Bacillus coagulans beneficial to thallus recovery and application of bacillus coagulans in lactic acid fermentation

Abstract

The invention discloses bacillus coagulans beneficial to thallus recovery and application of the bacillus coagulans in lactic acid fermentation, and belongs to the technical field of microorganisms and fermentation engineering. The method comprises the following steps: separating a batch of heat-resistant bacteria from metamorphic cow milk, culturing a small amount of thalli, adding a flocculating agent to observe the sedimentation condition, selecting a bacterial strain with the fastest sedimentation speed, and identifying to select bacillus coagulans in the bacterial strain. And a strain XQ20249 with fast cell sedimentation and high optical purity of L-lactic acid produced by fermentation is further screened out. Furthermore, nitrosoguanidine is adopted for mutagenesis and screening to obtain a mutant strain with faster cell sedimentation, wherein the flocculability of the mutant strain numbered as LL08 is greatly increased, and the growth speed and lactic acid fermentation speed of the mutant strain under the lactic acid fermentation condition are both higher than those of other mutant strains. When the LL08 strain is used for lactic acid fermentation, thalli can be collected through a thalli sedimentation method for secondary fermentation after fermentation is finished, and the use amount of an organic nitrogen source can be greatly reduced.

Description

Technical Field

[0001] The invention relates to a bacillus coagulans strain which is beneficial to bacterial cell recovery and application thereof in lactic acid fermentation, belonging to the technical field of microorganisms and fermentation engineering. Background Art

[0002] Bacillus coagulans is currently the main strain for industrial production of food-grade and polymer-grade L-lactic acid. Bacillus coagulans has the characteristics of high lactic acid production under specific fermentation conditions. The optical purity of lactic acid products of some strains is high, and its fermentation temperature can reach about 50°C. There is rarely any contamination by foreign bacteria during the fermentation process, so it has become one of the main strains for high optical purity lactic acid fermentation. There are also certain defects in the application of Bacillus coagulans. One of them is that its growth requires a certain amount of organic nitrogen source, and yeast extract is commonly used, which leads to an increase in production costs to a certain extent. The recycling and reuse of bacteria can reduce the amount of raw materials used in the bacterial culture stage, which is an effective way to reduce production costs for the fermentation of Bacillus coagulans.

[0003] Plate and frame filtration is the lowest cost method for collecting cells in the fermentation industry, but Bacillus coagulans cells are tiny and require the addition of filter aids to achieve cell recovery. Commonly used filter aids such as diatomaceous earth and perlite are mixed with the bacteria, which seriously affects the reuse of recovered cells in fermentation. After the fermentation is completed, when the cells are used as feed, the filter aids also have an adverse effect on the product quality. Centrifugation is another important method for recovering cells, but industrial centrifugation consumes a lot of power energy and has a large investment in equipment. Centrifugation is mainly used in the industry to recover cells of microorganisms with larger cells such as yeast. Bacterial cells are small and require a higher centrifugal force to effectively collect cells, so the cost of use is high. In the fermentation industry, there are some special microorganisms that have the characteristics of cell aggregation. After aggregation, these microorganisms can naturally settle to the bottom of the container. After collecting the cells at the bottom, these concentrated cell suspensions can be directly used for re-fermentation or centrifugation can be used to recover cells. Since the cells are concentrated at the bottom, it is only necessary to centrifuge the fermentation liquid with a smaller volume at the bottom to recover most of the cells, thereby greatly reducing the cost of centrifugation operation. This is of great significance in fermentation with bacteria as the strain, and can significantly reduce production costs. So far, no cell aggregation or natural sedimentation has been found in Bacillus coagulans used for lactic acid fermentation.

[0004] When Bacillus coagulans is used for lactic acid fermentation, the organic nitrogen source is the key factor causing the high cost of raw materials, and is also the key factor affecting the current production of high optical purity lactic acid and further production of biodegradable plastics by fermentation. Therefore, it is necessary to screen out Bacillus coagulans strains that are easy to flocculate and naturally settle, so as to be able to recover the bacteria through natural sedimentation and use them for the second lactic acid fermentation, thereby significantly reducing the use of organic nitrogen sources in lactic acid fermentation. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the present invention provides a Bacillus coagulans strain that is conducive to the recovery of bacteria and its application in lactic acid fermentation, aiming to solve the technical problems in the prior art that when the cells of the Bacillus coagulans strain are reused after fermentation, the cell recovery needs to be achieved by centrifugation, which results in excessively high production costs and affects the production of high optical purity lactic acid.

