Bacillus subtilis and application thereof

Abstract

The application provides a bacillus subtilis and application thereof, and relates to the field of microorganisms.The bacillus subtilis is named as bacillus subtilis BLDT-9, has been preserved in the China Center for Type Culture Collection located in Wuhan University in Wuhan City, China on February 17, 2025, and the preservation number is CCTCC NO: M2025228.The strain can significantly improve multiple performance indexes of poultry: reduce the unqualified egg rate by more than 50%, improve the egg production rate, and improve the feed-egg ratio; has the effect of prolonging the shelf life of egg products, keeps the AA grade egg product rate to be more than 90% after cold storage for 5 days; can improve the reproductive performance of breeding poultry, including improving the fertilization rate and improving the hatching rate; can maintain good production performance in the high age period of more than 380-day-old laying hens and more than 39-week-old breeding hens; significantly enhances the body's anti-stress ability, which is manifested as improving the antioxidant index, and the dead and removed rate in the high-temperature season is less than 1 ‰; at the same time, improves the feed utilization efficiency by improving the intestinal flora structure, and has a wide application prospect.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of microorganisms and fermentation engineering, and in particular to a Bacillus subtilis and its application. BACKGROUND

[0002] Any discussion of the prior art throughout the specification should in no way be considered as an acknowledgement that such prior art is widely known, or forms part of the common general knowledge in the field.

[0003] With the improvement of people's living standards and the enhancement of health consciousness, there is an increasing demand for high-quality, safe and healthy livestock and poultry products. As important livestock and poultry breeds, the production performance and health status of laying hens and breeding hens directly affect the quality of eggs and chicken meat. However, under the modern intensive farming mode, laying hens and breeding hens are faced with various stress factors, such as high-density feeding, environmental changes, pathogenic microorganism invasion, etc. These factors can lead to an increase in the level of oxidative stress, a large accumulation of free radicals, and further damage to cells and tissues, accelerate the aging of the body, reduce production performance and immunity, and ultimately affect the farming benefit.

[0004] In recent years, probiotics as a green and safe feed additive have been increasingly widely used in livestock and poultry farming. Bacillus subtilis as a common probiotic has the characteristics of 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. Studies have shown that Bacillus subtilis can improve the health of livestock and poultry and improve production performance through various ways such as regulating intestinal flora balance, enhancing immunity, and improving antioxidant capacity. However, different Bacillus subtilis strains have different biological characteristics, and their probiotic effects also differ. SUMMARY

[0005] The present application relates to a Bacillus subtilis and its application in improving the production performance of poultry, especially the improvement of the production performance, reproductive performance, and feed utilization efficiency of old laying hens and breeding hens. The present application further provides a feed additive comprising the Bacillus subtilis and a method of using the same.

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

[0007] In the first aspect of the present application, a Bacillus subtilis is provided, which is named Bacillus subtilis BLDT-9, and has been preserved in the China Center for Type Culture Collection located in Wuhan University, Wuhan, China on February 17, 2025, with the preservation number CCTCC NO: M 2025228.

[0008] The experiment proves that the strain has good colonization ability in the intestinal tract, 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, the strain has the characteristics of enhancing the body's antioxidant capacity, which is manifested as being able to improve the total antioxidant capacity and total superoxide dismutase activity, and reduce the content of malondialdehyde. In addition, the strain can also regulate endocrine function, increase the hormone level of serum progesterone, and improve the reproductive performance of poultry. These biological characteristics make it have significant effects in improving egg quality, improving production performance, and enhancing stress resistance, and it is a high-quality strain resource with important application value.

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

[0010] In some embodiments of the present application, wherein the feed additive is added to the feed (such as basal diet) in an amount of 1.0x10 5 to 1.0x10 7 CFU / g of bacillus subtilis.

[0011] In some embodiments of the present application, the poultry is a laying hen or a breeder. Wherein, the breeder includes but is not limited to an egg breeder (also known as an egg breeder) and a meat breeder (also known as a meat breeder).

[0012] In some embodiments of the present application, the poultry is a laying hen of 380 days of age and above and / or a breeder of 39 weeks of age and above, in particular a laying hen of 420 days of age and above and / or a breeder of 39 weeks to 50 weeks of age.

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

[0014] (1) improving production performance;

[0015] (2) improving the preservation effect of egg products;

[0016] (3) improving the reproductive performance of breeders;

[0017] (4) maintaining the egg-laying performance in the high age period;

[0018] (5) improving the stress resistance;

[0019] (6) improving the feed utilization efficiency.

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

[0021] Experimental studies have shown that the addition of the strain can significantly improve the production performance indicators of poultry: by regulating the intestinal microecological environment, improving the body's nutrient absorption and utilization efficiency, reducing the rate of substandard eggs, reducing the rate of substandard eggs from 4.62‰ to 0.58‰, and reducing the rate of substandard eggs by more than 80%; at the same time, the eggshell quality is improved, the eggshell surface is more smooth and uniform, and the production of substandard products such as sand eggs is reduced. In terms of egg production performance, it can increase the egg production rate by 1-5%, and still maintain a high level in the later production period; the improvement effect on the quality of hatching eggs is also very significant, and the rate of qualified hatching eggs is increased by 1-3%. These improvement effects have significance and persistence, and provide a strong guarantee for improving breeding efficiency.

