Enterococcus faecalis RS711 and application thereof

Abstract

The application discloses an enteric coagulase Weitzmann's bacteria RS711 and application thereof. The application provides the enteric coagulase Weitzmann's bacteria RS711 which can efficiently utilize beet sucrose, and the bacteria strain has certain temperature resistance and acid resistance, has better cellulase, xylanase and amylase and other polysaccharide hydrolytic enzyme activities, and can produce L-lactic acid through simultaneous saccharification and fermentation. In addition, the enteric coagulase Weitzmann's bacteria RS711 can also metabolically produce a certain content of bacteriocin, can effectively inhibit certain competitive bacteria strains such as pristinamycetin and bacillus subtilis, and is helpful to the prevention and treatment of fermentation contamination. The enteric coagulase Weitzmann's bacteria RS711 disclosed by the application has important significance for reducing the production cost of L-lactic acid fermentation and improving the L-lactic acid yield.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and relates to a strain of Bacillus coagulans RS711 derived from the gut and its applications; specifically, it relates to a strain of Bacillus coagulans RS711 that can produce L-lactic acid by fermenting beet sugar and its applications. Background Technology

[0002] Lactic acid, also known as α-hydroxypropionic acid, has the molecular formula CH3CHOHCOOH and a relative molecular mass of 900.8. It is a simple and common organic acid widely found in humans, animals, plants, and microorganisms. Due to the presence of an asymmetric carbon atom in the lactic acid molecule, it exhibits optical activity, with forms including L-lactic acid (dextral), D-lactic acid (levorotatory), and DL-lactic acid (racemic). As an important daily chemical product, it is one of the three major organic acids recognized worldwide, widely used in various industrial fields such as pharmaceuticals, food, chemicals, textiles, cosmetics, and environmental protection. In particular, polylactic acid (PLA), prepared from high-optical-purity L-lactic acid, is a novel non-petroleum material with biodegradability and biocompatibility, and is considered a promising recyclable polymer.

[0003] Currently, the main method for producing L-lactic acid is fermentation, with microorganisms from the genera *Lactobacillus* and *Weizmannia* being the most commonly used. *Bacillus coagulans* (formerly known as *Bacillus coagulans*) is widely recognized as one of the best strains for L-lactic acid bio-fermentation due to its ability to form spores, its strong resistance to adverse conditions, its well-developed amylase system, its rapid growth rate, and the high optical purity of its products. Lactic acid is primarily produced by microorganisms through the metabolic fermentation of glucose, starch, or sucrose as carbon sources. Reducing the production cost of lactic acid has become a recent research hotspot. Since the carbon source accounts for a large proportion of the production cost of L-lactic acid, using refined sugars to produce L-lactic acid, while reducing post-processing costs to some extent, results in higher overall costs due to the generally higher price of refined sugars. Furthermore, using refined sugars derived from traditional grain crops as substrates for lactic acid bio-refining creates a situation of competition with food production, making this method uneconomical. Sugar beets, as a renewable energy crop, are an inexhaustible renewable resource. Using sugar beet production (primarily sucrose) as a substrate for lactic acid fermentation can solve the problems of limited raw material sources and high raw material costs. Simultaneously, it can fully utilize sugar beet molasses, a byproduct of sugar beet production, thereby increasing the yield of L-lactic acid fermentation and the utilization rate of locally sourced raw materials. Based on these advantages, utilizing sugar beet fermentation to produce L-lactic acid is a promising direction for the industrial production of lactic acid.

[0004] While *Weizmannii coagulans* is a mainstream L-lactic acid fermenting strain with high yields, it still suffers from drawbacks such as long fermentation cycles, susceptibility to contamination by other spore-forming bacteria, low production efficiency, and low optical purity. Therefore, selecting strains that can efficiently utilize sugar beet products, tolerate high concentrations of substrate and end-product osmotic pressure, maintain cell growth and enzyme activity, and achieve high productivity has become crucial for L-lactic acid strain breeding research. Summary of the Invention

[0005] To address the aforementioned problems, the purpose of this invention is to provide a strain of Weizmannia coagulans that can efficiently utilize beet sugar fermentation to produce L-lactic acid and its applications in producing L-lactic acid.

[0006] To achieve the above objectives, the technical solution adopted by the present invention is as follows: a strain of Weizmannia coagulans RS711, characterized in that the Weizmannia coagulans RS711 was deposited at the Guangdong Provincial Center for Microbial Culture Collection on August 10, 2022, with accession number GDMCC No:62693, and the deposit address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

[0007] The *Weizmannii coagulans* RS711 provided by this invention was obtained by ARTP mutagenesis of protoplasts from the starting strain W103 isolated from the feces of healthy lactating sows. It grows rapidly, exhibits stable genetic traits, and possesses certain abilities to synthesize amylase, xylanase, and cellulase. Furthermore, it can efficiently utilize beet sugar as a carbon source for fermentation to produce L-lactic acid.

