A highly acid-tolerant bacillus velezensis strain lybv022-ac R and its progressive acid stress domestication screening method and application

Abstract

This invention discloses a highly acid-resistant Bacillus belye strain LYBV022-Ac R This invention relates to a method for screening and acclimating bacteria under progressive acid stress, and its application, belonging to the technical field of industrial microbial breeding and probiotic application. The disclosed high-acid-resistant Bacillus belyeis LYBV022-Ac... R By designing a pH gradient decreasing (6.5→3.0) acclimatization strategy and combining it with microplate rapid screening technology, a breakthrough improvement in acid resistance was achieved. After exposure to a simulated gastric acid environment (pH 3.0, containing pepsin) for 4 hours, the survival rate reached 48.52%, a 970-fold increase compared to the original strain (0.05%), while maintaining excellent growth performance and genetic stability. This invention also provides the application of this strain in the preparation of oral probiotic preparations for livestock and poultry, solving the common technical problem of insufficient potency in oral Bacillus preparations due to gastric acid damage.

Description

Technical Field

[0001] This invention relates to the field of industrial microbial breeding and probiotic application technology, and more specifically to a strain of Bacillus belyei LYBV022-Ac with high acid resistance. R Its targeted domestication and screening methods and their application in the preparation of acid-resistant oral probiotic preparations. Background Technology

[0002] Bacillus belesiensis ( Bacillus velezensis Bacillus belyss is a novel species of Bacillus that has shown great application potential in agriculture, animal husbandry, food and health products, and environmental management in recent years. During its growth, it produces various antimicrobial lipopeptides (such as surfactants, iturobrine, and fenestrator), hydrolytic enzymes, and vitamins, exhibiting multiple probiotic functions, including inhibiting animal and plant pathogens, promoting growth, and improving intestinal microecological balance. Therefore, Bacillus belyss is considered one of the ideal candidate species for developing probiotic preparations. On January 6, 2026, Bacillus belyss CGMCC 24752 strain was approved as a new feed additive.

[0003] Probiotics are a general term for a class of live microorganisms that are beneficial to the host. They have functions such as improving the balance of intestinal flora, enhancing immunity, and inhibiting pathogenic bacteria. They have shown significant effects in regulating intestinal function, promoting nutrient absorption, and improving animal production performance, and are widely used in food, medicine, and feed industries. The gastric acid barrier is the primary challenge faced by probiotics during oral administration. Human and animal gastric juices are typically highly acidic (pH 1.5–3.5) and contain digestive enzymes such as pepsin. This extreme environment easily leads to damage to microbial cell membranes, protein denaturation, and enzyme inactivation, resulting in a sharp decline in the number of live bacteria. Therefore, improving the gastric acid tolerance of probiotics is crucial for their effective function in the intestines when administered orally.

[0004] Currently, the main techniques for improving the gastric acid tolerance of probiotics include physical encapsulation (such as microencapsulation) and chemical protection (such as adding skim milk, trehalose, and other protective agents). While these methods have some effect, they also have limitations. Physical encapsulation is complex and costly, and the encapsulation material may affect the release and activity of the bacteria. Chemical protection may alter the formulation, increasing uncertainties and affecting product stability. Targeted domestication technology is a non-genetic engineering, phenotypic selection-based biofortification method. It typically involves applying gradual, stable, and controllable selection pressures (such as acid, heat, and bile salts) to a microbial population under controlled conditions, allowing more adaptable individuals to grow and accumulate. This gradually yields stable strains with significantly enhanced target tolerance without introducing exogenous genes. Compared to physical encapsulation or chemical protection, targeted domestication offers advantages such as fundamentally strengthening the strain's intrinsic resistance, relatively simple processing, lower cost, no introduction of exogenous substances, and ease of industrialization. Especially in the field of probiotics, this technology has been used to improve the stomach acid tolerance, bile salt tolerance and intestinal colonization ability of strains, and has important application prospects.

[0005] However, current conventional methods for targeted acid acclimatization still suffer from drawbacks such as broad gradient settings, reliance on manual experience in the screening process, low throughput, and difficulty in accurately obtaining strains that exhibit both high survival rates and good growth performance under extremely acidic conditions. Therefore, developing an efficient and controllable method for the acclimatization and screening of acid-resistant Bacillus belyssus, and obtaining a strain with high survival rates and excellent growth performance in gastric acid, is of great significance for promoting its application in oral formulations.

