Clostridium tyrobutyricum, culture conditions and application thereof

Abstract

This invention belongs to the field of microbial technology, specifically relating to a *Clostridium butyricum* strain, its culture conditions, and applications. This invention provides a novel *Clostridium butyricum* strain with strong resistance to high temperatures, acids, alkalis, and salts, exhibiting high biocompatibility and no significant toxic side effects or hemolytic activity. Therefore, the *Clostridium butyricum* H2-2 is a potential probiotic strain that can be used as feed or feed additive in pig, poultry, and aquatic feeds for animal husbandry, strengthening the intestinal barrier and enhancing animal immunity; it also holds promise for use as an additive in human food, pharmaceuticals, and health products.

Description

Technical Field

[0001] This invention belongs to the field of microbial technology, specifically relating to a Clostridium butyricum bacterium, its culture conditions, and its applications. Background Technology

[0002] Butyric acid (BFA) is a short-chain fatty acid that plays a crucial role in maintaining intestinal homeostasis. As the preferred energy source for colonic epithelial cells, BFA promotes the integrity of the intestinal epithelial barrier, downregulates the expression of inflammatory factors, regulates immune cell differentiation, and alleviates intestinal inflammation by inhibiting the NF-κB pathway. Therefore, BFA-producing bacteria, as natural producers of BFA, have attracted much attention in the regulation of the intestinal microecology and disease prevention and treatment. In addition, BFA-producing bacteria can also be used in the fermentation production of specialty foods, utilizing their BFA-producing properties to improve the flavor of these foods.

[0003] Among known butyric acid-producing bacteria, *Clostridium butyricum* is one of the most extensively studied and widely used species. This bacterium can inhibit pro-inflammatory factors and plays a positive role in preventing and alleviating intestinal inflammation such as ulcerative colitis, colitis, and colorectal cancer. It has been approved by many countries as a probiotic to be added to food, health products, and animal feed, and its safety, stability, and probiotic effects have been fully verified. Most existing research on butyric acid-producing bacteria and their metabolic mechanisms uses *C. butyricum* as the type strain, and high-density fermentation processes and industrial application systems have been established based on this.

[0004] However, not all butyrate-producing Clostridium species possess equivalent probiotic effects, and the widespread application of butyrate-producing Clostridium should not be simply generalized to the entire Clostridium genus. A recent landmark study revealed diametrically opposed effects and mechanisms of two butyrate-producing Clostridium species in a necrotizing enterocolitis (NEC) model (Tao et al., 2023). This study confirmed that Clostridium tyrobutyricum can restore damaged intestinal barrier integrity and alleviate inflammatory responses by significantly increasing the abundance of Akkermansia muciniphila in the gut. Conversely, Clostridium butyricum not only failed to show a protective effect in the same model but also further exacerbated intestinal barrier damage and inflammatory responses by reducing the abundance of A. muciniphila. This finding confirms the fundamental difference in their mechanisms of action and provides crucial experimental evidence for the "strain specificity" principle in probiotic development. *C. tyrobutyricum* is a naturally occurring and highly efficient butyrate-producing *C.* species that synthesizes butyrate via the butyryl-CoA / acetyl-CoA transferase pathway, achieving high yields and productivity (Lee et al., 2016). This bacterium possesses a unique oxidative stress defense system, ensuring its survival in the intestinal environment (Food Frontiers, 2023). Furthermore, a European patent (EP 3806651 A1) relates to feed ingredients containing *C. tyrobutyricum*. Therefore, *C. tyrobutyricum* is considered by the scientific community as a promising candidate for next-generation probiotics.

[0005] Although Clostridium butyricum shows promising application prospects, existing research still faces key bottlenecks, such as a mismatch between the breadth and depth of resource research, a relative lack of systematic reviews on its probiotic properties and safety, and a lack of systematic process parameters for high-density fermentation, as well as a lack of key technologies that can be converted into large-scale production, which seriously restricts its industrialization process. Summary of the Invention

[0006] The purpose of this invention is to provide a Clostridium butyricum bacterium, its culture conditions, and its applications.

[0007] To achieve the above-mentioned objectives, the technical solution adopted in this invention is: a strain of Clostridium tyrobutyricum H2-2, deposited at the Guangdong Provincial Center for Microbial Culture Collection, with a deposit date of April 7, 2026, and a deposit number of GDMCC No: 68056.

[0008] Accordingly, bacterial preparations made using Clostridium butyricum H2-2, or bacterial preparations containing Clostridium butyricum H2-2.

[0009] Accordingly, the application of Clostridium butyricum H2-2 or the bacterial preparation in the preparation of butyric acid.

[0010] Accordingly, the application of Clostridium butyricum H2-2 or the bacterial preparation in the preparation of feed, feed additives, food, food additives, pharmaceuticals, pharmaceutical additives, health products, and health product additives.

[0011] Accordingly, a method for culturing *Clostridium butyricum* H2-2 involves adding tryptophan during the culturing process. Preferably, the *Clostridium butyricum* H2-2 is cultured using RCM medium, to which tryptophan is additionally added. Preferably, the components of the RCM medium include: glucose, corn steep liquor, soluble starch, sodium chloride, anhydrous sodium acetate, L-cysteine ​​hydrochloride, and tryptophan. Preferably, the culture conditions are: a culture pH of 4.5–10.0; and / or; a culture salinity of 0–4.5%.

