Bacillus velezensis with high yield of hydrolytic enzymes and application thereof

Abstract

This invention belongs to the field of microbiology and discloses a strain of *Bacillus velezensis* that produces high levels of hydrolytic enzymes and its applications. The *Bacillus velezensis* strain is named *Bacillus velezensis* 172D4, with accession number GDMCC NO: 67771, and was deposited at the Guangdong Provincial Microbial Culture Collection Center on January 29, 2026. This *Bacillus velezensis* strain can simultaneously produce high levels of protease, amylase, and cellulase, possessing a complete enzyme system; it can be used for fermenting soybean meal, improving the nutritional quality and flavor of soybean meal; moreover, it is free of toxic factors and is a safe strain.

Description

Technical Field

[0001] This invention belongs to the field of microbiology, and specifically relates to a strain of Bacillus belye that produces high levels of hydrolytic enzymes and its applications. Background Technology

[0002] Bacillus velezensis is a Gram-positive bacterium with excellent characteristics such as strong metabolic activity, rapid growth rate, and good environmental tolerance. This strain has obtained GRAS (Generally Recognized As Safe) certification from the US FDA and is listed in the Ministry of Agriculture and Rural Affairs of the People's Republic of China's Announcement No. 2045, "Catalogue of Feed Additive Varieties," and related supplementary announcements. It is a microbial variety permitted for direct addition by the state, possessing clear legal basis and commercial application qualifications.

[0003] Bacillus bellis has broad application prospects in agricultural production, livestock and poultry breeding, industrial fermentation, and environmental remediation. In the fermentation of plant protein raw materials, this bacterium, due to its ability to secrete various extracellular enzymes, is widely used to improve the degradation efficiency of plant proteins, release nutrients, and improve the quality of fermentation products. Soybean meal, as the most important plant protein raw material, has fermentation quality that not only affects the feed industry but also directly impacts the economic benefits and product competitiveness of food products such as plant-based seasonings and fermented condiments.

[0004] A systematic analysis of recently granted patents related to Bacillus belyssus (including CN116515792B, CN115261316B, CN114908005B, CN113528414B, CN112831443B, CN115851561B, CN114196590B, CN116004473B, CN114107127B, CN115927085B, etc.) reveals the following common defects in the existing technology:

[0005] (1) The enzyme system is simple and lacks the ability of multiple enzymes to work together to degrade.

[0006] Existing reports mostly focus on single enzymatic functions or agricultural biocontrol. For example, CN116515792B discloses a strain of *Bacillus belyssus* that produces a high amount of neutral protease, mainly used for protein degradation; CN114908005B discloses a strain that produces cellulase, used for straw degradation; and CN113528414B discloses a strain that produces amylase. None of these strains simultaneously possess the ability to produce high amounts of protease, amylase, and cellulase. However, the proteins in soybean meal are tightly wrapped by cell wall polysaccharides (cellulose, hemicellulose), and a single protease can only degrade the exposed protein components, making it difficult to reach the wrapped parts, resulting in low overall degradation efficiency. Although CN114107127B reports a strain of *Bacillus belyssus* that produces three enzymes simultaneously, this patent only discloses enzyme activity data, does not apply its multi-enzyme synergistic ability to soybean meal fermentation and systematically evaluate the effect, nor does it elucidate the multi-enzyme genetic basis at the genomic level.

[0007] (2) The flavor and quality of fermentation products vary, making it difficult to meet the needs of condiments.

[0008] Soybean meal itself has an unpleasant beany odor, severely limiting its palatability in food and condiments. However, none of the aforementioned published patents address the analysis of volatile flavor compounds in soybean meal fermented by *Bacillus belyssioides*, nor do they report the formation of characteristic aroma compounds such as pyrazines and esters. For plant-based condiments, rich roasted, nutty, and savory aromas are key factors determining product quality and market acceptance. The lack of existing technology in this area severely limits the practical application value of *Bacillus belyssioides* in high-end condiments and plant-based flavoring ingredients.

[0009] (3) Neglecting peptide molecular weight distribution and bioactivity verification

[0010] Existing technologies often use the total amount of acid-soluble proteins as an indicator of protein degradation, but this indicator cannot reflect the molecular weight distribution of degradation products. Studies have shown that peptides in different molecular weight ranges have significant differences in nutrient absorption efficiency, flavor characteristics, and bioactivity, with peptides in the 189 Da to 3000 Da range considered to have the highest functional value. However, none of the aforementioned patents analyzed the molecular weight distribution of peptides in the fermentation products, nor did they achieve targeted enrichment of specific functional peptides. Even when some patents mention "small peptides," they did not verify through cell models whether they truly possess antioxidant, blood pressure-lowering, or other bioactivities.

[0011] (4) Phenotype-genotype association analysis was missing, and the genetic basis of enzyme production and antibacterial activity is unclear.

[0012] Current technologies primarily focus on enzyme production capacity or plant biocontrol functions, with limited systematic research on beneficial animal characteristics (such as inhibition of animal pathogens) and the potential for secondary metabolite synthesis. More importantly, most studies remain at the phenotypic level, lacking systematic verification from genotype to phenotype, resulting in an unclear genetic basis for strain function.

[0013] (5) Insufficient security verification

[0014] Although Bacillus belesiensis is generally recognized as a safe strain, the genomes of different strains vary, and some strains may still carry potential virulence factors or drug resistance genes. Most of the aforementioned patents rely solely on traditional phenotypic observations and do not conduct genome-wide virulence factor screening, resulting in a low level of safety evidence and limiting the commercial application of these strains in the food and condiment industries.

[0015] In summary, existing technologies urgently need to address the following technical issues: providing a safe-sourced Bacillus vesiculosus strain that can efficiently degrade soybean meal through multi-enzyme synergy, directionally produce functional peptides, and impart a rich roasted, nutty, and savory aroma to the fermentation products (suitable as a raw material for plant-based seasonings). Simultaneously, it should possess probiotic properties that inhibit animal pathogens, establish phenotypic-genotypic evidence linking enzyme production and antibacterial activity, and complete genome-wide screening of virulence factors and drug resistance genes. This would provide a complete scientific basis for the industrial application of soybean meal in high-end seasonings and plant-based flavoring ingredients. Summary of the Invention

[0016] To address the problems existing in the prior art, the primary objective of this invention is to provide a strain of Bacillus belesi that produces high levels of hydrolytic enzymes.

[0017] Another object of the present invention is to provide the application of the above-mentioned Bacillus belyssus.

[0018] The objective of this invention is achieved through the following technical solution:

[0019] A strain of Bacillus velezensis, which produces high levels of hydrolytic enzymes, named Bacillus velezensis 172D4, with accession number GDMCC NO: 67771, was deposited on January 29, 2026, at the Guangdong Provincial Microbial Culture Collection Center of the Institute of Microbiology, Guangdong Academy of Sciences, located on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0020] The application of the above-mentioned high-yield hydrolytic enzyme Bacillus belye in the preparation of hydrolytic enzymes.

