed utilization efficiency of old laying hens and breeding chickens. The present invention further provides a feed additive containing the Bacillus subtilis and its usage method.

[0006] Specifically, the present invention provides the following technical solutions.

[0007] In the first aspect of the present invention, a strain of Bacillus subtilis is provided, which is named Bacillus subtilis BLDT-9 and was deposited on February 17, 2025 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, and its deposit number is CCTCC NO: M 2025228.

[0008] Tests have shown that this strain has good colonization ability in the intestine, can effectively regulate the intestinal flora structure, increase the number of beneficial bacteria such as Lactobacillus, and reduce the number of conditional pathogenic bacteria such as Escherichia coli. At the same time, this strain has the characteristic of enhancing the body's antioxidant capacity, manifested as being able to increase the total antioxidant capacity and the activity of total superoxide dismutase, and reduce the content of malondialdehyde. In addition, this strain can also improve the reproductive performance of poultry by regulating endocrine function and increasing the levels of hormones such as serum progesterone. These biological characteristics make it show significant effects in improving egg quality, enhancing production performance, and strengthening stress resistance, and it is a high-quality strain resource with important application value.

[0009] In a second aspect of the present invention, there is provided the use of the Bacillus subtilis described in the first aspect in the preparation of a feed additive for improving poultry performance.

[0010] In some embodiments of the present invention, the feed additive is added to feed (such as a basal diet) in an amount of 1.0×10 5 to 1.0×10 7 CFU / g of Bacillus subtilis.

[0011] In some embodiments of the present invention, the poultry are laying hens or breeding hens. Among them, the breeding hens include but are not limited to egg breeding hens (also known as egg-type breeding hens) and meat breeding hens (also known as meat-type breeding hens).

[0012] In some embodiments of the present invention, the poultry are laying hens 380 days old and above and / or breeding hens 39 weeks old and above, especially laying hens 420 days old and above and / or breeding hens from 39 weeks to 50 weeks old.

[0013] In some embodiments of the present invention, the improvement of poultry performance includes one or more of the following:

[0014] (1) Improving production performance;

[0015] (2) Improving the freshness preservation effect of eggs;

[0016] (3) Improving the reproductive performance of breeding poultry;

[0017] (4) Maintaining egg-laying performance in old age;

[0018] (5) Improving stress resistance;

[0019] (6) Improving feed utilization efficiency.

[0020] Specifically, in some embodiments, the present invention provides the application of the Bacillus subtilis in improving the production performance of poultry. The improvement of the production performance of poultry includes one or more of the following: (1) reducing the rate of unqualified eggs; (2) improving the eggshell quality; (3) increasing the egg production rate; (4) increasing the qualification rate of breeding eggs.

[0021] Experimental studies have shown that adding this strain can significantly improve the production performance indicators of poultry: by regulating the intestinal microecological environment, improving the absorption and utilization efficiency of nutrients in the body, reducing the rate of unqualified eggs, which decreases from the initial 4.62‰ to 0.58‰, with a decrease rate of more than 80%; at the same time, it improves the eggshell quality, making the eggshell surface smoother and more uniform, and reducing the production of unqualified products such as sandy eggshells. In terms of egg production performance, it can increase the egg production rate by 1 - 5%, and still maintain a relatively high level in the later stage of production; the improvement effect on the quality of breeding eggs is also very significant, with the qualification rate of breeding eggs increasing by 1 - 3%. These improvement effects are significant and persistent, providing a strong guarantee for improving the breeding efficiency.

[0022] In some embodiments, the present invention provides the application of the Bacillus subtilis in improving the fresh-keeping effect of eggs. The addition of this strain significantly extends the shelf life of eggs, specifically manifested as: the experimental group added with this strain can still maintain a 100% AA-grade egg rate even after 5 days of refrigeration, higher than the 80% AA-grade ratio of the control group; by improving the uniformity of the eggshell thickness and the texture of the eggshell surface, it effectively improves the appearance quality of eggs; at the same time, it increases the Haugh unit and maintains a steadily increasing trend. This improvement in the fresh-keeping effect not only extends the shelf life of eggs, but also improves the market competitiveness of products.

[0023] In some embodiments, the present invention provides the application of the Bacillus subtilis in improving the reproductive performance of breeding poultry. The improvement of the reproductive performance of breeding poultry includes one or more of the following: (1) increasing the fertilization rate of breeding eggs; (2) increasing the hatching rate of breeding eggs; (3) reducing the rate of dead embryos; (4) increasing the serum progesterone level.

[0024] Research has shown that this strain can improve the reproductive performance of breeding poultry through multiple pathways: increasing the fertilization rate of breeding eggs by 1 - 3%, making the reproductive performance more stable; increasing the hatching rate of breeding eggs by 1 - 2%, increasing the utilization efficiency of breeding eggs; reducing the rate of dead embryos by 1 - 2%, reducing losses during the reproductive process. More importantly, by increasing the serum progesterone level by 5 - 12% and regulating the levels of other reproductive hormones, it improves the reproductive function of breeding poultry from the endocrine level. The combined effect of these effects significantly improves the reproductive efficiency of breeding poultry.

[0025] In some embodiments, the present invention provides the application of the Bacillus subtilis in maintaining laying performance during the advanced age period. The maintenance of laying performance during the advanced age period includes one or more of the following: (1) increasing the laying rate of laying hens at an advanced age (especially laying hens over 380 days old); (2) increasing the laying rate of breeding hens at an advanced age (especially breeding hens at 39 weeks old and above); (3) improving the feed-to-egg ratio. This application is particularly targeted at the critical period of poultry production: laying hens over 380 days old have completed their first peak laying period and usually face problems such as a decline in laying rate, a decrease in eggshell quality, and a reduction in feed conversion efficiency; breeding hens at 39 weeks old and above are in a stage of gradually declining production performance, and common challenges include a decrease in laying rate, fluctuations in the quality of breeding eggs, and a decline in reproductive performance. Experiments have shown that adding the Bacillus subtilis of the present invention at this stage can significantly improve the production status of poultry during the advanced age period: the laying rate of laying hens over 380 days old (especially 420 days old) can still be maintained above 89%, and the laying rate of breeding hens over 39 weeks old can be maintained above 80%; at the same time, it significantly improves the feed-to-egg ratio and reduces it by 2 - 5%. This continuous improvement effect has important practical significance for extending the efficient production period of poultry.

