Bacillus velezensis HALZ676 as well as application and method of bacillus velezensis HALZ676 in cowhide fermentative degradation

Abstract

The invention belongs to the technical field of biology, and particularly relates to bacillus velezensis HALZ676 as well as application and a method of the bacillus velezensis HALZ676 to cowhide fermentative degradation, the bacillus velezensis HALZ676 is separated from preserved pig trotters, and by analyzing biological characteristics, whole genome and metabonomics differences of the bacillus velezensis HALZ676, the cowhide can be efficiently degraded, and the cowhide can be efficiently degraded. It is found that peptide components smaller than 3000 Da in cow leather degradation liquid obtained through fermentation of the strain HALZ676 account for 68% or above, and the cowhide degradation liquid has remarkable antioxidant capacity and antibacterial capacity; the cowhide fermentation liquor is mainly used for promoting conversion and enrichment of antioxidant, anti-inflammatory and anti-cancer metabolites through a linoleic acid metabolic pathway, so that the antioxidant, anti-inflammatory and anti-cancer capacities of the cowhide fermentation liquor are enhanced; the cowhide fermented by the strain HALZ676 can enrich functional active components, and a theoretical basis and a promising strategy are provided for high-valued production of processing byproducts of livestock and poultry skin fermented by probiotic bacillus with high yield of collagenase.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and particularly relates to a strain of Bacillus velezinsis and an application and method of fermenting and degrading cowhide. Background Art

[0002] Cowhide accounts for 10.7% of the total live weight of a cow and is a major byproduct of the beef cattle farming industry, representing a significant portion of livestock byproducts. According to statistics from the Food and Agriculture Organization of the United Nations, the global meat and livestock industry produces 331.9 million pieces of cowhide annually, with only approximately 55% entering the leather processing industry. Cowhide is primarily composed of collagen, which accounts for over 90% of its dry weight. This collagen is not fully utilized during processing and is instead discarded as waste, resulting in a waste of biological resources and negative environmental impacts. Cowhide collagen, with a typical molecular weight of around 300 kDa, is a large protein with a unique triple helix structure, making it difficult for the human body to digest and absorb. Collagen peptides are small proteins formed by breaking the molecular chains within the triple helix structure of collagen. They are highly water-soluble, high in protein content, low in viscosity, and easily absorbed by the human body. They possess antioxidant, blood pressure, and lipid-lowering properties, and have great potential for application in food, cosmetics, and biomedicine. Using cowhide collagen as raw material to prepare collagen active peptides is an important way to realize the resource utilization, functionalization and high-value utilization of cowhide scraps.

[0003] Currently, the preparation of collagen peptides in industrial production commonly uses hot water extraction, acid-base hydrolysis, ultrasonic / microwave-assisted methods, enzymatic hydrolysis, and microbial fermentation. Among these, hot water extraction, acid-base hydrolysis, and ultrasonic / microwave-assisted methods have unavoidable disadvantages, including heavy pollution, low extraction yields, damage to the activity of small-molecule peptides, complex processes, and corrosion to equipment. Enzymatic hydrolysis is the most commonly used method for preparing collagen peptides, and enzymes commonly used for enzymatic extraction include trypsin, pepsin, and papain. Enzymatic hydrolysis offers advantages such as high safety and minimal damage to the activity of small-molecule peptides. However, these commercial enzyme preparations often have poor specificity for collagen, resulting in oligopeptides with a wide molecular weight range and high molecular weight. Chemical reagents must be introduced before raw material pretreatment, and enzymatic hydrolysis can easily produce bitter substances, resulting in an unpleasant odor. The debittering and deodorization process is cumbersome.

[0004] Microbial fermentation is a green and efficient method for producing collagen peptides under mild reaction conditions. The rich enzymes in these microorganisms not only enhance collagen degradation but also remove odors. Microbial fermentation allows collagen extraction to be completed in a single step, including defatting, protein removal, enzymatic hydrolysis, and collagen extraction, through inoculation and fermentation. This greatly simplifies the process and reduces production costs. Compared with other traditional methods, microbial fermentation does not require the addition of any chemical reagents, making it an environmentally and ecologically friendly strategy. Microbial fermentation holds great promise for producing aquatic collagen peptides. Currently, known collagenase-producing strains primarily come from pathogenic Vibrio (Vibrio alginolyticus and Vibrio vulnificus) and Clostridia (Clostridium histolyticum, Clostridium perfringens, and Clostridium tetani). However, these strains harbor virulence genes and are unable to meet fermentation requirements. Probiotic Bacillus sp., with their rich protease profiles, safety, beneficial properties, and ability to meet fermentation requirements, are potential candidates for collagenase production. Patent 202010651271.9 discloses a method for preparing collagen oligopeptides by hydrolyzing animal skins using Bacillus subtilis. The method involves fermenting Bacillus subtilis to obtain a Bacillus subtilis fermentation broth, which is then used to hydrolyze animal skins to obtain pig skin collagen oligopeptides with a molecular weight of 4311 Da and fish skin collagen oligopeptides with a molecular weight of 2721 Da. However, the degree of hydrolysis is not high and the mechanism of collagen hydrolysis by the Bacillus subtilis fermentation broth is not explored. Wu Qi et al. screened a strain of Bacillus pumilus, which had a collagenase activity of 35.97 u / mL and an enzyme production capacity that needs to be improved (Wu Qi, Li Jun, Li Chen, et al. Isolation and identification of a strain of Bacillus pumilus producing collagenase [J]. China Leather, 2007, (17): 16-19.). Therefore, screening a strain with the ability to efficiently degrade collagen and is non-toxic is the basis and key to achieving high-value utilization of cowhide through microbial fermentation. Summary of the Invention

[0005] To solve the above problems, the present invention provides a strain of Bacillus velez HALZ676 and its application and method for fermenting and degrading cowhide.

