Bacillus velezensis J2 for producing saline-alkaline resistant protease and application of bacillus velezensis J2

Abstract

The invention relates to the field of microbial technology and waste recycling, in particular to bacillus velezensis J2 and application thereof, the preservation number of the bacillus velezensis J2 is GDMCC No.66644, and saline-alkaline resistant protease secreted by the bacillus velezensis J2 has the advantages that (a) the optimum pH is 8.5; (b) the optimum temperature is 50 DEG C; (c) the enzyme activity retention rate in 4 mol / L NaCl is greater than or equal to 50%; and (d) maintaining more than 80% of enzyme activity within the pH range of 6-11. The method for degrading the insoluble protein by the saline-alkaline resistant protease comprises the following steps: directly adding the strain or the protease secreted by the strain into a culture medium containing soy sauce residues and / or feathers, fermenting for 24-48 hours under the conditions that the pH is 7.5-9.5 and the temperature is 30-40 DEG C, and realizing that the feather degradation rate is greater than or equal to 90% without adding an auxiliary nitrogen source. The obtained soy sauce residue protein hydrolysate and feather keratin degradation product can be used as feed protein sources or biological medicine raw materials. The strain can also be used for degrading protein industrial wastes, realizes high-efficiency resource utilization of wastes such as high-salt sauce residues and feathers, and has remarkable economic and environmental benefits.

Description

Technical Field

[0001] This invention relates to the field of microbial technology and utilization of biological resources, specifically to a strain of Bacillus velezensis J2 that produces salt- and alkaline-resistant proteases and its applications. Background Technology

[0002] The food, leather, and livestock industries generate large quantities of byproducts such as high-salt soy sauce residue and feathers, which contain abundant insoluble protein resources such as keratin and collagen. However, due to their unique physicochemical properties, related resource utilization technologies face multiple challenges. Taking high-salt soy sauce residue, a byproduct of soy sauce production, as an example, its salt content is typically as high as 10%-20%, and the proteins mostly exist in aggregated states. Traditional microbial growth is limited in high-salt environments, and conventional enzymatic hydrolysis methods require additional pH and salt concentration adjustments, resulting in high processing costs, low efficiency, and degradation rates generally below 60%. Keratin accounts for over 80% of feathers, and its dense structure formed by numerous disulfide bonds makes traditional physical methods such as high temperature and high pressure energy-intensive, while chemical methods such as strong acids / bases easily cause environmental pollution. Biological treatment relying on keratinase and its enzyme-producing microorganisms is a mild, efficient, green, and sustainable development direction. However, existing strains have insufficient activity under neutral / weakly acidic conditions, and the degradation cycle typically exceeds 48 hours, making it difficult to meet the needs of industrial production.

[0003] Chinese patent CN202411436428 discloses a strain of Bacillus velezensis phb02, whose secreted protease achieves a 35% degradation rate of trypsin inhibitors at a 3M salt concentration, but does not specify the optimal pH range or its adaptability to alkaline environments with pH > 8.0; Chinese patent CN202311693504 discloses a strain of Bacillus velezensis DLWHY-3, but does not disclose the salt and alkali tolerance parameters of its protease; Chinese patent CN202011585317 discloses a strain of Bacillus velezensis NLG1, which can degrade feathers, but its soluble protein yield is low. In addition, existing biological technologies for the resource utilization of protein-based waste have the following significant drawbacks: There is a lack of strains producing salt-tolerant alkaline proteases; most protein-degrading strains are only suitable for neutral environments with a pH of 6.0-7.5, and the activity of their secreted proteases decreases significantly under high-salt conditions (>3 mol / L NaCl) and alkaline conditions (>8.0), making them unable to directly process complex substrates such as high-salt soy sauce residue; Furthermore, they have poor compatibility across multiple application scenarios. Existing technologies are typically designed for single substrates and require frequent adjustments to parameters such as pH and temperature, making it difficult to form universal technical solutions and adapt to the actual needs of diversified utilization of industrial by-products. Summary of the Invention

