An enteric Weizmannella coagulans strain RS804 and its application

Abstract

The present invention discloses an enteric Weizmannella coagulans strain RS804 and its application. The present invention provides an enteric Weizmannella coagulans strain RS804 that can efficiently utilize sugar beet sucrose. The strain has certain heat and acid resistance, good polysaccharide hydrolase activities such as cellulase, xylanase and amylase, and can produce L-lactic acid by saccharification and fermentation. In addition, the enteric Weizmannella coagulans strain RS804 can also metabolize and produce a certain amount of bacteriocin, which can effectively inhibit strains with certain competitiveness such as Priesteria gigantea and Bacillus subtilis, and help prevent and control fermentation contamination. The enteric Weizmannella coagulans strain RS804 disclosed in the present invention is of great significance for reducing the production cost of L-lactic acid fermentation and increasing the yield of L-lactic acid.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to an intestinal-derived Bacillus coagulans RS804 and an application thereof; in particular, it relates to a Bacillus coagulans RS804 capable of producing L-lactic acid by fermenting beet sucrose and an application thereof. Background Art

[0002] Lactic acid, also known as α-hydroxypropionic acid, has a molecular formula of CH3CHOHCOOH and a relative molecular mass of 900.8. It is a simple and common organic acid widely found in the human body, animals, plants, and microorganisms. Due to the presence of an asymmetric carbon atom in the lactic acid molecule, it exhibits optical rotation, namely L-lactic acid (dextrorotatory), D-lactic acid (levorotatory), and DL-lactic acid (racemic). As an important daily chemical, it is currently recognized as one of the three major organic acids in the world and is widely used in various industrial fields such as medicine, food, chemicals, textiles, cosmetics, and environmental protection. In particular, polylactic acid (PLA), prepared from high-optical purity L-lactic acid, is a new non-petroleum material that is biodegradable and biocompatible, and is considered a promising recyclable polymer.

[0003] Currently, L-lactic acid is primarily produced by fermentation, with microorganisms of the genera Lactobacillus and Weizmannia being the most widely used. Weizmannia coagulans (formerly known as Bacillus coagulans) is recognized as one of the most superior strains for L-lactic acid biofermentation due to its ability to form spores, strong stress resistance, a well-developed amylase system, rapid growth, and high product optical purity. Lactic acid is primarily produced by microorganisms through metabolic fermentation using glucose, starch, or sucrose as a carbon source. Reducing the production cost of lactic acid has recently become a research hotspot. Because its carbon source raw materials account for a significant proportion of the production cost of L-lactic acid, using refined sugars to produce L-lactic acid can reduce the cost of post-processing to a certain extent. However, the generally high price of refined sugars not only increases overall costs, but also creates a "competition with the public for food" situation when using refined sugars from traditional grain crops as substrates for lactic acid biorefining, making this method uneconomical. As a renewable energy crop, sugar beets are an inexhaustible resource. Using sugar beet (primarily sucrose) as a substrate for lactic acid fermentation not only addresses the issues of limited raw material sources and high costs, but also fully utilizes sugar beet molasses, a byproduct of sugar production, to increase L-lactic acid fermentation yield and utilize local raw materials. Given these advantages, utilizing sugar beet fermentation to produce L-lactic acid is a promising development direction for industrial lactic acid production.

[0004] Weizmannella coagulans, a leading L-lactic acid fermentation strain, has high yields, but 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. Breeding strains that can efficiently utilize sugar beet sugar products, tolerate high substrate concentrations and the osmotic pressure of the final product, maintain cell growth and enzyme activity, and achieve high productivity is becoming a key issue in L-lactic acid strain breeding research. Summary of the Invention

[0005] In view of the above problems, the object of the present invention is to provide Weizmannia coagulans which can efficiently utilize sugar beet sucrose to ferment and produce L-lactic acid, and its application in producing L-lactic acid by fermentation.

[0006] To achieve the above-mentioned purpose, the technical solution adopted by the present invention is: a strain of enteric Weizmannia coagulans RS804, characterized in that the enteric Weizmannia coagulans RS804 was deposited in the Guangdong Provincial Microbiological Culture Collection Center on August 10, 2022, with the preservation number GDMCC No: 62692, and the preservation address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City.