[0006] The technical solution of the present invention is achieved as follows:

[0007] The present application isolates and screens a strain of Bacillus velez HALZ676 from fermented cured pig's trotters, and analyzes its biological characteristics and whole genome. Furthermore, the molecular weight composition, antioxidant capacity and antibacterial capacity of the cowhide fermentation degradation liquid obtained by inoculating Bacillus velez HALZ676 into cowhide fermentation are studied. Finally, the metabolomics differences of cowhide fermented by Bacillus velez HALZ676 are analyzed by non-targeted metabolomics technology. In order to provide theoretical support for the deep processing and high-value utilization of livestock and poultry skin by-product resources, thereby promoting the development of the livestock processing industry and reducing environmental pollution.

[0008] On the one hand, the present application provides a strain of Bacillus velezensis HALZ676, whose classification name is Bacillus velezensis, deposited in the China Center for Type Culture Collection on March 6, 2025, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCC M 2025388.

[0009] In a second aspect, a bacterial agent comprises the aforementioned Bacillus Velez subsp. HALZ676.

[0010] In a third aspect, the above-mentioned Bacillus Velez HALZ676 or the above-mentioned bacterial agent is used in fermentation and degradation of animal skins.

[0011] In a fourth aspect, the method for fermenting and degrading cowhide using the above-mentioned Bacillus Velezii HALZ676 comprises the following steps:

[0012] (1) inoculating a seed solution of Bacillus velez HALZ676 into a fermentation medium for culturing to obtain a fermentation solution of Bacillus velez HALZ676;

[0013] (2) Chopping raw cowhide from which surface impurities have been removed is mixed with water and sterilized to obtain a mixed solution; then adding fermentation liquid of Bacillus velezensis HALZ676, and fermenting and culturing until the cowhide is completely liquefied to obtain a cowhide fermentation liquid.

[0014] The volume ratio of the seed solution in the above step (1) is 3-5%, the fermentation culture conditions are 35-37°C, 140-180r / min, and culture for 22-26h. The OD 600 The value is 6.00-6.50.

[0015] Preferably, in the above step (2), raw cowhide and water are mixed at a material-liquid ratio of 1-3:7-9 g / mL; the amount of Bacillus velezensis HALZ676 fermentation broth added is 5-10% of the volume of the mixed liquid; and the fermentation culture conditions are 35-37° C., 140-180 r / min shaking culture for 22-26 h.

[0016] In a fifth aspect, the cowhide fermentation liquid is prepared by the above-mentioned method of fermenting and degrading cowhide with Bacillus Velez HALZ676.

[0017] Preferably, the mass proportion of lipids and lipid-like molecules in the above-mentioned cow hide fermentation broth is 25.80%, and the mass proportion of organic acids and derivatives is 20.35%.

[0018] Preferably, the functionally active metabolites in the above-mentioned bovine hide fermentation broth include linoleic acid, vernolic acid, coronaric acid and 11-Hpode (11(S)-HPODE).

[0019] In a sixth aspect, the cow hide fermentation liquid is used to inhibit common foodborne pathogens, including one or more of Listeria monocytogenes, Escherichia coli, Salmonella typhimurium and Staphylococcus aureus.

[0020] In a seventh aspect, the cow hide fermentation broth is used for preparing antioxidant, anti-inflammatory and anticancer drugs.

[0021] The present invention has the following beneficial effects:

[0022] 1. This application isolated a strain of Bacillus velezensis HALZ676 from cured pig's trotters. The construction of a phylogenetic tree showed that the sequence similarity between strain HALZ676 and B. velezensis (GCF-001461825.1) was 99.9%, and it was identified as B. velezensis. Through biological characteristic analysis, it was found that strain HALZ676 has the ability to produce collagenase, is non-hemolytic, and is sensitive to antibiotics. Therefore, strain HALZ676 is safe and reliable and has the potential to be used as a probiotic. Strain HALZ676 showed exponential growth after 2 hours of growth, reached a stable phase at 16 hours, and entered a decay phase at 26 hours; the enzyme activity reached a stable phase at 22 hours and lasted to 36 hours. Therefore, fermentation at 37°C for 22-26 hours is the optimal fermentation time for Bacillus velezensis HALZ676. The microorganism is in a stable growth phase and the OD 600 At 6.25-6.35, the enzyme activity was 14.50-15.00 U / mL.

[0023] 2. Whole genome analysis showed that the genome sequence of strain HALZ676 was 3,992,894 bp in size with an average GC content of 46.50%. COG annotation revealed that the most abundant category was amino acid metabolism and transport. Strain HALZ676 was annotated in 40 pathways across six major functions in the KEGG database, including environmental information processing, cellular processes, human diseases, metabolism, genetic information processing, and organic systems, further confirming its strong collagen degradation ability.

[0024] 3. The molecular weight distribution of the cowhide fermentation broth (CFDL) showed that peptides less than 3000 Da accounted for over 68%, indicating that fermentation of cowhide with strain HALZ676 to produce collagen peptides is a feasible and efficient biotransformation method. CFDL exhibited high inhibitory activity against the foodborne pathogen Staphylococcus aureus, with an inhibition diameter reaching 24.39±1.02 mm. The antioxidant activity of CFDL was significantly enhanced, with DPPH free radical scavenging activity increasing from 4.24±0.34% to 58.05±0.68%, and hydroxyl free radical scavenging activity increasing from 2.23±0.12% to 75.64±1.16%.