[0021] The hydrolytic enzyme is at least one of protease, amylase and cellulase.

[0022] Application of the above-mentioned high-yield hydrolytic enzyme Bacillus belyi in soybean meal fermentation.

[0023] A method for preparing fermented soybean meal includes the following steps:

[0024] (1) The above-mentioned Bacillus belye was activated and cultured, and the culture medium was separated into solid and liquid. The bacterial cells were taken, and the bacterial cells were resuspended to obtain the fermentation agent.

[0025] (2) Inoculate the fermenting agent into sterilized water-containing soybean meal for fermentation to obtain fermented soybean meal.

[0026] The culture medium used in the activation culture described in step (1) is preferably TSB medium.

[0027] The activation culture operation described in step (1) is preferably performed by first streaking the culture plate, and then inoculating the colonies grown on the plate into the liquid culture medium and shaking the culture.

[0028] The preferred conditions for streak culturing are inverted culture at 35-38℃ for 16-30 hours; more preferably, inverted culture at 37℃ for 24 hours.

[0029] The preferred conditions for the shaking culture are 35–38°C and 150–250 rpm for 36–72 h; more preferably, 37°C and 200 rpm for 48 h.

[0030] The preferred method for solid-liquid separation in step (1) is centrifugation.

[0031] The resuspension solution mentioned in step (1) is preferably sterile physiological saline.

[0032] The concentration of the fermenting agent mentioned in step (1) is 10. 7 ~10 8 CFU / g.

[0033] The water-containing soybean meal mentioned in step (2) is preferably obtained by mixing soybean meal and water at a material-to-liquid ratio of 1:0.5 to 1:2 (w / w); more preferably, it is obtained by mixing soybean meal and water at a material-to-liquid ratio of 1:1 (w / w).

[0034] The sterilization conditions described in step (2) are preferably 115-121℃ for 15-30 min; more preferably 121℃ for 15-20 min.

[0035] The amount of fermentation agent in step (2) is calculated as 1% to 10% of the mass (g) of the water-containing soybean meal in its volume (mL); preferably, it is calculated as 4% to 6% of the mass (g) of the water-containing soybean meal in its volume (mL); more preferably, it is calculated as 5% of the mass (g) of the water-containing soybean meal in its volume (mL).

[0036] The fermentation conditions described in step (2) are static culture at 30-40℃ for 24-72 h; preferably static culture at 37℃ for 48 h.

[0037] A fermented soybean meal is obtained by the above preparation method.

[0038] Compared to unfermented soybean meal, the fermented soybean meal exhibits improved nutritional quality and enhanced volatile flavor compounds. Specifically, it reduces trypsin inhibitors by 81.6%, increases reducing sugars by 10 times, and increases acid-soluble proteins by 122%. It also selectively enriches 500-189 Da active peptides (accounting for 44.29%), significantly improving cellular antioxidant capacity (SOD and CAT are restored, and MDA is reduced by 50%). Simultaneously, it produces a large amount of aroma compounds such as pyrazines and ketones, eliminating the beany taste.

[0039] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0040] (1) The Bacillus belyssus 172D4 disclosed in this invention has the characteristics of high production through enzyme synergy. It can simultaneously produce high levels of protease (24.67 U / mL), amylase (45.33 U / mL), and cellulase (20.33 U / mL), with a complete enzyme system. Furthermore, a multi-enzyme gene cluster has been identified at the gene level: a total of ≥54 protease-related genes (covering 6 major categories such as serine, metalloids, and threonine), 8 amylase-related genes (GH13 family), and 13 cellulase / hemicellulase-related genes (including GH1, GH5, GH51, GH3 families).

[0041] (2) The Bacillus vesiculosus 172D4 fermented soybean meal disclosed in this invention has excellent comprehensive performance. The nutritional factor trypsin inhibitor is reduced by 81.6%, reducing sugar is increased by 10 times, and acid-soluble protein is increased by 122%. It can selectively enrich 500-189Da active peptides (accounting for 44.29%), significantly improve cell antioxidant capacity (SOD and CAT are restored, and MDA is reduced by 50%). At the same time, it produces a large number of aroma substances such as pyrazines and ketones, eliminating the beany smell.

[0042] (3) The whole genome of Bacillus belyssus 172D4 disclosed in this invention contains 12 secondary metabolic gene clusters, of which 7 can clearly produce high levels of probiotic / antibacterial substances such as surfactants and cytokinins; and combined with the whole genome, it was found to be a safe strain with no virulence factors. Attached Figure Description

[0043] Figure 1 This is a colony morphology diagram of the Bacillus velezensis 172D4 strain of the present invention.

[0044] Figure 2This is a microscopic image of the Bacillus velezensis 172D4 strain of the present invention.

[0045] Figure 3 This is a plate image showing the initial screening results of Bacillus velezensis protease production in an embodiment of this invention; from left to right, they are 172D4 and LDH6.

[0046] Figure 4 The image shows the results of the initial screening of amylase-producing Bacillus velezensis according to an embodiment of this invention; from left to right, they are 172D4 and LDH6.

[0047] Figure 5 This is a diagram showing the results of the initial screening of cellulase production by Bacillus velezensis in an embodiment of this invention; from left to right, they are 172D4 and LDH6.

[0048] Figure 6 This is an SDS-PAGE image of fermented soybean meal from Bacillus velezensis 172D4 and the control strain Bacillus velezensis LDH6. Detailed Implementation

[0049] To better understand the present invention, it will be further described below with reference to the accompanying drawings and specific embodiments. However, the implementation of the present invention is not limited thereto. The described embodiments are some, but not all, of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0050] Example 1

[0051] (1) Isolation and purification of strains

[0052] First, 25g of fermented black soybeans was weighed into 225mL of sterile physiological saline. After homogenization, 1mL of the mixture was added to 9mL of sterile physiological saline, and the mixture was spread onto LB agar plates using a 10-fold serial dilution method. The plates were then incubated upside down at 37℃ for 24 hours. Colonies showing morphological differences and good growth on the LB agar plates after incubation were selected and streaked until uniform single colonies appeared. After activation with LB liquid medium, the colonies were stored at -80℃. Morphological observation yielded 80 Bacillus strains from the fermented black soybeans. 16S rDNA identification was performed on all 80 strains, further confirming their genus *Bacillus*. These 80 strains were then subjected to initial enzyme production screening (see Example 3 for details). The screening revealed that *Bacillus belyssiensis* 172D4 exhibited a large and clear clear zone on all three plates, indicating that 172D4 was the dominant strain.