[0022] In some embodiments, the present application provides the use of the Bacillus subtilis in improving the preservation effect of egg products. The addition of the strain significantly prolongs the shelf life of egg products, which is specifically manifested as: the test group with the addition of the strain can still maintain 100% of the AA grade egg product rate after 5 days of cold storage, which is higher than the 80% AA grade proportion of the control group; by improving the uniformity of eggshell thickness and the texture of eggshell surface, the appearance quality of egg products is effectively improved; at the same time, the Haugh unit is increased and maintains a steady upward trend. This improvement in preservation effect not only prolongs the shelf life of egg products, but also improves the market competitiveness of products.

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

[0024] Studies have shown that the strain can improve the reproductive performance of breeding poultry through multiple pathways: increasing the fertilization rate of hatching eggs by 1-3%, making the reproductive performance more stable; increasing the hatching rate of hatching eggs by 1-2%, increasing the utilization efficiency of hatching eggs; reducing the dead embryo rate by 1-2%, reducing the loss in the reproduction process. More importantly, by increasing the serum progesterone level by 5-12% and adjusting the levels of other reproductive hormones, the reproductive function of breeding poultry is improved from the endocrine level. The combined effect of these effects significantly improves the reproductive efficiency of breeding poultry.

[0025] In some embodiments, the present application provides the use of the Bacillus subtilis in maintaining high-age laying performance. The high-age laying performance includes one or more of the following: (1) increasing the laying rate of high-age hens (especially hens over 380 days old); (2) increasing the laying rate of high-age breeders (especially breeders over 39 weeks old); (3) improving the feed-to-egg ratio. This application is particularly aimed at the critical period of poultry production: hens over 380 days old have completed the first laying peak and usually face problems such as decreased laying rate, decreased eggshell quality, and decreased feed conversion efficiency; breeders over 39 weeks old are in the stage of gradually declining production performance, often facing challenges such as decreased laying rate, fluctuating egg quality, and decreased reproductive performance. Tests have shown that the addition of the Bacillus subtilis of the present application in this stage can significantly improve the production status of high-age poultry: the laying rate of hens over 380 days old (especially 420 days old) can still be maintained at over 89%, and the laying rate of breeders over 39 weeks old can be maintained at over 80%; at the same time, the feed-to-egg ratio is significantly improved, and is reduced by 2-5%. This sustained improvement has important practical significance for extending the high-efficiency production period of poultry.

[0026] In some embodiments, the present application provides the use 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 in high-temperature seasons; (2) increasing the total antioxidant capacity of the body; (3) increasing the total superoxide dismutase activity; (4) reducing the content of malondialdehyde.

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

[0028] In some embodiments, the present application provides the use 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 water content in feces.

[0029] The strain improves feed utilization efficiency through multiple pathways: significantly improving the intestinal flora structure, increasing the number of beneficial bacteria such as lactobacilli, and reducing the number of conditional pathogenic bacteria such as escherichia coli; at the same time, reducing the crude protein content in feces, indicating that the protein utilization rate is improved; maintaining stable fecal water content, reflecting the improvement of intestinal environment. These changes collectively promote the absorption and utilization of nutrients, and improve the feed conversion efficiency.

[0030] In a third aspect, the present invention also provides a feed additive comprising the aforementioned Bacillus subtilis. This feed additive is used to ensure that the content of Bacillus subtilis in the basal diet of poultry reaches 1.0 × 10⁻⁶. 5 Up to 1.0×10 7 The dosage range of CFU / g has been proven in trials to achieve optimal improvement. This additive exhibits good stability, is easy to use, readily integrates with various feeds, and is suitable for different types of poultry feed formulations.

[0031] In a fourth aspect, the present invention provides a method for using the above-mentioned feed additive. This method is simple to operate, comprising adding the additive to a basal diet at a prescribed dosage, and then feeding the diet containing the additive to poultry. This method not only facilitates large-scale production but also achieves stable improvement results.

[0032] Through the implementation of the above technical solutions, this invention not only effectively solves a number of technical problems in poultry farming, but also fully verifies the effects of each technology, and has important practical application value and promotion significance.

[0033] Compared with existing technologies, the Bacillus subtilis BLDT-9 and its application provided by this invention have significant technical effects.

[0034] In terms of improving egg quality, this invention significantly reduced the proportion of substandard eggs from 4.62‰ to 0.58‰, a reduction of over 80%. Simultaneously, by improving eggshell quality, making the eggshell surface smoother and more uniform, it effectively solved quality problems such as rough-shelled eggs. Particularly in terms of shelf life, the experimental group with this strain maintained a 100% AA-grade egg rate after 5 days of refrigeration, significantly extending the shelf life of the eggs.

[0035] This invention demonstrates excellent performance in maintaining productivity during advanced stages of poultry production. For laying hens over 380 days old (especially 420 days old), the egg production rate remains above 89%; for breeder hens over 39 weeks old, the egg production rate also remains above 80%. Simultaneously, the feed conversion ratio decreases by 2-5%, significantly improving feed utilization efficiency. These effects are of great significance for extending the high-efficiency production period of poultry.