[0004] To address the aforementioned technical bottlenecks, the present invention aims to provide a strain of Bacillus velezensis J2 that produces salt- and alkaline-tolerant proteases and its applications in the degradation of high-salt soy sauce residue and feather keratin. Furthermore, it provides an application for using this strain or its secreted salt- and alkaline-tolerant proteases to act on substrates to prepare high-value-added products rich in small molecule peptides and essential amino acids. This enables the strain to achieve highly efficient protein degradation under extreme environments and expands its applications in feed preparation, biomedicine, and environmental pollutant treatment, filling the gap in existing technologies regarding salt- and alkaline-tolerant strains and the resource utilization of protein-based wastes such as soy sauce residue and feathers.

[0005] To achieve the above objectives, the present invention adopts the following technical solution: a strain of Bacillus velezensis J2, which was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 10, 2025, with the accession number GDMCC No. 66644.

[0006] The salt-tolerant protease produced by Bacillus velezensis J2,

[0007] (a) The optimal pH value is 8.5;

[0008] (b) The optimal temperature is 50°C;

[0009] (c) The enzyme activity retention rate in 4 mol / L NaCl solution is ≥50%;

[0010] (d) Maintain 80% or more of the enzyme activity under optimal pH and temperature conditions within the pH range of 6-11.

[0011] The method for degrading poorly soluble proteins with salt-tolerant alkaline proteases includes:

[0012] The strain or its secreted protease is directly added to a culture medium containing soy sauce residue and / or feathers, and fermented for 24-48 hours at pH 7.5-9.5 and temperature 30-40 ℃.

[0013] The concentration of the sauce residue is 5% w / v, and the concentration of the feathers is 6.7% w / v, and no additional nitrogen source or physical / chemical pretreatment is required.

[0014] The method for degrading poorly soluble proteins using salt-tolerant alkaline proteases achieves a feather degradation rate of ≥90% within 48 hours, resulting in a soluble protein content of ≥17,000 μg / mL.

[0015] The protein hydrolysate obtained by the method is rich in small molecule peptides and essential amino acids.

[0016] Application of the protein hydrolysate in the preparation of animal feed.

[0017] Application of the protein hydrolysate in the preparation of functional foods, bioactive peptides, or cosmetic raw materials.

[0018] A strain of Bacillus velezensis J2 was isolated from high-salt soy sauce residue with a salinity ≥15% and obtained through targeted screening for salt-tolerant alkaline protease activity.

[0019] A strain of Bacillus velezensis J2, a Gram-positive bacillus, has white colonies with irregular edges, slightly raised surfaces, and a smooth, moist texture with mucus.

[0020] The 16S rRNA gene sequence of this strain is shown in SEQ ID No. 1 in the sequence listing. It has a high sequence identity (>99%) with Bacillus velezensis. Phylogenetic analysis shows that it clusters with the type strain Bacillus velezensis LHSB1.

[0021] This combination of properties makes the protease particularly suitable for industrial applications in high-salt, alkaline, or pH-fluctuating environments, such as high-salt food processing, textile scouring, or saline wastewater treatment.

[0022] Compared with the prior art, the outstanding advantages of this invention are:

[0023] 1. The salt-tolerant alkaline protease secreted by Bacillus velezensis J2 of the present invention has an optimal pH of 8.5 and a temperature of 50°C. The enzyme activity is still ≥50% in 4M NaCl and maintains more than 80% activity in the pH range of 6-11, which significantly broadens the boundary of enzyme action conditions.

[0024] 2. Regarding substrate adaptability, the Bacillus velezensis J2 of this invention has achieved a breakthrough in the simultaneous degradation of high-salt soy sauce residue (5% w / v) and feather keratin (6.7% w / v). The feather degradation rate reaches 90% in 48 hours, and the soluble protein content is as high as 17,223 μg / mL. Its soy sauce residue protein hydrolysis efficiency is better than that of commercial alkaline enzymes, and no auxiliary nitrogen source needs to be added.