[0053] (2) Colony morphology observation and microscopic examination

[0054] Colony morphology was observed on LB agar plates and then Gram-stained and examined under a microscope. Colony images are shown below. Figure 1 As shown, on TSB solid plates, the colonies appear milky white, smooth, moist, slightly raised in the center, and soft and viscous. Microscopic observation confirmed this. Figure 2 The bacteria were found to be Gram-positive, and the cells were rod-shaped.

[0055] (3) 16S rDNA sequencing identification

[0056] The commonly used method is to perform PCR using the most common universal primers for bacteria, 27F and 1492R. The PCR reaction system is as follows: 2 μL DNA template, 2 μL primer 27F (10 μmol / L), 2 μL primer 1492R (10 μmol / L), 20 μL 2×PCR Mix, and 24 μL ultrapure water. The PCR reaction program is as follows: 94℃ pre-denaturation for 5 min; 94℃ denaturation for 30 s, 46℃ annealing for 30 s, 72℃ extension for 1 min, for 35 cycles; 72℃ final extension for 10 min. The obtained PCR product was sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The 16S rDNA sequence is shown in SEQ ID NO.1. Homology search of the sequencing results was performed using the BLAST tool in NCBI, and a phylogenetic tree was constructed using MEGA 7.0, identifying it as Bacillus belesiensis.

[0057] The strain was named Bacillus belyssus 172D4. It was deposited at the Guangdong Institute of Microbiology Culture Collection Center on January 29, 2026, with accession number GDMCC NO: 67771.

[0058] 27F: 5'-AGAGTTTGATCCCTGGCTCAG-3';

[0059] 1492R: 5'-GGTTACCTTGTTCGACTT-3'.

[0060] 16S rDNA

[0061]

[0062] Example 2

[0063] (1) Whole genome analysis

[0064] Bacillus velezensis strain 172D4, stored at -80℃ for 100 μL, was inoculated into 20 mL of sterile TSB medium and cultured at 37℃ for 24 h. After two consecutive passages, the bacterial cells were collected, flash-frozen in liquid nitrogen, and then rapidly preserved on dry ice and sent to Shanghai Biomarker Sequencing Co., Ltd. for whole-genome sequencing.

[0065] (2) Analysis of protease-related genes

[0066] The results of whole-genome annotation of protease-related genes are shown in Table 1. Bacillus belyss 172D4, after whole-genome annotation, identified ≥54 protease-related genes, covering six major catalytic types: serine, metalloproteinases, threonine, cysteine, and aspartic acid. Compared with published patents / strains: strain X49 reported only 11 serine protease genes; strain WH-7 identified only 7 protease genes; and strain CN114107127B (G02) only reported enzyme activity phenotypes without whole-genome protease gene annotation. The present invention's 172D4 strain is superior to the comparative strains in terms of the number of protease genes, family diversity, and completeness of molecular evidence.

[0067] Table 1

[0068]

[0069] Annotation of cellulase and amylase-related genes: As shown in Table 2, the whole-genome annotation of 172D4 in this invention shows that, for amylase, there are 8 GH13 family genes; for cellulase, there are GH1 (8 genes), GH5 (2 genes), GH51 (2 genes), and GH3 (1 gene); for hemicellulase, there are GH43 (4 genes), GH30 (2 genes), GH26 (1 gene), GH11 (1 gene), GH16 (1 gene), and GH113 (1 gene). Compared with published patent applications: CN118599720A (ZM2024) only describes the "high-yield cellulase" phenotype and does not disclose the number of GH family genes; CN114058532A (NKY1) confirms multi-enzyme ability through enzyme activity, but does not perform CAZyme family system analysis; CN115637240A (FLU-1) has performed whole-genome sequencing, but does not classify or count the number of GH family genes. Existing patent applications have not systematically disclosed the specific number of GH family genes related to amylase and cellulase. This invention provides complete GH family gene distribution data for the first time in a Bacillus belysus patent, achieving a closed loop of evidence from phenotype to genotype.

[0070] Table 2

[0071]

[0072] (3) Analysis of secondary metabolic gene clusters

[0073] The antiSMASH software was used to predict the secondary metabolite synthesis gene clusters of the whole genome sequence of Bacillus bereaves 172D4. As shown in Table 3, the genome of this strain contains 12 secondary metabolite synthesis gene clusters, covering various types such as NRPS, transAT-PKS, T3-PKS, PKS-like, RiPP-like, and terpene. Among them, 7 gene clusters could clearly predict their metabolites with a similarity ≥82%, including Locillomycin (100%), Surfactin (82%), Difficidin (86%), Fengycin (93%), Bacillaene (100%), Macrolactin H (100%), and Bacillibactin (100%), indicating that this strain has good biocontrol and probiotic potential.

[0074] Compared to publicly disclosed Bacillus bereaves patent strains (such as CN114107127B, which only reports a total of 12 gene clusters without clearly distinguishing between known and unknown gene clusters; and ES2-4 strain, which has 14 gene clusters, of which 9 are clearly predicted), 7 out of the 12 gene clusters in this invention 172D4 can clearly predict metabolites with a similarity ≥82%, and 5 of them have a similarity of 100%, indicating higher reliability and abundance of predicted metabolites. Furthermore, among the 5 unknown gene clusters (2 RiPP-like, 2 terpene, and 1 PKS-like), the PKS-like cluster has only 7% similarity to the Butirosin-like cluster, suggesting the potential presence of novel active compounds with further development value.

[0075] Table 3

[0076]

[0077] (4) Virulence gene analysis: The genome data of Bacillus velezensis was compared with the VFDB database. With a similarity of more than 80% as the screening condition, no virulence factors were screened, indicating that the bacterium has good safety.

[0078] Example 3

[0079] (1) Determination of the protease production capacity of 172D4

[0080] 1) Initial screening of protease production capacity:

[0081] Seed culture preparation: Bacillus velezensis 172D4 stored at -80℃ was streaked on a TSB solid plate for activation. Single colonies were picked and inoculated into an Erlenmeyer flask containing 20mL of TSB liquid medium. The culture was carried out at 37℃ and 200r / min for 24h to obtain Bacillus velezensis seed culture.

[0082] Culture method: Using a sterile toothpick, inoculate *Bacillus belye* seed culture onto a protease selection medium plate. After incubation at 37°C for 48 hours, observe the clear zone around the colony and calculate the ratio of the clear zone diameter D to the colony diameter d. Protease selection medium: Contains 100 g / L skim milk powder and 20 g / L agar.