[0036] Regarding improving reproductive performance, this invention can increase the fertilization rate of hatching eggs by 1-3% and make the reproductive performance of the population more stable. By increasing the hatching rate of hatching eggs by 1-2%, reducing the embryonic mortality rate by 1-2%, and increasing serum progesterone levels by 5-12%, the reproductive performance of breeding poultry is comprehensively improved.

[0037] In terms of enhancing stress resistance, the application is outstanding. Even in the summer high temperature and rainy season, the mortality rate of the test group remains below 1 ‰. At the same time, the body's antioxidant indicators are significantly improved: total antioxidant capacity is increased by 15-35%, total superoxide dismutase activity is increased by 25-45%, and malondialdehyde content is reduced by 30-45%.

[0038] In terms of improving intestinal health, the application significantly increases the number of beneficial bacteria such as lactobacillus by regulating the intestinal flora structure, and effectively reduces the number of conditional pathogenic bacteria such as escherichia coli. 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 application has significant advantages. The strain has good stability and is suitable for large-scale production; the method is simple and easy to popularize and apply; the addition amount range is clear (1.0 x 10 5 -1.0 x 10 7 CFU / g), the effect is stable and continuous, and has significant economic benefits.

[0040] The above technical effects are fully supported by experimental data, indicating that the application not only solves many technical problems in the prior art, but also has significant technical effects and economic benefits in practical application. BRIEF DESCRIPTION OF DRAWINGS

[0041] The drawings accompanying the specification of this application are used to provide further understanding of the application, the illustrative embodiments of the application and their descriptions serve to explain the application, and do not constitute an improper limitation on the application. In the following, the embodiments of the application are described in detail in conjunction with the drawings, in which:

[0042] Figure 1 Figure 2 shows a comparison of egg sampling of the test group and the control group on the 10th day of the production performance test in Example 2, where the marked (black dots) are the control group, and the eggshell of the test group is uniform and smooth, while the eggshell of the control group has many sand points.

[0043] Figure 2 Figure 3 shows a comparison of egg sampling of the test group and the control group on the 24th day of the production performance test in Example 2, where the marked (black dots) are the control group, and the eggshell of the test group is uniform and smooth, while the eggshell of the control group has many sand points.

[0044] Figure 3 Figure 4 shows a comparison of egg sampling of the test group and the control group on the 38th day of the production performance test in Example 2, where the marked (black dots) are the control group, and the eggshell of the test group is uniform and smooth, while the eggshell of the control group has many sand points.

[0045] Figure 4The influence of adding Bacillus subtilis BLDT-9 in Example 2 on egg quality related indicators, including egg weight, egg yolk color, egg yolk specific gravity, eggshell thickness, Haugh unit, and AA grade proportion, is shown. The abscissa represents the number of test days, and the ordinate represents the numerical value of the corresponding detection index.

[0046] Figure 5 The influence of adding Bacillus subtilis BLDT-9 in Example 2 on the biomass changes of four bacterial groups (Escherichia coli, Clostridium perfringens, Lactobacillus, and Bacillus) is shown. The X-axis represents time (0d, 10d, 24d, 38d), and the Y-axis represents the lg value of the number of bacterial groups (CFU / g). DETAILED DESCRIPTION

[0047] The present application is further described in conjunction with specific examples. It should be understood that these examples are only used to illustrate the present application and not to limit its scope. Experimental methods not specified in the examples are generally carried out according to conventional conditions or manufacturer's recommended conditions.

[0048] Unless otherwise defined, all professional and scientific terms used in the present application 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 by conventional routes and used according to conventional methods or product instructions in the art. In addition, any similar or equivalent content 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 illustration.

[0049] Example 1 Study on efficacy in mice

[0050] 1 Test materials

[0051] 1.1 Test strains: Bacillus subtilis BLDT-9, Bacillus subtilis TH5, and Bacillus subtilis 9-1, all preserved and provided by the Strain Resource Library of the Innovation Center of Shandong Baolailailai Biological Engineering Co., Ltd.

[0052] 1.2 Test animals: male Kunming mice.

[0053] 1.3 Culture medium:

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

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

[0056] 1.4 Preparation of probiotic fermentation broth: Bacillus subtilis BLDT-9, Bacillus subtilis TH5 and Bacillus subtilis 9-1 lyophilized powder were inoculated on Bacillus subtilis solid slant medium, and cultured at 37°C for 24h. The cultured slant was inoculated with inoculation ring in 100mL Bacillus subtilis liquid medium under sterile conditions, and cultured at 37°C, 180rpm for 18h to prepare fermentation broth for standby.

[0057] 1.5 The kit for detecting serum IL-1β, IL-6, IL-10 and TNF-α levels was purchased from Shanghai Enzyme-linked Biotechnology Co., Ltd. The kit for detecting serum T-AOC, T-SOD and MDA levels, liver tissue T-AOC, T-SOD, MDA, GSH-PX and CAT levels was purchased from Nanjing Jianshen Biological Engineering Research Co., Ltd.

