High-density culture medium of lactobacillus buchneri and fermentation method

Abstract

The invention discloses a high-density culture medium of lactobacillus buchneri and a fermentation method. The high-density culture medium is prepared from 28-35 g / L of glucose, 20-30 g / L of corn steep liquor dry powder, 20-30 g / L of yeast extract, 5-9 g / L of sodium acetate trihydrate, 4-7 g / L of diammonium hydrogen citrate, 0.6-1 g / L of MgCl2, 0.8-1 g / L of CaCO3, 0.2-0.5 g / L of MnSO4 and 0.6-1 g / L of Tween-80; 2, a fermentation process: placing the seed solution at 37 DEG C, performing two-stage culture, namely first-stage culture for 24 hours and second-stage culture for 12 hours, inoculating the second-stage seed solution into a sterilized fermentation tank culture medium according to 3% (v / v), controlling the initial pH to be 7.0 and the fermentation pH to be 6.5, performing fermentation at 37 DEG C, supplementing glucose twice during the fermentation period, and performing culture until the fermentation is finished. The total fermentation time is 40-44 hours. The activity of the lactobacillus buchneri fermented by the method can reach 1.42 * 10 < 10 > CFU / mL, and the high-density culture technology of the lactobacillus buchneri is improved. The industrial pain point problem of low activity of the lactobacillus buchneri at present is successfully solved.

Description

Technical Field

[0001] The invention belongs to the technical field of microbial fermentation, and particularly relates to a high-density culture medium and a fermentation method for Lactobacillus buchneri. Background Art

[0002] Lactobacillus buchneri is a Gram-positive lactic acid bacterium that does not produce spores and is non-motile. It is short rod-shaped, and will become shorter in the later stage of growth, becoming an approximately elliptical shape. Lactobacillus buchneri is a heterotypic fermentative lactic acid bacterium, and the final product includes other compounds such as lactic acid and acetic acid. Lactobacillus buchneri is widely found in the human intestine and mouth, silage, and fermented foods such as kimchi juice. Studies have shown that Lactobacillus buchneri has the functions of lowering total cholesterol, protecting nerve cells, improving reproductive function, and inhibiting the growth of spoilage bacteria. Lactobacillus buchneri agents generally work with live bacteria. When the number of live bacteria in the agent reaches a certain level, it can play a probiotic role. Therefore, the effective number of live bacteria is an important indicator for measuring the quality of the agent. At present, the number of live bacteria in the Lactobacillus buchneri live bacterial preparation is generally 5×109CFU / mL, and after optimization, it can reach 1×1010CFU / mL. However, due to the shortcomings in fermentation technology and backward production equipment, it is difficult to increase the number of live bacteria.

[0003] Therefore, how to obtain highly active Lactobacillus buchneri products more efficiently is the core of the microbial preparation production process and also the most critical issue. Summary of the invention

[0004] The purpose of the present invention is to provide a high-density culture medium for Lactobacillus buchneri, and to control the growth of Lactobacillus buchneri by adjusting the formula of the culture medium and adjusting the fermentation process conditions, thereby achieving the purpose of improving the activity of Lactobacillus buchneri.

[0005] The above technical objectives of the present invention are achieved through the following technical solutions:

[0006] A high-density culture medium for lactobacillus buchneri. The fermentation culture medium consists of the following components and proportions: 28-35 g / L of glucose, 20-30 g / L of corn steep liquor dry powder, 20-30 g / L of yeast extract, 5-9 g / L of sodium acetate trihydrate, 4-7 g / L of diammonium hydrogen citrate, 0.6-1 g / L of MgCl2, 0.8-1 g / L of CaCO3, 0.2-0.5 g / L of MnSO4, 0.6-1 g / L of Tween-80, and the remainder is water.

[0007] Preferably, the following component ratio is adopted: 33 g / L glucose, 27 g / L corn steep liquor powder, 30 g / L yeast extract, 7 g / L sodium acetate trihydrate, 5 g / L diammonium hydrogen citrate, 0.8 g / L MgCl2, 0.8 g / L CaCO3, 0.4 g / L MnSO4, 1 g / L Tween-80, and the rest is water.