[0006] Therefore, a highly acid-resistant Bacillus berberis strain LYBV022-Ac is provided. R The methods and applications of gradual acid stress acclimatization screening are problems that urgently need to be solved by those skilled in the art. Summary of the Invention

[0007] In view of this, the present invention provides a strain of Bacillus belyei LYBV022-Ac with high acid resistance. R It also describes a targeted acclimatization method combining progressive acid stress and high-throughput screening, and its application in the preparation of acid-resistant oral probiotic formulations. This method can be used for the development of oral probiotics for animals.

[0008] The purpose of this invention is to overcome the problems of insufficient gastric acid tolerance of Bacillus belysin vegetative cells in the prior art, as well as the complexity, high cost, or potential impact on bacterial activity of physicochemical protection methods. The invention provides an acid-resistant Bacillus belysin strain obtained through directed domestication that has a high survival rate in a simulated gastric acid environment, and a simple, low-cost, and highly efficient screening method for the aforementioned acid-resistant Bacillus belysin.

[0009] To achieve the above objectives, the present invention adopts the following technical solution:

[0010] A strain of Bacillus belye ( Bacillus velezensis ) LYBV022-Ac R Its accession number is GDMCCNo: 66951, and it has been deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on September 11, 2025. The classification and naming are as follows: Bacillus velezensis The most significant technical feature of this strain is that, obtained through the specific acid stress induction and selection procedure of this invention, its survival rate remains at 48.52% after treatment in artificial gastric juice (containing pepsin) at pH 3.0 and conforming to the standards of the Chinese Pharmacopoeia for 240 min, which is significantly higher than that of the original strain (0.05%), representing a 970-fold increase; and it can still grow well in acidic culture media with pH 2.0-4.0, exhibiting a significant and stable acid-resistant phenotype.

[0011] Furthermore, a screening method for acid-resistant Bacillus belye is provided. The core of this method lies in combining a gradual acid stress acclimatization strategy with high-throughput screening technology, and mainly includes the following steps: (1) Gradual acid stress acclimatization: Using the original strain of Bacillus belyssus LYBV022 as the starting strain, the inoculum was successively transferred to LB liquid medium with gradually decreasing pH values ​​(pH 6.5 → 6.0 → 5.5 → 5.0 → 4.5 → 4.0 → 3.5 → 3.0) at an inoculation rate of 1% to 5%. Under each pH gradient, the culture was shaken at 37 ℃ and 180 r / min for 18 h to 72 h, and the culture was passaged three times. The third generation of bacterial culture that grew stably under the acclimatization pH conditions was selected for high-throughput screening in 96-well plates.

[0012] (2) High-throughput screening in 96-well plates: The concentration of the third-generation bacterial culture that had been stably grown under the pH conditions of this round of acclimatization was adjusted to 1×10⁻⁶ using sterile PBS. 8 CFU / mL, and serially diluted 10-fold to 10. -6 Power of 10, so that each dilution (10) 0 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 The corresponding 10 μL volume of bacterial culture contained 1×10⁻⁶ bacteria. 6 1×10 5 1×104 1×10 3 1×10 2 1×10 1 and 1×10 0 Approximately CFU. 90 μL of LB liquid medium (pH set to the acclimatization pH conditions) was added to each well of a 96-well sterile round-bottom plate. Then, 10 μL of bacterial suspension at different dilutions was added to wells 1-12 in each row. After incubation at 37°C for 24 h, OD was measured. 600 nm Value. Select those with bacterial growth, the highest dilution, and the highest OD. 600 nm The bacterial solution in the sample well with the highest pH value was taken as the domesticated strain obtained under the domestication pH conditions in this round.

[0013] (3) Verification of acclimatized strains: The acclimatized strains obtained under the current acclimatization pH conditions were transferred to LB liquid medium (pH value of the current acclimatization pH conditions) at a 1% inoculum concentration and cultured at 37 ℃ and 180 r / min for 18 h. The morphology of the bacteria was observed by Gram staining and microscopic examination; the bacterial species were confirmed by PCR using Bacillus-specific primers. The verified acclimatized strains were then transferred to LB liquid medium under the next acclimatization pH conditions at an inoculum concentration of 1% to 5%.