[0058] 2 Test design and grouping

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

[0060] Table 1 Test grouping and treatment

[0061]

[0062] 3 Test indexes

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

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

[0065] 3.3 Effect on serum cytokine level: At the end of the test, the mice in each test group were taken blood from the eyeball, centrifuged at 4000rpm for 10min, and the serum was collected. The effect of the test strain on serum IL-1β, IL-6, IL-10 and TNF-α levels was detected by kit;

[0066] 3.4 Anti-oxidation index: At the end of the experiment, the mice in each group were taken blood from the eyeball, centrifuged at 4000 rpm for 10 min, and the serum was collected. The effects of the test strain on the levels of T-AOC, T-SOD and MDA in the serum were detected by using a kit. At the end of the experiment, the mice in each group were killed, and the liver tissue was ground. The effects of the test strain on the levels of T-AOC, T-SOD, MDA, GSH-PX and CAT in the liver tissue were detected by using a kit.

[0067] 4 Results of the experiment

[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 column indicate significant differences (P<0.05), and the same or no letter indicates no significant difference (P>0.05).

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

[0073] 4.2 Effects on the organ index

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

[0075]

[0076] Note: Different lowercase letters in the same column indicate significant differences (P<0.05), and the same or no letter indicates no significant difference (P>0.05).

[0077] From the results of the organ index statistics in Table 3, it can be seen that:

[0078] (1) The organ indexes of the heart, liver, spleen, lung, kidney and thymus of the model group mice were reduced to different degrees compared with the CK group, and the organ indexes of the liver, spleen, lung, kidney and thymus of the model group were significantly different from those of the CK group (P<0.05).

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

[0080] (3) The spleen index of the BLDT-9 group was significantly different from that of the model group (P<0.05).

[0081] (4) Kidney index and thymus index, each probiotic group and model group were significantly different (P<0.05).

[0082] D-galactose neck injection treatment will cause mice atrophy of each organ, organ index decreased, gavage probiotic group can be in a certain extent to ease this change, help to maintain normal physiological function. Comprehensive consideration, the BLDT-9 group effect is optimal.

[0083] 4.3 Effect on the number of colon flora

[0084] Table 4: Colon flora number statistics (lg CFU / g)

[0085]

[0086]

[0087] Note: the same column with different lowercase letters indicate significant difference (P<0.05), the same or no letter shoulder mark indicates no significant difference (P>0.05).

[0088] From table 4, the colon flora number statistics results can be known:

[0089] (1) the model group of mice, the total number of bacteria, lactobacillus number were significantly lower than CK group (P<0.05), the number of escherichia coli was significantly higher than CK group (P<0.05).

[0090] (2) three probiotic groups, the total number of bacteria, lactobacillus number and lactobacillus / escherichia coli were significantly higher than model group (P<0.05), the number of escherichia coli was significantly lower than model group (P<0.05).

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

[0092] D-galactose injection treatment mice, lead to the diversity of colon flora decreased, the total number of bacteria and lactobacillus number decreased, the number of escherichia coli relatively increased, gavage probiotic group can be in a certain extent to ease the injection of D-galactose caused by changes in the colon flora of mice.

[0093] 4.4 Effect on serum antioxidant index

[0094] Table 5: serum antioxidant index of each group of mice

[0095]

[0096] Note: the same column with different lowercase letters indicate significant difference (P<0.05), the same or no letter shoulder mark indicates no significant difference (P>0.05).

[0097] From the results of Table 5:

[0098] (1) T-AOC and T-SOD of the model group were lower than those of the CK group, and MDA of the model group was higher than that of the CK group, and the differences were significant (P<0.05), and the model was successfully constructed;

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

[0100] Each probiotic group could significantly improve the oxidative stress index of the body and help maintain normal physiological functions, and the improvement effect of Bacillus subtilis BLDT-9 was the most significant.

[0101] 4.5 Effect on serum immune indexes

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

[0103]

[0104] Note: The same column with different lowercase letters indicates a significant difference (P<0.05), and the same or no letter indicates no significant difference (P>0.05).

[0105] From Table 6:

[0106] (1) IL-1β, IL-6 and TNF-α of the model group were significantly higher than those of the CK group (P<0.05), and IL-10 was significantly lower than that of the CK group (P<0.05), and the model was successfully constructed.

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

[0108] Among the three test strains, the BLDT-9 group could better improve the immune performance of the body.

[0109] 4.6 Effect on liver antioxidant indexes

[0110] Table 7 Antioxidant indexes of liver tissue of mice in each group

[0111]

[0112]

[0113] Note: The same column with different lowercase letters indicates significant difference (P<0.05), the same or no letter shoulder indicates no significant difference (P>0.05).

[0114] From Table 7, we can see that:

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

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

[0117] (3) The T-AOC and T-SOD of the BLDT-9 group were significantly higher than those of the model group (P<0.05), and the MDA was significantly lower than that of the model group (P<0.05), indicating that BLDT-9 had a better antioxidant effect in vivo.

