Compound probiotics with anti-oxidation effect and application of compound probiotics in laying hen breeding

Abstract

The invention relates to a compound probiotic with an antioxidant effect and application of the compound probiotic in laying hen breeding. The compound probiotic comprises a combination of a bacillus coagulans BC66 strain with the preservation number of CGMCC (China General Microbiological Culture Collection Center) 21879, a lactobacillus plantarum LP194 strain with the preservation number of CGMCC 21766 and a pediococcus pentosaceus PP106 strain with the preservation number of GDMCC No: 63093. The three probiotics can promote each other after being compounded and combined, and have a synergistic effect in the aspects of promoting the growth and development of laying hens and improving the egg yield and disease resistance, so that the immune system regulation ability of the laying hens is further improved, the antioxidant level of organisms is improved, and the laying rate and the yield rate are improved. Therefore, the compound probiotics have a good prospect when being used for preparing a laying hen feed additive or laying hen feed.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotics and relates to a composite probiotic with antioxidant effect and application thereof in laying hen breeding. Background Art

[0002] The laying hen industry is a multi-generational livestock and poultry industry with vertical blood relations as the bond. Laying hens, or laying-type chickens, are chickens that are raised specifically for laying eggs without considering their meat quality. The breeding environment of laying hens and some laying hens themselves carry a variety of conditional pathogens. When the external environment changes and the immunity of the chickens is greatly reduced, the conditional pathogens they carry can easily cause the chickens to become ill. The use of antibiotics in laying hen breeding is strictly controlled. In order to prevent the presence of residual drugs in eggs, most antibiotics are strictly prohibited during laying. Therefore, various additives that replace antibiotics on the market, such as acidifiers, Chinese herbal enzyme preparations or microbial preparations, are currently used in laying hen breeding.

[0003] As a type of animal health product that replaces antibiotics, microecological preparations can metabolize and produce organic acids, antibacterial substances, and some microecological preparations can also produce digestive enzymes, vitamins and other substances to promote the digestion and absorption of nutrients by animals. At present, there are more than 30 kinds of probiotics that can be used to prepare microecological preparations, and different probiotics have different metabolites or growth characteristics.

[0004] The laying hen breeding cycle is more than 500 days. During the breeding process, the chickens are affected by factors such as changes in the breeding environment, invasion by pathogenic microorganisms and decreased immunity, which will lead to a decrease in the antioxidant capacity of the chickens and premature aging of the chickens, which in turn leads to a decline in egg production performance and egg quality. It is of great significance to provide a feed additive product containing probiotics that can effectively inhibit the invasion of intestinal pathogens on the intestines of laying hens, enhance immunity, delay the aging and elimination of laying hens, and improve the production performance of laying hens. Summary of the invention

[0005] In view of the deficiencies in the prior art, the object of the present invention is to provide a composite probiotic with antioxidant effect and its application in laying hen farming.

[0006] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a composite probiotic with antioxidant effect, wherein the composite probiotic comprises a combination of Bacillus coagulans BC66 strain with a preservation number of CGMCC 21879, Lactobacillus plantarum LP194 strain with a preservation number of CGMCC 21766, and Pediococcus pentosaceus PP106 strain with a preservation number of GDMCC No: 63093.

[0008] The present invention creatively develops a new probiotic compounding method and a new strategy for increasing egg production and income of laying hens, that is, Bacillus coagulans BC66 strain, Lactobacillus plantarum LP194 strain and Pediococcus pentosaceus PP106 strain are compounded and used together to promote each other, and the effects of promoting the growth and development of laying hens, increasing egg production and disease resistance are synergistic, specifically manifested in (1) improving the immune system regulation ability of laying hens; (2) improving the body's antioxidant level; (3) improving egg production rate and qualified egg rate. When the amount of bacteria is consistent, the compounding of the three bacteria is significantly improved in the effect of related aspects compared with the single strain intervention method. Therefore, the composite probiotics are used to prepare laying hen feed additives or laying hen feeds, which has a good prospect. At the same time, the above three bacteria are all probiotics, the product is highly safe, and it is not easy to produce resistance.

