Probiotic characteristics, whole-genome sequence analysis of Bacillus with high collagenase production and its fermentation hydrolysis of cowhide

Probiotic characteristics, whole-genome sequence analysis of Bacillus with high collagenase production and its fermentation hydrolysis of cowhide | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Probiotic characteristics, whole-genome sequence analysis of Bacillus with high collagenase production and its fermentation hydrolysis of cowhide Shijie Liu, Qian Ding, Yueyv Bai, Lijun Zhao, Miaoyun Li, Jong-Hoon Lee, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7565406/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract In this study, strains with high collagenase producing enzyme production were isolated from traditional naturally fermented foods rich in collagen. The safety of these strains was evaluated through in vitro biological characteristics and whole-genome sequence analysis. The results indicated that strains HA3, HA5, HA7, and HA8 exhibited high collagenase production, lacked hemolytic activity, and were sensitive to most commonly used antibiotics. Comprehensive analysis of antibiotic resistance genes, virulence factor genes, and metabolic systems at the genomic level confirmed the safety of these four strains, which were identified as B. spizizenii HA3, B. velezensis HA5, B. safensis HA7, and B. velezensis HA8, respectively. Notably, B. velezensis HA5 and B. velezensis HA8 could completely degrade cowhide into a liquid state and the antioxidant activity of the resulting fermentation degradation liquid was evidently improved. This study provides theoretical support and technical reserves for the development and utilization of livestock and poultry skins. Collagenase producing enzyme Probiotic Bacillus Safety Whole-genome analysis Cowhide fermentation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1 Introduction With the development of the livestock industry, the annual production of beef, pork, and chicken is expanding, which has led to a large amount of by-products that are rich in collagen such as skin and cartilage [1]. However, due to the water-insoluble nature and complex triple helix structure of collagen, the collagen in animal skin is too difficult to degrade and has absolutely low bioavailability. This not only results in economic losses, but it poses a serious threat to the natural environment [2]. Under the limited environmental carrying capacity of livestock farming, how to achieve the refined deep processing and high-value utilization of livestock by-products is of great significance. It can promote the comprehensive utilization of livestock products, and is also important for the development of the livestock industry and environmental protection [3]. The preparation of collagen peptides is an effective way to achieve high-value utilization of collagen in livestock by-products. As a functional protein ingredient, collagen peptides are increasingly being used in food, nutritional supplements, pharmaceuticals, and cosmetics [4, 5]. With the continuous increase of people's consumption, especially the economic development of the Asia-Pacific region represented by China, consumers' demands and consumption for health, beauty, and wellness are constantly increasing. The global consumer demand for collagen peptide products continues to grow, and the transactions of the global collagen peptide products keep rising [6, 7]. Nowadays, the preparation methods for pig skin and fish skin collagen peptides are mostly enzymatic hydrolysis. However, use the enzymatic hydrolysis method for preparing collagen peptides requires the addition of chemical reagents for defatting and removing the miscellaneous proteins in the pre-treatment process. Moreover, after enzymatic hydrolysis, deodorization and bitterness removal are needed, which process is complex and poses serious environmental pollution issues [8, 9]. Microbial fermentation degradation is an emerging, environmentally friendly, and efficient biotechnological method for preparing collagen peptides. By using microbial fermentation to obtain collagen, the steps of defatting, removing miscellaneous proteins, enzymatic hydrolysis, and extracting collagen can be concentrated in a fermentation process. This greatly simplifies the process, reduces the use of toxic chemical reagents, and aligns with the trend of eco-friendly production, which is an environmentally friendly technology [10-12]. The complex enzyme systems secreted by microorganisms may produce a variety of functional small-molecule active peptides with potential activities such as antioxidant and anti-inflammatory. Microbial fermentation is a worthwhile method to promote the high-value utilization of livestock by-products, such as cowhide [13]. However, the safe and probiotic microbial strain with high collagenase production is the prerequisite and key to the degradation of collagen by microbial fermentation [14]. Up to now, collagenase-producing strains are mainly Clostridium histolyticum , Clostridium perfringens , and Vibrio alginolyticus , which are mostly pathogenic. While producing collagenase, they also produce toxins or pathogenic factors in varying degrees. This results in low safety and high production costs of the collagen peptide products obtained, which make against to further processing and utilization [15-17]. With the in-depth research on probiotics, probiotic bacilli have become a heat topic as the next-generation. Many microorganisms are adapted to their living environments. In China, there are a large number of naturally fermented foods rich in collagen raw materials, such as fermented pig's trotters and fermented sour fish, which not only have a history of thousands of years, but also serve as a treasure of probiotic resources [18, 19]. These probiotic bacilli are involved to varying degrees in traditionally natural fermented foods and have advantages of being diverse in species, fast in growth and reproduction, and rich in enzyme systems. This provides the possibility for screening safe strains that can produce a large amount of collagenase [20, 21]. In this study, strains with high collagenase production were screened from traditional natural fermented foods rich in collagen raw materials (fermented air-dried trotters and fermented sour fish), and their safety was evaluated through hemolysis and antibiotic susceptibility. Furthermore, the whole genome sequence of the organism was obtained from the screened strains with high safety by whole genome sequencing technology, and the organism was interpreted from the molecular level, and the related functional genes were mined to clarify the evolutionary relationship of species. Finally, the target strain was inoculated into cowhide fermentation medium for practical verification test, and the degradation effect and functional activity of cowhide fermented by the target strain were determined, which provided theoretical support and technical reserve for the development and utilization of livestock and poultry skin resources and had broad application prospects. 2 Materials and Methods 2.1 Experimental Materials Fresh raw cowhide, purchased from Henan Hengdu Food Co., Ltd .. Clean the impurities on the surface of the cowhide and cut it into pieces. Wash it with distilled water and drain it thoroughly and store it in a refrigerator at -20℃ for later use. Fermented air-dried trotters and fermented sour fish were sampled in Zigui County, Hubei Province and Longli County, Guizhou Province, respectively. Gelatin agar medium: gelatin 4%, glucose 5%, agar powder 1.5% KH 2 PO 4 0.05%, MgSO 4 ·7H 2 O 0.02%, pH value to 7.2~7.5. Fermentation medium: gelatin 0.5%, peptone 1%, sodium chloride 1%, glucose 1.5%, pH to 7.5. LB agar medium and LB liquid medium were purchased from Qingdao Haibo Biological Co., Ltd. . 2.2 Screening of collagenase producing strains Weigh 25 g of sample, suspend it in 225 mL sterile physiological saline and homogenize it, then coat it on gelatin culture medium, culture it at 37℃ for 24-48 h, and observe and select a single colony with obvious transparent circle. Single colony was selected and cultured in gelatin agar medium for three times. Acid mercury reagent (15g HgCl 2 , 20g HCl, titrate with water to 100mL) was dripped around the colony, and the formation of transparent circle was observed [22]. 2.3 Detection of collagenase activity The single colony of the target strain was inoculated into 50 mL LB broth, respectively, and cultured overnight at 37℃ and 160 r/min for 24 h to logarithmic phase. Inoculated with 1% inoculation amount into a 250 mL sterile triangular flask containing 50 mL fermentation medium, cultured for 48 h at 37℃ and 200 r/min, and centrifuged (8000 r/min, 20 min) to collect supernatant for enzyme activity determination. All experiments were designed in parallel for three times. Take a 5 mg/mL cowhide type I collagen solution as the substrate, add 1 mL of the collagenase solution to be detected, and react in a 37℃ water bath for 30 minutes. Add 300 μL of 30% trichloroacetic acid as the termination solution to terminate the reaction. At the same time, use the same reaction system containing inactivated collagenase as the control, and then add 600 μL of 2 mol/L acetic acid buffer and 600 μL of indene ketone colorimetric solution in sequence. Mix well and boil in a boiling water bath for 15 min. After cooling, add 1.8 mL of 60% ethanol to dilute the reaction solution. Use a multifunctional enzyme-linked immunosorbent assay reader to measure the absorbance at 570 nm. The enzyme activity unit is defined as the amount of enzyme that hydrolyzes collagen to produce 1 μL glycine per minute at 37℃ and pH 7.5, which is one enzyme activity unit (U) [23]. 2.4 Evaluation of microbial safety 2.4.1 Hemolytic evaluation After activating 4 strains of Bacillus, the bacterial solution was picked out using an inoculation ring and streaked on a blood agar plate (Guangdong Huankai Microbial Technology Co., Ltd. ). After culturing at 37℃ for 24 hours, the hemolytic activity of the strains was detected based on the formation of a transparent circle around the colony [24]. 2.4.2 Antibiotic sensitivity test The antibiotic sensitivity of four strains was determined using K-B paper agar diffusion method. Take 200 μL of bacterial growth solution from 4 strains of Bacillus subtilis and evenly spread it on LB agar medium. After the surface of the plate is slightly dry, place antibiotic paper strips evenly spaced on the surface and incubate at 37℃ for 24 h. Measure the diameter of the inhibition zone (mm). The testing scope of antibacterial drugs and drug sensitivity results refer to the CLSI (M100-S30) and EUCAST (2021 edition) judgment standards [25]. 2.5 Whole genome analysis Illumina sequencing platform was used to identify and analyze the whole genome of the high-yielding collagenase strains. The Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Groups (COG), Non Redundant Proteins (NR), Carbohydrate Active Enzymes (CAZy), Transporter Classification Database (TCBD), Swiss Prot, and Pfam databases were used to annotate the key functional genes of the high-yielding collagenase strains at the gene level. 2.6 Fermentation degradation of cowhide by bacterial strains 10 g cowhide and 90 mL distilled water were put into a 250 mL conical flask, sealed and sterilized at 121℃ for 15 min, which was used as the substrate culture medium for cowhide fermentation. Inoculate 5 mL of enrichment solution with the concentration of 10 6 CFU/mL, shake culture at 37℃ for 36 h and observe the degradation of cowhide every 2 h. 2.7 Biological activity of degradation solution 2.7.1 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity The DPPH radical scavenging rate was determined according to the method of Abbas et al. [26], and calculated according to eq. (1). DPPH radical scavenging rate =[1-( A i - A c )/ A 0 ]x100% eq. (1) Where, A i is the absorbance of 2 mL of 0.1mmol/L DPPH anhydrous ethanol solution and 2 mL of sample mixed solution, A c is the absorbance of 2 mL of sample solvent and 2ml of anhydrous ethanol mixed solution, A 0 is the absorbance of 2 mL sample solvent and 2 mL 0.1 mmol/L DPPH anhydrous ethanol mixture. 