[0083] 2) Secondary screening of protease production capacity:

[0084] Preparation of crude enzyme solution: The bacterial culture was inoculated into TSB medium at a rate of 1% v / v and cultured at 37℃ with shaking at 220 rpm for 24 h. The activated bacterial strain was then inoculated into protein fermentation medium at a rate of 5% v / v and cultured at 37℃ with shaking at 220 rpm for 48 h. The fermentation broth was collected and centrifuged at 4℃ and 8000 rpm for 15 min. The supernatant was filtered through a 0.22 μm filter membrane to obtain the crude enzyme solution. Protease fermentation medium: Casein 5 g / L, yeast extract 1 g / L, glucose 1 g / L, dipotassium hydrogen phosphate 4 g / L, potassium dihydrogen phosphate 0.5 g / L, magnesium sulfate 0.1 g / L.

[0085] Enzyme activity assay: The enzyme activity of the protease produced by the strain was determined using the Folin-phenol method. 1.0 mL of the crude enzyme solution to be tested and 2.0 mL of 100 g / L trichloroacetic acid solution were added to the blank tube. 1.0 mL of the crude enzyme solution to be tested and 1.0 mL of phosphate buffer (pH 7.0, 50 mmol / L) to prepare a 1 g / 100 mL casein solution were added to the sample tube. Both tubes were incubated at 40 °C for 10 min. After the reaction, 1.0 mL of casein reagent was added to the blank control tube, and 2.0 mL of 100 g / L trichloroacetic acid solution was added to the sample tube. After centrifugation, 1.0 mL of the supernatant was collected from each tube. 42 g / L sodium carbonate solution and 10-fold diluted Folin-phenol reagent were added to the supernatant. The mixture was incubated at 40 °C for 20 min. After the reaction was complete, 200 μL of the reaction solution was measured at 680 nm using a microplate reader.

[0086] Definition of protease activity: Under conditions of 40℃ and pH 7.0, the production of 1 μg of tyrosine per minute by hydrolyzing casein per mL of crude enzyme solution is defined as 1 unit of protease activity (U / mL).

[0087] Calculation formula: U = (ΔA × K × V1) / (V2 × t);

[0088] In the formula: U is the protease activity (U / mL); ΔA is the difference in absorbance between the sample tube and the blank control tube; K is the slope of the standard curve (μg / absorbance unit); V1 is the total volume of the reaction system (mL); V2 is the volume of crude enzyme solution taken during the determination (mL); t is the enzymatic hydrolysis reaction time (min).

[0089] (2) Determination of the amylase production capacity of 172D4

[0090] 1) Initial screening of amylase production capacity:

[0091] Seed liquid preparation: Same as the seed liquid preparation in step (5).

[0092] Culture method: Using a sterile toothpick, seed culture of *Bacillus velezensis* was inoculated onto an amylase selection medium plate. After incubation at 37°C for 48 hours, Lugol's iodine solution was added to the plate. Utilizing the colorimetric reaction between iodine and starch, strains secreting amylase hydrolyzed the surrounding starch, forming a transparent hydrolysis zone. This method allowed for rapid screening of enzyme-positive strains. The ratio of the transparent zone diameter D to the colony diameter d (D / d) was recorded. The amylase selection medium consisted of: 10 g / L soluble starch, 5.0 g / L peptone, 3.0 g / L beef extract, 5.0 g / L sodium chloride, and 20 g / L agar, with deionized water as the solvent.

[0093] 2) Secondary screening of amylase production capacity:

[0094] Preparation of crude enzyme solution: The preparation of crude enzyme solution is basically the same as in step (5), except that the protease fermentation medium is replaced with amylase fermentation medium. Amylase fermentation medium: soluble starch 20 g / L, peptone 20 g / L, disodium hydrogen phosphate 5 g / L, MgSO4·7H2O 0.1 g / L, sodium chloride 0.1 g / L.

[0095] Enzyme activity assay: The DNS method was used to determine the activity of amylase, as follows: 0.5 mL of sterile amylase fermentation medium was added to the blank tube, and 0.5 mL of crude enzyme solution was added to the experimental group. 1.0 mL of preheated citrate buffer (0.05 M, pH 5.0) at 40℃ was added to both tubes and mixed well. Then, 2.0 mL of starch solution with a mass-to-volume ratio of 1% (g / mL) was added. After incubating in a 40℃ water bath for 30 min, 2.0 mL of DNS reagent was added to each tube, and the mixture was boiled for 5 min. The tubes were then immediately cooled with ice water, 5.0 mL of distilled water was added, and the mixture was shaken well. The OD value was measured at 540 nm.

[0096] Enzyme activity unit (U): The amount of enzyme required to hydrolyze starch to produce 1 μg of glucose in 1 minute at 40℃ using 1 mL of crude enzyme solution is defined as 1 enzyme activity unit. The formula for calculating amylase activity is as follows:

[0097] The formula for calculating amylase activity is: U = (x × n × 1000) / (V × T);

[0098] In the formula: U is the amylase activity (U / mL); x is the glucose concentration in the standard curve corresponding to the absorbance value, mg / mL; n is the dilution factor of the fermentation supernatant; V is the volume of the supernatant, mL; T is the reaction time, min.

[0099] (3) Determination of 172D4 cellulase production capacity

[0100] 1) Initial screening of cellulase production capacity:

[0101] Seed liquid preparation: Same as the seed liquid preparation in step (5).

[0102] Culture method: Using a sterile toothpick, inoculate *Bacillus belye* seed culture onto a CMC-Na agar plate. After incubation at 37℃ for 48 h, stain with Congo red solution (1 mg / mL) for 1 h, then wash with NaCl (1 mol / L) solution. Calculate the ratio of the clear zone diameter D to the colony diameter d. The composition of the CMC-Na agar plate is as follows: CMC-Na 10 g / L, (NH4)2SO4 4.0 g / L, peptone 1.0 g / L, MgSO4·7H2O 0.5 g / L, KH2PO4 1.0 g / L, agar 20 g / L, and deionized water as the solvent.

[0103] 2) Secondary screening of cellulase production capacity:

[0104] Preparation of crude enzyme solution: The preparation of crude enzyme solution is basically the same as in step (5), except that the protease fermentation medium is replaced with cellulase fermentation medium. The composition of cellulase fermentation medium is as follows: CMC-Na 10 g / L, yeast extract 3 g / L, peptone 5 g / L, sodium chloride 1 g / L, magnesium sulfate 1 g / L, dipotassium hydrogen phosphate 1 g / L, and deionized water as solvent.

[0105] Cellulase activity assay: Take 0.5 mL of crude enzyme solution, mix with 1.0 mL of citrate buffer (0.05 M, pH 5.0), add 2.0 mL of 1% carboxymethyl cellulose solution (1 g sodium carboxymethyl cellulose / 100 mL water), react in a 50℃ water bath for 30 min, then add 2.0 mL of DNS reagent to terminate the reaction, boil in a boiling water bath for 5 min, cool in ice water, add 5 mL of distilled water and mix well. Take 200 μL of the mixture and measure the absorbance at 540 nm using a microplate reader. Plot a standard curve using glucose.

