An aquatic alkali-producing bacterium and its application in cottonseed meal fermentation

Abstract

This invention discloses an aquatic alkaloid bacterium and its application in cottonseed meal fermentation. The aquatic alkaloid bacterium is named *Alcaligenes aquatilis* C1, with accession number CCTCC NO: M20241716; the 16S rDNA gene sequence of this *Alcaligenes aquatilis* C1 strain is shown in SEQ ID No: 1. This strain can effectively degrade the content of anti-nutritional factors in cottonseed meal and improve its nutritional properties through solid-state fermentation. This invention solves the problem of unsatisfactory fermentation effects of existing feed fermentation strains and has great application potential for improving the nutritional quality of cottonseed meal feed and developing feed resources.

Description

Technical Field

[0001] This invention relates to the field of microbial technology, specifically to an alkaline bacillus produced in water and its application in cottonseed meal fermentation. Background Technology

[0002] Cottonseed meal, a high-quality protein source in my country, is rich in nutrients, with a high protein content, a comprehensive and well-proportioned amino acid composition, and is an excellent source of vitamins, biotin, and minerals. With the continuous expansion of the aquaculture industry and the soaring prices of fishmeal and soybean meal, cottonseed meal has potential economic value and application prospects as a novel alternative protein source. However, the high levels of anti-nutritional factors and low protein utilization in cottonseed meal significantly limit its application in aquatic feed. Gossypol, the most significant anti-nutritional factor in cottonseed meal, is the main reason limiting its use.

[0003] Currently, methods for reducing gossypol include solvent extraction, physical methods (radiation), chemical methods (ferrous sulfate), and microbial removal; the characteristics of these three methods are as follows:

[0004] 1. Solvent extraction methods use various chemical reagents, which increases the difficulty of separation and purification.

[0005] 2. Physical methods for processing cottonseed crude oil can lead to increased complexity in the process.

[0006] 3. Chemical treatment is quick, but it can cause the feed to turn black, as it does not remove gossypol but only passivates it.

[0007] Microbial fermentation offers advantages such as low cost, high nutritional value of fermentation products, safety and no pollution, and suitability for large-scale production. Therefore, microbial solid-state fermentation is an excellent solution to improve the feed value, increase nutrient utilization, and enhance the flavor of cottonseed meal. Summary of the Invention

[0008] The purpose of this invention is to overcome the shortcomings of existing technologies and provide an aquatic alkaloid bacterium and its application in cottonseed meal fermentation. This invention screened an aquatic alkaloid bacterium C1 from rapeseed rhizomes, which exhibits a significant degradation effect on anti-nutritional factors in cottonseed meal.

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

[0010] This invention provides an aquatic alkalobacterium C1, named Alcaligenes saquatilis C1, with accession number CCTCC NO: M 20241716.

[0011] Furthermore, the 16S rDNA sequence of the aquatic alkali-producing bacterium C1 is shown in SEQ ID NO: 1.

[0012] The present invention also provides the application of the aforementioned Alcaligenes aquaticus C1 in reducing the content of anti-nutritional factors and improving the nutritional value of cottonseed meal.

[0013] The present invention also provides a fermentation agent of *Alkalibium aquaponics*, wherein the fermentation agent comprises *Alkalibium aquaponics* C1 at a concentration of 4 × 10⁻⁶. 7 CFU / mL.

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

[0015] (1) Activate the aquatic alkalobacterium C1, and then inoculate the activated bacteria into liquid LB medium and culture with shaking to obtain the first-stage seed culture.

[0016] (2) Inoculate the primary seed culture into liquid LB medium and shake to obtain the secondary seed culture, which is the fermentation agent of Alkalobacterium tumefaciens.

[0017] Further, in step (1), the amount of activated bacterial strain inoculated into liquid LB medium is 1-4%, and the culture conditions are 30℃ shaking culture for 22-26h;

[0018] In step (2), the amount of primary seed culture inoculated into liquid LB medium is 4-6%, and the culture conditions are 30℃ shaking culture for 22-26 hours.

