Bacillus and application thereof in solid-state fermentation of soybean meal

Abstract

The invention provides a bacillus halotolans strain with high protease activity, and the bacillus halotolans strain is characterized in that the strain is preserved in Guangdong Microbial Culture Collection Center on July 15, 2024, and the preservation number is GDMCC (China General Microbiological Culture Collection Center) NO: 64868. The strain is named as CQPL9. The strain can efficiently secrete acidic, neutral and alkaline full-spectrum protease, and can efficiently decompose large protein molecules and anti-nutritional ingredients in soybean meal. The method is suitable for a solid-state fermentation process and can be used for improving the quality of soybean meal, increasing the nutritional value and developing functional products.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of microorganisms, and particularly relates to a bacillus strain and its application in soybean meal solid-state fermentation and the like. BACKGROUND

[0002] Soybean meal is a by-product after soybean is pressed, has high protein content, balanced amino acid composition, is rich in oligosaccharides, minerals and vitamins, is a high-quality high-protein low-fat plant protein source, and is widely used in the feed industry. However, soybean meal contains antinutritional factors such as trypsin inhibitor and phytic acid, and macromolecular allergen proteins such as glycinin and beta-conglycinin, which can easily interfere with the function of digestive enzymes in animals, reduce the biological utilization rate of protein in soybean meal, cause diarrhea in young animals such as piglets, and greatly limit the application of soybean meal in young animals.

[0003] Microbial fermentation is a common method for processing soybean meal at present. On the one hand, the antinutritional factors and allergen proteins in soybean meal can be degraded by microbial treatment, and on the other hand, the macromolecular proteins without biological functional activity in soybean meal can be converted into small molecular peptides with higher absorption efficiency and certain functional activity, which is an important means to improve the quality of soybean meal and improve the nutritional value and functional characteristics. CN111826295A discloses a bacillus velezensis DP-2 (Bacillus velezensis DP-2) for soybean meal fermentation, and the preservation number is CGMCC No. 17235. The strain can secrete neutral protease and alkaline protease, and has a degradation effect on antinutritional substances such as glycinin and beta-conglycinin in soybean meal. However, the strain still has the following problems: The liquid fermentation method has high requirements for equipment, site and energy consumption, and the cost of subsequent treatment (such as drying) is also high. Solid-state fermentation has the advantages of easy operation, low water consumption, and batch production, and is a lower investment threshold for soybean meal fermentation. The strain mainly produces neutral and alkaline proteases, lacks efficient acid protease activity, and the degradation capacity in the simulated animal stomach acid environment may be limited. The protein quality of the fermentation product still has room for improvement: the acid-soluble protein content of the fermented soybean meal is 11.81%, accounting for 23.2% of the crude protein. SUMMARY

[0004] Based on the above problems, the present application provides a bacillus strain with high protease activity, particularly capable of efficiently secreting acid, neutral and alkaline proteases of the whole spectrum, and suitable for solid-state fermentation process, for improving the quality of soybean meal, improving the nutritional value and developing functional products.

[0005] The purpose of the present application is achieved by the following measures: A Bacillus halotolerans is characterized in that the strain is preserved in the Guangdong Microbial Culture Collection Center on July 15, 2024, and the preservation number is GDMCC NO: 64868. The strain is self-named CQPL9. The Bacillus halotolerans has high-yield full-spectrum protease and can efficiently decompose large protein molecules and anti-nutritional components in soybean meal.

[0006] The application also provides application of the above-mentioned Bacillus halotolerans in solid-state fermentation of soybean meal.

[0007] A method for solid-state fermentation of soybean meal comprises the following steps: weighing 30 g of soybean meal in a 250 mL conical flask, sterilizing at 121 DEG C for 15 min, then adding 27 mL of sterilized distilled water, adding 3 mL of bacterial liquid, mixing uniformly, and then culturing in a 30 DEG C incubator for 72-96 h. After fermentation, drying or freeze-drying is performed, and the dried product is crushed to 60 mesh.

[0008] A soybean meal fermentation product is fermented by the above-mentioned Bacillus halotolerans. The functional characteristics of the fermentation product are high content of small molecular proteins, low content of anti-nutritional factors, far higher ratio of acid-soluble protein to crude protein than the provisions in GB / T43745-2024, and lower content of anti-nutritional factors stachydrine and beta-conglycinin than the provisions in GB / T43745-2024.

