CN112175856A

CN112175856A - Solid culture medium for bacillus subtilis fermentation, bacillus subtilis solid microbial inoculum and preparation method and application thereof

Info

Publication number: CN112175856A
Application number: CN201910606485.1A
Authority: CN
Inventors: 佟毅; 张德国; 卢宗梅; 陈影; 杨鑫; 范佳硕
Original assignee: Cofco Nutrition and Health Research Institute Co Ltd; Anhui BBCA Biochemical Co Ltd
Current assignee: Cofco Nutrition and Health Research Institute Co Ltd; Anhui BBCA Biochemical Co Ltd
Priority date: 2019-07-05
Filing date: 2019-07-05
Publication date: 2021-01-05
Anticipated expiration: 2039-07-05
Also published as: CN112175856B

Abstract

The invention relates to the field of microbial agents, and discloses a solid culture medium for bacillus subtilis fermentation, a bacillus subtilis solid microbial agent, and a preparation method and application thereof. The solid culture medium for fermenting the bacillus subtilis is prepared from the raw materials including a starchy raw material crushed product, starch residues, starchy raw material seed coats and yeast extract powder. The invention also provides a preparation method of the bacillus subtilis solid microbial inoculum, which comprises the following steps: inoculating bacillus subtilis strain to the solid culture medium for fermentation. The solid culture medium for fermenting the bacillus subtilis is used for producing the bacillus subtilis solid microbial inoculum by a solid fermentation method, the prepared bacillus subtilis solid microbial inoculum has strong thallus and low contamination rate, and the variation coefficient of the mixing uniformity is less than 5%; the preparation process of the microbial inoculum is simplified, equipment and energy consumption are reduced, and the generation of waste water is greatly reduced, so that the environmental burden is reduced.

Description

Solid culture medium for bacillus subtilis fermentation, bacillus subtilis solid microbial inoculum and preparation method and application thereof

Technical Field

The invention relates to the field of microbial agents, and particularly relates to a solid culture medium for bacillus subtilis fermentation, a bacillus subtilis solid microbial agent, and a preparation method and application thereof.

Background

Bacillus subtilis can synthesize digestive enzymes such as protease, amylase, lipase, etc.; during the fermentation and storage of the whole daily ration, the produced enzyme system can degrade fiber, starch and protein into micromolecular substances which are easier to be absorbed and utilized by animals, and the conversion rate of the feed are improved. The bacillus subtilis plays a role together with endogenous enzymes in the digestive tract, so that the digestibility of the feed is improved. The feed additive can grow and reproduce in animal intestinal tracts, can generate various nutrient substances such as vitamins, amino acids, organic acids, growth promoting factors and the like, participate in metabolism of animal organisms and provide nutrient substances for the animal organisms.

Along with the popularization of the application range of the fermented feed, the improvement of the yield of the fermented feed is urgent, and the preparation of liquid seed liquid is a key factor for restricting the yield; the preparation process of the liquid seed liquid is complex and the storage period is short; therefore, the application of the solid microbial inoculum to fermented feed is a breakthrough progress, can solve the limitations of equipment, technology, manpower and other directions, and is an important factor for stabilizing and improving the product quality and the yield.

At present, the preparation of the bacillus subtilis solid microbial inoculum generally comprises the steps of liquid fermentation, thallus collection, carrier addition, drying and the like, the operation flow and the use equipment are complex, a large amount of wastewater is generated in the production process, and the obtained product is easy to be infected with bacteria, uneven in product, high in mixed variation coefficient and the like.

Disclosure of Invention

The invention aims to overcome the problems in the prior art, and provides a solid culture medium for bacillus subtilis fermentation and a method for preparing a bacillus subtilis solid microbial inoculum by adopting the solid culture medium, so that the operation process is less, wastewater is not generated basically, the bacterial contamination rate of a product is reduced, the product quality is improved, and the equipment and the cost are saved; the mixing uniformity variation coefficient of the prepared probiotic agent is less than 5 percent.

In order to achieve the above object, the present invention provides a solid medium for fermentation of bacillus subtilis, wherein the raw materials for preparing the solid medium comprise a pulverized product of a starchy raw material, starch residues, a seed coat of the starchy raw material and yeast extract powder.