[0014] (4) Repeat steps (1), (2), and (3) until a strain that can survive stably at pH 3.0 is obtained, and named LYBV022-Ac. R .

[0015] (5) Acid resistance verification and strain identification: LYBV022-Ac R The strain was tested for tolerance to artificial gastric juice, and morphological, physiological, biochemical, and molecular biological identification was performed.

[0016] The Bacillus lybdenum LYBV022-Ac R Its applications in the preparation of acid-resistant oral probiotic preparations include livestock and poultry microecological preparations, probiotic feed additives, or oral antibacterial preparations for pharmaceutical use.

[0017] As can be seen from the above technical solution, compared with the prior art, the present invention discloses a strain of Bacillus belye LYBV022-Ac R Its domestication methods and application in the preparation of acid-resistant oral probiotic preparations have the following beneficial effects: (1) Excellent and specific strain performance: This invention obtained a new strain of Bacillus belyei with significant and stable acid resistance, LYBV022-Ac. RAfter being exposed to a simulated gastric acid environment for 4 hours, the survival rate of this strain increased significantly from 0.05% of the original strain to 48.52%, effectively solving the gastric acid barrier problem encountered when it is administered orally.

[0018] (2) The screening method is highly efficient and innovative: The screening method established in this invention has the advantages of strong directionality, high efficiency and simple operation, providing a reliable solution for industrial breeding of stress-resistant probiotics.

[0019] (3) Safety and broad application prospects: The strain of the present invention is selected by non-genetic engineering means, which is safe and reliable, and provides core strain resources for the development of highly efficient oral probiotic preparations. Attached Figure Description

[0020] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on the provided drawings without creative effort.

[0021] Figure 1 This is a flowchart of the screening method for acid-resistant Bacillus belye.

[0022] Figure 2 Acid-resistant Bacillus baileyi LYBV022-Ac R Morphological observation; where A: Colony morphology on LB agar plate; B: Gram staining microscopic image; C: Scanning electron micrograph.

[0023] Figure 3 Acid-resistant Bacillus baileyi LYBV022-Ac R 16S rRNA The identification results are shown in the image; where A: 16S rRNA PCR amplification electrophoresis results; M: DL 2 000 DNA Marker; 1: LYBV022-Ac R ;2: ddH2O; B: strain 16S rRNA Construct a phylogenetic tree from the sequence.

[0024] Figure 4 Acid-resistant Bacillus baileyi LYBV022-Ac R The results of the artificial gastric fluid tolerance test are shown in the figure; : P <0.01; : P<0.001.

[0025] Figure 5 Acid-resistant Bacillus baileyi LYBV022-Ac R The graph shows the growth ability of the original strain LYBV022 under pH 1-7 conditions; where A: acid-resistant Bacillus belyeis LYBV022-Ac R B: Wild-type Bacillus belyeis LYBV022.

[0026] Figure 6 The figure shows the results of the assay for the survival of the strain in the gastrointestinal tract of mice; where A: stomach, B: intestine.

[0027] Figure 7 The graph shows the results of the in vitro antibacterial activity assay for the strain; where A: 24 h of action; B: 48 h of action; C: 72 h; D: 96 h.

[0028] Figure 8 The strain is LYBV022-Ac R Safety evaluation results; where A: lung; B: liver; C: brain; D: spleen; E: kidney; F: heart; the challenge dose was 3.16 × 10⁻⁶. 7 CFU / mL. Detailed Implementation

[0029] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0030] Example 1: Domestication and screening of low-acid-tolerant Bacillus belye The flowchart for the domestication and screening of acid-resistant Bacillus belyi is shown below. Figure 1 .

[0031] (1) Recovery of the original strain: The original strain LYBV022 was streaked onto LB agar plates and incubated at 37 ℃ for 24 h. Single colonies were picked and inoculated into LB liquid medium and incubated at 37 ℃ with shaking at 180 rpm for 12 h. After Gram staining and microscopic examination and identification by Bacillus spp. atopic PCR, the strain was stored at 4 ℃ for later use.

[0032] Original strain Bacillus velezensisLYBV022, with accession number GDMCC No: 66952, is deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on September 11, 2025. It is classified and named as follows: Bacillus velezensis .