[0009] A feed comprises the above-mentioned soybean meal fermentation product.

[0010] The application also provides a method for preparing soybean peptides by using the above-mentioned Bacillus halotolerans.

[0011] A soybean peptide is fermented by the above-mentioned Bacillus halotolerans. The functional characteristics of the soybean peptide are strong antioxidant activity.

[0012] The preparation method of the above-mentioned functional soybean peptide comprises the following steps: weighing 10 g of soybean meal fermentation product in a 1 L conical flask, adding 200 mL of deionized water, stirring uniformly, adjusting pH to 4 by using 1 mol / L HCl or NaOH, placing on a shaker for 30-60 min at 180-200 r / min, fully mixing, then centrifuging at 4 DEG C and 5000 r / min for 10 min, collecting the supernatant, and freeze-drying.

[0013] Beneficial effects The Bacillus halotolerans CQPL9 provided by the application has more broad-spectrum and more powerful protease activity. Not only can it efficiently secrete neutral protease (223.36 U / mL) and alkaline protease (123.87 U / mL), but also has high-activity acid protease (40.96 U / mL); and it can better degrade anti-nutritional factors and large molecular protein in soybean meal into small molecular substances with biological activity.

[0014] The application provides a method for solid-state fermentation of soybean meal by using the CQPL9 strain, which is easier to operate, has lower requirements for equipment and site, consumes less water, and is easier to control in the fermentation process, and is more suitable for large-scale application.

[0015] The fermented soybean meal product fermented by the strain has better protein quality. The acid-soluble protein content of the fermented soybean meal of the application reaches 18.84%, and the proportion of acid-soluble protein in crude protein reaches 36%, which is significantly higher than 11.81% and 23.2% of CN111826295A.

[0016] The application proves that the polypeptide bioactivity function (such as antioxidant activity) in the soybean meal fermented by the strain of the application can be improved through the research on the preparation of functional soybean peptides by the fermented soybean meal of Bacillus CQPL9. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Colony morphology of CQPL9 strain Figure 2 Electron microscope image of CQPL9 strain Figure 3 Phylogenetic tree of CQPL9 strain Figure 4 SDS-Page electrophoresis diagram of fermented freeze-dried powder obtained by CQPL9 strain fermentation of soybean meal Figure 5 Actual picture of soybean peptide extracted from fermented freeze-dried powder obtained by CQPL9 strain fermentation of soybean meal DETAILED DESCRIPTION

[0018] The technical solutions of the application will be further described below in combination with specific examples, but should not be understood as limitations to the application.

[0019] The technical solutions of the application will be further described below in combination with specific examples, but should not be understood as limitations to the application.

[0020] Example 1: Screening and identification of Bacillus with high protease yield 1. Isolation and screening of Bacillus with high protease yield Take 25g of fecal sample of healthy sows and place it in 225mL of normal saline, shake at 30℃ and 150r / min for 30min, and then heat treat at 80℃ for 15min. Dilute 1mL of supernatant by gradient dilution, take 0.1mL and spread on NB culture medium, and incubate at 30℃ for 24-48h. Pick single colonies in each well containing 1mL of NB liquid medium in a 96-well deep well plate, and incubate at 30℃ and 220r / min for 24h. Centrifuge at 4000r / min for 10min, and reserve the supernatant.

[0021] Skim milk medium (20 g sterilized skim milk powder was added to nutrient agar) was prepared, and a puncher was used to punch holes on the skim milk plate. 50 μL of the supernatant was injected into the holes, and the holes were incubated at 30°C for 24 h. The size of the hydrolysis ring was observed, and the strains with larger hydrolysis rings were isolated and purified.

[0022] The purified strains were inoculated on skim milk plates, and the plates were incubated at 30°C for 24 h. The diameters of the hydrolysis rings and the colonies were measured, and the strains with a larger ratio of the diameter of the hydrolysis ring to the diameter of the colony were selected as the re-screening strains.

[0023] The protease activity of the re-screening strains was determined. Four strains with a larger ratio of the diameter of the hydrolysis ring to the diameter of the colony were selected from the re-screening strains, and single colonies were inoculated in NB medium and incubated at 30°C for 24 h. Then, 1% of the inoculum was inoculated in enzyme production medium, and the medium was incubated at 30°C and 200 r / min for 48 h. After centrifugation at 8000 r / min and 4°C for 10 min, the supernatant was obtained. The proteinase activity of the obtained fermentation supernatant was determined by the Folin-phenol method.