[0025] 3. In terms of process practicality, the present invention can be carried out under mild fermentation conditions of pH 7.5-9.5 and 30-40℃, and can directly process complex substrates without pretreatment, which significantly simplifies the operation process and reduces costs.

[0026] This invention achieves a substantial breakthrough over existing technologies in terms of enzymatic performance, substrate coverage, and industrial adaptability through the innovative coupling of the characteristic expression of salt-tolerant alkaline protease with a multi-substrate degradation process. Attached Figure Description

[0027] Figure 1 The diagram shows the isolation, identification, morphological and hemolytic analysis of Bacillus velezensis J2 as described in this invention.

[0028] Figure 2 Phylogenetic tree of Bacillus velezensis J2 constructed based on 16S rRNA gene sequence.

[0029] Figure 3 This is a diagram showing the optimization of fermentation conditions and growth monitoring of Bacillus velezensis J2 as described in this invention.

[0030] Figure 4 This is a preliminary purification diagram of the ammonium sulfate precipitation of Bacillus velezensis J2 protease as described in this invention.

[0031] Figure 5 This is a characterization diagram of the crude protease produced by Bacillus velezensis J2 as described in this invention.

[0032] Figure 6 The images show SEM images of the treatment of soy sauce residue by Bacillus velezensis J2 as described in this invention, and a comparison of the enzyme activity of crude protease on soy sauce residue protein.

[0033] Figure 7 The graph shows the degradation of feathers, enzyme activity, and soluble protein content of Bacillus velezensis J2 as described in this invention. Detailed Implementation

[0034] Example 1

[0035] This embodiment illustrates the isolation, culture, and preservation of the Bacillus velezensis J2 strain described in this invention, including the following steps:

[0036] ① Strains Isolation and Preliminary Screening

[0037] Weigh 1 gram of soy sauce residue sample and resuspend it in 100 ml of sterile physiological saline to obtain the bacterial community. Inoculate 1% (v / v) of this resuspended solution into sterile soy sauce residue culture medium and incubate at 37°C and 220 rpm with shaking for 12 hours to achieve moderate activation of the bacterial community. Serially dilute the activated fermentation broth, and evenly spread an appropriate amount of the diluted solution onto an agar plate containing 10 g / L (w / v) skim milk. Incubate the plates at 37°C until multiple bacterial colonies with clear hydrolysis zones appear on the plate. Select individual colonies exhibiting hydrolysis zones for subsequent experiments.

[0038] The results showed that after culturing on agar plates containing skim milk, a total of five strains exhibiting distinct hydrolysis zones were isolated, indicating that these strains all possessed protein hydrolytic activity. These five strains were identified as candidate strains with protein degradation potential.

[0039] ② Target strain selection, purification and phenotypic identification

[0040] The candidate strain from step ① was streaked onto LB agar plates and incubated at 37°C for 24 hours for isolation and purification. Hemolytic activity was tested on the purified strain: the strain was streaked onto blood agar plates containing 5% (v / v) defibrinated sheep blood and incubated at 37°C for 24-48 hours, observing hemolysis. Simultaneously, the colony morphology characteristics of the purified strain on LB plates, including color, shape, edge, surface elevation, and texture, were observed and recorded. The purified strain was stained using standard Gram staining, and the staining results were observed under an optical microscope. The target strain was observed using a scanning electron microscope, and electron micrographs were taken. Protease activity was determined using the Folin-Ciocalteu method. The reaction system contained 600 μL of 50 mM Tris-HCl buffer (pH 8.0), 200 μL of crude enzyme solution, and 200 μL of 2% casein solution. After reacting at 50°C for 25 minutes, 400 μL of 10% trichloroacetic acid was added to terminate the reaction. The reaction solution was centrifuged at 8000 rpm for 5 minutes at room temperature. 150 μL of the supernatant was collected, and 200 μL of Folin-Ciocalteu phenol reagent and 750 μL of 0.4 M Na₂CO₃ solution were added sequentially. After mixing, the mixture was incubated at 50°C for 20 minutes. The absorbance was measured at 680 nm using an Infinite M200 Pro microplate reader. One unit of enzyme activity (U) was defined as the amount of enzyme required to catalyze the production of 1 μg of tyrosine per minute.