[0008] A high-density fermentation method for Lactobacillus buchneri comprises the following steps: placing Lactobacillus buchneri seed liquid in a seed liquid culture medium at 37°C and culturing the liquid in two stages, wherein the first stage is cultured for 20-24 hours and the second stage is cultured for 10-12 hours; the second stage seed liquid is inoculated into the sterilized fermentation culture medium; the initial pH is controlled to be 7.0, the fermentation pH is controlled to be 6.5, the temperature is 37°C, and the rotation speed is 250 rpm; glucose is supplemented twice during the fermentation period; the culture is carried out until the fermentation is finished; and the total fermentation time is 40-44 hours.

[0009] Preferably, a single colony is picked up with a sterilized toothpick and inoculated into a seed liquid culture medium, and cultured at 37°C for 24 hours as a primary seed liquid; 2-5 mL of the primary seed liquid is inoculated into a fresh seed culture medium, and cultured at 37°C for 12 hours as a secondary seed liquid.

[0010] Preferably, the seed liquid culture medium is MRS culture medium.

[0011] Preferably, the above-mentioned seed liquid culture medium and fermentation culture medium are obtained by sterilizing at 121° C. for 30 minutes.

[0012] Preferably, the Lactobacillus buchneri seed solution is inoculated into a 5 L fermentation tank culture medium at an inoculation rate of 3% (v / v).

[0013] Preferably, the liquid volume of the fermentation tank culture medium is 70%-80% of the volume of the fermentation tank.

[0014] Preferably, nitrogen is used as the pressure-maintaining gas at the beginning of fermentation, and the tank pressure is 0.02-0.04 MPa. After the inoculation is completed, nitrogen is continuously introduced for 20-30 minutes to remove the remaining oxygen in the tank.

[0015] Preferably, the feeding time is 16h and 24h from the start of the fermentation timing, and 1% (w / w) glucose is supplemented respectively.

[0016] In summary, the present invention has the following beneficial effects:

[0017] 1. The present invention adopts a new liquid culture medium formula, which is rich in nutrients, so that the metabolism of Lactobacillus buchneri strain is accelerated, and most of the energy generated by consuming nutrients flows to its own growth rather than producing primary metabolites. The number of viable bacteria reaches 1.42×1010 CFU / mL after fermentation for 40 hours, which greatly improves the growth vitality of Lactobacillus buchneri.

[0018] 2. The present invention adopts a new fermentation process technology, and creates an anaerobic environment by adjusting fermentation process parameters and introducing nitrogen, thereby achieving the purpose of increasing the number of viable Lactobacillus buchneri.

[0019] 3. The present invention provides a technical method for the industrial production of Lactobacillus buchneri inoculant. The fermentation technology is easy to master and the operation is simple. The fermentation raw materials are cheap and easy to obtain. It can not only greatly increase the yield of the strain, but also reduce the cost to a minimum, save energy loss, and further improve the quality and economic value of the product. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a statistical graph of the effect of carbon source on the number of viable Lactobacillus buchneri;

[0021] Figure 2 It is a statistical chart of the effect of nitrogen source on the number of viable Lactobacillus buchneri;

[0022] Figure 3 It is a statistical graph of the effect of buffer salt on the number of viable Lactobacillus buchneri;

[0023] Figure 4 It is a statistical graph of the effect of metal ions on the number of viable Lactobacillus buchneri;

[0024] Figure 5 It is a statistical graph of the effect of growth factors on the number of viable Lactobacillus buchneri;

[0025] Figure 6 is the half-normal probability effect plot of the standardized effect values ​​of each factor;

[0026] Figure 7 It is a Pareto chart of the standardized effects of each factor;

[0027] Figure 8 is a correlation diagram between the model prediction value and the experimental value;

[0028] Fig. 9 is a scatter plot of residuals versus model predictions;

[0029] Fig.10 This is the effect of the interaction between A and C on the number of viable Lactobacillus buchneri;

[0030] Fig.11 This is the effect of the interaction between A and B on the number of viable Lactobacillus buchneri;

[0031] Fig.12 This is a graph showing the effect of the interaction between B and C on the number of live Lactobacillus buchneri.

