Alcaligenes alcaligenes and application of alcaligenes alcaligenes in cottonseed meal fermentation

Abstract

The invention discloses alcaligenes aqualis and application thereof in cottonseed meal fermentation. The bacillus alcaligenes sp. Is named as Alcaligenes sp. C1, and the preservation number of the bacillus alcaligenes sp. Is CCTCC (China Center for Type Culture Collection) NO: M20241716; the 16S rDNA (ribosomal deoxyribonucleic acid) gene sequence of the alcaligenes aquae C1 strain is as shown in SEQ ID No: 1. The strain can effectively degrade the content of antinutritional factors in the cottonseed meal, and the nutritional characteristics of the cottonseed meal are improved through solid fermentation of the cottonseed meal. The invention solves the problem that the fermentation effect of the existing feed fermentation strain raw material is not ideal, and has great application potential for improving the nutritional quality of cottonseed meal feed and developing feed resources.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a water-based alkali-producing bacillus and an application thereof in cottonseed meal fermentation. Background Art

[0002] As a high-quality protein raw material in my country, cottonseed meal is rich in nutrients, high in protein content, and has a comprehensive and reasonable amino acid composition. It is a good source of vitamins, biotin and minerals. With the continuous expansion of the scale of the aquatic industry and the soaring prices of fish meal and soybean meal, cottonseed meal has potential economic value and application prospects as a new alternative protein source. The high anti-nutritional factors and low protein utilization rate in cottonseed meal greatly limit the application of cottonseed meal in aquatic feed. Cottonpol is the most important anti-nutritional factor in cottonseed meal and is the main reason for limiting the application of cottonseed meal.

[0003] At present, there are three methods to reduce cottonpol: solvent extraction, physical method (radiation), chemical method (ferrous sulfate) and microbial removal. The characteristics of the three methods are as follows:

[0004] 1. Solvent extraction method Various chemical reagents increase the difficulty of separation and purification.

[0005] 2. Physical methods for treating cottonseed crude oil will lead to the complexity of the process.

[0006] 3. The chemical treatment method has a short treatment time, but it will cause the feed to turn black. It does not remove the cottonpol but only makes it passivated.

[0007] Microbial fermentation has the advantages of low cost, high nutritional value of fermentation products, safety and pollution-free, and suitability for large-scale production. Therefore, cottonseed meal can be a good solution to improve its feeding value, increase the utilization rate of nutrients, and increase the flavor of cottonseed meal through microbial solid fermentation. Summary of the invention

[0008] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a water-based alkalinity-producing bacillus and its application in cottonseed meal fermentation. The present invention screened a water-based alkalinity-producing bacillus C1 from the rhizomes of rapeseed plants, and the water-based alkalinity-producing bacillus C1 has a significant degradation effect on anti-nutritional factors in cottonseed meal.

[0009] To achieve the above purpose, the technical solution designed by the present invention is as follows:

[0010] The invention provides a water-alkali-producing bacillus C1. The water-alkali-producing bacillus is named as Alcaligenes aquatilis C1 and has a preservation number of CCTCC NO: M 20241716.

[0011] Furthermore, the 16S rDNA sequence of the water-producing alkaline bacteria C1 is shown as SEQ ID NO:1.

[0012] The present invention also provides an application of the water-producing alkalinitybacillus C1 in reducing the content of anti-nutritional factors in cottonseed meal and improving the nutritional value of cottonseed meal.

[0013] The present invention also provides a water-producing alkaline bacillus fermentation agent, wherein the fermentation agent comprises the water-producing alkaline bacillus C1, and the concentration thereof is 4×10 7 CFU / mL.

[0014] The present invention also provides a method for preparing the fermentation agent, comprising the following steps:

[0015] (1) activating Alkali-producing Bacillus C1, and then inoculating the activated bacteria into a liquid LB medium and culturing with shaking to obtain a primary seed solution;

[0016] (2) The first-level seed liquid is inoculated into a liquid LB culture medium and cultured by shaking to obtain a second-level seed liquid, which is the water-producing alkaline bacteria fermentation agent.

[0017] Furthermore, in the step (1), the activated bacteria are inoculated in a liquid LB culture medium at an inoculation rate of 1-4%, and the culture conditions are shaking culture at 30° C. for 22-26 hours;

[0018] In the step (2), the inoculation amount of the primary seed liquid in the liquid LB culture medium is 4-6%, and the culture condition is shaking culture at 30° C. for 22-26 hours.