2.7.2 Hydroxyl radical scavenging activity 0.1 mL of 9 mmol/L FeS0 4 ·7H 2 0, 0.1 mL of 9 mmol/L salicylic acid, 0.1 mL of sample to be tested and 0.1 mL 0.03% H 2 0 2 were mixed and incubated at 37℃ for 15 min, and the absorbance at 510 nm was determined [27]. The hydroxyl radical scavenging rate is calculated according to eq. (2). Hydroxyl radical scavenging rate= A -( B - C )/( A )×100% eq. (1) Where, A is the absorbance without sample, B is the absorbance after adding the sample, C is the absorbance of the blank reagent. 3 Results and discussion 3.1 Isolation of collagenase-producing strains and assay of enzymatic activity Using gelatin agar plates combined with the gradient dilution method and spot inoculation, a total of nine collagenase-producing strains with distinct hydrolysis zones were isolated and screened from fermented air-dried pork trotters and fermented sour fish. The hydrolysis zones produced by these strains on gelatin agar plates are shown in Fig. 1. According to Section 2.3, the nine strains were subjected to collagenase activity assay. As shown in Fig. S1, all nine screened strains were capable of producing collagenase, and their enzyme activity levels followed this order: HA5> HA7> HA8> HA9> HA3> HA6> HA4> HA2> HA1. Fig. 1 3.2 Evaluation of microbial safety As shown in Fig.2, strains HA1, HA2, HA4, HA6, and HA9 exhibited distinct hemolytic zones around their colonies, indicating the production of exoenzymes or exotoxins capable of lysing red blood cells or degrading proteins. Consequently, these five strains were excluded from further experiments. In contrast, HA3, HA5, HA7, and HA8 showed no hemolytic activity, as no clear zones were observed [28]. These four non-hemolytic strains were subsequently subjected to antibiotic susceptibility testing, with the results presented in Table S1. These four strains exhibited multidrug resistance, they were susceptible to Ampicillin (AMP), Penicillin (PEN), Tetracycline (TET), Gentamicin (GEN), Chloramphenicol (C), Sulfamethoxazole (SXT), and Vancomycin (Van), but showed varying degrees of resistance to Cefradine (CTR), Erythromycin (E), Clindamycin (MY), and Ciprofloxacin (CIP). Strain HA3 was intermediate in susceptibility to Cefradine (CTR), Erythromycin (E), and Ciprofloxacin (CIP). Strains HA5, HA7, and HA8 were susceptible to Cefradine (CTR), Ciprofloxacin (CIP), and Erythromycin (E). Strains HA5 and HA8 were intermediate in susceptibility to Clindamycin (MY), while strains HA3 and HA7 were susceptible to Clindamycin (MY). The results indicated that the four strains were susceptible to most commonly used antibiotics, ensuring their safety and controllability during application [29]. Fig. 2 3.4 Whole genome analysis 3.4.1 Genome-wide overview The genomic characteristics of the four strains are shown in Table S2 and Fig. 3. As shown in Fig. 3 (A), almost all the points in the GC-Depth distribution plots of the genomic data of the four strains are concentrated within a relatively narrow range. This indicates that there was no species contamination during the genome assembly process. Meanwhile, the GC-Depth is basically in accordance with the Poisson distribution, with a certain degree of GC bias. Overall, the low sequence redundancy and high confidence level suggest that the assembly results are normal. According to Fig.3 (B) and Table S2, the whole-genome sequence sizes of HA3, HA5, HA7, and HA8 are 4,048416 bp, 4,183,037 bp, 3714516 bp, and 4122232 bp, respectively. Their average GC contents are 43.68%, 45.62%, 41.61%, and 45.64%, respectively. The numbers of protein-coding genes (CDSs) are 4053, 4140, 3695, and 4156, respectively. Fig. 3 3.4.2 Phylogenetic tree of house-keeping genes By comparing with the local database based on 31 house-keeping 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 ), the 19 strains closest at the species level were selected. The NJ (Neighbor-Joining) method in MEGA 6.0 software was used to construct the phylogenetic tree. As shown in Fig. 4, strains HA3, HA5, HA7, and HA8 respectively had the highest phylogenetic affinity with Bacillus spizizenii (GCF 000227465.1), Bacillus velezensis (GCF 001461825.1), Bacillus safensis (GCF 000691165.1), and Bacillus velezensis (GCF 001461825.1), with sequence similarities of 99.4%. Therefore, strains HA3, HA5, HA7, and HA8 were identified as Bacillus spizizenii , Bacillus velezensis , Bacillus safensis, and Bacillus velezensis ,and were named B. spizizenii HA3, B. velezensis HA5, B. safensis HA7, and B. velezensis HA8. Fig. 4 3.4.3 Gene prediction and functional annotation COG annotation. The genes encoding functional proteins in the genomes of strains B. spizizenii HA3, B. velezensis HA5, B. safensis HA7, and B. velezensis HA8 were annotated with COG (as shown in Fig. S2). It was found that the number of protein-coding genes annotated with biological activity was 3282, 3201, 3026, and 3196. The gene function annotation information was divided into 25 categories, the most abundant category among the four strains are Aminoundefinedacid transport and metabolism, Carbohydrateundefinedtransportundefinedand metabolism and Transcription. KEGG functional annotation. KEGG is a database that systematically analyzes the metabolic pathways and functions of gene products in cells. The annotation results are shown in Fig. 5. In the KEGG database, 2476, 2440, 2314, and 2924 genes from B. spizizenii HA3, B. velezensis HA5, B. safensis HA7, and B. velezensis HA8 were annotated. These genes were annotated in 40 pathways across six major functional categories: environmental information processing, cellular processes, human diseases, metabolism, genetic information processing, and organismal systems. The number of genes involved in Metabolism was the highest for all four strains, with 1962, 1901, 1851 and 2201 genes, respectively. Among these, the number of genes annotated in the amino acid metabolism pathway was 208, 210, 205, and 234. The presence of a large number of genes related to amino acid metabolism pathways in the functional gene database suggests that the four strains have a strong capacity for protein utilization and metabolism [30]. Fig. 5 3.4.4 Safety evaluation The amino acid sequences of the target strains were analyzed using the VFDB (Virulence Factors of Pathogenic Bacteria) and CARD databases to assess the safety of the four strains, with the results shown in Fig. 6 and Table S3. As shown in Fig. 6 (A)–(D), compared with the VFDB database using an E-value ≤1e-5 as the screening criterion, 459, 486, 454, and 472 virulence genes were identified in strains B. spizizenii HA3, B. velezensis HA5, B. safensis HA7, and B. velezensis HA8, respectively. The genes related to Nutritional/Metabolic factor, Immune modulation, Motility, and Exotoxin were the most abundant, and no genes with identity ≥80% and coverage ≥80% were detected. Through sequence alignment-based targeted analysis, hemolytic enterotoxin virulence genes ( hblA , hblC , hblD ), non-hemolytic enterotoxin virulence genes ( nheA , nheB , nheC ), and enterotoxin virulence genes ( entFM ) were not detected in the genomes of the four strains. Moreover, the genomes did not contain enterotoxin virulence genes ( bceT ), cytotoxin virulence genes ( cytK ), or emetic toxin virulence genes ( ces ). Comparison with the CARD database revealed that strains B. spizizenii HA3, B. velezensis HA5, B. safensis HA7, and B. velezensis HA8 possessed 282, 290, 266, and 276 resistance genes, respectively, accounting for 6.96%, 7.00%, 7.20%, and 6.64% of the total genes. The most abundant resistance genes in the genomes of the four strains were related to peptide antibiotics, representing 12.81%, 12.61%, 11.48%, and 11.62% of the total resistance genes, respectively. Table S3 showed the prediction results of resistance genes in the genomes of the four strains with identity ≥90% and coverage ≥80%. In all four strains, genes related to peptide antibiotics were detected, namely gene3176 in B. spizizenii HA3, gene3570 in B. velezensis HA5, gene3622 in B. safensis HA7, and gene4010 in B. velezensis HA8. This is consistent with the in vitro antibiotic resistance test results showing that the four strains were susceptible to Vancomycin (Van). However, in vitro studies do not fully support these findings. For instance, resistance genes related to macrolide antibiotics, lincosamide antibiotics, aminoglycoside antibiotics, and fluoroquinolone antibiotics were detected in the genomes of the four strains, but in vitro tests still indicated that they were susceptible to these antibiotics in varying degrees. This suggests that not all predicted genes in the genome are expressed. Many commercial Bacillus strains have been proven to be resistant to erythromycin, clindamycin, penicillin, streptomycin, etc., yet they are widely used as probiotics [31, 32]. Fig. 6 3.4.5 Metabolic system analysis CAZY function annotation results. Comparing the genome sequences of strains B. spizizenii HA3, B. velezensis HA5, B. safensis HA7 and B. velezensis HA8 with CAZY database, as shown in Table S4. Protein domains encoded by 148, 134, 121 and 133 genes in the genome sequences of four strains belong to CAZY database, among which the number of genes related to Carbohydrate Esterases, Glycohydrolases and Glycotransferases is rich, and the sum of the annotation numbers of these three kinds of hydrolases accounts for 87.84%, 88.81%, 89.26% and 88.72% respectively. Cluster analysis of secondary metabolites synthesis gene. Table S5 and Fig. 7 summarized the secondary metabolite synthesis gene clusters with similarity > 90% in the gene sequences of four kinds of Bacillus . Therefore, the gene sequence of B. spizizenii HA3 had the largest number of genes in the biosynthetic gene cluster with bacillaene, and the similarity was 100%. Bacillane is a polyketide produced by bacteria, which can inhibit the protein synthesis of other bacteria and has antibacterial activity. Bacillane usually participates in the competition or signal transmission among microorganisms as a secondary metabolite [33]. The gene sequence of B. safensis HA7 had the largest number of genes with the biosynthetic gene cluster of lichenysin, and the similarity is 92%. Lichenysin gene is an antibacterial peptide gene, which can inhibit the growth of fungi by producing metabolites or competitive strategies [34]. In the gene sequences of B. velezensis HA5 and B. velezensis HA8, the number of genes in the biosynthetic gene cluster of bacillibactin was the largest and the similarity was 100%. The core function of Bacillibactin gene ( dhb gene cluster) is to encode iron carrier synthesis pathway, help bacteria survive in iron-deficient environment and compete for resources, and then inhibit other pathogenic bacteria [35]. As a way to compete for nutrition, bacteria belonging to the genus Bacillus dedicate an important part of their genome to coding antibiotic molecules with various structures [36]. Therefore, the analysis of antibiotic resistance gene, virulence factor gene, metabolic system analysis and in vitro safety test showed that these four strains were safe. Fig. 7 3.5 Fermentation degradation of cowhide by strains The appearance of the degradation effect of the four strains on cowhide fermentation is shown in Fig. 8 (A)–(D). As shown in Fig. 8 (A) and (C), B. spizizenii HA3 and B. safensis HA7 cannot completely degrade, with