Application of ergothioneine in increasing viable count of bacillus coagulans in high-density fermentation culture

Abstract

The invention discloses application of ergothioneine in increasing the viable count of bacillus coagulans in high-density fermentation culture, and belongs to the technical field of biological fermentation. According to the invention, by adding an antioxidant ergothioneine (EGT), the effects of relieving oxidative stress, enhancing the metabolic activity of thalli and improving the fermentation efficiency are discussed. Meanwhile, by combining culture medium optimization and amplification culture of a 5L fermentation tank, process parameters are optimized, so that the viable count is increased, and a scientific basis is provided for industrial production. The invention shows that the ergothioneine can effectively remove active oxygen, remarkably relieve oxidative stress and improve the survival rate of thalli, fermentation process optimization and 5L fermentation tank amplification are combined, the fermentation efficiency of the bacillus coagulans is greatly improved, and the viable count is further increased to 1.28 * 10 < 10 > CFU / mL. The optimized process is suitable for large-scale production, and important reference is provided for industrial application and product development of bacillus coagulans.

Description

Technical Field

[0001] The present invention belongs to the technical field of biological fermentation, and particularly relates to the application of ergothioneine in increasing the viable cell count of Bacillus coagulans in high-density fermentation culture. Background Art

[0002] Bacillus coagulans is a facultative anaerobic bacterium that can form spores. Based on its probiotic characteristics [1] and safety [2] , Bacillus coagulans has been widely used in the livestock industry [3] , aquaculture [4] , agriculture and forestry [5] and the field of medical health [6] . Although the benefits of Bacillus coagulans in different industries have been proven, its application still faces certain limitations. Some studies have failed to link culture conditions such as temperature and inorganic salts with the growth of Bacillus coagulans [7,8] , and there are also differences in the biomass of the fermentation broth. Secondly, although studies have shown that Bacillus coagulans can resist the damage of free radicals by accelerating metabolism and producing substances such as lactic acid [9] , this method may consume its lifespan, resulting in the viable cell count not meeting expectations. It has also been found that when the freeze-dried powder of Bacillus coagulans is added to chocolate and refrigerated for three months, the viable cell count significantly decreases

[10] . Although chocolate contains antioxidant substances such as phenols, its protective effect on the target strain in resisting oxidative stress still needs to be further improved.

[0003] Ergothioneine is a natural sulfur-containing amino acid, mainly derived from edible fungi and can also be synthesized by microorganisms. Due to its strong ability to scavenge reactive oxygen species and chelate metal ions, ergothioneine is regarded as a new type of natural antioxidant. It has been found that adding ergothioneine to pig semen stored at room temperature can improve the quality of pig sperm

[11] ; ergothioneine can effectively inhibit the browning of the surface and internal tissues of the caps of Agaricus bisporus

[12] . In addition, experiments have verified that it can also be used as a strain protectant to improve the survival rate and metabolic activity of probiotics and improve the quality of probiotic products

[13] . Therefore, ergothioneine has great potential in promoting the fermentation culture of Bacillus coagulans.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] In order to overcome the disadvantages and deficiencies of the prior art, the primary object of the present invention is to provide the application of ergothioneine in increasing the viable cell count of Bacillus coagulans in high-density fermentation culture.

[0006] Another object of the present invention is to provide a culture medium for high-density fermentation of Bacillus coagulans with a high viable cell count.

[0007] Still another object of the present invention is to provide a method for high-density fermentation of Bacillus coagulans with a high viable cell count.

[0008] The object of the present invention is achieved by the following technical solutions:

[0009] Application of ergothioneine in increasing the viable cell count of Bacillus coagulans in high-density fermentation culture.

[0010] Preferably, in the above application, the concentration of ergothioneine in the culture system is 0.8 - 1.5 mg / L; further preferably 0.8 - 1.2 mg / L; and still further preferably 1 ± 0.1 mg / L.

[0011] A culture medium for high-density fermentation of Bacillus coagulans with a high viable cell count, comprising components with the following concentrations: carbon source 8 - 15 g / L, nitrogen source 35 - 45 g / L, K2HPO4 5 - 15 g / L, ergothioneine 0.8 - 1.5 mg / L, pH 6.5 - 7.

[0012] Preferably, the culture medium comprises components with the following concentrations: carbon source 10 - 15 g / L, nitrogen source 35 - 40 g / L, K2HPO4 10 - 15 g / L, ergothioneine 0.8 - 1.2 mg / L, pH 6.5 - 7.

[0013] Preferably, the carbon source is selected from at least one of glucose and soluble starch; further preferably glucose.

[0014] Preferably, the nitrogen source is a composite nitrogen source, which is selected from at least one of dried corn steep liquor, corn steep liquor protein, soy peptone, tryptone and yeast powder for compounding; further, the mass proportion of yeast powder in the composite nitrogen source is 75%; still further preferably, it is selected from the compounding of soy peptone and yeast powder in a mass ratio of 1:3.