[0009] Preferably, the ratio of the viable counts of the Bacillus coagulans BC66 strain, the Lactobacillus plantarum LP194 strain and the Pediococcus pentosaceus PP106 strain is (1-10):(1-10):(1-10);

[0010] Among them, the above-mentioned "1-10" can be, for example, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, etc., and other specific point values ​​within the numerical range can be selected, which will not be described one by one here.

[0011] Based on the potential interaction between the three strains, the present invention also found that when the three strains are compounded and used in the above-mentioned specific ratio of viable bacteria count, the effect in improving the level of laying hen farming is more significant.

[0012] In a second aspect, the present invention provides a feed additive for laying hen farming, wherein the feed additive comprises the composite probiotics described in the first aspect.

[0013] Preferably, the total number of viable bacteria in the feed additive is not less than 1×10 8CFU / mL or 1×10 8 CFU / g, for example 1×10 8 CFU / mL (CFU / g), 2×10 8 CFU / mL (CFU / g), 5×10 8 CFU / mL (CFU / g), 8×10 8 CFU / mL (CFU / g), 1×10 9 CFU / mL (CFU / g), 5×10 9 CFU / mL (CFU / g), 1×10 10 CFU / mL (CFU / g), etc. Other specific point values ​​within this numerical range can be selected and will not be described in detail here.

[0014] Preferably, the dosage form of the feed additive includes solution, lyophilized powder, granules or tablets.

[0015] Preferably, the feed additive is in the form of a powder, which is prepared by a preparation method comprising the following steps:

[0016] The BC66 strain, LP194 strain and PP106 strain are inoculated into the culture medium respectively for activation and fermentation culture in sequence, and the obtained fermentation liquids are centrifuged respectively, mixed with a lyophilization protective agent for freeze drying, and the obtained BC66 bacterial powder, LP194 bacterial powder and PP106 bacterial powder are mixed according to a ratio to obtain the feed additive.

[0017] Preferably, the lyoprotectant is selected from any one or a combination of at least two of skim milk, gelatin, dextrin, gum arabic, dextran, sodium alginate, polyvinyl pyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol.

[0018] In a third aspect, the present invention provides a use of the composite probiotics according to the first aspect or the feed additive according to the second aspect in laying hen feed.

[0019] In a fourth aspect, the present invention provides a laying hen feed, comprising a basic feed and the feed additive according to the second aspect.

[0020] In a fifth aspect, the present invention provides a use of the composite probiotics according to the first aspect or the feed additive according to the second aspect in the preparation of a product for promoting the growth and development and disease resistance of laying hens.

[0021] Compared with the prior art, the present invention has the following beneficial effects:

[0022] The present invention creatively develops a new probiotic compounding method and a new strategy for increasing egg production and income of laying hens, that is, Bacillus coagulans BC66 strain, Lactobacillus plantarum LP194 strain and Pediococcus pentosaceus PP106 strain are compounded and used together to promote each other, and the effects of promoting the growth and development of laying hens, increasing egg production and disease resistance are synergistic, specifically manifested in (1) improving the immune system regulation ability of laying hens; (2) improving the body's antioxidant level; (3) improving egg production rate and qualified egg rate. When the amount of bacteria is consistent, the compounding of the three bacteria is significantly improved in the effect of related aspects compared with the single strain intervention method. Therefore, the composite probiotics are used to prepare laying hen feed additives or laying hen feeds, which has a good prospect. At the same time, the above three bacteria are all probiotics, the product is highly safe, and it is not easy to produce resistance.

[0023] The BC66 strain involved in the present invention is classified and named Bacillus coagulans, the preservation unit is the General Microbiology Center of China Microorganism Culture Collection Administration, the preservation number is CGMCC 21879, the preservation date is March 8, 2021, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0024] The LP194 strain involved in the present invention is classified and named Lactobacillus plantarum, the preservation unit is the General Microbiology Center of China Microorganism Culture Collection Administration, the preservation number is CGMCC 21766, the preservation date is January 29, 2021, and the preservation address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing.

[0025] The PP106 strain involved in the present invention is classified and named Pediococcus pentosaceus, the preservation unit is Guangdong Provincial Microbiological Culture Collection Center, the preservation number is GDMCC No: 63093, the preservation date is December 26, 2022, and the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou. DETAILED DESCRIPTION

[0026] In order to further explain the technical means and effects adopted by the present invention, the technical solution of the present invention is further described below in conjunction with the preferred embodiments of the present invention, but the present invention is not limited to the scope of the embodiments.