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

[0119] 5 Summary

[0120] 5.1 Successful construction of D-galactose model in mice

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

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

[0123] D-galactose injection in the neck and back can cause changes in various physiological indicators of mice, including decreased organ index, decreased diversity of colon flora, and increased oxidative stress level. Gavage with probiotics can improve these indicators to some extent: maintain normal organ index (alleviate organ atrophy caused by D-galactose injection); improve intestinal flora structure; improve antioxidant capacity of the body; enhance immune function; combined with the detection results of serum antioxidant indicators, immune indicators and liver antioxidant indicators, 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 Test materials and methods

[0126] 1.1 Test animals

[0127] 420-day-old post-egg laying hens (variety: Dayu Jinfeng), two chicken houses, each with about 14,000 chickens. One was the control group, fed with basic daily feed (purchased from Taian Xinnongzhou Feed Co., Ltd.). The other was the test group, Bacillus subtilis BLDT-9 was fermented and prepared into a powder, with a viable bacterial count of about 100 billion / g. 1.0 x 10 6 CFU / g was added to the basic daily feed, and other management methods were consistent with the conventional feeding method of the chicken farm. The test period was 38 days.

[0128] 1.2 Test indicators

[0129] 1.2.1 Production performance

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

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

[0132] 1.2.2 Egg quality

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

[0134] (1) Use an egg quality analyzer to analyze Haugh units and egg yolk color;

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

[0136] (3) Use the hemagglutination inhibition method to detect Newcastle disease (ND) and different virus subtypes of avian influenza (H5-13, H5-14, H7-4, H9) antibody titers in chicken eggs. Hemagglutination inhibition antigens were purchased from Haishenyanweike Biological.

[0137] 1.2.3 Fecal microorganisms and protein level detection

[0138] On the 10th, 24th, and 38th days of the test, 1 kg of fecal samples was collected from each house, 50-100 g each, and immediately tested for viable bacterial counts of Lactobacillus, Clostridium perfringens, and Escherichia coli. The remaining samples were dried to calculate the moisture content and prepare dried samples for testing.

[0139] 2 Test results

[0140] 2.1 Effects on production performance of laying hens

[0141] Table 8 Egg production

[0142]

[0143] As shown in Table 8:

[0144] (1) During the whole experiment, the laying rate of the control group did not fluctuate significantly, remaining between 87.29% and 88.04%, while the laying rate of the experimental group increased slightly from 87.31% to 89.61%.

[0145] (2) The unqualified egg rate of the control group remained at a 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 sand-shell eggs in the experimental group were significantly reduced to half of the original amount, accounting for 2.34‰ of the number of eggs laid, and the eggshell fragility was 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 sand-shell eggs in the experimental group were significantly reduced, accounting for 1.17‰ of the number of eggs laid, and the eggshell quality was improved. From the sampling situation, the eggshell of the experimental group was smooth and uniform, while the control group had more sand-shell points (see Figure 2 ).

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

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

[0150] 2.2 Effects on egg quality of laying hens

[0151] As shown in Table 8: Figure 4As shown, the egg weight of both groups showed an upward trend during the test period, and the test group had a greater increase in egg weight. After 24 days, the egg weight of the test group was higher than that of the control group, and this advantage was maintained. The test group showed a stable trend in egg yolk color, with a small fluctuation range, indicating good stability. The control group showed a significant downward trend after reaching a peak at 24 days. This stable color performance is conducive to consistent quality control of the product. In terms of egg yolk specific gravity, although both groups showed a normal downward trend in the later laying period, the test group showed a significant rebound at 24 days, indicating that the addition of Bacillus subtilis BLDT-9 helps maintain egg yolk quality. Eggshell thickness is an important indicator of egg quality. The test group maintained stable eggshell thickness throughout the test period, with little fluctuation. The control group showed a fluctuating trend, first decreasing and then increasing, and the measured value was not uniform at 38 days due to the sandiness of the eggshell, resulting in a higher measured result. In terms of Haugh units, a key indicator of egg quality, the test group showed a consistent upward trend. In particular, after 5 days of cold storage, 100% of the test group's eggs reached AA grade, which was better than the 80% AA grade of the control group, demonstrating the positive effect of the present application on extending the shelf life of egg products. This data fully demonstrates that the use of Bacillus subtilis BLDT-9 not only improves egg quality, but more importantly, maintains the stability of various quality indicators, which is of great significance for improving the market competitiveness of products.

[0152] 2.3 Effect on antibody titer in chicken 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 of the test group and the control group at different periods (0d, 10d, 24d, 38d). From the antibody titer detection results, both the test group and the control group showed good immune response ability. During the whole test period, the antibody titers of the two groups remained at normal levels, among which the H9 antibody titer was the highest, basically maintaining at 10-11.3. In terms of population uniformity, the test group showed a significant advantage. Taking the ND antibody titer as an example, the dispersion of the test group was 0.82-0.5, which was significantly lower than the dispersion range of the control group 2.17-1.6. In terms of H7-4 antibody titer, the dispersion of the test group was stable at 0.84-0.9, which was also better than the level of the control group 1.17-1.1. In the later detection of H9 antibody titer, the dispersion of the test group (0.7-0.5) was also significantly lower than that of the control group (1.1-0.8). The dispersion of H5-13 and H5-14 was not much different between the two groups. These data fully proved that the use of Bacillus subtilis BLDT-9 could significantly improve the uniformity of antibody titer and reduce the difference between individuals. This effect of improving the uniformity of population immunity has important practical application value for large-scale breeding production.