[0015] Still further preferably, the culture medium comprises components with the following concentrations: glucose 10 - 15 g / L, nitrogen source 35 - 40 g / L, K2HPO4 10 - 15 g / L, ergothioneine 0.8 - 1.2 mg / L, pH 6.5 - 7; the nitrogen source is selected from the compounding of soy peptone and yeast powder in a mass ratio of 1:3.

[0016] Most preferably, the culture medium comprises components with the following concentrations: glucose 15 g / L, nitrogen source 35 g / L, K2HPO4 15 g / L, ergothioneine 1 mg / L, pH 7; the nitrogen source is selected from the compounding of soy peptone and yeast powder in a mass ratio of 1:3.

[0017] A culture method for high-density fermentation of Bacillus coagulans with a high viable cell count, comprising the following steps:

[0018] (1) Inoculate a single colony of Bacillus coagulans into an LB liquid medium for activation culture to obtain a seed solution;

[0019] (2) Inoculate the seed solution obtained in step (1) into the medium for high-density fermentation of Bacillus coagulans with a high viable cell count for fermentation culture;

[0020] (3) Inoculate the seed solution obtained in step (1) into the medium for high-density fermentation of Bacillus coagulans with a high viable cell count for scale-up culture.

[0021] Preferably, the conditions for the activation culture in step (1) are shaking culture at 35-45°C and 150-200 r / min for 20-24 h; more preferably, shaking culture at 37°C and 180 r / min for 24 h.

[0022] Preferably, the inoculation amount of the seed solution in step (2) is 2-10%; more preferably, 6±0.5%.

[0023] Preferably, the conditions for the fermentation culture in step (2) are shaking culture at 35-45°C and 100-200 r / min for 10-24 h; more preferably, shaking culture at 45°C and 200 r / min for 24 h.

[0024] Preferably, the inoculation amount of the seed solution in step (3) is 2-10%; more preferably, 6±0.5%.

[0025] Preferably, the conditions for the scale-up culture in step (3) are shaking culture at 35-45°C and 100-250 r / min for 24-48 h, with an air flow rate of 0.6-1.0 L / min; more preferably, shaking culture at 45°C and 200 r / min for 48 h, with an air flow rate of 0.7 L / min.

[0026] The present invention has the following advantages and effects compared with the prior art:

[0027] In this invention, by adding the antioxidant ergothioneine (EGT), its effects of alleviating oxidative stress, enhancing the metabolic activity of bacteria, and improving fermentation efficiency are explored. Meanwhile, combined with medium optimization and scale-up cultivation in a 5L fermenter, process parameters are optimized to increase the viable cell count and provide a scientific basis for industrial production. This invention shows that ergothioneine can effectively scavenge reactive oxygen species, significantly alleviate oxidative stress, improve the survival rate of bacteria, and combined with fermentation process optimization and scale-up in a 5L fermenter, greatly improve the fermentation efficiency of Bacillus coagulans, and the viable cell count is further increased to 1.28×1010 CFU / mL. The optimized process is applicable to larger-scale production, providing an important reference for the industrial application and product development of Bacillus coagulans. Description of the Drawings

[0028] Figure 1 It is the determination of the growth curve of Bacillus coagulans.

[0029] Figure 2 It is the influence of different single factors on the viable cell count during the fermentation of Bacillus coagulans; among them, A: inoculation amount %; B: initial pH value; C: liquid loading volume / mL; D: rotation speed / (r / min).

[0030] Figure 3 It is the influence of different carbon sources and different concentrations of the optimal carbon source on the fermentation of Bacillus coagulans; among them, A: different carbon sources, G - Gluoose: glucose, ss - Soluble starch: soluble starch, s - Sucrose: sucrose, M - Maltose: maltose, T - Trehalose: trehalose, sor - Sorbitol: sorbitol; B: different glucose concentrations.

[0031] Figure 4 It is the influence of the compounding of yeast powder and different nitrogen sources at a mass ratio of 3:1 on the fermentation of Bacillus coagulans; among them, Ammonium citrate dibasic: dibasic ammonium citrate, Soybean meal: soybean meal, CO(NH2)2: urea, Beefextract powder: beef powder, Corn syrup dry powder: dry corn syrup, Corn syrup proteinpowder: corn syrup protein, Soya peptone: soy peptone, Tryptone: tryptone.

[0032] Figure 5 It is the influence of different inorganic salts on the fermentation of Bacillus coagulans.

[0033] Figure 6Effects of different antioxidants on the fermentation of Bacillus coagulans; among them, Grape seed extract: grape seed extract, Cysteine: cysteine, Aspartate: aspartic acid, Vitamin C: vitamin C, EGT: ergothioneine.

[0034] Figure 7 Effects of different concentrations of ergothioneine on Bacillus coagulans.

[0035] Figure 8 Effects of different concentrations of ergothioneine on the reactive oxygen species level of Bacillus coagulans.

[0036] Figure 9 Results of experimental optimization; among them, A: results of orthogonal experiments; B: growth curves before and after optimization.

[0037] Figure 10 Effects of different culture conditions on Bacillus coagulans in a 5L fermenter; among them, A: effects of different rotation speeds on the viable cell count; B: effects of different aeration rates on the viable cell count.

