Laying hen feed additive and preparation method thereof

Abstract

The invention relates to a laying hen feed additive and a preparation method thereof, and belongs to the technical field of feed additives. The preparation method of the laying hen feed additive comprises the following steps: putting mixed powder into an acetic acid solution, heating and stirring, then carrying out ultrasonic treatment, dropwise adding the mixed powder into the mixed solution for soaking, centrifuging to take solids, freeze-drying, putting the solids into an ethanol solution, stirring, adding glutaraldehyde, continuously stirring, centrifuging to remove a solvent, washing, and drying to obtain the laying hen feed additive. And after freeze drying, adding a compound bacteria solution, stirring at low temperature, filtering, and washing to obtain the product. The prepared additive can effectively reduce contact between probiotics and adverse factors in the external environment, so that the survival rate of the probiotics in various environments is remarkably improved. When the feed additive is applied to broiler feed, the nutritional value of the feed can be increased, the digestion and absorption time is prolonged, and the feed utilization rate is improved, so that the weight and muscle growth of broilers are increased, and the feed conversion rate is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of feed additives and relates to a laying hen feed additive and a preparation method thereof. Background Art

[0002] In contemporary animal husbandry practices, broiler feeds are usually formulated to contain a variety of nutrients, aiming to ensure the healthy growth of chickens and achieve the expected production benefits. Probiotics, as a key feed additive, have been widely used in broiler farming. Probiotics are active microorganisms, and moderate intake can have a positive effect on the health of broilers. However, due to the influence of processing, transportation, storage and other links, as well as the harsh gastrointestinal environment of broilers (the presence of gastric acid and proteases), probiotics are very easy to lose their activity. Specifically, probiotics are consumed by gastric acid in the stomach of broilers, and their residence time in the intestines is short. Most probiotics are excreted from the body with feces, resulting in low utilization and no significant effect on improving the digestive ability of broilers.

[0003] Chitosan is a product of chitin deacetylation. Its natural form is a powdery mesh structure with a large specific surface area, good mechanical strength and stability. The amino groups on the surface of chitosan can adsorb with the amino groups on proteins. Due to its wide source, low cost and simple preparation, chitosan is regarded as a probiotic carrier with broad development potential. However, the existing technology encounters problems when processing chitosan powder into millimeter-sized pellets: the moist chitosan pellets contain a large amount of water, and the structure is prone to collapse during the drying process. Summary of the invention

[0004] The purpose of the present invention is to provide a laying hen feed additive and a preparation method thereof. The additive prepared by the present invention can effectively reduce the contact between probiotics and adverse factors in the external environment, thereby significantly improving their survival rate in various environments. When applied to broiler feed, it can increase the nutritional value of the feed, prolong its digestion and absorption time, and improve feed utilization, thereby increasing the weight and muscle growth of broilers and improving feed conversion rate.

[0005] The purpose of the present invention can be achieved by the following technical solutions:

[0006] A method for preparing a laying hen feed additive, the preparation method comprising the following steps:

[0007] 1) placing the mixed powder in an acetic acid solution, heating and stirring, and then ultrasonically treating the mixed powder, dripping the mixed powder into the mixed solution drop by drop, soaking the mixed powder, centrifuging the solid, and freeze-drying the solid to obtain a preform;

[0008] 2) placing the preform in an ethanol solution and stirring it, adding glutaraldehyde and continuing to stir it, removing the solvent by centrifugation, washing it, and freeze-drying it to obtain a composite carrier;

[0009] 3) Add the composite bacterial solution to the composite carrier, stir at low temperature, filter, and wash to obtain the composite carrier.

[0010] As a preferred technical solution of the present invention, in step 1), the heating and stirring is stirring at a temperature of 50-60° C. for 30-45 minutes.

[0011] As a preferred technical solution of the present invention, in step 1), the ultrasonic treatment is performed at a frequency of 500-600W for 10-15min.

[0012] As a preferred technical solution of the present invention, in step 1), the soaking time is 3-4 hours.

[0013] As a preferred technical solution of the present invention, in step 1), the mixed powder, acetic acid solution and mixed solution are used in a ratio of 4-5g:120-150mL:160-180mL; the acetic acid solution is prepared by mixing acetic acid and deionized water in a ratio of 1g:80mL.