[0012] The present invention has the following beneficial effects:

[0013] This invention isolates a novel Clostridium butyricum strain from nature. Whole-genome sequencing confirmed that this strain possesses avirulence genes and transferable antibiotic resistance genes, and its probiotic potential (including acid tolerance, bile salt tolerance, high and low temperature tolerance, high salt tolerance, and hemolytic activity) was systematically evaluated. Based on this, its cultivation process was optimized, and a high-density fermentation method was established, providing key process parameters and theoretical basis for subsequent large-scale production and industrial application.

[0014] Specifically:

[0015] The Clostridium butyricum H2-2 obtained by screening in this invention has excellent stress resistance and biosafety, specifically: (1) wide pH adaptability: it can grow normally at pH=4.5~10.0 and sodium chloride content of 4.5%; (2) good osmotic pressure tolerance: it maintains its proliferation ability under 0~4.5% NaCl conditions, which is conducive to industrial fermentation production; (3) stable temperature tolerance: it has excellent high temperature resistance, which is convenient for formulation processing and storage and transportation; (4) outstanding gastrointestinal fluid tolerance: after treatment with simulated artificial gastric fluid (pH=2.0) and simulated artificial intestinal fluid (pH=8.0, bile salt 3.0g / L) for 2h, the survival rate reached 92.17% and 95.28% respectively, which can ensure that the live bacteria can pass through the stomach and colonize the intestine smoothly; (5) reliable biosafety: the hemolysis test is negative; there is no acute toxicity reaction, no toxicity gene and no transferable antibiotic resistance gene, which is suitable for development as a probiotic preparation.

[0016] Therefore, the Clostridium butyricum H2-2 is a potential probiotic strain that can be added to pig, poultry, and aquatic feed as feed or feed additive for animal husbandry, strengthening the intestinal barrier and enhancing animal immunity; it also holds promise for use as an additive in food, medicine, and health products. Attached Figure Description

[0017] Figure 1 This is a colony morphology diagram of Clostridium butyricum H2-2;

[0018] Figure 2 This is a scanning electron microscope image of Clostridium butyricum strain H2-2.

[0019] Figure 3 Phylogenetic tree of Clostridium butyricum H2-2;

[0020] Figure 4 A schematic diagram illustrating the production of butyric acid and other metabolites by Clostridium butyricum H2-2.

[0021] Figure 5 Image of blood agar plate culture of Clostridium butyricum H2-2;

[0022] Figure 6 A schematic diagram showing the growth of Clostridium butyricum H2-2 using different carbon sources;

[0023] Figure 7 A schematic diagram showing the growth of Clostridium butyricum H2-2 using different concentrations of glucose;

[0024] Figure 8 A schematic diagram showing the growth of Clostridium butyricum H2-2 using different nitrogen sources;

[0025] Figure 9 A schematic diagram showing the growth of Clostridium butyricum H2-2 using corn steep liquor of different concentrations;

[0026] Figure 10 A schematic diagram showing the effect of different amino acids on the growth of Clostridium butyricum H2-2;

[0027] Figure 11 This is a schematic diagram showing the effect of different concentrations of tryptophan on the growth of Clostridium butyricum H2-2. Detailed Implementation

[0028] This invention provides a novel Clostridium tyrobutyricum H2-2 strain, deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on April 7, 2026, with accession number GDMCC No: 68056.

[0029] The 16S rDNA sequence of Clostridium butyricum H2-2 is shown in SEQ ID NO: 1.

[0030] This invention also provides a method for culturing *Clostridium butyricum* H2-2, using an optimized RCM medium. The optimized RCM medium consists of: 21 g / L glucose, 18 g / L corn steep liquor, 1 g / L soluble starch, 5 g / L sodium chloride, 3 g / L anhydrous sodium acetate, 0.5 g / L L-cysteine ​​hydrochloride, and 1 g / L tryptophan. The initial pH is 6.3, the inoculum size is 4%, and the culture is statically incubated.

[0031] 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. Unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art, and the data obtained are all average values ​​obtained after at least three repetitions, and each repetition yields valid data.

[0032] Example 1: Screening, Identification and Preservation of Strains

[0033] 1. Screening

[0034] Prepare RCM medium, purge with nitrogen for 2 minutes to remove dissolved oxygen from the water, maintain anaerobic sealing, and sterilize at 115℃ for 20 minutes. After the medium cools, add diluted pit mud to the RCM medium and incubate statically at 37℃ for 7 days. Take the enriched culture medium and incubate in an anaerobic chamber at a ratio of 10... -2 10 -3 10 -4 10 -5 10 -6 10 -7 Serial dilutions were performed, and the diluted solutions were spread onto prepared RCM medium plates and anaerobically incubated at 37°C. Once single colonies grew on the plates, they were streaked for purification. The resulting colonies were white, and butyrate-producing single colonies were selected as candidate strains. Physiological and biochemical characteristics and 16S rRNA sequencing were then performed for identification.