[0027] ① The BC66 strain involved in the following examples is classified as Bacillus coagulans, with a deposit number of CGMCC 21879;

[0028] ② The LP194 strain involved in the following examples is classified and named as Lactobacillus plantarum, and its deposit number is CGMCC 21766;

[0029] ③ The PP106 strain involved in the following examples is classified and named Pediococcus pentosaceus, and its deposit number is GDMCC No: 63093;

[0030] ④ATCC 7050 strain is Bacillus coagulans ATCC 7050 strain;

[0031] ⑤The CICC 21812 strain is the Lactobacillus plantarum CICC 21812 strain.

[0032] The culture medium involved in the following examples is as follows:

[0033] MRS medium (g / L): peptone 10g / L, beef extract 10g / L, glucose 15g / L, lactose 15g / L, yeast powder 5g / L, diammonium hydrogen citrate 2g / L, K2PO4·3H2O 2.6g / L, MgSO4·7H2O 0.1g / L, MnSO4 0.05g / L, Tween 80 1mL / L, cysteine ​​salt 0.5g / L.

[0034] Liquid thioglycollate medium (FTG) concentrations were: 29.25 g / L;

[0035] The concentration of tryptone-sulfite-cycloserine agar medium (TSC) is: 47 g / L.

[0036] The following method for preparing bacterial suspension and bacterial powder is as follows: after activating the strain, inoculate it into a culture medium for cultivation to obtain a culture solution; centrifuge the culture solution, resuspend the bacteria, mix the bacterial suspension according to the corresponding ratio of live bacterial count, or further add a protective agent for freeze-drying to obtain a freeze-dried bacterial powder product.

[0037] Example 1

[0038] To explore the inhibitory effect of compound probiotics on common pathogenic bacteria in laying hens

[0039] (1) Pathogen test liquid culture: 100 μL each of Salmonella, Escherichia coli and Staphylococcus aureus preserved in fresh glycerol tubes were selected and inoculated into 5 mL of sterilized LB liquid culture medium. After the inoculation, the test tubes were fixed in a shaker at 37°C, 200 rpm, and cultured for 18 h to obtain Salmonella suspension, Escherichia coli suspension and Staphylococcus aureus suspension, respectively.

[0040] Pick a fresh glycerol tube to preserve 100 μL of Clostridium perfringens, inoculate it into 5 mL of sterilized liquid thioglycollate medium, and after the inoculation, place the test tube in a shaker and fix it at 37° C., 200 rpm, and culture for 18 h to obtain a Clostridium perfringens bacterial suspension.

[0041] The four cultured pathogenic bacteria suspensions were diluted with blank LB liquid medium to make 10-fold increasing sample homogenates until the bacterial suspension was 10 7 CFU / mL, and obtain the pathogenic bacteria suspension test solution.

[0042] (2) Grouping: S1 group (BC66 bacterial suspension + LP194 bacterial suspension + PP106 bacterial suspension, the ratio of live bacteria count was 5:1:1), S2 group (BC66 bacterial suspension + LP194 bacterial suspension + PP106 bacterial suspension, the ratio of live bacteria count was 1:1:3), S3 group (BC66 bacterial suspension + LP194 bacterial suspension + PP106 bacterial suspension, the ratio of live bacteria count was 2:5:4), S4 group (BC66 bacterial suspension + LP194 bacterial suspension, the ratio of live bacteria count was 5:1), S5 group (LP194 bacterial suspension + PP106 bacterial suspension, the ratio of live bacteria count was 1:1), S6 group (BC66 bacterial suspension + PP106 bacterial suspension, the ratio of live bacteria count was 5:1), S7 group (ATCC 7050 bacterial suspension + LP194 bacterial suspension + PP106 bacterial suspension, the ratio of viable bacteria was 5:1:1), S8 group (BC66 bacterial suspension + CICC 21812 bacterial suspension + PP106 bacterial suspension, the ratio of viable bacteria was 5:1:1), the total viable bacteria count in each group of mixed bacterial suspension was 1×10 11 CFU / mL.