[0014] As a preferred technical solution of the present invention, in step 1), the mixed powder is prepared by mixing chitosan, sodium carboxymethyl cellulose and glucomannan oligosaccharide in a mass ratio of 3.0-3.3:0.6-0.8:0.40-0.48.

[0015] As a preferred technical solution of the present invention, the addition of glucomannan oligosaccharides can significantly increase the relative weight of the bursa of Fabricius, an immune organ of broilers, significantly improve the intestinal morphology of broilers and increase the number of goblet cells, improve the intestinal barrier integrity of broilers, and improve the antioxidant capacity and immunoglobulin secretion level of broilers.

[0016] As a preferred technical solution of the present invention, sodium carboxymethyl cellulose contains a large number of hydroxyl groups on its skeleton, which can form a dense hydrogen bond network, so that the sodium carboxymethyl cellulose carrier has higher stability, and a large number of strong hydrogen bonds make it insoluble in water. Adding sodium carboxymethyl cellulose can increase the porosity of the carrier, increase the surface area of ​​the carrier, and make its contact and combination with beneficial bacteria closer.

[0017] As a preferred technical solution of the present invention, the lattice structure of glucomannan oligosaccharide not only wraps water molecules, but also connects with the grid through hydrogen bonds, further stabilizing the gel structure.

[0018] As a preferred technical solution of the present invention, in step 1), the mixed solution is prepared by mixing sodium hydroxide and anhydrous ethanol in a mass ratio of 3:1.

[0019] As a preferred technical solution of the present invention, in step 2), the stirring treatment is stirring at a temperature of 20-23° C. and a rotation speed of 300-400 r / min for 30-45 min.

[0020] As a preferred technical solution of the present invention, in step 2), the stirring time is continued for 3-4 hours; and the washing is performed by using Tris-HCl with a pH value of 6 and a concentration of 1 mol / L.

[0021] As a preferred technical solution of the present invention, in step 2), the usage ratio of the preform, the ethanol solution and the glutaraldehyde is 9.5 g: 90-100 mL: 10-12 mL.

[0022] As a preferred technical solution of the present invention, in step 2), the ethanol solution is prepared by mixing ethanol and deionized water in a mass ratio of 1:8.

[0023] As a preferred technical solution of the present invention, in step 3), the low-temperature stirring is stirring at a temperature of 20-23° C. for 10-12 hours.

[0024] As a preferred technical solution of the present invention, in step 3), the usage ratio of the composite carrier and the composite bacterial solution is 1.0-1.2 g: 20 mL.

[0025] As a preferred technical solution of the present invention, in step 3), the bacterial sludge precipitate is suspended in sterile physiological saline to prepare a composite bacterial solution; the concentration of Bacillus subtilis in the composite bacterial solution is 1-2×10 9 CFU / mL, lactic acid bacteria concentration 2-3×10 7 CFU / mL, the concentration of yeast is 1-2×10 7 CFU / mL; Adding compound probiotics to the feed can slow down uterine tissue damage, regulate the expression of biomineralization-related genes in the uterus, improve the microbial flora in the uterus, and thus improve the quality of laying eggs and the ultrastructure of eggshells.

[0026] Beneficial effects of the present invention:

[0027] The present invention significantly improves the survival rate of beneficial bacteria by compounding multiple natural polysaccharides to prepare a composite polysaccharide carrier to fix beneficial bacteria; compared with unfixed beneficial bacteria, the temperature stability and pH stability of the polysaccharide carrier-fixed beneficial bacteria are significantly improved. That is, the additive prepared by the present invention can effectively reduce the contact between the probiotics and the adverse factors in the external environment, thereby significantly improving their survival rate in various environments.

[0028] The additive of the present invention is applied to broiler feed, which can increase the nutritional value of the feed, prolong its digestion and absorption time, and improve feed utilization, thereby increasing the weight and muscle growth of broilers and improving feed conversion rate. In addition, the beneficial bacteria on the polysaccharide carrier can also play a role in regulating the balance of intestinal microecology and enhancing immunity in the feed, which helps to reduce the adaptation pressure of broilers to the external environment, thereby promoting growth. DETAILED DESCRIPTION

[0029] In order to further illustrate the technical means and effects adopted by the present invention to achieve the predetermined invention purpose, the specific implementation methods, structures, features and effects of the present invention are described in detail below in combination with the embodiments.