[0019] The present invention also provides an application of the aquaculture alkali-producing bacillus fermentation agent in preparing fermented cottonseed meal with low anti-nutritional factor content and high nutritional value.

[0020] A method for preparing fermented cottonseed meal with low anti-nutritional factor content and high nutritional value comprises the following steps:

[0021] (1) Grind and sieve cottonseed meal, sterilize under high pressure for later use;

[0022] (2) adding the cottonseed meal treated in step (1) to the fermentation agent of Alcaligenes aquaticus, mixing, oscillating and culturing for solid-state fermentation to obtain fermented cottonseed meal.

[0023] Furthermore, in the step (2), the solid-liquid ratio of cottonseed meal to the water-producing alkaline bacteria fermentation agent is 10:0.6-0.8, and the fermentation is carried out at 30°C for 45-50 hours.

[0024] The present invention also provides an application of the fermented cottonseed meal prepared by the method in preparing largemouth bass feed replacing fish meal.

[0025] Beneficial effects of the present invention:

[0026] 1. The present invention screens out a strain of Alcaligenes aquaticus that has a significant degradation effect on anti-nutritional factors in cottonseed meal; the present invention can effectively reduce the cottonphenol content, and the degradation rate of C1 glucosinolate, the degradation rate of phytic acid, and the degradation rate of cottonphenol by the screened strain is 31.51%, 12.22%, and 59.09%, thereby achieving the purpose of detoxification.

[0027] 2. The cottonseed meal is fermented with the water-producing alkaline bacteria C1 screened by the present invention. While converting cottonphenol into other substances, the nutritional value of the cottonseed meal is improved. The antioxidant properties, crude protein, acid-soluble protein, etc. are improved during the fermentation process, which can improve the nutritional value of the cottonseed meal and increase the proportion of fermented cottonseed meal in feed.

[0028] 3. The method of the present invention is used to ferment cottonseed meal, which has low production cost, does not require the use of expensive equipment, has no chemical residue, and is safe to use; the fermented cottonseed meal has reduced free cottonpol, which is beneficial to the balance of animal intestinal flora, helps animals digest and absorb, and improves feed utilization, which has important practical significance for alleviating the shortage of protein feed resources, reducing breeding costs, and producing green and healthy meat products. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a colony morphology diagram of the water-producing alkalinitybacillus C1 described in Example 1;

[0030] Figure 2 This is a phylogenetic tree diagram of the water-producing alkaline bacteria C1 described in Example 1;

[0031] Figure 3 This is the growth curve of the water-producing alkalinity bacteria C1 described in Example 1. DETAILED DESCRIPTION

[0032] The present invention is further described in detail below in conjunction with specific embodiments so that those skilled in the art can understand.

[0033] Use and preparation of culture medium in this example

[0034] LB liquid medium / enriched medium: 10 g peptone, 5 g yeast powder, 10 g sodium chloride, distilled water to 1000 mL, pH 7.0 ± 0.2, sterilized at 121 °C for 20 min; used for seed liquid culture.

[0035] LB solid medium: peptone 10g, yeast powder 5g, sodium chloride 10g, agar powder 18g, distilled water to 1000mL, pH 7.0±0.2, sterilized at 121℃ for 20min; used for bacterial preservation.

[0036] Skim milk screening medium: 5g yeast powder, 5g NaCl, 18g agar powder, 10g skim milk powder, add distilled water to 1000mL, sterilize at 121℃ for 20min; used to separate protease.

[0037] Protease-producing fermentation medium: glucose 5g, peptone 10g, KH2PO4 0.5g, MgSO4 0.3g, (NH4)2SO41g, CaCl2 1g, NaCl 1g, pH 7.2, add distilled water to 1000mL, sterilize at 121℃ for 20min; used for shake flask fermentation of the strain.

[0038] Solid fermentation medium for meal: 30g sterilized feed, 18mL distilled water, 2.4mL strain; fermented feed is used for anti-nutritional factor determination.

[0039] Example 1

[0040] Isolation and identification of Alcaligenes spp. C1

[0041] 1. Isolation of Alcaligenes C1

[0042] (1) Source: The fungus was collected from the rhizomes of rapeseed flowers at the rapeseed breeding base of Huazhong Agricultural University.