obvious cowhide particles remaining. Fig. 8 (B) and (D) show that B. velezensis HA5 and B. velezensis HA8 can completely degrade into a liquid state. The results indicate that the degradation effect of B. velezensis HA5 and B. velezensis HA8 on cowhide is superior to that of B. spizizenii HA3 and B. safensis HA7. The antioxidant activities of unfermented cowhide, bacterial culture broth, and fermented cowhide are shown in Fig. 8 (A-1)–(D-1). Fig. 8 (A-1) and (C-1) show the antioxidant activities of cowhide fermented and degraded by B. spizizenii HA3 and B. safensis HA7, which are inferior to those of cowhide fermented and degraded by B. velezensis HA5 and B. velezensis HA8. As shown in Fig. 8 (B-1) and (D-1), the DPPH free radical scavenging activity and hydroxyl radical scavenging activity of the degradation solution of cowhide fermented and degraded by B. velezensis HA5 and B. velezensis HA8 were significantly improved. When B. velezensis HA5 was used to ferment and degrade cowhide, the DPPH free radical scavenging activity increased from 9.56±0.42% before fermentation to 64.36±0.78% after fermentation, and the hydroxyl radical scavenging activity increased from 5.21±0.29% before fermentation to 91.69±1.05% after fermentation. When B. velezensis HA8 was used to ferment and degrade, the DPPH free radical scavenging activity increased from 9.56±0.42% before fermentation to 61.26±0.84% after fermentation, and the hydroxyl radical scavenging activity increased from 5.21±0.29% before fermentation to 92.96±0.82% after fermentation. The results indicate that the fermentation and degradation of leather by B. velezensis HA5 and B. velezensis HA8 to prepare collagen peptides is a feasible and efficient method of bioconversion. Fig. 8 4 Conclusion Nine strains with high collagenase production (HA1-HA9) were isolated from traditional naturally fermented foods rich in collagen. The safety of these strains was evaluated through hemolysis and drug resistance tests. HA3, HA5, HA7, and HA8 were identified as having high collagenase production capabilities, with no hemolytic activity and sensitivity to most commonly used antibiotics. The genomic results indicated that these four strains were identified as B. spizizenii HA3, B. velezensis HA5, B. safensis HA7, and B. velezensis HA8, respectively. These four strains have no hemolytic enterotoxin virulence genes, and the resistance genes were consistent with the in vitro study results, showing potential to resist pathogenic bacteria. The in vitro safety tests and genomic analysis results demonstrated that these four strains were safe. Then, these four strains were inoculated into cowhide fermentation medium to observe the degradation effects on cowhide and the functional activities of the fermentation products. The results showed that B. velezensis HA5 and B. velezensis HA8 could completely degrade cattle hide into a liquid state, and the antioxidant activity of the resulting fermentation degradation liquid was significantly enhanced. Therefore, the fermentation degradation of cowhide to prepare collagen peptides using B. velezensis HA5 and B. velezensis HA8 is a feasible, eco-friendly, and efficient biotransformation method. It provides microbial strain reserves and theoretical support for the development and utilization of livestock and poultry bone resources, and holds broad application prospects. Declarations CRediT authorship contribution statement Shijie Liu: Writing - original draft, Data curation. Qian Ding: Investigation, Validation. Yueyu Bai: Supervision, Investigation. Lijun Zhao: Formal analysis, Writing–review & editing. Miaoyun Li: Resources, Funding acquisition, Writing–review & editing. Jong-Hoon Lee: Methodology. Yijing Ding: Data curation. Yaodi Zhu: Supervision. Yanxia Liu: Supervision. Lingxia Sun: Methodology. Yangyang Ma: Validation. Gaiming Zhao: Formal analysis. Dong Liang: Supervision. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This research was supported by Zhongyuan Sci-Tech Innovation Leading Talents Support Program (254000510015), General Program of National Natural Science Foundation of China (32472417), the Major science and technology projects in Henan province (231100110400). References Grasso S., Estévez M., Lorenzo J.M., Pateiro M., Ponnampalam E.N., The utilisation of agricultural by-products in processed meat products: Effects on physicochemical, nutritional and sensory quality – Invited Review Meat Science, 211 (2024) 109451. https://doi.org/10.1016/j.meatsci.2024.109451. Zhang X., Gao M., Zhang C., Peng B., Enzymatic processes for animal hide/skin collagen fiber purification processing: Recent progress, challenges and recommendations, Bioresource Technology, 418 (2025) 131955. https://doi.org/10.1016/j.biortech.2024.131955. Zhao Y., Zhang M., Law C.L., Yang C., New technologies and products for livestock and poultry bone processing: Research progress and application prospects: A review, Trends in Food Science & Technology, 144 (2024) 104343-. https://doi.org/10.1016/j.tifs.2024.104343. Ren B., Yue K., Zhang Y., Fu Y., Collagen-derived peptides as prebiotics to improve gut health, Current Opinion in Food Science, 55 (2024) 101123. https://doi.org/10.1016/j.cofs.2024.101123. Zhou Y., Jiang Y., Zhang Y., Yokoyama W., Wu J., Chang S.K.C., Hong H., Luo Y., Li B., Tan Y., Unveiling a new chapter for collagen peptides: Comprehensive insights into oral bioavailability and the enhancement via encapsulation systems, Trends in Food Science & Technology, 156 (2025) 104849. https://doi.org/10.1016/j.tifs.2024.104849. Hong H., Fan H., Chalamaiah M., Wu J., Preparation of low-molecular-weight, collagen hydrolysates (peptides): Current progress, challenges, and future perspectives, Food Chemistry, 301 (2019) 125222. https://doi.org/10.1016/j.foodchem.2019.125222. Li C., Fu Y., Dai H., Wang Q., Gao R., Zhang Y., Recent progress in preventive effect of collagen peptides on photoaging skin and action mechanism, Food Science and Human Wellness, 11 (2022) 218-229. https://doi.org/10.1016/j.fshw.2021.11.003. Leticia M., Fidel T., Advanced enzymatic hydrolysis of food proteins for the production of bioactive peptides, Current Opinion in Food Science, 49 (2023) 100973. https://doi.org/10.1016/J.COFS.2022.100973. Bi Y., Zhang M., Liu K., Lv X., Li X., Gao X., Cui F., Zhang D., Li J., Optimization of enzymatic hydrolysis of collagen and screening and application of hypolipidemic peptide, Food Bioscience, 66 (2025) 106268. https://doi.org/10.1016/j.fbio.2025.106268. Ma T., Li C., Zhao F., Cao J., Zhang X., Shen X., Effects of co-fermented collagen peptide-jackfruit juice on the immune response and gut microbiota in immunosuppressed mice, Food Chemistry, 365 (2021) 130487-130487. https://doi.org/10.1016/J.FOODCHEM.2021.130487. Alzira A.P., Mario A.Y.G., Mayara C.M., Brenda A.S., Rakesh K.B., Anuj K.C., Precision fermentation in the realm of microbial protein production: State-of-the-art and future insights, Food Research International, 200 (2025) 115527. https://doi.org/10.1016/j.foodres.2024.115527. María L.-P., M. L.J., Ricard B., Antonio V.J., Jesús V., Mònica T., Daniel F., Valorisation of pork by-products to obtain antioxidant and antihypertensive peptides, Food Chemistry, 423 (2023) 136351. https://doi.org/10.1016/j.foodchem.2023.136351. Oliveira R.R., Diniz C., Ferreira I.M.P.L.V.O., In vitro strategies to understand the impact of oral intake on the bioavailability and bioactivity of peptides from brewing by-products, Critical reviews in food science and nutrition, (2024) 1-9. https://doi.org/10.1080/10408398.2024.2362410. Luo W., Zhang J., Ahmmed M.K., Sakai K., Shahidi F., Zhi Z., Wu H., Valorization of animal by-product enzymes: Advancing sustainable food processing through innovative extraction, purification, and application strategies, Trends in Food Science & Technology, 156 (2025) 104870. https://doi.org/10.1016/j.tifs.2025.104870. Kim S.-K., Yang J.-Y., Cha J., Cloning and sequence analysis of a novel metalloprotease gene from Vibrio parahaemolyticus, Gene, 283 (2002) 277-286. https://doi.org/10.1016/S0378-1119(01)00882-4. Ganeshpurkar A., Kumar D., Singh S.K., Design, synthesis and collagenase inhibitory activity of some novel phenylglycine derivatives as metalloproteinase inhibitors, International Journal of Biological Macromolecules, 107 (2018) 1491-1500. https://doi.org/10.1016/j.ijbiomac.2017.10.008. Hu Y., Webb E., Singh J., Morgan B.A., Gainor J.A., Gordon T.D., Siahaan T.J., Rapid Determination of Substrate Specificity of Clostridium histolyticumβ-Collagenase Using an Immobilized Peptide Library, Journal of Biological Chemistry, 277 (2002) 8366-8371. https://doi.org/10.1074/jbc.M111042200. Liu S., Zhao L., Li M., Zhu Y., Liang D., Ma Y., Sun L., Zhao G., Tu Q., Probiotic Bacillus as fermentation agents: Status, potential insights, and future perspectives, Food Chemistry: X, 22 (2024) 101465-. https://doi.org/10.1016/j.fochx.2024.101465. Zhao N., Yu T., Yan F., Probiotic role and application of thermophilic Bacillus as novel food materials, Trends in Food Science & Technology, 138 (2023) 1-15. https://doi.org/10.1016/j.tifs.2023.05.020. Li Z., Zheng M., Zheng J., Gänzle M.G., Bacillus species in food fermentations: an underappreciated group of organisms for safe use in food fermentations, Current Opinion in Food Science, 50 (2023) 101007. https://doi.org/10.1016/j.cofs.2023.101007. Yang X., Mao Y., Chen L., Guan X., Wang Z., Huang T., Structural characteristics, biotechnological production and applications of exopolysaccharides from Bacillus sp.: A comprehensive review, Carbohydrate Polymers, 355 (2025) 123363. https://doi.org/10.1016/j.carbpol.2025.123363. Supaluk S., Punnanee S., Soottawat B., Pussadee T., Collagenolytic proteases from Bacillus subtilis B13 and B. siamensis S6 and their specificity toward collagen with low hydrolysis of myofibrils, LWT, 126 (2020) 109307. https://doi.org/10.1016/j.lwt.2020.109307. Warinda S., Benjamas C., Akkasit J., Production and properties of two collagenases from bacteria and their application for collagen extraction, New BIOTECHNOLOGY, 28 (2011) 649-655. https://doi.org/10.1016/j.nbt.2011.04.003. Klichko V.I., Miller J., Wu A., Popov S.G., Alibek K., Anaerobic induction of Bacillus anthracis hemolytic activity, Biochemical and biophysical research communications, 303 (2003) 855-862. https://doi.org/10.1016/S0006-291X(03)00440-6. Na H.-E., Heo S., Kim Y.-S., Kim T., Lee G., Lee J.-H., Jeong D.-W., The safety and technological properties of Bacillus velezensis DMB06 used as a starter candidate were evaluated by genome analysis, LWT, 161 (2022) 113398. https://doi.org/10.1016/j.lwt.2022.113398. Abbas Z., Zhang J., Tong Y., Ahmad B., Wang J., Zhang H., Li Z., Wei X., Zhang R., Si D., Production and characterization of novel antioxidant peptides from mulberry leaf ferment using B. subtilis H4 and B. amyloliquefaciens LFB112, Food Chemistry, (2025) 144022. https://doi.org/10.1016/j.foodchem.2025.144022. Wang D., Xu Z., Wang Y., Li Y., Zheng W., Chai Y., Wei G., Huang A., Identification and characterization of novel antioxidant peptides from Yunnan dry-cured beef: A combined in silico and in vitro study, Food chemistry, 477 (2025) 143485. https://doi.org/10.1016/j.foodchem.2025.143485. Chaudhari D.N., Ahire J.J., Kulthe A.A., Ghodke S., Complete Genome Sequence and In Vitro Probiotic Assessment of Bacillus subtilis DC-11 Isolated from Traditionally Fermented Idli Batter, Current microbiology, 82 (2024) 35. https://doi.org/10.1007/s00284-024-04014-y. Chelliah R., Kim N.H., Rubab M., Yeon S.J., Barathikannan K., Vijayalakshmi S., Hirad A.H., Oh D.H., Robust and safe: Unveiling Bacillus clausii OHRC1's potential as a versatile probiotic for enhanced food quality and safety, LWT, 203 (2024) 116291. https://doi.org/10.1016/j.lwt.2024.116291. Gauri K., Rachatida D.