[0025] 4. Inoculation with strain HALZ676 increased the metabolites of CFDL. The key differential metabolites were mainly lipids and lipidoids, accounting for 25.80% by mass, and organic acids and their derivatives, accounting for 20.35% by mass. The main upregulated metabolites were precocious phenotype II, desobutamide, fumonisin, N-lauroylphenylalanine, Pgd2 etheramide, and Gly-Asn-Val. Analysis revealed five key metabolic pathways: linoleic acid metabolism, glycerophospholipid metabolism, starch and sucrose metabolism, nucleotide metabolism, and α-linolenic acid metabolism. The nitrite metabolism pathway was the most critical pathway for the production of differentiated metabolites during cow hide fermentation. Upregulated metabolites in this pathway included linoleic acid, vernolic acid, corosolic acid, and 11-Hpode, which together promoted the conversion and enrichment of antioxidant, anti-inflammatory, and anticancer metabolites in CFDL, thereby enhancing its antioxidant, anti-inflammatory, and anticancer abilities. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0027] Figure 1 Analysis of the biological characteristics of B. velezensis HALZ676; (A) shows the collagen degradation ability of B. velezensis HALZ676, (B) shows the hemolytic activity evaluation of B. velezensis HALZ676, (C) and (D) show the antibiotic sensitivity evaluation of B. velezensis HALZ676, (E) shows the growth curve of B. velezensis HALZ676, and (F) shows the enzyme activity curve of B. velezensis HALZ676.

[0028] Figure 2 This is the circular genome map of B. velezensis HALZ676.

[0029] Figure 3 Whole-genome analysis of B. velezensis HALZ676; (A) is COG annotation classification statistics, (B) is KEGG functional classification, (C) is phylogenetic analysis, (D) is virulence gene analysis, and (E) is drug resistance gene analysis.

[0030] Figure 4 This is the biological activity determination of cowhide fermentation products; (A) is the actual picture of unfermented cowhide (UFC) and cowhide fermentation degradation liquid (CFDL), (B) is the molecular weight distribution of B. velezensis HALZ676 fermented cowhide degradation products, (C) is the antibacterial activity of unfermented cowhide treatment liquid, B. velezensis HALZ676 enrichment liquid and fermented cowhide degradation liquid, (D) is the antioxidant activity of unfermented cowhide treatment liquid, B. velezensis HALZ676 enrichment liquid and fermented cowhide degradation liquid.

[0031] Figure 5 An overview of the OPLS-DA model; (A) is an overview of the OPLS-DA model in positive ion mode, and (B) is an overview of the OPLS-DA model in negative ion mode.

[0032] Figure 6 PCA, OPLS-DA, and permutation test analysis of unfermented cowhide (UFC) and fermented degradation liquor of cowhide (CFDL); (A) is the PCA score graph in positive ionization mode, (B) is the PCA score graph in negative ionization mode, (C) is the OPLS-DA score graph in positive ionization mode, (D) is the OPLS-DA score graph in negative ionization mode, (E) is the OPLS-DA permutation test graph in positive ionization mode, and (F) is the OPLS-DA permutation test graph in negative ionization mode.

[0033] Figure 7 Metabolic networks and metabolite enrichment in the KEGG pathway; (A) is the volcano plot of differential metabolite screening between the unfermented cowhide treatment liquid group and the fermented cowhide degradation liquid group, (B) is the agglomerative hierarchical clustering heat map, (C) is the bubble map of KEGG enrichment, (D) is the five key metabolic pathways screened, and (E) is the linoleic acid metabolic network. DETAILED DESCRIPTION

[0034] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0035] Unless otherwise specified, the experimental methods used in the following experimental examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0036] A strain of Bacillus velezensis HALZ676, whose classification name is Bacillus velezensis, was deposited in the China Center for Type Culture Collection on March 6, 2025, with the deposit address being Wuhan University, Wuhan, China, and the deposit number being CCTCCM2025388.

[0037] Main reagents: Gelatin was purchased from Sigma;

[0038] Beef extract, peptone, sodium chloride, sucrose, glucose, casein, yeast powder, disodium hydrogen phosphate dodecahydrate, dipotassium hydrogen phosphate trihydrate, sodium hydroxide, agar, magnesium sulfate heptahydrate, trichloroacetic acid, casein, n-propanol, mercuric chloride, etc., are all domestically produced analytical grade or biochemical grade.

[0039] Gelatin plate culture medium: gelatin 4%, glucose 5%, agar powder 1.5%, KH2PO4 0.05%, MgSO4·7H2O0.02%, adjust the pH to 7.2-7.5.

[0040] Fermentation medium: gelatin 0.5%, peptone 1.0%, sodium chloride 1.0%, glucose 1.5%, adjust pH to 7.5.

[0041] Pretreatment of cowhide and preparation of fermentation samples:

[0042] Pretreatment: Fresh raw cowhide was purchased from Henan Hengdu Food Co., Ltd. The fat on the inside of the cowhide was scraped clean with a knife, and cut into 1 cm × 1 cm × 1 cm pieces with a scalpel. The pieces were stored in a refrigerator at -20°C until use.

[0043] Preparation of fermented cowhide sample: 5 g of treated raw cowhide and 45 mL of deionized water were placed in a conical flask to obtain unfermented cowhide pretreatment solution (UFC). After sterilization at 115 °C for 15 min, 5 mL of Bacillus velezensis HALZ676 was added and fermented at 37 °C with shaking (120 rpm / min) for 18 h until the cowhide was completely liquefied to prepare cowhide fermentation liquid (CFDL).

[0044] Example 1: Analysis of biological characteristics of Bacillus velezini HALZ676

[0045] (1) Determination of collagenase production capacity

[0046] The Bacillus velezensis HALZ676 (collected in Zigui County, Wuhan City in October 2023) stored at -80℃ was activated and cultured for three generations on nutrient agar medium. Then, a single colony was inoculated into LB liquid culture medium and cultured in a shaker at 37℃ for 18-24h (containing 10 sterile glass beads). Take 2μL of the enriched bacterial solution (OD 600 The cells were spotted onto gelatin plate culture medium and cultured at 37°C for 24 h. 35% trichloroacetic acid solution was added dropwise and the size of the transparent zone was observed.

[0047] like Figure 1 As shown in (A), after adding 35% trichloroacetic acid solution to the gelatin plate culture medium, a clear transparent zone was observed around the colony of Bacillus velez HALZ676, indicating that Bacillus velez HALZ676 has the ability to produce collagenase.