[0106] Enzyme activity unit definition: The amount of enzyme required to hydrolyze CMC-Na to produce 1 μg of glucose in 1 min at 40℃ using 1 mL of crude enzyme solution is defined as 1 enzyme activity unit (U). The formula for calculating cellulase activity is as follows:

[0107] Cellulase activity U = (x × n × 1000) / (V × T);

[0108] x represents the glucose concentration in the standard curve corresponding to the absorbance value, in mg / mL; n represents the dilution factor of the fermentation supernatant; V represents the volume of the supernatant, in mL; and T represents the reaction time, in min.

[0109] (4) Test results

[0110] Using a previously screened Bacillus belye LDH6 strain as a control strain, the detection of its protease, amylase, and cellulase production was the same as that of 172D4. The results are as follows:

[0111] The initial screening results for protease production by Bacillus velezensis 172D4 are as follows: Figure 3 As shown, a clear and distinct transparent zone can be seen around the strain, indicating that the strain can secrete proteases to degrade the skim milk powder around the strain, and the D / d ratio reached 1.98±0.05.

[0112] The initial screening results for amylase production by Bacillus velezensis 172D4 are as follows: Figure 4 As shown, a clear transparent zone was observed to form around the strain after treatment with iodine solution, and the D / d ratio reached 2.17±0.05, indicating that the strain has a good ability to produce amylase.

[0113] Preliminary screening results for cellulase production by Bacillus velezensis 172D4 are as follows: Figure 5 As shown, after Congo red staining and NaCl washing, a clear zone was observed around the strain, with a D / d value of 4.36±0.13. In contrast, the clear zone D / d of strain LDH6 was 3.43±0.36. This indicates that 172D4 has a stronger cellulose degradation ability and can secrete cellulase to degrade carboxymethyl cellulose into cellobiose and glucose, which cannot bind with Congo red, thus forming a clear zone.

[0114] The clear zone sizes of the comparative strain LDH6 on the three primary screening plates for protease, amylase, and cellulase were 1.98±0.1, 1.34±0.3, and 3.43±0.36, respectively. It can be seen that the protein production capacity of the comparative strain LDH6 in the primary screening is not much different from that of the strain 172D4 of the present invention, but its capacity to produce cellulase and amylase is significantly lower than that of the strain 172D4 of the present invention.

[0115] Enzyme activity assays showed that *Bacillus belyssiensis* 172D4 possessed protease, amylase, and cellulase activities, with protease activity of 24.67±0.33 U / mL, amylase activity of 45.33±0.58 U / mL, and cellulase activity of 20.33±0.58 U / mL. In comparison, the protease, amylase, and cellulase activities of the control strain LDH6 were 21.33±0.03 U / mL, 26.33±0.1 U / mL, and 15.33±0.23 U / mL, respectively. This indicates that strain 172D4 of the present invention has a strong ability to produce multiple enzymes, synergistically degrading proteins, polysaccharides, and cellulose substrates, providing a foundation for soybean meal fermentation and plant raw material degradation, while also improving the nutritional composition and flavor characteristics of the fermentation products.

[0116] Example 4

[0117] (1) Preparation and detection of fermented soybean meal samples

[0118] 1) Preparation of Bacillus belysin seed culture

[0119] First, using a sterile inoculation loop, Bacillus belye was streaked onto sterile TSB solid medium and incubated upside down at 37°C for 24 hours. Single colonies were then picked and inoculated into TSB liquid medium and incubated at 37°C with shaking at 200 rpm for 48 hours. Afterward, the bacterial culture was centrifuged at 8000 rpm for 5 minutes, the supernatant was discarded, and the bacterial sludge was collected. The sludge concentration was adjusted to 10% using sterile physiological saline. 8 Fermentation agent with CFU / g.

[0120] The Bacillus species used were Bacillus 172D4 and Bacillus LDH6 for comparison.

[0121] 2) Solid-state fermented soybean meal

[0122] Accurately weigh 30g of soybean meal and add it to an Erlenmeyer flask containing 30g of sterile water. Stir well and sterilize at 121℃ for 15 minutes to obtain sterilized soybean meal with moisture. Inoculate with 5.0% bacterial fermentation inoculum based on the initial weight of the soybean meal (i.e., add 5mL of 10% bacterial inoculum per 100g of soybean meal). 8The starter culture was prepared in CFU / g, with 3 replicates per group. The fermentation was carried out at a constant temperature of 37℃ for 48 hours. After fermentation, the fermented feed was dried at 60℃ and then passed through a 60-mesh sieve to obtain fermented soybean meal samples.

[0123] The experimental group consisted of samples fermented with a starter culture prepared from Bacillus belyssus 172D4, the control group consisted of samples fermented with a starter culture prepared from Bacillus belyssus LDH6 isolated in the laboratory, and the control group consisted of unfermented soybean meal.

[0124] 3) The assay method for trypsin inhibitors shall be performed in accordance with GB / T21498-2008.

[0125] 4) The reducing sugar determination method uses the DNS method, and the specific method is as follows:

[0126] Resuspend 0.4 g of sample in 4 mL of sterile distilled water, extract by shaking at 37°C and 150 rpm for 30 min, then centrifuge at 6000 rpm for 2 min. Filter the supernatant through a 0.45 μm filter membrane as the test solution. Take 1 mL of the test solution, mix thoroughly with 1.5 mL of DNS reagent, heat in a boiling water bath for 5 min, rapidly cool to room temperature, dilute with 7.5 mL of ultrapure water, and measure the absorbance at 540 nm. For the standard curve, use glucose as the standard. Prepare solutions of concentrations of 0, 0.2, 0.4, 0.6, 0.8, and 1.0 g / L, and measure the absorbance using the same DNS method. Perform linear regression of the absorbance against glucose concentration to obtain the standard curve equation for sample quantification.

[0127] The reducing sugar content is calculated as follows:

[0128] Reducing sugar content (%) = (C×V)×100% / (m×1000);

[0129] In the formula, C is the concentration of the test solution calculated based on the glucose standard curve (unit: mg / mL); V is the total volume of the extract (mL); and m is the weight of the sample (g).

[0130] 5) The method for determining acid-soluble proteins is as follows:

[0131] Weigh 0.50 g of the sample into a 10 mL stoppered test tube, add 5 mL of ultrapure water, vortex to mix for 1 min, then place in a 45℃ constant temperature water bath shaker and extract at 150 rpm for 1 h. After extraction, add an equal volume (5 mL) of 10% v / v trichloroacetic acid (TCA) solution to a centrifuge tube, vortex to mix for 30 s, let stand at room temperature for 30 min, then centrifuge the mixture at 25℃ and 8000×g for 20 min. Filter the supernatant through a 0.22 μm aqueous filter to obtain the acid-soluble protein test solution. Prepare the working solution and plot the bovine serum albumin (BSA) standard curve according to the BCA protein quantification kit instructions. After performing the colorimetric reaction, measure the absorbance at 562 nm wavelength. Three replicates were set for each sample.