[0026] In some embodiments, the present invention provides the application of the Bacillus subtilis in improving stress resistance. The improvement of stress resistance includes one or more of the following: (1) reducing the mortality rate during the high-temperature season; (2) increasing the total antioxidant capacity of the body; (3) enhancing the activity of total superoxide dismutase; (4) reducing the content of malondialdehyde.

[0027] Research has shown that this strain can significantly enhance the stress resistance of poultry: during the high-temperature and rainy season in summer, the mortality rate of the experimental group remains below 1‰; at the same time, it significantly improves the antioxidant indicators of the body, including increasing the total antioxidant capacity by 15 - 35%, enhancing the activity of total superoxide dismutase by 25 - 45%, and reducing the content of malondialdehyde by 30 - 45%. Through the improvement of these indicators, the survival ability and the stability of production performance of poultry under stress conditions are significantly enhanced.

[0028] In some embodiments, the present invention provides the application of the Bacillus subtilis in improving feed utilization efficiency. The improvement of feed utilization efficiency includes one or more of the following: (1) reducing the crude protein content in feces; (2) stabilizing the moisture content of feces.

[0029] This strain improves feed utilization efficiency through multiple pathways: significantly improving the intestinal flora structure, increasing the number of beneficial bacteria such as Lactobacillus, and reducing the number of conditional pathogenic bacteria such as Escherichia coli; at the same time, reducing the crude protein content in feces, indicating an increase in protein utilization rate; maintaining a stable moisture content of feces, reflecting the improvement of the intestinal environment. These changes together promote the absorption and utilization of nutrients and improve the feed conversion efficiency.

[0030] In the third aspect of the present invention, the present invention also provides a feed additive comprising the above-mentioned Bacillus subtilis. This feed additive is used to make the content of the Bacillus subtilis in the basal diet fed to poultry reach 1.0×10 5 to 1.0×10 7 CFU / g. This dosage range has been proven by experiments to be able to obtain better improvement effects. This additive has good stability, is convenient to use, is easy to be combined with various feeds, and is applicable to different types of poultry feed formulations.

[0031] In the fourth aspect of the present invention, the present invention provides a method for using the above feed additive. This method is simple to operate and includes adding the additive to the basal diet according to the specified dosage, and then feeding the diet added with this additive to poultry. This method of use is not only convenient for large-scale production implementation, but also can obtain stable improvement effects.

[0032] By implementing the above technical solutions, the present invention not only effectively solves a number of technical problems in poultry farming, but also all the effects have been fully verified by experiments, and has important practical application value and popularization significance.

[0033] Compared with the prior art, the Bacillus subtilis BLDT-9 and its applications provided by the present invention have significant technical effects.

[0034] In terms of improving egg quality, the present invention significantly reduces the proportion of unqualified eggs, from 4.62‰ to 0.58‰, with a reduction rate of more than 80%. At the same time, by improving the eggshell quality, the eggshell surface becomes smoother and more uniform, effectively solving quality problems such as sandy eggs. Especially in terms of the shelf life, the experimental group added with this strain can still maintain a 100% AA-grade egg rate after being refrigerated for 5 days, significantly extending the shelf life of eggs.

[0035] In terms of maintaining the production performance in the advanced age period, the present invention shows excellent effects. For laying hens over 380 days old (especially 420 days old), the egg production rate can still be maintained above 89%; the egg production rate of breeding hens over 39 weeks old can also be maintained above 80%. At the same time, the feed-to-egg ratio is reduced by 2-5%, significantly improving the feed utilization efficiency. These effects are of great significance for extending the high-efficiency production period of poultry.

[0036] In terms of improving the reproductive performance, the present invention can increase the fertilization rate of breeding eggs by 1-3%, and the reproductive performance of the population is more stable. By increasing the hatching rate of breeding eggs by 1-2%, reducing the dead embryo rate by 1-2%, and increasing the serum progesterone level by 5-12%, the reproductive performance of breeding poultry is comprehensively improved.

[0037] In terms of enhancing stress resistance, the present invention performs outstandingly. Even in the hot and rainy summer season, the mortality and culling rate of the experimental group remains below 1‰. At the same time, it significantly improves the antioxidant indexes of the body: the total antioxidant capacity increases by 15 - 35%, the activity of total superoxide dismutase increases by 25 - 45%, and the content of malondialdehyde decreases by 30 - 45%.

[0038] In terms of improving intestinal health, the present invention significantly increases the number of beneficial bacteria such as Lactobacillus and effectively reduces the number of conditional pathogenic bacteria such as Escherichia coli by regulating the intestinal flora structure. This improvement not only optimizes the intestinal microecological environment but also improves the absorption and utilization rate of nutrients.

[0039] In terms of practical application, the present invention has significant advantages. This strain has good stability and is suitable for large-scale production; the usage method is simple and easy to promote and apply; the addition amount range is clear (1.0×10 5 -1.0×10 7 CFU / g), the effect is continuously stable, and it has significant economic benefits.

[0040] The above technical effects are all supported by sufficient experimental data, indicating that the present invention not only solves multiple technical problems in the prior art but also has significant technical effects and economic benefits in practical applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] The specification drawings forming a part of this application are used to provide a further understanding of this application. The schematic embodiments of this application and their descriptions are used to explain this application and do not constitute an improper limitation of this application. Hereinafter, the implementation embodiments of this application will be described in detail with reference to the drawings, where:

[0042] Figure 1 It shows the comparison diagram of egg sampling of the experimental group and the control group on the 10th day of production performance test in Example 2, where the marked (black dots) are the control group. It can be seen that the eggshell color of the experimental group is uniform, and there are sand-like spots on the eggshell surface of the control group.

[0043] Figure 2 It shows the comparison diagram of egg sampling of the experimental group and the control group on the 24th day of production performance test in Example 2, where the marked (black dots) are the control group. It can be seen that the eggshell of the experimental group is smooth and uniform, and there are multiple sand-like spots on the eggshell of the control group.

[0044] Figure 3 It shows the comparison diagram of egg sampling of the experimental group and the control group on the 38th day of production performance test in Example 2, where the marked (black dots) are the control group. It can be seen that the eggshell of the experimental group is uniform and smooth, and there are multiple sand-like spots on the eggshell of the control group.