[0024] Protease activity determination: GB / T 23527.1-2023 Enzyme preparations - Requirements - Part 1: Determination of protease activity in protease preparations - Folin method.

[0025] The protease activities of the 6 Bacillus strains are shown in the following table. CQPL9 has the highest protease activity, and the neutral protease activity, alkaline protease activity, and acid protease activity of CQPL9 are the highest, which are 223.36 U / mL, 123.87 U / mL, and 40.96 U / mL, respectively. The results are shown in Table 1.

[0026]

[0027] 2. Identification of strains The CQPL9 strain has irregular-shaped colonies with jagged edges and a rough surface with wrinkles. It does not produce pigments and is a gram-positive bacterium with rod-shaped cells. The results are shown in Figure 1 and Figure 2 .

[0028] The 16S rDNA sequencing results were compared with the NCBI database, and a phylogenetic tree was constructed using MEGA 7. The results are shown in Figure 3 . The strain is a salt-tolerant Bacillus strain (Bacillus halotolerans strain).

[0029] Example 2: Application in fermented soybean meal Select a single colony of strain CQPL9 and inoculate it into NB medium. After culturing for 12-16 hours, it becomes a first-grade seed culture. Then, inoculate it into NB liquid medium at a rate of 1% and culture for 12-16 hours to obtain a second-grade seed culture. Finally, inoculate it into soybean meal fermentation medium at a rate of 10%.

[0030] Soybean meal fermentation culture: Weigh 30g of soybean meal into a 250mL Erlenmeyer flask, sterilize at 121℃ for 15min, then add 27mL of sterilized distilled water and 3mL of bacterial solution, mix well, and ferment in a 30℃ incubator for 72-96h. After fermentation, freeze-dry, then pulverize through a 60-mesh sieve to obtain fermented soybean meal powder.

[0031] The obtained lyophilized fermentation powder was subjected to SDS-Page polyacrylamide gel electrophoresis. The specific steps were as follows: 1.0 g of lyophilized fermentation powder sample (passed through a 60-mesh sieve) was added to 20 mL of 0.03 mol / L Tris-HCl solution and soaked for 1 h (shaking once every 20 min). Then, it was centrifuged at 10000 r / min for 10 min. 80 μL of the supernatant was taken, and 20 μL of 5× sample buffer (sample and sample buffer were mixed at a 4:1 volume ratio) was added. After boiling in a water bath for 30 min, 10 μL of each sample solution was added to the gel bath. The regulated power switch was turned on, and the voltage was set to 180V for vertical electrophoresis. Electrophoresis was stopped when the blue band of the sample buffer migrated to the bottom of the gel. The gel was then carefully removed using a gel removal tool and placed in a container containing staining solution. It was stained on a shaker at 40-60 r / min for 30 min, then transferred to destaining solution and soaked overnight, changing the destaining solution 2-3 times until the gel background color returned to the initial white and the bands were clearly visible. Figure 4 The distribution and color intensity of protein bands on the electrophoresis results are used for qualitative analysis.

[0032] Depend on Figure 4 It can be seen that after fermentation, β-conglobulin and soy globulin in soybean meal are basically degraded, and the large molecular proteins are transformed into small molecular proteins of less than 10kD that are more easily digested and absorbed. The anti-nutritional factors in soybean meal are also degraded.

[0033] Example 3: Conventional Nutritional Indicators of Fermented Soybean Meal Crude protein, water-soluble protein, acid-soluble protein, volatile basic nitrogen, soy globulin, β-congruin, stachyose, and raffinose were determined using fermented soybean meal (fermented freeze-dried powder of Example 2) and soybean meal raw material before fermentation, which were passed through a 60-mesh sieve.

[0034] The specific methods for each indicator are as follows: Crude protein: The crude protein content was tested according to GB / T 6432-1994, "Test Method for Crude Protein in Feed"; Soluble protein: 1.5 g of ground (passed through a 60-mesh sieve) sample was weighed into a 250-mL beaker, 75 mL of 0.2% KOH solution was added, stirred on a magnetic stirrer for 20 min, 50 mL of the liquid was transferred into a centrifuge tube, centrifuged at 2700 r / min for 10 min, 15 mL of supernatant was aspirated, and the protein content in the supernatant was determined by the Kjeldahl method.