[0048] (2) Hemolysis test

[0049] After the strain was activated, the bacterial liquid was picked up with an inoculating loop and streaked on a blood agar plate (Guangdong Huankai Microbiology Technology Co., Ltd.). After incubation at 37°C for 24 h, the hemolytic activity of the strain was detected based on the formation of a transparent zone around the colony.

[0050] The results of hemolytic activity of Bacillus velez HALZ676 are as follows Figure 1 As shown in (B), no hemolytic zone appeared around the colonies of Bacillus velez HALZ676, indicating that Bacillus velez HALZ676 had no hemolytic activity.

[0051] (3) Antibiotic sensitivity test

[0052] The KB paper agar diffusion method was used to determine the antibiotic susceptibility of Bacillus velez HALZ676. That is, 200 μL of Bacillus velez HALZ676 bacterial suspension was evenly spread on LB solid culture medium. After the surface of the plate was slightly dry, antibiotic paper discs were evenly spaced on the surface. A total of 10 antibiotics, including ampicillin (AMP), erythromycin (E), tetracycline (TET), gentamicin (GEN), clindamycin (MY), penicillin (PEN), chloramphenicol (C), cephradine (CTR), ciprofloxacin (CIP), and sulfamethoxazole (SXT), were incubated at 37°C for 24 hours and then the diameter of the inhibition zone (mm) was measured. The antimicrobial drug test range and drug susceptibility results were based on the CLSI (M100-S30) and EUCAST (2021 edition) criteria.

[0053] Table 1 Antibiotic sensitivity test results of strain B. velezensis HALZ676

[0054]

[0055] Note: The diameter of the drug susceptibility plate is 6 mm. R (resistant) indicates the presence of antibiotic resistance, I (intermediate) indicates moderate antibiotic resistance, S (sensitive) indicates the presence of resistance to sensitive antibiotics, and “-” indicates no antibiotic sensitivity zone.

[0056] The results of antibiotic sensitivity test of Bacillus velez HALZ676 are as follows Figure 1 As shown in (C), (D) and Table 1, Bacillus velez HALZ676 was sensitive to ampicillin (AMP), tetracycline (TET), gentamicin (GEN), chloramphenicol (C), cefradine (CTR), ciprofloxacin (CIP), and succinate-trimethoprim-sulfamethoxazole (SXT). Therefore, Bacillus velez HALZ676 is safe and reliable and has the potential to be used as a probiotic.

[0057] Example 2: Growth curve and enzyme activity curve of Bacillus velez HALZ676

[0058] Standard curve: Prepare six test tubes of the same specifications and add 1.0 mL of glycine standard solution at concentrations of 0, 0.06, 0.12, 0.18, 0.24, and 0.30 mmol / L, respectively. Next, add 1 mL of acetate buffer and mix thoroughly. Add 1 mL of ninhydrin colorimetric solution and, after mixing, boil in a boiling water bath for 15 minutes. Cool in tap water for 5 minutes. Add 3 mL of 60% ethanol and mix thoroughly. Measure absorbance at 570 nm using a spectrophotometer. Draw a standard curve with glycine concentration as the horizontal axis and absorbance as the vertical axis.

[0059] Pick the above-mentioned well-grown single colony and inoculate it into the seed culture medium, and culture it in an air bath shake flask at 37°C and 180 rpm until the OD 600 After the value reaches 1.0, the seed culture solution is obtained. Then take 5% of the seed solution as an inoculum and transfer it to the fermentation medium (0.5% gelatin, 1.0% peptone, 1.0% sodium chloride, 1.5% glucose), and continue to culture under the same conditions. The first sampling is taken from the time of inoculation, and then sampling is taken every 2 hours. Each time 4mL of fermentation liquid is sampled for testing: 2mL of sample is used to detect the growth of the strain, and the fresh culture medium is used as a reference to measure the bacterial solution at OD 570 The absorbance value under 40°C was 0.0447 nm. 2 mL of sample was used to detect enzyme production in the fermentation broth, i.e., enzyme activity in the fermentation broth was determined using the ninhydrin method. Three parallel experiments were performed for each sample group.

[0060] Determine the OD of glycine solutions with different concentrations 570 value, made of Figure 1(E) shows the glycine standard curve. This standard curve can be used to calibrate collagenase activity. The linear standard equation obtained by curve fitting is y = 0.66x - 0.01, R 2 =0.99, and the enzyme activity (U / mL) was calculated as shown in formula (1). Figure 1 (F) is the growth curve of Bacillus velez HALZ676. It can be seen that Bacillus velez HALZ676 showed an obvious exponential growth trend after 2 hours of culture. The growth rate gradually slowed down near 16 hours and then reached a stable period. At 26 hours, the growth of Bacillus velez HALZ676 entered the decline period. The enzyme activity of Bacillus velez HALZ676 reached a stable period at 22 hours and lasted until 36 hours. Therefore, the optimal fermentation time for Bacillus velez HALZ676 was 22-26 hours at 37°C. The microorganism was in a stable growth period and the OD 600 At 6.25-6.35, the enzyme activity was 14.50-15.00 U / mL.

[0061]

[0062] In formula (1), 75 is the relative molecular mass of glycine, N is the dilution factor, and 30 is the reaction time.

[0063] Example 3: Whole genome analysis of Bacillus velezini HALZ676

[0064] 1. Whole genome analysis of Bacillus velezensis HALZ676

[0065] Bacillus velez HALZ676 was inoculated into LB liquid medium and cultured in a 37°C incubator for 24 hours. The culture broth was centrifuged at 4°C and 6000 rpm for 15 minutes, and the supernatant was discarded to obtain the Bacillus velez HALZ676 cell pellet. The pellet was then sent to Shanghai Meiji Biotechnology Co., Ltd. for quality inspection. Passing samples were sequenced using the Illumina HiSeq high-throughput sequencing platform. The whole genome sequence was annotated and functionally predicted by NCBI and IMG / M (Integrated Microbial Genomes & Microbiomes, https: / / img.jgi.doe.gov / ), respectively.