[0008] Furthermore, the present invention also claims protection for the use of the aforementioned enterocoagulant Weizmannii RS711 in fermentation.

[0009] Since the enterogenic coagulant strain RS711 provided by this invention has a certain ability to synthesize amylase, xylanase and cellulase, its characteristics can be utilized in various corresponding fermentation production processes.

[0010] In a preferred embodiment of the present invention, the enteric-derived Weizmann's coagulant is used for fermentation to produce L-lactic acid.

[0011] As a preferred embodiment of the present invention, the process of producing L-lactic acid by fermentation of the enterogenic Weizmann's coagulant includes the following steps: inoculating the enterogenic Weizmann's coagulant into a fermentation medium with beet sugar as the main carbon source, and producing L-lactic acid by fermentation.

[0012] Using beet sugar as a specific carbon source, the selection of superior strains that are heat-resistant and produce antibacterial substances can effectively reduce the risk of contamination during fermentation. Furthermore, the high-temperature fermentation conditions help reduce energy consumption and save costs in producing high-yield lactic acid. Under the conditions of this invention, the *Weizmannii* RS711 strain exhibits excellent L-lactic acid fermentation production capacity, reaching 195.6 g / L, representing an increase of approximately 21.8% compared to the starting strain.

[0013] In a preferred embodiment of the present invention, the fermentation medium with beet sucrose as the main carbon source comprises the following components by weight and volume percentage: yeast extract 0.25%, beet molasses 5%, dipotassium hydrogen phosphate 0.05%, potassium dihydrogen phosphate 0.025%, ammonium dihydrogen phosphate 0.18%, ammonium sulfate 0.5%, zinc sulfate 0.01%, folic acid 0.02%, biotin 0.02%, and calcium carbonate 10.0%, and the following components by volume percentage: beet extract sugar 75%; deionized water to make up the balance.

[0014] As a preferred embodiment of the present invention, the process of fermentation to produce L-lactic acid is as follows: static culture at 50-55℃ for 40-48 hours.

[0015] The fermentation conditions are simple and require minimal equipment. Since no stirring is needed, production costs are further reduced.

[0016] Furthermore, the fermentation process also includes strain activation, shake-flask seed culture, and seed scale-up culture.

[0017] As a preferred embodiment of the present invention, the process of activating the strain is as follows: the enterogenic Weizmann's coagulant is inoculated onto a modified MRS agar slant and cultured at 50-55°C for 48 hours; then the strain on the slant is washed off with sterile physiological saline to prepare a bacterial suspension, which is then transferred to a culture medium containing modified MRS medium and cultured at 50-55°C for 24 hours.

[0018] The process of seed culture in shake flasks is as follows: the strain obtained from activation culture is added to sterile water for elution to prepare a bacterial suspension, which is then transferred to a shake flask containing modified MRS medium and cultured at 100-150 r / min and 50-55℃ for 12-18 h with shaking, and then cultured statically at 50-55℃ for 12-18 h.

[0019] The seed culture expansion process is as follows: 20% of the inoculum from the seed shake flask is transferred to a seed tank containing seed culture medium. Under a tank pressure of 0.06 MPa, the culture is carried out at 50-55℃ with stirring at 100-150 rpm for 6-10 hours. Then, stirring is stopped, and the tank is kept under positive pressure for further static incubation at 50-55℃ for 12-18 hours. The transfer standard is a colony count > 10. 9 CFU / mL;

[0020] The modified MRS medium contains the following components by weight / volume percentage: 1% peptone, 1% beef extract, 0.5% yeast extract, 0.2% diammonium hydrogen citrate, 0.5% sodium acetate, 0.2% dipotassium hydrogen phosphate, 0.06% magnesium sulfate, 0.025% manganese sulfate, and 0.6% calcium carbonate, and the following components by volume percentage: 75% beet extract and 0.1% Tween-80; adjust the pH to approximately 6.5; and replenish the balance with deionized water.

[0021] The modified MRS agar medium is based on the modified MRS medium with the following components added in weight-volume percentages: 2% agar powder; deionized water to make up the balance.

[0022] The seed culture medium contains the following components by weight and volume percentage: 1.5% yeast extract, 0.2% ammonium dihydrogen phosphate, 0.5% ammonium sulfate, 1.0% light calcium carbonate, 0.02% magnesium sulfate, 0.01% zinc sulfate, and the following component by volume percentage: 75% beet extract sugar; the pH is adjusted to 6.0-6.5, and deionized water is used to make up the balance.

[0023] Strain activation, shake-flask seed culture, and seed expansion culture can increase the enzyme activity and bacterial count of the gut-derived Weizmann's coagulant RS711, providing favorable support for subsequent inoculation into a fermenter for large-scale fermentation.

