Salivaria lactobacillus d3-8 and application thereof in preparation of agelatin peptide by fermentation

Abstract

This invention belongs to the field of intestinal microbial technology, and specifically relates to a strain of *Lactobacillus salivarius* D3-8 and its application in the fermentation preparation of donkey-hide gelatin peptides. This strain was isolated from the intestines of healthy humans and is deposited at the China Center for Type Culture Collection (CCTCC), accession number CCTCC NO: M 20251921. *Lactobacillus salivarius* D3-8 can ferment and degrade donkey-hide gelatin, a large-molecule traditional Chinese medicine, into a series of low-molecular-weight donkey-hide gelatin peptides, showing broad application prospects in the fermentation and processing of donkey-hide gelatin and the preparation of donkey-hide gelatin peptides.

Description

Technical Field

[0001] This invention belongs to the field of intestinal microbial technology, and in particular relates to the application of a strain of Lactobacillus salivarius D3-8 in the fermentation preparation of donkey-hide gelatin peptides. Background Technology

[0002] *Lactobacillus salivarius* is a rod-shaped, Gram-positive bacterium initially isolated from the human oral cavity by M. Rogosa et al., who named it *Lactobacillus salivarius* (PMID: 13069442). Jinshui Zheng et al. revised its taxonomic status in 2020, renaming it *Ligilactobacillus salivarius* (PMID: 32293557). Previous studies have shown that *Lactobacillus salivarius* possesses antibacterial, anti-inflammatory, anticancer, and gut microbiota-regulating effects (PMID: 34889985). On August 18, 2022, the National Health Commission of China updated and released the latest "List of Bacterial Strains that Can Be Used in Food," which includes *Lactobacillus salivarius*, indicating that it is a safe probiotic for use in food.

[0003] Donkey-hide gelatin, a traditional Chinese medicine, is a solid gelatin made from the dried or fresh skin of the donkey (Equus asinus L.) through decoction and concentration (PMID: 32495607). According to the 2020 edition of the Chinese Pharmacopoeia, donkey-hide gelatin has the functions of nourishing blood and yin, moisturizing dryness, and stopping bleeding. Previous studies have found that donkey-hide gelatin possesses various biological activities, including anti-anemia, immune regulation, hemostasis, anti-inflammation, anti-oxidation, and antibacterial effects (PMID: 25382554). Donkey-hide gelatin contains abundant proteins, polypeptides, fatty acids, polysaccharides, and other components, with proteins and polypeptides accounting for 70%-90% of its composition (DOI: 10.1016 / j.jff.2025.106678).

[0004] Studies have shown that donkey-hide gelatin peptides are key molecules in the pharmacological effects of donkey-hide gelatin (PMID: 23561109). Furthermore, within a specific range, the smaller the molecular weight of the donkey-hide gelatin peptide, the stronger its activity (PMID: 23561109). Compared to donkey-hide gelatin, donkey-hide gelatin peptides have the characteristics of low molecular weight and high absorption and utilization rate. Therefore, the market demand for health products containing donkey-hide gelatin peptides has been strong in recent years. However, at present, different donkey-hide gelatin peptides are mainly prepared through enzymatic hydrolysis, with commonly used enzyme preparations including alkaline protease, papain, thermophilic protease, and neutral protease (PMID: 35359251). Although there are many types of donkey-hide gelatin peptide products on the market, the similarity of enzymatic hydrolysis processes leads to severe product homogenization. Therefore, developing unique donkey-hide gelatin peptide products using innovative processes has become an urgent need. To address these issues, this study is the first to discover that *Lactobacillus salivarius* D3-8 can degrade large molecular proteins in donkey-hide gelatin into low molecular weight oligopeptides, showing broad application prospects in the fermentation and processing of donkey-hide gelatin and the preparation of donkey-hide gelatin peptides. Summary of the Invention

[0005] To achieve the above objectives, the technical solution of the present invention is implemented as follows: In a first aspect, the present invention provides a strain of *Ligilactobacillus salivarius* D3-8, deposited at the China Center for Type Culture Collection (CCTCC) with accession number CCTCC NO: M 20251921.

[0006] Secondly, the present invention provides a compound microbial agent, wherein the compound microbial agent includes the *Lactobacillus saliva-associated* and other probiotic lactic acid bacteria described in the first aspect.

[0007] Thirdly, the present invention provides the application of the saliva-associated lactobacillus D3-8 described in the first aspect in the fermentation preparation of donkey-hide gelatin peptides.