[0038] Figure 11 Effects of changes in aeration rate in a 5L fermenter; among them, A: dissolved oxygen changes under different aeration rates; B: results of alkali supplementation amounts under different aeration rates.

[0039] Note: Different letters indicate significant differences, and the same letters indicate no significant differences. Detailed implementation manners

[0040] The present invention will be further described in detail below in conjunction with the embodiments and the accompanying drawings, but the implementation manners of the present invention are not limited thereto. For the test methods without specific experimental conditions noted in the following embodiments, they are generally carried out according to conventional experimental conditions or according to the experimental conditions recommended by the manufacturer. The materials, reagents, etc. used, unless otherwise specified, are reagents and materials obtained from commercial channels.

[0041] Example 1

[0042] 1. Materials and methods

[0043] 1.1 Materials and instruments

[0044] Bacillus coagulans BNCC192399, purchased from Beina Biological Co., Ltd. (provided by the national preservation center, the recommended culture temperature of this strain is 45°C), and stored at -80°C using a freeze-dried glycerol tube.

[0045] Culture medium: The seed culture medium is LB medium (10 g / L peptone, 5 g / L yeast extract, 10 g / L NaCl, pH 7.4). The fermentation medium is the optimized formula, and the specific components are shown in Table 1 for details.

[0046] Reagents: Glucose, soluble starch, sucrose, maltose, trehalose, sorbitol, yeast powder, diammonium citrate, soybean meal, (NH4)2SO4, urea, beef powder, dried corn steep liquor, corn steep liquor protein, soy peptone, tryptone, grape seed extract, produced by Guangzhou Chemical Reagent Company and purchased from Yuanye Reagent Co., Ltd.; reagents such as MgSO4, CaCl2, MnSO4, K2SO4, Na2PO4, KH2PO4, K2HPO4, NaCl, cysteine, aspartic acid, vitamin C and ergothioneine were produced by Macklin Reagent Company and purchased from Haige Biotechnology Co., Ltd. The reactive oxygen species detection kit S0033S was purchased from Beyotime Co., Ltd. PBS buffer was produced by Bio-sharp Reagent Co., Ltd. and purchased from Haige Biotechnology Co., Ltd.

[0047] 1.2 Main Instruments

[0048] Laminar flow hood, Sujing Antai Air Technology Co., Ltd.; Microplate reader SAF-680T, Shanghai Bajiu Technology Co., Ltd.; High-pressure steam autoclave BXM-30R, Shanghai Boxun Medical Biological Instrument Co., Ltd. Ultra-low temperature freezer U410, Eppendorf Company, Germany; Biochemical incubator LRH-70, Shanghai Yiheng Scientific Instrument Co., Ltd. Continuous wavelength multi-functional microplate detection platform SpectraMax i3x, produced by Molecular Devices. 5L glass fermenter BRM-A5G, Jiangsu Longteng Biological Equipment Co., Ltd.

[0049] 1.3 Activation and Cultivation of Bacterial Strains

[0050] The Bacillus coagulans BNCC192399 stored in a glycerol tube (-80 °C) was thawed to room temperature, and single colonies were isolated on LB solid medium by the streak plate method. Single colonies were picked and inoculated into LB liquid medium, and cultured with shaking at 37 °C and 180 r / min for 24 h to obtain the second-activated seed liquid, which was stored short-term at 4 °C. Subsequently, the seed liquid was cultured to the logarithmic growth phase and inoculated into LB medium at an inoculation amount of 2% (v / v). The corresponding viable cell count of the seed liquid was about 1×10 6 CFU / mL, and fermentation culture was carried out under the same conditions

[14] , and the fermentation conditions were shaking culture at 37 °C and 180 r / min for 18 h.

[0051] 1.4 Determination of the Growth Curve of Bacillus coagulans

[0052] During the fermentation culture process, starting from 0 h, three parallels were set for each sample, and samples were taken every 2 h, with 3 parallel samples taken each time. The uninoculated medium was used as a blank control, and the absorbance value (OD 600). Using the cultivation time (h) as the abscissa and OD 600 as the ordinate, the growth curve of Bacillus coagulans was plotted.

[0053] 1.5 Single-factor experiment on the cultivation conditions of Bacillus coagulans

[0054] According to the suggestions of the national preservation center for the cultivation of this strain (the optimal temperature is 45°C), this experiment will not explore the temperature in depth, but only compare the normal cultivation temperature (37°C), the temperature lower than the cultivation temperature (30°C), and the recommended temperature (45°C). Therefore, the initial inoculation amount was set at 2% (v / v), natural pH, the liquid loading volume was 100 mL / 250 mL, the shaker speed was 150 r / min, and the cultivation was carried out for 18 h. The effects of inoculation amount (2%, 4%, 6%, 8%, 10%), pH (6.0, 6.5, 7.0, 7.5, 8.0), liquid loading volume (30 mL / 250 mL, 50 mL / 250 mL, 70 mL / 250 mL, 90 mL / 250 mL), and shaker speed (120 r / min, 140 r / min, 160 r / min, 180 r / min, 200 r / min) on the viable cell count of the strain were investigated respectively.