[0043] (3) 1 mL of each pathogen test solution prepared in (1) was taken and added to the surface of the MRS solid culture medium plate. The bacterial solution was evenly spread with an applicator. An Oxford cup was placed vertically on the surface of each culture medium and gently pressed to make it contact with the culture medium without any gaps. 150 μL of the mixed bacterial suspension of each group in (2) was added to the cup. At the same time, a blank control group was set up. 150 μL of sterile PBS solution was added to the cup. After culturing at 37°C for 12 h, the diameter of the inhibition zone in each group of culture dishes was measured. The results are shown in Table 1.

[0044] Table 1

[0045]

[0046] From the data in the table, we can see that:

[0047] (1) It can be seen from groups S1 to S3 that the composite probiotics provided by the present invention, including Bacillus coagulans BC66 strain, Lactobacillus plantarum LP194 strain and Pediococcus pentosaceus PP106 strain, have certain antibacterial properties against four common pathogens in laying hen farming, namely Escherichia coli, Salmonella, Staphylococcus aureus and Clostridium perfringens, and effectively improve the immune level of laying hens.

[0048] (2) By comparing the S1 group with the S4 to S8 groups, it can be seen that the present invention can effectively improve the immune level of laying hens, enhance resistance to common pathogens, and promote the healthy level of laying hens by combining the three strains of Bacillus coagulans BC66, Lactobacillus plantarum LP194 and Pediococcus pentosaceus PP106. When any of the three strains is missing in the S4 to S6 groups, and the ATCC 7050 strain and CICC 21812 strain are used to replace the corresponding BC66 and LP194 strains in the S7 to S8 groups, the inhibitory effect of the mixed bacterial suspension in the corresponding groups on the four common pathogens of laying hens is reduced compared with the S1 group, indicating that the three bacteria of Bacillus coagulans BC66, Lactobacillus plantarum LP194 and Pediococcus pentosaceus PP106 have a synergistic effect in improving the disease resistance of laying hens and improving the quality of breeding.

[0049] Example 2

[0050] (1) Test samples: The grouping was the same as that in Example 1, except that the total number of viable bacteria in the mixed bacterial suspension in each group was 1×10 8 CFU / mL.

[0051] (2) Experimental animals and intervention methods

[0052] One-day-old newborn Jianghan native chicks were randomly divided into 10 groups, with 16 chickens in each group, and divided into S1-S8 groups, immune control group and blank control group. The chicks in each group had free access to food and water, and were fed with conventional commercially available egg chick feed every day. The chicks in S1-S8 groups were gavaged with the corresponding group mixed bacterial suspension once a day from 1 to 7 days of age, and the gavage dose was 2 mL each time. The immune control group and the blank control group were gavaged with an equal dose of normal saline every day. On the 7th day of gavage, one dose of Newcastle disease (La Sota strain) attenuated virus vaccine was eye-dotted into the chicks in S1-S8 groups and the immune control group.

[0053] (3) Determination of immune organ index

[0054] When the chicks were 28 days old, 5 chickens were randomly selected from each group and weighed. After being killed, the spleen, thymus and bursa of Fabricius were taken to determine the immune organ index. The results are shown in Table 2.

[0055] Table 2

[0056]

[0057]

[0058] It can be seen from the data in the table that the combined use of Bacillus coagulans BC66, Lactobacillus plantarum LP194 and Pediococcus pentosaceus PP106 in the present invention can effectively improve the organ indexes of the spleen, thymus and bursa of Fabricius. It can be seen that the combination of the three promotes the immune development of these organs, slows down the atrophy rate of the bursa of Fabricius, can effectively improve the immune level of laying hens, delay the culling years of laying hens, and reduce the mortality rate.

[0059] (4) Determination of cytokine and serum antibody levels

[0060] When the chicks were 28 days old, 2 mL of blood was collected from the hearts of 5 chickens in each group. The contents of cytokine gamma-interferon (IFN-γ), interleukin 6 (IL-6), immunoglobulin IgA, and immunoglobulin IgG in the serum of laying hens were determined using ELISA kits. At the same time, the titer of Newcastle disease (ND) antibody was measured by hemagglutination inhibition test. The results are shown in Table 3.

[0061] Table 3

[0062]

[0063]

[0064] It can be seen from the data in the table that the combined use of Bacillus coagulans BC66, Lactobacillus plantarum LP194 and Pediococcus pentosaceus PP106 in the present invention can effectively increase the levels of IgA, IgG, IFN-γ and IL-6 in serum. It can be seen that the combination of the three improves the humoral immunity level of laying hens and can promote T lymphocyte differentiation.