[0008] Furthermore, the fermentation of *Lactobacillus saliva-associated* D3-8 was a liquid fermentation, with a viable cell concentration in the fermentation broth of (1~10)×10⁻¹⁰. 9 CFU / mL.

[0009] Furthermore, the donkey-hide gelatin used is the traditional Chinese medicinal material, donkey-hide gelatin.

[0010] Furthermore, the donkey-hide gelatin mentioned above is in powder form.

[0011] Fourthly, the present invention provides a method for preparing fermented donkey-hide gelatin peptides using the strain described in the first aspect, the preparation method comprising the following steps: a. Inoculate Lactobacillus saliva D3-8 into MRS medium for activation; b. Inoculate the activated saliva-containing Lactobacillus D3-8 into the gelatin aqueous solution and perform anaerobic culture; c. After fermentation and growth of Lactobacillus saliva-associated D3-8, the supernatant is collected by centrifugation; d. After pre-freezing the supernatant overnight in a -20 ℃ freezer, it was freeze-dried to obtain donkey-hide gelatin peptide.

[0012] Furthermore, the activation conditions in step a are growth at 35~38℃ for 20~26h; preferably, growth at 37℃ for 24h.

[0013] Furthermore, the concentration of the donkey-hide gelatin aqueous solution in step b is 4~8 g / L.

[0014] Furthermore, the growth in step c is carried out at 35~38℃ for 20~26 hours; preferably, it is carried out at 37℃ for 24 hours.

[0015] Fifthly, the present invention also provides fermented donkey-hide gelatin peptides prepared by the method described in the fourth aspect.

[0016] Sixthly, the present invention also provides the application of the fermented donkey-hide gelatin peptide described in the fifth aspect in the preparation of health products.

[0017] The beneficial effects of this invention include: 1) This invention obtained the anaerobic bacteria with the strongest ability to decompose and degrade donkey-hide gelatin by screening the intestines of healthy human subjects. It was identified as Ligilactobacillus salivarius, named D3-8, and deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M 20251921. 2) Experiments have shown that the saliva-associated lactobacillus D3-8 can ferment and degrade donkey-hide gelatin, a large molecular weight traditional Chinese medicine, into a series of low molecular weight donkey-hide gelatin peptides, which show broad application prospects in the fermentation and processing of donkey-hide gelatin and the preparation of donkey-hide gelatin peptides. Attached Figure Description

[0018] Figure 1 Phylogenetic evolutionary tree diagram of 11 strains of donkey-hide gelatin degrading bacteria.

[0019] Figure 2 Growth curves of 11 strains of donkey-hide gelatin-degrading bacteria.

[0020] Figure 3 The graph shows the lactic acid content of donkey-hide gelatin after fermentation by 11 strains of donkey-hide gelatin-degrading bacteria.

[0021] Figure 4 This is a complete genome map (chromosomal genes) of Lactobacillus salivarius D3-8.

[0022] Figure 5This is a complete genome loop of Lactobacillus saliva-associated D3-8 (plasmid gene).

[0023] Figure 6 This is a COG (Cluster of Orthologous Groups of Proteins) functional annotation diagram of the whole genome of Lactobacillus salivarius D3-8.

[0024] Figure 7 Principal component analysis (PCA) diagram of oligopeptides produced during the fermentation of donkey-hide gelatin by Lactobacillus saliva-associated D3-8.

[0025] Figure 8 This figure shows the content changes of the characteristic oligopeptide subcluster 1 component during the fermentation of donkey-hide gelatin by Lactobacillus saliva-associated D3-8.

[0026] Figure 9 This is a graph showing the content changes of the characteristic oligopeptide subcluster 10 component during the fermentation of donkey-hide gelatin by Lactobacillus saliva-associated D3-8.

[0027] Figure 10 This figure shows the content changes of the characteristic oligopeptide subcluster 11 component during the fermentation of donkey-hide gelatin by Lactobacillus saliva-associated D3-8.

[0028] Figure 11 This figure shows the content changes of the characteristic oligopeptide subcluster 17 component during the fermentation of donkey-hide gelatin by Lactobacillus saliva-associated D3-8.

[0029] Figure 12 This figure shows the content changes of the characteristic oligopeptide subcluster 20 component during the fermentation of donkey-hide gelatin by Lactobacillus saliva-associated D3-8.