[0055] 1.6 Single-factor experiment on the optimization of the culture medium of Bacillus coagulans

[0056] According to the suggestions of the national preservation center for the cultivation of this strain, the original medium was LB basal medium. Based on the experimental results of the optimization of the fermentation conditions of Bacillus coagulans above, by changing a single variable, the effects of carbon source (1% glucose, soluble starch, sucrose, maltose, trehalose, sorbitol, and the optimal carbon source was selected for exploration at five concentrations of 0.5%, 1%, 1.5%, 2%, 2.5%), nitrogen source (the optimal combination of yeast powder: other nitrogen sources = 3:1, 3% yeast powder was compounded with 1% diammonium citrate, soybean meal, (NH4)2SO4, urea, beef powder, dried corn steep liquor, corn steep liquor protein, soy peptone, and tryptone), inorganic salts (1% MgSO4, CaCl2, MnSO4, K2SO4, Na2PO4, KH2PO4, K2HPO4, and NaCl), and growth factors with antioxidant effects (grape seed extract, cysteine, aspartic acid, vitamin C, and ergothioneine were screened from concentrations of 1 mg / L, 10 mg / L, 100 mg / L respectively, and the optimal group was selected for exploration with a concentration difference of 0.5 mg / L) on the viable cell count of Bacillus coagulans were investigated.

[0057] 1.7 Orthogonal experiment design

[0058] Orthogonal experiment is an experimental method for multi-factor analysis by selecting representative level combinations from all levels. Based on the results of the single-factor experiments on the fermentation conditions and culture medium of Bacillus coagulans mentioned above, an orthogonal experiment of four factors, namely carbon source, nitrogen source, inorganic salts, and antioxidant growth factors, was designed. The maximum viable cell count was measured to determine the culture plan, and three replicates were made for each group.

[0059] 1.8 Detection of reactive oxygen species (ROS)

[0060] To explore the effect of ergothioneine on the ROS level of Bacillus coagulans, the fluorescence probe method was used in this study to detect the accumulation of ROS in the cells during fermentation. The activated Bacillus coagulans was inoculated into the culture medium at an inoculation amount of 6% (v / v). A control group without ergothioneine and experimental groups with ergothioneine (0.5, 1, 1.5, 2, 2.5 mg / L) were set up, and samples were taken at 12 h of fermentation. Dichlorodihydrofluorescein diacetate (DCFH-DA) in the ROS kit was used as a fluorescence probe. After preparing the working solution according to the instructions, the fermentation broth was centrifuged to collect the cells (5000 rpm, 5 min), washed with PBS, and then added to the DCFH-DA working solution (10 μM). The cells were incubated at 37 °C in the dark for 30 min, and then washed again with PBS to remove the unbound probe. The fluorescence intensity of the samples was detected with a microplate reader at an excitation wavelength of 485 nm and an emission wavelength of 525 nm. The ROS level was represented by the fluorescence intensity, and LB medium was used as a blank control. The experiment was repeated three times, and the data were expressed as "mean ± standard deviation".

[0061] 1.9 Scale-up culture in a 5 L fermenter with different parameters

[0062] According to the experimental results of the optimized culture medium above, 2 L of culture medium was prepared, and inoculation was carried out by the flame method with an inoculation amount of 6% (v / v). Different fermenter conditions (rotation speeds of 50 r / min, 100 r / min, 200 r / min, and 300 r / min and different aeration rates) were set, and scale-up culture was carried out at 45 °C and pH = 7 for 48 h. The scale-up culture fermenter parameters were determined by specific total biomass, dissolved oxygen, and alkali supplementation amount.

[0063] 1.10 Data statistical analysis

[0064] All experimental data were repeated three times, and the results were expressed as "mean ± standard deviation". Graphpad Prism 9.5 was used for drawing and variance analysis, and the significance levels were set as P < 0.05 (significant difference) and P < 0.01 (extremely significant difference). Orthogonal Design Assistant II V3.1 was used to process the orthogonal experiment data.

[0065] 2. Results and analysis

[0066] 2.1 Determination of the growth curve of Bacillus coagulans

[0067] According to the method in 1.4, the OD of the fermentation broth was measured every 2 h 600 , and the growth curves of Bacillus coagulans at 30 °C, 37 °C and 45 °C were plotted (see Figure 1 ). It can be seen from the growth curves that there are certain differences in the growth trends of Bacillus coagulans at different temperatures. Within 0 - 6 h, the bacteria are in the lag phase with a low growth rate, and there is no obvious difference among the three temperature conditions at this stage. After 6 - 8 h, the bacteria enter the logarithmic phase, and the growth rate is faster at 45 °C, showing a steeper growth trend compared with 30 °C and 37 °C. Within 10 - 14 h, the bacteria enter the stationary phase. The maximum OD 600 value of the 45 °C group reaches 0.919, while those of the 37 °C and 30 °C groups are 0.870 and 0.793 respectively, indicating that 45 °C is more conducive to the bacteria reaching a higher biomass level in a short time. However, after 14 h, the bacteria enter the decline phase, and the OD 600 of the bacteria in the 45 °C group decreases faster than the other two groups. It may be that the metabolic activity of the bacteria is higher under high temperature conditions, resulting in faster consumption of nutrients, so that they enter the decline stage earlier, shortening the fermentation time and reducing the fermentation cost. The maximum viable count of Bacillus coagulans cultured at 45 °C is 3.5×10 6 CFU / mL, which is the result before optimization. And 45 °C was selected for the subsequent investigation of the inoculation amount.