[0024] As a preferred embodiment of the present invention, the fermentation conditions are as follows: 20-50% of the seed culture solution is transferred to a fermenter containing fermentation medium; the aeration rate of the fermenter is controlled at 150-200 mL / min; the initial total sugar concentration is controlled at 100-160 g / L; the pH value is 5.5-6.0; and the culture is carried out statically at 50-55℃; the total sugar content in the fermenter is maintained at 16-20 g / L; the fermenter is kept under positive pressure; and the fermentation is carried out for 40-48 hours.

[0025] During the initial stage of fermentation, the aeration rate of the fermenter is controlled at 150-200 mL / min. Maintaining a certain amount of dissolved oxygen without stirring is mainly to promote the early reproduction of the microorganisms. After fermentation, the residual sugar content in the fermentation broth is less than 5 g / L.

[0026] More preferably, the pH is adjusted during fermentation by controlling the addition of 25% lime milk.

[0027] This invention provides a *Weizmannii coagulans* strain RS711 capable of efficiently producing L-lactic acid through fermentation of beet sugar. This strain exhibits certain temperature and acid tolerance, and possesses good activity of polysaccharide hydrolytic enzymes such as cellulase, xylanase, and amylase, enabling simultaneous saccharification and fermentation to produce L-lactic acid. Furthermore, *Weizmannii coagulans* RS711 can also metabolize and produce a certain amount of bacteriocins, effectively inhibiting competing strains such as *Priestia megaterium* and *Bacillus subtilis*, thus helping to prevent contamination during fermentation. The *Weizmannii coagulans* RS711 disclosed in this invention is of great significance for reducing the production cost of L-lactic acid fermentation and increasing the yield of L-lactic acid. Attached Figure Description

[0028] Figure 1 The results show the enzyme production characteristics of the enterogenic Weizmannii RS711 strain described in this invention.

[0029] Figure 2 This is a phylogenetic tree of the *Weizmannii coagulans* strain RS711 described in this invention, based on its 16S rDNA sequence. Detailed Implementation

[0030] To better illustrate the purpose, technical solution, and advantages of the present invention, the present invention will be further described below in conjunction with the accompanying drawings and specific embodiments.

[0031] Unless otherwise specified, the experimental methods used in the following examples are performed under conventional conditions. Unless otherwise specified, all materials and reagents used in the following examples are commercially available. All quantitative experiments in the following examples were performed in triplicate, and the results were averaged.

[0032] Example 1: Screening of L-lactic acid producing starting strain W103

[0033] 1. Pretreatment and bacterial isolation of fecal samples from healthy lactating sows

[0034] Pretreatment: In a sterile operating table, 10g of feces from healthy lactating sows at different time points was taken and added to an Erlenmeyer flask containing 50mL of sterile physiological saline (pH 2.5, adjusted by adding 3mol / L hydrochloric acid). The flask was thoroughly vortexed, filtered through gauze, and the filtrate was transferred to a new sterile Erlenmeyer flask. The pH was then adjusted to 5.5-6.0 using sterile 20% sodium hydroxide. To specifically screen for strains that can tolerate certain temperatures, the Erlenmeyer flask containing the contents was further incubated in an 80℃ water bath with shaking at 100rpm for 10min.

[0035] After pretreatment, the water bath solution was serially diluted 10-fold with sterile physiological saline to a final concentration of 10. -5 Spread the diluted solution onto a plate containing 1.5% calcium carbonate and 1.5% calcium lactate (i.e., add 1.5% (w / w) calcium carbonate and 1.5% calcium lactate to the isolation medium) and incubate at 55°C for at least 48 hours until single colonies grow.

[0036] The separation culture medium comprises the following components at mass-volume concentrations: 10 g / L tryptone, 10 g / L yeast extract, 10 g / L beef extract, 2 g / L K₂HPO₄, 0.2 g / L MgSO₄, 0.1 g / L MnSO₄, 0.03 g / L NaCl, 0.01 g / L FeSO₄, and 20 g / L agar powder, and the following components at volume percentages: 75% (v / v) beet syrup sugar, 0.1% Tween-80, pH 6.5 ± 0.2, with deionized water to make up the balance. Sterilize at 121°C for 15 min before use.

[0037] The aforementioned beet sugar refers to a concentrated solution prepared according to the method described in the literature "Guo Chengyu. Modern Beet Sugar Making Technology [M]. China Light Industry Press, 2015," with a total sugar content of approximately 20%, including approximately 18.0% sucrose and approximately 2.0% monosaccharides.

[0038] 2. Specific screening of strains

[0039] (1) Selective primary screening

[0040] Ten strains exhibiting rapid growth, large colonies, a milky-white color, raised surfaces, a distinct calcium dissolution zone, and Gram-positive staining were selected from isolation medium plates. These strains were repeatedly streaked onto isolation medium plates and incubated at 50-55°C for purification and preservation. Simultaneously, purified strains were separately cultured on enzyme-selective media containing different substrates at 50-55°C for 24-36 hours. Then, 0.1% Congo red or iodine solution was added to each medium to determine the activities of cellulase (CMC), xylanase (XYL), and amylase (AMY). The ratio of the diameter of the enzyme digestion zone (H) to the colony diameter (C) (H / C) was recorded.