[0030] Biological Preservation Instructions: The *Lactobacillus salivarius* D3-8 provided by this invention is deposited at the China Center for Type Culture Collection (CCTCC), with accession number CCTCC NO: M 20251921, deposit date of September 1, 2025, and address of the collection center: China Center for Type Culture Collection, No. 299 Bayi Road, Wuchang District, Wuhan City, Hubei Province, within the campus of Wuhan University. Detailed Implementation

[0031] The following detailed embodiments further illustrate the concept and technical effects of the present invention to fully understand its purpose, features, and effects. Unless otherwise specified, all methods described are conventional methods. Unless otherwise specified, all materials are available from publicly available commercial sources. The illustrative embodiments and descriptions of the present invention are used to explain the invention and do not constitute an undue limitation thereof. It should be noted that, unless otherwise specified, the embodiments and features described in the present invention can be combined with each other.

[0032] Example 1: Isolation and purification of donkey-hide gelatin-degrading bacteria The main instrument used in this embodiment is the AW500SG anaerobic workstation (Electrotek, UK).

[0033] (1) Preparation of culture medium Prepare a liquid culture medium for donkey-hide gelatin with the following components: 8 g / L of powdered donkey-hide gelatin and distilled water as the solvent. Pour the culture medium into anaerobic vials, fill with nitrogen, seal and store, and then autoclave at 121 °C for 20 min.

[0034] Prepare a solid culture medium for donkey-hide gelatin with the following components: 8 g / L of powdered donkey-hide gelatin, distilled water as the solvent, and 1.2% (w / v) agar. After dissolving in an Erlenmeyer flask, autoclave the medium at 121 °C for 20 min.

[0035] (2) Source of fecal samples Stool samples were taken from 10 healthy adults, and none of the participants had recently used antibiotics.

[0036] (3) Isolation and purification of donkey-hide gelatin degrading bacteria Fecal samples from 10 volunteers were aliquoted in an anaerobic incubator. After aliquoting, an appropriate amount of fecal sample was dissolved in 10 mL of sterile water, thoroughly mixed, and allowed to stand for 5 minutes. Then, 1 mL of the supernatant was aspirated using a 1 mL syringe and inoculated into 50 mL of donkey-hide gelatin liquid culture medium. After incubation at 37 ℃ for 24 h in an anaerobic incubator, the culture medium was aspirated and purified using the serial dilution plate method.

[0037] (3) Experimental results and analysis A total of 134 strains belonging to 7 genera and 10 species (including 2 subspecies) were obtained through enrichment culture on donkey-hide gelatin medium. All obtained strains were preserved in a laboratory strain library at -80 ℃. Eleven strains were randomly selected from the purified strain library, one strain from each species / subspecies, and a phylogenetic tree was constructed based on 16S rDNA. The results are as follows: Figure 1 As shown.

[0038] Table 1. Results of bacterial identification for donkey-hide gelatin degrading bacteria Example 2: Growth curve determination of 11 strains of donkey-hide gelatin degrading bacteria The main instrument used in this embodiment is the ReadMax 1200 full-wavelength microplate reader (Shanghai Flash Spectrum Biotechnology Co., Ltd.).

[0039] (1) Preparation of culture medium Prepare liquid anaerobic donkey-hide gelatin culture medium according to the method in Example 1.

[0040] Prepare MRS liquid anaerobic culture medium with the following components: 10 g / L peptone, 5 g / L beef extract, 4 g / L yeast extract, 2 g / L dipotassium hydrogen phosphate, 20 g / L glucose, 2 g / L triammonium citrate, 5 g / L sodium acetate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1 g / L Tween 80, and distilled water as the solvent. The pH value is 6.2 ± 0.2. Pour the culture medium into anaerobic vials, purge with nitrogen, seal, and autoclave at 121 °C for 15 min.

[0041] (2) Activation of strain Eleven strains of donkey-hide gelatin-degrading bacteria were activated using fresh MRS medium and cultured at 37 °C for 24 h before being transferred to 20 mL of fresh donkey-hide gelatin medium.

[0042] (3) Growth curve determination Eleven activated strains of donkey-hide gelatin-degrading bacteria were placed in a 37°C incubator for static culture. After 12 h, 24 h, 48 h, and 72 h of culture, 200 μL of bacterial culture was aspirated and the OD values ​​were measured using a 96-well plate and a microplate reader. 600nm Value. Take 200 μL of fresh liquid culture medium and measure its absorbance (OD). 600nm () as the original control.