[0041] By measuring the protease activity of the fermentation broth of candidate strains, it was found that strain J2 had significantly higher protease activity than other candidate strains. Therefore, strain J2 was selected as the subject of subsequent research. Figure 1As shown in A, strain J2 was identified as a Gram-positive bacterium, as... Figure 1 As shown in Figure B, its colonies on LB agar plates are white with irregular edges, slightly raised surfaces, and a smooth, moist texture covered with mucus. Scanning electron microscopy clearly reveals the rod-shaped morphology and surface structure characteristics of strain J2, as shown in Figure B. Figure 1 As shown in C. After culturing on blood agar plates, no transparent or green hemolytic zone was observed around the colonies of strain J2, as shown in Figure C. Figure 1 As shown in D, this indicates that the strain exhibits γ-hemolysis under the tested conditions, meaning it does not hemolyze and has no hemolytic activity.

[0042] ③ Molecular biological identification

[0043] The 16S rRNA gene of strain J2 was amplified by PCR using universal primers 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and 1492R (5′-TACGGYTACCTTGTTACGACTT-3′). The PCR reaction program was set as follows: 95℃ pre-denaturation for 10 minutes; followed by 35 cycles, each cycle consisting of 95℃ denaturation for 10 seconds, 55℃ annealing for 15 seconds, 72℃ extension for 100 seconds, and a final extension at 72℃ for 5 minutes. The PCR amplified products were purified and sent to Shanghai Sangon Biotech Co., Ltd. for bidirectional sequencing. The obtained sequences were uploaded to the NCBI database and homologous nucleotide sequence alignment was performed using BLAST. Clustal W software was used to perform multiple sequence alignment of the 16S rRNA nucleotide sequences of strain J2 and several related reference strains. A phylogenetic tree was constructed using MEGA-X software based on the Neighbor-Joining (NJ) method.

[0044] The 16S rRNA gene sequence of strain J2 was successfully amplified and sequenced, with a sequence length of 1451 bp, as shown in SEQ ID: No. 1. This sequence information has been submitted to the GenBank database, obtaining accession number PV156326. BLAST homology sequence alignment analysis showed that the 16S rRNA gene sequence of strain J2 exhibited >99% high homology with several strains of Bacillus velezensis, such as GenBank accessions KP851947.1 and KM596517.1. Phylogenetic tree analysis based on the 16S rRNA sequence showed that strain J2 closely clustered with known strains of Bacillus velezensis within the same evolutionary branch, such as... Figure 2 As shown.

[0045] ④ Strain identification conclusions and preservation

[0046] Based on the above-mentioned morphological characteristics—Gram-positive, rod-shaped, with white, irregularly edged, mucous-like colonies; strong proteolytic activity; non-hemolytic physiological and biochemical properties; and 16S rRNA gene sequence homology and phylogenetic classification—strain J2 was identified as *Bacillus velezensis*. This *Bacillus velezensis* J2 strain was deposited at the Guangdong Provincial Microbial Culture Collection Center on July 10, 2025, with accession number GDMCC No. 66644.

[0047] Example 2

[0048] This embodiment is an example of fermentation condition optimization and growth monitoring of Bacillus velezensis J2 strain described in this invention, including the following steps:

[0049] ① Dynamic monitoring of the fermentation process and determination of the optimal fermentation time

[0050] Bacillus velezensis J2 was inoculated into LB broth and cultured at 37°C with shaking for a total of 54 hours. Samples were taken every 6 hours during fermentation. The OD value of the fermentation broth was measured after each sampling. 600 The values ​​represent cell concentration and protease activity, which represent the level of extracellular protease secretion, to dynamically monitor the growth of the strain and its protease production capacity.