[0068] 2.2 Maximum viable count of Bacillus coagulans at different inoculation amounts

[0069] The inoculation amount is the volume ratio of the seed liquid to the medium, which directly affects the growth and reproduction space of the bacteria. An excessive inoculation amount may lead to the accumulation of harmful metabolites in the medium, thus inhibiting the normal growth of the bacteria, and at the same time increasing the preparation cost of the seed liquid; while a too low inoculation amount will prolong the fermentation cycle

[15] . The results of the effects of different inoculation amounts on the growth of Bacillus coagulans are shown in Figure 2 A. It can be observed from Figure 2 A that when the inoculation amount is 1.0% - 5.0%, the effect on fermentation is small, but as the inoculation amount increases (5.0% - 10.0%), the fermentation speed significantly accelerates and the lag phase shortens. Therefore, selecting an inoculation amount of 6% can balance the growth of viable counts and production efficiency on the premise of ensuring fermentation stability, and has certain practical application value. Select 45 °C and 6% inoculation amount for the subsequent investigation of pH.

[0070] 2.3 Maximum viable count of Bacillus coagulans at different initial pH values

[0071] During the cultivation process of microorganisms, the pH value of the cultivation environment affects their growth activity by influencing the microbial metabolic process.

[16] . Bacillus coagulans is a homofermentative lactic acid bacterium that can produce a large amount of lactic acid. During the cultivation process, a large amount of acid is produced, which causes the pH value of the cultivation environment to change. Therefore, the impact of pH on its growth is particularly prominent in this experiment. The results are as Figure 2 shown in B of

[0072] As can be seen from Figure 2 B of 7 , when the pH value is set to 6.0 - 6.5, the change in the viable count of Bacillus coagulans is small; when the pH value is 7, the viable count of Bacillus coagulans is the largest, reaching 6.2×10

[0073] 2.4 Maximum viable count of Bacillus coagulans under different liquid volumes and shaker speeds

[0074] Bacillus coagulans is a facultative anaerobic microorganism. The amount of liquid volume in the fermentation process affects the oxygen transfer rate. When the shaker speed remains the same, when the liquid volume is less, the fermentation broth is prone to tumbling, increasing the dissolved oxygen content; conversely, when the liquid volume is more, the dissolved oxygen content decreases. The exploration results of this condition are as Figure 2 shown in C of 7 . When the liquid volume is 30 mL / 250 mL, the strain obtains the maximum viable count of 3×10

[0075] CFU / mL, and the difference under this condition reaches an extremely significant level (P < 0.01). Therefore, 30 mL / 250 mL is used as the optimal liquid volume. Select 45 °C, 6% inoculum amount, pH 7, and 30 mL / 250 mL liquid volume for subsequent investigation of the liquid volume. Figure 2 As shown in D of 8 CFU / mL, which is the maximum viable count of Bacillus coagulans at this time. Therefore, 200 r / min is selected as the optimal shaker speed. Select 45 °C, 6% inoculum amount, pH 7, 30 mL / 250 mL liquid volume, and 200 r / min shaker speed for subsequent fermentation optimization experiments.CFU / mL, significantly higher than the results at other rotation speeds, reaching an extremely significant level (P < 0.01). Excessively high rotation speeds may cause bacteria to aggregate at the bottom or result in violent bubbles in the culture medium, thus restricting growth. Therefore, 200 r / min was determined as the optimal shaker rotation speed for Bacillus coagulans.

[0076] In summary, the results of the single-factor experiment on the culture conditions of Bacillus coagulans are as follows: 45 °C, inoculation amount of 6% (v / v), initial pH 7.0, liquid loading of 30 mL / 250 mL, rotation speed of 200 r / min; under these optimized culture conditions, the maximum viable cell count of Bacillus coagulans is 1.28×10 8 CFU / mL, which is two orders of magnitude higher than before optimization (3.5×10 6 CFU / mL).

[0077] 2.5 Effects of different carbon sources on the growth of Bacillus coagulans

[0078] Carbon sources provide the main energy source during the growth of microorganisms, but different carbon sources can affect the growth rate of cells and the structure of the microbial community

[17] . Different strains have different utilization efficiencies for various carbon sources. Therefore, it is of great significance to study the effects of different carbon sources on the growth of Bacillus coagulans. Based on the experimental results of the optimization of the fermentation conditions of Bacillus coagulans above, the relevant experimental results are shown in Figure 3 A. Glucose has the most significant effect on the cultivation of the strain, and the viable cell count reaches 1.49×10 8 CFU / mL. According to the significant difference analysis, the results of the glucose group reach an extremely significant level (P < 0.01); as a monosaccharide that can quickly enter the metabolic pathway of Bacillus coagulans, glucose directly provides power for cells, and the metabolites are stable without producing excessive by-products or harmful substances; glucose is inexpensive and highly available. Therefore, glucose was used as the optimal carbon source for this strain in this experiment.