[0043] (4) Experimental results and analysis The results are as follows Figure 2 As shown, the 11 strains of donkey-hide gelatin-degrading bacteria reached their maximum concentration after 12 h of growth at 37 ℃, and tended to stabilize after 24 h. Among the 11 strains of donkey-hide gelatin-degrading bacteria, strain D3-8 of *Lactobacillus salivarius* had the highest OD value. 600nm The value is the highest, which initially indicates that it has the strongest fermentation ability for donkey-hide gelatin.

[0044] Example 3: Determination of short-chain fatty acid content in donkey-hide gelatin fermented by 11 strains of donkey-hide gelatin-degrading bacteria. The main instrument used in this embodiment is an Agilent 1260 high-performance liquid chromatograph (Agilent Technologies).

[0045] (1) Pretreatment of fermentation broth While measuring the growth curve in Example 2, after culturing for 72 h, 500 μL of bacterial culture was taken and mixed with 500 μL of 1% H2SO4. After thorough mixing, the mixture was stored in a -20 ℃ refrigerator for later use.

[0046] (2) Detection method of fermentation broth The chromatographic column used was an Aminex HPX-87H Exclusion Column. The mobile phase was 5 mM H₂SO₄, and the elution flow rate was 0.6 mL / min with a gradient flow rate. The column temperature was controlled at 50 ℃, and the RID detector was used at a wavelength of 210 nm. The standard curve was established as follows: 100 mM stock solutions were prepared from formic acid, acetic acid, propionic acid, butyric acid, lactic acid, and succinic acid standards. These stock solutions were then diluted to prepare standard solutions of different concentrations (0 mM, 5 mM, 10 mM, 20 mM, 40 mM, 50 mM, 70 mM, and 100 mM). The injection volume was 20 μL, and a standard curve was plotted using peak area versus concentration. After centrifuging the fermentation broth at 12000 rpm for 15 min, 20 μL of the sample was loaded. The content of various short-chain fatty acids in the fermentation broth was calculated by comparing the results with the standards.

[0047] (3) Results and Analysis like Figure 3 As shown, in the culture medium for pure donkey-hide gelatin, 11 strains of donkey-hide gelatin-degrading bacteria mainly produced lactic acid. Among them, the fermentation broth of *Lactobacillus salivarius* D3-8 had the highest lactic acid content, further indicating its strongest fermentation ability for donkey-hide gelatin.

[0048] Example 4: Whole genome sequencing of Lactobacillus saliva-associated salivarius D3-8 (1) Preparation of culture medium Prepare 1 L of liquid anaerobic donkey-hide gelatin culture medium according to the method in Example 1. Prepare anaerobic MRS liquid culture medium according to the method in Example 2.

[0049] (2) Activation and scale-up culture of strains Lactobacillus salivae D3-8 was activated using fresh MRS medium and cultured at 37 °C for 24 h before being transferred to 1 L of fresh donkey-hide gelatin medium.

[0050] (3) Whole genome sequencing After inoculating Lactobacillus saliva-associated D3-8 into anaerobic donkey-hide gelatin medium and culturing at 37 ℃ for 24 h, the culture was centrifuged at 12000 rpm for 10 min. The supernatant was discarded, and the bacterial precipitate was collected into cryovials. The cryovials were then rapidly frozen in liquid nitrogen and stored at -20 ℃. Finally, the samples were sent to Shanghai Meiji Biomedical Technology Co., Ltd. for sequencing.

[0051] (4) Results and Analysis As shown in Table 2 and Figure 4-5 As shown, the complete genome of *Lactobacillus salivarius* D3-8 contains one chromosomal gene and one plasmid gene. Figure 6 As shown in Tables 3-4, COG functional annotation indicates the presence of 25 protease-related genes and 38 peptidase-related genes in the genome, suggesting that Lactobacillus salivae D3-8 can degrade large-molecule donkey-hide gelatin protein into small-molecule donkey-hide gelatin peptides.

[0052] Table 2. Genome information of Lactobacillus salivae D3-8 Table 3 COG annotation results - information on protease-related genes Table 4 COG Annotation Results - Peptidase-Related Gene Information Example 5: Peptide Omics Analysis of Donkey-hide Gelatin Fermented by Lactobacillus D3-8 in Saliva The main instruments and equipment used in this embodiment are: PD-2 freeze dryer (Shanghai Bilang Instrument Manufacturing Co., Ltd.); ReadMax 1200 full-wavelength microplate reader (Shanghai Flash Spectrum Biotechnology Co., Ltd.).