[0156] 2.4 Effect on the number of bacterial flora in the feces of laying hens

[0157] As Figure 5 shown, in terms of harmful bacterial flora, the test group with the addition of Bacillus subtilis BLDT-9 showed a significant inhibitory effect. The content of Escherichia coli remained at a low level in the test group and showed a stable downward trend, which was significantly lower than that of the control group on the 38th day of the test. Similarly, Clostridium perfringens also showed a consistent downward trend in the test group, which was significantly lower than that of the control group on the 24th and 38th days. The use of Bacillus subtilis BLDT-9 showed a good effect of promoting the proliferation of beneficial bacterial flora. The content of Lactobacillus was higher than that of the control group during the whole test period. The addition of Bacillus subtilis BLDT-9 in the feed of the test group reduced the excretion of Bacillus in the feces. In summary, Bacillus subtilis BLDT-9 effectively inhibited the growth of harmful bacteria and promoted the proliferation of beneficial bacteria by regulating the structure of intestinal flora, thereby establishing a healthier intestinal microecological environment. This improvement is of great significance for improving the production performance of poultry.

[0158] 2.5 Effect on the moisture and protein levels in the feces of laying hens

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

[0160]

[0161] Table 10 shows the fecal moisture and crude protein content data, and the changes in fecal moisture and crude protein content reflect the influence of Bacillus subtilis BLDT-9 on nutrient utilization. From the test data, the following characteristics can be observed: in terms of fecal moisture content, the test group showed better stability. The moisture content of the test group remained in the range of 76-80%, with a small fluctuation range. In contrast, the moisture content of the control group fluctuated greatly, from the initial 81.03% to between 70-74%. This result shows 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 test group showed a clear downward trend. From 25.61% at the beginning of the test to 21.32% at the end of the test, the decrease was 4.29 percentage points. The crude protein content of the control group remained basically at about 23%, with little change. The decrease in crude protein content in feces indicates that the addition of Bacillus subtilis BLDT-9 helps to improve the digestibility and utilization of dietary protein. In summary, Bacillus subtilis BLDT-9 not only maintains the stability of the intestinal environment, but more importantly, it improves the utilization efficiency of protein, which has important practical application value for improving feed conversion rate and reducing breeding costs.

[0162] Summary: The application test of Bacillus subtilis BLDT-9 on commercial laying hens was carried out at 420 days of age, which is a key production stage. Generally speaking, laying hens start laying eggs at 140-150 days of age, and 300-380 days of age is the peak period of the first laying period, while 420 days of age belongs to the post-laying period, at which time the laying capacity usually starts to decline. Laying hens at this stage face many challenges, such as problems with eggshell quality, a decline in egg production rate, a decrease in feed conversion efficiency, an increase in substandard eggs, etc., which not only increase the difficulty of breeding, but also have a serious impact on production efficiency.

[0163] In such a challenging production stage, the application effect of Bacillus subtilis BLDT-9 on laying hens, especially on 420-day-old post-laying hens, mainly reflects the following aspects:

[0164] First, significant improvement in egg quality. The test results show that the proportion of substandard eggs decreased from the initial 4.62‰ to 0.58‰, a decrease of more than 80%. At the same time, the eggshell quality was significantly improved, and substandard products such as sand eggs were significantly reduced. More importantly, in terms of egg quality performance, even after 5 days of cold storage, the test group still maintained a 100% AA grade egg rate, significantly extending the egg preservation period.

[0165] Secondly, all production performance indicators are optimized. The test group shows a stable trend of egg weight increase, stable egg yolk quality, more uniform eggshell thickness distribution, and a continuous upward trend of Haugh units. These improvements have special practical significance for laying hens in the later laying period.

[0166] In terms of immune function, the test group shows excellent performance. It improves the uniformity of group immunity and significantly reduces the dispersion of multiple antibody indicators, effectively maintaining the stability of group immunity.

[0167] The intestinal health status is also significantly improved. Bacillus subtilis BLDT-9 can effectively inhibit the growth of conditional pathogenic bacteria such as Escherichia coli, while promoting the proliferation of beneficial bacteria such as Lactobacillus, showing good intestinal colonization ability and helping to maintain a stable intestinal microecological environment.

[0168] In terms of feed utilization efficiency, the test group performs outstandingly. The feces detection results show that the crude protein content is significantly reduced, indicating that the protein utilization rate has been improved; the stable feces moisture content reflects the stability of the intestinal environment, which is conducive 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 rate of the test group remains below 1‰, and the production indicators fluctuate less, showing excellent stress resistance.