[0043] (2) A method for isolating a water-producing alkalinity-producing bacillus strain, comprising the following steps:

[0044] Step S1: collecting rhizomes of rapeseed planted in the rapeseed cultivation base of Huazhong Agricultural University, grinding and crushing, mixing evenly, putting into sterilization bags, and refrigerating for later use;

[0045] Step S2: Weigh 5 g of the ground mixture obtained in step S1 and place it in a 100 mL conical flask containing 45 mL of sterile water, shake it at 160 r / min for 30 min, and then let it stand; take 2 mL of the supernatant and place it in a 250 mL conical flask containing 45 mL of enrichment culture medium, and shake and culture it at 160 r / min for 2 days;

[0046] Step S3: dilute the bacterial culture solution obtained in step S2 to 10 -3 , 10 -4 , 10 -5 times, 30 μL of each gradient was spread on skim milk screening medium and incubated at 30°C for 2 days;

[0047] Step S4: According to the size of the transparent zone of the colonies of different shapes on the plate, record the diameter of the hydrolysis transparent zone d1 and the diameter of the single bacterial colony d2, calculate the ratio of d1 / d2, and select the single colony with strong protease transparent zone activity and streak it on LB solid medium for preservation. Through the initial screening and observation of the size of the hydrolysis zone, the hydrolysis zone of strain C1 on the skim milk powder plate is 3.20±0.42cm.

[0048] 2. Identification of Alcaligenes C1

[0049] (1) Identification of colony morphology and physiological and biochemical characteristics

[0050] The strain C1 isolated in Example 2 was subjected to colony morphology observation and physiological and biochemical characteristics identification.

[0051] like Figure 1 Shown: The strain is small, lobed, flat and dark yellow.

[0052] (2) Molecular Biological Identification

[0053] The strain C1 isolated in Example 1 was subjected to 16S rDNA molecular biological identification. The genomic DNA of the single colony culture was extracted as a template, and PCR amplification was performed using the bacterial 16S universal primer 27F / 1492R. The product was sent to Qingke Biotechnology Co., Ltd. for sequencing, and the sequence was compared with the known sequences in the GenBank database by BLAST analysis, and a phylogenetic tree was constructed.

[0054] According to the nucleotide and amino acid comparison results of strain C1, strain C1 has a high degree of match with Alcaligenes aquatilis, and its physiological characteristics are highly similar to those of water-producing alkaline bacteria. Figure 2 and Figure 3 , named it Alcaligenes aquatilis C1; it was deposited on July 31, 2024, and its preservation number is CCTCCNO: M 20241716. The preservation unit is China Center for Type Culture Collection, and the preservation address is Wuhan University, Wuhan, China, and the postal code is 430072.

[0055] Example 2

[0056] Determination of the growth curve of Alkali-producing Bacillus C1

[0057] Pick a ring of Alkali-producing Bacillus C1 and inoculate it into a 250mL Erlenmeyer flask containing 50mL LB medium, and culture it in a shaking incubator at 180r / min and 39°C. Take 200μL of the culture solution every 3h to measure OD600, and use the blank medium without bacteria as blank calibration, repeat three times, and draw a growth curve. When OD600 reaches the highest point, the biological activity is the highest, which is the most suitable strain culture time.

[0058] Results Figure 3 The optimal culture time of water-producing alkaline bacteria C1 is 27 to 33 hours.

[0059] Example 3

[0060] Preparation of fermentation agent of water-producing alkaline bacillus

[0061] Step S1: First, scrape 2-3 loops of the single alkaline-producing bacillus C1 preserved in LB solid culture medium with an inoculation loop, inoculate it into 15 mL of liquid LB culture medium placed in a 50 mL centrifuge tube, and culture it at 30° C. with shaking for 24 hours to obtain a primary seed solution.

[0062] Step S2: The first-level seed liquid is inoculated into the liquid LB medium at an inoculation rate of 5%, and the culture is shaken at 30°C for 24 hours to obtain the second-level seed liquid, i.e., the fermentation agent of Alkali-producing Bacillus aquatica. The concentration of Alkali-producing Bacillus aquatica C1 is 4×10 7 CFU / mL.