[0045] Figure 4Shows the effects of adding the Bacillus subtilis BLDT-9 in Example 2 on the egg quality-related indicators, including: egg weight, egg yolk color, egg yolk specific gravity, eggshell thickness, Haugh unit, and the proportion of grade AA eggs. The abscissa represents the number of experimental days, and the ordinate represents the values of the corresponding detection indicators.

[0046] Figure 5 Shows the effects of adding the Bacillus subtilis BLDT-9 in Example 2 on the biomass changes of four kinds of bacteria (Escherichia coli, Clostridium perfringens, Lactobacillus, and Bacillus). The X-axis represents time (0d, 10d, 24d, 38d), and the Y-axis represents the lg value of the number of bacteria (CFU / g). Detailed implementation manners

[0047] The present application is further described in combination with specific embodiments. It should be understood that these embodiments are only used to illustrate the present application and not to limit its scope. Experimental methods without specific conditions noted in the embodiments are generally carried out according to conventional conditions or conditions recommended by the manufacturer.

[0048] Unless otherwise defined, all professional terms and scientific terms used in the present application should have the meanings familiar to those skilled in the art. Unless otherwise specified, the reagents or raw materials used in the present application can be obtained through conventional channels and used according to the conventional methods in the art or the product instructions. In addition, any content similar or equivalent to the described methods or materials can be applied to the methods of the present application. The preferred embodiments and materials described in the present application are only for illustrative purposes.

[0049] Example 1 Study on the efficacy for mice

[0050] 1 Experimental materials

[0051] 1.1 Experimental strains: Bacillus subtilis BLDT-9, Bacillus subtilis TH5, and Bacillus subtilis 9-1, all preserved and provided by the strain resource library of the Scientific and Technological Innovation Center of Shandong Bailailai Biotechnology Co., Ltd.

[0052] 1.2 Experimental animals: Male Kunming mice.

[0053] 1.3 Culture media:

[0054] Bacillus liquid medium: Peptone 10g, yeast extract 5g, sodium chloride 5g, glucose 2g, distilled water 1000 mL, pH 7.0, sterilized at 121 °C for 30 min.

[0055] Bacillus solid medium: Peptone 10g, yeast extract 5g, sodium chloride 5g, glucose 2g, agar 15g, distilled water 1000 mL, pH 7.0, sterilized at 121 °C for 30 min.

[0056] 1.4 Preparation method of probiotic fermentation broth: The freeze-dried strains of Bacillus subtilis BLDT-9, Bacillus subtilis TH5 and Bacillus subtilis 9-1 were respectively inoculated on the solid slant medium of Bacillus, and cultured at 37 °C for 24 h. The cultured slants were respectively inoculated with 2 loops into 100 mL of Bacillus liquid medium under sterile conditions, and cultured at 37 °C and 180 rpm for 18 h to obtain the fermentation broth for standby.

[0057] 1.5 The kits for detecting the levels of serum IL-1β, IL-6, IL-10 and TNF-α were purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd., and the kits for detecting the levels of serum T-AOC, T-SOD and MDA, and the levels of T-AOC, T-SOD, MDA, GSH-PX and CAT in liver tissues were all purchased from Nanjing Jiancheng Bioengineering Research Institute Co., Ltd.

[0058] 2 Experimental design and grouping

[0059] 150 healthy male Kunming mice with a body weight of 20 ± 2 g were selected, pre-fed for 3 d, and randomly divided into 5 groups according to the principle of insignificant body weight difference, with 3 replicates in each group and 10 mice in each replicate. The experimental period was 12 weeks (a total of 87 days including 3 days of pre-feeding), and the experimental grouping and treatment are shown in Table 1.

[0060] Table 1 Experimental grouping and treatment

[0061]

[0062] 3 Detection indexes

[0063] 3.1 Body weight of mice: The mice in each group were weighed at 3 d, 10 d, 17 d, 24 d, 31 d, 38 d, 45 d, 52 d, 59 d, 66 d, 73 d, 80 d and 87 d at the beginning of the experiment to compare their body weight conditions.

[0064] 3.2 Organ index: At the end of the experiment, the mice in each group were weighed and sacrificed, and the weights of the heart, liver, kidney, spleen, lung and thymus of the mice were respectively measured, and the organ index was calculated. Organ index % = organ weight / mouse body weight × 100%.

[0065] 3.3 Effects on serum cytokine levels: At the end of the experiment, the mice in each experimental group were bled from the eyeballs, centrifuged at 4000 rpm for 10 min, the serum was collected, and the effects of the test strains on the levels of serum IL-1β, IL-6, IL-10 and TNF-α were detected using the kit;

[0066] 3.4 Antioxidant indices: At the end of the experiment, blood was collected from the eyes of the mice in each experimental group, centrifuged at 4000 rpm for 10 min, and the serum was collected. A kit was used to detect the effects of the test strains on the levels of serum T-AOC, T-SOD, and MDA. At the end of the experiment, the mice in each experimental group were sacrificed, and liver tissues were taken for grinding. A kit was used to detect the effects of the test strains on the levels of T-AOC, T-SOD, MDA, GSH-PX, and CAT in the liver tissues.

[0067] 4 Experimental results

[0068] 4.1 Effects on the body weight of mice

[0069] Table 2 Changes in the body weight of mice in each group (unit: g)

[0070]

[0071] Note: Different lowercase letters in the same row with superscripts indicate significant differences (P < 0.05), while the same or no letters with superscripts indicate no significant differences (P > 0.05).

[0072] At 87 d, the average weight gain of the mice in each group was about 24 - 27 g compared with the initial weight. The CK group had the heaviest weight, followed by the 9 - 1 group. There were no significant differences in the body weights of the mice in each group (P > 0.05).

[0073] 4.2 Effects on the organ indices

[0074] Table 3 Statistics of the organ indices of mice in each group (%)

[0075]

[0076] Note: Different lowercase letters in the same row with superscripts indicate significant differences (P < 0.05), while the same or no letters with superscripts indicate no significant differences (P > 0.05).

[0077] It can be seen from the statistical results of the organ indices in Table 3 that:

[0078] (1) The organ indices of the heart, liver, spleen, lung, kidney, and thymus of the mice in the model group were all decreased to varying degrees compared with the CK group. Among them, the organ indices of the liver, spleen, lung, kidney, and thymus in the model group were significantly different from those in the CK group (P < 0.05).