[0035] 2. Identification

[0036] (1) Identification of physiological and biochemical characteristics

[0037] The colony morphology of strain H2-2 obtained in this invention is as follows: Figure 1 As shown, the scanning electron microscope image of the strain is as follows: Figure 2As shown, the colonies of strain H2-2 are white, round, smooth, and moist, and Gram-positive. Under scanning electron microscopy, strain H2-2 appears as rod-shaped cells, 1–5 μm in length, and can utilize glucose and fructose as carbon sources for growth. The main metabolite from glucose fermentation is butyric acid, followed by acetic acid. Laboratory analysis of colony morphology and metabolites after 20 consecutive generations on agar plates revealed that strain H2-2 exhibits stable colony morphology and butyric acid production performance.

[0038] The API characteristics of strain H2-2 are shown in Table 1. In Table 1, the control is sterile culture medium, "-" indicates negative, that is, strain H2-2 has no activity of the enzyme to be measured; "+" indicates positive, that is, strain H2-2 has the activity of the enzyme to be measured; "W" indicates weak positive, that is, strain H2-2 has very weak activity of the enzyme to be measured.

[0039] Table 1. API characteristics of strain H2-2

[0040]

[0041] (2) Molecular identification

[0042] 16S rRNA gene sequencing was performed on strain H2-2, and the results are shown in SEQ ID NO: 1. Sequence alignment results showed high homology (>99%) with the standard strain Clostridium tyrobutyricum ATCC 25755. The phylogenetic tree is shown below. Figure 3 As shown in Table 2, to further confirm its taxonomic status, the whole genome of the target strain H2-2 was sequenced, and the mean nucleotide identity (ANI) between the target strain and six known Clostridium butyricum reference strains was analyzed using the JSpeciesWS online server (Richter et al., 2016).

[0043] Table 2 ANI Analysis Table

[0044]

[0045] The ANI analysis results showed that the ANI values ​​between the target strain and the reference strain ranged from 98.36% to 98.93%, which is higher than the internationally recognized prokaryotic species definition threshold of 95% to 96% (Richter & Rosselló-Móra, 2009; Ciufo et al., 2024).

[0046] Based on the combined results of 16S rRNA gene sequence analysis and whole-genome ANI analysis, the target strain H2-2 was identified as Clostridium tyrobutyricum.

[0047] Based on whole-genome information, this invention uses three databases—Pathogen-Host Interaction (PHI), Virulence Factor Database (VFDB), and Comprehensive Antibiotic Resistance Database (CARD)—to perform homology comparison analysis on the whole genome of the strain, and systematically predict pathogen genes, virulence factors, and drug resistance genes.

[0048] Pathogen gene prediction (PHI database): Comparative analysis using the PHI database showed that no known typical exotoxin genes (such as botulinum toxin, tetanus toxin, enterotoxin, etc.) were matched. The matched genes were mainly conserved metabolic, regulatory, and stress-related genes (such as gyrA, Mfd, ptsI, clpB, recA, etc.). Their deletion leading to "reduced virulence" is a common bacterial phenomenon and does not specifically indicate pathogenicity. No complete secretion system gene clusters were found. These results indicate that the strain does not possess the typical genetic characteristics of a pathogen.

[0049] Virulence factor prediction (VFDB database): Comparative analysis using the VFDB database showed that no known highly pathogenic virulence factors (such as hemolysins, cytolysins, and the genotoxic substance Colibactin) were matched, and the homology of related toxins was less than 35%. The matched virulence-related genes were mainly conserved bacterial functional systems, including iron uptake systems (HitABC, FbpABC, Shu, Enterobactin, etc.), magnesium ion transport systems (MgtBC), capsule / LPS synthases, and stress proteins (ClpC, ClpP), with homology generally less than 50%. No complete secretion system (TTSS, T4SS, T6SS, etc.) gene clusters were found. These systems are widely present in non-pathogenic bacteria and do not constitute evidence of pathogenicity.

[0050] Drug resistance gene prediction (CARD database): Comparative analysis using the CARD database showed that no transferable high-risk drug resistance genes were matched (such as vancomycin resistance gene cluster vanA / vanB, carbapenemase genes blaKPC / blaNDM, methicillin resistance gene mecA, etc.); there were fluoroquinolone resistance-related homologous mutations (gyrA / gyrB) consistent with Clostridium characteristics. These mutations are widespread in Clostridium and represent inherent resistance rather than acquired transferable risk; the remaining matched drug resistance genes were conserved efflux pump components (efrA / B, macB, TaeA, etc.) or metabolic enzyme mutations (ptsI, murA, fusA, etc.), with homology generally below 50%, and did not constitute clinical drug resistance risk.

[0051] Genome-wide predictive analysis based on the PHI, VFDB, and CARD databases revealed no typical highly pathogenic virulence factors, intact secretion system gene clusters, or transferable high-risk drug resistance genes in strain H2-2. The fluoroquinolone resistance-related mutations it carries are inherent to the Clostridium genus and do not pose a transferable resistance risk. Bioinformatics assessment indicates that this strain poses an extremely low risk of pathogenicity to humans and animals and exhibits good biosafety.

[0052] (3) Preservation

[0053] Strain H2-2 was deposited at the Guangdong Provincial Microbial Culture Collection Center (GDMCC) on April 7, 2026, with accession number GDMCC No: 68056.

[0054] Example 2: Basic performance demonstration of strain H2-2

[0055] Since strain H2-2 is an anaerobic bacterium, all cultures involving strain H2-2 were conducted in an anaerobic environment.