The second aspect of the invention provides a preparation method of a bacillus subtilis solid microbial inoculum, which comprises the following steps: inoculating bacillus subtilis strain into the solid culture medium for fermentation.

Preferably, the method of vaccination comprises: and mixing the bacillus subtilis strain with water to obtain a seed inoculation liquid, and then mixing the seed inoculation liquid with the solid culture medium to obtain a mixed material.

Preferably, the inoculation liquid is mixed with the solid culture medium in a spraying manner.

The third aspect of the present invention provides the bacillus subtilis solid microbial inoculum prepared by the preparation method of the bacillus subtilis solid microbial inoculum.

The fourth aspect of the invention provides the application of the bacillus subtilis solid microbial inoculum in preparing animal feed.

Through the technical scheme, the solid culture medium for fermenting the bacillus subtilis is used for producing the bacillus subtilis solid microbial inoculum by a solid fermentation method, so that the microbial inoculum preparation process is simplified, the equipment and energy consumption are reduced, the generation of waste water is greatly reduced, and the environmental burden is reduced. The bacillus subtilis solid microbial inoculum prepared by the invention has the advantages of strong thallus, low contamination rate and mixing uniformity variation coefficient less than 5%.

Detailed Description

The following describes in detail specific embodiments of the present invention. It should be understood that the detailed description and specific examples, while indicating the present invention, are given by way of illustration and explanation only, not limitation.

The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value, and such ranges or values should be understood to encompass values close to those ranges or values. For ranges of values, between the endpoints of each of the ranges and the individual points, and between the individual points may be combined with each other to give one or more new ranges of values, and these ranges of values should be considered as specifically disclosed herein.

In the present invention, the pulverized product of a starchy material refers to a product obtained by directly pulverizing a starchy material, such as a pulverized product of a seed or a rhizome containing starch. For example, corn flour refers to a product obtained by directly pulverizing corn seeds, and sweet potato flour refers to a product obtained by directly pulverizing sweet potato roots.

In the invention, the starch residue refers to a byproduct or leftover obtained in the process of producing starch from a starchy raw material. For example, corn grits refer to by-products or offal from the production of corn starch.

In the present invention, the seed coat of the starchy material refers to a part of the seed coat of the starchy material. Specifically, taking corn bran as an example, the preparation method of the corn bran is to separate the corn bran after soaking and crushing the corn.

In a first aspect, the invention provides a solid culture medium for fermentation of bacillus subtilis, wherein raw materials for preparing the solid culture medium comprise a starch raw material crushed product, starch residues, a starch raw material seed coat and yeast extract powder.

In the invention, the mass ratio of the starchy raw material crushed product, the starch residue, the starchy raw material seed coat and the yeast extract powder can be selected in a wide range. Preferably, in the raw materials for preparing the solid culture medium, the mass ratio of the starchy raw material crushed product, the starch residue, the starchy raw material seed coat and the yeast extract powder can be (25-35): (15-25): (5-15): (1-5); further preferably, the mass ratio of the starchy raw material crushed product, the starch residue, the starchy raw material seed coat and the yeast extract powder can be (28-32): (18-22): (8-13): (1.5-4).

In the present invention, the source of the pulverized product of the starchy raw material may not be particularly limited, and may be derived from one or more of rice, wheat, barley, corn, sweet potato, and combinations thereof, preferably corn.

In the present invention, the source of the starch residue may not be particularly limited, such as may be derived from one or more of rice, wheat, barley, corn, sweet potato, and combinations thereof, preferably corn.

In the present invention, the source of the starchy material seed coat may not be particularly limited, and may be derived from one or more of rice, wheat, barley, corn and combinations thereof, preferably corn.

The inventor of the invention finds that when the solid culture medium consists of corn flour, corn starch residue, corn bran and yeast extract powder and is used for solid fermentation, the solid culture medium is beneficial to the growth of thalli, and particularly, the growth speed is high, the thalli are more robust, the bacterial contamination rate is lower, and the finally obtained thalli are higher in density.

In the present invention, the solid medium may further contain water, and the water is added in an unlimited manner, for example, directly to the solid medium, mixed with the bacillus subtilis cells and added to the solid medium, or a combination of both methods. When the water is directly added to the solid medium, the water may be sterile water or sterilized water; when the water is mixed with the bacillus subtilis cells, the water is sterile water. The amount of the water added is based on the degree of wetting of the solid medium, and specifically, the water may be 20 to 40 parts by weight relative to 100 parts by weight of the solid medium.