[0053] (1) Preparation of culture medium Prepare liquid anaerobic donkey-hide gelatin culture medium according to the method in Example 1. Prepare anaerobic MRS liquid culture medium according to the method in Example 2.

[0054] (2) Activation of strain Lactobacillus salivae D3-8 was activated using fresh MRS medium and cultured at 37 °C for 24 h before being transferred to fresh donkey-hide gelatin medium.

[0055] (3) Sampling points and sample delivery process Based on the growth curve in Example 2, it was determined that *Lactobacillus salivarius* D3-8 reached the mid-logarithmic growth phase after 6 hours of growth in the donkey-hide gelatin culture medium. Five mL of the fermentation broth was collected at each time point (0 h, 6 h, 24 h, and 72 h) after *Lactobacillus salivarius* D3-8 fermentation in the donkey-hide gelatin culture medium. The samples were then frozen overnight at -20 °C and freeze-dried using a vacuum freeze dryer. The obtained samples were sent to Shanghai Meiji Biomedical Technology Co., Ltd. for analysis to determine the sequences and contents of oligopeptides produced during the fermentation of donkey-hide gelatin by *Lactobacillus salivarius* D3-8.

[0056] (4) Results and Analysis like Figure 7 As shown, the types and abundance of peptides in donkey-hide gelatin changed significantly before and after fermentation by *Lactobacillus salivarius* D3-8, indicating that *Lactobacillus salivarius* D3-8 can degrade large molecular proteins in donkey-hide gelatin. Figures (8-12) and Table 5 show that *Lactobacillus salivarius* D3-8 can degrade and metabolize large molecular proteins in donkey-hide gelatin, converting them into small oligopeptides.

[0057] Table 5. Information on Characteristic Oligopeptides In summary, Lactobacillus saliva-associated D3-8 can ferment and degrade donkey-hide gelatin, a macromolecular traditional Chinese medicine with protein as its main component, into a series of low molecular weight donkey-hide gelatin peptides, showing broad application prospects in the fermentation and processing of donkey-hide gelatin and the preparation of donkey-hide gelatin peptides.

[0058] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A strain of *Ligilactobacillus salivarius* D3-8, characterized in that, It is deposited at the China Center for Type Culture Collection, with accession number CCTCC NO: M 20251921.

2. A compound microbial agent, characterized in that, The compound microbial agent includes the *Lactobacillus saliva-associated* as described in claim 1, as well as other probiotic lactic acid bacteria.

3. The application of the salivary-associated lactobacillus D3-8 as described in claim 1 in the fermentation preparation of donkey-hide gelatin peptide.

4. The application according to claim 3, characterized in that, The fermentation of *Lactobacillus salivarius* D3-8 was a liquid fermentation, with a viable cell concentration in the fermentation broth of (1~10) 10. 9 CFU / mL.

5. The application according to claim 3, characterized in that, The donkey-hide gelatin used is the traditional Chinese medicinal material, donkey-hide gelatin.

6. The application according to claim 5, characterized in that, The donkey-hide gelatin mentioned above is in powder form.

7. A method for preparing donkey-hide gelatin peptides using the saliva-associated Lactobacillus D3-8 as described in claim 1, characterized in that, The method includes the following steps: a. Inoculate Lactobacillus saliva D3-8 into MRS medium for activation; b. Inoculate the activated saliva-containing Lactobacillus D3-8 into the gelatin aqueous solution and perform anaerobic culture; c. After fermentation and growth of Lactobacillus saliva-associated D3-8, the supernatant is collected by centrifugation; d. After pre-freezing the supernatant overnight in a -20 ℃ freezer, it was freeze-dried to obtain donkey-hide gelatin peptide.

8. The method according to claim 7, characterized in that, in, The activation conditions in step a are growth at 35-38℃ for 20-26 hours; the concentration of the donkey-hide gelatin aqueous solution in step b is 4-8 g / L; the growth in step c is growth at 35-38℃ for 20-26 hours; preferably, growth at 37℃ for 24 hours.

9. The fermented donkey-hide gelatin peptide prepared by the method of claim 7 or 8.

10. The application of the fermented donkey-hide gelatin peptide according to claim 9 in the preparation of health products.

Images