[0170] These test results fully prove that Bacillus subtilis BLDT-9 of the present application has significant effects on improving the production performance of laying hens in the later laying period, especially in improving egg quality, enhancing immune function and improving intestinal health, providing an effective technical solution to solve 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 Test materials and methods

[0173] Randomly select 360 39-week-old laying hens, randomly divide them into 2 groups (control group and test group), 4 replicates in each group, 45 chickens in each replicate, and arrange the test according to the random grouping. The control group is fed with the basic diet (the composition and nutritional level of the basic diet are shown in Table 11); the test group is fed with the basic diet + Bacillus subtilis BLDT-9, Bacillus subtilis BLDT-9 is fermented and dried to prepare a bacteria powder, the number of live bacteria is about 100 billion / g, 1.0 x 10 6 CFU / g is added to the basic diet. Free access to feed and water, and other management methods are consistent with the conventional feeding method of the farm.

[0174] At 50 weeks of age, 3 chickens were randomly selected from each replicate, blood was collected from the wing vein, centrifuged at 3000 r / min for 10 min, and the upper serum was absorbed. The serum antioxidant indexes were determined. The levels of serum estradiol (E2), progesterone (P4), follicle stimulating hormone (FSH), and luteinizing hormone (LH) were detected by chicken serum ELISA kit, which was purchased from Nanjing Jiancheng Biological Engineering Institute.

[0175] Table 11 Basic diet composition and nutritional level

[0176]

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

[0178] 2 Test results

[0179] 2.1 Effect on the production performance of breeders

[0180] Table 12 Effect on the production performance of 39-50 week-old breeders

[0181]

[0182] Note: The same row data with different letters indicates significant difference (P<0.05), and no annotation or the same letter indicates no significant difference (P>0.05).

[0183] As shown in Table 12, during the 39-44 week-old stage, the test group showed good production performance improvement effect. Compared with the control group, the egg production of the test group was significantly increased by 3.37% (P<0.05), and the feed egg ratio was significantly reduced by 2.53% (P<0.05). At the same time, the egg production rate and the qualified rate of breeding eggs of the test group also increased, but the difference was not significant (P>0.05). These data show that the addition of Bacillus subtilis BLDT-9 can improve feed utilization efficiency and improve egg production performance in the early stage.

[0184] The effect of Bacillus subtilis BLDT-9 was more significant at the stage of 45-50 weeks of age. The laying rate of the test group was significantly increased by 4.80% (P<0.05) than the control group, the feed-egg ratio was significantly reduced by 4.88% (P<0.05), and the rate of qualified eggs was significantly increased by 2.20% (P<0.05). The data show that Bacillus subtilis BLDT-9 can not only maintain high production performance, but also significantly improve feed utilization efficiency and egg quality in the later stage of breeder production.

[0185] The above test results show that Bacillus subtilis BLDT-9 has a significant effect on improving the production performance of breeders, especially in the later stage of breeder production, which can significantly improve the laying rate, feed efficiency and egg quality. This sustained improvement effect has important practical application value for extending the high production period of breeders.

[0186] 2.2 Effect on hatching of eggs

[0187] Table 13 Effect on hatching of 50-week-old eggs

[0188]

[0189]

[0190] Note: The same column data with different letters indicates significant difference (P<0.05), and no letter indicates no significant difference (P>0.05).

[0191] Table 13 shows that the test group with Bacillus subtilis BLDT-9 added reached 97.35%, which was 1.58 percentage points higher than the control group of 95.77%. In terms of hatching rate, the test group reached 91.37%, which was 1.23 percentage points higher than the control group of 90.14%. Although the improvement effect was not significant (P>0.05), it still maintained a high level of hatching rate in the later stage of breeder production, which had important production significance. In terms of dead embryo rate, the test group decreased to 2.03%, which was 1.63 percentage points lower than the control group of 3.66%. The decrease in dead embryo rate indicates that the present application 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 eggs, not only increasing the fertilization rate and hatching rate, but also reducing the dead embryo rate, which has important practical application value for improving the reproductive efficiency of breeders.

[0192] 2.3 Effect on serum antioxidant indexes of breeders

[0193] Table 14 Effect on serum antioxidant indexes of 50-week-old breeders

[0194]

[0195] Note: The same column data shoulder note letters different significant difference (P <0.05), no marked or the same letter indicates no significant difference (P> 0.05).

[0196] From table 14, in terms of total antioxidant capacity (T-AOC), the test group added Bacillus subtilis BLDT-9 reached 17.24 U / mL, significantly higher than the control group of 13.76 U / mL (P <0.05), the increase of 25.3%. In terms of total superoxide dismutase (T-SOD) level, the test group reached 106.26 U / mL, significantly higher than the control group of 78.39 U / mL (P <0.05), the increase of 35.6%. This significant increase shows that Bacillus subtilis BLDT-9 can effectively enhance the antioxidant capacity of the body. In terms of malondialdehyde (MDA) content, the test group decreased to 2.03 nmol / mL, significantly lower than the control group of 3.26 nmol / mL (P <0.05), the decrease of 37.7%. The significant decrease of MDA content shows that the application can effectively reduce the degree of oxidative damage of 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 reflected not only in the improvement of antioxidant enzyme activity, but also in the reduction of oxidative damage. This comprehensive effect is of great significance to maintain the health status and production performance of breeding hens.