[0079] Based on the selection of the optimal carbon source, this experiment continued to explore the optimal carbon source concentration and investigated the effects of different concentrations of glucose on the maximum viable cell count of Bacillus coagulans. The results are shown in Figure 3 B. The viable cell count reached the maximum when the glucose concentration was 1%, reaching 1.49×10 8 CFU / mL. Since too low a concentration of glucose cannot be fully utilized by Bacillus coagulans, it restricts its growth rate. Too high a concentration of glucose will produce a substrate inhibition effect, affecting the catalytic reaction rate of enzymes, thus restricting the growth of the bacteria. Therefore, 1% glucose was used as the optimal carbon source for this strain in this experiment.

[0080] 2.6 Effects of different nitrogen sources on Bacillus coagulans

[0081] Yeast powder contains rich nutrients such as amino acids, peptides, and minerals. The small-molecule nutrients it contains can be rapidly absorbed by cells, promoting cell reproduction and division, and achieving a relatively high cell density in a short time. The original nitrogen source based on LB is a complex nitrogen source: a ratio of three times yeast powder and one time peptone. Therefore, in this experiment, yeast powder is not tested, but the nitrogen source with a relatively small proportion is explored. Among them, the nitrogen source is divided into organic nitrogen source and inorganic nitrogen source for testing, and the results are as Figure 4 shown.

[0082] Compared with the organic nitrogen source, the growth trend of the bacteria in the inorganic nitrogen source is relatively poor. When yeast powder and soy peptone are combined, the number of viable bacteria reaches the maximum, up to 1.085×10 9 CFU / mL, and the difference reaches an extremely significant level (P<0.01). Yeast powder and soy peptone, as organic nitrogen sources, are rich in various amino acids and trace elements and are relatively economical. Therefore, in this experiment, yeast powder + soy peptone is used as the best complex nitrogen source.

[0083] 2.7 Results of inorganic salt optimization

[0084] The addition of inorganic salts is crucial for cell growth during microbial fermentation and cultivation. Different strains have different requirements for inorganic salts, and different inorganic salts play key roles in cell metabolism, division, activity, and the overall growth environment. Therefore, this experiment explores various inorganic salts. The results are as Figure 5 shown. When K2HPO4 is added, the growth trend of Bacillus coagulans is the best, and the number of viable bacteria reaches 8.35×10 7 CFU / mL. According to the significant difference analysis, Bacillus coagulans cannot effectively utilize the seven inorganic salts of MgSO4, CaCl2, MnSO4, K2SO4, Na2PO4, KH2PO4, and NaCl, and the number of its viable bacteria is much lower than that of K2HPO4, and the difference reaches an extremely significant level (P<0.01). K2HPO4 provides a source of phosphorus and potassium ions for the culture medium, which is beneficial to the growth of Bacillus coagulans. At the same time, K2HPO4 can be used as a buffer to maintain the stability of the fermentation broth. Therefore, in this experiment, K2HPO4 is used as the best inorganic salt for this strain.

[0085] 2.8 Effects of adding different antioxidants on Bacillus coagulans

[0086] Cells may face oxidative damage, environmental stress, etc. during growth, which affect their own reproduction and growth. The addition of antioxidants can reduce the oxidative stress in cells and maintain the balanced metabolism of cells

[18] . The effects of different antioxidants on Bacillus coagulans are as Figure 6 shown. When 1mg / L ergothioneine is added, the number of viable bacteria reaches the maximum, 8.16×10 8CFU / mL. Therefore, based on this, the optimal concentration of ergothioneine was further explored, and according to the results Figure 7 shown, 1 g / mL of ergothioneine was still the optimal concentration and was significant (P < 0.01).

[0087] Ergothioneine is a natural antioxidant that can protect cells from excessive oxidative stress

[19] . Its safety in the food field has also been recognized by the European Food Safety Authority

[20] . Therefore, it is very necessary to use ergothioneine as an antioxidant to reduce the oxidative stress of Bacillus coagulans, prevent the oxidation of nutrients in the culture medium, and its spore formation rate.