[0007] The Weizmannella coagulans RS804 strain provided by the present invention was obtained by ARTP-induced protoplast mutagenesis of the original strain W103, isolated from the feces of healthy lactating sows. It has rapid growth, stable genetic traits, and a certain ability to synthesize amylase, xylanase, and cellulase. It can also efficiently produce L-lactic acid using beet sugar as a carbon source.

[0008] Furthermore, the present invention also claims protection for the use of the enteric Weizmannella coagulans RS804 in fermentation.

[0009] Since the intestinal Weizmannella coagulans RS804 provided by the present invention has certain amylase, xylanase and cellulase synthesis capabilities, its characteristics can be utilized in a variety of corresponding fermentation productions.

[0010] As a preferred embodiment of the present invention, the enteric Weizmannella coagulans 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 the enteric Weizmannella coagulans comprises the following steps: inoculating the enteric Weizmannella coagulans into a fermentation medium with beet sucrose as the main carbon source, and producing L-lactic acid by fermentation.

[0012] By specifically using beet sugar as a carbon source and selecting strains that are heat-resistant and capable of producing antibacterial substances, the risk of bacterial contamination during the fermentation process is effectively reduced. Furthermore, high-temperature fermentation conditions help reduce fermentation energy consumption, saving costs and achieving high lactic acid yields. Under the conditions of this invention, the enteric Weizmannia coagulans RS804 strain exhibits excellent L-lactic acid fermentation production, reaching 194.8 g / L, an increase of approximately 21.3% compared to the starting strain.

[0013] As a preferred embodiment of the present invention, the fermentation medium with beet sucrose as the main carbon source includes the following components in mass 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, as well as the following components in volume percentage: 75% beet liquid sugar; deionized water makes up the balance.

[0014] As a preferred embodiment of the present invention, the process of producing L-lactic acid by fermentation is: static culture at 50-55° C. for 40-48 hours.

[0015] The fermentation conditions are simple and the requirements for equipment are low. Since stirring is not required, production costs are further saved.

[0016] Furthermore, the fermentation process also includes the processes of strain activation, shake flask seed culture and seed expansion culture.

[0017] As a preferred embodiment of the present invention, the process of strain activation is: inoculating the enteric Weizmannella coagulans onto a modified MRS agar medium slant and culturing at 50-55°C for 48 hours; then washing the slant strain with sterile saline to prepare a bacterial suspension, which is transferred to a culture medium containing a modified MRS medium and cultured at 50-55°C for 24 hours.

[0018] The shake flask seed culture process is as follows: adding sterile water to wash the bacteria obtained by activation culture to prepare a bacterial suspension, transferring it to a shake flask filled with improved MRS culture medium, shaking culture at 100-150 r / min and 50-55° C. for 12-18 hours, and then static culture at 50-55° C. for 12-18 hours.

[0019] The process of seed expansion culture is as follows: the seed shake flask is transferred to the seed tank containing the seed culture medium at an inoculum volume of 20%, and the culture is stirred at 50-55°C and 100-150r / min for 6-10 hours under the condition of a tank pressure of 0.06Mpa, and then the stirring is stopped, the tank is kept under positive pressure, and the culture is continued at 50-55°C for 12-18 hours. The transplanting standard is that the number of colonies is greater than 10 9 CFU / mL;

[0020] The modified MRS medium contains the following components by mass 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, 0.6% calcium carbonate, and the following components by volume percentage: 75% beet liquid sugar and 0.1% Tween-80; the pH value is adjusted to about 6.5; and deionized water is used to make up the balance;

[0021] The improved MRS agar medium is prepared by adding the following components in percentage by weight: 2% agar powder; and deionized water to make up the balance.

[0022] The seed culture medium contains the following components in percentage by mass and volume: 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 components in percentage by volume: 75% beet liquid sugar; the pH value is adjusted to 6.0-6.5, and deionized water is used to make up the balance.

[0023] The enzyme activity and bacterial population of the enteric Weizmannella coagulans RS804 can be improved by strain activation, shake flask seed culture and seed expansion culture, providing favorable support for subsequent inoculation in a fermenter for large-scale fermentation.

[0024] As a preferred embodiment of the present invention, the fermentation conditions are as follows: transplanting 20-50% of the seed culture solution into a fermentation tank filled with fermentation medium, controlling the fermentation tank ventilation rate to 150-200 mL / min, controlling the initial total sugar concentration to 100-160 g / L, the pH value to 5.5-6.0, and culturing at 50-55° C.; maintaining the total sugar content in the fermentation tank at 16-20 g / L, positive pressure in the fermentation tank, and fermenting for 48 hours.