[0066] The basic information of the B. velezensis HALZ676 genome is as follows Figure 2As shown, a full-genome map was generated using Circos software. The genome sequence is 3,992,894 base pairs (bp) with an average GC content of 46.50%. It comprises a 3,989,424-bp chromosome and a 932,595-bp plasmid. The entire genome contains 3,925 protein-coding genes (CDSs), seven rRNA genes (six 5S rRNAs and one 23S rRNA), 82 tRNA genes, and 81 sRNA genes.

[0067] Phylogenetic analysis: By comparing with the local database, the 19 strains closest to each other at the species level were selected based on 31 housekeeping genes (dnaG, frr, infC, nusA, pgk, pyrG, rplA, rplB, rplC, rplD, rplE, rplF, rplK, rplL, rplM, rplN, rplP, rplS, rplT, rpmA, rpoB, rpsB, rpsC, rpsE, rpsI, rpsJ, rpsK, rpsM, rpsS, smpB, tsf), and the phylogenetic tree was constructed using the Neighbor-Joining method using MEGA6.0 software. Figure 2 (C) The results showed that the sequence similarity between strain HALZ676 and B. velezensis (GCF-001461825.1) was 99.9%. Therefore, strain HALZ676 was identified as B. velezensis and named B. velezensis HALZ676.

[0068] COG annotation results: COG annotation was performed on genes encoding functional proteins in the B. velezensis HALZ676 genome, such as Figure 3 As shown in (A), a total of 3491 protein-coding genes were annotated to have biological activity. The gene function annotation information was divided into 25 categories, of which the most abundant category was amino acid metabolism and transport (Amino acid transport and metabolism), with a total of 304 genes, accounting for 8.71% of the total annotated genes.

[0069] KEGG functional annotation: KEGG is a database that systematically analyzes the metabolic pathways of gene products in cells and the functions of gene products. Functional annotation results are as follows Figure 3As shown in (B), a total of 2,673 genes in B. velezensis HALZ676 were annotated in the KEGG database for 40 pathways across six major functions: environmental information processing, cellular processes, human diseases, metabolism, genetic information processing, and organic systems. The largest number of genes, 1,872, were involved in metabolism, with 731 of these annotated to global and overview maps. Furthermore, 208 genes were annotated to amino acid metabolism, and 167 genes were associated with amino acid metabolism. This further confirms that B. velezensis HALZ676 possesses strong collagen degradation capabilities.

[0070] 2. Virulence gene and drug resistance gene analysis:

[0071] The safety of B. velezensis HALZ676 was evaluated by analyzing its genome using the VFDB database. Figure 3 As shown in (D). Using an E-value ≤ 1e-5 as a screening criterion, a total of 462 virulence genes were identified in B. velezensis HALZ676, none of which shared greater than 70% similarity with known virulence genes. No genes encoding hemolysin (hly), cytotoxin K (cyt), diarrheal toxin (bce), hemolytic enterotoxin (hbl), or non-hemolytic enterotoxin (nhe) were detected. None of the strains formed pericolony zones on Columbia agar or MYP agar, and hemolytic and lecithinase activities were negative, respectively.

[0072] Comparison with the CARD database revealed that 276 drug-resistant genes were identified in the B. velezensis HALZ676 genome, accounting for only 7.03% of the total number of genes. Figure 3(E) and Table 2 show the statistical results of the predicted resistance gene classification. Among them, the most common resistance genes were associated with peptide antibiotics, tetracycline antibiotics, macrolide antibiotics, fluoroquinolones, penicillins, disinfectants and antiseptics, and glycopeptide antibiotics, with 52, 45, 36, 34, 29, 29, and 20 genes, respectively. This is consistent with the in vitro results that showed B. velezensis HALZ676 to be resistant to penicillin, erythromycin, and clindamycin. However, in vitro studies do not fully support these findings. For example, while resistance genes to β-lactams, tetracyclines, aminoglycosides, fluoroquinolones, and sulfonamides were found in the genome of B. velezensis HALZ676, in vitro results showed that it was sensitive to ampicillin, cefradine, tetracycline, gentamicin, and ciprofloxacin, indicating that not all predicted genes in the genome are expressed. In many cases, the presence of antibiotic resistance genes facilitates the use of probiotics, as these antibiotics are used to treat common infections. Many commercial Bacillus strains have been shown to be resistant to erythromycin, lincomycin, penicillin, streptomycin, etc., yet they are widely used as probiotics.

[0073] Table 2 Prediction and classification statistics of antibiotic resistance genes

[0074]

[0075]

[0076] 3. Molecular weight (MW) distribution of cowhide fermentation broth

[0077] The degraded bovine hide solution was filtered through a 0.22 μm pore size membrane and ultrasonically degassed for 15 min before being fixed to volume. The molecular weight distribution of the degraded bovine hide solution was measured using an HPLC SCL-10AVP (Shimadzu Co., Ltd., Tokyo, Japan). The analytical conditions were a TSK gel G2000 SWXL column (7.8 mm × 300 mm, 5 μm); a mobile phase of acetonitrile:water:trifluoroacetic acid (20:80:0.1 v / v); a detection wavelength of 220 nm; and a flow rate of 0.5 mL / min. -1 ; The column temperature is 30℃.

[0078] Figure 4 (A) is a physical picture of unfermented cowhide (UFC) and cowhide fermentation degradation liquid (CFDL). Figure 4(B) shows the molecular weight distribution curve of CFDL, where the relative contents of peptides <1000 Da, peptides 1000-3000 Da, peptides 3000 < 5000 Da, and peptides >5000 Da were 36.68 ± 0.31%, 32.26 ± 0.21%, 13.38 ± 0.26%, and 17.69 ± 0.22%, respectively. These results indicate that peptides less than 3000 Da account for over 68% of the fermented CFDL, demonstrating that fermentation of bovine hide with B. velezensis HALZ676 to produce collagen peptides is a feasible and efficient bioconversion method.