In the present invention, the preparation method of the solid medium is not particularly limited, and can be prepared using a method that is conventional in the art. Preferably, the components of the solid medium are uniformly mixed together, the order of addition of the components is not particularly limited, and the solid medium may be either aqueous or non-aqueous, and then subjected to a sterilization treatment. In the present invention, the solid medium may be sterilized or sterilized by a means conventional in the art, and for example, dry heat sterilization, ozone sterilization, moist heat sterilization, irradiation sterilization, ultra-high pressure sterilization, microwave sterilization, and the like may be mentioned. After the sterilization is finished, when the solid culture medium reaches the condition of being capable of being inoculated, inoculating the bacillus subtilis strain into the solid culture medium. In the case of moist heat sterilization, inoculation may be carried out after the temperature is lowered to room temperature after sterilization.

In a second aspect, the invention provides a preparation method of a bacillus subtilis solid microbial inoculum, which comprises the following steps: the bacillus subtilis strain was inoculated on the solid medium as described above for fermentation.

In the present invention, the inoculation may be carried out by means of techniques conventional in the art. Preferably, the method of inoculation may comprise: and mixing the bacillus subtilis strain with water to obtain a seed inoculation liquid, and then mixing the seed inoculation liquid with the solid culture medium to obtain a mixed material. Preferably, the inoculation liquid is mixed with the solid culture medium in a spraying manner.

In the present invention, the fermentation may be carried out in a vessel conventionally used in the art, and for example, may be a fermentation cart, a fermenter, a respiration bag.

In the present invention, the amount of the Bacillus subtilis inoculum per gram of the solid medium can be selected within a wide range, and is preferably 1X 10⁵-1×10⁹cfu, more preferably 1X 10⁶-1×10⁸cfu。

In the present invention, the bacillus subtilis strain can be obtained by a conventional technical means in the field, for example, the bacillus subtilis strain can be a seed solution obtained by liquid fermentation, can be thalli collected from the seed solution, and can also be a bacterial suspension obtained by diluting the collected thalli to a certain concentration.

In the present invention, conditions of the fermentation process, such as fermentation temperature, etc., may not be particularly limited and may be selected within the conditions conventional in the art. Preferably, the temperature of fermentation during the fermentation is 28-38 ℃, more preferably 30-35 ℃. The fermentation time is preferably the fermentation end point when the bacillus subtilis spore rate in the material reaches more than 95%. Wherein the spore rate can be determined by sampling microscopic count. Preferably, during the fermentation, the material is stirred or turned over.

In the present invention, the method may further comprise subjecting the fermentation product to a drying treatment, which may be performed by a means conventional in the art. Preferably, the temperature of the drying is 80-105 ℃, preferably 90-100 ℃; the drying time is 1-30min, preferably 10-20 min. The fermentation product can be dried for a single time or multiple times, so that the fermentation product can be conveniently crushed.

In the present invention, the method may further comprise subjecting the dried fermentation product to a pulverization treatment, which may be carried out by a conventional technique in the art, for example, a pulverization treatment using a solid pulverizer. The fermentation product may be subjected to a single or multiple pulverization treatments as required. Preferably, the particle size of the fermentation product after said drying and crushing treatment is less than 2mm, more preferably 0.001-1 mm.

In the present invention, in order to obtain a more uniform fermentation product, it is also possible to sieve the pulverized material, then to pulverize the oversize material again to obtain a desired particle size, and then to mix the pulverized oversize material with the undersize material. Wherein, the oversize product refers to a fermentation product in a sieve after sieving, and the undersize product refers to a fermentation product which is sieved due to the particle size smaller than the sieve mesh after sieving. The size of the screen is selected so that the undersize can reach a desired particle size, and may be, for example, 10 to 200 mesh. The screening treatment can be combined with the steps of drying, crushing and the like, and can be carried out once or for multiple times so as to achieve the level of low variation coefficient of mixing uniformity.

In a third aspect, the invention provides a bacillus subtilis solid microbial inoculum prepared by the method.

In a fourth aspect, the invention provides an application of the bacillus subtilis solid microbial inoculum in preparing animal feed.

The present invention will be described in detail below by way of examples.