[0063] Example 4

[0064] Identification of enzyme activity in fermentation broth

[0065] Step S1: Inoculate the alkaline water-producing bacillus C1 into a 15 mL centrifuge tube with a liquid volume of 4.5 mL of protease fermentation medium, culture at 30°C, 2000r / min for 12-16 hours, inoculate the cultured seed liquid with a 10% inoculation amount into a 15 mL centrifuge tube with a liquid volume of 6 mL of protease fermentation medium, and culture for 24 hours. Centrifuge at 4°C, 10000r / min for 5 minutes to remove the bacteria and precipitate, and absorb the supernatant to obtain the fermentation supernatant.

[0066] Step S2: Take 3 test tubes, number them 1, 2, and 3 respectively, add 2 mL of fermentation supernatant into each tube, preheat it in a 40°C water bath for 2 min, then add 1 mL of the preheated 20 g / L casein solution, and keep the temperature precisely for 10 min.

[0067] Step S3: After the incubation is completed, 2 mL of 0.4 mol / L trichloroacetic acid is immediately added to each tube to terminate the reaction. The incubation is continued in a water bath for 20 min to precipitate the residual protein, and the tube is centrifuged at 4000 rpm for 20 min.

[0068] Step S4: Take another 3 test tubes, numbered 1, 2, and 3, add 1 mL of the corresponding numbered supernatant, then add 5 mL of 0.4 mol / L sodium carbonate solution and 1 mL of Folin reagent, shake well, keep in a 40°C water bath for 20 min to develop color, take out and cool to room temperature.

[0069] Step S5: Determine OD680. For the blank control, add 2 mL of trichloroacetic acid before adding the substrate casein solution to inactivate the enzyme. Determine the OD680 value of the sample, calculate the corresponding tyrosine amount (μg) according to the standard curve, and then calculate the enzyme activity.

[0070] Protease activity (U / g) = sample tyrosine content (μg) * dilution factor / 10min;

[0071] The protease activity in the fermentation broth of Alkali-producing Bacillus C1 was calculated by repeated screening to be 140.85±0.34U / g.

[0072] Example 5

[0073] Fermentation of cottonseed meal

[0074] Step S1: Grind the cottonseed meal with a grinder, pass it through a 60-mesh sieve, and sterilize it under high pressure at 120° C. for 20 min for later use.

[0075] Step S2: Add 2.4 mL of Alkali-producing Bacillus fermentation agent and 30 g of cottonseed meal to each fermentation glass bottle, mix well, and solid-state ferment for 48 hours at 30° C. in an oscillating incubator. Put the fermented cottonseed meal into an oven for drying, crush it with a grinder, and pass it through a 40-mesh sieve. Store the fermented cottonseed meal in a dryer.

[0076] Example 6

[0077] Detection of anti-nutritional factors in fermented cottonseed meal

[0078] 1. Determination of glucosinolates:

[0079] Step S1: Accurately weigh 0.1 g of crushed and sieved cottonseed meal into a 10 mL test tube, add an equal amount of 80% ethanol solution, shake well, place on a 40° C. shaker for 4 h, and centrifuge at 4000 r / min for 5 min.

[0080] Step S2: Take 0.5 mL of the supernatant after centrifugation into a 10 mL test tube, add 2 mL of 0.1% sodium carboxymethyl cellulose solution and 1 mL of 0.7 mg / mL palladium chloride color development solution, shake well and place in a 24°C constant temperature box for 1.5 to 2 hours for color development reaction.

[0081] Step S3: using a spectrophotometer to measure the absorbance of the reaction solution at a wavelength of 540 nm, and obtaining the content of glucosinolates through conversion.

[0082] 2. Detection of free gossypol content:

[0083] Step S1: Accurately weigh 1 g of sample (fermented cottonseed meal) into a 250 mL stoppered conical flask, add glass beads, accurately add 50 mL of reagent A (500 mL of isopropanol-n-hexane mixed solution with a volume ratio of isopropanol to n-hexane of 6:4, add 2 mL of aminopropanol, 8 mL of glacial acetic acid, 50 mL of distilled water, and then make up to 1000 mL with isopropanol-n-hexane mixed solution), shake and extract at room temperature for 3 h, filter with filter paper, and obtain the filtrate as the sample to be tested.

[0084] Step S2: Pipette two equal parts of 10 mL of the sample to be tested into 25 mL brown volumetric flasks a and b, take two equal parts of 10 mL of solvent A into 25 mL volumetric flasks a0 and b0, use isopropanol-n-hexane mixed solution to make a and a0 to scale as reference solution, add 2 mL of aniline to b and b0, boil in water bath for 30 min, cool to room temperature, and then make up to scale with the mixed solution. Use an enzyme marker to measure the absorbance values ​​at 440 nm, and then calculate according to the calculation formula in the national standard method.