[0132] The calculation of acid-soluble proteins is as follows:

[0133] Acid-soluble protein content (mg / g): X = C × V / m;

[0134] In the formula: X - acid-soluble protein content in the sample (mg / g); C - acid-soluble protein concentration in the test solution obtained from the standard curve (mg / mL); V - total volume of the sample after extraction (mL); m - sample weight (g).

[0135] 6) The changes in protein molecular weight before and after soybean meal fermentation were determined by SDS-PAGE. The specific steps are as follows:

[0136] Preparation of fermented soybean meal protein sample solution: Fermented soybean meal protein was extracted using an alkali dissolution and acid precipitation method. The extracted protein was quantified using a BCA protein quantification kit to obtain a protein solution of 2 mg / mL. 10 μL of protein solution was added to 40 μL of loading buffer, reacted in a boiling water bath for 5-10 minutes, and then centrifuged for later use.

[0137] Electrophoresis gel preparation: Prepare the electrophoresis gel according to the 12.5% ​​PAGE gel rapid preparation kit. Sample loading: Add 10 μL of the test solution to the sample well. Gel running: After loading the sample, run at 80V for 30 min, and then run at 120V for the entire process. After gel running, stain with Coomassie Brilliant Blue R-250 for 30 min, then destain overnight at room temperature with destaining solution until the background color is clear, and then observe the bands.

[0138] 7) The relative molecular weight and distribution range of peptides are determined using high gel filtration chromatography, which mainly includes the following steps:

[0139] First, regarding the instrumentation and chromatographic conditions, an Agilent 1260 Infinity II high-performance liquid chromatograph was used, equipped with a variable wavelength detector (VWD) and OpenLab CDS Data Analysis 2.7 software. A TSK gel G2000SWXL (300mm × 7.8mm, 0.5μm) column was used. The mobile phase was prepared by mixing acetonitrile, water, and trifluoroacetic acid in a volume ratio of 45:55:0.1. The flow rate of the mobile phase was set to 1 mL / min, and the injection volume was 20 μL. The absorbance was monitored in the wavelength range of 200–350 nm, and the absorbance value at 214 nm was recorded. Calibration curves were plotted using co-albumin (75000 Da), ovalbumin (43000 Da), cytochrome C (12384 Da), aprotinin (6512 Da), and vitamin B12. 12 Six substances, including (1855 Da) and glutathione (307 Da), were used as external standards. A standard solution of 1.0 mg / mL was prepared using the mobile phase and filtered through a 0.22 μm filter. A relative molecular mass calibration curve and its regression equation were established with the logarithm of the relative molecular mass (lgMr) of each standard as the ordinate and the corresponding retention time (tR) as the abscissa. Sample preparation: 20.0 mg of the sample to be tested was accurately weighed into a volumetric flask, diluted to the mark with the mobile phase, and sonicated for 10 min to ensure complete dissolution. The sample was then filtered through a 0.22 μM filter to obtain the test solution. The concentration of the test solution was adjusted to 1.0 mg / mL using the mobile phase. Finally, the molecular weight distribution of the peptides was calculated based on the elution volume.

[0140] 8) Establish an antioxidant model using Caco-2 cells to evaluate the antioxidant activity of the fermentation extract, following these steps.

[0141] A. Preparation of extract: Take 1g of soybean meal, add it to 9mL of water, and extract by shaking at 150rpm for 30min at 37℃. After centrifugation at 10000rpm for 15min, collect the supernatant and filter it through a 0.22μm filter membrane to obtain the extract. Extraction was performed using unfermented soybean meal to obtain the SBM extract; extraction was performed using soybean meal fermented with strain 172D4 to obtain the 172D4 extract; and extraction was performed using soybean meal fermented with strain LDH6 to obtain the LDH6 extract.

[0142] B. Culture of the human clonal colon adenocarcinoma cell line Caco-2: Caco-2 cells were removed from the liquid nitrogen tank and quickly transferred to a preheated 37°C water bath for thawing. After thawing, the cells were transferred to a 15mL centrifuge tube containing 5mL of complete culture medium (DMEM containing 20% ​​v / v fetal bovine serum and 1% penicillin-streptomycin mixture). The cells were centrifuged at 1000rpm for 3 minutes, and the supernatant was discarded. 1mL of complete culture medium was added to the cell pellet, and the cells were gently pipetted using a dropper. The pellet was then transferred to a T25 cell culture flask containing 4mL of complete culture medium and cultured in a 37°C, 5% CO2 incubator. The medium was changed every 48 hours. Cells were passaged when they reached 80-90% confluence.

[0143] C. MTT toxicity test of fermented soybean meal extract

[0144] Group setup: The intervention group consisted of fermented soybean meal aqueous extract FSBM172D4, FSBMLDH61, and the control group (normal cultured cells without any treatment) and the negative control group consisting of unfermented soybean meal SBM. Eight different concentration gradients were set for each group: 0.5, 5, 50, 200, 500, 1000, 2000 and 5000 μg / mL.

[0145] The method for determining the antiproliferative effect of fermented soybean meal on Caco-2 was as follows: Cells grown to 80-90% confluence were digested with trypsin, centrifuged and resuspended, and seeded into 96-well plates to achieve a cell density of 2 × 10⁻⁶ cells / well. 4 Cells were cultured in 96-well plates for 24 hours. After culturing, the culture medium was discarded, and 100 μL of the pre-concentrated sample was added to each well. After 24 hours of culture, the culture medium was aspirated, and the plates were washed with 100 μL of PBS in each well. After removing the PBS, 100 μL of DMEM was added, followed by 20 μL of 0.5 mg / mL MTT in each well. The plates were then incubated at 37°C in a 5% CO2 incubator for 4 hours in the dark. The culture medium was discarded, and 150 μL of DMSO was added to each well to dissolve the cells. After dissolution, the absorbance was measured at 490 nm using a microplate reader. The experiment was performed in 6 replicates. Cell viability was calculated as follows: Cell viability (%) = (ODs - ODb) / (ODc - ODb) × 100. In this formula, ODs represents the absorbance at 570 nm in the intervention group, ODb represents the absorbance at 570 nm in the control group, and ODc represents the absorbance at 570 nm in the negative control group.