-u., Pinidphon P., Cheunjit P., Probiogenomic analysis and safety assessment of Bacillus isolates using Omics approach in combination with In-vitro, LWT, 159 (2022). https://doi.org/10.1016/j.lwt.2022.113216. Zhou Y., Tu T., Yao X., Luo Y., Yang Z., Ren M., Zhang G., Yu Y., Lu A., Wang Y., Pan-genome analysis of Streptococcus suis serotype 2 highlights genes associated with virulence and antibiotic resistance, Frontiers in Microbiology, 15 (2024) 1362316. https://doi.org/10.3389/FMICB.2024.1362316. Liang A., Wang J., Ding L., Zou L., Wang D., Zhu C., Tang J., Probiotic properties, whole-genome sequence analysis, and safety assessment of BreviBacillus borstelensis S8, LWT, 210 (2024) 116800. https://doi.org/10.1016/j.lwt.2024.116800. Jason J.R., Stephanie A.A., Gregory M.R., PksS from Bacillus subtilis is a cytochrome P450 involved in bacillaene metabolism, Biochemical and Biophysical Research Communications, 358 (2007) 363-367. https://doi.org/10.1016/j.bbrc.2007.04.151. Czinkóczky R., Németh Á., Techno-economic assessment of Bacillus fermentation to produce surfactin and lichenysin, Biochemical Engineering Journal, 163 (2020) 107719. https://doi.org/10.1016/j.bej.2020.107719. Dimopoulou A., Theologidis I., Benaki D., Koukounia M., Zervakou A., Tzima A., Diallinas G., Hatzinikolaou D.G., Skandalis N., Direct Antibiotic Activity of Bacillibactin Broadens the Biocontrol Range of Bacillus amyloliquefaciens MBI600, mSphere, 6 (2021) e0037621. https://doi.org/10.1128/mSphere.00376-21. Luigia S., Elda R., Margherita C., Damiano B., Giulia M., Paolo C., G. G.C., Thomas L., Maurizio S., J.V. P.L., François F., Sally E., Elena C., Alessia S., Evelina T., Systematic review and meta-analysis of in vitro efficacy of antibiotic combination therapy against carbapenem-resistant Gram-negative bacilli, International Journal of Antimicrobial Agents, 57 (2021) 106344. https://doi.org/10.1016/j.ijantimicag.2021.106344. Additional Declarations No competing interests reported. Supplementary Files SupportingMaterial.doc Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-7565406","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":514874401,"identity":"93cf131d-a1b3-457e-ba67-6337b20b0e2a","order_by":0,"name":"Shijie Liu","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Shijie","middleName":"","lastName":"Liu","suffix":""},{"id":514874402,"identity":"548978c4-66ba-4458-af97-28e1993c8c94","order_by":1,"name":"Qian Ding","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Qian","middleName":"","lastName":"Ding","suffix":""},{"id":514874403,"identity":"b5fe28d4-e5d6-4497-9308-bf77cb7b9d5e","order_by":2,"name":"Yueyv Bai","email":"","orcid":"","institution":"Henan University","correspondingAuthor":false,"prefix":"","firstName":"Yueyv","middleName":"","lastName":"Bai","suffix":""},{"id":514874404,"identity":"bcd12773-24f0-4b01-b37f-9f89bc862828","order_by":3,"name":"Lijun Zhao","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Lijun","middleName":"","lastName":"Zhao","suffix":""},{"id":514874405,"identity":"8efac2d1-3b20-4e28-8497-9ee358dd36f8","order_by":4,"name":"Miaoyun Li","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAwElEQVRIiWNgGAWjYBACA2YGBuY/PP/l+JmZDz4gWgsDjwyzsWQ7W7IBcVpABI8Nc6LBeR4zAeK0sHOnSUjksCUYH2YwY2CosYkmwmG82yQMzvDkmR1mSHvAcCwtt4EoLYk9EsVALccNGBsOE6nl4D+DxM3NjG0SRGuRbOBJSNzAzMxGtJbN1gw8B4wlDrMxGyQQ4xf7/rMbbwO1yPH3n//44EONDWEtQMAiAWcmEKEcBJg/EKlwFIyCUTAKRioAAB+WN0eQzaUEAAAAAElFTkSuQmCC","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":true,"prefix":"","firstName":"Miaoyun","middleName":"","lastName":"Li","suffix":""},{"id":514874406,"identity":"4a6321e5-0db7-42f0-b483-8f3687758a9e","order_by":5,"name":"Jong-Hoon Lee","email":"","orcid":"","institution":"Kyonggi University","correspondingAuthor":false,"prefix":"","firstName":"Jong-Hoon","middleName":"","lastName":"Lee","suffix":""},{"id":514874407,"identity":"89d1961a-e215-4262-b18f-e6ca60e94526","order_by":6,"name":"Yijing Ding","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yijing","middleName":"","lastName":"Ding","suffix":""},{"id":514874408,"identity":"a0d45061-10f4-4428-8047-240118e14558","order_by":7,"name":"Yaodi Zhu","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yaodi","middleName":"","lastName":"Zhu","suffix":""},{"id":514874409,"identity":"2826dd16-86a5-424d-81cd-a8c7efb0b8c5","order_by":8,"name":"Yanxia Liu","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yanxia","middleName":"","lastName":"Liu","suffix":""},{"id":514874410,"identity":"bd46a643-0d39-4ae8-8910-5c57952f0bd3","order_by":9,"name":"LingXia Sun","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"LingXia","middleName":"","lastName":"Sun","suffix":""},{"id":514874411,"identity":"3fd2bfc8-4356-4649-accc-d2832a961337","order_by":10,"name":"Yangyang Ma","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Yangyang","middleName":"","lastName":"Ma","suffix":""},{"id":514874412,"identity":"7e8c62a0-30bb-46fc-b27f-43bdf0e99f4a","order_by":11,"name":"Gaiming Zhao","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Gaiming","middleName":"","lastName":"Zhao","suffix":""},{"id":514874413,"identity":"c58380a3-1b80-4173-9db0-2517f2777fe5","order_by":12,"name":"Dong Liang","email":"","orcid":"","institution":"Henan Agricultural University","correspondingAuthor":false,"prefix":"","firstName":"Dong","middleName":"","lastName":"Liang","suffix":""}],"badges":[],"createdAt":"2025-09-08 14:38:33","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7565406/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7565406/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91524458,"identity":"8bb9f3de-9666-43b2-846a-f243c230d7a7","added_by":"auto","created_at":"2025-09-17 10:58:03","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":120730,"visible":true,"origin":"","legend":"\u003cp\u003eHydrolysis zones of the nine strains isolated from two fermented foods on gelatin agar plates\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7565406/v1/b91f95ffbb55998fa0d3c3a7.jpg"},{"id":91525347,"identity":"cba463fc-d0d2-43e2-ade4-5820fade0369","added_by":"auto","created_at":"2025-09-17 11:06:03","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":110497,"visible":true,"origin":"","legend":"\u003cp\u003eEvaluation of hemolytic activity of nine strains\u003c/p\u003e","description":"","filename":"2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7565406/v1/4c6e287ece6a585b73b1d1c9.jpg"},{"id":91524467,"identity":"898e674a-b30a-45fc-b05c-c5e093719824","added_by":"auto","created_at":"2025-09-17 10:58:03","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":3517381,"visible":true,"origin":"","legend":"\u003cp\u003e(A)-(D), GC-Depth distribution plots of the whole genomes of HA3, HA5, HA7, and HA8; (A-1)-(D-1), Whole-genome maps of HA3, HA5, HA7, and HA8.\u003c/p\u003e","description":"","filename":"3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7565406/v1/dd081c4dd528212e176ed6c7.jpg"},{"id":91524460,"identity":"ccd07f29-ef26-4b0c-b002-e97aff8fb5fb","added_by":"auto","created_at":"2025-09-17 10:58:03","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1293907,"visible":true,"origin":"","legend":"\u003cp\u003ePhylogenetic tree construction based on housekeeping genes in various samples\u003c/p\u003e","description":"","filename":"4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7565406/v1/5e49b700db7fd1b81ef5929a.jpg"},{"id":91524462,"identity":"61c20ea7-00af-4da8-9610-1b2449f77d42","added_by":"auto","created_at":"2025-09-17 10:58:03","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1184853,"visible":true,"origin":"","legend":"\u003cp\u003eFunctional classification statistics of encoded proteins in KEGG database of four strains\u003c/p\u003e","description":"","filename":"5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7565406/v1/4f6b2a1c908ee4233208cca2.jpg"},{"id":91524463,"identity":"91221ddf-dd4b-4450-bc8e-e12c874139dc","added_by":"auto","created_at":"2025-09-17 10:58:03","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":2172400,"visible":true,"origin":"","legend":"\u003cp\u003e(A)-(D), the virulence gene prediction analysis of \u003cem\u003eB. spizizenii\u003c/em\u003e HA3,\u003cem\u003e B. velezensis\u003c/em\u003e HA5, \u003cem\u003eB. safensis \u003c/em\u003eHA7 and \u003cem\u003eB. velezensis \u003c/em\u003eHA8, respectively; (A-1)-(D-1), the prediction and analysis of drug resistance genes of \u003cem\u003eB. spizizenii\u003c/em\u003e HA3,\u003cem\u003e B. velezensis\u003c/em\u003e HA5, \u003cem\u003eB. safensis \u003c/em\u003eHA7 and \u003cem\u003eB. velezensis \u003c/em\u003eHA8, respectively.\u003c/p\u003e","description":"","filename":"6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7565406/v1/dfdc6b381877c7abf583a597.jpg"},{"id":91524461,"identity":"fa4aa0d1-99ce-4183-94d7-bb4de5553832","added_by":"auto","created_at":"2025-09-17 10:58:03","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":845642,"visible":true,"origin":"","legend":"\u003cp\u003eMain secondary metabolite synthesis gene clusters in the gene sequences of four kinds of \u003cem\u003eBacillus\u003c/em\u003e. (A), the linear map of the biosynthetic gene cluster with bacillaene in the gene sequences of \u003cem\u003eB. spizizenii \u003c/em\u003eHA3; (B), the linear map of the biosynthetic gene cluster with bacillibactin in the gene sequences of \u003cem\u003eB. velezensis \u003c/em\u003eHA5; (C), the linear map of the biosynthetic gene cluster with lichenysin in the gene sequences of \u003cem\u003eB. spizizenii \u003c/em\u003eHA7; (D), the linear map of the biosynthetic gene cluster with bacillibactin in the gene sequences of \u003cem\u003eB. velezensis \u003c/em\u003eHA8.\u003c/p\u003e","description":"","filename":"7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7565406/v1/37818791947b9b95cac6b160.jpg"},{"id":91525348,"identity":"53b9b326-1faa-4703-b283-8e53c520dc81","added_by":"auto","created_at":"2025-09-17 11:06:03","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":1001669,"visible":true,"origin":"","legend":"\u003cp\u003eFermentation degradation and antioxidant effect of strains on cowhide. (A)-(D), the physical pictures of the effects \u003cem\u003eB. spizizenii \u003c/em\u003eHA3, \u003cem\u003eB. velezensis\u003c/em\u003e HA5, \u003cem\u003eB. safensis\u003c/em\u003eHA7, and \u003cem\u003eB. velezensis \u003c/em\u003eHA8 on the fermentation degradation of cowhide respectively.\u003c/p\u003e\n\u003cp\u003e(A-1)-(D-1), the antioxidant activities of cowhide before and after fermentation by \u003cem\u003eB. spizizenii \u003c/em\u003eHA3, \u003cem\u003eB. velezensis\u003c/em\u003e HA5, \u003cem\u003eB. safensis\u003c/em\u003e HA7, and \u003cem\u003eB. velezensis \u003c/em\u003eHA8, respectively.\u003c/p\u003e","description":"","filename":"8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-7565406/v1/bde2d984b489d7f8e67ac5ee.jpg"},{"id":94026186,"identity":"81996304-b4cf-420d-a1c6-4c9f6617730d","added_by":"auto","created_at":"2025-10-21 13:31:40","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":11087096,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7565406/v1/6c90200a-c25d-4e90-a68c-630ad3bceddf.pdf"},{"id":91524465,"identity":"53ccaf0f-a1fc-4753-af23-580d474b1129","added_by":"auto","created_at":"2025-09-17 10:58:03","extension":"doc","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":2535864,"visible":true,"origin":"","legend":"","description":"","filename":"SupportingMaterial.doc","url":"https://assets-eu.researchsquare.com/files/rs-7565406/v1/7a597d28257a3fee4d2a20ac.