[0006] The first technical solution provided by the present invention is a strain of Bacillus coagulans LL08, which has been deposited in the China Center for Type Culture Collection on November 2, 2021, with a deposit number of CCTCCNO: 20211356.

[0007] The second technical solution provided by the present invention is a microbial preparation containing the Bacillus coagulans LL08 described in the first technical solution.

[0008] In certain embodiments, the bacterial concentration of Bacillus coagulans LL08 in the microbial preparation is at least 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0009] The third technical solution provided by the present invention is a fermentation agent, which contains the Bacillus coagulans LL08 described in the first technical solution or the microbial preparation described in the second technical solution.

[0010] In certain embodiments, the amount of Bacillus coagulans LL08 added to the starter is at least 1×10 6 CFU / mL or 1×10 6 CFU / g.

[0011] The fourth technical solution provided by the present invention is the use of the Bacillus coagulans LL08 described in the first technical solution, or the microbial preparation described in the second technical solution, or the starter described in the third technical solution in the preparation of lactic acid or products containing lactic acid.

[0012] The fifth technical scheme provided by the present invention is a method for producing optically pure L-lactic acid by recycling fermentation, wherein the method comprises: introducing the Bacillus coagulans LL08 described in the first technical scheme, or the microbial preparation described in the second technical scheme, or the starter described in the third technical scheme into a fermentation medium for fermentation, and after the fermentation is completed, obtaining a fermentation liquid containing high optical purity L-lactic acid, recovering the bacterial cells and using the fermentation liquid as a seed culture to introduce the fermentation liquid into the next batch of fermentation medium for fermentation culture, and then performing a bacterial cell recovery operation on the fermentation liquid, and obtaining a fermentation liquid containing high optical purity L-lactic acid again; and repeating this cycle for multiple times to obtain optically pure L-lactic acid.

[0013] In certain embodiments, the first batch of fermentation medium contains 15 g / L yeast powder, and the amount of yeast powder added to the fermentation medium after recovery is 10% of the amount of yeast powder added to the first batch of fermentation medium.

[0014] In certain embodiments, the fermentation conditions are as follows: after the Bacillus coagulans to be tested is streaked and separated on a solid enriched LB medium, a single colony is taken and inoculated into the enriched LB medium, and the medium is shaken and cultured at 50°C and 200 r / min for 24 hours, and the medium is sealed with gauze for ventilation fermentation. The shake flask fermentation medium is inoculated at an inoculum of 10% to 15%, and the medium is sealed with aluminum foil for anaerobic fermentation, the culture temperature is 45 to 50°C, and the shake flask is shaken slowly (generally 80 to 100 r / min).

[0015] In certain embodiments, the shake flask fermentation medium includes: 80 g / L glucose, 15 g / L yeast powder, 2 g / L corn steep liquor powder, 0.2 g / L anhydrous magnesium sulfate, 25 g / L calcium carbonate, and the pH is adjusted to about 7.0 with hydrochloric acid or sodium hydroxide.

[0016] In certain embodiments, a flocculant is used to coagulate and settle the bacteria to achieve recovery of the bacteria.

[0017] In certain embodiments, the specific method of bacterial cell recovery is as follows: after the last batch of fermentation is completed, the fermentation liquid is placed at 50°C for 15 minutes, a flocculant is added, and the amount is 10mL in 1L of fermentation liquid, and then slowly shaken at 50°C for 10 minutes (generally to control the shaker at 75 rpm), and then the fermentation liquid is allowed to stand for natural sedimentation of the bacteria, generally standing for 45 minutes, most of the cells settle to the bottom of the container, the upper clear liquid is taken out, and the fermentation liquid with concentrated bacteria in the lower layer is left. The fermentation liquid containing the concentrated bacteria liquid is generally 10% of the total volume of the fermentation liquid, and a special shake flask fermentation medium for recovering bacteria is added to the concentrated bacteria liquid to restore the fermentation liquid to its original volume, and the anaerobic fermentation is carried out by sealing with aluminum foil, the culture temperature is 50°C, and the shake flask is slowly shaken (generally 80-100r / min). When LL08 is used as the strain, the fermentation is generally fermented for 12-14 hours, and the lactic acid concentration can reach 46-48g / L.