[0019] The present invention also provides the application of the aforementioned Alcaligenes aquaticus fermentation agent in the preparation of fermented cottonseed meal with low content of anti-nutritional factors and high nutritional value.

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

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

[0022] (2) Add the cottonseed meal treated in step (1) to the aquatic alkaloid bacterium fermentation agent, mix well, and shake to carry out solid-state fermentation to obtain fermented cottonseed meal.

[0023] Furthermore, in step (2), the solid-liquid ratio of cottonseed meal to Alcaligenes aquatic fermentation agent is 10:0.6-0.8, and 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 described above in the preparation of a fishmeal substitute for largemouth bass feed.

[0025] The beneficial effects of this invention are:

[0026] 1. This invention screens out strains of Alkalobacterium aquaponics that have a significant degradation effect on anti-nutritional factors in cottonseed meal; this invention can effectively reduce the content of gossypol. The screened strain C1 has a glucosinolate degradation rate of 31.51%, a phytic acid degradation rate of 12.22%, and a gossypol degradation rate of 59.09%, achieving the purpose of detoxification.

[0027] 2. The cottonseed meal fermented with the Alkalibacterium alkali-producing C1 strain obtained by the present invention not only converts gossypol into other substances, but also improves the nutritional value of the cottonseed meal. During the fermentation process, the antioxidant properties, crude protein, acid-soluble protein and other components are improved, which can enhance the nutritional value of the cottonseed meal and increase the proportion of fermented cottonseed meal used in feed.

[0028] 3. The method of this invention for fermenting cottonseed meal has low production costs, does not require expensive equipment, leaves no chemical residues, and is safe to use. The fermented cottonseed meal has reduced free gossypol, which is beneficial to the balance of animal intestinal flora, helps animals digest and absorb nutrients, and improves feed utilization. It has important practical significance for alleviating the shortage of protein feed resources, reducing breeding costs, and producing green and healthy meat products. Attached Figure Description

[0029] Figure 1 This is a colony morphology diagram of *Alkalibacterium alkali* C1 described in Example 1;

[0030] Figure 2 This is the phylogenetic tree diagram of the aquatic alkaloid bacillus C1 described in Example 1;

[0031] Figure 3 This is a growth curve of the aquatic alkaloid bacillus C1 described in Example 1. Detailed Implementation

[0032] The present invention will now be described in further detail with reference to specific embodiments, so that those skilled in the art can understand it.

[0033] The culture medium used and prepared in this embodiment

[0034] LB liquid medium / enrichment medium: 10g peptone, 5g yeast extract, 10g sodium chloride, add distilled water to a final volume of 1000mL, pH 7.0±0.2, sterilize at 121℃ for 20min; used for seed culture.

[0035] LB solid medium: 10g peptone, 5g yeast extract, 10g sodium chloride, 18g agar powder, add distilled water to a final volume of 1000mL, pH 7.0±0.2, sterilize 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 isolate protease production.

[0037] Fermentation medium for protease production: 5g glucose, 10g peptone, 0.5g KH2PO4, 0.3g MgSO4, 1g (NH4)2SO4, 1g CaCl2, 1g NaCl, pH 7.2, add distilled water to 1000mL, sterilize at 121℃ for 20min; used for shake-flask fermentation of the inoculum.

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

[0039] Example 1

[0040] Isolation and identification of Alcaligenes aquaticus C1

[0041] 1. Isolation of Alcaligenes C1 from water

[0042] (1) Source: The strains were collected from the rapeseed rhizomes of the rapeseed breeding base of Huazhong Agricultural University.

[0043] (2) A method for isolating aquatic alkaloid bacteria strains, comprising the following steps:

[0044] Step S1: Collect the rootstocks of rapeseed plants from the rapeseed cultivation base of Huazhong Agricultural University, grind and crush them, mix them evenly, pack them into sterilized bags, and refrigerate them for later use.