[0041] The enzyme production screening medium is based on the modified MRS agar medium, with the following components added in weight-volume percentages: 0.5% sodium carboxymethyl cellulose (cellulase screening), 0.5% xylan (xylanase screening), or 1.0% soluble starch (amylase screening) for selective screening.

[0042] The modified MRS agar medium contains the following components by weight percentage: 2% agar powder, 1% peptone, 1% beef extract, 0.5% yeast extract, 0.2% diammonium citrate, 0.5% sodium acetate, 0.2% dipotassium hydrogen phosphate, 0.06% magnesium sulfate, 0.025% manganese sulfate, and 0.6% calcium carbonate, and the following components by volume percentage: 75% beet extract and 0.1% Tween-80; deionized water is added to make up the balance, and the pH is approximately 6.5.

[0043] Based on the differences in enzyme production ability among different strains, bacteria capable of forming distinct enzyme digestion zones on various substrates were selected. Their enzyme production ability was preliminarily ranked according to the ratio of the diameter of the enzyme digestion zone to the colony diameter; the larger the ratio, the stronger the enzyme activity. The results of the enzyme production screening are shown in Table 1.

[0044] Table 1 Comparison of enzyme production capacity of different strains

[0045] W01 1.0 1.0 1.0 W12 1.0 1.21 1.0 W23 1.20 1.0 1.0 W29 1.16 1.12 1.0 W58 1.22 1.0 1.0 W77 1.0 1.0 1.21 W103 1.45 1.30 1.25 W109 1.11 1.0 1.10 W116 1.15 1.0 1.10 W121 1.20 1.0 1.0

[0046] The initial screening results of enzyme-producing fermentation in Table 1 show that the polysaccharide hydrolysis ability of the strains obtained in the initial screening is not strong. However, compared with other strains, strain W103 has the enzyme activities of cellulase, xylanase and amylase, which shows that it has a better polysaccharide hydrolysis and utilization ability.

[0047] (2) Shake-flask fermentation and double screening

[0048] The L-lactic acid synthesis capacity of the above-mentioned strains with different enzyme-producing abilities was compared and the antibacterial properties of the fermentation broth were determined by shake-flask fermentation:

[0049] The strains with different substrate hydrolysis capabilities obtained above were transferred to test tube slant agar (containing modified MRS agar, 18×180mm test tubes) and incubated at 50-55℃ for 24 h. Then, 5 mL of sterile physiological saline was added for elution to prepare a bacterial suspension (colony count 1×10⁻⁶). 8 (CFU / mL) Finally, the bacterial suspension was transferred to a shake flask for fermentation culture. The shake flask fermentation conditions were as follows: 100 mL of shake flask fermentation medium was added to a 250 mL Erlenmeyer flask, and the mixture was incubated at 50-55℃ for 48 h.

[0050] The shake-flask fermentation medium described above comprises the following components by weight / volume percentage: 0.25% yeast extract, 5% beet molasses, 0.05% dipotassium hydrogen phosphate, 0.025% potassium dihydrogen phosphate, 0.18% ammonium dihydrogen phosphate, 0.5% ammonium sulfate, 0.01% zinc sulfate, 0.02% folic acid, 0.02% biotin, and 10.0% calcium carbonate, and the following component by volume percentage: 75% beet extract sugar; deionized water is added to make up the balance. Autoclave at 121°C for 15 minutes for later use.

[0051] The above-mentioned beet molasses contains 45% (w / v) sucrose, 0.5% (w / v) nitrogen, and 2.6% betaine. It was purchased from Xinjiang Kuitun Sugar Factory (No. 169, Aksu East Road, Kuitun City, Xinjiang).

[0052] After fermentation, the supernatant was collected by centrifugation at 10,000 r / min for 2 min. The content of L-lactic acid in the fermentation medium was quantitatively analyzed by using an SBA-40D biosensor analyzer (purchased from Jinan Yanke Experimental Instrument Co., Ltd.) with enzymatic reaction.

[0053] In addition, the antibacterial properties of the shake-flask fermentation supernatant were tested using the agar diffusion method: the fermentation supernatant collected by centrifugation was adjusted to pH 5.5-6.0 with 10% sodium hydroxide solution to eliminate the interference of acid on subsequent measurements. Using *Priscilla megaterium* as the indicator bacterium, inhibition plates were prepared, and the diameter of the inhibition zone was measured. The size of the inhibition zone determined the strength of the inhibitory effect of the supernatant on the indicator bacterium. The aforementioned inhibition plates refer to sterilized LB solid medium that has been heated and then cooled to 45-55°C at room temperature, with an appropriate amount of *Priscilla megaterium* or *Bacillus subtilis* suspension added (the final concentration of the indicator bacterium is 1×10⁻⁶). 5 Mix the agar (CFU / mL) thoroughly and pour into a plate. Use a 6.0 mm diameter sterile punch to make holes in the plate. Remove the agar block and add 50 μL of fermentation broth to the wells. Let stand at 4°C for about 1 hour, then incubate at 37°C for 24 hours. Remove the plate and measure the diameter of the inhibition zone.