[0051] The results are as follows Figure 3 As shown in Figure A, Bacillus velezensis J2 exhibited strong protease synthesis capabilities in the early stages of fermentation: significant protease activity was detected only 6 hours after inoculation. The protease activity showed a typical single-peak curve over time, reaching a peak of 590.26 ± 46.02 U / mL at 24 hours of culture. Notably, after 30 hours of culture, the protease activity began to decline significantly, retaining only about 23% of the peak activity by 36 hours. Based on the principle of maximizing protease activity, 24 hours was determined as the optimal fermentation endpoint.

[0052] ② Optimization of the effects of initial culture temperature and pH on protease activity

[0053] To systematically evaluate the effects of initial culture temperature and initial pH on the J2 protease production capacity of Bacillus velezensis, a single-factor experimental method was used for optimization, with protease activity as the key evaluation index. Temperature optimization: Six culture temperature gradients were set: 25℃, 30℃, 37℃, 40℃, 45℃, and 50℃. pH optimization: Six initial pH gradients were set: 5.0, 6.0, 7.0, 8.0, 9.0, and 10.0. Fermentation was carried out under the above experimental conditions, and samples were taken at the previously determined optimal fermentation time point, i.e., 24 hours, to measure the protease activity of the fermentation broth, thereby quantifying the effect of different fermentation conditions on protease secretion levels.

[0054] The results are as follows Figure 3 B and Figure 3 As shown in Figure C, Bacillus velezensis J2 exhibited optimal protease production capacity under the conditions of a culture temperature of 37°C and an initial pH of 7.0.

[0055] ③ Plotting the growth curve of the strain under optimized conditions

[0056] Bacillus velezensis J2 was inoculated into LB broth at a 1% (v / v) inoculum and cultured under optimized conditions: 37°C, initial pH 7.0, and constant temperature shaking. Samples were taken every 2 hours during culture. The absorbance of the culture medium was measured at 600 nm using a microplate reader. OD was monitored in real time. 600 By analyzing the changes in values, we can plot the bacterial growth curve and obtain its growth dynamics, including key information such as growth rate and growth cycle.

[0057] The growth curve of Bacillus velezensis J2 was monitored at 37°C and an initial pH of 7.0, as shown below. Figure 3 As shown in D. The results showed that the strain exhibited a typical three-stage growth pattern: after a lag phase of about 2 hours, it entered the exponential growth phase and reached the stationary phase after about 20 hours of culture. As nutrients in the culture medium were continuously consumed, the strain entered the death phase after about 54 hours of culture.

[0058] Example 3

[0059] This embodiment is an example of partial purification and enzymatic property analysis of the protease from Bacillus velezensis strain J2 described in this invention, including the following steps:

[0060] ① Partial purification of protease

[0061] The LB medium fermentation broth was centrifuged at 12,000 rpm for 20 minutes at 4°C to remove bacterial cells, and the supernatant was collected. Ammonium sulfate of varying saturations (20%–100%) was gradually added to the supernatant, and the mixture was incubated overnight at 4°C for protein precipitation. The precipitate was collected after centrifugation at 12,000 rpm for 20 minutes and redissolved in 20 mM Tris-HCl buffer (pH 8.0). Subsequently, the enzyme solution was concentrated and residual salt ions were removed by centrifugation at 5,000 rpm for 30 minutes at 4°C using a Millipore ultrafiltration column with a molecular weight cutoff of 3000 Da.

[0062] By measuring the protease activity of precipitates at different ammonium sulfate saturations, it was found that no protease activity was detected when the saturation was below 40%; the activity increased significantly when the saturation reached 60%, and peaked at 90% saturation. Figure 4 As shown in Figure A. Combined with SDS-PAGE analysis, as... Figure 4 As shown in Figure B, the optimal purification conditions were determined to be within the 60%-90% saturation range. Ultrafiltration concentration significantly increased protease activity, and SDS-PAGE showed a clear main band at 25 kDa. Figure 4 As shown in C. Enzyme spectrum analysis is as follows. Figure 4 As shown in Figure D, multiple white hydrolysis bands appear on a blue background, confirming that the strain secretes a variety of proteases, among which the 25 kDa protease is the main component of the enzyme system.