[0033] (2) Acclimation of low-acid-tolerant strains: The original strain LYBV022 was transferred to LB liquid medium (pH 7.0) at an inoculum of 1% and cultured at 37 ℃ and 180 r / min until OD. 600 ≈1. Subsequently, the inoculum was transferred to LB liquid medium (pH 6.5) at a ratio of 1% and cultured at 37 °C and 180 r / min for 18 h. After that, the inoculum was continuously transferred for 3 generations at the same pH conditions at a ratio of 1%.

[0034] Take the bacterial culture that has grown stably at pH 6.5 and adjust the concentration to 1×10⁻⁶ with PBS. 8 CFU / mL and diluted 10-fold to 10. -6 To the power of the power, 90 μL of LB liquid medium (pH 6.5) was added to each well of a 96-well round-bottom plate. Then, 10 μL of bacterial suspension at different dilutions was added to wells 1-12 in each row. After incubation at 37 ℃ for 24-72 h, the OD was measured. 600 Value. Select those with bacterial growth, the highest dilution, and the highest OD. 600 The bacterial culture from the sample well with the highest pH value was used as the acid-resistant strain under pH 6.5 conditions, and was transferred to LB liquid medium (pH 6.5) at an inoculum of 1% and cultured at 37 ℃ and 180 r / min for 18 h. The morphology of the bacteria was observed by Gram staining and microscopic examination, and the bacterial species was confirmed by PCR using Bacillus-specific primers (rpoB-F: 5'-GCGGCGTGCCTAATACATGC-3'; SEQ ID NO.1; rpoB-R: 5'-CTCAGGTCGGCTACGCATCG-3'; SEQ ID NO.2).

[0035] The confirmed strains were transferred to LB liquid medium at pH 6.0 at a ratio of 1%-5% and acclimatized according to the aforementioned procedures. Strains that could stably survive at pH 5.5, 5.0, 4.5, 4.0, 3.5, and 3.0 were obtained sequentially. The strain that could stably survive at pH 3.0 was named the acid-tolerant strain LYBV022-Ac. R .

[0036] (3) Morphological observation of acid-resistant strains: colony, staining microscopy, electron microscopy strain LYBV022-Ac R Streak inoculation was performed on LB agar plates, and the colonies were observed after incubation at 37 °C for 24 h. The colonies were round, milky white, with a dry, rough, and opaque surface and irregular edges. Figure 2 A); Gram staining microscopy ( Figure 2 B) and electron microscopy ( Figure 2 C) The results showed that strain LYBV022-Ac R It is a Gram-positive large bacillus. This indicates that the morphology of the acid-resistant strain is consistent with the original Bacillus belyssus LYBV022.

[0037] (4) Acid-resistant strains 16S rRNA Identification: Sequence amplification and sequencing To study the acid-resistant strain LYBV022-Ac obtained through domestication R Precise taxonomic identification was performed, and its phylogenetic relationship with the original strain was confirmed. Based on... 16S rRNA Molecular biological analysis of gene sequences. The specific steps are as follows: ①Genomic DNA extraction: Take an appropriate amount of strain LYBV022-Ac R Freshly cultured bacterial cells were used to extract total genomic DNA using a bacterial genomic DNA extraction kit, strictly following the instructions. The purity and concentration of the extracted DNA were determined by agarose gel electrophoresis and a nucleic acid protein analyzer, and the DNA was stored at -20 °C for later use.

[0038] ② 16S rRNA Gene PCR amplification: Using extracted genomic DNA as a template, PCR was performed using universal primers for bacterial 16S rRNA gene amplification (27F: 5'-AGAGTTTGATCCTGGCTCAG-3'; SEQ ID NO.3; 1492R: 5'-GGTTACCTTGTTACGACTT-3'; SEQ ID NO.4). The PCR amplification products were detected by agarose gel electrophoresis, and the target band (approximately 1500 bp in size) was analyzed. Figure 3 A) Gel extraction, recovery, and purification were performed. The purified product was cloned using TA and sent to Sangon Biotech Co., Ltd. for sequencing to obtain strain LYBV022-Ac. R of 16S rRNA Gene fragment sequence (as shown in SEQ ID NO.5).