[0025] During the initial fermentation phase, the fermenter's ventilation rate is controlled at 150-200 mL / min. Agitation is not initiated to maintain a certain level of dissolved oxygen, primarily for the purpose of bacterial growth. At the end of fermentation, the residual sugar content in the fermentation broth is less than 5 g / L.

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

[0027] The present invention provides an enteric Weizmannella coagulans RS804 that can efficiently utilize beet sucrose to ferment and produce L-lactic acid. The strain has certain heat and acid resistance, good enzymatic activity of polysaccharide hydrolases such as cellulase, xylanase, and amylase, and can produce L-lactic acid by saccharification and fermentation. In addition, the enteric Weizmannella coagulans RS804 can also metabolize and produce a certain amount of bacteriocin, which can effectively inhibit strains with certain competitiveness such as Priestia megaterium and Bacillus subtilis, thereby helping to prevent and control fermentation contamination. The enteric Weizmannella coagulans RS804 disclosed in the present invention is of great significance for reducing the production cost of L-lactic acid fermentation and increasing the yield of L-lactic acid. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 This is the enzyme production characteristic test result of the intestinal Weizmannella coagulans RS804 described in the present invention.

[0029] Figure 2 This is a phylogenetic tree of the enteric Weizmannella coagulans RS804 strain described in the present invention based on the 16s rDNA sequence. DETAILED DESCRIPTION

[0030] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below with reference to the accompanying drawings and specific embodiments.

[0031] Unless otherwise specified, the experimental methods used in the following examples were generally based on conventional conditions. The materials and reagents used in the following examples were all commercially available unless otherwise specified. The quantitative experiments in the following examples were all repeated three times, and the results were averaged.

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

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

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

[0035] After the pretreatment, the bath solution was diluted 10-fold to 10 -5 The diluted solution was spread on a separation medium plate containing 1.5% calcium carbonate and 1.5% calcium lactate (i.e., 1.5% (w / w) calcium carbonate and 1.5% calcium lactate were added to the separation medium), and cultured at 55°C for more than 48 hours until a single colony grew.

[0036] The separation medium comprises the following components in 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, as well as the following components in volume percentages: 75% (v / v) beet liquid sugar, 0.1% Tween-80, pH 6.5 ± 0.2, with deionized water making up the balance. The medium is sterilized at 121°C for 15 minutes before use.

[0037] The beet liquid sugar mentioned above refers to a concentrated liquid prepared according to the method of "Guo Chengyu. Modern Beet Sugar Refining Technology [M]. China Light Industry Press, 2015" with a total sugar content of about 20%, including about 18.0% sucrose and about 2.0% monosaccharides.

[0038] 2. Specific screening of strains

[0039] (1) Selective initial screening

[0040] Ten strains with rapid colony growth, large colonies, milky white color, raised surfaces, distinct calcium-dissolving zones, and Gram-positive colonies were selected from the isolation medium plates. These strains were repeatedly streaked onto the isolation medium plates and cultured at 50-55°C for strain purification and preservation. Simultaneously, purified strains were individually spotted onto enzyme-producing screening media containing different substrates and cultured at 50-55°C for 24-36 hours. 0.1% Congo red or iodine solution was then added to measure cellulase (CMC), xylanase (XYL), and amylase (AMY) activities. The ratio (H / C) of the enzymatic zone diameter (H) to the colony diameter (C) was recorded.

[0041] Among them, the enzyme production screening medium is based on the improved MRS agar medium, and the following components are added in mass volume percentage: 0.5% sodium carboxymethyl cellulose (cellulase screening) or 0.5% xylan (xylanase screening) or 1.0% soluble starch (amylase screening) for selective screening.

[0042] The improved MRS agar medium contains the following components by mass percentage: 2% agar powder, 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 liquid sugar and 0.1% Tween-80; deionized water makes up the balance, and the pH value is about 6.5.

[0043] Based on the differences in enzyme production capacity among strains, we selected strains that formed distinct enzymatic zones for various substrates. We then used the ratio of the enzymatic zone diameter to the colony diameter to preliminarily rank their enzyme production abilities. A larger ratio indicates stronger 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] strain number CMC H / C XYL H / C AMY H / C 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 for enzyme production (Table 1) show that none of the strains screened had strong polysaccharide hydrolysis capabilities. However, compared to other strains, strain W103 possessed cellulase, xylanase, and amylase activities, demonstrating its superior polysaccharide hydrolysis and utilization capabilities.