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"Probiotic characteristics, whole-genome sequence analysis of Bacillus with high collagenase production and its fermentation hydrolysis of cowhide","fulltext":[{"header":"1 Introduction","content":"\u003cp\u003eWith the development of the livestock industry, the annual production of beef, pork, and chicken is expanding, which has led to a large amount of by-products that are rich in collagen such as skin and cartilage [1]. However, due to the water-insoluble nature and complex triple helix structure of collagen, the collagen in animal skin is too difficult to degrade and has absolutely low bioavailability. This not only results in economic losses, but it poses a serious threat to the natural environment [2]. Under the limited environmental carrying capacity of livestock farming, how to achieve the refined deep processing and high-value utilization of livestock by-products is of great significance. It can promote the comprehensive utilization of livestock products, and is also important for the development of the livestock industry and environmental protection [3].\u003c/p\u003e\n\u003cp\u003eThe preparation of collagen peptides is an effective way to achieve high-value utilization of collagen in livestock by-products. As a functional protein ingredient, collagen peptides are increasingly being used in food, nutritional supplements, pharmaceuticals, and cosmetics [4, 5]. With the continuous increase of people\u0026apos;s consumption, especially the economic development of the Asia-Pacific region represented by China, consumers\u0026apos; demands and consumption for health, beauty, and wellness are constantly increasing. The global consumer demand for collagen peptide products continues to grow, and the transactions of the global collagen peptide products keep rising [6, 7].\u003c/p\u003e\n\u003cp\u003eNowadays, the preparation methods for pig skin and fish skin collagen peptides are mostly enzymatic hydrolysis. However, use the enzymatic hydrolysis method for preparing collagen peptides requires the addition of chemical reagents for defatting and removing the miscellaneous proteins in the pre-treatment process. Moreover, after enzymatic hydrolysis, deodorization and bitterness removal are needed, which process is complex and poses serious environmental pollution issues [8, 9]. Microbial fermentation degradation is an emerging, environmentally friendly, and efficient biotechnological method for preparing collagen peptides. By using microbial fermentation to obtain collagen, the steps of defatting, removing miscellaneous proteins, enzymatic hydrolysis, and extracting collagen can be concentrated in a fermentation process. This greatly simplifies the process, reduces the use of toxic chemical reagents, and aligns with the trend of eco-friendly production, which is an environmentally friendly technology [10-12]. The complex enzyme systems secreted by microorganisms may produce a variety of functional small-molecule active peptides with potential activities such as antioxidant and anti-inflammatory. Microbial fermentation is a worthwhile method to promote the high-value utilization of livestock by-products, such as cowhide [13]. However, the safe and probiotic microbial strain with high collagenase production is the prerequisite and key to the degradation of collagen by microbial fermentation [14].\u003c/p\u003e\n\u003cp\u003eUp to now, collagenase-producing strains are mainly \u003cem\u003eClostridium histolyticum\u003c/em\u003e, \u003cem\u003eClostridium perfringens\u003c/em\u003e, and \u003cem\u003eVibrio alginolyticus\u003c/em\u003e, which are mostly pathogenic. While producing collagenase, they also produce toxins or pathogenic factors in varying degrees. This results in low safety and high production costs of the collagen peptide products obtained, which make against to further processing and utilization [15-17]. With the in-depth research on probiotics, probiotic bacilli have become a heat topic as the next-generation. Many microorganisms are adapted to their living environments. In China, there are a large number of naturally fermented foods rich in collagen raw materials, such as fermented pig\u0026apos;s trotters and fermented sour fish, which not only have a history of thousands of years, but also serve as a treasure of probiotic resources [18, 19]. These probiotic bacilli are involved to varying degrees in traditionally natural fermented foods and have advantages of being diverse in species, fast in growth and reproduction, and rich in enzyme systems. This provides the possibility for screening safe strains that can produce a large amount of collagenase [20, 21].\u003c/p\u003e\n\u003cp\u003eIn this study, strains with high collagenase production were screened from traditional natural fermented foods rich in collagen raw materials (fermented air-dried trotters and fermented sour fish), and their safety was evaluated through hemolysis and antibiotic susceptibility. Furthermore, the whole genome sequence of the organism was obtained from the screened strains with high safety by whole genome sequencing technology, and the organism was interpreted from the molecular level, and the related functional genes were mined to clarify the evolutionary relationship of species. Finally, the target strain was inoculated into cowhide fermentation medium for practical verification test, and the degradation effect and functional activity of cowhide fermented by the target strain were determined, which provided theoretical support and technical reserve for the development and utilization of livestock and poultry skin resources and had broad application prospects.\u003c/p\u003e"},{"header":"2 Materials and Methods","content":"\u003cp\u003e\u003cstrong\u003e2.1 Experimental Materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFresh raw cowhide, purchased from Henan Hengdu Food \u003cem\u003eCo., Ltd\u003c/em\u003e.. Clean the impurities on the surface of the cowhide and cut it into pieces. Wash it with distilled water and drain it thoroughly and store it in a refrigerator at -20℃\u0026nbsp;for later use.\u003c/p\u003e\n\u003cp\u003eFermented air-dried trotters and fermented sour fish were sampled in Zigui County, Hubei Province and Longli County, Guizhou Province, respectively.\u003c/p\u003e\n\u003cp\u003eGelatin agar medium: gelatin 4%, glucose 5%, agar powder 1.5% KH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e 0.05%, MgSO\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003eO 0.02%, pH value to 7.2~7.5. Fermentation medium: gelatin 0.5%, peptone 1%, sodium chloride 1%, glucose 1.5%, pH to 7.5. LB agar medium and LB liquid medium were purchased from Qingdao Haibo Biological \u003cem\u003eCo., Ltd.\u003c/em\u003e.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.2 Screening of collagenase producing strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWeigh 25 g of sample, suspend it in 225 mL sterile physiological saline and homogenize it, then coat it on gelatin culture medium, culture it at 37℃ for 24-48 h, and observe and select a single colony with obvious transparent circle. Single colony was selected and cultured in gelatin agar medium for three times. Acid mercury reagent (15g HgCl\u003csub\u003e2\u003c/sub\u003e, 20g HCl, titrate with water to 100mL) was dripped around the colony, and the formation of transparent circle was observed [22].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.3 Detection of collagenase activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe single colony of the target strain was inoculated into 50 mL LB broth, respectively, and cultured overnight at 37℃\u0026nbsp;and 160 r/min for 24 h to logarithmic phase. Inoculated with 1% inoculation amount into a 250 mL sterile triangular flask containing 50 mL fermentation medium, cultured for 48 h at 37℃\u0026nbsp;and 200 r/min, and centrifuged (8000 r/min, 20 min) to collect supernatant for enzyme activity determination. All experiments were designed in parallel for three times.\u003c/p\u003e\n\u003cp\u003eTake a 5 mg/mL cowhide type I collagen solution as the substrate, add 1 mL of the collagenase solution to be detected, and react in a 37℃\u0026nbsp;water bath for 30 minutes. Add 300 \u0026mu;L of 30% trichloroacetic acid as the termination solution to terminate the reaction. At the same time, use the same reaction system containing inactivated collagenase as the control, and then add 600 \u0026mu;L of 2 mol/L acetic acid buffer and 600 \u0026mu;L of indene ketone colorimetric solution in sequence. Mix well and boil in a boiling water bath for 15 min. After cooling, add 1.8 mL of 60% ethanol to dilute the reaction solution. Use a multifunctional enzyme-linked immunosorbent assay reader to measure the absorbance at 570 nm. The enzyme activity unit is defined as the amount of enzyme that hydrolyzes collagen to produce 1 \u0026mu;L glycine per minute at 37℃\u0026nbsp;and pH 7.5, which is one enzyme activity unit (U) [23].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4 Evaluation of microbial safety\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.1 Hemolytic evaluation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter activating 4 strains of Bacillus, the bacterial solution was picked out using an inoculation ring and streaked on a blood agar plate (Guangdong Huankai Microbial Technology \u003cem\u003eCo., Ltd.\u003c/em\u003e). After culturing at 37℃\u0026nbsp;for 24 hours, the hemolytic activity of the strains was detected based on the formation of a transparent circle around the colony [24].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4.2 Antibiotic sensitivity test\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe antibiotic sensitivity of four strains was determined using K-B paper agar diffusion method. Take 200 \u0026mu;L of bacterial growth solution from 4 strains of Bacillus subtilis and evenly spread it on LB agar medium. After the surface of the plate is slightly dry, place antibiotic paper strips evenly spaced on the surface and incubate at 37℃\u0026nbsp;for 24 h. Measure the diameter of the inhibition zone (mm). The testing scope of antibacterial drugs and drug sensitivity results refer to the CLSI (M100-S30) and EUCAST (2021 edition) judgment standards [25].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.5 Whole genome analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIllumina sequencing platform was used to identify and analyze the whole genome of the high-yielding collagenase strains. The Kyoto Encyclopedia of Genes and Genomes (KEGG), Clusters of Orthologous Groups (COG), Non Redundant Proteins (NR), Carbohydrate Active Enzymes (CAZy), Transporter Classification Database (TCBD), Swiss Prot, and Pfam databases were used to annotate the key functional genes of the high-yielding collagenase strains at the gene level.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.6 Fermentation degradation of cowhide by bacterial strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e10 g cowhide and 90 mL distilled water were put into a 250 mL conical flask, sealed and sterilized at 121℃ for 15 min, which was used as the substrate culture medium for cowhide fermentation. Inoculate 5 mL of enrichment solution with the concentration of 10\u003csup\u003e6\u0026nbsp;\u003c/sup\u003eCFU/mL, shake culture at 37℃ for 36 h and observe the degradation of cowhide every 2 h.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.7 Biological activity of degradation solution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e2.7.1 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity\u003c/p\u003e\n\u003cp\u003eThe DPPH radical scavenging rate was determined according to the method of Abbas et al. [26], and calculated according to \u003cem\u003eeq.\u003c/em\u003e (1).\u003c/p\u003e\n\u003cp\u003eDPPH radical scavenging rate =[1-(\u003cem\u003eA\u003csub\u003ei\u003c/sub\u003e\u003c/em\u003e-\u003cem\u003eA\u003csub\u003ec\u003c/sub\u003e\u003c/em\u003e)/\u003cem\u003eA\u003csub\u003e0\u003c/sub\u003e\u003c/em\u003e]x100% \u0026nbsp; \u0026nbsp; \u003cem\u003eeq.