[0030] Example 1

[0031] A method for preparing a laying hen feed additive, the preparation method comprising the following steps:

[0032] 1) placing the mixed powder in an acetic acid solution, heating and stirring, and then ultrasonically treating the mixed powder, dripping the mixed powder into the mixed solution drop by drop, soaking the mixed powder, centrifuging the solid, and freeze-drying the solid to obtain a preform;

[0033] 2) placing the preform in an ethanol solution and stirring it, adding glutaraldehyde and continuing to stir it, removing the solvent by centrifugation, washing it, and freeze-drying it to obtain a composite carrier;

[0034] 3) Add the composite bacterial solution to the composite carrier, stir at low temperature, filter, and wash to obtain the composite carrier.

[0035] In step 1), the heating and stirring is stirring at 50° C. for 30 minutes; the ultrasonic treatment is ultrasonic treatment at a frequency of 500 W for 10 minutes; and the soaking time is 3 hours;

[0036] In step 1), the ratio of the mixed powder, acetic acid solution and mixed solution is 4g:120mL:160mL; the mixed powder is formed by mixing chitosan, sodium carboxymethyl cellulose and glucomannan oligosaccharide in a mass ratio of 3.0:0.6:0.40; the mixed solution is formed by mixing sodium hydroxide and anhydrous ethanol in a mass ratio of 3:1;

[0037] In step 2), the stirring treatment is stirring at 20°C for 30 minutes at a speed of 300 r / min; the stirring time is continued for 3 hours; the washing is washing with Tris-HCl having a pH value of 6 and a concentration of 1 mol / L;

[0038] In step 2), the ratio of the preform, the ethanol solution and the glutaraldehyde is 9.5 g:90 mL:10 mL; the ethanol solution is prepared by mixing ethanol and deionized water in a mass ratio of 1:8;

[0039] In step 3), the low temperature stirring is stirring at 20° C. for 10 h;

[0040] In step 3), the ratio of the composite carrier to the composite bacterial solution is 1.0 g: 20 mL; the concentration of Bacillus subtilis in the composite bacterial solution is 1×10 9 CFU / mL, lactic acid bacteria concentration 2×10 7 CFU / mL, the concentration of yeast is 1×10 7 CFU / mL.

[0041] Example 2

[0042] A method for preparing a laying hen feed additive, the preparation method comprising the following steps:

[0043] 1) placing the mixed powder in an acetic acid solution, heating and stirring, and then ultrasonically treating the mixed powder, dripping the mixed powder into the mixed solution drop by drop, soaking the mixed powder, centrifuging the solid, and freeze-drying the solid to obtain a preform;

[0044] 2) placing the preform in an ethanol solution and stirring it, adding glutaraldehyde and continuing to stir it, removing the solvent by centrifugation, washing it, and freeze-drying it to obtain a composite carrier;

[0045] 3) Add the composite bacterial solution to the composite carrier, stir at low temperature, filter, and wash to obtain the composite carrier.

[0046] In step 1), the heating and stirring is stirring at 52° C. for 34 minutes; the ultrasonic treatment is ultrasonic treatment at a frequency of 520 W for 11 minutes; and the soaking time is 3.2 hours;

[0047] In step 1), the ratio of the mixed powder, acetic acid solution and mixed solution is 4.2 g: 128 mL: 165 mL; the mixed powder is formed by mixing chitosan, sodium carboxymethyl cellulose and glucomannan oligosaccharide in a mass ratio of 3.1: 0.65: 0.42; the mixed solution is formed by mixing sodium hydroxide and anhydrous ethanol in a mass ratio of 3: 1;

[0048] In step 2), the stirring treatment is stirring at 21°C and 320 r / min for 34 min; the stirring time is 3.2 h; the washing is washing with Tris-HCl having a pH value of 6 and a concentration of 1 mol / L;

[0049] In step 2), the ratio of the preform, the ethanol solution and the glutaraldehyde is 9.5 g:92 mL:10.5 mL; the ethanol solution is prepared by mixing ethanol and deionized water in a mass ratio of 1:8;

[0050] In step 3), the low temperature stirring is stirring at 21° C. for 10.5 h;

[0051] In step 3), the ratio of the composite carrier to the composite bacterial solution is 1.05 g: 20 mL; the concentration of Bacillus subtilis in the composite bacterial solution is 1.2×10 9 CFU / mL, lactic acid bacteria concentration 2.2×10 7 CFU / mL, the concentration of yeast was 1.2×10 7 CFU / mL.