[0032] Fig.13 is the number of viable Lactobacillus buchneri under different culture conditions. DETAILED DESCRIPTION

[0033] The present invention is further described in detail below in conjunction with the embodiments, and the technical solutions in the embodiments of the present invention are clearly and completely described. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0034] A high-density culture medium for lactobacillus buchneri. The fermentation culture medium consists of the following components and proportions: 28-35 g / L of glucose, 20-30 g / L of corn steep liquor dry powder, 20-30 g / L of yeast extract, 5-9 g / L of sodium acetate trihydrate, 4-7 g / L of diammonium hydrogen citrate, 0.6-1 g / L of MgCl2, 0.8-1 g / L of CaCO3, 0.2-0.5 g / L of MnSO4, 0.6-1 g / L of Tween-80, and the remainder is water.

[0035] Preferably, the following components and proportions are used: 33 g / L glucose, 27 g / L corn steep liquor powder, 30 g / L yeast extract, 7 g / L sodium acetate trihydrate, 5 g / L diammonium hydrogen citrate, 0.8 g / L MgCl2, 0.8 g / L CaCO3, 0.4 g / L MnSO4, 1 g / L Tween-80, and the remainder is water.

[0036] The invention discloses a high-density fermentation method of lactobacillus buchneri. The method comprises the following steps: taking out a strain (number: HEW-A666) frozen in a -80 DEG C refrigerator and bringing it to room temperature; dipping the bacterial liquid in a blank MRS solid culture medium and culturing at 37 DEG C until a single colony grows; picking up the single colony with a sterilized toothpick and inserting it into an MRS liquid culture medium; and culturing at 37 DEG C for 20-24 hours as a primary seed liquid; taking 2-5 mL of the primary seed liquid and inserting it into a fresh MRS liquid culture medium, and culturing at 37 DEG C for 10-12 hours as a secondary seed liquid; inoculating the secondary seed liquid into the sterilized fermentation culture medium; controlling the initial pH to 7.0, the fermentation pH to 6.5, the temperature to 37 DEG C, and the rotation speed to 250 rpm for fermentation; supplementing glucose twice during the fermentation; and culturing until the fermentation is finished. The total fermentation time is 40-44 hours.

[0037] Preferably, the above-mentioned seed liquid culture medium and fermentation culture medium are obtained by sterilizing at 121° C. for 30 minutes.

[0038] Preferably, the Lactobacillus buchneri seed solution is inoculated into a 5 L fermentation tank culture medium at an inoculation rate of 3% (v / v).

[0039] Preferably, the liquid volume of the fermentation tank culture medium is 70%-80% of the volume of the fermentation tank.

[0040] Preferably, nitrogen is used as the pressure-maintaining gas at the beginning of fermentation, and the tank pressure is 0.02-0.04 MPa. After the inoculation is completed, nitrogen is continuously introduced for 20-30 minutes to remove the remaining oxygen in the tank.

[0041] Preferably, the feeding time is 16h and 24h from the start of the fermentation timing, and 1% (w / w) glucose is supplemented respectively.

[0042] 1. Single factor optimization of culture medium formulation

[0043] (I) Carbon source

[0044] The basic formula is MRS culture medium, and equal proportions of glucose, sucrose, maltose, fructose and soluble starch are used to replace the carbon source in the original culture medium. The optimal carbon source is screened out based on the number of viable bacteria, and the optimal addition amount of the screened carbon source is selected according to the mass fraction of 1%, 2%, 3%, 4% and 5%.