[0047] (2) Shake flask fermentation rescreening

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

[0049] The strains with different substrate hydrolysis abilities obtained above were transferred to the slant of test tubes (containing modified agar MRS medium, 18×180 mm test tubes), cultured at 50-55°C for 24 h, and then eluted with 5 mL of sterile saline to prepare bacterial suspensions (the number of colonies was 1×10 8 The bacterial suspension was finally transferred to a shake flask for fermentation. The shake flask fermentation conditions were as follows: 100 mL of shake flask fermentation medium was placed in a 250 mL Erlenmeyer flask and cultured at 55°C for 48 h.

[0050] The shake flask fermentation medium comprises the following components by weight and volume: 0.25% yeast extract, 5% beet molasses, 0.05% dipotassium hydrogen phosphate, 0.025% potassium dihydrogen phosphate, 0.18% monoammonium phosphate, 0.5% ammonium sulfate, 0.01% zinc sulfate, 0.02% folic acid, 0.02% biotin, and 10.0% calcium carbonate, and the following components by volume: 75% beet liquid sugar; the balance is made up with deionized water. The medium is autoclaved at 121°C for 15 minutes and set aside.

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

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

[0053] In addition, the agar diffusion method was used to detect the antibacterial properties of the shake flask fermentation supernatant: the fermentation supernatant collected by centrifugation was adjusted to a pH of 5.5-6.0 with 10% sodium hydroxide solution to eliminate the interference of acid on subsequent determinations. Antibacterial plates were prepared using Priesteria gigantea as an indicator bacterium, and the diameter of the inhibition zone was measured. The size of the inhibition zone was used to determine the inhibitory effect of the supernatant on the indicator bacteria. The above-mentioned antibacterial plate refers to a sterilized LB solid culture medium that is heated and then cooled to 45-55°C at room temperature, and an appropriate amount of Priesteria gigantea or Bacillus subtilis suspension is added (the final concentration of the indicator bacteria is 1×10 5 After mixing, pour the mixture into the plate, punch holes on the plate with a 6.0 mm diameter sterile puncher, remove the agar block, take 50 μL of fermentation liquid and add it to the sample well, let it stand at 4 ° C for about 1 hour, and then culture it in a 37 ° C incubator for 24 hours, take out the plate, and measure the diameter of the inhibition zone.

[0054] Inhibition zone diameter = inhibition zone outer diameter - pore diameter (6.0 mm). The results of shake flask fermentation rescreening are shown in Table 2.

[0055] Table 2 Shake flask fermentation rescreening results

[0056] strain number L-lactic acid (%) Diameter of inhibition zone (mm) 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] As shown in Table 2, strain W103 has the strongest ability to produce L-lactic acid in shake flask fermentation. It also metabolizes and produces certain antibacterial substances, demonstrating a certain degree of inhibitory activity, effectively reducing the risk of contamination by Bacillus species that can also produce spores and compete with fermentation feedstock. Strain W103 was stored on slant plates and in glycerol stock, resulting in the isolation of the high-L-lactic acid-producing starting strain W103.

[0058] Example 2: Breeding of superior mutant strain Weizmannella coagulans RS804

[0059] 1. Protoplast Preparation

[0060] (1) Activation of strains

[0061] The W103 strain isolated above was used as the starting strain, and its glycerol bacteria were inoculated on the slant of the modified MRS agar medium and cultured at 50-55°C for 48 hours. After the culture was completed, a loop of the strain was streaked onto another fresh modified MRS slant medium and cultured at 50-55°C for 24 hours to further enhance the strain vigor, rejuvenate the strain, and achieve the purpose of strain activation.

[0062] (2) Preparation of strain suspension

[0063] Wash the activated strain from the slant with sterile saline to prepare a slant strain suspension, and thoroughly vortex to mix. Collect the bacterial cells by centrifugation at 8000 rpm and 4°C for 5 minutes. Wash twice with sterile saline, then wash twice with hypertonic SMM solution, centrifuge and remove the supernatant. Finally, resuspend the cells in SMM solution to obtain a bacterial suspension, and adjust the colony count to approximately 2.0 × 10 6 CFU / mL (SMM hypertonic solution composition includes: sucrose 0.5 mol / L, MgCl2·6H2O 0.02 mol / L, maleic acid 0.02 mol / L, distilled water, pH 7.0, 121°C, sterilized for 22 min, stored at 4°C until use).