[0056] 1. Effect of salt concentration on the growth of strain H2-2

[0057] In RCM medium with a glucose concentration of 25 g / L, sodium chloride was added to achieve concentrations of 0%, 0.5%, 1.0%, 2.0%, 3.0%, 4.0%, 4.5%, and 5.0% (w / L, mass-volume ratio) to obtain RCM medium with different salt concentrations. In subsequent experiments, unless otherwise specified, RCM medium with a glucose concentration of 25 g / L was used. Higher glucose concentrations can increase biomass and viable cell count.

[0058] Each group of RCM medium was inoculated with 10% (v / v) Clostridium butyricum H2-2 seed culture (viable count 1.0 × 10⁻⁶). 6 CFU / mL, OD 600 =1.3), and after being incubated at 37℃ for 3 days, the OD was measured. 600 The bacterial growth was observed under different salt concentrations. The results are shown in Table 3.

[0059] Table 3. Comparison of bacterial cell growth after 3 days of culture at different salt concentrations.

[0060]

[0061] The results showed that the culture medium became turbid and produced bubbles at salt concentrations ranging from 0% to 4.5%, and the OD at a salt concentration of 4.5% was [missing value]. 600 The value reached 0.69, demonstrating that this Clostridium butyricum bacterium has a high salt tolerance.

[0062] 2. Effect of pH on the growth of strain H2-2

[0063] The *Clostridium butyricum* H2-2 seed culture, prepared using the culture medium from step 1, was inoculated at a rate of 10% (v / v) into culture media with pH values ​​ranging from 4.0 to 11.0 (pH adjusted with 1M hydrochloric acid or 1M sodium hydroxide). The media were then anaerobically cultured at 37°C for 3 days, and the OD values ​​were measured. 600 The results are shown in Table 4.

[0064] Table 4. Effects of pH on bacterial cell growth (Comparison Table)

[0065]

[0066] The results showed that strain H2-2 could grow normally at pH values ​​ranging from 4.5 to 10.0. In an acidic environment, the growth of strain H2-2 was only slightly delayed at pH 4.5, but the OD (Organization Rate) increased after 3 days. 600 It can still reach above 2.0. In an alkaline environment (pH 8.0–10.0), strain H2-2 grows rapidly, with an OD value exceeding 2.0 after 1 day. 600 It can reach 1.3 to 1.8, and after 3 days it can reach 1.9 to 2.3.

[0067] 3. Effect of temperature on the growth of strain H2-2

[0068] The seed culture of strain H2-2 was inoculated into the culture medium at a rate of 10%. After incubation at 37℃ for 3 days, 1 mL of the bacterial culture was taken and treated at 80℃ for 10 min, 85℃ for 10 min, 90℃ for 5 min, 95℃ for 1 min, and 100℃ for 1 min and 3 min, respectively. The colony counts after each high-temperature treatment were determined by the dilution plate method. The results are shown in Table 5.

[0069] Table 5. Effects of Temperature on the Growth of Strains (Comparison Table)

[0070]

[0071] The results showed that strain H2-2 could withstand temperatures above 80℃. As the temperature increased, the shorter the exposure time, the higher the survival rate; even after exposure to 100℃ for 1 minute, the viable count still reached 10-1. 6 CFU / mL or higher.

[0072] 4. Antibiotic resistance of strain H2-2

[0073] The gradient diffusion method (E-test) was used. Antibiotic test strips with a continuous gradient were purchased. The H2-2 bacterial suspension was evenly spread on RCM agar plates, and the test strip was placed in the center of the plate. The plates were anaerobically incubated at 37°C for 2 days. The MIC (microinhibition limit) was determined by the measurement at the junction of the inhibition zone and the test strip. Chloramphenicol, tetracycline, erythromycin, ampicillin, gentamicin, streptomycin, clindamycin, vancomycin, linezolid, trimethoprim, rifampin, and ciprofloxacin were selected to test the antibiotic resistance of strain H2-2. According to EFSA (Enhanced Antibiotic Resistant Study), a MIC value higher than the breakpoint value indicates drug resistance. The results are shown in Table 6.

[0074] Table 6. Comparison of H2-2 antibiotic resistance status

[0075]

[0076] The results showed that strain H2-2 was resistant to streptomycin, trimethoprim, and gentamicin, but sensitive to vancomycin, chloramphenicol, clindamycin, erythromycin, and tetracycline. Furthermore, this strain was sensitive to other antibiotics (ampicillin, levozolam, and rifampin) for which EPSA breakpoint values ​​were not specified.

[0077] 5. Butyric acid production capacity

[0078] Clostridium butyricum is recognized as capable of producing short-chain fatty acids, typically acetate and butyrate. For example... Figure 4 As shown, the batch fermentation results of strain H2-2 showed that when the initial glucose concentration was 20 g / L (RCM medium), no acetic acid was produced after 24 h, while the butyric acid yield was 4.11 ± 0.20 g / L. After 96 h, the acetic acid yield was only 1.58 ± 0.09 g / L, while the butyric acid yield reached 8.46 ± 0.36 g / L, indicating that the main metabolite of strain H2-2 is butyric acid.

[0079] Example 3: Demonstration of the potential of strain H2-2 as a probiotic

[0080] 1. Survival status of strain H2-2 in simulated gastric and intestinal fluids

[0081] Fermentation broth was obtained after culturing strain H2-2 in RCM medium with a glucose concentration of 25 g / L at 37°C for 3 days, and the OD value was... 600 =2.0, viable count is 5.4×10 7 CFU / mL.