[0052] Example 3

[0053] A method for preparing a laying hen feed additive, the preparation method comprising the following steps:

[0054] 1) placing the mixed powder in an acetic acid solution, heating and stirring, and then ultrasonically treating the mixed powder, dripping the mixed powder into the mixed solution drop by drop, soaking the mixed powder, centrifuging the solid, and freeze-drying the solid to obtain a preform;

[0055] 2) placing the preform in an ethanol solution and stirring it, adding glutaraldehyde and continuing to stir it, removing the solvent by centrifugation, washing it, and freeze-drying it to obtain a composite carrier;

[0056] 3) Add the composite bacterial solution to the composite carrier, stir at low temperature, filter, and wash to obtain the composite carrier.

[0057] In step 1), the heating and stirring is stirring at 55° C. for 38 minutes; the ultrasonic treatment is ultrasonic treatment at a frequency of 550 W for 12 minutes; and the soaking time is 3.5 hours;

[0058] In step 1), the ratio of the mixed powder, acetic acid solution and mixed solution is 4.5 g: 135 mL: 170 mL; the mixed powder is formed by mixing chitosan, sodium carboxymethyl cellulose and glucomannan oligosaccharide in a mass ratio of 3.15: 0.7: 0.44; the mixed solution is formed by mixing sodium hydroxide and anhydrous ethanol in a mass ratio of 3: 1;

[0059] In step 2), the stirring treatment is stirring at 21.5°C for 38 minutes at a speed of 350 r / min; the stirring time is continued for 3.5 hours; the washing is washing with Tris-HCl with a pH value of 6 and a concentration of 1 mol / L;

[0060] In step 2), the ratio of the preform, the ethanol solution and the glutaraldehyde is 9.5 g:95 mL:11 mL; the ethanol solution is prepared by mixing ethanol and deionized water in a mass ratio of 1:8;

[0061] In step 3), the low temperature stirring is stirring at 21.5° C. for 11 h;

[0062] In step 3), the ratio of the composite carrier to the composite bacterial solution is 1.1 g: 20 mL; the concentration of Bacillus subtilis in the composite bacterial solution is 1.5×10 9 CFU / mL, lactic acid bacteria concentration 2.5×10 7 CFU / mL, the concentration of yeast was 1.5×10 7 CFU / mL.

[0063] Example 4

[0064] A method for preparing a laying hen feed additive, the preparation method comprising the following steps:

[0065] 1) placing the mixed powder in an acetic acid solution, heating and stirring, and then ultrasonically treating the mixed powder, dripping the mixed powder into the mixed solution drop by drop, soaking the mixed powder, centrifuging the solid, and freeze-drying the solid to obtain a preform;

[0066] 2) placing the preform in an ethanol solution and stirring it, adding glutaraldehyde and continuing to stir it, removing the solvent by centrifugation, washing it, and freeze-drying it to obtain a composite carrier;

[0067] 3) Add the composite bacterial solution to the composite carrier, stir at low temperature, filter, and wash to obtain the composite carrier.

[0068] In step 1), the heating and stirring is stirring at 58° C. for 40 min; the ultrasonic treatment is ultrasonic treatment at a frequency of 570 W for 14 min; and the soaking time is 3.8 h;

[0069] In step 1), the ratio of the mixed powder, acetic acid solution and mixed solution is 4.8 g: 140 mL: 175 mL; the mixed powder is formed by mixing chitosan, sodium carboxymethyl cellulose and glucomannan oligosaccharide in a mass ratio of 3.2: 0.75: 0.45; the mixed solution is formed by mixing sodium hydroxide and anhydrous ethanol in a mass ratio of 3: 1;

[0070] In step 2), the stirring treatment is stirring at 22.5°C and 370r / min for 41min; the stirring time is 3.8h; the washing is washing with Tris-HCl having a pH value of 6 and a concentration of 1mol / L;

[0071] In step 2), the ratio of the preform, the ethanol solution and the glutaraldehyde is 9.5 g:98 mL:11.5 mL; the ethanol solution is prepared by mixing ethanol and deionized water in a mass ratio of 1:8;

[0072] In step 3), the low temperature stirring is stirring at 22.5° C. for 11.5 h;

[0073] In step 3), the ratio of the composite carrier to the composite bacterial solution is 1.15 g: 20 mL; the concentration of Bacillus subtilis in the composite bacterial solution is 1.8×10 9 CFU / mL, lactic acid bacteria concentration 2.8×10 7 CFU / mL, the concentration of yeast was 1.8×10 7 CFU / mL.