[0085] 3. Determination of phytic acid:

[0086] Step S1: weigh 0.5 g of sample (fermented cottonseed meal), add 10 mL of 2.4% HCl solution, extract at 200 rpm / min at room temperature for 16 h, centrifuge for 20 min and take the supernatant.

[0087] Step S2: Add 1.0 g of NaCl to the supernatant, shake to dissolve, treat at -20°C for 20 min, centrifuge for 20 min, take the supernatant and dilute, mix 3 mL of the diluted sample with 1 mL of modified Wade reagent (0.03% FeCl3·6H2O+0.3% sulfosalicylic acid), mix thoroughly in a vortex, and centrifuge for 10 min.

[0088] Step S3: Using sodium phytate as a raw material, prepare a standard product containing 0.00, 1.12, 2.24, 3.36, 5.60, 7.84 and 11.20 mg / L of phytic acid, and use absorbance as the ordinate and phytic acid content as the abscissa to prepare a standard curve. Use a spectrophotometer to read the absorbance value at 500 nm, and substitute it into the formula to obtain the phytic acid content.

[0089] The contents of anti-nutritional factors related to fermented and unfermented cottonseed meal were determined. The results are shown in Table 1. The degradation rate of glucosinolate, phytic acid and gossypol of fermented cottonseed meal was 31.51%, 12.22% and 59.09%, respectively.

[0090] Table 1 Effect of fermentation of Alcaligenes aquaticus C1 on degradation of anti-nutritional factors in cottonseed meal

[0091]

[0092]

[0093] Example 7

[0094] Detection of nutritional indexes of fermented cottonseed meal

[0095] 1. Crude protein content is determined using GB / T 6432-2018

[0096] 2. The determination of acid-soluble protein is carried out according to NY / T 3801-2020. The operation steps are as follows:

[0097] Weigh 2g of fermented cottonseed meal into a 50mL centrifuge tube, add 40mL of 20% trichloroacetic acid solution, shake at 150r / min for 30min, let stand for 5min, and centrifuge at 4000r / min for 5min. Take 10mL of supernatant and add it to a digestion tube, add a catalyst, and then determine the crude protein content according to the crude protein content determination method.

[0098] After cottonseed meal was fermented by Alcaligenes C1 microorganism, the nutritional indicators were shown in Table 2. The crude protein and acid-soluble protein contents of the fermented cottonseed meal were significantly increased by 5.76% and 118.69% respectively.

[0099] Table 2 Effect of fermentation of Alcaligenes aquatica C1 on the nutritional components of cottonseed meal

[0100]

[0101] Example 8

[0102] Detection of Antioxidant Properties of Fermented Cottonseed Meal

[0103] 1. Determination of DPPH free radical scavenging ability:

[0104] Step S1: Take 1 g of sample (fermented cottonseed meal) and mix and shake it in 10 mL of distilled water at 100 r / min for 30 min, dilute it 1 times, take 2 mL of the diluted sample and put it in 2 mL of 0.2 mmol / L DPPH-anhydrous ethanol solution, mix well to obtain a sample dilution, and place it in a dark place for 30 min.

[0105] Step S2: Determine the absorbance of the sample diluent at 517 nm (A0); mix 2 mL of the sample diluent with 2 mL of 95% ethanol solution and determine the absorbance (A1); mix 2 mL of 95% ethanol solution with 2 mL of DPPH-anhydrous ethanol solution and determine the absorbance (A2); calculate the DPPH free radical scavenging rate Y1, the formula is as follows:

[0106]

[0107] 2. Determination of hydroxyl free radical scavenging ability:

[0108] Step S1: Take 1 g of sample (fermented cottonseed meal) and mix and shake it in 10 mL of distilled water at 100 r / min for 30 min, dilute it by 1 time, take 1 mL of the diluent, 1 mL of ferrous sulfate solution (9 mmol / L, diluted 2 times before use), 1 mL of 0.3% H2O2 solution, and 1 mL of salicylic acid-ethanol solution (9 mmol / L, diluted 2 times before use) respectively, mix to obtain a sample reaction solution, and let it stand at 37°C for 0.5 h.