[0079] Example 4: Bioactivity of Fermented Cowhide

[0080] 1. Antibacterial activity analysis

[0081] The agar well diffusion method was used to test the antimicrobial activity of unfermented cowhide pretreatment solution, enrichment solution of Bacillus Velez HALZ676, and degradation solution of cowhide fermented with Bacillus Velez HALZ676. 100 μL of each sample was added to plates coated with pathogens (Listeria monocytogenes ATCC 19115, Escherichia coli ATCC 25922, Salmonella typhimurium ATCC 14028, and Staphylococcus aureus ATCC 49230). The plates were incubated at 37°C for at least 24 hours. The antimicrobial activity was then observed, and the diameter of the zone of inhibition was measured using a vernier caliper.

[0082] The antimicrobial activities of unfermented cattle hide treatment solution, Bacillus velezensis HALZ676 enrichment solution and fermented cattle hide degradation solution against common foodborne pathogens are shown in Figure 4 (C). The results showed that the unfermented cowhide treatment liquid had no antibacterial activity against four common foodborne pathogens. However, the fermented cowhide degradation liquid had significant antibacterial activity against common foodborne pathogens and had a significant inhibitory effect on the four pathogens. Among them, the inhibitory activity of the fermented cowhide degradation liquid against Staphylococcus aureus ATCC 49230 (G) was significantly higher than that of the Bacillus Velez HALZ676 enrichment liquid. Among them, the fermented cowhide degradation liquid had the highest inhibitory activity against Staphylococcus aureus ATCC 49230 (G), with an inhibition diameter of 24.39 ± 1.02 mm.

[0083] 2. Determination of antioxidant activity

[0084] The antioxidant activity was determined according to the instructions of the DPPH free radical scavenging activity kit (Beijing Solebold Technology Co., Ltd., BC4750) and the hydroxyl free radical scavenging activity kit (Beijing Solebold Technology Co., Ltd., BC1320).

[0085] Figure 4 (D) Shows the antioxidant activities of unfermented cattle hide treatment fluid, Bacillus Velez HALZ676 enrichment fluid, and fermented cattle hide degradation fluid. The results showed that both antioxidant activities of CFDL were significantly increased (p < 0.05). DPPH free radical scavenging activity increased from 4.24 ± 0.34% (UFC) before fermentation to 58.05 ± 0.68% (CFDL) after fermentation, and hydroxyl free radical scavenging activity increased from 2.23 ± 0.12% (UFC) before fermentation to 75.64 ± 1.16% (CFDL) after fermentation.

[0086] Example 5: Metabolite Analysis of Unfermented Cowhide (UFC) and Cowhide Fermented Degraded Liquor (CFDL)

[0087] 1. Metabolite Analysis: UFC and CFDL were sent to Shanghai Meiji Biopharmaceutical Technology Co., Ltd. (Shanghai, China) for nontargeted metabolomics analysis, with four replicates per sample. The following procedures were performed: 50 mg of each sample was mixed with 400 μL of extraction solution (methanol:water = 4:1), ground in a cryo-tissue grinder for 6 min (-10°C, 50 Hz), and subjected to cryo-ultrasonic extraction for 30 min (5°C, 40 kHz). After stabilization, the supernatant was centrifuged for 15 min (4°C, 13,000 g). The supernatant was then analyzed by LC-MS / MS using a UHPLC Q Exactive HF-X system (Thermo Fisher Scientific). Chromatographic conditions: 3 μL of sample was separated on an HSS T3 column (100 mm × 2.1 mm ID, 1.8 μm) before mass spectrometry. Mobile phase A was 95% water + 5% acetonitrile (containing 0.1% formic acid), and mobile phase B was 47.5% acetonitrile + 47.5% isopropanol + 5% water (containing 0.1% formic acid). The flow rate was 0.40 mL / min, and the column temperature was 40°C. Mass spectrometry conditions: Mass spectrometry signals were acquired using both positive and negative ion scanning methods, with a mass spectrometry scan range of 70-1050 m / z. The sheath gas flow rate was 50 psi, the auxiliary gas flow rate was 13 psi, the temperature was 425°C, the positive mode ion spray voltage was 3500 V, the negative mode ion spray voltage was -3500 V, the ion transfer tube temperature was 325°C, the primary mass spectrometer resolution was 60,000, the secondary mass spectrometer resolution was 7500, and data were collected in DDA mode.

[0088] Key differential metabolite analysis: multivariate analysis of non-targeted metabolomics based on LC / MS

[0089] Figure 6(A) and (B) depict principal component analysis (PCA) plots of metabolite data from the unfermented cowhide treatment liquid group and the fermented cowhide degradation liquid group collected in positive and negative ion modes, respectively. In positive ion mode, PCA results showed that the contribution rates of PC1 and PC2 were 85.50% and 3.19%, respectively, with a cumulative contribution rate of 88.69%. In negative ion mode, PCA results showed that the contribution rates of PC1 and PC2 were 87.00% and 2.98%, respectively, with a cumulative contribution rate of 89.98%. In both positive and negative ion modes, the metabolomic data of the two groups of samples showed a clustered distribution of samples within the group, with clear separation between the groups, clearly reflecting the differences in metabolites before and after fermentation, indicating that fermentation significantly changed the types and contents of metabolites in CFDL.