[0063] ② Enzymatic property analysis

[0064] The protease activity was determined using a casein hydrolysis method. Optimal temperature testing was performed in 50 mM Tris-HCl buffer at pH 8.0–9.0 at a temperature gradient of 30–70 °C. Thermal stability was assessed by measuring residual activity after incubation at 30–70 °C for 1 hour. Optimal pH testing was performed at 55 °C in the following buffer systems: 50 mM citrate buffer (pH 4.0–5.5), 50 mM sodium phosphate buffer (pH 5.5–8.5), 50 mM Tris-HCl buffer (pH 8.0–9.5), and 50 mM carbonate buffer (pH 9.0–11.0). pH stability was assessed by measuring residual activity after pre-incubation at 4 °C for 24 hours in pH 4.0–11.0 buffer. Tolerance to metal ions and chemical reagents was tested by co-incubating the protease with 5 mM metal ions or 1% (v / v) surfactant at room temperature for 1 hour to determine activity. Salt tolerance was assessed by immediate activity at 0–4 M NaCl concentrations and residual activity after 48 hours of pre-incubation. Substrate specificity was determined using casein, bovine serum albumin (BSA), type I / II collagen, and keratin as substrates, with a difference in absorbance of 0.001 denoted as one unit (U) of protease activity.

[0065] The results are as follows Figure 5 As shown in A, the optimal temperature for the crude protease is 50℃, where the enzyme activity reaches 284 U / mL; at 70℃, the activity decreases to 30%. Figure 5 As shown in Figure B, incubation at 30-50℃ retains more than 90% of the activity, while incubation at 70℃ leaves only 10% residual activity. Figure 5 As shown in C, the optimal pH is 8.5, and it maintains greater than 85% activity within the pH range of 7.0-11.0; Figure 5 As shown in D, the residual activity after pre-incubation at pH 5.5-11.0 is greater than 80%. Salt tolerance characteristics are as follows: Figure 5 As shown in Figure E, the results indicate that more than 50% of the activity was retained under 4 M NaCl conditions. Substrate specificity is as follows: Figure 5 As shown in F, casein exhibits the highest hydrolytic activity, followed by keratin, while the hydrolytic activity for type II collagen, BSA, and type I collagen is relatively low.

[0066] Table 1. Effects of metal ions on the J2 protease activity of Bacillus velezensis

[0067] metal ions Working concentration Relative enzyme activity (%) control group - 100 <![CDATA[Li + ]]> 5mM <![CDATA[77.92±6.65 *** ]]> <![CDATA[Na + ]]> 5mM <![CDATA[70.15±1.13 *** ]]> <![CDATA[K + ]]> 5mM <![CDATA[90.32±1.30 * ]]> <![CDATA[Rb + ]]> 5mM <![CDATA[89.74±0.11 * ]]> <![CDATA[Cs + ]]> 5mM <![CDATA[91.19±0.63 * ]]> <![CDATA[Cu 2+ ]]> 5mM <![CDATA[57.98±0.74 *** ]]> <![CDATA[Mg 2+ ]]> 5mM <![CDATA[85.73±1.13 *** ]]> <![CDATA[Ca 2+ ]]> 5mM <![CDATA[83.00±2.44 *** ]]> <![CDATA[Mn 2+ ]]> 5mM <![CDATA[16.97±5.88 *** ]]> <![CDATA[Zn 2+ ]]> 5mM <![CDATA[16.19±2.57 *** ]]> <![CDATA[Ni 2+ ]]> 5mM <![CDATA[54.80±2.28 *** ]]> <![CDATA[Fe 2+ ]]> 5mM <![CDATA[86.50±4.03 *** ]]> <![CDATA[Fe 3+ ]]> 5mM <![CDATA[82.42±1.38 *** ]]>

[0068] Data are expressed as mean ± standard deviation of three independent studies, *p<0.05, **p<0.01, ***p<0.001.