[0146] D. Establishment of the hydrogen peroxide damage model

[0147] Reference (Song JL, Gao Y. Effects of methanolic extract form Fuzhuanbrick-tea on hydrogen peroxide-induced oxidative stress in human intestinal epithelial adenocarcinoma Caco-2 cells[J]. Molecular Medicine Reports, 2014,9(3): 1061-1067.), and made corresponding modifications, the hydrogen peroxide model was constructed according to the following method: according to 6×10 4 Cells were seeded into 96-well plates and cultured to the logarithmic growth phase (density 70-80%). Then, the cells were treated with hydrogen peroxide diluted in DMEM medium (concentration gradients of 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000 μM). The specific preparation method was as follows: using serum-free medium (DMEM formulation) as the solvent, the 3% H2O2 stock solution was serially diluted to prepare the working solution. First, 113.6 μL of 3% H2O2 stock solution was added to 886.4 μL of serum-free medium and mixed to prepare 1 mL of 100 mM H2O2 intermediate solution. Then, 100 μL of the 100 mM intermediate solution was added to 900 μL of serum-free medium and mixed to prepare 1 mL of 10 mM H2O2 intermediate solution. Subsequently, using a 10 mM intermediate solution as the stock solution, further dilution with serum-free medium was carried out to prepare H2O2 working solutions with final concentrations of 100, 200, 300, 400, 500, 600, 700, 800, 900, and 1000 μM, respectively. The specific preparation method was as follows: 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 μL of the 10 mM intermediate solution were taken and brought to a final volume of 1 mL with serum-free medium. Each concentration of working solution was prepared fresh and used immediately. The cell group treated in the same way but without hydrogen peroxide served as the control group, and the group without cells and hydrogen peroxide served as the blank group. After 4 hours of treatment, cell viability was determined using the MTT assay. The hydrogen peroxide concentration corresponding to a cell viability of approximately 60% was set as the modeling concentration.

[0148] E. Effects of the sample on oxidative damage in Caco-2 cells

[0149] Protection group: 100 μL of Caco-2 cells in logarithmic growth phase were taken and administered at 6 × 10⁻⁶ doses. 4The cells were seeded at a density of / wells in 96-well plates and cultured routinely in a constant temperature incubator at 37°C and 5% CO2 for 12 h. After observing cell adhesion under an inverted fluorescence microscope, the cells were treated with complete culture medium containing different concentrations of the sample for 24 h. After the treatment, the cells were modeled with 850 μmol / L hydrogen peroxide (serum-free culture medium) for 4 h. Cell viability was then measured by the MTT assay.

[0150] F. Measurement of oxidative indicators in cells

[0151] In a 6-well plate, press 2×10 5 Cells were seeded at a density of cells / well and cultured routinely in a 37°C, 5% CO2 incubator for 12 h. After observing cell adhesion under an inverted fluorescence microscope, 100 μL of a 500 μg / mL sample was added to each well (obtained by serial dilution of the extracted fermentation supernatant, see the dilution steps above, but this time it was obtained by dilution with complete culture medium). The samples (FSBM172D4, FSBMLDH61, and the control group, as well as the fermented soybean meal negative control group SBM) were treated for 24 h. After treatment, the cells were used to establish a cell model with 850 μmol / L hydrogen peroxide (serum-free medium) for 4 h. Cells were then collected using the following kit for MDA, SOD, and CAT assays. The complete culture medium consisted of 79% DMEM high-glucose medium, 20% fetal bovine serum, 1% penicillin (1000 U / mL), and 1% streptomycin (1000 μg / mL), and needed to be preheated to 37°C.

[0152] Determination of malondialdehyde (MDA) in cells: The Solarbio MDA content assay kit was used. Principle: Based on the TBARS assay: Under acidic and high-temperature conditions, malondialdehyde (MDA) can combine with thiobarbituric acid (TBA) to form a brownish-red trimethylolpropionate (3,5,5-trimethyloxazol-2,4-dione), with a maximum absorption wavelength at 532 nm. The MDA content in the sample can be estimated after colorimetric analysis.

[0153] Determination of total superoxide dismutase (SOD) in cells: After the experiment, the cells were washed twice with PBS, collected with a cell scraper, and then fully lysed on an ice box with the cell lysis buffer provided with the kit. The SOD activity in the cells was then detected according to the steps of the kit.

[0154] Assay of catalase (CAT) in cells: After treating cells in the experimental and control groups, the activity of CAT was measured according to the operation steps of the CAT kit, and the absorbance was measured at a wavelength of 405 nm using an ELISA reader.

[0155] 9) The volatile flavor compounds in fermented soybean meal were identified using static headspace-solid phase microextraction-gas chromatography-mass spectrometry (HS-SPME-GC-MS). The determination method is as follows:

[0156] Accurately weigh 2.00 g of fermented soybean meal powder sample into a 20 mL headspace vial, add 6 mL of ultrapure water and 3 g of sodium chloride sequentially, and stir magnetically until homogeneous. Equilibrate at 60 °C and 500 rpm for 15 min, place a 50 / 30 μm DVB / CAR / PDMS extraction head in the injection port, and age at 250 °C for 5 min. Extract at 60 °C and 500 rpm for 30 min, then desorb at 250 °C for 5 min. Volatile compounds are then separated using a 60 m × 0.25 mm × 0.25 μm DB-5 column. The temperature program is as follows: hold at 40 °C for 10 min, then increase to 240 °C at a rate of 5 °C / min, and hold at this temperature for 5 min. High-pressure helium is used as the carrier gas at a flow rate of 1.0 mL / min, and the injection method is splitless injection. The mass spectrometry conditions were: EI ion source, temperature 230℃, electron energy 70eV, transfer line temperature 280℃, and mass scan range 33-500m / z.

[0157] (2) Test results

[0158] 1) As shown in Table 4, after fermentation of soybean meal by Bacillus vesiculosus 172D4 of this invention, the trypsin inhibitor activity decreased to 295.00 TIU / g, which is 81.6% lower than that of unfermented soybean meal (SBM) (1602.77 TIU / g) and 55.4% lower than that of the control strain LDH6 (662.00 TIU / g), indicating a significant effect on the degradation of anti-nutritional factors. Simultaneously, the reducing sugar content reached 5.21%, approximately 10 times higher than that of unfermented soybean meal (0.48%) and approximately 7.9% higher than that of the control strain LDH6 (4.83%); the acid-soluble protein content reached 57.80 mg / g, approximately 122% higher than that of unfermented soybean meal (25.99 mg / g) and slightly higher than that of the control strain LDH6 (57.14 mg / g). These results indicate that strain 172D4 is significantly superior to both unfermented soybean meal and strain LDH6 in terms of anti-nutritional factor degradation and nutritional quality improvement.