[0082] Take 10 mL of fermentation broth, centrifuge at 3000 rpm for 10 min, remove the supernatant, add physiological saline to resuspend, centrifuge and wash again, remove the supernatant, and repeat once more (i.e. wash twice with physiological saline).

[0083] Then, 1 mL of sterile water was added, and the cells were resuspended to form a bacterial suspension. 9 mL of simulated gastric fluid (pH=2.0, containing dilute acid and pepsin, purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number R28616, pH=2.0±0.5, sterile) was added to the bacterial suspension and mixed thoroughly. The initial sample (N0, the bacterial suspension sampled immediately after adding the simulated gastric fluid) was taken at 0 h, and the viable bacteria count was determined using the dilution-spreading method. The tubes containing the simulated gastric fluid were then placed in a 37℃ incubator for 2 h, and a second sample (N1, the 2 h sample) was taken. The viable bacteria count after 2 h in the simulated gastric fluid was determined using the dilution-spreading method, and the survival rate was calculated. Simultaneously, the bacterial suspension treated with simulated gastric fluid for 2 hours was centrifuged again, the supernatant was removed, and 1 mL of sterile water was added for resuspending. After mixing thoroughly, 9 mL of simulated intestinal fluid (pH=8.0, containing 0.3% porcine bile salts, purchased from Shanghai Yuanye Biotechnology Co., Ltd., catalog number R29306, containing 0.3% porcine bile salts, pH=8.0, sterile) was added. The initial sample (N0, the bacterial suspension immediately after adding the simulated intestinal fluid and mixing after treatment with simulated gastric fluid) was taken, and the viable count was counted. Then, the test tubes containing the simulated intestinal fluid were placed in a 37℃ incubator for 2 hours, and a second sample (N1) was taken. The viable count was calculated using the dilution-spreading method, and the survival rate was calculated. The above experiment was conducted in triplicate, and the results were averaged.

[0084] Survival rate (%) = N1 / N0 × 100. Where: N1 is the number of viable bacteria (CFU / mL) after treatment with simulated gastric or intestinal fluid; N0 is the number of viable bacteria (CFU / mL) in the control.

[0085] The results showed that after 2 hours in gastric juice at pH 2.0, the average survival rate of strain H2-2 was 92.17%. After 2 hours in simulated intestinal juice, the average survival rate of strain H2-2 was 95.28% (Table 7). This indicates that strain H2-2 has a very high survival rate in both gastric acid and intestinal juice, and has strong potential for utilization as a probiotic for the gut.

[0086] Table 7 Results of H2-2 Artificial Gastrointestinal Fluid Tolerance Test

[0087]

[0088] 2. Hemolytic activity of strain H2-2

[0089] Blood agar plates are nutrient culture media containing animal blood. When hemolysin-producing bacteria grow on blood agar plates, the surrounding red blood cells are destroyed, forming a characteristic hemolytic ring.

[0090] This embodiment uses Columbia agar plates for verification, employing the dilution plating method, with the bacterial suspension of strain H2-2 diluted to 10⁻⁶. 5Blood agar plates were spread and inoculated, then incubated at 37°C for 2 days. The presence of hemolytic zones was observed. Images of the obtained blood agar plates are shown below. Figure 5 As shown.

[0091] Blood agar observations showed that strain H2-2 did not produce a hemolytic zone on the agar plate, indicating that strain H2-2 lacks hemolytic activity, does not produce hemolysin, and does not damage erythrocytes. This demonstrates that it does not possess hemolytic virulence factors, and its biosafety characteristics are clearly defined, meeting the basic requirements for probiotic safety evaluation. Since strain H2-2 lacks hemolytic activity, its use as a probiotic can effectively avoid damage to host erythrocytes and intestinal mucosa, reducing potential virulence and invasion risks. This facilitates the strain's gentle colonization in the intestine and its stable exertion of probiotic effects, providing important evidence for its safe application in food, feed, and microecological preparations.

[0092] 3. Animal pathogenicity test of strain H2-2

[0093] This experiment was commissioned to the Guangdong Provincial Center for Microbiology Analysis and Testing. The testing methods and basis were Appendix A of the "Technical Guidelines for Safety Testing and Evaluation of Microbial Strains Used in Health Food Raw Materials (2020 Edition)".

[0094] (1) Experimental preparation

[0095] Preparation of bacterial suspension: After activating strain H2-2, it was inoculated into reinforced Clostridium perfringens (RCM) medium (5 g / L glucose) plates and anaerobically cultured at 37°C for 2 days. Colonies were scraped from the plates and suspended in sterile physiological saline. The mixture was thoroughly mixed, and the bacterial concentration was adjusted to 5.0 × 10⁻⁶ using sterile physiological saline. 7 CFU / mL was used to obtain a bacterial suspension for intraperitoneal injection experiments; the bacterial concentration was adjusted to 2.5 × 10⁻⁶. 8 CFU / mL, centrifuged, and the supernatant was concentrated 5 times by freezing to adjust the bacterial concentration to 1.25 × 10⁻⁶. 9 CFU / mL was used to obtain a bacterial suspension, which was then used as the bacterial suspension for oral gavage experiments.