[0018] In certain embodiments, the recovered bacterial shake flask fermentation medium includes: 80 g / L glucose, 1.5 g / L yeast powder, 2 g / L corn steep liquor powder, 0.2 g / L anhydrous magnesium sulfate, 25 g / L calcium carbonate, and the pH is adjusted to about 7.0 with hydrochloric acid or sodium hydroxide.

[0019] The technical effects of the present invention are as follows:

[0020] The present invention isolates a batch of heat-resistant bacteria from spoiled cow's milk, and after a small amount of bacterial cell culture, a flocculant is added to observe the sedimentation, and the strain with the fastest sedimentation rate is selected, which is identified as Bacillus coagulans. Nitrosoguanidine is further used to induce a mutant strain with enhanced flocculation, among which the mutant numbered LL08 has the largest increase in flocculation, and after adding a small amount of special flocculation promoter, the cell sedimentation rate is significantly faster than that of the control bacteria. The above-mentioned LL08 strain is named Bacillus coagulans LL08, which has been deposited in the China Center for Type Microorganism Collection with a deposit number of CCTCC NO: 20211356 and a deposit time of November 2, 2021. When the LL08 strain is used for lactic acid fermentation, the bacteria can be collected by the method of bacterial sedimentation after the fermentation is completed for secondary fermentation, which can greatly reduce the use of organic nitrogen sources.

[0021] Biomaterial sample storage

[0022] Bacillus coagulans LL08, whose classification name is Bacillus coagulans, has been deposited in the China Center for Type Culture Collection, with the deposit number CCTCC NO: M20211356. The deposit date is November 2, 2021, and the deposit address is: Wuhan University, Wuhan, China. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Diagram of the lactic acid fermentation process of different mutant strains. DETAILED DESCRIPTION

[0024] The preferred embodiments of the present invention are described below. It should be understood that the embodiments are for better explaining the present invention and are not used to limit the present invention.

[0025] The method involved in the embodiment:

[0026] 1. Fermentation method: After the Bacillus coagulans to be tested is streaked and separated on a solid enriched LB medium, a single colony is taken and inoculated into the enriched LB medium, and the medium is cultured in a shaking incubator at 50°C and 200r / min for 24 hours, and the medium is sealed with gauze for ventilation and fermentation. The shake flask fermentation medium is inoculated at 10% volume, and the flask is sealed with aluminum foil for anaerobic fermentation. The culture temperature is 50°C, and the shake flask is shaken slowly (generally 80-100r / min). When conducting the bacterial cell recycling experiment, the amount of yeast powder is changed, and the other ingredients remain unchanged. The fermentation or culture temperature of the Bacillus coagulans used shows the best growth or fermentation performance at 45-50°C. In actual work, a higher temperature in this range should generally be used to reduce contamination by miscellaneous bacteria.

[0027] 2. The detection methods of bacterial mass, lactic acid content and optical purity are the same as those in the literature: [Tian Kangming, Shi Guiyang, Wang Zhengxiang. Study on the production of L-lactic acid by fermentation of Bacillus coagulans CICIM1821. Food and Fermentation Industries, 2011, 32(10): 245-248, 276].

[0028] 3. Method for identification of bacterial species: The method for 16s rDNA gene sequence analysis first obtains the PCR product of the 16srDNA gene of the strain according to the literature [Sui Xinhua. Experimental Tutorial on Prokaryotic Evolution and Systematic Taxonomy. Beijing: Science Press, 2011: 62-69 pages]. The PCR product is sent to Shanghai Shenggong Biotechnology Service Company for sequencing. After the sequence is obtained, the sequence homology analysis is performed according to the method in the above literature.

[0029] 4. Calculation of the remaining calcium carbonate in the fermentation broth: Take 100mL of fermentation broth and centrifuge at 8,000r / min, remove the supernatant, suspend the bacteria with pure water and restore the volume to 100mL and transfer to a conical flask. Use a burette to drop hydrochloric acid with a concentration of 3mol / L into the above suspension, shake the conical flask while dropping, hydrochloric acid reacts with calcium carbonate to produce bubbles, drop until the suspension no longer produces bubbles, and record the volume of hydrochloric acid consumed. If the volume of hydrochloric acid consumed is X liters, the remaining calcium carbonate in the fermentation broth can be roughly estimated to be (3X / 2)×100.1g.