[0054] The diameter of the inhibition zone = the outer diameter of the inhibition zone - the pore size (6.0 mm). The results of the shake-flask fermentation and re-screening are shown in Table 2.

[0055] Table 2 Results of secondary screening during shake-flask fermentation

[0056] W01 21.0 2.5mm W12 11.9 2.2mm W23 32.6 2.3mm W29 11.6 1.8mm W58 22.2 1.6mm W77 21.0 1.5mm W103 44.5 2.6mm W109 11.1 1.1mm W116 11.5 2.0mm W121 22.0 1.4mm

[0057] Table 2 shows that strain W103 exhibits the strongest ability to produce L-lactic acid during shake-flask fermentation. Furthermore, it can metabolize and produce certain antibacterial substances, possessing inhibitory activity and effectively reducing the risk of contamination from Bacillus species that can also produce spores and compete for fermentation feedstock. Strain W103 was preserved by slant culture and glycerol inoculum preservation; this is the originating strain W103 for high L-lactic acid production obtained through isolation.

[0058] Example 2: Selection of superior mutant strain *Weizmannii coagulans* RS711

[0059] 1. Protoplast preparation

[0060] (1) Activation of the strain

[0061] The W103 strain obtained above was used as the starting strain. Its glycerol bacteria were inoculated onto the slant of a modified MRS agar medium and cultured at 50-55℃ for 48 hours. After the culture was completed, a loopful of the strain was streaked into another fresh modified MRS slant medium and cultured at 50-55℃ for 24 hours to further enhance the viability of the strain, rejuvenate the strain, and achieve the purpose of strain activation.

[0062] (2) Preparation of strain suspension

[0063] The activated bacterial strains from the slant culture were washed off with sterile physiological saline to prepare a slant culture suspension, which was then thoroughly vortexed to mix. The bacterial cells were collected by centrifugation at 8000 rpm and 4°C for 5 min, and washed twice with sterile physiological saline, followed by two washes with hypertonic solution SMM. The supernatant was discarded by centrifugation, and the final bacterial suspension was resuspended in SMM solution to obtain a bacterial cell suspension. The colony count was adjusted to approximately 2.0 × 10⁻⁶. 6 CFU / mL (SMM hypertonic solution composition includes: 0.5 mol / L sucrose, 0.02 mol / L MgCl2·6H2O, 0.02 mol / L maleic acid, prepared with distilled water, pH 7.0, sterilized at 121℃ for 22 min, and stored at 4℃ for later use).

[0064] (3) Preparation of protoplasts

[0065] The preparation of protoplasts refers to first activating the bacterial strain on an agar slant, then preparing a bacterial suspension, and finally performing enzymatic hydrolysis to break the cell wall under certain conditions to prepare protoplasts.

[0066] Cell digestion was performed using a combination of enzymes. Specifically, lysozyme and cell wall lysin were used for combined digestion. A certain amount of filtered and sterilized enzyme stock solution (prepared with SMM solution) was added to the bacterial suspension to prepare an enzyme-containing bacterial suspension. The suspension was placed on a shaker and reacted at 80 rpm. The cells were examined under a microscope every 5 minutes to observe the formation of protoplasts. When about 90% of the cells were transformed into protoplasts, the cells were collected by centrifugation at 2000 rpm, washed twice with SMM, and resuspended in an appropriate amount of SMM. The concentration of lysozyme (purchased from Sangon Biotech (Shanghai) Co., Ltd.) was 0.05%-0.5%, with an optimal concentration of 0.05%-0.15%; the concentration of lysozyme (purchased from Guangdong Institute of Microbiology) was 0.01%-0.1%, with an optimal concentration of 0.01%-0.05%; the enzymatic hydrolysis temperature range was 24-35℃, with an optimal temperature of 26-30℃; the hydrolysis time was 10-90 min, with an optimal time of 10-30 min. Under optimized conditions, the final protoplast concentration reached 1.9 × 10⁻⁶. 6 The number of cells / mL was 95.0%, and the protoplast formation rate reached 95.0%, and the protoplast regeneration rate was also the best, reaching 20.5%.