\u0026nbsp;\u003c/em\u003e(1)\u003c/p\u003e\n\u003cp\u003eWhere, \u003cem\u003eA\u003csub\u003ei\u003c/sub\u003e\u003c/em\u003e is the absorbance of 2 mL of 0.1mmol/L DPPH anhydrous ethanol solution and 2 mL of sample mixed solution, \u003cem\u003eA\u003csub\u003ec\u003c/sub\u003e\u003c/em\u003e is the absorbance of 2 mL of sample solvent and 2ml of anhydrous ethanol mixed solution, \u003cem\u003eA\u003csub\u003e0\u003c/sub\u003e\u0026nbsp;\u003c/em\u003eis the absorbance of 2 mL sample solvent and 2 mL 0.1 mmol/L DPPH anhydrous ethanol mixture.\u003c/p\u003e\n\u003cp\u003e2.7.2 Hydroxyl radical scavenging activity\u003c/p\u003e\n\u003cp\u003e0.1 mL of 9 mmol/L FeS0\u003csub\u003e4\u003c/sub\u003e\u0026middot;7H\u003csub\u003e2\u003c/sub\u003e0, 0.1 mL of 9 mmol/L salicylic acid, 0.1 mL of sample to be tested and 0.1 mL 0.03% H\u003csub\u003e2\u003c/sub\u003e0\u003csub\u003e2\u003c/sub\u003e were mixed and incubated at 37℃ for 15 min, and the absorbance at 510 nm was determined [27]. The hydroxyl radical scavenging rate is calculated according to \u003cem\u003eeq.\u0026nbsp;\u003c/em\u003e(2).\u003c/p\u003e\n\u003cp\u003eHydroxyl radical scavenging rate=\u003cem\u003eA\u003c/em\u003e-(\u003cem\u003eB\u003c/em\u003e-\u003cem\u003eC\u003c/em\u003e)/(\u003cem\u003eA\u003c/em\u003e)\u0026times;100% \u0026nbsp; \u0026nbsp; \u003cem\u003eeq.\u0026nbsp;\u003c/em\u003e(1)\u003c/p\u003e\n\u003cp\u003eWhere, \u003cem\u003eA\u003c/em\u003e is the absorbance without sample, \u003cem\u003eB\u003c/em\u003e is the absorbance after adding the sample, \u003cem\u003eC\u003c/em\u003e is the absorbance of the blank reagent.\u003c/p\u003e"},{"header":"3 Results and discussion","content":"\u003cp\u003e\u003cstrong\u003e3.1 Isolation of collagenase-producing strains and assay of enzymatic activity\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing gelatin agar plates combined with the gradient dilution method and spot inoculation, a total of nine collagenase-producing strains with distinct hydrolysis zones were isolated and screened from fermented air-dried pork trotters and fermented sour fish. The hydrolysis zones produced by these strains on gelatin agar plates are shown in Fig. 1. According to Section 2.3, the nine strains were subjected to collagenase activity assay. As shown in Fig. S1, all nine screened strains were capable of producing collagenase, and their enzyme activity levels followed this order: HA5\u0026gt; HA7\u0026gt; HA8\u0026gt; HA9\u0026gt; HA3\u0026gt; HA6\u0026gt; HA4\u0026gt; HA2\u0026gt; HA1.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 1\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Evaluation of microbial safety\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in Fig.2, strains HA1, HA2, HA4, HA6, and HA9 exhibited distinct hemolytic zones around their colonies, indicating the production of exoenzymes or exotoxins capable of lysing red blood cells or degrading proteins. Consequently, these five strains were excluded from further experiments. In contrast, HA3, HA5, HA7, and HA8 showed no hemolytic activity, as no clear zones were observed [28]. These four non-hemolytic strains were subsequently subjected to antibiotic susceptibility testing, with the results presented in Table S1. These four strains exhibited multidrug resistance, they were susceptible to Ampicillin (AMP), Penicillin (PEN), Tetracycline (TET), Gentamicin (GEN), Chloramphenicol (C), Sulfamethoxazole (SXT), and Vancomycin (Van), but showed varying degrees of resistance to Cefradine (CTR), Erythromycin (E), Clindamycin (MY), and Ciprofloxacin (CIP). Strain HA3 was intermediate in susceptibility to Cefradine (CTR), Erythromycin (E), and Ciprofloxacin (CIP). Strains HA5, HA7, and HA8 were susceptible to Cefradine (CTR), Ciprofloxacin (CIP), and Erythromycin (E). Strains HA5 and HA8 were intermediate in susceptibility to Clindamycin (MY), while strains HA3 and HA7 were susceptible to Clindamycin (MY). The results indicated that the four strains were susceptible to most commonly used antibiotics, ensuring their safety and controllability during application [29].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 2\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Whole genome analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e3.4.1 Genome-wide overview\u003c/p\u003e\n\u003cp\u003eThe genomic characteristics of the four strains are shown in Table S2 and Fig. 3. As shown in Fig. 3 (A), almost all the points in the GC-Depth distribution plots of the genomic data of the four strains are concentrated within a relatively narrow range. This indicates that there was no species contamination during the genome assembly process. Meanwhile, the GC-Depth is basically in accordance with the Poisson distribution, with a certain degree of GC bias. Overall, the low sequence redundancy and high confidence level suggest that the assembly results are normal. According to Fig.3 (B) and Table S2, the whole-genome sequence sizes of HA3, HA5, HA7, and HA8 are 4,048416 bp, 4,183,037 bp, 3714516 bp, and 4122232 bp, respectively. Their average GC contents are 43.68%, 45.62%, 41.61%, and 45.64%, respectively. The numbers of protein-coding genes (CDSs) are 4053, 4140, 3695, and 4156, respectively.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 3\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e3.4.2\u0026nbsp;Phylogenetic tree of house-keeping genes\u003c/p\u003e\n\u003cp\u003eBy comparing with the local database based on 31 house-keeping genes (\u003cem\u003ednaG\u003c/em\u003e, \u003cem\u003efrr\u003c/em\u003e, \u003cem\u003einfC\u003c/em\u003e, \u003cem\u003enusA\u003c/em\u003e, pgk, pyrG, \u003cem\u003erplA\u003c/em\u003e, \u003cem\u003erplB\u003c/em\u003e, \u003cem\u003erplC\u003c/em\u003e, \u003cem\u003erplD\u003c/em\u003e,\u003cem\u003e\u0026nbsp;rplE\u003c/em\u003e, \u003cem\u003erplF\u003c/em\u003e,\u003cem\u003e\u0026nbsp;rplK\u003c/em\u003e, \u003cem\u003erplL\u003c/em\u003e,\u003cem\u003e\u0026nbsp;rplM\u003c/em\u003e, \u003cem\u003erplN\u003c/em\u003e, \u003cem\u003erplP\u003c/em\u003e, \u003cem\u003erplS\u003c/em\u003e, \u003cem\u003erplT\u003c/em\u003e, \u003cem\u003erpmA\u003c/em\u003e, \u003cem\u003erpoB\u003c/em\u003e, \u003cem\u003erpsB\u003c/em\u003e, \u003cem\u003erpsC\u003c/em\u003e, \u003cem\u003erpsE\u003c/em\u003e, \u003cem\u003erpsI\u003c/em\u003e, \u003cem\u003erpsJ\u003c/em\u003e, \u003cem\u003erpsK\u003c/em\u003e, \u003cem\u003erpsM\u003c/em\u003e, \u003cem\u003erpsS\u003c/em\u003e,\u003cem\u003e\u0026nbsp;smpB\u003c/em\u003e, \u003cem\u003etsf\u003c/em\u003e), the 19 strains closest at the species level were selected. The NJ (Neighbor-Joining) method in MEGA 6.0 software was used to construct the phylogenetic tree. As shown in Fig. 4, strains HA3, HA5, HA7, and HA8 respectively had the highest phylogenetic affinity with \u003cem\u003eBacillus spizizenii\u0026nbsp;\u003c/em\u003e(GCF 000227465.1), \u003cem\u003eBacillus velezensis\u003c/em\u003e (GCF 001461825.1), \u003cem\u003eBacillus safensis\u0026nbsp;\u003c/em\u003e(GCF 000691165.1), and \u003cem\u003eBacillus velezensis\u003c/em\u003e (GCF 001461825.1), with sequence similarities of 99.4%. Therefore, strains HA3, HA5, HA7, and HA8 were identified as \u003cem\u003eBacillus spizizenii\u003c/em\u003e, \u003cem\u003eBacillus velezensis\u003c/em\u003e, \u003cem\u003eBacillus safensis,\u003c/em\u003e and \u003cem\u003eBacillus velezensis\u003c/em\u003e,and were named \u003cem\u003eB. spizizenii\u003c/em\u003e HA3, \u003cem\u003eB. velezensis\u003c/em\u003e HA5, \u003cem\u003eB. safensis\u003c/em\u003e HA7, and \u003cem\u003eB. velezensis\u0026nbsp;\u003c/em\u003eHA8.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 4\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e3.4.3 Gene prediction and functional annotation\u003c/p\u003e\n\u003cp\u003eCOG annotation. The genes encoding functional proteins in the genomes of strains \u003cem\u003eB. spizizenii\u003c/em\u003e HA3, \u003cem\u003eB. velezensis\u003c/em\u003e HA5, \u003cem\u003eB. safensis\u003c/em\u003e HA7, and \u003cem\u003eB. velezensis\u0026nbsp;\u003c/em\u003eHA8 were annotated with COG (as shown in Fig. S2). It was found that the number of protein-coding genes annotated with biological activity was 3282, 3201, 3026, and 3196. The gene function annotation information was divided into 25 categories, the most abundant category among the four strains are Aminoundefinedacid transport and metabolism, Carbohydrateundefinedtransportundefinedand metabolism and Transcription.\u003c/p\u003e\n\u003cp\u003eKEGG functional annotation. KEGG is a database that systematically analyzes the metabolic pathways and functions of gene products in cells. The annotation results are shown in Fig. 5. In the KEGG database, 2476, 2440, 2314, and 2924 genes from \u003cem\u003eB. spizizenii\u003c/em\u003e HA3, \u003cem\u003eB. velezensis\u003c/em\u003e HA5, \u003cem\u003eB. safensis\u003c/em\u003e HA7, and \u003cem\u003eB. velezensis\u003c/em\u003e HA8 were annotated. These genes were annotated in 40 pathways across six major functional categories: environmental information processing, cellular processes, human diseases, metabolism, genetic information processing, and organismal systems. The number of genes involved in Metabolism was the highest for all four strains, with 1962, 1901, 1851 and 2201 genes, respectively. Among these, the number of genes annotated in the amino acid metabolism pathway was 208, 210, 205, and 234. The presence of a large number of genes related to amino acid metabolism pathways in the functional gene database suggests that the four strains have a strong capacity for protein utilization and metabolism [30].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 5\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e3.4.4 Safety evaluation\u003c/p\u003e\n\u003cp\u003eThe amino acid sequences of the target strains were analyzed using the VFDB (Virulence Factors of Pathogenic Bacteria) and CARD databases to assess the safety of the four strains, with the results shown in Fig. 6 and Table S3. As shown in Fig. 6 (A)–(D), compared with the VFDB database using an E-value ≤1e-5 as the screening criterion, 459, 486, 454, and 472 virulence genes were identified in strains \u003cem\u003eB. spizizenii\u0026nbsp;\u003c/em\u003eHA3, \u003cem\u003eB. velezensis\u0026nbsp;\u003c/em\u003eHA5, \u003cem\u003eB. safensis\u0026nbsp;\u003c/em\u003eHA7, and \u003cem\u003eB. velezensis\u003c/em\u003e HA8, respectively. The genes related to Nutritional/Metabolic factor, Immune modulation, Motility, and Exotoxin were the most abundant, and no genes with identity ≥80% and coverage ≥80% were detected. Through sequence alignment-based targeted analysis, hemolytic enterotoxin virulence genes (\u003cem\u003ehblA\u003c/em\u003e, \u003cem\u003ehblC\u003c/em\u003e, \u003cem\u003ehblD\u003c/em\u003e), non-hemolytic enterotoxin virulence genes (\u003cem\u003enheA\u003c/em\u003e, \u003cem\u003enheB\u003c/em\u003e, \u003cem\u003enheC\u003c/em\u003e), and enterotoxin virulence genes (\u003cem\u003eentFM\u003c/em\u003e) were not detected in the genomes of the four strains. Moreover, the genomes did not contain enterotoxin virulence genes (\u003cem\u003ebceT\u003c/em\u003e), cytotoxin virulence genes (\u003cem\u003ecytK\u003c/em\u003e), or emetic toxin virulence genes (\u003cem\u003eces\u003c/em\u003e).