[0090] To more clearly characterize the differences in metabolites, an orthogonal least partial squares discriminant analysis (OPLS-DA) model was used for in-depth analysis. OPLS-DA is a supervised analysis method that effectively reduces intra-group differences, amplifies inter-group differences, eliminates the influence of irrelevant variables on experimental data, and improves the model's effectiveness and analytical capabilities. Figure 6 (C) and (D) are the OPLS-DA scores of the metabolite data of the UFC group and CFDL group collected in positive and negative ion modes, respectively. The OPLS-DA model effectively distinguished the metabolites of the unfermented cowhide treatment liquid group and the fermented cowhide degradation liquid group, confirming the significant metabolic differences between the two. Figure 5 (A) and (B) are overviews of the OPLS-DA model in positive and negative ion modes, respectively. 2 Y and Q 2 They are all close to 1, indicating that the model is stable and reliable with good predictive ability. Figure 6 (E) and (F) are the OPLS-DA permutation test diagrams of the metabolite data of the non-fermented cowhide treatment liquid group and the fermented cowhide degradation liquid group under positive and negative ion modes, respectively. The results show that R 2 X, R 2 Y and Q 2 They are all close to 1, indicating that the model is stable and reliable with good predictive ability, and the variable projection importance (VIP) method can be used to screen differential metabolites.

[0091] 2. Analysis of differential metabolites

[0092] We screened the differential metabolites using p-value ≤ 0.05 and VIP ≥ 1 as criteria, and obtained a total of 1558 differential metabolites. Compared with the unfermented cowhide treatment liquid group, 1069 differential metabolites were upregulated and 489 were downregulated in the fermented cowhide degradation liquid group, indicating that inoculation with Bacillus Velezii HALZ676 can increase the metabolites of CFDL. The main types of key differential metabolites were lipids and lipid-like molecules (25.80%), organic acids and derivatives (20.35%), organic heterocyclic compounds (9.37%), organic oxygen compounds (5.78%), benzene ring compounds (4.04%), phenylpropanoids and polyketides (3.34%), alkaloids and derivatives (1.54%), nucleosides, nucleotides and analogues (1.48%), organic nitrogen compounds (0.77%) and others (27.54%). We combined the above results with fold change (FC>3 and FC<0.3) to further screen the metabolites with significant differences between the non-fermentation group and the fermentation group. The results are shown in Table 3.

[0093] Figure 7(B) Agglomerative hierarchical clustering using a heatmap visualization of the top 30 differentially abundant metabolites. As shown in Table 3, there were 33 significantly differentially expressed metabolites between the fermented and unfermented bovine hide degradation liquid groups. Of these, 23 were significantly upregulated in the fermented bovine hide degradation liquid group relative to the unfermented bovine hide treatment liquid group, while 10 were significantly downregulated. Substances that increased in the fermented bovine hide degradation liquid group included: Precocene II (329.41-fold increase), Disobutamide (11.65-fold increase), Fumifugin (10.58-fold increase), N-Lauroyl Phenylalanine (8.16-fold increase), Pgd2Ethanolamide (6.31-fold increase), and Gly-Asn-Val (5.91-fold increase). Precocene II has excellent antioxidant activity and demonstrates enhanced cytotoxicity and apoptosis-inducing potential against cervical cancer cell lines (HeLa). Disobutamide is primarily used as an antiarrhythmic agent and is used to treat cardiac arrhythmias, particularly cardiac depressants. Fumifungin is an antifungal antibiotic that kills yeast, mold, and fungi by interfering with cell membrane ion exchange. N-lauroyl phenylalanine significantly increases the intensity and duration of umami flavor, masks bitterness, and reduces the inherent fishy odor of cowhide. Pgd2 ethanoylamide is a bioactive lipid that induces apoptosis in skin cancer cells by inhibiting the activity of cellular antioxidants.

[0094] Table 3 Significant differences in metabolites between the UFC group and the CFDL group

[0095]

[0096] 3. Metabolic pathway analysis

[0097] Figure 7 (C) shows the bubble diagram of KEGG enrichment (top 20), and the results show that the change of CFDL has a significant impact on metabolic pathways such as Cholinemetabolismin cancer and Fc epsilon RI signaling pathway (P<0.01). KEGG topology analysis was performed based on the impact value and P value (P<0.01). The results are shown in Figure 2. Figure 7As shown in (D), the five most significant key metabolic pathways screened out were: linoleic acid metabolism, glycerophospholipid metabolism, starch and sucrose metabolism, nucleotide metabolism, and alpha-linolenic acid metabolism. Among them, the nitrite metabolism pathway had the largest bubbles and the darkest color, indicating that the nitrite metabolism pathway is the most critical pathway for the production of differentiated metabolites during cowhide fermentation.

[0098] Table 4 Metabolites enriched in the linoleic acid metabolic pathway

[0099]

[0100] Figure 7 (E) and Table 4 depict the metabolic network of the nitrite metabolic pathway, as well as the metabolites enriched and differentiated in this pathway. The results showed that linoleic acid (Linoleate), vernolic acid (12(13)-EpOME), corosolic acid (9(10)-EpOME) and 11-Hpode (11(S)-HPODE) were the main metabolites upregulated in the nitrite metabolic pathway in this study. Linoleic acid (LA) is an omega-6 polyunsaturated fatty acid and an essential fatty acid. This fatty acid plays a vital role in maintaining health. LA shows anti-inflammatory effects, skin whitening and moisturizing properties on the skin. The combination of LA and conjugatedlinoleic acid (CLA) showed a strong synergistic effect on inflammatory macrophage RAW264.7 cells by inhibiting proliferation and stimulating cell death, resulting in a decrease in cell number in vitro, and a low-content combination of LA and CLA showed an effective anti-inflammatory effect on the skin. LA enhances CD8 +T cell metabolic adaptability and antitumor immunity were significantly improved, significantly enhancing antitumor responses in various tumor types. Upregulation of LA promoted the synthesis of methyl-cis-9-octadecene-12-ynoate (crepenynate) and 9C,11TR-conjugated linoleic acid methyl ester (9cis,11-trans-octadecadienoate). Crepenynate has potential anti-inflammatory and antibacterial properties and may have adjuvant therapeutic effects in certain inflammatory diseases and bacterial infections. Its antioxidant and anti-inflammatory properties may make it an active ingredient in cosmetics. 9C,11TR-conjugated linoleic acid methyl ester (9cis,11-trans-octadecadienoate) is one of the main active isomers of conjugated linoleic acid (CLA) and has important value in antioxidant, cardiovascular protection, metabolic regulation, and immune enhancement. Vernolic acid has significant antioxidant properties, neutralizing free radicals, reducing oxidative stress, and protecting cells from damage. In addition, it also has anti-inflammatory and antibacterial activity, can alleviate inflammatory symptoms, and has important application value in food preservation and drug development. Coronaric acid has potential anti-inflammatory and antioxidant properties, and may have an adjuvant therapeutic effect on certain inflammatory diseases (such as cardiovascular disease and skin diseases). Antioxidant, anti-inflammatory, blood sugar regulation, cardiovascular protection and digestion promotion. In summary, in this application, Bacillus Velez fermented cowhide mainly promotes the conversion and enrichment of antioxidant, anti-inflammatory and anti-cancer metabolites in CFDL through the linoleic acid metabolic pathway, thereby enhancing its antioxidant, anti-inflammatory and anti-cancer abilities.