[0079] (2) The organ indices of the three probiotic groups were not significantly different from those of the CK group (P > 0.05).

[0080] (3) For the spleen index, there was a significant difference between the BLDT - 9 group and the model group (P < 0.05).

[0081] (4) The kidney index and thymus index showed significant differences between each probiotic group and the model group (P < 0.05).

[0082] Intramuscular injection of D-galactose in the nape of the neck of mice can lead to atrophy of various organs and a decrease in organ index. Administration of probiotics by gavage can alleviate this change to a certain extent and help maintain normal physiological functions. Considering comprehensively, the BLDT-9 group has the best effect.

[0083] 4.3 Effects on the number of colonic flora

[0084] Table 4 Statistics of the number of colonic flora (lg CFU / g)

[0085]

[0086]

[0087] Note: Different lowercase letters in the same column with superscripts indicate significant differences (P < 0.05), while the same or no superscript letters indicate no significant differences (P > 0.05).

[0088] It can be seen from the statistical results of the number of colonic flora in Table 4 that:

[0089] (1) The total number of bacteria and the number of Lactobacillus in the model group were significantly lower than those in the CK group (P < 0.05), and the number of Escherichia coli was significantly higher than that in the CK group (P < 0.05).

[0090] (2) The total number of bacteria, the number of Lactobacillus, and the ratio of Lactobacillus to Escherichia coli in the three probiotic groups were significantly higher than those in the model group (P < 0.05), and the number of Escherichia coli was significantly lower than that in the model group (P < 0.05).

[0091] (3) The total number of bacteria and the number of Lactobacillus in each probiotic group and the CK group were at the same order of magnitude.

[0092] Intramuscular injection of D-galactose in the nape of the neck of mice led to a decrease in the diversity of colonic flora, a decrease in the total number of bacteria and the number of Lactobacillus, and a relative increase in the number of Escherichia coli. Administration of probiotics by gavage can alleviate the changes in the colonic flora of mice caused by injection of D-galactose to a certain extent.

[0093] 4.4 Effects on serum antioxidant indexes

[0094] Table 5 Serum antioxidant indexes of mice in each group

[0095]

[0096] Note: Different lowercase letters in the same column with superscripts indicate significant differences (P < 0.05), while the same or no superscript letters indicate no significant differences (P > 0.05).

[0097] As can be seen from the results in Table 5:

[0098] (1) For T-AOC and T-SOD, both were lower in the model group than in the CK group, while MDA was higher in the model group than in the CK group, and the differences were all significant (P < 0.05), indicating successful model construction.

[0099] (2) For T-AOC and T-SOD in each probiotic group, both were significantly higher than those in the model group (P < 0.05), and MDA was significantly lower than that in the model group (P < 0.05). Each probiotic could play a certain antioxidant role, and among them, the BLDT-9 group had the best antioxidant effect.

[0100] Each probiotic group could significantly improve the body's oxidative stress indicators and help maintain normal physiological functions, and among them, Bacillus subtilis BLDT-9 had the most significant improvement effect.

[0101] 4.5 Effects on serum immune indicators

[0102] Table 6 Serum immune indicators of mice in each group

[0103]

[0104] Note: Different lowercase letters in the same column with superscripts indicate significant differences (P < 0.05), and the same or no letter superscripts indicate no significant differences (P > 0.05).

[0105] As can be seen from Table 6:

[0106] (1) In the model group, IL-1β, IL-6, and TNF-α were significantly higher than those in the CK group (P < 0.05), and IL-10 was significantly lower than that in the CK group (P < 0.05), indicating successful model construction.

[0107] (2) In each probiotic group, IL-1β, IL-6, and TNF-α were all lower than those in the model group, and IL-10 was higher than that in the model group. Each probiotic could improve the body's immune performance. Among them, in the BLDT-9 group, IL-1β, IL-6, and TNF-α were all significantly lower than those in the model group (P < 0.05), and IL-10 was significantly higher than that in the model group (P < 0.05).

[0108] Among the three tested strains, the BLDT-9 group could better improve the body's immune performance.

[0109] 4.6 Effects on liver antioxidant indicators

[0110] Table 7 Antioxidant indicators of liver tissues of mice in each group

[0111]

[0112]

[0113] Note: Different lowercase letters in the same column of shoulder marks indicate significant differences (P < 0.05), while the same or no letter shoulder marks indicate no significant differences (P > 0.05).

[0114] As can be seen from Table 7:

[0115] (1) T-AOC, T-SOD, GSH-PX and CAT in the model group were significantly lower than those in the CK group (P < 0.05), while MDA was significantly higher than that in the CK group (P < 0.05), indicating that the model was successfully constructed.

[0116] (2) T-AOC, T-SOD, GSH-PX and CAT in each probiotic group were higher than those in the model group, while MDA was lower than that in the model group, indicating that each probiotic had antioxidant effects.

[0117] (3) T-AOC and T-SOD in the BLDT-9 group were significantly higher than those in the model group (P < 0.05), while MDA was significantly lower than that in the model group (P < 0.05), indicating that it had good in vivo antioxidant effects.

[0118] Among the three tested strains, the Bacillus subtilis BLDT-9 group could better improve the antioxidant performance of the body.

[0119] 5 Summary

[0120] 5.1 Successfully constructed a D-galactose model in mice

[0121] By treating mice with subcutaneous injection of 200 mg / kg D-galactose in the neck and back every day, oxidative stress and immune function changes occurred in the body. The organ indices of the liver, spleen, lung, kidney and thymus in the model group were significantly lower than those in the CK group (P < 0.05). The contents of T-AOC, T-SOD, GSH-PX and CAT in the liver tissue of the model group were significantly lower than those in the CK group (P < 0.05), while the MDA content was significantly higher than that in the CK group (P < 0.05), indicating that subcutaneous injection of 200 mg / kg D-galactose in the neck and back every day significantly affected the physiological state of mice and successfully constructed a D-galactose model in mice.

[0122] 5.2 Strain BLDT-9 has a significant effect on improving the body function

[0123] Intraperitoneal injection of D-galactose in the neck and back can cause changes in various physiological indexes of mice, including decreased organ index, reduced diversity of colonic flora and increased oxidative stress level. Oral administration of probiotics can improve these indexes to a certain extent: maintaining normal organ index (alleviating organ atrophy caused by injection of D-galactose); improving the intestinal flora structure; enhancing the antioxidant ability of the body; enhancing immune function; combining the detection results of serum antioxidant indexes, immune indexes and liver antioxidant indexes, Bacillus subtilis BLDT-9 has the most significant improvement effect on improving the antioxidant performance and immune regulation ability of the body.