[0069] As can be seen from the experimental results in Table 1, in the metal ion experiment, K⁺, Rb⁺, and Cs⁺ have a weak effect on the activity, with residual activity greater than 89%, while Cu²⁺ and Ni²⁺ have inhibition rates greater than 40%.

[0070] Table 2. Effects of chemical reagents on the J2 protease activity of Bacillus velezensis.

[0071] Chemical reagents Working concentration Relative enzyme activity (%) control group - 100 EDTA 5mM <![CDATA[67.07±0.99 *** ]]> EGTA 5mM <![CDATA[67.18±1.36 *** ]]> DTT 5mM 98.89±4.69 β-Mercaptoethanol 5mM 90.94±9.54 PMSF 5mM <![CDATA[2.71±0.58 *** ]]> Triton-X100 1% (w / v) 87.71±7.13 Tween20 1% (w / v) 88.30±6.29 Tween80 1% (w / v) <![CDATA[86.00±1.80 * ]]> SDS 1% (w / v) <![CDATA[81.70±2.75 *** ]]> DMSO 50% (v / v) <![CDATA[83.06±3.15 ** ]]> Methanol 50% (v / v) <![CDATA[82.88±0.66 ** ]]> Acetone 50% (v / v) 95.04±2.30 Ethyl alcohol 50% (v / v) 98.46±3.13

[0072] Data are expressed as mean ± standard deviation of three independent studies, *p<0.05, **p<0.01, ***p<0.001.

[0073] The experimental results in Table 2 show that EDTA, EGTA, and PMSF strongly inhibit enzyme activity, while DTT and organic solvents have a relatively low impact.

[0074] Example 4

[0075] This embodiment illustrates the use of Bacillus velezensis J2 strain described in this invention for degrading soy sauce residue, and includes the following steps:

[0076] ① Observation of the effects of bacterial strains on soy sauce residue and morphological changes

[0077] Bacillus velezensis J2 was inoculated into a sterile culture medium containing 5% (w / v) soy sauce residue and cultured at 50°C and 220 rpm for 48 hours with shaking. After culture, the residue precipitate was collected by centrifugation, washed, and freeze-dried for storage. Uninoculated soy sauce residue served as a control group and was treated simultaneously. The freeze-dried samples were sputter-coated with gold and their microstructure was observed using scanning electron microscopy (SEM).

[0078] The results are as shown in the table. Figure 6 A and SEM Figure 6 As shown in Figure B, untreated soy sauce residue was dark in color and clearly clumped together. SEM revealed that its proteins and fibers were tightly cross-linked, forming dense aggregates. After treatment with Bacillus velezensis J2 strain, the soy sauce residue became significantly lighter in color and looser in structure. SEM confirmed that a large amount of protein bound to the fiber surface dissociated, and the original complex structure was destroyed. This indicates that Bacillus velezensis J2 can effectively dissociate the protein-fiber complex in soy sauce residue.

[0079] ② Protein extraction from soy sauce residue

[0080] After pulverizing the washed sauce residue, add distilled water at a ratio of 1:5 (w / v) to adjust the pH to 10.0, and stir at a constant temperature of 50°C for 2.5 hours. Separate the supernatant from the alkaline extract by centrifugation, wash the precipitate twice with water, and combine the washes. Adjust the pH of the mixture to 4.0–4.5 to precipitate the protein, collect the precipitate by centrifugation, and freeze-dry.

[0081] ③ The hydrolytic efficiency of proteases on the protein in fermented soybean residue

[0082] According to the enzyme activity assay in Example 2.6, using extracted soy sauce residue protein as a substrate, the protease activity of crude enzyme solution from Bacillus velezensis J2 was determined. Commercially available pepsin, trypsin, papain, and alkaline protease were used as controls, and the enzymatic hydrolysis efficiency was measured in parallel under the same conditions of pH 8.0 and 50°C.

[0083] The results are as follows Figure 6 As shown in C, the protease of Bacillus velezensis J2 has a significantly higher hydrolysis efficiency for soy sauce residue protein than pepsin, trypsin and papain, and its activity is comparable to that of commercial alkaline protease, but superior to that of commercial alkaline protease.

