Method for improving methionine content by fermenting cottonseed meal with bacillus subtilis

Abstract

The invention discloses a method for increasing the content of methionine in cottonseed meal, raw materials are composed of cottonseed meal, and microbial fermentation probiotics are composed of pHY300PLK-Z recombinant bacillus subtilis. According to the invention, the safety and nutritional value of the cottonseed meal as the feed raw material are improved.

Description

Technical Field

[0001] The present invention relates to the technical field of feed processing, and particularly relates to a method for improving the methionine content by fermenting cottonseed meal with Bacillus subtilis. Background Art

[0002] Methionine is an essential amino acid in livestock and poultry feed. Methionine in cottonseed meal is the first limiting amino acid for poultry and the second limiting amino acid for pigs. In addition, due to the presence of the antinutritional factor gossypol in cottonseed meal, the digestibility of methionine is greatly reduced. Therefore, adopting specific strain fermentation technology to increase the methionine content and methionine digestibility in cottonseed meal is an important way to improve the utilization rate of methionine in livestock and poultry diets and reduce production costs. Summary of the Invention

[0003] The present invention aims to provide a method for increasing the methionine content in cottonseed meal based on the fermentation of Bacillus subtilis, so as to solve the drawbacks of the existing low methionine content and low utilization rate in cottonseed meal. This method utilizes the metabolic activity of Bacillus subtilis to reduce the influence of antinutritional factors on the utilization rate of methionine and improve the safety and nutritional value of cottonseed meal as a feed raw material. Specifically, it includes the following steps:

[0004] Technical Solution

[0005] Screening and Activation of Bacillus subtilis

[0006] Inoculate the pure strain screened from the Cell Biochemistry Laboratory 617, No. 5 Teaching Building, College of Animal Science and Technology, Yangtze University onto an LB solid medium and culture it in an incubator at 37°C for 12 - 16 hours for activation.

[0007] Preparation of Seed Liquid

[0008] Pick a single colony of activated Bacillus subtilis and inoculate it into a 250 ml Erlenmeyer flask containing 50 ml of LB liquid medium. Culture it on a shaker at 37°C and 180 r / min for 12 - 16 hours (primary strain). Transfer 200 μl of the cultured bacterial liquid into 50 mL of LB liquid medium and continue to culture it at 37°C and 180 r / min for 12 - 16 h (secondary strain) to obtain the seed liquid.

[0009] Fermentation of Cottonseed Meal

[0010] Treatment of Fermentation Substrate Experimental Group Substrate Treatment: Weigh 40 g of cottonseed meal, sterilize it with steam at 121°C for 20 min, cool it, and then inoculate an appropriate amount of recombinant Bacillus subtilis, molasses, and sterilized water under sterile conditions. Stir evenly, put it into a sealed bag, and place it in an incubator at 37°C for fermentation for 48 h. After fermentation is completed, sterilize it, put the fermentation product into an oven and dry it at 45°C for 48 h, cool it, crush it, pass it through a 60-mesh sieve, put it into a sample bag, and store it in a refrigerator at 4°C for testing.

[0011] Control group substrate treatment: Weigh 40 g of cottonseed meal, sterilize it with steam at 121 °C for 20 min, cool it, then add liquid medium (without strains), molasses, and sterilized water, stir evenly, without fermentation, directly put it into an oven, dry it at 45 °C for 48 h, cool it, crush it, sieve it through a 60-mesh sieve, put it into a sample bag, and store it in a 4 °C refrigerator for later measurement.

[0012] Determination of methionine content

[0013] The methionine content was determined by the method of GBT 17810-1999.

[0014] Beneficial effects

[0015] The present invention uses the screened Bacillus subtilis with the ability to increase the methionine content for fermentation, which can significantly increase the methionine content in cottonseed meal. After detection, the methionine content in the fermented cottonseed meal can be increased by 30-50%. During the fermentation process, the metabolic activities of Bacillus subtilis not only degrade gossypol but also produce various beneficial substances such as enzymes and vitamins, improving the nutritional value of cottonseed meal and increasing the digestibility and utilization rate of methionine. This fermentation method uses solid-state fermentation, with a simple process, low equipment requirements, easy industrial production, and no secondary pollution during the fermentation process, meeting the concept of green environmental protection.

[0016] Test cases

[0017] Ferment cottonseed meal with recombinant Bacillus subtilis;

[0018] This test was divided into six groups: unfermented cottonseed meal group, 5% fermented cottonseed meal group, 8% fermented cottonseed meal group, 11% fermented cottonseed meal group, 14% fermented cottonseed meal group, 17% fermented cottonseed meal group, with each group repeated 3 times.