[0064] (3) Preparation of protoplasts

[0065] The protoplast preparation mentioned above refers to first activating the strain on a slant, then preparing a bacterial suspension, and finally performing enzymatic cell wall breaking under certain conditions to prepare the protoplast.

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

[0067] 2. ARTP mutagenesis of protoplasts

[0068] (1) Determination of ARTP mutagenesis parameters

[0069] ARTP mutagenesis breeding refers to the use of an atmospheric and room temperature plasma (ATP) mutagenesis breeding machine (Wuxi Yuanqing Tianmu Biotechnology Co., Ltd.). This breeding machine generates a plasma jet with a temperature of 25-40°C under atmospheric pressure and a high concentration of active particles (including excited helium atoms, oxygen atoms, nitrogen atoms, OH free radicals, etc.). When acting on microorganisms, it can change the structure and permeability of the microbial wall / membrane and cause genetic damage, thereby significantly changing the microbial gene sequence and its metabolic network, and ultimately leading to mutations in the microorganisms.

[0070] Adopt above-mentioned mutation breeding machine to the optimum condition of protoplast mutation breeding: get the protoplast suspension of 10-20 μ L, evenly coat on the upper surface of metal slide, dry after the protoplast suspension slide is transferred to the stage with tweezers. Adopt high-purity helium as the working gas of plasma, set power 80W, irradiation distance 4mm, the temperature of plasma 26-30 ℃, gas flow 10L / min, process bacteria slide, set different treatment groups, the processing time of each group is respectively 0 (control), 10, 20, 30, 40, 50, 60s, and every group arranges three repetitions. After processing, slide is transferred in the EP pipe that contains SMM solution, concussion elution forms new protoplast suspension, is placed in 50-55 ℃ of incubator and is cultivated to having single bacterium colony to grow, counting after coating regeneration flat board. Calculate lethality, lethality calculation method is as follows:

[0071] Lethality rate % = (number of colonies without mutation treatment - number of colonies with mutation treatment) / number of colonies without mutation treatment × 100%

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

[0073] The results showed that, with the strain not treated with ARTP (treatment time 0s) as the control, the lethality of the W103 bacterial suspension after ARTP mutagenesis was 54.1% (treatment time 10s), 79.8% (treatment time 20s), and 89.8% (treatment time 30s), respectively. Moreover, after treatment for more than 40s, the lethality reached more than 99.9%, and the cell survival rate was basically 0. Therefore, in order to ensure a certain cell survival rate while ensuring the mutagenic damage, the conditions with a lethality of about 90% were selected for ARTP treatment, that is, the optimal treatment time of ARTP for the W103 strain was determined to be 30s.

[0074] (3) Screening of mutagenic strains

[0075] 96-well plate initial screening: The protoplast suspension after ARTP mutagenesis was spread on a regeneration plate containing 1% bromocresol green-methyl red. According to the size of the colony color circle on the plate, 245 colonies with larger light blue to light red circles were picked and transferred to a 96-well plate containing microplate screening medium. The plates were cultured at 50-55°C for 48 hours, and the color change of the culture medium was observed every 2 hours after 24 hours. The 10 strains with faster culture solution color change and darker red were selected for further preservation and standby use.

[0076] The bromocresol green-methyl red was prepared as follows: 0.1 g bromocresol green and 0.06 g methyl red were accurately weighed, dissolved in 95% ethanol, and the volume was adjusted to 100 mL with 95% ethanol.

[0077] The above-mentioned regeneration plate culture medium is prepared as follows: 20 g of glucose, 10 g of peptone, 10 g of beef extract, 5 g of yeast extract powder, 2 g of diammonium hydrogen citrate, 1 g of NaCl, 2 g of K2HPO4, 0.6 g of MgSO4, 0.25 g of MnSO4, 171 g of sucrose, 2.5 g of CaCl2, 4.066 g of MgCl26H2O, and 2.32 g of maleic acid are weighed, and 1 ml of Tween-80 and 2 mL of bromocresol green-methyl red solution are measured, the volume is made up to 1 L with deionized water, and the mixture is sterilized by high pressure at 121°C for 20 min.