[0039] >LYBV022-Ac R 16S rRNA :

[0040] ③ Homology alignment and phylogenetic tree construction: The measured 16S rRNA Gene sequences were compared for homology in the National Center for Biotechnology Information (NCBI) database using the BLAST program (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) to obtain sequences of highly homologous related type strains. From the comparison results, several strain sequences with the highest homology were selected, along with the present invention strain LYBV022-Ac. R The sequence of the original strain LYBV022 was obtained, and a phylogenetic tree was constructed using MEGA software (version 11.0) via the Neighbor-Joining method. Bootstrap tests with 1000 replicates were performed to assess the confidence of each branch.

[0041] ④ Testimonial results: Sequence alignment results showed that strain LYBV022-Ac R of 16S rRNA The gene sequence showed 99.99% homology with the original strain LYBV022, except that the G at position 189 was changed to A. Based on 16S rRNA The constructed phylogenetic tree ( Figure 3 B) shows that LYBV022-Ac R With LYBV022 and Bacillus velezensis The reference strain FZB42 (NR_075005.2) is on the same branch, and its phylogenetic relationship is much higher than that of other Bacillus species and their closely related species.

[0042] Conclusion: Based on the high homology of the 16S rRNA gene sequences and the structure of the phylogenetic tree, and according to the classification criteria of Bergey's Manual of Systematic Bacteriology, the acid-resistant strain LYBV022-Ac obtained through domestication in this invention is clearly identified as such. R Identified as Bacillus belye ( Bacillus velezensis This identification result confirms its species stability during the domestication process, providing an accurate taxonomic basis for its subsequent application.

[0043] Bacillus belesiensis ( Bacillus velezensis ) LYBV022-Ac R Its accession number is GDMCC No: 66951, and it has been deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC), located at the Institute of Microbiology, Guangdong Academy of Sciences, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, on September 11, 2025. The classification and naming are as follows: Bacillus velezensis .

[0044] Example 2: Validation of tolerance to artificial gastric juice The selected strain LYBV022-Ac was selected separately. R The bacterial concentration of the original strain LYBV022 was adjusted to 5×10⁻⁶. 8 CFU / mL. Take 1 mL of bacterial culture, centrifuge, discard the supernatant, and resuspend the precipitate in 1 mL of artificial gastric fluid (purchased from Regen Biosciences, catalog number CZ0213). Incubate at 37℃ for 0, 30, 60, 120, and 240 min respectively. Take samples at each time point for viable bacterial count. Viability (%) = (N) t / N0) ×100%, where N t The viable cell counts were denoted as N1, N2, and N3, respectively, after 30, 60, 120, and 240 min of treatment. N0 represents the viable cell count after 0 min of treatment. Results are shown below. Figure 4 After being exposed to artificial gastric juice for 30 min, 60 min, 120 min, and 240 min, LYBV022-Ac R The survival rates were 60.87%, 60.15%, 56.28%, and 48.52%, respectively, all significantly higher than the original strain's 1.34%, 0.56%, 0.23%, and 0.05%. P <0.01).

[0045] Example 3: Determination of the growth ability of the strain under pH 1-7 conditions The acid-resistant strain LYBV022-Ac R The original strain LYBV022 was inoculated into LB liquid medium with pH ranging from 1.0 to 7.0, and cultured with shaking at 37°C and 180 rpm. The OD values ​​of the culture medium were measured at 0, 1, 2, 4, 8, 12, 16, and 24 h of culture. 600 nm The values ​​were calculated, and a growth curve was plotted.

[0046] See results Figure 5 LYBV022-Ac R ( Figure 5 A) and the original strain LYBV022 ( Figure 5 B) Growth characteristics differ significantly under different pH conditions.

[0047] Under neutral to slightly acidic conditions (pH 5.0-7.0), both strains were able to grow normally and enter the logarithmic growth phase, with the final bacterial OD... 600 nm The values ​​showed no significant difference, indicating that acid tolerance acclimatization did not impair its normal growth ability in normal and weakly acidic environments.

[0048] Under acidic conditions (pH 4.0), LYBV022-Ac R Its growth advantages are beginning to emerge. LYBV022-AcR After culturing in a medium at pH 4.0 for 24 h, OD 600 nm The value reached 1.862, while the OD of the original strain under the same conditions was... 600 nm The value is 0.950. This indicates that LYBV022-Ac R It exhibits significantly stronger growth capacity under pH 4.0 conditions.