In the following examples and comparative examples:

the liquid culture medium contains: 1 wt% peptone, 1 wt% yeast extract powder, 1 wt% sodium chloride, 0.5 wt% glucose; the pH was adjusted to 7.2. + -. 0.2.

The effective viable count refers to the number of bacillus subtilis in feed liquid, and comprises a nutrient body and spores, and the determination method comprises the following steps: referring to the detection of the bacillus subtilis in the GBT 26428-.

The mixed bacteria rate is the percentage of the mixed bacteria number in the bacillus subtilis solid microbial inoculum to the total bacteria number, wherein all bacteria except the target bacteria are regarded as the mixed bacteria.

The shelf life refers to the time when the effective viable count of the bacillus subtilis preparation still meets the standard requirement after the bacillus subtilis preparation is placed under certain conditions. In the embodiment of the invention, the effective viable count is more than 200 hundred million, namely in the shelf life.

Mixing uniformity: expressed as coefficient of variation CV%, CV% ÷ standard deviation ÷ mean × 100%.

At least 10 representative samples are taken from each batch of feed, the effective viable count of the bacillus subtilis is determined in each sample, and the count is X₁、X₂、X₃、X₄、X₅、X₆、X₇、X₈、X₉、X₁₀The mean value X and standard deviation S are calculated.

Bacillus subtilis is obtained from the university of agriculture of Anhui, Life sciences college, number 951NA 4.

Preparation example 1

This preparation example was used to prepare Bacillus subtilis seed solutions used in the following examples

Sterilizing the liquid culture medium at 121 deg.C for 15min, and cooling to room temperature. Inoculating 2 vol% of Bacillus subtilis solution into 200mL of the sterilized liquid culture medium, and culturing at 30 deg.C and 100rpm for 18h to obtain seed solution.

Wherein the viable count of the seed liquid is 2 × 10¹⁰cfu/mL。

Example 1

This example is provided to illustrate the preparation method of Bacillus subtilis solid microbial inoculum

Taking 300g of corn flour, 200g of corn starch residue, 100g of corn bran and 20g of yeast extract powder, mixing uniformly, and sterilizing for 10min by using an ultraviolet lamp (40 w); taking 50ml diluted seed liquid, adding 330ml water, mixing well, spraying into solid culture medium, mixing well to make viable count in mixed material 1 × 10⁸cfu/g; freely placing the fermentation starting material into a fermentation barrel, covering filter cloth on the surface, and placing into a constant temperature incubator of 35 ℃ for fermentation. In the fermentation process, the materials are stirred.

And (5) performing microscopic examination on the materials, recording the fermentation days when the spore rate is more than 95 percent and the fermentation is finished.

Drying and crushing the materials at 100 ℃, sieving the materials by a 80-mesh sieve, repeating the steps of drying, crushing and sieving the oversize materials, and finally, allowing all the material bacteria to pass through the 80-mesh sieve to obtain the solid microbial inoculum.

The effective viable count, the mixed bacteria rate, the variation Coefficient (CV) of the mixing uniformity and the shelf life of the solid microbial inoculum are measured, and the results are shown in table 1.

Example 2

Taking 280g of corn flour, 220g of corn starch residue, 80g of corn bran and 40g of yeast extract powder, uniformly mixing, and performing moist heat sterilization at 121 ℃ for 20 min; taking 50ml diluted seed liquid, adding 330ml water, mixing well, spraying into solid culture medium, mixing well to make viable count in mixed material 1 × 10⁷cfu/g; freely placing fermentation starting material into a fermentation barrel, covering filter cloth on the surface, placing at constant temperature of 32 deg.C, and culturingAnd (5) cultivating in a box for fermentation. In the fermentation process, the materials are stirred.

And drying the materials at 80 ℃, sieving the materials by a 20-mesh sieve, repeating the steps of drying, crushing and sieving the oversize materials, and finally, allowing all the material bacteria to pass through the 20-mesh sieve to obtain the solid microbial inoculum.