[0030] The raw materials used in the examples are:

[0031] 1. Enriched LB medium: 10g / L yeast powder, 10g / L peptone, 0.1g / L corn steep liquor powder, 0.5g / L anhydrous calcium carbonate, 2g / L glucose, sterilize at 110℃ for 10min. Add 15g / L agar powder to make a solid enriched LB medium.

[0032] 2. LB medium: 5 g / L yeast powder, 10 g / L peptone, 10 g / L sodium chloride, sterilize at 115°C for 15 min and set aside.

[0033] 3. Shake flask fermentation medium: 80g / L glucose, 15g / L yeast powder, 2g / L corn steep liquor powder, 0.2g / L anhydrous magnesium sulfate, 25g / L calcium carbonate, adjust the pH to about 7.0 with hydrochloric acid or sodium hydroxide.

[0034] 4. Shake flask fermentation medium for recovering bacterial cells: 80 g / L glucose, yeast powder (see Example), 2 g / L corn steep liquor powder, 0.2 g / L anhydrous magnesium sulfate, 25 g / L calcium carbonate, pH adjusted to about 7.0 with hydrochloric acid or sodium hydroxide. Note: The composition of the shake flask fermentation medium for recovering bacterial cells is consistent with that of the shake flask fermentation medium, the only difference is the amount of yeast powder added, the specific amount of addition is shown in "Example 4 Mutant LL08 for bacterial cell recovery and re-fermentation", and the calcium carbonate needs to be tested for residual calcium carbonate in the fermentation liquid before adding fresh calcium carbonate to 25 g / L.

[0035] 5. Flocculation accelerator: Add 800 mL of warm water to a 1 L graduated beaker, add anhydrous ethanol to 30 g / L, add polyethyleneimine to 50 mg / L, add cationic polyacrylamide to 20 mg / L, add KBr to 0.5 g / L, and stir continuously in a 65°C water bath for 12 hours.

[0036] 6. MRS solid culture medium: Take 90 mL of pure water, add 2.5 g of MRS broth powder and 1.5 g of agar powder, heat and boil to dissolve all the powder, add water to 100 mL, sterilize at 115°C for 15 minutes for later use. MRS broth powder is a product of Qingdao Haibo Biotechnology Co., Ltd., product number HB0384-1. MRS is a special culture medium for lactic acid bacteria.

[0037] 7. Bacillus coagulans strains CICIM B0599, CICIM B1353, CICIM B1562, and CICIM B1821 were purchased from the China University Industrial Microbiology Resource Information Platform of Jiangnan University, and Bacillus coagulans ATCC7050 was purchased from Beijing Baozang Biotechnology Co., Ltd. The above 5 strains were used as common Bacillus coagulans for control experiments.

[0038] 8. Sources of raw materials and reagents: Corn syrup powder is a product of Beijing Hongrun Baoshun Co., Ltd. (Y042C); yeast powder is a product of Beijing Hongrun Baoshun Co., Ltd. (Y017C, yeast extract powder); glucose is a product of Xiwang Pharmaceutical Co., Ltd., which is food-grade anhydrous glucose. Calcium carbonate is a product of Jiangxi Mingyuan High-tech Materials Co., Ltd., which is a food additive light calcium carbonate; tap water is used to prepare the fermentation medium, and the rest are pure water. The rest of the reagents are domestic analytical grade or biochemical reagents purchased from Sinopharm Chemical Reagent Co., Ltd.

[0039] Example 1 Screening of Highly Flocculating Bacillus Coagulans

[0040] In July of each year (2016, 2017, and 2019), various discarded foods with high protein content, including dairy products, meat, and soy products, were searched in Turpan. The samples were refrigerated and taken back to the laboratory for separation. After the sample was crushed, it was suspended with physiological saline, and a small amount of liquid was streaked on the MRS solid culture medium for separation, and cultured at 50°C for 36 hours to form colonies. Preliminary experiments found that the colony morphology of the control bacteria Bacillus coagulans standard strain ATCC7050 and several other control bacteria were very similar. Based on this, strains with similar colony morphology were found. After streaking on the MRS culture medium, single colonies were taken and placed in 15% glycerol tubes and stored at -70°C for later use. A total of more than 3,000 strains were collected.