[0084] Example 5

[0085] This embodiment illustrates the use of Bacillus velezensis J2 strain described in this invention for degrading feathers, and includes the following steps:

[0086] Feather culture medium: chicken feathers 6.7% w / v, 0.5g / L NaCl, 0.15g / L MgSO4·H2O, 0.7g / L KH2PO4, 1.4g / L K2HPO4, pH 7.0-7.5, autoclaved at 121℃ for 15min.

[0087] 5 mL of Bacillus velezensis J2 seed culture was inoculated into an Erlenmeyer flask containing 30 mL of feather culture medium and incubated at 40 °C and 150 rpm for 72 hours. The degradation of feathers was observed, the degradation rate was calculated, and the protease activity and soluble protein content were determined.

[0088] The formula for calculating the feather degradation rate is as follows: Degradation rate = (M1-M2) / M1*100%. Where M1 is the dry weight of the feathers before degradation, and M2 is the dry weight of the feathers after degradation.

[0089] The results are as follows Figure 7 As shown in Figure A, the feather degradation rate of Bacillus velezensis J2 reached 47% in the first 12 hours of fermentation; it reached 90% in 48 hours, after which the degradation rate leveled off. Figure 7 As shown in Figure B, the protease activity reached as high as 1050 U / mL after 60 hours of fermentation. After 72 hours of fermentation, the soluble protein content reached 17223 μg / mL, and the feather degradation rate reached 91.2%.

[0090] The above embodiments are merely specific implementations that have been explored and have achieved good results in this invention. However, the scope of protection of this invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in this invention, based on the technical solution and inventive concept of this invention, should be covered within the scope of protection of this invention.

Claims

1. A strain of Bacillus velezensis J2, characterized in that, This strain was deposited at the Guangdong Provincial Center for Microbial Culture Collection on July 10, 2025, with accession number GDMCC No. 66644.

2. A salt- and alkaline-resistant protease produced by Bacillus velezensis J2 as described in claim 1, characterized in that: (a) The optimal pH value is 8.5; (b) The optimal temperature is 50°C; (c) The enzyme activity retention rate in 4 mol / L NaCl solution is ≥50%; (d) Maintain 80% or more of the enzyme activity under optimal pH and temperature conditions within the pH range of 6-11.

3. The Bacillus velezensis J2 according to claim 1, characterized in that, The 16S rRNA of this strain is shown in the sequence listing SEQ ID No.

1.

4. The Bacillus velezensis J2 according to claim 1, characterized in that, This strain is a Gram-positive bacillus. Its colonies are white in appearance, with irregular edges, slightly raised surfaces, and a smooth, moist texture with mucus.

5. A method for degrading poorly soluble proteins using Bacillus velezensis J2 as described in claim 1 or the salt-tolerant alkaline protease as described in claim 2, characterized in that, include: The strain or its secreted protease is directly added to a culture medium containing soy sauce residue and / or feathers, and fermented for 24-48 hours at pH 7.5-9.5 and temperature 30-40 ℃.

6. The method according to claim 5, characterized in that, The concentration of the sauce residue is 5% w / v, and the concentration of the feathers is 6.7% w / v, and no additional nitrogen source or physical / chemical pretreatment is required.

7. The method according to claim 5 or 6, characterized in that, The degradation rate of feathers was ≥90% within 48 hours, and the content of soluble protein obtained was ≥17,000 μg / mL.

8. A protein hydrolysate obtained by the method of any one of claims 5-7, which is rich in small molecule peptides and essential amino acids.

9. The use of the protein hydrolysate of claim 8 in the preparation of animal feed.

10. The use of the protein hydrolysate of claim 8 in the preparation of functional foods, bioactive peptides or cosmetic raw materials.

11. The Bacillus velezensis J2 according to claim 1, characterized in that, This strain was isolated from high-salt soy sauce residue with a salinity ≥15% and obtained through targeted screening for salt-tolerant alkaline protease activity.

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