[0049] Under strongly acidic conditions (pH 3.0), the growth of the original strain LYBV022 was completely inhibited, with an OD of [value missing] within 24 h. 600 nm The value did not exceed 0.1. In contrast, LYBV022-Ac R After culturing at pH 3.0 for 24 h, OD 600 The value reached 1.774, proving that it can achieve good growth in a highly acidic environment where the original strain cannot grow.

[0050] Under extremely acidic conditions (pH 2.0), the growth of the original strain LYBV022 was completely inhibited (OD). 600 nm Value <0.12). Acid-resistant strain LYBV022-Ac R It showed clear signs of growth at pH 2.0, with its OD... 600 nm The value increased from an initial 0.010 to 1.612 after 24 h, demonstrating that the strain acquired an excellent ability to grow in an extremely acidic environment at pH 2.0.

[0051] No growth was observed in either strain at pH 1.0, and OD... 600 The value did not increase significantly throughout the culture period, indicating that the bacteria could not proliferate under this extreme environment.

[0052] Example 4: Determination of the survival rate of the strain in the gastrointestinal tract of mice Sixty-three SPF-grade female KM mice, weighing 20-25 g, were purchased and acclimatized for 4 days. The mice were divided into three groups: an acid-resistant strain group (LYBV022-Ac...). R The study included three groups of bacteria: the acid-resistant strain group (LYBV022), the original bacterial strain group, and the blank control group (PBS). Each of the acid-resistant strain group and the original bacterial strain group contained 27 bacteria, and the blank control group contained 9 bacteria. The concentration of the bacterial suspension administered via gavage to both the acid-resistant strain group and the original bacterial strain group was 1×10⁻⁶. 9 CFU / mL, gavage volume 0.3 mL / animal, dose 3×10 8CFU / mouse; blank control group received 0.3 mL / mouse of sterile PBS via gavage. Mice were allowed free access to food and water via gavage. Three mice were sacrificed at 0.5, 2, 4, 6, 8, 12, 24, 36, and 60 h post-gavage, with samples collected at each time point. Gastric and intestinal tissues were aseptically isolated, weighed, and homogenized using sterile PBS at a 1:4 (w / v) ratio. The homogenate was serially diluted 10-fold and spread onto LB agar containing ampicillin (50 µg / mL) and polymyxin B (15 µg / mL). After incubation at 37°C for 24 h, colonies were counted, and the viable cell count in the tissue homogenate was calculated. Data are expressed as logarithmic values. 10 CFU indicates.

[0053] The survival rates of acid-resistant strains and the original strain in mouse gastric tissue are as follows: Figure 6 As shown in Figure A, 0.5 h after gavage, LYBV022-Ac R The bacterial count of the strain was 6.58 log. 10 The CFU count for strain LYBV022 was 4.23 log. 10 CFU; 2 h, LYBV022-Ac R The bacterial count of the strain was 5.83 log. 10 The CFU count of strain LYBV022 decreased to 2.56 log. 10 CFU; sterility was detected in the LYBV022 group after 4 h; LYBV022-Ac R At 4, 6, 8, and 12 hours, CFU levels were still detected at 3.49, 3.07, 2.59, and 1.87 log10 CFU, respectively. After 24 hours, LYBV022-Ac... R The group was found to be sterile.

[0054] The survival rates of acid-resistant strains and the original strains in mouse intestinal tissue are as follows: Figure 6 As shown in Figure B, no live bacteria were detected in the intestinal tissue of either group of mice 0.5 h after gavage; at 2 h, LYBV022-Ac R The bacterial count of the strain was 5.14 log. 10 The CFU, LYBV022 strain count was 2.14 log. 10 CFU;LYBV022 was still detected at 1.65, 1.76, 1.34, and 0.967 log at 4, 6, 8, and 12 hours, respectively. 10 CFU was detected in the LYBV022 group after 24 hours; at 4 hours, LYBV022-Ac... R The bacterial count of the strain was 4.48 log. 10 CFU; 6 h was 4.29 log 10 CFU; 8 h was 3.92 log 10 CFU; 12 h was 4.06 log.10 CFU; 24 h was 3.15 log. 10 CFU; viable bacteria were still detected at 36 h and 60 h (1.83 and 0.65 log, respectively). 10 CFU). Septicemia was detected in gastric and intestinal tissues at all time points in the PBS group.