[0124] Example 2 Application of Bacillus subtilis BLDT-9 in Commercial Laying Hens

[0125] 1 Experimental Materials and Methods

[0126] 1.1 Experimental Animals

[0127] Laying hens at the late laying stage of 420 days old (breed: Dawu Jinfeng), two chicken houses, with about 14,000 chickens in each house. One house was used as the control group, fed with the basal diet (purchased from Tai'an Xinnong Fangzhou Feed Co., Ltd.). The other house was used as the experimental group. Bacillus subtilis BLDT-9 was fermented and then spray-dried to prepare bacterial powder with a viable count of about 100 billion / g, and added to the basal diet at a dose of 1.0×10 6 CFU / g by mixing with the feed. Other management methods were the same as the conventional feeding methods in the chicken farm. The experimental period was 38 days.

[0128] 1.2 Detection Indexes

[0129] 1.2.1 Production Performance

[0130] (1) Count the number of eggs laid every day and calculate the egg production rate.

[0131] (2) Record the number of unqualified eggs: soft eggs, broken eggs, sandy eggs, small eggs, double-yolk eggs, etc.

[0132] 1.2.2 Egg Quality

[0133] On the 10th, 24th, and 38th days of the experiment, 10 eggs were randomly collected from different positions in each chicken house, a total of 20 eggs:

[0134] (1) Analyze the Haugh unit and egg yolk color using an egg quality analyzer.

[0135] (2) Weigh the egg weight and yolk weight; measure the thickness of the three ends of the eggshell using a vernier caliper.

[0136] (3) Detect the antibody titers of Newcastle disease (ND) and different avian influenza virus subtypes (H5-13, H5-14, H7-4, H9) in eggs using the hemagglutination inhibition method. The hemagglutination inhibition antigens were all purchased from Harbin Veterinary Research Institute Weike Biology.

[0137] 1.2.3 Detection of Fecal Microorganisms and Protein Levels

[0138] On the 10th, 24th, and 38th days of the experiment, 1 fecal sample was collected from each house, each sample was 50 - 100 g, and immediately detected for the viable counts of Lactobacillus, Clostridium perfringens, Escherichia coli, etc. The remaining samples were air-dried, the moisture content was calculated, and air-dried samples were prepared for testing.

[0139] 2 Experimental Results

[0140] 2.1 Influence on the production performance of laying hens

[0141] Table 8 Laying performance

[0142]

[0143] As shown in Table 8:

[0144] (1) During the entire experimental period, the laying rate of the control group did not show obvious fluctuations and remained between 87.29% and 88.04%. The laying rate of the experimental group increased slightly during the experimental period, from 87.31% to 89.61%.

[0145] (2) The unqualified egg rate of the control group always maintained at the level of 4.62‰ - 4.68‰; while the unqualified egg rate of the experimental group showed a continuous downward trend.

[0146] (3) By the 10th day of the experiment, the unqualified eggs such as sandy eggs in the experimental group decreased significantly, dropping to half of the original, accounting for 2.34‰ of the laying quantity, and the phenomenon of brittle eggshells improved. The eggshell color of the experimental group was significantly more uniform than that of the control group (see Figure 1 ).

[0147] (4) By the 24th day of the experiment, the unqualified eggs such as sandy eggs in the experimental group decreased significantly, accounting for 1.17‰ of the laying quantity, and the eggshell quality improved. From the sampling situation, the eggshells of the experimental group were uniform and smooth, while the control group had more sandy spots (see Figure 2 ).

[0148] (5) On the 38th day of the experiment, the unqualified eggs such as sandy eggs in the experimental group continued to decrease, from the initial 4.62‰ to 0.58‰, and the eggshell quality improved. From the sampling situation, the eggshells of the experimental group were uniform and smooth, while the control group had more sandy spots( Figure 3 ).

[0149] (6) Mortality and culling rate: In the early stage of the experiment, during the high-temperature and rainy summer season, the mortality and culling rate of the experimental group under heat stress was less than 1‰.

[0150] 2.2 Influence on the egg quality of laying hens

[0151] Such as Figure 4As shown, during the test period, the egg weights of both groups showed an upward trend, and the increase in egg weight of the experimental group was greater. After 24 days, the egg weight of the experimental group was higher than that of the control group and could continuously maintain this advantageous level. The change trend of the egg yolk color in the experimental group was stable with a small fluctuation range, showing good stability; while the control group showed an obvious downward trend after reaching the peak at 24 days. This stable chromaticity performance is conducive to the consistency control of product quality. In terms of the yolk specific gravity, although both groups showed a normal downward trend in the late laying period, the experimental group showed an obvious rebound at 24 days, indicating that the addition of Bacillus subtilis BLDT-9 helps to maintain the yolk quality. The eggshell thickness is an important indicator to measure the egg quality. The eggshell thickness of the experimental group remained stable throughout the process with small fluctuations; while the control group showed a fluctuating trend of first decreasing and then increasing, and the measurement results were on the high side at 38 days due to uneven measurement values caused by the sanding of the eggshell. In terms of the key index of egg quality, the Haugh unit, the experimental group showed a continuous and stable upward trend. Especially in the test at 24 days, after 5 days of refrigeration, 100% of the eggs in the experimental group reached the AA grade standard, which was better than the 80% AA grade ratio of the control group, reflecting the positive effect of the present invention in extending the shelf life of eggs. This set of data fully proves that the use of Bacillus subtilis BLDT-9 can not only improve the egg quality, but more importantly, can maintain the stability of various quality indicators, which is of great significance for improving the market competitiveness of products.