[0096] Select healthy SPF-grade Kunming mice, 20 males and 20 females, with a body weight range of 18-22 g. They are from the Guangdong Provincial Medical Experimental Animal Center. The animal production license number is: SCXK(Yue)2022-0002, and the animal certificate number is SCXK(Yue)2022-0002202557981. The animal use license number is: SYXK(Yue)2021-0156. Mouse rearing environment: 20-26°C, relative humidity 40%-70%, with free access to water. The feed is provided by the Guangdong Provincial Medical Experimental Animal Center. The feed production license number is: Yue Si Zheng(2024)05073, and the feed certificate number is: 2512160028. The experimental animals and animal laboratories comply with relevant national regulations.

[0097] (2) Intraperitoneal injection experiment

[0098] According to Appendix A5.1 of the Technical Guidelines for the Safety Inspection and Evaluation of Strains Used as Health Food Ingredients (2020 Edition), male and female mice are randomly divided into: male mouse sterile saline control group, male mouse bacterial suspension group; female mouse sterile saline control group, female mouse bacterial suspension group. Weigh the initial body weight of each group of mice, and then intraperitoneally inject 0.2 mL of bacterial suspension into each mouse in the bacterial suspension group, and intraperitoneally inject an equal amount of sterile saline into each mouse in the control group at the same time.

[0099] After the intraperitoneal injection treatment of the mice, observe the poisoning manifestations, number of deaths and death time of the mice, and continuously observe for 21 days. Weigh all surviving mice on the 0th day (D0, before dosing) and the 21st day (D21) of dosing, and record the death time and body weight of the mice during the test period. The results are shown in Table 8.

[0100] Table 8 Comparison of mouse body weight and death

[0101]

[0102] The results show that: Strain H2-2 has no adverse effects on the health of mice. During the test period, the mice did not show poisoning symptoms and adverse symptoms, did not have an obvious impact on the drinking water and food intake of the mice, and did not affect the body weight of the mice.

[0103] (3) Oral gavage experiment

[0104] According to Appendix A5.2 of the Technical Guidelines for the Safety Inspection and Evaluation of Strains Used as Health Food Ingredients (2020 Edition), male and female mice are randomly divided into: male mouse culture medium control group, male mouse bacterial suspension group, male mouse 5-fold concentrated culture medium control group, male mouse 5-fold concentrated bacterial suspension group; female mouse culture medium control group, male and female mouse bacterial suspension group, female mouse 5-fold concentrated culture medium control group, female mouse 5-fold concentrated bacterial suspension group.

[0105] Before gavage, mice in each group were fasted for 16 hours but had free access to water. Each group of mice was orally administered the corresponding substance at a standard dose of 20 mL / kg body weight (BW). Specifically, the culture medium control group was administered RCM culture medium, while the bacterial suspension group was administered 2.5 × 10⁻⁶ BW culture medium. 8 CFU / mL bacterial suspension, 5-fold concentrated bacterial solution group administered by gavage 1.25×10 9 CFU / mL bacterial suspension. Patients were kept NPO (nothing by mouth) for 3 hours after gavage, followed by a normal diet. This was repeated once daily for 3 consecutive days.

[0106] After gavage administration, the toxic symptoms, number of deaths, and time of death of mice were observed for 21 consecutive days. On day 0 (D0, before gavage) and day 21 (D21) after administration, the body weight of all surviving mice was measured, and the time of death and body weight of mice during the experiment were recorded. The results are shown in Table 9.

[0107] Table 9 Comparison of mouse weight and mortality

[0108]

[0109] The results showed that no mice showed signs of poisoning during the experiment, no animals died, and their body weight was not significantly different from that of the control group.

[0110] The results show that strain H2-2 has no obvious toxic side effects or pathogenicity, does not cause significant adverse effects on animal health, and has high biosafety.

[0111] Example 4: Condition optimization for high-density fermentation culture of strain H2-2

[0112] 1. Based on RCM medium, the composition of the medium was optimized.

[0113] In this embodiment of the invention, unless otherwise specified, the RCM culture medium consists of: 10g peptone, 10g beef meal, 3g yeast extract, 1g starch, 0.5g L-cysteine ​​hydrochloride, 5g NaCl, 3g sodium acetate, and 5g glucose. In this embodiment, the following optimizations are made to this RCM culture medium:

[0114] (1) Optimization of carbon source

[0115] The carbon sources in the RCM medium were replaced with 10 g / L of glucose, xylose, fructose, sucrose, mannitol, maltose, and lactose, respectively. The seed culture of strain H2-2 (with a viable count of 1.0 × 10⁻⁶) was then used. 6 The CFU / mL concentration was 10% by volume. The culture was inoculated into each of the adjusted culture media and anaerobic cultured at 37°C for 3 days. The viable count of strain H2-2 in each culture medium was then determined.

[0116] like Figure 6 As shown, the results indicate that strain H2-2 cannot utilize xylose, sucrose, mannose, maltose, and lactose (strain H2-2 did not grow in these corresponding media), and can only grow using glucose and fructose. The highest viable count, reaching 9.0 ± 0.40 × 10⁻⁶, was achieved when glucose was used as the carbon source. 6 CFU / mL; When fructose was used as the carbon source, the viable count after 3 days of culture was 3.7 ± 0.26 × 10⁻⁶. 6 CFU / mL.