[0074] Example 5

[0075] A method for preparing a laying hen feed additive, the preparation method comprising the following steps:

[0076] 1) placing the mixed powder in an acetic acid solution, heating and stirring, and then ultrasonically treating the mixed powder, dripping the mixed powder into the mixed solution drop by drop, soaking the mixed powder, centrifuging the solid, and freeze-drying the solid to obtain a preform;

[0077] 2) placing the preform in an ethanol solution and stirring it, adding glutaraldehyde and continuing to stir it, removing the solvent by centrifugation, washing it, and freeze-drying it to obtain a composite carrier;

[0078] 3) Add the composite bacterial solution to the composite carrier, stir at low temperature, filter, and wash to obtain the composite carrier.

[0079] In step 1), the heating and stirring is stirring at 60° C. for 45 minutes; the ultrasonic treatment is ultrasonic treatment at a frequency of 600 W for 15 minutes; and the soaking time is 4 hours;

[0080] In step 1), the ratio of the mixed powder, acetic acid solution and mixed solution is 5g:150mL:180mL; the mixed powder is formed by mixing chitosan, sodium carboxymethyl cellulose and glucomannan oligosaccharide in a mass ratio of 3.3:0.8:0.48; the mixed solution is formed by mixing sodium hydroxide and anhydrous ethanol in a mass ratio of 3:1;

[0081] In step 2), the stirring treatment is stirring at 23°C and 400 r / min for 45 min; the stirring time is 4 h; the washing is washing with Tris-HCl having a pH value of 6 and a concentration of 1 mol / L;

[0082] In step 2), the ratio of the preform, the ethanol solution and the glutaraldehyde is 9.5 g: 100 mL: 12 mL; the ethanol solution is prepared by mixing ethanol and deionized water in a mass ratio of 1:8;

[0083] In step 3), the low temperature stirring is stirring at 23° C. for 12 h;

[0084] In step 3), the ratio of the composite carrier to the composite bacterial solution is 1.2 g: 20 mL; the concentration of Bacillus subtilis in the composite bacterial solution is 2×10 9 CFU / mL, lactic acid bacteria concentration 3×10 7 CFU / mL, the concentration of yeast is 2×10 7 CFU / mL.

[0085] Comparative Examples 1-3

[0086] Compared with Example 4, the difference between Comparative Examples 1-3 is that the amounts of chitosan, sodium carboxymethyl cellulose and glucomannan oligosaccharide are as shown in Table 1; the other components, preparation steps and parameters are the same.

[0087] Table 1 (Unit: parts by weight)

[0088] Chitosan Sodium Carboxymethyl Cellulose Glucomannan oligosaccharide Example 4 3.2 0.75 0.45 Comparative Example 1 0 2.35 2.05 Comparative Example 2 3.5 0 0.9 Comparative Example 3 3.4 1 0

[0089] Comparative Example 4

[0090] Compared with Example 4, Comparative Example 4 is different in that glutaraldehyde is not added, and the remaining components, preparation steps and parameters are the same.

[0091] Comparative Example 5

[0092] Compared with Example 4, Comparative Example 5 is different in that no ultrasonic treatment is performed, and the remaining components, preparation steps and parameters are the same.

[0093] Stability test: stored at 4°C for 30 days. At predetermined time intervals (0 and 30 days), the feed additives prepared in Examples 1-5 and Comparative Examples 1-5 were added to PBS (0.01 mol / L, pH 7.4), stirred and incubated at 150 r / min for 24 hours, and the live bacterial counts on day 0 and after 30 days were recorded, and the survival rate of the live bacteria was calculated. The test results are shown in Table 2.