[0045] Step S2: Weigh 5g of the ground and pulverized mixture obtained in step S1 and place it in a 100mL Erlenmeyer flask containing 45mL of sterile water. Shake at 160r / min for 30min and let stand. Take 2mL of the supernatant and place it in a 250mL Erlenmeyer flask containing 45mL of enrichment medium. Shake at 160r / min and incubate for 2d.

[0046] Step S3: Serially dilute the bacterial culture medium obtained in step S2 to 10⁻⁶. -3 10 -4 10 -5 Each gradient was multiplied by 30 μL and spread onto skim milk screening medium, and incubated at 30°C upside down for 2 days.

[0047] Step S4: Based on the size of the clear zone of different colonies on the plate, record the diameter of the hydrolysis clear zone (d1) and the diameter of a single colony (d2), calculate the ratio of d1 / d2, and screen single colonies with strong protease clear zone activity for streaking and preservation on LB solid medium. Through initial screening and observation of the hydrolysis zone size, the hydrolysis zone of strain C1 on the skim milk powder plate was 3.20 ± 0.42 cm.

[0048] 2. Identification of Alcaligenes C1 in Aquatic Organisms

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

[0050] The colony morphology and physiological and biochemical characteristics of strain C1 isolated in Example 2 were observed.

[0051] like Figure 1 As shown: The strain is small, lobed, flat, and deep yellow.

[0052] (2) Molecular biological identification

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

[0054] Based on the nucleotide and amino acid alignment results of strain C1, strain C1 shows a high degree of matching with *Alcaligenes aquatilis*, and its physiological characteristics are highly similar to those of *Alcaligenes aquatilis*. (See attached figures.) Figure 2 and Figure 3 It was named *Alcaligenes aquatilis* C1 and deposited on July 31, 2024, with accession number CCTCCNO: M 20241716. The depositary institution is the China Center for Type Culture Collection, located at Wuhan University, Wuhan, China, postal code 430072.

[0055] Example 2

[0056] Determination of the growth curve of Alcaligenes aquaticus C1

[0057] One loopful of *Alcaligenes aquaticus* C1 was inoculated into a 250 mL Erlenmeyer flask containing 50 mL of LB medium and cultured in a shaking incubator at 180 rpm and 39 °C. OD600 was measured every 3 hours using 200 μL of culture medium, with uninoculated blank medium used as a calibration. This process was repeated three times, and a growth curve was plotted. The optimal culture time for the strain was defined as the point where OD600 reached its maximum, indicating the highest biological activity.

[0058] See results Figure 3 The optimal culture time for Alcaligenes aquaticus C1 is 27–33 h.

[0059] Example 3

[0060] Preparation of Alcaligenes aquatic fermentation inoculum

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

[0062] Step S2: Inoculate the primary seed culture at a rate of 5% into liquid LB medium and incubate at 30°C with shaking for 24 hours to obtain the secondary seed culture, which is the fermentation agent for *Alkalibium aquaponics*. The concentration of *Alkalibium aquaponics* C1 is 4 × 10⁻⁶. 7 CFU / mL.

[0063] Example 4

[0064] Identification of enzyme activity in fermentation broth

[0065] Step S1: Inoculate *Alkalibacterium aquaponics* C1 into a 15 mL centrifuge tube containing 4.5 mL of protease-producing fermentation medium and incubate at 30°C and 2000 rpm for 12–16 h. Inoculate the prepared seed culture at a 10% inoculation rate into a 15 mL centrifuge tube containing 6 mL of protease-producing fermentation medium and incubate for 24 h. Centrifuge at 4°C and 10000 rpm for 5 min to remove bacterial cells and precipitate, and collect the supernatant as the fermentation supernatant.

[0066] Step S2: Take 3 test tubes and label them 1, 2 and 3 respectively. Add 2 mL of fermentation supernatant to each tube and preheat in a 40℃ water bath for 2 min. Then add 1 mL of the same preheated 20 g / L casein solution and keep it at the correct temperature for 10 min.