[0117] Therefore, glucose was continued to be selected as the carbon source, and the glucose concentration was adjusted to 5 g / L, 10 g / L, 15 g / L, 20 g / L, 25 g / L and 30 g / L, respectively. The cultures were anaerobic at 37℃ for 3 days, and the number of viable bacteria of strain H2-2 in each culture medium was measured.

[0118] like Figure 7 As shown in the results, within a certain range, the number of viable bacteria gradually increased with increasing glucose concentration, reaching its highest level of 1.32 × 10⁻⁶ at a glucose concentration of 25 g / L. 8 The CFU / mL concentration showed a slight decrease in viable bacteria count when the glucose concentration was 30 g / L. Therefore, a glucose concentration of 25 g / L is preferred as the carbon source.

[0119] (2) Optimization of nitrogen source

[0120] The compound nitrogen source in the RCM medium was replaced with 23 g / L of beef meal, soybean peptone, peptone, yeast powder, and corn steep liquor (the corn steep liquor was purchased from Shandong and obtained by grinding ordinary corn kernels). The seed culture of strain H2-2 (with a viable count of 1.0 × 10⁻⁶) was then used. 6 CFU / mL) was inoculated into each adjusted culture medium at a volume ratio of 10%, and anaerobic cultured at 37℃ for 3 days. The viable number of strain H2-2 in each group of culture medium was then determined.

[0121] like Figure 8 As shown, the results indicate that when the three compound nitrogen sources were replaced with seven different single nitrogen sources, the number of viable bacteria was highest when corn steep liquor was used as the nitrogen source, which was significantly higher than that of the other six nitrogen sources (P < 0.05).

[0122] Therefore, corn steep liquor was continued as the nitrogen source, and the concentrations were adjusted to 10 g / L, 15 g / L, 20 g / L, 25 g / L, and 30 g / L. The cultures were then anaerobic at 37°C for 3 days, and the viable count of strain H2-2 in each culture medium was determined. Figure 9 As shown, the results indicated that when the corn steep liquor concentration was 10–15 g / L, the viable bacterial count increased significantly, with the highest viable bacterial count (1.76 × 10⁻⁶) observed at a concentration of 15 g / L. 7The optimal concentration of corn steep liquor is 15 g / L, but the viable count begins to decrease when it exceeds 15 g / L.

[0123] (3) Optimization of inorganic salts

[0124] Ca 2+ Mg 2+ K + Fe 2+ It is the most common metal ion that affects bacterial growth. It can not only participate in cell formation, but also act as a coenzyme to activate biochemical reactions, maintain the life activities of microorganisms, and promote the formation of spores.

[0125] On RCM medium, 1 g / L of CaCl2, FeSO4, KH2PO4, and MgSO4 were exogenously added, respectively. The seed culture of strain H2-2 (with a viable count of 1.0 × 10⁻⁶) was then used. 6 CFU / mL) was inoculated into each adjusted culture medium at a volume ratio of 10%, and anaerobic cultured at 37℃ for 3 days. The viable number of strain H2-2 in each group of culture medium was then determined.

[0126] The results showed that there was no significant difference in the growth and viable cell count of strain H2-2 between the addition and non-addition of metal ions (P>0.05).

[0127] (4) Optimization of growth factors

[0128] On RCM medium, 1 g / L of glutamic acid, valine, lysine, cysteine, glycine, leucine, tryptophan, phenylalanine, and methionine were exogenously added respectively. The seed culture of strain H2-2 (with a viable count of 1.0 × 10⁻⁶) was then used. 6 CFU / mL) was inoculated into each adjusted culture medium at a volume ratio of 10%, and anaerobic cultured at 37℃ for 3 days. The viable number of strain H2-2 in each group of culture medium was then determined.

[0129] like Figure 10 As shown, the results revealed that the tryptophan group had significantly higher viable counts than other groups (P < 0.05), with a viable count of 1.03 × 10⁻⁶ under the condition of adding this substance. 7 The CFU / mL concentration was significantly higher than that of the group without added amino acids (1.84 × 10⁻⁶). 6 CFU / mL.

[0130] Further experiments were conducted using tryptophan, adjusting the concentration to 0 g / L, 0.5 g / L, 1 g / L, 1.5 g / L, and 2 g / L. Seed culture of strain H2-2 (with a viable count of 1.0 × 10⁻⁶) was then added. 6CFU / mL) was inoculated into each adjusted culture medium at a volume ratio of 10%, and anaerobic cultured at 37℃ for 3 days. The viable count of strain H2-2 in each group of culture medium was then determined. Figure 11 As shown, the results indicate that there was no significant difference in tryptophan concentration between 0.5 and 1 g / L (P > 0.05).

[0131] 2. Optimize the cultivation conditions

[0132] In this embodiment, the inoculation amount of the H2-2 seed liquid was 10%, and unless otherwise specified, the culture conditions were: cultured at 37°C for 3 days.

[0133] (1) pH optimization

[0134] The pH of the optimized RCM medium was adjusted to 5.0–7.0 using NaOH or HCl, and the effects of different pH conditions on the number of viable bacteria were compared. The results are shown in Table 10.

[0135] Table 10 Viable bacterial counts after 3 days of culture at different pH levels

[0136]

[0137] The results showed that the optimal growth pH was 6.0.

