Method for reducing content of free gossypol by fermenting cottonseed meal with bacillus subtilis

Abstract

The invention discloses a method for reducing gossypol content 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 method, the gossypol content in the cottonseed meal is reduced in an efficient, environment-friendly and low-cost manner, and the safety and nutritional value of the cottonseed meal as a 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 reducing gossypol content by fermenting cottonseed meal with Bacillus subtilis. Background Art

[0002] Cottonseed meal is a by-product obtained after extracting cottonseed oil. Due to its high protein content, it is an important feed raw material. However, cottonseed meal contains gossypol, which is toxic to animals and can affect their growth performance, reproductive performance, immune function, etc.; animals fed with cottonseed meal with high gossypol content may encounter problems such as slow growth and damaged reproductive systems.

[0003] Currently, there are various methods for reducing the gossypol content in cottonseed meal. For example, physical de-phenol methods, such as adsorption and heating, remove gossypol by physical means, but this method has a high cost and limited de-phenol effect; chemical de-phenol methods use acids, alkalis, etc. to react with gossypol to remove it, but it is easy to cause environmental pollution and may affect the nutritional components of cottonseed meal; microbial fermentation method is a method that has been studied more in recent years. Compared with physical and chemical methods, the microbial fermentation method has the advantages of low cost, ecological environment protection, and little damage to the nutritional components of cottonseed meal. Among various microorganisms, Bacillus subtilis has the characteristics of fast growth, strong adaptability, safety and harmlessness, and can secrete various enzymes and metabolites during fermentation, which may play a positive role in the degradation of gossypol. However, the existing methods for fermenting cottonseed meal with Bacillus subtilis to reduce gossypol still have room for optimization in terms of fermentation conditions and bacterial strain characteristics, resulting in insufficient gossypol reduction rate and limiting the wide application of cottonseed meal in the feed industry. Summary of the Invention

[0004] The present invention aims to provide a method for degrading gossypol in cottonseed meal based on the fermentation of Bacillus subtilis to solve the deficiencies of the existing methods for reducing the gossypol content in cottonseed meal. This method utilizes the metabolic activity of Bacillus subtilis to convert gossypol into soluble substances or energy substances, thereby efficiently, environmentally friendly and at low cost reducing the gossypol content in cottonseed meal and improving the safety and nutritional value of cottonseed meal as a feed raw material. Specifically, it includes the following steps:

[0005] Technical Solution

[0006] Screening and Activation of Bacillus subtilis

[0007] The pure strain screened from the Cell Biochemistry Laboratory 617 of the No. 5 Teaching Building of the College of Animal Science and Technology, Yangtze University is inoculated on an LB solid medium and cultured in an incubator at 37°C for 12 - 16 hours for activation.

[0008] Preparation of Seed Liquid

[0009] Pick a single colony of activated Bacillus subtilis and inoculate it into a 250 ml Erlenmeyer flask containing 50 ml of LB liquid medium. Incubate 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 incubate at 37 °C and 180 r / min for 12 - 16 h (secondary strain) to obtain the seed liquid.

[0010] Cottonseed meal fermentation

[0011] Treatment of the fermentation substrate in the experimental group: 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 aseptic conditions. Stir well, put it into a sealed bag, and ferment it in a 37 °C constant temperature incubator for 48 h. After fermentation, sterilize it, put the fermentation product into an oven and 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 testing.

[0012] Treatment of the control group substrate: Weigh 40 g of cottonseed meal, sterilize it with steam at 121 °C for 20 min, cool it, and then inoculate liquid medium (without bacteria), molasses, and sterilized water. Stir well, 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 testing.

[0013] Determination of free gossypol content

[0014] Free gossypol (FG) was determined by the method of GB 13086—1991.

[0015] Beneficial effects

[0016] In the present invention, Bacillus subtilis with high gossypol degradation ability obtained by screening is used for fermentation, which can significantly reduce the gossypol content in cottonseed meal. After detection, the free gossypol content in the fermented cottonseed meal can be reduced to below the national standard, improving the safety of cottonseed meal. 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 enhancing the protein digestibility and utilization rate. 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. Description of the drawings

[0017] Figure 1 To compare with the CK group (unfermented cottonseed meal group), the reduction of free gossypol content by adding 5%, 8%, 11%, 14%, and 17% recombinant Bacillus subtilis.

[0018] Test cases

[0019] Use recombinant Bacillus subtilis to ferment cottonseed meal;

[0020] This experiment was divided into six groups: the unfermented cottonseed meal group, the 5% fermented cottonseed meal group, the 8% fermented cottonseed meal group, the 11% fermented cottonseed meal group, the 14% fermented cottonseed meal group, and the 17% fermented cottonseed meal group. Each group was replicated 3 times.