[0067] Step S3: After the incubation period, immediately add 2 mL of 0.4 mol / L trichloroacetic acid to each tube to terminate the reaction. Continue incubation in a water bath for 20 min to precipitate any remaining protein, then centrifuge at 4000 rpm for 20 min.

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

[0069] Step S5: Measure OD680. For the blank control, add 2 mL of trichloroacetic acid before adding the casein substrate solution to inactivate the enzyme. Measure the OD680 value of the sample, calculate the corresponding amount of tyrosine (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 Alkalobacterium tumefaciens C1 was calculated to be 140.85 ± 0.34 U / g through repeated screening.

[0072] Example 5

[0073] Fermentation of cottonseed meal

[0074] Step S1: Crush the cottonseed meal with a pulverizer, pass it through a 60-mesh sieve, and autoclave it at 120℃ for 20 minutes. Set aside for later use.

[0075] Step S2: Add 2.4 mL of water to each fermentation glass bottle to produce *Alcaligenes alkali* fermentation inoculum, and add 30 g of cottonseed meal. Mix well and ferment in a shaking incubator at 30°C for 48 hours. After fermentation, dry the cottonseed meal in an oven, pulverize it with a pulverizer, and pass it through a 40-mesh sieve. Store the fermented cottonseed meal in a desiccator.

[0076] Example 6

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

[0078] 1. Determination of glucosinolates:

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

[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 colorimetric solution, shake well and place in a 24℃ constant temperature incubator for 1.5 to 2 hours to carry out the colorimetric reaction.

[0081] Step S3: Measure the absorbance of the reaction solution at a wavelength of 540 nm using a spectrophotometer, and calculate the content of glucosinolates.

[0082] 2. Detection of free gossypol content:

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

[0084] Step S2: Pipette 10 mL of two equal portions of the sample to be tested into two 25 mL brown volumetric flasks, a and b. Pipette 10 mL of two equal portions of solvent A into two 25 mL volumetric flasks, a0 and b0. Dilute a0 and a0 to the mark with isopropanol-n-hexane mixed solution as a reference solution. Add 2 mL of aniline to b and b0. Incubate in a boiling water bath for 30 min. After cooling to room temperature, dilute to the mark with the mixed solution. Measure the absorbance at 440 nm using a microplate reader, and then calculate according to the formula in the national standard method.

[0085] 3. Determination of phytic acid:

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

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

[0088] Step S3: Prepare standards containing 0.00, 1.12, 2.24, 3.36, 5.60, 7.84, and 11.20 mg / L phytic acid using sodium phytate as the raw material. Plot absorbance on the ordinate and phytic acid content on the abscissa to construct a standard curve. The phytic acid content can be obtained by substituting the absorbance value at 500 nm using a spectrophotometer into the formula.

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

[0090] Table 1. Effect of Alcaligenes aquaticus C1 fermentation on the degradation of antinutritional factors in cottonseed meal.

[0091]

[0092]

[0093] Example 7

[0094] Nutritional index testing of fermented cottonseed meal

[0095] 1. Crude protein content was determined according to GB / T 6432-2018.

[0096] 2. The determination of acid-soluble proteins was performed according to NY / T 3801-2020. The general procedure is 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 then centrifuge at 4000r / min for 5min. Take 10mL of the supernatant and add it to a digestion tube, add the catalyst, and then determine the crude protein content according to the method for crude protein content determination.

[0098] The nutritional indicators of cottonseed meal after fermentation by Alkalobacterium alkali-producing C1 microorganism are shown in Table 2. The crude protein and acid-soluble protein content of fermented cottonseed meal increased significantly by 5.76% and 118.69%, respectively.

[0099] Table 2. Effects of Alcaligenes aquaticus C1 fermentation on the nutrient composition of cottonseed meal.