[0152] 2.3 Influence on the antibody titer in eggs

[0153] Table 9 Mean antibody titer (log2)

[0154]

[0155] Table 9 shows the five antibody titer levels (ND, H7-4, H9, H5-13, H5-14) and their dispersion changes in the experimental group and the control group at different time points (0d, 10d, 24d, 38d). Judging from the antibody titer detection results, both the experimental group and the control group showed good immune response capabilities. During the entire experiment, the antibody titers of both groups remained at normal levels, among which the H9 antibody titer was the highest, basically remaining within the range of 10-11.3. In terms of flock uniformity, the experimental group showed significant advantages. Taking the ND antibody titer as an example, the dispersion of the experimental group was 0.82-0.5, significantly lower than the dispersion range of 2.17-1.6 in the control group. In terms of the H7-4 antibody titer, the dispersion of the experimental group was stable between 0.84-0.9, also superior to the level of 1.17-1.1 in the control group. In the later detection of the H9 antibody titer, the dispersion of the experimental group (0.7-0.5) was also significantly lower than that of the control group (1.1-0.8). There was little difference in the dispersion between H5-13 and H5-14 in the two groups. These data fully demonstrate that the use of Bacillus subtilis BLDT-9 can significantly improve the uniformity of antibody titers and reduce the degree of individual differences. This effect of improving the group immunity uniformity has important practical application value for large-scale breeding production.

[0156] 2.4 Effects on the number of bacteria in laying hens' feces

[0157] As Figure 5 shown, in terms of harmful bacteria, the experimental group supplemented with Bacillus subtilis BLDT-9 showed a significant inhibitory effect. The content of Escherichia coli remained at a low level in the experimental group and showed a stable downward trend, being significantly lower than that in the control group on the 38th day of the experiment. Similarly, Clostridium perfringens also showed a continuous downward trend in the experimental group and was significantly lower than that in the control group on the 24th and 38th days. The use of Bacillus subtilis BLDT-9 demonstrated a good role in promoting the proliferation of beneficial bacteria. The content of Lactobacillus was higher than that in the control group throughout the experiment. Adding Bacillus subtilis BLDT-9 to the feed of the experimental group reduced the excretion amount of Bacillus in the feces. To sum up, Bacillus subtilis BLDT-9 effectively inhibited the growth of harmful bacteria and promoted the proliferation of beneficial bacteria by regulating the intestinal flora structure, thus establishing a healthier intestinal microecological environment. This improvement effect is of great significance for improving the production performance of poultry.

[0158] 2.5 Effects on the moisture and protein levels in laying hens' feces

[0159] Table 10 Detection of moisture and crude protein content in the feces of two chicken houses

[0160]

[0161] Table 10 shows the data of fecal moisture and crude protein content. The changes in fecal moisture and crude protein content reflect the impact of Bacillus subtilis BLDT-9 on nutrient utilization. The following characteristics can be observed from the experimental data: In terms of fecal moisture content, the experimental group showed better stability. The moisture content of the experimental group was always maintained within the range of 76 - 80%, with a smaller fluctuation range. In contrast, the moisture content of the control group fluctuated greatly, decreasing from the initial 81.03% to between 70 - 74%. This result indicates that the addition of Bacillus subtilis BLDT-9 helps to maintain the stability of the intestinal environment. In terms of fecal crude protein content, the experimental group showed an obvious downward trend. It gradually decreased from 25.61% at the beginning of the experiment to 21.32% at the end of the experiment, with a decrease of 4.29 percentage points. While the crude protein content of the control group remained basically around 23%, with little change. The decrease in the crude protein content in feces indicates that the addition of Bacillus subtilis BLDT-9 helps to improve the digestion and utilization rate of dietary protein. To sum up, Bacillus subtilis BLDT-9 can not only maintain the stability of the intestinal environment, but more importantly, can improve the utilization efficiency of protein, which has important practical application value for improving feed conversion rate and reducing breeding costs.

[0162] Summary: The application experiment of Bacillus subtilis BLDT-9 of the present invention on commercial laying hens was carried out at the critical production stage of 420 days old. Generally speaking, laying hens start laying eggs from 140 - 150 days old, the peak period of the first laying cycle is from 300 - 380 days old, and 420 days old belongs to the late laying period, when the production capacity usually begins to decline. Laying hens in this stage face many challenges in production, such as problems with eggshell quality, a decline in laying rate, a reduction in feed conversion efficiency, an increase in unqualified eggs, etc. These problems not only increase the breeding difficulty, but also have a serious impact on production efficiency.

[0163] In such a challenging production stage, the application effects of Bacillus subtilis BLDT-9 on laying hens, especially laying hens at 420 days old in the late laying period, are mainly reflected in the following aspects:

[0164] First of all, significant improvements have been achieved in egg quality. The experimental results show that the proportion of unqualified eggs has been greatly reduced from the initial 4.62‰ to 0.58‰, with a reduction rate of more than 80%. At the same time, the eggshell quality has been significantly improved, and unqualified products such as sandy eggshells have significantly decreased. More importantly, in terms of the preservation performance of eggs, even after 5 days of refrigeration, the experimental group can still maintain a 100% AA-grade egg rate, significantly extending the fresh-keeping period of eggs.

[0165] Secondly, all production performance indicators have been optimized. The experimental group showed a stable upward trend in egg weight, the egg yolk quality remained stable, the eggshell thickness distribution was more uniform, and the Haugh unit showed a continuous upward trend. These improvement effects have special practical significance for laying hens in the late laying period.

[0166] In terms of immune function, the experimental group showed excellent performance. The uniformity of group immunity was improved, the dispersion of multiple antibody indicators was significantly reduced, and the stability of the group immune level was effectively maintained.

[0167] The intestinal health status has also been significantly improved. Bacillus subtilis BLDT-9 can effectively inhibit the growth of conditional pathogenic bacteria such as Escherichia coli, and at the same time promote the proliferation of beneficial bacteria such as Lactobacillus, showing good intestinal colonization ability, which helps to maintain a stable intestinal microecological environment.

[0168] In terms of feed utilization efficiency, the experimental group performed outstandingly. The fecal test results showed that the crude protein content was significantly reduced, indicating an increase in protein utilization rate; the stable fecal moisture content reflected the stability of the intestinal environment, all of which were beneficial to improving feed conversion efficiency.

[0169] It is particularly noteworthy that under the stress conditions of high temperature and heavy rain in summer, the mortality and culling rate of the experimental group remained below 1‰, and the fluctuations of various production indicators were small, showing excellent stress resistance ability.

[0170] These experimental results fully prove that the Bacillus subtilis BLDT-9 of the present invention has significant effects in improving the production performance of laying hens in the late laying period, especially outstanding in improving egg quality, enhancing immune function and improving intestinal health, providing an effective technical solution for solving the production problems in this critical period.