[0197] 2.4 Effect on serum reproductive hormones of breeding hens

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

[0199]

[0200] Note: The same column data shoulder note letters different significant difference (P <0.05), no marked or the same letter indicates no significant difference (P> 0.05).

[0201] From table 15, in terms of progesterone (P4) level, the test group added Bacillus subtilis BLDT-9 reached 0.208 ng / mL, significantly higher than the control group of 0.192 ng / mL ((P <0.05), the increase of 8.3%. The estradiol (E2), follicle stimulating hormone (FSH), luteinizing hormone (LH) levels of the test group showed an upward trend compared with the control group, and the difference was not significant (P> 0.05). The above data show that the addition of Bacillus subtilis BLDT-9 can significantly improve the progesterone level of breeding hens, and show a positive effect in maintaining the level of other reproductive hormones.

[0202] Summary: The Bacillus subtilis BLDT-9 of the present application shows significant effect in improving the production performance of breeding hens. The test results show that by 45-50 weeks of age, the egg laying rate of the test group is significantly increased by 4.80%, the feed-egg ratio is significantly reduced 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 period, reflecting a sustained improvement effect.

[0203] In terms of reproductive performance, the addition of Bacillus subtilis BLDT-9 increases the fertilization rate of breeding hens 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 dead embryo rate is reduced by 1.63 percentage points, which improves the hatching effect of breeding eggs as a whole.

[0204] The improvement of the body function is particularly obvious, the total antioxidant capacity of the test group is increased by 25.3%, the total superoxide dismutase activity is increased by 35.6%, and the malondialdehyde content is reduced by 37.7%. The significant improvement of these indicators shows that the present application can effectively enhance the body function of breeding hens.

[0205] In terms of endocrine regulation, the progesterone level of the test group is significantly increased by 8.3%, and the levels of estradiol, follicle stimulating hormone and luteinizing hormone are also showing an increasing trend, indicating that the present application helps to maintain the reproductive endocrine function of breeding hens.

[0206] The above test results show that the Bacillus subtilis BLDT-9 of the present application can effectively solve the main problems faced by breeding hens in the later production period. Especially in improving the production performance, increasing the reproductive efficiency, enhancing the body function, etc. It has important practical application value for prolonging the high-efficiency production period of breeding hens.

[0207] The above only describes the preferred embodiments of the present application and does not limit the scope of the present application. Although the present application has been described in detail with reference to the above embodiments, those skilled in the art can make various modifications or equivalent replacements of part of the technical features after reading the description. Any modification, equivalent replacement or improvement made within the spirit and principle of the present application shall be considered to fall within the protection scope of the present application.

Claims

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

2. The use of Bacillus subtilis as described in claim 1 in the preparation of feed additives for improving poultry performance; The improvement in poultry performance is comprised of one or more of the following: (1) Improve production performance; (2) Improves the preservation effect of eggs; (3) Improve the reproductive performance of breeding poultry; (4) Maintain egg production performance in older egg-laying adults; (5) Improve stress resistance; (6) Improve feed utilization efficiency; The improvement in production performance comprises one or more of the following: (1) Reduce the rate of substandard eggs; (2) Improve eggshell quality; (3) Increase egg production rate; (4) Improve the qualification rate of hatching eggs; The improvement in the reproductive performance of breeding poultry comprises one or more of the following: (1) Improve the fertilization rate of hatching eggs; (2) Improve the hatching rate of hatching eggs; (3) Reduce the rate of dead embryos; (4) Increase serum progesterone levels; The maintenance of egg production performance in older larvae consists of one or more of the following: (1) Increase the egg production rate of laying hens over 380 days old; (2) Improve the egg production rate of breeder chickens aged 39 weeks and above; (3) Improve the feed conversion ratio; The improvement in stress resistance is composed of one or more of the following: (1) Reduce the mortality rate of fish during the high-temperature season; (2) Improve the body's total antioxidant capacity; (3) Increase total superoxide dismutase activity; (4) Reduce malondialdehyde content; The improvement in feed utilization efficiency comprises one or more of the following: (1) Reduce the crude protein content in feces; (2) Stabilize fecal moisture content; The improvement in gut microbiota structure comprises one or more of the following: (1) Increase the number of beneficial bacteria; (2) Reduce the number of opportunistic pathogens; The beneficial bacteria are lactobacilli; the conditionally pathogenic bacteria are Escherichia coli.

3. The application according to claim 2, characterized in that, The feed additive is used to ensure that the content of Bacillus subtilis in the basal diet of poultry reaches 1.0 × 10⁻⁶. 5 Up to 1.0×10 7 CFU / g.

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

5. The application according to claim 2, characterized in that, The poultry referred to are laying hens aged 380 days or older and / or breeding hens aged 39 weeks or older.

6. A feed additive, characterized in that, It includes Bacillus subtilis as described in claim 1.

7. A feed, characterized in that, It contains Bacillus subtilis as described in claim 1 or a feed additive as described in claim 6.

8. The feed according to claim 7, characterized in that, The content of Bacillus subtilis in the feed is 1.0 × 10⁻⁶. 5 Up to 1.0×10 7 The amount of CFU / g.

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