[0065] (5) Determination of antioxidant index

[0066] When the chicks were 35 days old, 2 mL of blood was collected from the hearts of 5 chickens in each group at random to determine the antioxidant index data in the serum, including the content levels of superoxide dismutase (SOD), peroxide dismutase (CAT), malondialdehyde (MAD), and total antioxidant capacity (T-AOC). The determination method and steps were carried out according to the operating instructions of the ELISA kit and the total antioxidant capacity detection kit (colorimetric method). The determination results are shown in Table 4.

[0067] Table 4

[0068] SOD(U / mL) CAT (U / mL) MAD (U / mL) AOC (U / mL) S1 Group 118.69 0.82 9.3 16.13 S2 Group 116.54 0.79 8.7 14.88 S3 Group 117.99 0.80 9.0 15.68 S4 Group 111.45 0.64 7.5 14.23 S5 Group 92.26 0.46 6.0 11.64 S6 Group 90.04 0.42 5.7 11.24 S7 Group 85.34 0.39 4.5 9.55 S8 Group 101.23 0.52 6.8 12.38 Blank control group 63.48 0.26 3.9 7.46

[0069] It can be seen from the data in the table that the combined use of Bacillus coagulans BC66, Lactobacillus plantarum LP194 and Pediococcus pentosaceus PP106 in the present invention can effectively increase the secretion of related superoxide dismutase, peroxide dismutase and malondialdehyde, and improve the body's antioxidant capacity.

[0070] Example 3

[0071] (1) Test samples and groups: Group S1 (BC66 powder + LP194 powder + PP106 powder, the ratio of live bacteria count was 5:1:1), Group S2 (BC66 powder + LP194 powder + PP106 powder, the ratio of live bacteria count was 1:1:3), Group S3 (BC66 powder + LP194 powder + PP106 powder, the ratio of live bacteria count was 2:5:4), Group S4 (BC66 powder + LP194 powder, the ratio of live bacteria count was 5:1), Group S5 (LP194 powder + PP106 powder, the ratio of live bacteria count was 1:1), Group S6 (BC66 powder + PP106 powder, the ratio of live bacteria count was 5:1), Group S7 (ATCC 7050 powder + LP194 powder + PP106 powder, the ratio of live bacteria count was 5:1:1), Group S8 (BC66 powder + CICC 21812 bacterial powder + PP106 bacterial powder, the ratio of live bacteria count was 5:1:1), and each group of mixed bacterial powder was mixed with maltodextrin at a mass ratio of 1:9, loaded into a clean feed mixer, and mixed at a frequency of 10 Hz for 10 min to prepare feed additives. The total live bacteria count in each group of feed additives was 1×10 8 CFU / g.

[0072] (2) Experimental animals: 40-week-old Hy-Line brown laying hens were divided into 9 groups, each with 30 hens, namely Group S1-S8 and a blank control group. The laying hens in each group were allowed free access to food and water, and were fed with conventional commercially available laying hen feed daily. The feed of Group S1-S8 was mixed with 2% of the feed additives prepared in (1) for each group. After feeding for 60 days, the average egg production rate, average egg weight, average good egg rate and mortality rate of various laying hens were calculated. The calculation formula is as follows. The statistical results are shown in Table 5.

[0073] Average egg production rate = total number of eggs produced during the statistical period / (total number of feeding days during the statistical period × average number of stocks) × 100%;

[0074] Average egg weight = total egg weight / total number of eggs;

[0075] Average good quality egg rate = (number of good quality eggs in the statistical period / total number of eggs laid in the statistical period) × 100%;

[0076] Cumulative mortality rate = (number of dead chickens / total number of laying hens) × 100%;

[0077] Among them, good quality eggs refer to qualified eggs weighing 50-65g, which are not too large, too small or deformed; dead chickens refer to eliminated laying hens (laying hens that have exhausted their energy), that is, chickens that are close to the end of laying eggs or chickens that have died.