[0171] Example 3 Effects on the production performance of breeding hens and the hatching effect of breeding eggs

[0172] 1 Experimental materials and methods

[0173] Randomly select 360 39-week-old breeding hens, randomly divide them into 2 groups (control group and experimental group), with 4 replicates in each group and 45 chickens in each replicate, and conduct the experiment according to the random grouping arrangement. The control group is the group fed with the basal diet (the composition and nutritional level of the basal diet are shown in Table 11); the experimental group is the group fed with the basal diet + Bacillus subtilis BLDT-9. The Bacillus subtilis BLDT-9 was fermented and then spray-dried to prepare a bacterial powder, and the viable bacteria count was about 100 billion / g, and it was added to the basal diet at 1.0×10 6 CFU / g by mixing with the feed. Feed and water were provided ad libitum, and other management methods were the same as the conventional breeding methods in the chicken farm.

[0174] At 50 weeks of age, three chickens were randomly selected from each replicate, and blood was collected from the wing vein. After centrifugation at 3000 r / min for 10 min, the upper serum was aspirated, and serum antioxidant indexes were measured. The levels of estradiol (E2), progesterone (P4), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) in the serum were detected using a chicken serum ELISA kit, and all the kits were purchased from Nanjing Jiancheng Bioengineering Institute.

[0175] Table 11 Composition and nutrient levels of the basal diet

[0176]

[0177] Note: 1. The premix can provide per kilogram of diet: VA 12,500 IU, VB1 1 mg, VB2 8.5 mg, calcium pantothenate 50 mg, VB6 2 mg, VB12 0.01 mg, VD3 4,125 IU, VE 20 IU, VK3 30 mg, pantothenic acid 4.5 mg, niacin 20 mg, folic acid 1 mg, biotin 1 mg, choline 450 mg, copper 5 mg, iodine 0.5 mg, iron 40 mg, manganese 75 mg, selenium 0.2 mg, zinc 50 mg. 2. The crude protein and calcium in the nutrient levels are measured values, and the rest are calculated values.

[0178] 2 Experimental results

[0179] 2.1 Effects on the production performance of breeding chickens

[0180] Table 12 Effects on the production performance of breeding chickens at 39 - 50 weeks of age

[0181]

[0182] Note: In the same row, data with different superscript letters indicate significant differences (P < 0.05), while no superscript or the same letter indicates no significant differences (P > 0.05).

[0183] As can be seen from Table 12, in the stage of 39 - 44 weeks of age, the experimental group showed good improvement in production performance. Compared with the control group, the egg production of the experimental group increased significantly by 3.37% (P < 0.05), and the feed-to-egg ratio decreased significantly by 2.53% (P < 0.05). At the same time, the laying rate and the qualified rate of breeding eggs in the experimental group also increased, but the differences were not significant (P > 0.05). These data indicate that the addition of Bacillus subtilis BLDT-9 can improve feed utilization efficiency and egg production performance in the early stage.

[0184] At the age of 45 - 50 weeks, the effect of Bacillus subtilis BLDT - 9 was more significant. The laying rate of the experimental group was significantly increased by 4.80% compared with the control group ((P < 0.05), the feed - to - egg ratio was significantly decreased by 4.88% ((P < 0.05), and the qualified rate of breeding eggs was also significantly increased by 2.20% ((P < 0.05). This set of data shows that in the late stage of breeding chicken production, Bacillus subtilis BLDT - 9 can not only maintain high production performance, but also significantly improve feed utilization efficiency and the quality of breeding eggs.

[0185] The above experimental results show that Bacillus subtilis BLDT - 9 has a significant effect on improving the production performance of breeding chickens. Especially in the late stage of breeding chicken production, its role in increasing the laying rate, improving feed efficiency and enhancing the quality of breeding eggs is more obvious. This continuous improvement effect has important practical application value for extending the high - efficiency production period of breeding chickens.

[0186] 2.2 Influence on the hatching effect of breeding eggs

[0187] Table 13 Influence on the hatching effect of breeding eggs at 50 weeks of age

[0188]

[0189]

[0190] Note: Data in the same column with different superscript letters indicate significant differences (P < 0.05), while those without superscript or with the same letter indicate no significant differences (P > 0.05).

[0191] The results in Table 13 show that in terms of fertilization rate, the experimental group with the addition of Bacillus subtilis BLDT - 9 reached 97.35%, which was 1.58 percentage points higher than 95.77% of the control group. In terms of hatching rate, the experimental group reached 91.37%, which was 1.23 percentage points higher than 90.14% of the control group. Although this improvement effect was not significant (P > 0.05), it could still maintain a relatively high hatching rate level in the late stage of breeding chicken production, which had important production significance. In terms of the rate of dead embryos, the experimental group decreased to 2.03%, which was 1.63 percentage points lower than 3.66% of the control group. The decrease in the rate of dead embryos indicates that the present invention helps to improve the survival rate of embryo development. The above data show that the addition of Bacillus subtilis BLDT - 9 can comprehensively improve the hatching performance of breeding eggs, not only increasing the fertilization rate and hatching rate, but also reducing the rate of dead embryos, which has important practical application value for improving the reproductive efficiency of breeding chickens.

[0192] 2.3 Influence on the serum antioxidant indexes of breeding chickens

[0193] Table 14 Influence on the serum antioxidant indexes of breeding chickens at 50 weeks of age

[0194]

[0195] Note: Different superscript letters in the same column of data indicate significant differences (P<0.05), while no superscript or the same superscript letters indicate no significant differences (P>0.05).

[0196] As can be seen from Table 14, in terms of total antioxidant capacity (T-AOC), the experimental group supplemented with Bacillus subtilis BLDT-9 reached 17.24 U / mL, significantly higher than 13.76 U / mL of the control group (P<0.05), with an increase of 25.3%. In terms of total superoxide dismutase (T-SOD) level, the experimental group reached 106.26 U / mL, significantly higher than 78.39 U / mL of the control group (P<0.05), with an increase of 35.6%. This significant increase indicates that Bacillus subtilis BLDT-9 can effectively enhance the antioxidant capacity of the body. In terms of malondialdehyde (MDA) content, the experimental group decreased to 2.03 nmol / mL, significantly lower than 3.26 nmol / mL of the control group (P<0.05), with a decrease of 37.7%. The significant decrease in MDA content indicates that the present invention can effectively reduce the degree of oxidative damage to the body. The above data show that the addition of Bacillus subtilis BLDT-9 can significantly improve the antioxidant status of breeding hens, which is manifested not only in the increase of antioxidant enzyme activity but also in the reduction of the degree of oxidative damage. This comprehensive effect is of great significance for maintaining the health status and production performance of breeding hens.