[0067] 2. ARTP mutagenesis in protoplasts

[0068] (1) Determination of ARTP mutagenesis parameters

[0069] ARTP mutagenesis breeding refers to the use of an Atmospheric and Room Temperature Plasma (ARTP) mutagenesis breeding machine (Wuxi Yuanqing Tianmu Biotechnology Co., Ltd.). This breeding machine generates a plasma jet with a temperature between 25-40℃ at atmospheric pressure and a high concentration of active particles (including excited-state helium atoms, oxygen atoms, nitrogen atoms, OH free radicals, etc.). This plasma jet acts on microorganisms, altering the structure and permeability of the microbial wall / membrane and causing gene damage. Consequently, it significantly alters the microbial gene sequence and metabolic network, ultimately leading to mutations in the microorganisms.

[0070] The optimal conditions for protoplast mutagenesis using the aforementioned mutagenesis breeding machine are as follows: Take 10-20 μL of protoplast suspension, evenly spread it on the upper surface of a metal slide, and after drying, transfer the protoplast suspension slide to the stage using tweezers. Use high-purity helium as the working gas for the plasma, setting the power supply to 80W, irradiation distance to 4mm, plasma temperature to 26-30℃, and gas flow rate to 10L / min. Different treatment groups are set up, with treatment times of 0 (control), 10, 20, 30, 40, 50, and 60 s for each group, and three replicates for each group. After treatment, transfer the slides to EP tubes containing SMM solution, shake to elute and form a new protoplast suspension, spread it on regeneration plates, and incubate at 50-55℃ until single colonies grow, then count them. Calculate the lethality rate using the following method:

[0071] Lethality % = (Number of unmutated colonies - Number of mutagenized colonies) / Number of unmutated colonies × 100%

[0072] By statistically analyzing the lethality of each treatment group, we selected an irradiation treatment time with a lethality of approximately 90% for formal experiments. This ensured a certain level of mutation richness while also providing a certain survival rate for subsequent screening.

[0073] The results showed that, compared with the untreated strain (treatment time 0 s), the lethality rates of W103 bacterial suspension after ARTP mutagenesis were 54.1% (treatment time 10 s), 79.8% (treatment time 20 s), and 89.8% (treatment time 30 s), respectively. Furthermore, after treatment of more than 40 s, the lethality rate reached over 99.9%, and the cell viability was essentially zero. Therefore, to ensure sufficient cell viability while preventing mutagenesis damage, subsequent screening was conducted. ARTP treatment was performed under conditions with a lethality rate of approximately 90%, meaning the optimal treatment time for W103 strain using ARTP was determined to be 30 s.

[0074] (3) Screening of mutant strains

[0075] Primary screening in 96-well plates: The protoplast suspension induced by ARTP was spread on a regeneration plate containing 1% bromocresol green-methyl red. Based on the size of the colony color rings on the plate, 245 colonies with larger light blue to light red rings were selected and transferred to 96-well microplates containing microplate selection medium. The plates were incubated at 50-55℃ for 48 hours, and the color change of the medium was observed every 2 hours after 24 hours. Ten strains with rapid color change and deeper red color were selected for further preservation and use.

[0076] The bromocresol green-methyl red mixture was prepared as follows: accurately weigh 0.1 g of bromocresol green and 0.06 g of methyl red, dissolve them in 95% ethanol, and dilute to 100 mL with 95% ethanol.

[0077] The above-mentioned method for preparing regenerated plate culture medium is as follows: Weigh 20g of glucose, 10g of peptone, 10g of beef extract, 5g of yeast extract, 2g of diammonium hydrogen citrate, 1g of NaCl, 2g of K2HPO4, 0.6g of MgSO4, 0.25g of MnSO4, 171g of sucrose, 2.5g of CaCl2, 4.066g of MgCl26H2O, and 2.32g of maleic acid. Measure 1ml of Tween-80 and 2mL of bromocresol green-methyl red solution, and bring the volume to 1L with deionized water. Autoclave at 121℃ for 20 minutes.

[0078] The preparation method of the microplate screening medium is as follows: Weigh 10g of peptone, 10g of beef extract, 5g of yeast extract, 2g of diammonium hydrogen citrate, 5g of NaCl, 2g of K2HPO4, 0.6g of MgSO4, 0.25g of MnSO4 and 10g of sucrose, and measure 1ml of Tween-80, 2mL of bromocresol green-methyl red solution and 75% beet sugar by volume. Add deionized water to 1000mL and autoclave at 121℃ for 20min.

[0079] The obtained strains were further screened by shake-flask fermentation: Nine mutant strains selected from the initial screening were transferred to an agar slant for activation along with the starting strain W103. A bacterial suspension was then prepared and inoculated into Erlenmeyer flasks containing 150 mL of shake-flask fermentation medium. The flasks were incubated at 50-55°C for 48 hours. After fermentation, the L-lactic acid content was detected using a biosensor analyzer. The results of the shake-flask fermentation screening are shown in Table 3.