\u003c/p\u003e\n\u003cp\u003eComparison with the CARD database revealed that strains \u003cem\u003eB. spizizenii\u0026nbsp;\u003c/em\u003eHA3, \u003cem\u003eB. velezensis\u003c/em\u003e HA5, \u003cem\u003eB. safensis\u003c/em\u003e HA7, and \u003cem\u003eB. velezensis\u003c/em\u003e HA8 possessed 282, 290, 266, and 276 resistance genes, respectively, accounting for 6.96%, 7.00%, 7.20%, and 6.64% of the total genes. The most abundant resistance genes in the genomes of the four strains were related to peptide antibiotics, representing 12.81%, 12.61%, 11.48%, and 11.62% of the total resistance genes, respectively. Table S3 showed the prediction results of resistance genes in the genomes of the four strains with identity ≥90% and coverage ≥80%. In all four strains, genes related to peptide antibiotics were detected, namely gene3176 in \u003cem\u003eB. spizizenii\u0026nbsp;\u003c/em\u003eHA3, gene3570 in \u003cem\u003eB. velezensis\u0026nbsp;\u003c/em\u003eHA5, gene3622 in \u003cem\u003eB. safensis\u0026nbsp;\u003c/em\u003eHA7, and gene4010 in \u003cem\u003eB. velezensis\u003c/em\u003e HA8. This is consistent with the in vitro antibiotic resistance test results showing that the four strains were susceptible to Vancomycin (Van). However, in vitro studies do not fully support these findings. For instance, resistance genes related to macrolide antibiotics, lincosamide antibiotics, aminoglycoside antibiotics, and fluoroquinolone antibiotics were detected in the genomes of the four strains, but in vitro tests still indicated that they were susceptible to these antibiotics in varying degrees. This suggests that not all predicted genes in the genome are expressed. Many commercial Bacillus strains have been proven to be resistant to erythromycin, clindamycin, penicillin, streptomycin, etc., yet they are widely used as probiotics [31, 32].\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 6\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e3.4.5 Metabolic system analysis\u003c/p\u003e\n\u003cp\u003eCAZY function annotation results. Comparing the genome sequences of strains \u003cem\u003eB. spizizenii\u003c/em\u003e HA3,\u003cem\u003e\u0026nbsp;B. velezensis\u003c/em\u003e HA5, \u003cem\u003eB. safensis\u0026nbsp;\u003c/em\u003eHA7 and \u003cem\u003eB. velezensis\u0026nbsp;\u003c/em\u003eHA8 with CAZY database, as shown in Table S4. Protein domains encoded by 148, 134, 121 and 133 genes in the genome sequences of four strains belong to CAZY database, among which the number of genes related to Carbohydrate Esterases, Glycohydrolases and Glycotransferases is rich, and the sum of the annotation numbers of these three kinds of hydrolases accounts for 87.84%, 88.81%, 89.26% and 88.72% respectively.\u003c/p\u003e\n\u003cp\u003eCluster analysis of secondary metabolites synthesis gene. Table S5 and Fig. 7 summarized the secondary metabolite synthesis gene clusters with similarity \u0026gt; 90% in the gene sequences of four kinds of \u003cem\u003eBacillus\u003c/em\u003e. Therefore, the gene sequence of\u003cem\u003e\u0026nbsp;B. spizizenii\u0026nbsp;\u003c/em\u003eHA3 had the largest number of genes in the biosynthetic gene cluster with bacillaene, and the similarity was 100%. Bacillane is a polyketide produced by bacteria, which can inhibit the protein synthesis of other bacteria and has antibacterial activity. Bacillane usually participates in the competition or signal transmission among microorganisms as a secondary metabolite [33]. The gene sequence of \u003cem\u003eB. safensis\u0026nbsp;\u003c/em\u003eHA7 had the largest number of genes with the biosynthetic gene cluster of lichenysin, and the similarity is 92%. Lichenysin gene is an antibacterial peptide gene, which can inhibit the growth of fungi by producing metabolites or competitive strategies [34]. In the gene sequences of\u003cem\u003e\u0026nbsp;B. velezensis\u003c/em\u003e HA5 and \u003cem\u003eB. velezensis\u0026nbsp;\u003c/em\u003eHA8, the number of genes in the biosynthetic gene cluster of bacillibactin was the largest and the similarity was 100%. The core function of Bacillibactin gene (\u003cem\u003edhb\u0026nbsp;\u003c/em\u003egene cluster) is to encode iron carrier synthesis pathway, \u003cem\u003ehelp\u003c/em\u003e bacteria survive in iron-deficient environment and compete for resources, and then inhibit other pathogenic bacteria [35]. As a way to compete for nutrition, bacteria belonging to the genus Bacillus dedicate an important part of their genome to coding antibiotic molecules with various structures [36]. Therefore, the analysis of antibiotic resistance gene, virulence factor gene, metabolic system analysis and in vitro safety test showed that these four strains were safe.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 7\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.5 Fermentation degradation of cowhide by strains\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe appearance of the degradation effect of the four strains on cowhide fermentation is shown in Fig. 8 (A)–(D). As shown in Fig. 8 (A) and (C), \u003cem\u003eB. spizizenii\u0026nbsp;\u003c/em\u003eHA3 and \u003cem\u003eB. safensis\u0026nbsp;\u003c/em\u003eHA7 cannot completely degrade, with obvious cowhide particles remaining. Fig. 8 (B) and (D) show that\u003cem\u003e\u0026nbsp;B. velezensis\u003c/em\u003e HA5 and \u003cem\u003eB. velezensis\u0026nbsp;\u003c/em\u003eHA8 can completely degrade into a liquid state. The results indicate that the degradation effect of \u003cem\u003eB. velezensis\u003c/em\u003e HA5 and\u003cem\u003e\u0026nbsp;B. velezensis\u0026nbsp;\u003c/em\u003eHA8 on cowhide is superior to that of \u003cem\u003eB. spizizenii\u003c/em\u003e HA3 and \u003cem\u003eB. safensis\u003c/em\u003e HA7. The antioxidant activities of unfermented cowhide, bacterial culture broth, and fermented cowhide are shown in Fig. 8 (A-1)–(D-1). Fig. 8 (A-1) and (C-1) show the antioxidant activities of cowhide fermented and degraded by \u003cem\u003eB. spizizenii\u0026nbsp;\u003c/em\u003eHA3 and \u003cem\u003eB. safensis\u003c/em\u003e HA7, which are inferior to those of cowhide fermented and degraded by \u003cem\u003eB. velezensis\u0026nbsp;\u003c/em\u003eHA5 and \u003cem\u003eB. velezensis\u003c/em\u003e HA8. As shown in Fig. 8 (B-1) and (D-1), the DPPH free radical scavenging activity and hydroxyl radical scavenging activity of the degradation solution of cowhide fermented and degraded by \u003cem\u003eB. velezensis\u0026nbsp;\u003c/em\u003eHA5 and\u003cem\u003e\u0026nbsp;B. velezensis\u003c/em\u003e HA8 were significantly improved. When \u003cem\u003eB. velezensis\u003c/em\u003e HA5 was used to ferment and degrade cowhide, the DPPH free radical scavenging activity increased from 9.56±0.42% before fermentation to 64.36±0.78% after fermentation, and the hydroxyl radical scavenging activity increased from 5.21±0.29% before fermentation to 91.69±1.05% after fermentation. When \u003cem\u003eB. velezensis\u003c/em\u003e HA8 was used to ferment and degrade, the DPPH free radical scavenging activity increased from 9.56±0.42% before fermentation to 61.26±0.84% after fermentation, and the hydroxyl radical scavenging activity increased from 5.21±0.29% before fermentation to 92.96±0.82% after fermentation. The results indicate that the fermentation and degradation of leather by \u003cem\u003eB. velezensis\u003c/em\u003e HA5 and \u003cem\u003eB. velezensis\u003c/em\u003e HA8 to prepare collagen peptides is a feasible and efficient method of bioconversion.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFig. 8\u003c/strong\u003e\u003c/p\u003e"},{"header":"4 Conclusion","content":"\u003cp\u003eNine strains with high collagenase production (HA1-HA9) were isolated from traditional naturally fermented foods rich in collagen. The safety of these strains was evaluated through hemolysis and drug resistance tests. HA3, HA5, HA7, and HA8 were identified as having high collagenase production capabilities, with no hemolytic activity and sensitivity to most commonly used antibiotics. The genomic results indicated that these four strains were identified as \u003cem\u003eB. spizizenii\u0026nbsp;\u003c/em\u003eHA3, \u003cem\u003eB. velezensis\u003c/em\u003e HA5, \u003cem\u003eB. safensis\u003c/em\u003e HA7, and \u003cem\u003eB. velezensis\u0026nbsp;\u003c/em\u003eHA8, respectively. These four strains have no hemolytic enterotoxin virulence genes, and the resistance genes were consistent with the in vitro study results, showing potential to resist pathogenic bacteria. The in vitro safety tests and genomic analysis results demonstrated that these four strains were safe. Then, these four strains were inoculated into cowhide fermentation medium to observe the degradation effects on cowhide and the functional activities of the fermentation products. The results showed that\u003cem\u003e\u0026nbsp;B. velezensis\u003c/em\u003e HA5 and\u003cem\u003e\u0026nbsp;B. velezensis\u0026nbsp;\u003c/em\u003eHA8 could completely degrade cattle hide into a liquid state, and the antioxidant activity of the resulting fermentation degradation liquid was significantly enhanced. Therefore, the fermentation degradation of cowhide to prepare collagen peptides using \u003cem\u003eB. velezensis\u003c/em\u003e HA5 and \u003cem\u003eB. velezensis\u0026nbsp;\u003c/em\u003eHA8 is a feasible, eco-friendly, and efficient biotransformation method. It provides microbial strain reserves and theoretical support for the development and utilization of livestock and poultry bone resources, and holds broad application prospects.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCRediT authorship contribution statement\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eShijie Liu:\u0026nbsp;\u003c/strong\u003eWriting - original draft, Data curation. \u003cstrong\u003eQian Ding:\u003c/strong\u003e Investigation, Validation. \u003cstrong\u003eYueyu Bai:\u003c/strong\u003e Supervision, Investigation. \u003cstrong\u003eLijun Zhao:\u003c/strong\u003e Formal analysis, Writing\u0026ndash;review \u0026amp; editing. \u003cstrong\u003eMiaoyun Li:\u003c/strong\u003e Resources, Funding acquisition, Writing\u0026ndash;review \u0026amp; editing. \u003cstrong\u003eJong-Hoon Lee:\u003c/strong\u003e Methodology. \u003cstrong\u003eYijing Ding:\u003c/strong\u003e Data curation. \u003cstrong\u003eYaodi Zhu:\u003c/strong\u003e Supervision. \u003cstrong\u003eYanxia Liu:\u0026nbsp;\u003c/strong\u003eSupervision. \u003cstrong\u003eLingxia Sun:\u0026nbsp;\u003c/strong\u003eMethodology. \u003cstrong\u003eYangyang Ma:\u003c/strong\u003e Validation. \u003cstrong\u003eGaiming Zhao:\u0026nbsp;\u003c/strong\u003eFormal analysis. \u003cstrong\u003eDong Liang:\u0026nbsp;\u003c/strong\u003eSupervision.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of Competing Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by Zhongyuan Sci-Tech Innovation Leading Talents Support Program (254000510015), General Program of National Natural Science Foundation of China (32472417), the Major science and technology projects in Henan province (231100110400).