[0197] 2.4 Effects on reproductive hormones in the serum of breeding hens

[0198] Table 15 Effects on reproductive hormones in the serum of 50-week-old breeding hens

[0199]

[0200] Note: Different superscript letters in the same column of data indicate significant differences (P<0.05), while no superscript or the same superscript letters indicate no significant differences (P>0.05).

[0201] As can be seen from Table 15, in terms of progesterone (P4) level, the experimental group supplemented with Bacillus subtilis BLDT-9 reached 0.208 ng / mL, significantly higher than 0.192 ng / mL of the control group ((P<0.05), with an increase of 8.3%. The levels of estradiol (E2), follicle-stimulating hormone (FSH), and luteinizing hormone (LH) in the experimental group showed an upward trend compared with the control group, but the differences were not significant (P>0.05). The above data show that the addition of Bacillus subtilis BLDT-9 can significantly increase the progesterone level of breeding hens and play a positive role in maintaining the levels of other reproductive hormones.

[0202] Summary: Bacillus subtilis BLDT-9 of the present invention shows significant effects in improving the production performance of breeding chickens. The test results show that by 45 - 50 weeks of age, the laying rate of the experimental group is significantly increased by 4.80%, the feed - to - egg ratio is significantly decreased by 4.88%, and the qualified rate of breeding eggs is significantly increased by 2.20%. These improvement effects are more significant in the later stage, demonstrating a continuous improvement effect.

[0203] In terms of reproductive performance, the addition of Bacillus subtilis BLDT-9 increases the fertilization rate of breeding chickens by 1.58 percentage points, and the population reproductive performance is more stable. At the same time, the hatching rate is increased by 1.23 percentage points, and the rate of dead embryos is decreased by 1.63 percentage points, overall improving the hatching effect of breeding eggs.

[0204] The improvement of body functions is particularly obvious. The total antioxidant capacity of the experimental group is increased by 25.3%, the activity of total superoxide dismutase is increased by 35.6%, and the content of malondialdehyde is decreased by 37.7%. The significant improvement of these indicators shows that the present invention can effectively enhance the body functions of breeding chickens.

[0205] In terms of endocrine regulation, the progesterone level of the experimental group is significantly increased by 8.3%. At the same time, the levels of estradiol, follicle - stimulating hormone, and luteinizing hormone all show an upward trend, indicating that the present invention helps to maintain the reproductive endocrine function of breeding chickens.

[0206] The above - mentioned test results show that Bacillus subtilis BLDT-9 of the present invention can effectively solve the main problems faced in the later stage of breeding chicken production. Especially in improving production performance, increasing reproductive efficiency, and enhancing body functions, it shows outstanding performance and has important practical application value for extending the high - efficiency production period of breeding chickens.

[0207] The above is only the preferred embodiment of the present application and is not used to limit the scope of the present application. Although the present application has been described in detail with reference to the above - mentioned embodiments, after reading this specification, those skilled in the art can still make various modifications to the technical solutions or equivalent replacements of some technical features. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present application shall be regarded as falling within the protection scope of the present application.

Claims

1. A strain of Bacillus subtilis named Bacillus subtilis BLDT-9 was deposited on February 17, 2025 at the China Center for Type Culture Collection, Wuhan University, Wuhan, China, with the deposit number CCTCC NO: M 2025228.

2. Use of the Bacillus subtilis according to claim 1 in the preparation of a feed additive for improving poultry performance.

3. The application according to claim 2, characterized in that, The feed additive is used to bring the content of the Bacillus subtilis in the basal diet fed to poultry to 1.0×10 5 to 1.0×10 7 CFU / g.

4. The application according to claim 2, characterized in that The poultry is laying hens or breeding hens.

5. The application according to claim 2, wherein The poultry is laying hens over 380 days old and / or breeding hens at 39 weeks old or above.

6. The application according to claim 2, characterized in that The improvement of the poultry performance includes one or more of the following: (1) Improving production performance; (2) Improving the fresh-keeping effect of eggs; (3) Improving the reproductive performance of breeding poultry; (4) Maintaining egg-laying performance in old age; (5) Improving stress resistance; (6) Improving feed utilization efficiency.

7. The application according to claim 6, characterized in that The improvement of the production performance includes one or several of the following: (1) Reducing the rate of unqualified eggs; (2) Improving eggshell quality; (3) Increasing the egg-laying rate; (4) Increasing the qualification rate of breeding eggs; Preferably, the improvement of the reproductive performance of breeding poultry includes one or several of the following: (1) Increasing the fertilization rate of breeding eggs; (2) Increasing the hatching rate of breeding eggs; (3) Reducing the rate of dead embryos; (4) Increasing the serum progesterone level; Preferably, the maintenance of egg-laying performance in old age includes one or several of the following: (1) Increasing the egg-laying rate of laying hens over 380 days old; (2) Increasing the egg-laying rate of breeding hens at 39 weeks old or above; (3) Improving the feed-to-egg ratio; Preferably, the improvement of stress resistance includes one or several of the following: (1) Reducing the death and culling rate in high-temperature seasons; (2) Increasing the total antioxidant capacity of the body; (3) Enhancing the activity of total superoxide dismutase; (4) Reducing the malondialdehyde content; Preferably, the improvement of feed utilization efficiency includes one or several of the following: (1) Reducing the crude protein content in feces; (2) Stabilizing the moisture content of feces; Preferably, the improvement of the intestinal flora structure includes one or several of the following: (1) Increasing the number of beneficial bacteria; (2) Reducing the number of conditional pathogenic bacteria; Preferably, the beneficial bacteria are Lactobacillus; the conditional pathogenic bacteria are Escherichia coli.

8. A feed additive, characterized in that, Containing the Bacillus subtilis according to claim 1.

9. A feed, characterized in that, Containing the Bacillus subtilis according to claim 1 or the feed additive according to claim 8.

10. The feed according to claim 9, characterized in that, The content of the Bacillus subtilis in the feed is 1.0×10 5 to 1.0×10 7 CFU / g.

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