[0078] The microplate screening medium is prepared as follows: 10 g of peptone, 10 g of beef extract, 5 g of yeast extract powder, 2 g of diammonium hydrogen citrate, 5 g of NaCl, 2 g of K2HPO4, 0.6 g of MgSO4, 0.25 g of MnSO4, and 10 g of sucrose are weighed, and 1 ml of Tween-80, 2 ml of bromocresol green-methyl red solution, and 75% by volume of beet liquid sugar are measured, and the mixture is made up to 1000 ml with deionized water, and sterilized under high pressure at 121°C for 20 min.

[0079] The resulting strains were further screened using shake flask fermentation. Nine mutant strains from the initial screening were selected, along with the starting strain W103, and activated on a slant. A bacterial suspension was then prepared and inoculated into a triangular flask containing 150 mL of shake flask fermentation medium. The suspension was incubated at 50-55°C for 48 hours. After fermentation, L-lactic acid content was measured using a biosensor analyzer. The results of the shake flask fermentation rescreening are shown in Table 3.

[0080] Table 3 Shake flask fermentation rescreening of ARTP mutagenic strains

[0081]

[0082]

[0083] The results in Table 3 show that the RS804 strain produced 62.5 g / L of L-lactic acid, which was significantly higher than that of the other strains. The RS804 strain was stored on slant plates and in glycerol stock, and was designated RS804.

[0084] RS804, which has the highest acid production, 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 (Among them, AMY is amylase; CMC is cellulase; XYL is xylanase). Figure 1 It was found that on the enzyme production screening plates containing different substrates, the colony size of the RS804 strain was significantly larger than that of the starting strain W103, and its enzyme production characteristics (AMY, CMC and XYL enzyme activities) were also significantly better than those of the starting strain W103.

[0085] Example 3 Molecular Identification

[0086] First, a bacterial genomic DNA extraction kit (purchased from Sangon Biotech (Shanghai) Co., Ltd.) was used to extract the genome of the strain RS804 with good L-lactic acid production ability isolated and screened above, and 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°C pre-denaturation for 5 min; 30 cycles of denaturation at 94°C for 40 s, annealing at 57°C for 40 s, and extension at 72°C for 90 s, followed by a final extension at 72°C for 10 min, and storage at 4°C. After obtaining the specific amplified 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 RS804 is shown in SEQ ID NO: 3.

[0092] The sequencing results were subjected to Blast analysis and comparison in the NCBI nucleic acid library (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi). The results showed that the closest relative of the RS804 strain was the genus Weizmannia of the Bacillaceae family. The 16S rDNA sequences of homologous strains with high similarity were selected for phylogenetic analysis, and the Neighbor-Joining method (NJ) in MEGA4.0 was used to construct a phylogenetic evolutionary tree, as shown in Figure 2. Figure 2 .

[0093] RS804 clustered with strains of the genus Bacillus and was closest to the Weizmannia coagulans clade, indicating that RS804 and Weizmannia coagulans are evolutionarily related. Based on its colony and somatic morphology, RS804 was identified as Weizmannia coagulans and designated Weizmannia coagulans RS804, or simply Weizmannia coagulans RS804.

[0094] Example 4 Genetic stability verification

[0095] The superior mutant strain obtained above, Weizmannella coagulans RS804, was subcultured every three days for 15 generations. Shake flask fermentation was performed after each generation, and the L-lactic acid content of the fermentation broth was measured to investigate the strain's stability during the subculture process. The results showed that the L-lactic acid content in the fermentation broth of Weizmannella coagulans RS804 did not change significantly during the subculture process, indicating good genetic stability.

[0096] The genetically stable superior mutant strain Weizmannella coagulans RS804 was deposited in the Guangdong Provincial Microbial Culture Collection Center (GDMCC for short; address: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province, Guangdong Institute of Microbiology, Postal Code: 510075) on August 11, 2022. The deposit number of the RS804 strain is GDMCC No: 62692.

[0097] Example 5 Fermentation Application of Weizmannella coagulans RS804

[0098] 1. Seed cultivation

[0099] (1) Strain activation

[0100] First, the Weizmannia coagulans RS804 glycerol stock was inoculated onto a modified MRS agar medium slant (18×180 mm) and cultured in an incubator at 55° C. for 48 h, which was the F1 generation.