[0080] Table 3. Shake-flask fermentation secondary screening of ARTP mutagenized strains

[0081]

[0082]

[0083] As shown in Table 3, the L-lactic acid yield of strain RS711 was 62.7 g / L, significantly higher than that of other strains. Strain RS711 was preserved by slant culture and glycerol culture, and designated RS711.

[0084] The strain with the highest acid production, RS711, was selected and used together with the starting strain to verify its enzyme production characteristics using the spot colonization method. The results are as follows: Figure 1 (Where, AMY is amylase; CMC is cellulase; XYL is xylanase). From Figure 1 It can be seen that on enzyme production screening plates containing different substrates, the colony size of strain RS711 is significantly larger than that of the starting strain W103, and its enzyme production characteristics (AMY, CMC and XYL enzyme activities) are also significantly better than those of the starting strain W103.

[0085] Example 3 Molecular Identification

[0086] First, the genome of the strain RS711, which exhibits good L-lactic acid production ability, was extracted using a bacterial genomic DNA extraction kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.). Then, its 16S rDNA partial sequence was amplified using universal primers. The universal primer sequences are as follows:

[0087] 27F: 5'-AGAGTTTGATCCTGGCTCAG-3' (SEQ ID NO: 1)

[0088] 1492R: 5'-TACGGCTACCTTGTTACGACTT-3' (SEQ ID NO: 2)

[0089] The reaction system is as follows:

[0090] Pre-mix Ex Taq (Takara) 12.5 μL, 27F primer (10 μmol / L) 1 μL, 1492R primer (10 μmol / L) 1 μL, LA18 genomic DNA template 0.5 μL, ultrapure water 10 μL.

[0091] PCR conditions: 95℃ pre-denaturation for 5 min; 94℃ denaturation for 40 s, 57℃ annealing for 40 s, 72℃ extension for 90 s, for 30 cycles, with a final extension at 72℃ for 10 min, and storage at 4℃. After obtaining the specific amplification product, the target fragment was recovered using a PCR recovery kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.) and sent to Sangon Biotech (Shanghai) Co., Ltd. for sequencing. The sequence of strain RS711 is shown in SEQ ID NO: 3.

[0092] Sequencing results were analyzed and aligned using BLAST in the NCBI nucleic acid library (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The results showed that the closest phylogenetic relationship to strain RS711 was with *Bacillaceae* (Weizmannia). Phylogenetic analysis was performed on the 16S rDNA sequences of homologous strains with high similarity. A phylogenetic tree was constructed using the Neighbor-Joining method in MEGA 4.0. Figure 2 .

[0093] RS711 clusters with strains of the genus *Bacillus* and is most closely related to the genus *Weizmannia coagulans* in terms of evolutionary distance, indicating that RS711 and *Weizmannia coagulans* are evolutionarily closely related. Based on its colony and cell morphology characteristics, RS711 is identified as *Weizmannia coagulans*, designated as *Weizmannia coagulans* RS711, or simply *Weizmannia coagulans* RS711.

[0094] Example 4: Verification of Genetic Stability

[0095] The superior mutant strain obtained above, *Weizmannia coagulans* RS711, was subcultured every 3 days for 15 generations. Shake-flask fermentation was performed every other generation, and the L-lactic acid content of the fermentation broth was measured to assess the stability of the strain during subculturing. The results showed that the L-lactic acid content in the fermentation broth of *Weizmannia coagulans* RS711 did not change significantly during subculturing, indicating good genetic stability.

[0096] The genetically stable superior mutant strain, *Weizmannii coagulans* RS711, was deposited on August 11, 2022, at the Guangdong Provincial Microbial Culture Collection Center (GDMCC; address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou, Guangdong Province, 510075, China). The accession number for strain RS711 is GDMCC No: 62693.

[0097] Example 5: Fermentation application of *Weizmannii coagulates* RS711

[0098] 1. Seed culture

[0099] (1) Activation of strains

[0100] First, inoculate Weizmannii coagulate RS711 glycerol bacteria onto a modified MRS agar slant (18×180mm) and incubate at 55℃ for 48h. This is the F1 generation.

[0101] Then, the slant strain was washed with 5 mL of sterile physiological saline to prepare a bacterial suspension, which was then transferred to an eggplant flask containing modified MRS medium and incubated at 55°C for 24 h. This is the F2 generation activated strain.

[0102] The modified MRS medium is a modified MRS agar medium without agar.

[0103] (2) Shake flask seed culture

[0104] The activated culture of *Cyclocarya paliurus* was washed with 20 mL of sterile water to prepare a bacterial suspension, which was then transferred to a 5 L shake flask containing 2000 mL of modified MRS medium. The suspension was then incubated in a constant temperature shaker at 100 rpm and 55 °C for 12 h, followed by static incubation at 55 °C for 12 h to further increase the total number of colonies and enhance the viability of the strain.