[0138] (2) Optimization of inoculation amount

[0139] Inoculum size, by regulating the initial bacterial cell mass, affects bacterial growth, growth cycle, and accumulation of metabolites, and is an important parameter in the culture process. The inoculum size of strain H2-2 seed culture was controlled at 1%, 4%, 7%, 10%, and 15% (v / v) of RCM medium, respectively. After 3 days of culture, the results are shown in Table 11.

[0140] Table 11 Effects of different inoculum sizes on bacterial growth

[0141]

[0142] The results showed that when the inoculum size was only 1%, the bacteria were not limited by nutrients on the third day of culture, and remained in the exponential growth phase. When the inoculum size was 4%–10%, there was no significant difference (P > 0.05). However, when the inoculum size increased to 15%, a significant difference in viable bacterial count occurred, possibly due to nutrient depletion in the culture medium and bacterial death caused by nutrient limitation. Considering factors such as cost, 4% was selected as the optimal inoculum size.

[0143] (3) Optimization of rotational speed

[0144] The rotation speed was adjusted to 0 rpm, 150 rpm, and 250 rpm respectively, and the effect of different rotation speeds on the number of viable bacteria was compared. The results are shown in Table 12.

[0145] Table 12 Effect of different rotation speeds on bacterial cell culture

[0146]

[0147] In microbial culture, increasing the appropriate rotation speed can make the bacteria more evenly contacted with the surrounding nutrients. However, for strain H2-2, there was no significant difference in the number of viable bacteria at rotation speeds of 0 rpm and 150 rpm, and the number of viable bacteria actually decreased significantly after further increasing the rotation speed. Therefore, static culture is preferred.

[0148] Example 5: Response surface optimization of culture medium conditions

[0149] Based on single-factor experiments, five factors significantly affecting viable cell count were screened from multiple culture factors: glucose, corn steep liquor, pH, tryptophan, and inoculum size. Further Plackett-Burman and steepest climb experiments were used to determine the principal factors as pH, corn steep liquor, and glucose. Using viable cell count as the response value, glucose (A), corn steep liquor (B), and pH (C) were selected as the three principal factors. A Box-Behnken design was used to establish a multiple quadratic regression model to fit the functional relationship between each factor and the effect value, and the process parameters were optimized accordingly. The experiment included 17 operating groups, of which groups 1-12 were factorial experiments, and groups 13-17 were central experiments (repeated 5 times). Specific experimental design and results are shown in Table 13.

[0150] Table 13 Experimental Design and Box-Behnken Results

[0151]

[0152] The results in Table 13 were analyzed using Design-Expert software through multiple regression fitting, yielding the following multiple quadratic regression equations for viable cell count (Y) on glucose (A), corn steep liquor (B), and pH (C): Viable cell count (Y) = +4.54 + 0.1363A + 0.0575B + 0.0337C + 0.0800AB + 0.0625AC - 0.0250BC - 0.2943A 2 -0.3018B 2 -0.2092C 2The optimal culture conditions for *Clostridium butyricum* H2-2 were determined using Design-Expert 13.0 software to solve the multiple quadratic regression equation: glucose 21 g / L, corn steep liquor 18 g / L, soluble starch 1 g / L, sodium chloride 5 g / L, anhydrous sodium acetate 3 g / L, L-cysteine ​​hydrochloride 0.5 g / L, and tryptophan 1 g / L; initial pH = 6.3, and inoculum size 4%. Three validation cultures were conducted under these conditions. The results showed that after 3 days of culture, the viable count of *Clostridium butyricum* H2-2 reached 4.6 ± 0.36 × 10⁻⁶. 7 CFU / mL is the value obtained using traditional RCM medium before optimization (1.84 ± 0.09 × 10⁻⁶). 6 25 times that of CFU / mL.

[0153] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Any modifications, alterations, substitutions, or variations made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.

Claims

1. A strain of Clostridium tyrobutyricum H2-2, deposited at Guangdong Provincial Microbial Culture Collection Center on April 7, 2026, with accession number GDMCC No: 68056.

2. A bacterial preparation made using Clostridium butyricum H2-2 as described in claim 1, or a bacterial preparation containing Clostridium butyricum H2-2 as described in claim 1.

3. The use of Clostridium butyricum H2-2 as described in claim 1 or the bacterial preparation as described in claim 2 in the preparation of butyric acid.

4. The application of Clostridium butyricum H2-2 as described in claim 1 or the bacterial preparation as described in claim 2 in the preparation of feed, feed additives, food, food additives, pharmaceuticals, pharmaceutical additives, health products, and health product additives.

5. A method for culturing Clostridium butyricum H2-2 as described in claim 1, characterized in that: Tryptophan was added during the cultivation of Clostridium butyricum H2-2.

6. The method according to claim 5, characterized in that: The Clostridium butyricum H2-2 was cultured in RCM medium supplemented with tryptophan.

7. The method according to claim 6, characterized in that: The components of the RCM medium include: glucose, corn steep liquor, soluble starch, sodium chloride, anhydrous sodium acetate, L-cysteine ​​hydrochloride, and tryptophan.

8. The method according to any one of claims 5 to 7, characterized in that: The culture conditions are as follows: culture pH 4.5–10.0; and / or culture salinity 0–4.5%.

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