[0109] Step S2: Measure the absorbance value (A0) of the sample reaction solution at 510 nm, replace the diluent with distilled water, and keep other conditions unchanged, and measure the absorbance value (A1); keep the diluent, ferrous sulfate, and salicylic acid unchanged, and do not add the color developer H2O2 to measure the absorbance value (A). Calculate the hydroxyl radical scavenging rate Y, the formula is as follows:

[0110]

[0111] The DPPH free radical scavenging ability and hydroxyl free radical scavenging ability represent the antioxidant capacity of the substance. The antioxidant performance is shown in Table 3. After fermentation, cottonseed meal eliminated the influence of active oxygen in the feed, and the oxidation performance was significantly improved (P<0.05). Compared with the control group, the DPPH free radical scavenging rate of fermented cottonseed meal increased by 19.61%. The hydroxyl free radical scavenging rate of fermented cottonseed meal increased by 62.40%.

[0112] Table 3 Analysis of antioxidant properties of cottonseed meal after fermentation with Alcaligenes aquatica C1

[0113]

[0114] In summary, the present invention provides a water-based alkaline-producing bacillus and its application in cottonseed meal fermentation. The strain can increase the protein content of fermented cottonseed meal, significantly reduce the content of anti-nutritional factors, improve antioxidant properties, and significantly improve the nutritional value of the fermented cottonseed meal prepared. The present invention solves the problem that the fermentation effect of existing feed fermentation strain raw materials is not ideal, and has great application potential for improving the nutritional quality of cottonseed meal and developing feed resources.

[0115] Although the above embodiments have been described in detail, they are only a part of the embodiments of the present invention, not all of them. People can also obtain other embodiments based on this embodiment without creativity, and these embodiments all belong to the protection scope of the present invention.

Claims

1. A water-producing alkalinitybacillus C1, characterized in that: The water-based alkali-producing bacillus is named Alcaligenes aquatilis C1, and its preservation number is CCTCC NO: M 20241716.

2. The water-producing alkalinitybacillus C1 according to claim 1, characterized in that: The 16S rDNA sequence of the water-alkali-producing bacillus C1 is shown in SEQ ID NO:

1.

3. Use of the alkaline-producing bacillus C1 as claimed in claim 1 in reducing the content of anti-nutritional factors in cottonseed meal and improving the nutritional value of cottonseed meal.

4. A water-producing alkaline bacillus fermentation agent, characterized in that: The fermentation agent comprises the water-producing alkaline bacillus C1 as claimed in claim 1, and its concentration is 4×10 7 CFU / mL.

5. A method for preparing the fermentation agent as claimed in claim 4, characterized in that: The following steps are involved: (1) activating Alkali-producing Bacillus C1, and then inoculating the activated bacteria into a liquid LB medium and culturing with shaking to obtain a primary seed solution; (2) The first-level seed liquid is inoculated into a liquid LB culture medium and cultured by shaking to obtain a second-level seed liquid, which is the water-producing alkaline bacteria fermentation agent.

6. The preparation method according to claim 5, characterized in that: In the step (1), the activated bacteria are inoculated in a liquid LB culture medium at an inoculation rate of 1-4%, and the culture conditions are shaking culture at 30° C. for 22-26 hours; In the step (2), the inoculation amount of the primary seed liquid in the liquid LB culture medium is 4-6%, and the culture condition is shaking culture at 30° C. for 22-26 hours.

7. Use of the aquaculture bacteria fermentation agent as claimed in claim 4 in the preparation of fermented cottonseed meal with low anti-nutritional factor content and high nutritional value.

8. A method for preparing fermented cottonseed meal with low anti-nutritional factor content and high nutritional value, characterized in that: The following steps are involved: (1) Grind and sieve cottonseed meal, sterilize under high pressure for later use; (2) adding the cottonseed meal treated in step (1) to the fermentation agent of Alcaligenes aquaticus, mixing, oscillating and culturing for solid-state fermentation to obtain fermented cottonseed meal.

9. The method according to claim 8, characterized in that: In the step (2), the solid-liquid ratio of cottonseed meal to the water-producing alkaline bacteria fermentation agent is 10:0.6-0.8, and the fermentation is carried out at 30°C for 45-50 hours.

10. Use of the fermented cottonseed meal prepared by the method according to claim 8 in preparing a largemouth bass feed replacing fish meal.

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