Example 3

Taking 320g of corn flour, 180g of corn starch residue, 130g of corn bran and 15g of yeast extract powder, uniformly mixing, and carrying out dry heat sterilization at 200 ℃ for 40 min; taking 50ml of seed liquid, adding 330ml of water, uniformly mixing, spraying into a solid culture medium, and uniformly mixing to ensure that the number of viable bacteria in the mixed material is 1 multiplied by 10⁶cfu/g; freely placing the fermentation starting material into a fermentation barrel, covering filter cloth on the surface, and placing into a constant-temperature incubator at 38 ℃ for fermentation. And (5) performing microscopic examination on the materials, recording the fermentation days when the spore rate is more than 95 percent and the fermentation is finished.

And drying the material at 105 ℃, sieving the material by using a 100-mesh sieve, and repeating the steps of drying, crushing and sieving the oversize material, so that all material bacteria pass through the 100-mesh sieve finally to obtain the solid microbial inoculum.

Example 4

Example 4-1 the procedure of example 1 was followed except that the solid medium contained 250g of corn flour, 250g of corn starch residue, 150g of corn bran, 10g of yeast extract powder.

Example 4-2 the procedure of example 1 was followed except that the solid medium contained 350g of corn flour, 150g of corn starch residue, 50g of corn bran, 50g of yeast extract powder.

The fermentation days are recorded, and the effective viable count, the mixed bacteria rate, the variation Coefficient (CV) of the mixing uniformity and the shelf life of the solid microbial inoculum are measured, and the results are shown in table 1.

Example 5

Example 5-1 the procedure of example 1 was followed except that the viable count in the blended material was 1X 10⁵cfu/g。

Example 5-2 the procedure of example 1 was followed except that the viable count in the blended material was 1X 10⁹cfu/g。

Example 6

Example 6-1 the procedure of example 1 was followed except that the solid medium contained 300g of wheat flour, 200g of wheat starch residue, 100g of wheat bran, 20g of yeast extract, 50ml of diluted seed solution and 330ml of water.

Example 6-2 the procedure of example 1 was followed except that the solid medium comprised 300g of sweet potato flour, 200g of sweet potato starch residue, 100g of corn bran, 20g of yeast extract flour, 50ml of diluted seed solution and 330ml of water.

Examples 6-3 the procedure of example 1 was followed except that the solid medium contained 300g of wheat flour, 200g of corn starch residue, 100g of corn bran, 20g of yeast extract, 50ml of diluted seed solution and 330ml of water.

Example 7

The procedure of example 1 was followed except that no stirring treatment was performed during the fermentation.

Comparative example 1

This comparative example is used to illustrate the effect of solid medium composition on solid microbial inoculum

Comparative example 1-1: the conditions of this comparative example were the same as in example 1, except that the solid medium had a composition of 400g of corn starch residue, 200g of corn bran and 40g of yeast extract powder.

Comparative examples 1 to 2: the conditions of this comparative example were the same as in example 1, except that the composition of the solid medium was 450g of corn flour, 150g of corn bran, and 30g of yeast extract powder.

Comparative examples 1 to 3: the conditions of this comparative example were the same as example 1, except that the solid medium had a composition of 360g of corn flour, 240g of corn starch residue, and 24g of yeast extract powder.

Comparative example 2

This comparative example is used to illustrate the preparation of a Bacillus subtilis solid inoculum by liquid fermentation

The liquid medium was sterilized at 121 ℃ for 15min and cooled to room temperature. 2 vol% of the seed solution prepared in preparation example 1 was inoculated, and cultured at 30 ℃ and 100rpm for 15 hours. After the fermentation is finished, the number of viable bacteria in the obtained fermentation liquor is 200 hundred million cfu/ml, and spores account for more than 95%.

And (3) centrifugally separating the fermentation liquor to obtain bacterial sludge with the water content of 90%, adding 640g of corn flour and 360mL of water into the bacterial sludge, and uniformly mixing to ensure that the final effective viable count is over 500 hundred million cfu/g.

Drying and crushing the materials at 100 ℃, sieving the materials by a 80-mesh sieve, repeating the steps of drying, crushing and sieving, and finally mixing oversize materials and undersize materials after crushing to obtain the solid microbial inoculum.

Comparative example 3

The solid microbial inoculum is prepared according to the method of the comparative example 2, except that the fermentation liquor is centrifugally separated to obtain bacterial sludge with the water content of 90 percent, 300g of corn flour, 200g of corn starch residue, 100g of corn bran, 20g of yeast extract powder and 360mL of water are added into the bacterial sludge and evenly mixed, so that the final effective viable count is more than 500 hundred million cfu/g.