[0035] Water-soluble protein: 2 g of ground (passed through a 60-mesh sieve) sample was weighed into a 150-mL triangular flask, 25 mL of boric acid-phosphoric acid buffer (pH = 6.75) was added, and extraction was performed at 39°C for 1 h, then filtered with a rapid filter paper, 5 mL of filtrate was aspirated, and the protein content in the supernatant was determined by the Kjeldahl method.

[0036] Acid-soluble protein: The acid-soluble protein content was detected according to the method for detecting acid-soluble protein content in Appendix B of GB / T 22492-2008 Peptide Powder of Soybean; Volatile nitrogen: The volatile nitrogen content was detected according to GB / T 5009228-2016 Determination of Volatile Nitrogen; Glycinin: detected according to the glycinin detection kit; Beta-conglycin: detected according to the beta-conglycin detection kit; Stachyose: detected according to NY / T2218-2022 Fermented Soybean Meal as Feedstuff Raw Material; Raffinose: detected according to NY / T2218-2022 Fermented Soybean Meal as Feedstuff Raw Material.

[0037] As shown in Table 2, the contents of crude protein, water-soluble protein, and acid-soluble protein were increased by 8.15%, 667.95%, and 653.48%, respectively, the protein hydrolysis degree (soluble protein / crude protein) was increased by 20.85%, the ratio of acid-soluble protein / crude protein was increased by 591.29%, the volatile nitrogen was increased by 3.89 times, and the protein quality was improved. The anti-nutritional factors glycinin and beta-conglycin were degraded by 80.94% and 62.3%, respectively, and stachyose and raffinose were completely degraded after fermentation, reducing the damage of anti-nutritional factors to the animal body. It is shown that the use of CQPL9 Bacillus can effectively solve the problems of large molecules, poor solubility, and anti-nutritional factors in soybean meal, and has great application potential for improving the utilization value of feed.

[0038]

[0039] Example Four: Extraction and Preparation of Soybean Peptide Take 10 g of fermented soybean meal freeze-dried powder (fermented soybean meal made in Example Two) in a 1 L conical flask, add 200 mL of deionized water, stir evenly, and adjust the pH to 4 with 1 mol / L HC1 or NaOH, place it on a shaker at 180-200 r / min for 30-60 min, mix thoroughly, then centrifuge at 4°C and 5000 r / min for 10 min, collect the supernatant, and freeze-dry. The obtained soybean peptide sample is shown in Figure 5 The crude protein and acid-soluble protein contents are 88.5% and 75.5%, respectively.

[0040] Example Five: Antioxidant Activity of Soybean Peptide Take 1 g of soybean peptide sample prepared in Example Four and dissolve it in 100 mL of deionized water, then use the DPPH method and ATBS method in GB / T39100-2020 to determine the DPPH free radical scavenging rate and ABTS free radical scavenging rate of the soybean peptide, and the results are shown in Table 3. It is shown that the soybean peptide extracted from fermented soybean meal by CQPL9 strain has good antioxidant function.

[0041]

Claims

1. A Bacillus sp. characterized by: The strain was deposited in Guangdong Microbial Culture Collection Center on July 15, 2024, and the deposit number is GDMCC NO: 64868.

2. The bacillus of claim 1 in the application of solid-state fermentation of soybean meal.

3. A soybean meal fermentate fermented by the bacillus of claim 1.

4. A method of solid state fermentation of soybean meal comprising the steps of: Weigh the soybean meal and sterilize it at 121℃ for 15 minutes, then add a small amount of distilled water, add the bacterial solution of the bacillus of claim 1, mix well, and ferment for 72-96 hours; after the fermentation is completed, dry or freeze-dry, and then crush to 60 mesh after drying.

5. A feed comprising the soybean meal fermentate of claim 3.

6. A soybean peptide prepared by fermenting the bacillus of claim 1.

7. A method for preparing functional soybean peptides, comprising the following steps: weighing the soybean meal fermentate of claim 3, adding water, stirring uniformly, adjusting the pH to 4, placing on a shaker at 180-220 r / min for 30-60 min, mixing thoroughly, then centrifuging at 4℃ and 5000 r / min for 10 min, collecting the supernatant, and freeze-drying.

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