[0088] 2.9 Effect of ergothioneine on the reactive oxygen species level of Bacillus coagulans

[0089] To investigate the effect of ergothioneine on the reactive oxygen species (ROS) level of Bacillus coagulans, fluorescence probe method was used for detection in this study. The research results Figure 8 showed that the ROS level was the highest (4.07 ± 0.12) in the control group (Control), indicating that the bacteria experienced a certain degree of oxidative stress during fermentation. After adding 1 mg / L of ergothioneine, the ROS level decreased to 2.90 ± 0.10, which was about 28.7% lower than that of the control group (P < 0.0001), indicating that at this concentration, ergothioneine could effectively alleviate oxidative damage and improve the redox state of the bacteria. There were certain differences in the effects of different concentrations of ergothioneine on the ROS level. The ROS level in the 0.5 mg / L group was similar to that of the control group, and no obvious antioxidant effect was shown (P > 0.05). When the concentration of ergothioneine increased to 1.5 mg / L and above, the ROS level showed an upward trend (3.15 - 4.23), indicating that higher concentrations did not further enhance the antioxidant effect and may even impose a certain metabolic burden on the bacteria. This phenomenon may be related to the adaptive feedback mechanism of the bacteria itself or the dynamic regulation of ergothioneine metabolism, which is worthy of further study.

[0090] Generally speaking, 1 mg / L of ergothioneine had the most obvious effect on reducing ROS under the experimental conditions, indicating that an appropriate amount of antioxidant could effectively alleviate the damage of bacteria caused by oxidative stress during fermentation. However, higher concentrations did not bring more obvious improvement, suggesting that the antioxidant effect of ergothioneine may be concentration-dependent, and its regulatory mechanism can be further explored in combination with transcriptomics or metabolomics studies in the future.

[0091] 2.10 Orthogonal optimization results of the culture medium of Bacillus coagulans

[0092] Based on the previous experiments on medium optimization, an orthogonal experiment with four factors and three levels was designed to further optimize the medium formulation for the test strain. The factor levels of the orthogonal experiment are shown in Table 1, and the results of the orthogonal experiment are shown in Figure 9 A in , and the analysis of the orthogonal experiment results is shown in Table 2.

[0093] From the range analysis in Table 2, it can be seen that the primary and secondary order of factors affecting the maximum viable count of Bacillus coagulans is: A > C > B > D, and the optimal combination is A3B1C3D2, which is consistent with the optimal combination obtained from the orthogonal experiment results. The optimal medium combination is: 1.5% glucose, 2.625% yeast powder, 0.875% soy peptone, 1.5% K2HPO4, and 1 mg / L ergothioneine.

[0094] Table 1 Factor level table of the orthogonal experiment

[0095]

[0096] Table 2 Orthogonal experiment analysis table

[0097]

[0098] 2.11 Verification of the optimization results

[0099] To verify the optimization results, the strain was cultured according to the results of the single-factor experiment of the fermentation process and the orthogonal experiment of the medium, and compared with the results before optimization. The results are as Figure 9 shown in B in . After culturing for 24 h, the maximum OD value after optimization was 1.66, and the maximum OD 600 value before optimization was 0.919, an increase of 80.63%, indicating a significant optimization effect. The maximum viable count after optimization reached 5.1×10 9 CFU / mL, and the maximum viable count before optimization was 3.5×10 6 CFU / mL. LB medium was used for strain activation, and an optimized medium with greater industrial application value (15 g / L glucose, 26.25 g / L yeast powder, 8.75 g / L soy peptone, 15 g / L K2HPO4, 1 mg / L ergothioneine) was used during the fermentation process to reduce production costs and improve the growth efficiency of the bacteria.

[0100] 2.12 Scale-up culture in a 5 L fermenter

[0101] In the scale-up culture experiment in a 5 L fermenter, the rotation speed, aeration rate, dissolved oxygen level, and alkali addition amount have important effects on the growth and metabolism of Bacillus coagulans. By optimizing these factors, the growth of the bacteria was effectively improved. As Figure 10 shown by the results in A in , the rotation speed has a significant effect on the growth of the bacteria. When the rotation speed was increased from 50 r / min to 200 r / min, the viable count increased significantly, reaching 1.57×109 CFU / mL. Moderately increasing the rotation speed helps to increase the dissolved oxygen content and improve the transport of nutrients. However, when the rotation speed is further increased to 250 r / min and 300 r / min, the viable cell count decreases, indicating that high shear force may cause cell damage and inhibit the growth of bacteria [21,22] . A rotation speed of 200 r / min is selected for the subsequent investigation of the ventilation volume

[0102] The change in the ventilation volume also affects the growth of bacteria. The results are as Figure 10 shown in B below. At a ventilation volume of 0.7 L / min, the viable cell count reaches 1.28×10 10 CFU / mL, which is significantly higher than that in the 0 L / min (P<0.01) and 0.4 L / min (P<0.05) groups, indicating that an appropriate ventilation volume can increase the dissolved oxygen level and promote the metabolism and proliferation of bacteria. Although a ventilation volume of 1.3 L / min provides more oxygen, excessive oxygen may cause oxidative stress and inhibit the growth of bacteria

[23] .