[0159] Compared with the published Bacillus belyss patent applications, the strain 172D4 of this invention also exhibits significant advantages. For example, the DP-2 strain disclosed in CN111826295A only has protease capabilities and lacks amylase and cellulase, thus failing to achieve multi-enzyme synergistic degradation; the LB-Y-1 strain disclosed in CN114107127B, although possessing three enzyme capabilities, only qualitatively describes "high yield" without disclosing specific enzyme activity values, nor does it conduct a systematic evaluation of the soybean meal fermentation effect; the ZM2024 strain disclosed in CN118599720A focuses only on cellulase, with a single enzyme system. The strain 172D4 of this invention is the first to fully disclose quantitative enzyme activity data of high yield of three enzymes—protease (24.67 U / mL), amylase (45.33 U / mL), and cellulase (20.33 U / mL)—in a patent for Bacillus belyi. It has also achieved systematic verification in soybean meal fermentation that it can efficiently degrade anti-nutritional factors (TI reduction of 81.6%) and significantly improve nutritional quality (reducing sugar increased by 10 times and acid-soluble protein increased by 122%).

[0160] Table 4

[0161]

[0162] 2) Peptide distribution and antioxidant activity in Caco-2 cells

[0163] As shown in Table 5 and Figure 6 As shown, after fermentation, Bacillus bellis 172D4 contained 44.29% peptides in the 500-189 Da molecular weight range, significantly higher than unfermented soybean meal SBM (20.72%) and the control strain LDH6 (40.00%). This range represents a concentrated distribution area of ​​antioxidant peptides. Strain 172D4 achieved targeted enrichment of functional peptides.

[0164] Based on the data from the Caco-2 cell oxidative stress model (Table 6), it was confirmed that the fermentation product of strain 172D4 has good antioxidant activity. The fermented soybean meal of strain 172D4 of this invention can restore the SOD and CAT activities in oxidative stress cells to 73.7% and 88.4% of the blank group, respectively, and reduce the MDA content to 50.0% of the model group. All three indicators are significantly better than unfermented soybean meal and the control strain LDH6.

[0165] Table 5

[0166]

[0167] Table 6

[0168]

[0169] (3) As shown in Table 7, compared with unfermented soybean meal (SBM), the types and contents of flavor compounds changed significantly after fermentation with Bacillus vesiculosus 172D4. The contents of key aroma compounds such as pyrazines (tetramethylpyrazine, trimethylpyrazine, 2,3-dimethylpyrazine, etc.), phenols (2-methoxyphenol), organic acids (lactic acid, 3-methylvaleric acid, 3-methyl-2-butenoic acid), ketones (2,3-butanedione, 2-octanone) and trimethyloxazole in the 172D4 group were greatly increased, while furfural, an undesirable flavor compound present in unfermented soybean meal, was completely eliminated. Compared to LDH6, the 172D4 fermentation product showed that tetramethylpyrazine (37 μg / g) was 3.36 times that of LDH6, trimethylpyrazine (49 μg / g) was 1.48 times that of LDH6, 2,3-dimethylpyrazine (0.17 μg / g) was 5.67 times that of LDH6, 2,6-diethylpyrazine (0.98 μg / g) was 1.96 times that of LDH6, and 2,3-dimethyl-5-propylpyrazine (0. The content of 172D4 is 2.36 times that of LDH6 (13 μg / g); 2-methoxyphenol (1.1 μg / g) is 1.53 times that of LDH6; lactic acid (6.6 μg / g) is 1.47 times that of LDH6; and trimethyloxazole (4.5 μg / g) is 3.0 times that of LDH6. Furthermore, 172D4 uniquely produces 2,3-butanedione (1.3 μg / g) with a creamy aroma and 2-octanone (0.20 μg / g) with a fruity aroma. Compared with published Bacillus bereaves flavor patents (such as CN120888447B, which only provides qualitative descriptions without content data, and CN120536317A, which only focuses on tetramethylpyrazine and is based on liquid fermentation), this invention, 172D4, is the first to systematically and quantitatively produce multiple pyrazines, phenols, ketones, and organic acid flavor substances in solid-state fermentation, with a significant advantage in content, making it suitable for flavor enhancement and quality improvement of plant-based seasonings.

[0170] Table 7

[0171]

[0172] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A Bacillus velezensis strain producing high levels of hydrolytic enzymes, characterized in that: The high-yield hydrolytic enzyme-producing Bacillus velezensis is named Bacillus velezensis 172D4, with accession number GDMCC NO: 67771. It was deposited on January 29, 2026, at the Guangdong Provincial Microbial Culture Collection Center of the Institute of Microbiology, Guangdong Academy of Sciences, located on the 5th floor of Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The application of Bacillus belye, which produces high levels of hydrolases according to claim 1, in the preparation of hydrolases.

3. The application of *Bacillus belye*, a high-yield hydrolase-producing bacterium according to claim 2, in the preparation of hydrolases, characterized in that: The hydrolytic enzyme is at least one of protease, amylase and cellulase.

4. The application of the high-yield hydrolytic enzyme-producing Bacillus belye as described in claim 1 in soybean meal fermentation.

5. A method for preparing fermented soybean meal, characterized in that... Includes the following steps: (1) The Bacillus berberis described in claim 1 is activated and cultured, the culture medium is separated into solid and liquid, the bacterial cells are taken, the bacterial cells are resuspended, and the fermentation agent is obtained; (2) Inoculate the fermenting agent into sterilized water-containing soybean meal for fermentation to obtain fermented soybean meal.

6. The method for preparing fermented soybean meal according to claim 5, characterized in that: The culture medium used in the activation culture described in step (1) is TSB medium; The activation culture operation described in step (1) involves first streaking the culture plate, and then inoculating the colonies that grow on the plate into a liquid culture medium and shaking the culture.

7. The method for preparing fermented soybean meal according to claim 5, characterized in that: The solid-liquid separation method described in step (1) is centrifugation; The resuspension solution mentioned in step (1) is sterile physiological saline.

8. The method for preparing fermented soybean meal according to claim 5, characterized in that: The bacterial concentration of the starter culture in step (1) is 10 7 ~ 10 8 CFU / g; The water-containing soybean meal mentioned in step (2) is obtained by mixing soybean meal and water at a material-to-liquid ratio of 1:0.5 to 1:2 (w / w); The inoculation amount of the fermentation agent mentioned in step (2) is calculated as 1% to 10% of the mass of the water-containing soybean meal. The fermentation conditions described in step (2) are static culture at 30-40℃ for 24-72 h.

9. A fermented soybean meal, characterized in that: Obtained by the fermentation method according to any one of claims 5 to 8.

10. The fermented soybean meal according to claim 9, characterized in that: Compared to unfermented soybean meal, the fermented soybean meal exhibits an 81.6% reduction in trypsin inhibitors, a 10-fold increase in reducing sugars, and a 122% increase in acid-soluble proteins. Furthermore, it contains 44.29% 500-189 Da active peptides, significantly enhancing cellular antioxidant capacity. Simultaneously, it generates a large amount of pyrazines, ketones, and other aroma compounds, eliminating the beany odor.

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