[0041] After the above strains were taken out one by one and separated by streaking, a single colony was inoculated with enriched LB and cultured in a flask at 45°C for 24 hours. After the culture was completed, the above culture solution was placed at room temperature for 30 minutes. After observing that the bacteria in some of the culture solutions aggregated, a flocculation promoter was added. The amount was 10 mL added to 1L of fermentation solution, and then slowly shaken at 45°C for 10 minutes (generally controlling the shaker to 75 rpm). Take an appropriate amount of culture solution and pour it into an 18×180CM glass test tube, place it in a 50°C constant temperature box for 60 minutes, and observe the fermentation solution. The results showed that most of the control bacteria and the bacteria of most of the screened strains were dispersed and suspended in the fermentation solution, and the bacteria of a few strains were obviously settled to the bottom (the remaining calcium carbonate in the fermentation solution generally settled to the bottom within 20 minutes). 21 strains with obvious sedimentation were selected for subsequent experiments. The 16s rDNA sequence of these 21 strains was analyzed and compared with the 16s rDNA sequence of the standard strain 7050. The results showed that the 16s rDNA sequence of 6 strains was more than 98.5% similar to the standard strain, and they can be identified as Bacillus coagulans. The above 21 strains have been deposited in the strain library of Wuxi Xianqin Biotechnology Co., Ltd., a cooperative enterprise of this project, and are numbered XQ20241-XQ20261. The supernatant was taken to detect OD 600 The results showed that the supernatants of XQ20244, XQ20249, and XQ20258 had the lowest turbidity, and their OD 600 The OD values ​​of the supernatants of the five strains of Bacillus coagulans used as controls under the same experimental conditions were 1.3, 0.9 and 1.2 respectively. Low turbidity indicates that there are the fewest bacteria in the supernatant, which means that the sedimentation rate of the bacteria is the fastest. 600 The calculated turbidities were all above 5.0.

[0042] Since L-lactic acid is easily metabolized in animals, it is more beneficial to health as food or feed. The above 21 strains of Bacillus coagulans were subjected to shake flask lactic acid fermentation and their lactic acid production and L-lactic acid content were detected. The results showed that the lactic acid of the strain numbered XQ20249 reached the highest value after 48 hours of fermentation, which was about 46g / L. Although it was not the highest, the optical purity of its L-lactic acid reached 99.3%, while the other strains were all below 98.5%. Bacillus coagulans can almost achieve high lactic acid production under appropriate fermentation tank fermentation technology, so the lactic acid production problem can be solved by improving the fermentation tank process conditions in the later stage, while the optical purity is generally difficult to improve. At the same time, considering that the goal of the present invention is to obtain a high flocculation strain, Bacillus coagulans XQ20249 was selected as the starting strain for subsequent experiments.

[0043] Table 1 Lactic acid yield and optical purity of different Bacillus coagulans

[0044]

[0045] Example 2 Mutagenesis screening of highly flocculating mutant strains

[0046] The starting strain of Bacillus coagulans XQ20249 was induced to mutagenesis using nitrosoguanidine. The mutagenesis method is as follows: XQ20249 was cultured in enriched LB medium for about 6 hours. When it reached the logarithmic growth phase, 10 mL of the bacteria was taken, centrifuged, and the supernatant was discarded. The bacteria were suspended in an equal volume of 2 mmol / L pH 6.0 phosphate buffer, and nitrosoguanidine pre-dissolved in phosphate buffer was added to about 1 mg / 10 mL. The culture was shaken at 42°C for 3 hours. Centrifuged, the supernatant was discarded, and the bacteria were resuspended in phosphate buffer. The bacteria were transferred to 30 mL of LB liquid culture medium and shaken at 45°C for 16 hours. The bacterial solution was diluted to about 10 8 After counting the cells (using a blood cell counting plate), place them in a test tube and let them settle for about 90 minutes. Use a blood collection needle to take the cells at the bottom. Place the cells in 50 mL of liquid culture medium and shake and culture at 45°C for 16 hours. Dilute the culture solution to about 10 8 After counting the bacteria (using a blood cell counting plate), put them into a test tube. After settling for about 90 minutes, use a blood collection needle to take the bacteria at the bottom layer. Repeat this method for 5 times in a row, which is a round of mutagenesis and screening. After the end, take the bacteria taken out by the last sedimentation and put them into LB medium to culture to the logarithmic phase, and then perform nitrosoguanidine mutagenesis and screening according to the above method. The above mutagenesis and screening were carried out 10 times in total. The bacteria obtained in the last time were diluted and coated on LB plates, and the number of colonies on each plate was controlled to be below 400. After preserving single colonies, take them out one by one, culture them according to Example 1, and add flocculants to do bacterial sedimentation experiments. A total of 846 mutant strains were screened. Among them, the supernatants of the strains numbered LL08, LL243, and LL891 were measured by OD600 The calculated turbidity was the lowest, namely 0.54, 0.52 and 0.60 respectively, indicating that its sedimentation performance was significantly better than other strains, so it was used in subsequent experiments.