[0021] Table 1 Free gossypol content in each group (mg / kg)

[0022] Group a b c Mean Standard Deviation CK Group 1229.852 1199.724 1208.511 1212.69567 15.49379 5% Group 1105.312 1116.443 1152.31 1124.68833 24.55997 8% Group 1046.863 1043.114 1065.609 1051.862 12.05192 11% Group 1009.338 1036.728 993.152 1013.07267 22.02675 14% Group 1001.573 1030.524 1012.902 1014.99967 14.58905

[0023] From Figure 1 It can be seen that Example 1 of the present invention has a good effect on the method of reducing the free gossypol content. Compared with the CK group (unfermented cottonseed meal group), adding 5%, 8%, 11%, 14%, and 17% recombinant Bacillus subtilis can significantly reduce the free gossypol content.

[0024] The method of using Bacillus subtilis to ferment cottonseed meal adopted by the present invention can effectively reduce the free gossypol content in cottonseed meal, has the characteristics of adjusting 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, the safety and nutritional value of cottonseed meal are increased, providing strong support for the wider and more efficient application of cottonseed meal in the feed field.

[0025] 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 reducing the cottonphenol content in cottonseed meal, wherein the ratio of fermentation substrate to fermentation agent is 0.66:0.

34. According to the percentage of addition, 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 reducing the gossypol content in cottonseed meal, the fermentation substrate (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 reducing the cottonphenol content in cottonseed meal, the fermentation bacteria agent, the mass ratio of the total amount of recombinant Bacillus subtilis added to molasses and purified water is 1:0.4:

9.

4. The fermentation preparation for reducing the cottonphenol 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 reducing the gossypol 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 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 triangular flask containing 50ml LB liquid 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 medium and cultured on a shaker at 37°C and 180r / min for 12-16h (secondary strain) to obtain a seed solution (1.5×10 8 cfu). 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% of recombinant Bacillus subtilis and 2% of 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 reducing the gossypol 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 reducing the gossypol 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. Shelling: The cleaned cotton seeds are shelled by a shelling machine to separate the cottonseed hulls from the cotton kernels. The purpose of shelling is to increase the oil yield, because the cottonseed hulls have a low oil content and will affect the subsequent pressing or leaching effect. At the same time, the separated cottonseed hulls can be used for other purposes, such as fuel or for the production of activated carbon. Crushing: crush the cottonseed into particles of a certain size to increase the contact area between the oil and the solvent or pressing equipment, which is beneficial to the extraction of oil. The particle size of the crushed cottonseed is generally controlled at about 2-4 mm. Softening: Depending on the moisture and temperature of the cottonseed, it is sometimes necessary to soften the crushed cottonseed. By adjusting the moisture and temperature, the hardness of the cottonseed is reduced and the plasticity is increased, which is convenient for the subsequent flaking operation and also helps to improve the efficiency of oil extraction. The softening temperature is generally 50-70℃, and the moisture content is controlled at 12%-14%. Flaking: The softened cotton kernels are rolled into thin sheets using a flaking machine, which are called flaking sheets. The thickness of the flaking sheets is generally around 0.3-0.5 mm, which can greatly increase the specific surface area of ​​the oil, making it easier to extract oil from the oil. Oil Extraction Pressing method: mechanical pressure is used to squeeze out the oil in the cotton germ. Pressing equipment usually includes a screw oil press, etc. During the pressing process, the oil seeps out of the cotton germ under pressure and flows out through the oil outlet of the oil press, while the remaining solid matter forms a cake, called cotton cake. The residual oil rate of the cotton cake obtained by the pressing method is relatively high, generally around 5%-10%, but the process is simple and the investment cost is low. Extraction method: Soak cotton germs in organic solvents (such as n-hexane) to dissolve the oil in the solvent to form a mixed oil. Then, the solvent and oil are separated by evaporation, steam stripping and other operations to obtain crude oil, and the remaining solid matter is desolventized and dried to become cottonseed meal. The extraction method has a high oil yield, and the residual oil rate of cottonseed meal can be as low as below 1%, but attention must be paid to solvent recovery and safety issues. Post-processing Desolventization: For cottonseed meal obtained by leaching method, desolventization treatment is required to remove the residual organic solvent. Usually, heating and ventilation are used to evaporate the solvent under certain temperature and time conditions to reach the safe residual amount standard. Drying: Cottonseed meal may contain a certain amount of moisture after desolventizing, and needs to be dried to reduce the moisture content to an appropriate level, generally controlled below 12%, to prevent the cottonseed meal from mold and deterioration during storage. 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 fermented preparation for reducing the gossypol 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 reducing the gossypol 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.

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