\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGrasso S., Est\u0026eacute;vez M., Lorenzo J.M., Pateiro M., Ponnampalam E.N., The utilisation of agricultural by-products in processed meat products: Effects on physicochemical, nutritional and sensory quality \u0026ndash; Invited Review Meat Science, 211 (2024) 109451. https://doi.org/10.1016/j.meatsci.2024.109451. \u003c/li\u003e\n\u003cli\u003eZhang X., Gao M., Zhang C., Peng B., Enzymatic processes for animal hide/skin collagen fiber purification processing: Recent progress, challenges and recommendations, Bioresource Technology, 418 (2025) 131955. https://doi.org/10.1016/j.biortech.2024.131955. \u003c/li\u003e\n\u003cli\u003eZhao Y., Zhang M., Law C.L., Yang C., New technologies and products for livestock and poultry bone processing: Research progress and application prospects: A review, Trends in Food Science \u0026amp; Technology, 144 (2024) 104343-. https://doi.org/10.1016/j.tifs.2024.104343. \u003c/li\u003e\n\u003cli\u003eRen B., Yue K., Zhang Y., Fu Y., Collagen-derived peptides as prebiotics to improve gut health, Current Opinion in Food Science, 55 (2024) 101123. https://doi.org/10.1016/j.cofs.2024.101123. \u003c/li\u003e\n\u003cli\u003eZhou Y., Jiang Y., Zhang Y., Yokoyama W., Wu J., Chang S.K.C., Hong H., Luo Y., Li B., Tan Y., Unveiling a new chapter for collagen peptides: Comprehensive insights into oral bioavailability and the enhancement via encapsulation systems, Trends in Food Science \u0026amp; Technology, 156 (2025) 104849. https://doi.org/10.1016/j.tifs.2024.104849. \u003c/li\u003e\n\u003cli\u003eHong H., Fan H., Chalamaiah M., Wu J., Preparation of low-molecular-weight, collagen hydrolysates (peptides): Current progress, challenges, and future perspectives, Food Chemistry, 301 (2019) 125222. https://doi.org/10.1016/j.foodchem.2019.125222. \u003c/li\u003e\n\u003cli\u003eLi C., Fu Y., Dai H., Wang Q., Gao R., Zhang Y., Recent progress in preventive effect of collagen peptides on photoaging skin and action mechanism, Food Science and Human Wellness, 11 (2022) 218-229. https://doi.org/10.1016/j.fshw.2021.11.003. \u003c/li\u003e\n\u003cli\u003eLeticia M., Fidel T., Advanced enzymatic hydrolysis of food proteins for the production of bioactive peptides, Current Opinion in Food Science, 49 (2023) 100973. https://doi.org/10.1016/J.COFS.2022.100973. \u003c/li\u003e\n\u003cli\u003eBi Y., Zhang M., Liu K., Lv X., Li X., Gao X., Cui F., Zhang D., Li J., Optimization of enzymatic hydrolysis of collagen and screening and application of hypolipidemic peptide, Food Bioscience, 66 (2025) 106268. https://doi.org/10.1016/j.fbio.2025.106268. \u003c/li\u003e\n\u003cli\u003eMa T., Li C., Zhao F., Cao J., Zhang X., Shen X., Effects of co-fermented collagen peptide-jackfruit juice on the immune response and gut microbiota in immunosuppressed mice, Food Chemistry, 365 (2021) 130487-130487. https://doi.org/10.1016/J.FOODCHEM.2021.130487. \u003c/li\u003e\n\u003cli\u003eAlzira A.P., Mario A.Y.G., Mayara C.M., Brenda A.S., Rakesh K.B., Anuj K.C., Precision fermentation in the realm of microbial protein production: State-of-the-art and future insights, Food Research International, 200 (2025) 115527. https://doi.org/10.1016/j.foodres.2024.115527. \u003c/li\u003e\n\u003cli\u003eMar\u0026iacute;a L.-P., M. L.J., Ricard B., Antonio V.J., Jes\u0026uacute;s V., M\u0026ograve;nica T., Daniel F., Valorisation of pork by-products to obtain antioxidant and antihypertensive peptides, Food Chemistry, 423 (2023) 136351. https://doi.org/10.1016/j.foodchem.2023.136351. \u003c/li\u003e\n\u003cli\u003eOliveira R.R., Diniz C., Ferreira I.M.P.L.V.O., In vitro strategies to understand the impact of oral intake on the bioavailability and bioactivity of peptides from brewing by-products, Critical reviews in food science and nutrition, (2024) 1-9. https://doi.org/10.1080/10408398.2024.2362410. \u003c/li\u003e\n\u003cli\u003eLuo W., Zhang J., Ahmmed M.K., Sakai K., Shahidi F., Zhi Z., Wu H., Valorization of animal by-product enzymes: Advancing sustainable food processing through innovative extraction, purification, and application strategies, Trends in Food Science \u0026amp; Technology, 156 (2025) 104870. https://doi.org/10.1016/j.tifs.2025.104870. \u003c/li\u003e\n\u003cli\u003eKim S.-K., Yang J.-Y., Cha J., Cloning and sequence analysis of a novel metalloprotease gene from Vibrio parahaemolyticus, Gene, 283 (2002) 277-286. https://doi.org/10.1016/S0378-1119(01)00882-4. \u003c/li\u003e\n\u003cli\u003eGaneshpurkar A., Kumar D., Singh S.K., Design, synthesis and collagenase inhibitory activity of some novel phenylglycine derivatives as metalloproteinase inhibitors, International Journal of Biological Macromolecules, 107 (2018) 1491-1500. https://doi.org/10.1016/j.ijbiomac.2017.10.008. \u003c/li\u003e\n\u003cli\u003eHu Y., Webb E., Singh J., Morgan B.A., Gainor J.A., Gordon T.D., Siahaan T.J., Rapid Determination of Substrate Specificity of Clostridium histolyticum\u0026beta;-Collagenase Using an Immobilized Peptide Library, Journal of Biological Chemistry, 277 (2002) 8366-8371. https://doi.org/10.1074/jbc.M111042200. \u003c/li\u003e\n\u003cli\u003eLiu S., Zhao L., Li M., Zhu Y., Liang D., Ma Y., Sun L., Zhao G., Tu Q., Probiotic Bacillus as fermentation agents: Status, potential insights, and future perspectives, Food Chemistry: X, 22 (2024) 101465-. https://doi.org/10.1016/j.fochx.2024.101465. \u003c/li\u003e\n\u003cli\u003eZhao N., Yu T., Yan F., Probiotic role and application of thermophilic Bacillus as novel food materials, Trends in Food Science \u0026amp; Technology, 138 (2023) 1-15. https://doi.org/10.1016/j.tifs.2023.05.020. \u003c/li\u003e\n\u003cli\u003eLi Z., Zheng M., Zheng J., G\u0026auml;nzle M.G., Bacillus species in food fermentations: an underappreciated group of organisms for safe use in food fermentations, Current Opinion in Food Science, 50 (2023) 101007. https://doi.org/10.1016/j.cofs.2023.101007. \u003c/li\u003e\n\u003cli\u003eYang X., Mao Y., Chen L., Guan X., Wang Z., Huang T., Structural characteristics, biotechnological production and applications of exopolysaccharides from Bacillus sp.: A comprehensive review, Carbohydrate Polymers, 355 (2025) 123363. https://doi.org/10.1016/j.carbpol.2025.123363. \u003c/li\u003e\n\u003cli\u003eSupaluk S., Punnanee S., Soottawat B., Pussadee T., Collagenolytic proteases from Bacillus subtilis B13 and B. siamensis S6 and their specificity toward collagen with low hydrolysis of myofibrils, LWT, 126 (2020) 109307. https://doi.org/10.1016/j.lwt.2020.109307. \u003c/li\u003e\n\u003cli\u003eWarinda S., Benjamas C., Akkasit J., Production and properties of two collagenases from bacteria and their application for collagen extraction, New BIOTECHNOLOGY, 28 (2011) 649-655. https://doi.org/10.1016/j.nbt.2011.04.003. \u003c/li\u003e\n\u003cli\u003eKlichko V.I., Miller J., Wu A., Popov S.G., Alibek K., Anaerobic induction of Bacillus anthracis hemolytic activity, Biochemical and biophysical research communications, 303 (2003) 855-862. https://doi.org/10.1016/S0006-291X(03)00440-6. \u003c/li\u003e\n\u003cli\u003eNa H.-E., Heo S., Kim Y.-S., Kim T., Lee G., Lee J.-H., Jeong D.-W., The safety and technological properties of Bacillus velezensis DMB06 used as a starter candidate were evaluated by genome analysis, LWT, 161 (2022) 113398. https://doi.org/10.1016/j.lwt.2022.113398. \u003c/li\u003e\n\u003cli\u003eAbbas Z., Zhang J., Tong Y., Ahmad B., Wang J., Zhang H., Li Z., Wei X., Zhang R., Si D., Production and characterization of novel antioxidant peptides from mulberry leaf ferment using B. subtilis H4 and B. amyloliquefaciens LFB112, Food Chemistry, (2025) 144022. https://doi.org/10.1016/j.foodchem.2025.144022. \u003c/li\u003e\n\u003cli\u003eWang D., Xu Z., Wang Y., Li Y., Zheng W., Chai Y., Wei G., Huang A., Identification and characterization of novel antioxidant peptides from Yunnan dry-cured beef: A combined in silico and in vitro study, Food chemistry, 477 (2025) 143485. https://doi.org/10.1016/j.foodchem.2025.143485. \u003c/li\u003e\n\u003cli\u003eChaudhari D.N., Ahire J.J., Kulthe A.A., Ghodke S., Complete Genome Sequence and In Vitro Probiotic Assessment of Bacillus subtilis DC-11 Isolated from Traditionally Fermented Idli Batter, Current microbiology, 82 (2024) 35. https://doi.org/10.1007/s00284-024-04014-y. \u003c/li\u003e\n\u003cli\u003eChelliah R., Kim N.H., Rubab M., Yeon S.J., Barathikannan K., Vijayalakshmi S., Hirad A.H., Oh D.H., Robust and safe: Unveiling Bacillus clausii OHRC1\u0026apos;s potential as a versatile probiotic for enhanced food quality and safety, LWT, 203 (2024) 116291. https://doi.org/10.1016/j.lwt.2024.116291. \u003c/li\u003e\n\u003cli\u003eGauri K., Rachatida D.-u., Pinidphon P., Cheunjit P., Probiogenomic analysis and safety assessment of Bacillus isolates using Omics approach in combination with In-vitro, LWT, 159 (2022). https://doi.org/10.1016/j.lwt.2022.113216. \u003c/li\u003e\n\u003cli\u003eZhou Y., Tu T., Yao X., Luo Y., Yang Z., Ren M., Zhang G., Yu Y., Lu A., Wang Y., Pan-genome analysis of Streptococcus suis serotype 2 highlights genes associated with virulence and antibiotic resistance, Frontiers in Microbiology, 15 (2024) 1362316. https://doi.org/10.3389/FMICB.2024.1362316. \u003c/li\u003e\n\u003cli\u003eLiang A., Wang J., Ding L., Zou L., Wang D., Zhu C., Tang J., Probiotic properties, whole-genome sequence analysis, and safety assessment of BreviBacillus borstelensis S8, LWT, 210 (2024) 116800. https://doi.org/10.1016/j.lwt.2024.116800. \u003c/li\u003e\n\u003cli\u003eJason J.R., Stephanie A.A., Gregory M.R., PksS from Bacillus subtilis is a cytochrome P450 involved in bacillaene metabolism, Biochemical and Biophysical Research Communications, 358 (2007) 363-367. https://doi.org/10.1016/j.bbrc.2007.04.151. \u003c/li\u003e\n\u003cli\u003eCzink\u0026oacute;czky R., N\u0026eacute;meth \u0026Aacute;., Techno-economic assessment of Bacillus fermentation to produce surfactin and lichenysin, Biochemical Engineering Journal, 163 (2020) 107719. https://doi.org/10.1016/j.bej.2020.107719. \u003c/li\u003e\n\u003cli\u003eDimopoulou A., Theologidis I., Benaki D., Koukounia M., Zervakou A., Tzima A., Diallinas G., Hatzinikolaou D.G., Skandalis N., Direct Antibiotic Activity of Bacillibactin Broadens the Biocontrol Range of Bacillus amyloliquefaciens MBI600, mSphere, 6 (2021) e0037621. https://doi.org/10.1128/mSphere.00376-21. \u003c/li\u003e\n\u003cli\u003eLuigia S., Elda R., Margherita C., Damiano B., Giulia M., Paolo C., G. G.C., Thomas L., Maurizio S., J.V. P.L., Fran\u0026ccedil;ois F., Sally E., Elena C., Alessia S., Evelina T., Systematic review and meta-analysis of in vitro efficacy of antibiotic combination therapy against carbapenem-resistant Gram-negative bacilli, International Journal of Antimicrobial Agents, 57 (2021) 106344. https://doi.org/10.1016/j.ijantimicag.2021.106344. \u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Collagenase producing enzyme, Probiotic Bacillus, Safety, Whole-genome analysis, Cowhide fermentation","lastPublishedDoi":"10.21203/rs.3.rs-7565406/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7565406/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"In this study, strains with high collagenase producing enzyme production were isolated from traditional naturally fermented foods rich in collagen. The safety of these strains was evaluated through in vitro biological characteristics and whole-genome sequence analysis. The results indicated that strains HA3, HA5, HA7, and HA8 exhibited high collagenase production, lacked hemolytic activity, and were sensitive to most commonly used antibiotics. Comprehensive analysis of antibiotic resistance genes, virulence factor genes, and metabolic systems at the genomic level confirmed the safety of these four strains, which were identified as B. spizizenii HA3, B. velezensis HA5, B. safensis HA7, and B. velezensis HA8, respectively. Notably, B. velezensis HA5 and B. velezensis HA8 could completely degrade cowhide into a liquid state and the antioxidant activity of the resulting fermentation degradation liquid was evidently improved. This study provides theoretical support and technical reserves for the development and utilization of livestock and poultry skins.","manuscriptTitle":"Probiotic characteristics, whole-genome sequence analysis of Bacillus with high collagenase production and its fermentation hydrolysis of cowhide","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-17 10:57:58","doi":"10.21203/rs.3.rs-7565406/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"d390cff9-5207-41a4-818f-00c5755c8f57","owner":[],"postedDate":"September 17th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-10-21T13:23:30+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-17 10:57:58","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7565406","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7565406","identity":"rs-7565406","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}