[0047] Example 3 Basic fermentation performance of mutant strains

[0048] The mutant strains LL08, LL243, and LL891 obtained by mutagenesis screening were used to study the lactic acid fermentation performance. Figure 1 As shown. From the shake flask fermentation results, the final lactic acid production of the three strains was about 50-60g / L, and there was no great difference in the production and lactic acid optical purity (Table 2), but the bacterial volume and lactic acid production of the LL08 strain at 12hr were close to the maximum value and significantly higher than the other strains, with the characteristics of rapid fermentation. Obviously, LL08 has greater potential for subsequent process optimization or further genetically modified chassis cells, so LL08 was selected as the basis for subsequent work. After identification, the genus LL08 was Bacillus coagulans, and its 16s rDNA sequence was completely consistent with the starting strain XQ20249. The 16s rDNA sequence of LL08 is listed in: SEQ ID NO: 1.

[0049] Table 2 Results of lactic acid fermentation of Bacillus coagulans mutants LL08, LL243, and LL891

[0050] Strain No. Fermentation time (hr) Lactic acid content of fermentation broth (g / L) Lactic acid optical purity LL08 48 46 99.3 LL243 48 51 99.3 LL891 48 48 99.4

[0051] Example 4: Use of mutant LL08 for bacterial cell recovery and re-fermentation

[0052] The strain LL08, the starting strain XQ20249 and five control strains were all used for lactic acid fermentation, and the fermentation ended at 48h. After the fermentation, the fermentation liquid was placed at 50℃ for 15min, and a flocculant was added, with the amount of 10mL added to 1L fermentation liquid, and then slowly shaken at 50℃ for 10min (generally controlled to control the shaker at 75 rpm), and the sedimentation was observed. The results showed that all control strains did not show bacterial aggregation after standing for 2hr. After standing for 45min, most of the LL08 bacteria sank to the bottom, and its upper fermentation liquid became translucent. XQ20249 had obvious bacterial sedimentation, but the turbidity of the upper fermentation liquid was significantly higher than that of LL08. The upper liquid was carefully sucked away with a pipette. Among them, the five control bacteria could not be used for further bacterial recovery experiments because the bacteria did not show obvious sedimentation and the upper liquid could not be separated. LL08 and XQ20249 can remove about 90% of the upper liquid. The amount of cell in the fermentation broth was tested, and the results showed that the cell concentration of the original fermentation broth of LL08 was 5.8 g / L (calculated by cell dry weight DCW), and the cell concentration in the remaining liquid obtained after removing the supernatant was 50 g / L. According to the cell amount and volume, the cell recovery rate in the remaining fermentation broth reached 86%. The cell concentration of the original fermentation broth of XQ20249 was 6.2 g / L (DCW), and the cell concentration in the remaining liquid obtained after removing the supernatant was 36 g / L. According to the cell amount and volume, the cell recovery rate in the remaining fermentation broth was 58%.

[0053] The remaining culture liquid containing concentrated cells of the above two strains was supplemented with a fermentation medium for recovering cells (the composition of the culture medium is shown in the Materials and Methods section, in which the amount of yeast powder added reached a final concentration of 0.5g / L, 1g / L, 1.5g / L, 2g / L, 3g / L, 4g / L, and 5g / L, respectively) to restore the original volume and carry out shake flask fermentation. The results showed that for the LL08 strain, only an organic nitrogen source equivalent to 10% of the original fermentation liquid was added to the fermentation medium for recovering cells to obtain a lactic acid fermentation yield of about 46g / L or more. The formula of the recovered cell lactic acid shake flask fermentation medium used is as follows: 80g / L glucose, 1.5g / L yeast powder, 2g / L corn steep liquor dry powder, 0.2g / L anhydrous magnesium sulfate, 25g / L calcium carbonate, and the pH was adjusted to about 7.0 with hydrochloric acid or sodium hydroxide. The lactic acid yield was 46-48g / L. The optical purity was between 99.3% and 99.4%. The time to reach the highest level of lactic acid is 12 to 14 hours. The level of lactic acid fermentation is comparable to the fermentation level of the initial cells of LL08 in Example 3, which may be due to the fact that the cell growth time is not required and the fermentation time after the first cell recovery is greatly shortened. Table 3 shows the results of lactic acid fermentation of two strains of Bacillus coagulans using recovered cells. The fermentation broth obtained from the second fermentation can also be fermented using the method of recovering cells from the first fermentation broth, and the third fermentation is performed after the cells are obtained. The culture medium and fermentation method are the same as those of the second fermentation, and the results obtained are also similar to those listed in Table 3.