[0103] In addition, the change in the dissolved oxygen content directly affects the amount of alkali supplementation. The results are as Figure 11 shown. In the 0.7 L / min group, the amount of alkali supplementation increases with the growth of bacteria. When the viable cell count reaches the peak value, the amount of alkali supplementation is the highest (112 mL), indicating that the demand for alkali increases during the efficient metabolism of bacteria. As the dissolved oxygen content tends to be stable, the amount of alkali supplementation maintains a stable level, indicating that the bacteria enter the steady-state metabolism stage. Generally speaking, the optimized rotation speed and ventilation volume not only increase the dissolved oxygen level, but also promote the growth and metabolism of bacteria

[24] . Reasonably regulating the stirring rate and ventilation conditions of the fermenter can further improve the growth efficiency of bacteria and provide an experimental basis for industrial fermentation

[0104] In summary, in the 5 L fermenter scale-up experiment, after optimizing the ventilation volume (0.7 L / min) and rotation speed (200 r / min), the viable cell count is further increased to 1.28×10^10 CFU / mL, which is 2.51 times higher than that in the shake flask culture

[0105] 3. Conclusion

[0106] This study shows that ergothioneine plays a positive role in alleviating oxidative stress and improving the survival rate of bacteria, and thus has a certain promoting effect on the fermentation process of Bacillus coagulans. By optimizing the fermentation conditions and the medium formula, the viable cell count reaches 5.1×10^9 CFU / mL, which is significantly higher than 3.5×10 6 CFU / mL before optimization. In the 5 L fermenter scale-up experiment, the viable cell count is further increased to 1.28×10 10CFU / mL, which is about four orders of magnitude higher than that in shake flask culture, showing its application potential in industrial production. In addition, in this study, relatively economical culture medium components (such as glucose, soy peptone, yeast powder, etc.) were used to replace the traditional LB medium to control the industrial production cost while maintaining a high cell growth efficiency. The results of fluorescence probe detection showed that the addition of ergothioneine could reduce the intracellular ROS level by 28.7% (P<0.0001), effectively reducing oxidative damage.

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[0132] The above embodiments are the preferred embodiments of the present invention. However, the embodiments of the present invention are not limited to the above embodiments. Any other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Application of ergothioneine in increasing viable cell count of Bacillus coagulans in high-density fermentation culture.

2. The application according to claim 1, wherein: In the said application, the concentration of ergothioneine in the culture system is 0.8 - 1.5 mg / L; further preferably 0.8 - 1.2 mg / L; still further preferably 1 ± 0.1 mg / L.

3. A medium for high-density fermentation culture of Bacillus coagulans with a high viable cell count, characterized in that: The said culture medium comprises components with the following concentrations: carbon source 8 - 15 g / L, nitrogen source 35 - 45 g / L, K2HPO4 5 - 15 g / L, ergothioneine 0.8 - 1.5 mg / L, pH 6.5 - 7.

4. The culture medium according to claim 3, wherein: The said culture medium comprises components with the following concentrations: carbon source 10 - 15 g / L, nitrogen source 35 - 40 g / L, K2HPO4 10 - 15 g / L, ergothioneine 0.8 - 1.2 mg / L, pH 6.5 - 7.

5. The culture medium according to claim 3 or 4, wherein: The said carbon source is selected from at least one of glucose and soluble starch; The said nitrogen source is a composite nitrogen source, selected from the compounding of at least one of dried corn steep liquor, corn steep liquor protein, soy peptone and tryptone and yeast powder.

6. The culture medium according to claim 5, wherein: The said carbon source is selected from glucose; the said nitrogen source is selected from the compounding of soy peptone and yeast powder in a mass ratio of 1:

3.

7. The culture medium according to claim 6, wherein: The said culture medium comprises components with the following concentrations: glucose 15 g / L, nitrogen source 35 g / L, K2HPO4 15 g / L, ergothioneine 1 mg / L, pH 7; the said nitrogen source is selected from the compounding of soy peptone and yeast powder in a mass ratio of 1:

3.

8. A culture method for high-density fermentation of Bacillus coagulans with a high viable cell count, characterized in that, Comprises the following steps: (1) Inoculate a single colony of Bacillus coagulans into LB liquid medium for activation culture to obtain a seed solution; (2) Inoculate the seed solution obtained in step (1) into the culture medium according to any one of claims 3 - 7 for fermentation culture; (3) Inoculate the seed solution obtained in step (1) into the culture medium according to any one of claims 3 - 7 for scale-up culture.

9. The culture method according to claim 8, wherein: The conditions of the activation culture in step (1) are shaking culture at 35 - 45 °C, 150 - 200 r / min for 20 - 24 h; The inoculation amount of the seed solution in steps (2) and (3) is 2 - 10%; The conditions of the fermentation culture in step (2) are shaking culture at 35 - 45 °C, 100 - 200 r / min for 10 - 24 h; The conditions of the scale-up culture in step (3) are shaking culture at 35 - 45 °C, 100 - 250 r / min for 24 - 48 h, and the ventilation rate is 0.6 - 1.0 L / min.

10. The culture method according to claim 9, wherein: The conditions of the activation culture in step (1) are shaking culture at 37 °C, 180 r / min for 24 h; The inoculation amount of the seed solution in steps (2) and (3) is 6 ± 0.5%; The conditions for the fermentation culture described in step (2) are shaking culture at 45°C and 200 r / min for 24 h; The conditions for the scale-up culture described in step (3) are shaking culture at 45°C and 200 r / min for 48 h with an aeration rate of 0.7 L / min.

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