[0055] The above results indicate that the *Bacillus belyssus* LYBV022-Ac obtained through domestication in this invention is effective. R The number of surviving strains and the duration of survival in the stomach and intestinal tissues were significantly better than those of the original strain LYBV022, demonstrating excellent gastric acid tolerance and gastrointestinal survival ability.

[0056] Example 5: Determination of in vitro antibacterial activity strain LYBV022-Ac R Inoculated into LB liquid medium, and cultured at 37℃ and 180 rpm for 24 h, 48 h, 72 h, and 96 h, respectively. The OD values ​​of the bacterial culture at each time point were recorded. 600 nm The values ​​were 1.75±0.05, 2.38±0.05, 2.29±0.05, and 2.17±0.05, respectively. *Staphylococcus aureus* (… Staphylococcus aureus ATCC 25923 is the indicator bacterium. 200 μL of bacterial suspension (approximately 1 × 10⁻⁶) 8 The bacterial culture (CFU / mL) was evenly spread onto LB agar plates and incubated at 37 °C for 30 min to ensure complete absorption. Using sterile Oxford cups (6 mm in diameter), the bacterial strain LYBV022-Ac was placed in wells. R After centrifuging the bacterial culture, collect the supernatant, and then add 150 μL of LYBV022-Ac culture solution with different incubation times to each well. R Supernatant. After incubation at 37 ℃ for 24 h, the diameter of the inhibition zone was measured.

[0057] See results Figure 7 LYBV022-Ac R It exhibited significant inhibitory activity against Staphylococcus aureus ATCC 25923. Clear inhibition zones were formed in the supernatant after 24 h, 48 h, 72 h, and 96 h of culture, suggesting that the antibacterial substance produced by this strain has good persistence.

[0058] Example 6: Strain LYBV022-Ac R Safety assessment The strain LYBV022-Ac was tested using healthy SPF-grade ICR mice. RSafety was assessed. Twenty mice were randomly divided into four groups of five each, labeled AD group. Group A received an intraperitoneal injection of 1 mL PBS, while groups B-D received an injection of 1 mL LYBV022-Ac. R The bacterial suspension had a concentration of 3.16 × 10⁻⁶. 7 6.3×10 6 and 6.3×10 5 Mice were monitored daily for 7 days, with a concentration of CFU / mL, to observe their behavior, diet, and health status. After the observation period, three mice from each group were randomly selected for necropsy to observe changes in tissues and organs.

[0059] Animal experiment results are shown in Figure 8 LYBV022-Ac was injected intraperitoneally with 1 mL of different concentrations. R After the strain was administered, no mice died, no pathological changes were observed upon necropsy, and there was no significant difference in weight gain, suggesting that the strain has good safety.

[0060] Example 7 Preparation of oral formulation Bacillus lybditis elegans stored at 4℃ LYBV022-Ac R The bacterial culture was transferred to 5 mL of LB liquid medium at a ratio of 1:100 and incubated at 37°C and 180 rpm for 18 h. The next day, it was transferred to 250 mL of LB liquid medium in Erlenmeyer flasks at the same ratio (the bacterial morphology was confirmed to be correct by Gram staining and microscopic examination before each transfer), and incubated at 37°C and 180 rpm with shaking until OD was reached. 600 ≈1.0, after confirming the bacterial morphology again under a microscope, collect the bacterial culture. Take 200 mL of the culture medium, centrifuge at 8000 rpm for 20 min, discard the supernatant, and wash the bacterial pellet twice with sterile PBS. Centrifuge to collect the bacterial cells, and adjust the bacterial concentration to 5 × 10⁻⁶ with sterile PBS. 8 CFU / mL. Take the adjusted bacterial culture and perform a 10-fold serial dilution to 10⁻⁶. -8 Select 10 -5 10 -6 10 -7 10 -8 Dilutions were plate-counted, and plate-counts were performed the following day to calculate the actual bacterial count. Results showed that each mL of bacterial culture contained 6.17 × 10⁻⁶ bacteria before lyophilization. 8 CFU bacteria.