[0054] Table 3 Results of lactic acid fermentation of Bacillus coagulans LL08 and XQ20249 using recovered cells

[0055] Strain No. Fermentation time (hr) Lactic acid content of fermentation broth (g / L) Lactic acid optical purity LL08 12～14 46～48 99.3～99.4％ XQ20249 18～20 40～42 99.3～99.4％

[0056] As shown in Table 3, the fermentation time of XQ20249 for recovering the bacterial cells is significantly longer than that of LL08, and the lactic acid production of the fermentation broth is also lower than that of LL08. Further experiments show that XQ20249 needs to add about 4g / L of yeast powder to achieve a fermentation lactic acid production of more than 46g / L. Since yeast powder is a high-cost raw material in lactic acid fermentation, it is obvious that LL08 has a better performance in the secondary fermentation of bacterial cell recovery.

[0057] After the lactic acid fermentation is completely completed, LL08 cells can be naturally settled to the bottom of the container by adding a small amount of flocculation promoter, and then the settled cells can be collected and recovered by centrifugation. Since the cells are concentrated at the bottom, only 10% of the volume of the fermentation liquid at the bottom needs to be centrifuged to collect nearly 90% of the bacteria, which can greatly reduce the workload of centrifugation operation and significantly reduce the cost of cell collection. The obtained bacteria do not contain filter aids and are more suitable for use as feed.

[0058] Although the present invention has been disclosed as above in the form of a preferred embodiment, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A strain of Bacillus coagulans LL08, characterized in that: The Bacillus coagulans LL08 was deposited in the China Center for Type Culture Collection on November 2, 2021, with the deposit number being CCTCC NO: 20211356.

2. A microbial preparation containing the Bacillus coagulans LL08 according to claim 1.

3. The microbial preparation according to claim 2, characterized in that The bacterial concentration of Bacillus coagulans LL08 in the microbial preparation is at least 1×10 6 CFU / mL or 1×10 6 CFU / g.

4. A leavening agent, characterized in that The fermentation agent contains the Bacillus coagulans LL08 according to claim 1 or the microbial preparation according to claim 2 or 3.

5. Use of the Bacillus coagulans LL08 according to claim 1, or the microbial preparation according to claim 2 or 3, or the starter according to claim 4 in the preparation of lactic acid or products containing lactic acid.

6. A method for producing optically pure L-lactic acid by recycling fermentation, characterized in that: The method comprises the steps of introducing the Bacillus coagulans LL08 of claim 1, or the microbial preparation of claim 2 or 3, or the starter of claim 4 into a fermentation medium for fermentation, and obtaining a fermentation liquid containing high optical purity L-lactic acid after the fermentation is completed, recovering the bacterial cells and introducing the fermentation liquid as a seed culture into the next batch of fermentation medium for fermentation culture, and then recovering the bacterial cells of the fermentation liquid; and repeating the process for multiple times to obtain optically pure L-lactic acid.

7. The method according to claim 6, characterized in that The amount of yeast powder added to the fermentation medium after recovery is not less than 10% of the amount of yeast powder added to the first batch of fermentation medium.

8. The method according to claim 6, characterized in that The fermentation conditions are: anaerobic fermentation, inoculation amount of 10%-15%, temperature of 45-50°C, and rotation speed of 80-100r / min.

9. The method according to claim 6, characterized in that The flocculant is used to coagulate and settle the bacteria to achieve bacterial recovery.

10. The method according to claim 9, characterized in that The specific method of bacterial cell recovery is as follows: after the last batch of fermentation is completed, place it at 50℃ for 15 minutes, add a flocculation promoter, and then slowly shake it at 50℃ and 75 rpm for 10 minutes. Then, let the fermentation liquid stand and wait for the bacteria to settle naturally. Remove the upper clear liquid and leave the fermentation liquid with concentrated bacteria in the lower layer for the next batch of fermentation.

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