[0101] Then, the slant strain was washed with 5 mL of sterile saline to prepare a bacterial suspension, which was transferred to an eggplant flask culture medium containing a modified MRS medium and cultured in an incubator at 55°C for 24 hours. 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 cultured eggplant bottle strain was added with 20 mL of sterile water to elute and prepare a bacterial suspension, which was then transferred to a 5 L shake flask containing 2000 mL of modified MRS culture medium. The suspension was cultured in a constant temperature shaking incubator at 100 r / min and 55°C for 12 h, and then statically cultured at 55°C for 12 h to further increase the total colony count and enhance the activity of the strain.

[0105] (3) Seed expansion culture

[0106] The seed tank was used for the expansion culture of the shake flask seeds. The seed shake flask was transferred to the seed tank containing the seed culture medium at 20% of the inoculation amount. The control process was as follows: tank pressure: 0.06 MPa, tank temperature: 55 ° C, initial stirring: 100 r / min, dissolved oxygen naturally decreased, cultured for 6 hours, then stopped stirring, maintained the tank positive pressure, and continued to culture at 55 ° C for 12 hours. The transplanting standard was the number of colonies > 10 9 CFU / mL.

[0107] The seed culture medium comprises the following components by mass 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 components by volume percentage: 75% beet liquid sugar; deionized water makes up the balance; the pH value is adjusted to 6.0-6.5. Autoclave sterilization is carried out at 121°C for 20 minutes.

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

[0109] The seed culture solution was transplanted to a fermentation tank filled with fermentation medium, with the transplant volume being 50%. At the initial stage of fermentation, the fermentation tank ventilation was controlled at 150-200 mL / min. Stirring was not started to control a certain amount of dissolved oxygen, mainly for the early growth of the bacteria. The temperature was controlled at 50-55°C, the initial total sugar concentration was controlled at 100-160 g / L, and the dissolved oxygen was controlled naturally. Throughout the process, the pH value of the fermentation liquid was controlled to 5.5-6.0 by controlling the flow of 25% lime milk, and the total sugar content in the fermentation tank was maintained at 16-20 g / L by adding concentrated beet liquid sugar (40-50%) solution. Positive pressure was maintained in the fermentation tank, and fermentation was stopped for 48 hours. At the end of fermentation, the residual sugar content in the fermentation liquid was less than 5 g / L, and the L-lactic acid content in the fermentation medium was quantitatively analyzed by enzymatic reaction using an SBA-40D biosensor analyzer (purchased from Jinan Yanke Laboratory Instrument Co., Ltd.).

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

[0111] 3. Fermentation of control strain

[0112] The starting strain W103 was used instead of RS804 for fermentation under the same conditions, and the same method was used to detect the production of L-lactic acid.

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

[0114] The results showed that the L-lactic acid content in the fermentation broth of the RS804 strain reached 194.8g / L, while the L-lactic acid production of the starting strain W103 was only 160.6g / L. The superior mutant strain RS804 had better L-lactic acid synthesis and accumulation capabilities, and its L-lactic acid production was 21.3% higher than that of the starting strain, reflecting 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 following parts:

[0117] The culture and fermentation temperature is 50°C;

[0118] During the shake flask seed culture, the bacterial suspension was transferred to a shake flask containing a modified MRS medium, cultured at 150 r / min for 18 h, and then cultured statically for another 18 h.

[0119] During the seed expansion culture, the stirring culture condition is 150 r / min stirring culture for 10 hours, and the static culture condition is culture for 18 hours.

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

[0121] Under the conditions of this example, the RS804 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, rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. A strain of enteric Weizmannella coagulans ( Weizmannia coagulans ) RS804, characterized in that, The enteric Weizmannella coagulans RS804 was deposited in the Guangdong Provincial Microbial Culture Collection Center on August 10, 2022, with the collection number GDMCCNo: 62692, and the collection address is 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou.

2. Use of the enteric Weizmannella coagulans according to claim 1 in the fermentation production of L-lactic acid using sucrose as the main carbon source.

3. The use according to claim 2, characterized in that The method comprises the following steps: inoculating the enteric Weizmannella coagulans into a fermentation medium with beet sucrose as a main carbon source, and producing L-lactic acid through fermentation.

4. The use according to claim 3, characterized in that The process of producing L-lactic acid by fermentation is as follows: static culture at 50-55° C. for 40-48 hours.

5. The use according to 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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