[0019] Table 1 Methionine content of each group (%)

[0020] Group a b c Mean Standard Deviation CK group 0.52 0.58 0.54 0.55 0.02 5% group 0.68 0.71 0.65 0.68 0.03 8% group 0.74 0.73 0.78 0.78 0.03 11% group 0.72 0.76 0.75 0.75 0.02 14% group 0.76 0.78 0.81 0.79 0.02

[0021] It can be seen from Table 1 that Example 1 of the present invention has a good effect on the method for increasing the methionine content in fermented cottonseed meal. Compared with the CK group (unfermented cottonseed meal group), adding 5%, 8%, 11%, 14%, and 17% of recombinant Bacillus subtilis can significantly increase the methionine content in fermented cottonseed meal.

[0022] The method for fermenting cottonseed meal using Bacillus subtilis adopted in the present invention can effectively increase the methionine content of the fermented cottonseed meal, and has the characteristics of regulating the proportion of chemical components in cottonseed meal, optimizing the nutritional structure of cottonseed meal, and promoting the degradation of anti-nutritional factors in cottonseed meal. Through the action of microbial fermentation, the quality of cottonseed meal is significantly improved, and the safety and nutritional value of cottonseed meal are enhanced, providing strong support for the wider and more efficient application of cottonseed meal in the feed field.

[0023] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, improvements, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A fermentation preparation for increasing the methionine content in cottonseed meal, wherein the ratio of fermentation substrate to fermentation agent is 0.66:0.

34. Calculated by percentage of added amount, the compatibility and content of the fermentation substrate (raw materials and other components) are: 65.79% cottonseed meal, the ratio and content of the fermentation agent are: 3.29% recombinant Bacillus subtilis, 1.31% molasses, and 29.61% purified water.

2. According to claim 1, the fermentation preparation for increasing the methionine content in cottonseed meal, the fermentation matrix (raw materials and other components), the mass ratio of the total amount of cottonseed meal added is 1:

1.

3. According to claim 1, the fermentation preparation for increasing the methionine content in cottonseed meal, the fermentation bacteria agent, the total amount of recombinant Bacillus subtilis added, molasses, and the mass ratio of purified water is 1:0.4:

9.

4. The fermentation preparation for increasing the methionine content in cottonseed meal according to claim 1, wherein the ratio of the fermentation substrate to the fermentation agent is 0.66:0.

34. Calculated by percentage of added amount, the compatibility and content of the fermentation substrate (raw materials and other components) are: 65.79% cottonseed meal, the ratio and content of the fermentation agent are: 3.29% recombinant Bacillus subtilis, 1.31% molasses, and 29.61% purified water.

5. A method for preparing a fermentation preparation for increasing the methionine content in cottonseed meal as claimed in any one of claims 1 to 4, characterized in that: The following steps are involved: The recombinant Bacillus subtilis was determined to be pure bacteria and inoculated on LB solid culture medium, placed in a 37°C incubator for 12-16 hours for activation, and the activated single colony was picked and inoculated into a 250ml Erlenmeyer flask containing 50ml LB liquid culture medium, and cultured on a shaker at 37°C and 180r / min for 12-16h (primary strain). After completion, 200ul of the cultured bacterial solution was transferred to 50ml liquid culture medium and cultured on a shaker at 37°C and 180r / min for 12-16h (secondary strain) to obtain a seed solution (1.5×108cfu). The cottonseed meal was crushed and passed through a 60-mesh sieve, 40 g of the cottonseed meal was weighed, and steam sterilized at 121°C for 20 min. After cooling, 5% recombinant Bacillus subtilis and 2% molasses were inoculated under sterile conditions, with a material-water ratio of 1:0.

5. The mixture was stirred evenly and put into a fermentation bag, and placed in a 37°C constant temperature incubator for fermentation for 48 h. After the fermentation was completed, the fermentation product was placed in an oven at 45°C for drying for 10-12 h, placed in a sample bag, and stored in a 4°C refrigerator for testing, thereby obtaining a fermentation preparation for reducing the cottonphenol content in cottonseed meal.

6. The method for preparing a fermentation preparation for increasing the methionine content in cottonseed meal according to claim 5, characterized in that: The recombinant Bacillus subtilis is obtained by cultivation and identification.

7. The method for preparing a fermentation preparation for increasing the methionine content in cottonseed meal according to claim 6, characterized in that: The raw cottonseed meal is obtained by the following steps: Preprocessing: Cleaning: The cotton seeds are screened, winnowed, magnetically separated, and other operations are performed to remove impurities such as soil, stones, metal objects, lint, etc., to ensure the smooth progress of subsequent processing and reduce equipment wear and failure. Post-processing: Crushing: According to the experimental requirements, the dried cottonseed meal is crushed to meet certain particle size requirements for subsequent processing and use. Screening: The crushed extract powder is screened through a sieve to remove larger particles so that the powder particle size meets the requirements.

8. The method for preparing a fermentation preparation for increasing the methionine content in cottonseed meal according to claim 7, characterized in that: The raw material powder is obtained through the process of drying, crushing and screening cottonseed meal.

9. The method for preparing a fermentation preparation for increasing the methionine content in cottonseed meal according to claim 8, characterized in that: The drying temperature is 45-65° C., the drying time is 10-12 hours, and the particle size of the obtained raw material powder passes through a 60-mesh sieve.