[0105] (3) Seed expansion culture

[0106] Seed culture was expanded using seed tanks in shake flasks. 20% of the seed culture was transferred from the shake flasks to seed tanks containing seed culture medium. The control procedures were as follows: tank pressure: 0.06 MPa, tank temperature: 55℃, initial stirring: 100 rpm, dissolved oxygen allowed to decrease naturally, incubation for 6 hours, then stirring stopped, maintaining positive pressure in the tank, and continued static incubation at 55℃ for 12 hours. The transfer criterion was a colony count >10. 9 CFU / mL.

[0107] The seed culture medium described above comprises the following components by weight / volume percentage: 1.5% yeast extract, 0.2% ammonium dihydrogen phosphate, 0.5% ammonium sulfate, 1.0% light calcium carbonate, 0.02% magnesium sulfate, and 0.01% zinc sulfate, and the following components by volume percentage: 75% beet extract sugar; deionized water to make up the balance; adjust the pH to 6.0-6.5. Autoclave at 121℃ for 20 minutes.

[0108] 2. Fermentation to produce L-lactic acid

[0109] The seed culture was transferred to a fermenter containing fermentation medium, with a transfer volume of 50%. During the initial fermentation stage, the aeration rate in the fermenter was controlled at 150-200 mL / min. A certain amount of dissolved oxygen was maintained without stirring, primarily to facilitate early cell proliferation. The temperature was controlled at 55℃, and the initial total sugar concentration was controlled at 100-160 g / L, with dissolved oxygen controlled naturally. Throughout the process, the pH of the fermentation broth was controlled to 5.5-6.0 by adding 25% lime milk, and the total sugar content in the fermenter was maintained at 16-20 g / L by supplementing with concentrated beet extract sugar (40-50%) solution. Positive pressure was maintained in the fermenter, and fermentation was stopped after 48 hours. At the end of fermentation, the residual sugar content in the fermentation broth was less than 5 g / L. The L-lactic acid content in the fermentation medium was quantitatively analyzed using an SBA-40D biosensor analyzer (purchased from Jinan Yanke Experimental Instrument Co., Ltd.) via enzymatic reaction.

[0110] The fermentation medium described above comprises the following components by weight / volume percentage: 0.25% yeast extract, 5% beet molasses, 0.05% dipotassium hydrogen phosphate, 0.025% potassium dihydrogen phosphate, 0.18% ammonium dihydrogen phosphate, 0.5% ammonium sulfate, 0.01% zinc sulfate, 0.02% folic acid, 0.02% biotin, and 10.0% calcium carbonate, and the following component by volume percentage: 75% beet extract sugar; deionized water is used to make up the balance. The medium is autoclaved at 121°C for 15 min.

[0111] 3. Fermentation of control strain

[0112] The starting strain W103 was used instead of RS711 for fermentation under the same conditions, and the yield of L-lactic acid was detected using the same method.

[0113] 4. Comparison of L-lactic acid production

[0114] The results showed that the L-lactic acid content in the fermentation broth of strain RS711 reached 195.6 g / L, while the L-lactic acid yield of the original strain W103 was only 160.6 g / L. The superior mutant strain RS711 has a better ability to synthesize and accumulate L-lactic acid, and its L-lactic acid yield is 21.8% higher than that of the original strain, demonstrating a very good fermentation ability to produce L-lactic acid.

[0115] Example 6

[0116] This embodiment is the same as Embodiment 5 except for the parts mentioned below:

[0117] The culture and fermentation temperature is 50℃;

[0118] During the shake-flask seed culture process, the bacterial suspension is transferred to a shake flask containing modified MRS medium and cultured with shaking at 150 r / min for 18 h, followed by static culture for another 18 h.

[0119] During the seed expansion culture, the stirring culture conditions were 150 r / min for 10 h, and the static culture conditions were 18 h.

[0120] In the L-lactic acid production fermentation process, the transplanting amount was 20%, and the culture time was 40 hours.

[0121] Under the conditions described in this embodiment, the RS711 strain can effectively convert beet sucrose in the culture medium into L-lactic acid.

[0122] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A strain of enterogenic Weizmann's coagulant ( Weizmannia coagulans RS711, characterized in that, The *Weizmannii* strain RS711 was deposited on August 10, 2022, at the Guangdong Provincial Center for Microbial Culture Collection (GDMCC No: 62693), located at 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. The application of the enterogenic Weizmann's bacterium as described in claim 1 in the fermentation production of L-lactic acid.

3. The application as described in claim 2, characterized in that, The process includes the following steps: inoculating the intestinal-derived Weizmann's coagulant into a fermentation medium with beet sugar as the main carbon source, and producing L-lactic acid through fermentation.

4. The application as described in claim 3, characterized in that, The process for producing L-lactic acid by fermentation is as follows: static culture at 50-55℃ for 40-48 h.

5. The application as described in claim 3, characterized in that, It also includes the processes of strain activation, shake-flask seed culture, and seed expansion culture.

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