[0094] Table 2

[0095]

[0096]

[0097] As can be seen from the test results in Table 2, compared with Comparative Examples 1-5, the survival rate of beneficial bacteria on the feed additives prepared by the present invention is significantly better than that of Comparative Examples 1-5. This is because the present invention prepares a novel polysaccharide carrier by using chitosan, sodium carboxymethyl cellulose and glucomannan oligosaccharide, and a large number of hydroxyl groups are contained on the sodium carboxymethyl cellulose skeleton, which can form a dense hydrogen bond network, improve the porosity of the composite carrier, increase the surface area of ​​the composite carrier, and make it more closely contacted and combined with beneficial bacteria, and glucomannan oligosaccharide can be connected to the grid through hydrogen bonds, further stabilizing the gel network structure, and ultrasonic treatment can promote the binding of polysaccharides, and can increase the number of free amino groups of chitosan, thereby increasing the grafting effect with glutaraldehyde, so that the temperature stability of the polysaccharide carrier fixing beneficial bacteria is significantly improved, effectively reducing the contact of probiotics with adverse factors in the external environment, thereby significantly improving its survival rate in various environments.

[0098] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any form. Although the present invention has been disclosed as a preferred embodiment as above, it is not used to limit the present invention. Any technical personnel in this field can make some changes or modify the technical contents disclosed above into equivalent embodiments without departing from the scope of the technical solution of the present invention. However, any brief modifications, equivalent changes and modifications made to the above embodiments based on the technical essence of the present invention without departing from the content of the technical solution of the present invention are still within the scope of the technical solution of the present invention.

Claims

1. A method for preparing a laying hen feed additive, characterized in that: The preparation method comprises the following steps: 1) placing the mixed powder in an acetic acid solution, heating and stirring, and then ultrasonically treating the mixed powder, dripping the mixed powder into the mixed solution drop by drop, soaking the mixed powder, centrifuging the solid, and freeze-drying the solid to obtain a preform; 2) placing the preform in an ethanol solution and stirring it, adding glutaraldehyde and continuing to stir it, centrifuging to remove the solvent, washing it, and freeze-drying it to obtain a composite carrier; 3) Add the composite bacterial solution to the composite carrier, stir at low temperature, filter, and wash to obtain the composite carrier.

2. The method for preparing a laying hen feed additive according to claim 1, characterized in that: In step 1), the heating and stirring is stirring at a temperature of 50-60° C. for 30-45 minutes; the ultrasonic treatment is ultrasonic treatment at a frequency of 500-600 W for 10-15 minutes; and the soaking time is 3-4 hours.

3. The method for preparing a laying hen feed additive according to claim 1, characterized in that: In step 1), the usage ratio of the mixed powder, the acetic acid solution and the mixed solution is 4-5 g: 120-150 mL: 160-180 mL.

4. The method for preparing a laying hen feed additive according to claim 1 or 3, characterized in that: In step 1), the mixed powder is prepared by mixing chitosan, sodium carboxymethyl cellulose and glucomannan oligosaccharide in a mass ratio of 3.0-3.3:0.6-0.8:0.40-0.48; and the mixed solution is prepared by mixing sodium hydroxide and anhydrous ethanol in a mass ratio of 3:

1.

5. The method for preparing a laying hen feed additive according to claim 1, characterized in that: In step 2), the stirring treatment is stirring at a temperature of 20-23° C. and a rotation speed of 300-400 r / min for 30-45 min; the stirring is continued for 3-4 h.

6. The method for preparing a laying hen feed additive according to claim 1, characterized in that: In step 2), the ratio of the preform, the ethanol solution and the glutaraldehyde is 9.5 g: 90-100 mL: 10-12 mL; the ethanol solution is prepared by mixing ethanol and deionized water in a mass ratio of 1:

8.

7. The method for preparing a laying hen feed additive according to claim 1, characterized in that: In step 3), the low-temperature stirring is stirring at 20-23° C. for 10-12 h.

8. The method for preparing a laying hen feed additive according to claim 1, characterized in that: In step 3), the usage ratio of the composite carrier and the composite bacterial solution is 1.0-1.2 g: 20 mL.

9. The method for preparing a laying hen feed additive according to claim 1, characterized in that: In step 3), the concentration of Bacillus subtilis in the composite bacterial solution is 1-2×10 9 CFU / mL, lactic acid bacteria concentration 2-3×10 7 CFU / mL, the concentration of yeast is 1-2×10 7 CFU / mL.

10. A laying hen feed additive prepared by the preparation method according to any one of claims 1 to 9.

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