[0061] Prepare the freeze-drying protectant according to the following formula: each 100 mL of protectant contains 20% skim milk powder and 10% sucrose. Take 1 mL of the adjusted bacterial solution and mix thoroughly with 1 mL of sterile freeze-drying protectant. Dispense 2 mL into sterile vials. Add rubber stoppers, leaving a gap for water sublimation. Pre-freeze the vials at -80℃ for at least 12 hours. Turn on the freeze dryer and preheat for 20 minutes. Quickly transfer the pre-frozen vials to the freeze dryer and start the freeze-drying program for 48 hours. After freeze-drying, immediately seal the vials under vacuum, remove them, and store them at -20℃ for long-term storage. Reconstitute the freeze-dried powder with sterile PBS, perform 10-fold serial dilutions, spread on LB agar plates, and incubate at 37℃ for 18–24 hours. Count the viable bacteria and calculate the freeze-drying survival rate. The results showed that the bacterial count per vial after freeze-drying was 5.47 × 10⁻⁶. 8 CFU.

[0062] The freeze-drying survival rate was calculated as follows: (Number of bacteria after freeze-drying / Number of bacteria before freeze-drying) × 100%. The result showed that the freeze-drying survival rate was 5.47 × 10⁻⁶. 8 / 6.17×10 8 ) × 100% ≈ 88.7%.

[0063] The results show that, using the freeze-drying protectant and freeze-drying process of the present invention, the freeze-drying survival rate of the strain can reach 88.7%, demonstrating excellent freeze-drying protection effect.

[0064] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A strain of highly acid-resistant Bacillus belye ( Bacillus velezensis ) LYBV022-Ac R Its characteristics are, Its accession number is GDMCC No: 66951.

2. The method for progressive acid stress acclimatization and screening of a highly acid-tolerant Bacillus belye strain as described in claim 1, characterized in that, Includes the following steps: (1) Gradual acid stress acclimatization: Using the original strain of Bacillus belyssus LYBV022 as the starting strain, the strain was successively transferred to LB liquid medium with gradually decreasing pH values ​​at an inoculum of 1% to 5%: pH 6.5 → 6.0 → 5.5 → 5.0 → 4.5 → 4.0 → 3.5 → 3.0; under each pH gradient, the strain was shaken and cultured at 35-39 ℃ and 150-200 r / min for 18 h to 72 h, and then passaged 3 or more times; the 3rd generation or higher bacterial culture that grew stably under the acclimatization pH conditions was selected for high-throughput screening in 96-well plates. (2) High-throughput screening in 96-well plates: The concentration of the third-generation bacterial culture that had been stably grown under the pH conditions of this round of acclimatization was adjusted to 1×10⁻⁶ using sterile PBS. 8 CFU / mL, and serially diluted 10-fold to 10. -6 Power of 10, so that each dilution is: 10 0 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 The corresponding bacterial counts in a 10 μL volume of bacterial culture were 1 × 10⁻⁶. 6 1×10 5 1×10 4 1×10 3 1×10 2 1×10 1 and 1×10 0 CFU; 90 μL of LB liquid medium was added to each well of a 96-well sterile round-bottom plate, with the pH set to the acclimatization pH conditions for this round. Then, 10 μL of bacterial suspension at different dilutions was added to wells 1-12 in each row. After incubation at 37 ℃ for 24 h, the OD was measured. 600 nm Value; select those with bacterial growth, highest dilution, and highest OD. 600 nm The bacterial culture in the sample well with the highest pH value was taken as the domesticated strain obtained under the domestication pH conditions in this round. (3) Verification of domesticated strains: The domesticated strains obtained under the current domestication pH conditions were transferred to LB liquid medium at a concentration of 1% of the strains. The pH value was the same as the current domestication pH conditions. The strains were cultured at 37 ℃ and 180 r / min for 18 h. The morphology of the strains was observed by Gram staining and microscopic examination. Bacillus-specific primer identification PCR was used to confirm the bacterial species; the verified acclimatized strains were then transferred to LB liquid under the next round of acclimatization pH conditions at an inoculation rate of 1% to 5%. (4) Repeat steps (1), (2), and (3) until a strain that can survive stably at pH 3.0 is obtained.

3. The method according to claim 2, characterized in that, The criteria for determining stable growth in step (1) are: the absorbance of the culture medium for three or more consecutive generations at a wavelength of 600 nm is >1.

4. The Bacillus belyssus LYBV022-Ac as described in claim 1 R Application in the preparation of acid-resistant oral probiotic formulations.

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