[0078] Table 5

[0079]

[0080]

[0081] It can be seen from the data in the table that the combined use of Bacillus coagulans BC66, Lactobacillus plantarum LP194 and Pediococcus pentosaceus PP106 in the present invention can significantly improve the egg production rate, egg weight and good quality egg rate of laying hens, reduce the mortality rate, fully improve the production performance of laying hens, and improve the efficiency of laying hen farming.

[0082] In summary, the present invention creatively develops a brand-new probiotic compounding method and a brand-new strategy for increasing the egg production of laying hens, that is, Bacillus coagulans BC66 strain, Lactobacillus plantarum LP194 strain and Pediococcus pentosaceus PP106 strain are compounded and used in combination, and promote each other, and synergize in promoting the growth and development of laying hens, improving egg production and disease resistance, further improving the immune system regulation ability of laying hens, improving the body's antioxidant level, and improving egg production rate and good egg rate, etc. And when the amount of bacteria is consistent, compared with the single strain intervention method, the compounding of the three bacteria is significantly improved in the effect of related aspects. Therefore, the composite probiotics are used to prepare laying hen feed additives or laying hen feeds with good prospects.

[0083] The applicant declares that the present invention illustrates the technical solution of the present invention through the above-mentioned embodiments, but the present invention is not limited to the above-mentioned embodiments, that is, it does not mean that the present invention must rely on the above-mentioned embodiments to be implemented. Those skilled in the art should understand that any improvement of the present invention, equivalent replacement of various raw materials of the product of the present invention, addition of auxiliary components, selection of specific methods, etc., all fall within the protection scope and disclosure scope of the present invention.

[0084] The preferred embodiments of the present invention are described in detail above. However, the present invention is not limited to the specific details in the above embodiments. Within the technical concept of the present invention, a variety of simple modifications can be made to the technical solution of the present invention, and these simple modifications all belong to the protection scope of the present invention.

[0085] It should also be noted that the various specific technical features described in the above specific embodiments can be combined in any suitable manner without contradiction. In order to avoid unnecessary repetition, the present invention will not further describe various possible combinations.

Claims

1. A composite probiotic with antioxidant effect, characterized in that: The composite probiotics include a combination of Bacillus coagulans BC66 strain with a preservation number of CGMCC 21879, Lactobacillus plantarum LP194 strain with a preservation number of CGMCC 21766, and Pediococcus pentosaceus PP106 strain with a preservation number of GDMCC No: 63093.

2. The composite probiotic according to claim 1, characterized in that The ratio of the number of live bacteria of the Bacillus coagulans BC66 strain, the Lactobacillus plantarum LP194 strain and the Pediococcus pentosaceus PP106 strain is (1-10):(1-10):(1-10).

3. A feed additive for laying hen breeding, characterized in that: The feed additive includes the composite probiotics according to claim 1 or 2.

4. The feed additive for laying hen breeding according to claim 3, characterized in that: The total number of viable bacteria in the feed additive is not less than 1×10 8 CFU / mL or 1×10 8 CFU / g.

5. The feed additive for laying hen breeding according to claim 3, characterized in that: The dosage form of the feed additive includes solution, freeze-dried powder, granules or tablets.

6. The feed additive for laying hen breeding according to claim 5, characterized in that: The feed additive is in the form of a powder, which is prepared by a preparation method comprising the following steps: The BC66 strain, LP194 strain and PP106 strain are inoculated into the culture medium respectively for activation and fermentation culture in sequence, and the obtained fermentation liquids are centrifuged respectively, mixed with a lyophilization protective agent for freeze drying, and the obtained BC66 bacterial powder, LP194 bacterial powder and PP106 bacterial powder are mixed according to a ratio to obtain the feed additive.

7. The feed additive for laying hen breeding according to claim 6, characterized in that: The lyoprotectant is selected from any one or a combination of at least two of skim milk, gelatin, dextrin, gum arabic, dextran, sodium alginate, polyvinyl pyrrolidone, sucrose, lactose, trehalose, sorbitol or xylitol.

8. Use of the composite probiotic according to claim 1 or 2 or the feed additive according to any one of claims 3 to 7 in laying hen feed.

9. A laying hen feed, characterized in that: The laying hen feed comprises a basic feed and the feed additive described in any one of claims 3-7.

10. Use of the composite probiotic according to claim 1 or 2 or the feed additive according to any one of claims 3 to 7 in preparing a product for promoting the growth and disease resistance of laying hens.

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