[0100]

[0101] Example 8

[0102] Antioxidant performance testing of fermented cottonseed meal

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

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

[0105] Step S2: Measure the absorbance of the sample dilution at 517 nm (A0); mix 2 mL of sample dilution with 2 mL of 95% ethanol solution and measure the absorbance (A1); mix 2 mL of 95% ethanol solution with 2 mL of DPPH-anhydrous ethanol solution and measure the absorbance (A2); calculate the DPPH free radical scavenging rate Y1 using the following formula:

[0106]

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

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

[0109] Step S2: Measure the absorbance of the sample reaction solution at 510 nm (A0). Replace the diluent with distilled water, keeping other conditions unchanged, and measure the absorbance (A1). Keep the diluent, ferrous sulfate, and salicylic acid unchanged, and do not add the colorimetric reagent H2O2, then measure the absorbance (A). Calculate the hydroxyl radical scavenging rate Y using the following formula:

[0110]

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

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

[0113]

[0114] In summary, this invention provides an aquatic alkaloid bacterium and its application in cottonseed meal fermentation. This strain can increase the protein content of fermented cottonseed meal, significantly reduce the content of anti-nutritional factors, and enhance antioxidant properties, resulting in a significant improvement in the nutritional value of the fermented cottonseed meal. This invention solves the problem of unsatisfactory fermentation effects of existing feed fermentation strains and has great application potential for improving the nutritional quality of cottonseed meal and developing feed resources.

[0115] All other parts not described in detail are existing technologies. Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. An aquatic alkaloid-producing bacterium C1, characterized in that: The water-producing alkalibacter ( Alcaligenes aquatilis The accession number is CCTCC NO: M 20241716.

2. The application of *Alcaligenes aquaticus* C1 as described in claim 1 in reducing the content of anti-nutritional factors and improving the nutritional value of cottonseed meal, characterized in that: The anti-nutritional factors are glucosinolates, phytic acid, and gossypol.

3. A fermentation agent for Alcaligenes in water production, characterized in that: The fermentation agent comprises *Alcaligenes aquaticus* C1 as described in claim 1, at a concentration of 4 × 10⁻⁶. 7 CFU / mL.

4. A method for preparing the fermentation agent as described in claim 3, characterized in that: Includes the following steps: (1) Activate the aquatic alkalobacterium C1, and then inoculate the activated strain into liquid LB medium and shake to culture, which is the primary seed culture; (2) Inoculate the primary seed culture into liquid LB medium and shake to obtain the secondary seed culture, which is the fermentation agent of Alkalobacterium tumefaciens.

5. The preparation method according to claim 4, characterized in that: In step (1), the amount of activated bacteria inoculated into liquid LB medium is 1-4%, and the culture conditions are 30℃ shaking culture for 22-26 h; In step (2), the amount of primary seed culture inoculated into liquid LB medium is 4-6%, and the culture conditions are 30℃ shaking culture for 22-26 h.

6. The application of the *Alcaligenes aquaticus* fermentation agent as described in claim 3 in the preparation of fermented cottonseed meal with low anti-nutritional factor content and high nutritional value, characterized in that: The anti-nutritional factors are glucosinolates, phytic acid, and gossypol.

7. A method for preparing fermented cottonseed meal with low anti-nutritional factor content and high nutritional value using the *Alcaligenes aquaponics* fermentation agent described in claim 3, characterized in that: Includes the following steps: (1) Crush and sieve the cottonseed meal, and sterilize it under high pressure for later use; (2) Add the cottonseed meal treated in step (1) to the aquatic alkaloid bacterium fermentation agent, mix well, and carry out solid-state fermentation by shaking culture to obtain fermented cottonseed meal; The anti-nutritional factors are glucosinolates, phytic acid, and gossypol.

8. The method according to claim 7, characterized in that: In step (2), the solid-liquid ratio of cottonseed meal to Alcaligenes aquatic fermentation agent is 10:0.6~0.8, and fermentation is carried out at 30℃ for 45~50 h.

9. The application of fermented cottonseed meal prepared by the method described in claim 7 in the preparation of fishmeal substitute feed for largemouth bass.

Images