TABLE 1

As can be seen from the comparison of the results of the examples and the comparative example 1 in the table 1, the solid medium of the present invention, and the ratio of each component is controlled within a certain range, can obtain higher effective viable count in a shorter time, has lower mixed bacteria rate, lower variation coefficient of mixing uniformity, and has prolonged shelf life.

According to the data of example 1, example 4 and example 6, it can be seen that the product quality can be further improved under the condition that the solid medium comprises corn flour, corn starch residue, corn bran and yeast extract powder, and the thallus obtained by culturing with the solid medium is stronger according to the microscopic examination result.

As can be seen by comparing the data of example 1 and example 5, the different inoculation amounts have different performances on the thallus in the solid fermentation process, and when the inoculation amount is in an appropriate range, a faster growth rate can be obtained, so that the fermentation period is shortened; and the thallus grows vigorously, so that the rate of mixed bacteria is reduced, and the quality guarantee period is further prolonged.

As can be seen from the comparison of the results of examples 1 and 7, the stirring or turnover treatment during the solid-state fermentation process contributes to the growth of the cells, thereby shortening the fermentation period.

Compared with the solid microbial inoculum prepared by the liquid fermentation method, the solid microbial inoculum prepared by the solid fermentation method has the advantages of lower microbial inoculum rate, obviously reduced variation coefficient of mixing uniformity and certain prolonged shelf life as can be seen by comparing the data of the example 1 and the comparative examples 2 and 3.

The preferred embodiments of the present invention have been described above in detail, but the present invention is not limited thereto. Within the scope of the technical idea of the invention, many simple modifications can be made to the technical solution of the invention. Including each of the specific features, are combined in any suitable manner. The invention is not described in detail in order to avoid unnecessary repetition. Such simple modifications and combinations should be considered within the scope of the present disclosure as well.

Claims

1. The solid culture medium for fermentation of the bacillus subtilis is characterized in that raw materials for preparing the solid culture medium comprise a starchy raw material crushed product, starch residues, starchy raw material seed coats and yeast extract powder.

2. The solid culture medium according to claim 1, wherein in the raw materials for preparing the solid culture medium, the mass ratio of the starchy raw material crushed product, the starch residue, the starchy raw material seed coat and the yeast extract powder is (25-35): (15-25): (5-15): (1-5); preferably, the mass ratio of the starchy raw material crushed product, the starch residue, the starchy raw material seed coat and the yeast extract powder is (28-32): (18-22): (8-13): (1.5-4).

3. The solid medium according to claim 1 or 2, wherein the starchy feedstock pulverised product is derived from one or more of rice, wheat, barley, corn, sweet potato, potato and combinations thereof, preferably corn; and/or

The starch residue is derived from one or more of rice, wheat, barley, corn, sweet potato, potato and their combination, preferably corn; and/or

The starchy seed coat is derived from one or more of rice, wheat, barley, corn and combinations thereof, preferably corn.

4. The solid medium according to any one of claims 1 to 3, wherein the raw material for the preparation of the solid medium further comprises water.

5. A preparation method of a bacillus subtilis solid microbial inoculum is characterized by comprising the following steps: inoculating bacillus subtilis strain into the solid culture medium of any one of claims 1 to 4 for fermentation.

6. The method of claim 5, wherein the method of seeding comprises: mixing the bacillus subtilis strain with water to obtain a seed inoculation liquid, and then mixing the seed inoculation liquid with the solid culture medium to obtain a mixed material;

7. The method according to claim 5 or 6, wherein the amount of Bacillus subtilis inoculated is 1X 10 per gram of the solid medium⁵-1×10⁹cfu。

8. The method according to claim 5, wherein during the fermentation, the temperature of the fermentation is 28-38 ℃, preferably 30-35 ℃;

preferably, during the fermentation, the material is stirred or turned over.

9. The method of claim 5, wherein the method further comprises drying and pulverizing the fermentation product;

preferably, the drying temperature is 80-105 ℃, and the drying time is 1-30 min;

preferably, the particle size of the fermentation product after the comminution treatment is less than 2mm, preferably 0.001 to 1 mm.

10. A bacillus subtilis solid microbial agent prepared by the method of any one of claims 5 to 9.

11. The use of the bacillus subtilis solid preparation according to claim 10 in the preparation of animal feed.