[0101] in conclusion

[0102] This application addresses the current situation of resource waste in which cowhide collagen is not fully utilized during processing, and constructs a strategy for high-value utilization of livestock and poultry skin by-products through microbial fermentation.

[0103] We screened a strain of Bacillus Velez HALZ676 with high collagenase production from traditional Chinese fermented meat products (cured pig's trotters), and verified its safety and probiotic properties through its biological characteristics and whole genome. Furthermore, Bacillus Velez HALZ676 was inoculated into cowhide for fermentation and the differences in samples before and after fermentation and degradation of cowhide by Bacillus Velez HALZ676 were compared through bioactivity indicators and metabolomics analysis. The results of the study showed that Bacillus Velez HALZ676 is a safe and reliable strain with probiotic potential. The cowhide degradation liquid obtained by fermenting cowhide with Bacillus Velez HALZ676 has significant antioxidant and antibacterial properties, especially against Staphylococcus aureus.

[0104] Untargeted metabolomics analysis based on LC / MS revealed significant differences in metabolites before and after fermentation, with increased abundance of metabolites associated with antioxidant, anti-inflammatory, and anti-cancer properties. Key differential metabolites were primarily lipids and lipid-like molecules (25.80%) and organic acids and derivatives (20.35%). Metabolites that increased in the fermented bovine hide degradation liquid group included precocene II, disobutamide, fumifungin, N-lauroyl phenylalanine, Pgd2 ethanoylamide, and Gly-Asn-Val. KEGG pathway enrichment analysis showed that linoleic acid metabolism was the main pathway for producing differentially abundant metabolites, indicating that Bacillus velezini HALZ676 promoted linoleic acid metabolism and conversion during the fermentation of cowhide. The main functionally active metabolites enriched included linoleic acid, vemolic acid, coronaric acid, and 11-Hpode (11(S)-HPODE). This application shows that Bacillus velezini HALZ676 fermentation of cowhide can obtain rich functional active ingredients, providing a theoretical basis and data support for the functionalization and high-value utilization of cowhide scraps, and providing a promising strategy for the high-value production of livestock and poultry skin processing by-products fermented with high-yield collagenase probiotic Bacillus velezini HALZ676.

[0105] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A strain of Bacillus velez HALZ676, whose taxonomic name is Bacillus velezensis , deposited in the China Center for Type Culture Collection on March 6, 2025, with the deposit address at Wuhan University, Wuhan, China, and the deposit number is CCTCC M2025388.

2. A bacterial agent, characterized in that: The method comprises the Bacillus velez HALZ676 according to claim 1.

3. Use of the Bacillus Velezii HALZ676 according to claim 1 or the bacterial agent according to claim 2 in fermentation and degradation of animal skins.

4. The method for fermenting and degrading cowhide using Bacillus Velezii HALZ676 according to claim 1, characterized in that: The steps are: (1) inoculating a seed solution of Bacillus velez HALZ676 into a fermentation medium for culturing to obtain a fermentation solution of Bacillus velez HALZ676; (2) Chop the raw cowhide from which surface impurities have been removed, mix it with water, and sterilize it to obtain a mixed solution; then add the fermentation liquid of Bacillus velezensis HALZ676, and ferment and culture until the cowhide is completely liquefied to obtain the cowhide fermentation liquid.

5. The method for fermenting and degrading cowhide using Bacillus Velez subtilis HALZ676 according to claim 4, characterized in that: The volume ratio of the seed solution in step (1) is 3-5%, the fermentation culture conditions are 35-37°C, 140-180 r / min, and culturing for 22-26 h. The OD of the fermentation liquid of Bacillus velezensis HALZ676 is 600 The value is 6.00-6.50; in step (2), raw cowhide and water are mixed at a material-liquid ratio of 1-3:7-9 g / mL, the amount of Bacillus velezensis HALZ676 fermentation liquid added is 5-10% of the volume of the mixed liquid, and the fermentation culture conditions are 35-37°C, 140-180 r / min shaking culture for 22-26 h.

6. The cowhide fermentation liquid prepared by the method for fermenting and degrading cowhide using Bacillus Velez subtilis HALZ676 according to claim 4 or 5.

7. The cowhide fermented liquid according to claim 6, characterized in that: The mass proportion of lipids and lipid molecules in the cowhide fermentation liquid is 25.80%, and the mass proportion of organic acids and their derivatives is 20.35%.

8. The cowhide fermented liquid according to claim 6, characterized in that: The functionally active metabolites in the cow hide fermentation liquid include linoleic acid, vernolic acid, corosolic acid and 11-Hpode.

9. Use of the cow hide fermented liquid according to any one of claims 6 to 8 in inhibiting common foodborne pathogens, characterized in that: The foodborne pathogens include one or more of Listeria monocytogenes, Escherichia coli, Salmonella typhimurium and Staphylococcus aureus.

10. Use of the cow hide fermentation broth according to any one of claims 6 to 8 in the preparation of antioxidant, anti-inflammatory and anticancer drugs.

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