[0045] Results and analysis: Figure 1 As shown in (a), in the medium containing glucose and soluble starch, the activity of Lactobacillus buchneri was significantly higher than that of other species; Figure 1 As shown in (b), when the glucose concentration is 30g / L, Lactobacillus buchneri has the highest activity, and when the soluble starch concentration is 20g / L, Lactobacillus buchneri has the highest activity. Studies have shown that composite carbon sources are more suitable for slower growing strains. In the early stage of fermentation, fast carbon sources such as glucose are used to generate energy, and in the middle and late stages of fermentation, slow carbon sources such as soluble starch are used for slow metabolism to maintain the number of viable bacteria at a stable level.

[0046] (ii) Nitrogen source

[0047] The basic formula is MRS culture medium, and equal proportions of corn steep liquor powder, soy peptone, yeast extract, yeast extract powder, beef extract powder and trypsin are used to replace the nitrogen source in the original culture medium. The optimal nitrogen source is screened out based on the number of viable bacteria, and the optimal addition amount of the screened nitrogen source is selected according to the mass fraction of 1%, 2%, 3%, 4% and 5%.

[0048] Results and analysis: Figure 2 As shown in (a), in the culture medium containing yeast extract and corn steep liquor powder, the activity of Lactobacillus buchneri was significantly higher than that of other species; Figure 2 As shown in (b), when the yeast extract concentration is 30 g / L, the activity of Lactobacillus buchneri is the highest, and when the corn syrup dry powder concentration is 30 g / L, the activity of Lactobacillus buchneri is the highest. Studies have shown that composite nitrogen sources are more suitable for lactic acid bacteria than single nitrogen sources. Fast nitrogen sources are used to generate energy in the early stage of fermentation, and slow nitrogen sources are used to metabolize slowly in the middle and late stages of fermentation to maintain the number of live bacteria at a relatively stable level.

[0049] (III) Buffer salt

[0050] The basic formula is MRS culture medium, and equal proportions of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, sodium acetate trihydrate, diammonium hydrogen citrate, and ammonium sulfate are used to replace the buffer salts in the original culture medium. The optimal buffer salt is screened out based on the number of viable bacteria as the assessment indicator, and the optimal addition amount of the screened buffer salt is selected according to the mass fraction of 0.1%, 0.3%, 0.5%, 0.7%, and 0.9%.

[0051] Results and analysis: Figure 3 As shown in (a), in the culture medium containing sodium acetate trihydrate and diammonium hydrogen citrate, the activity of Lactobacillus buchneri was significantly higher than that of other species; Figure 3 As shown in (b), when the concentration of sodium acetate trihydrate is 7 g / L, the activity of Lactobacillus buchneri is the highest, and when the concentration of diammonium hydrogen citrate is 3 g / L, the activity of Lactobacillus buchneri is the highest. Studies have found that adding citrate to the culture medium can increase the bacterial density during lactic acid bacteria fermentation, which may be related to the growth metabolic pathway of lactic acid bacteria.

[0052] (IV) Metal ions

[0053] The basic formula is MRS culture medium, and equal proportions of magnesium chloride, ferrous sulfate, manganese sulfate, and calcium carbonate are used to replace the metal ions in the original culture medium. The optimal metal ions are screened out based on the number of viable bacteria, and the optimal addition amount of the screened metal ions is selected according to the mass fraction of 0.02%, 0.04%, 0.06%, 0.08%, and 0.10%.

[0054] Results and analysis: Figure 4 As shown, in the culture medium containing magnesium chloride, manganese sulfate and calcium carbonate, the strain growth activity is higher; when magnesium chloride, manganese sulfate and calcium carbonate are added at 0.8g / L, 0.4g / L and 0.8g / L respectively, the strain activity is highest, which is also consistent with the effect of metal ions on the physiological activity of microorganisms, low concentrations stimulate growth, and high concentrations inhibit growth.

[0055] (V) Growth Factors

[0056] The basic formula is MRS culture medium, and equal proportions of L-cysteine ​​hydrochloride, arginine, guanylate, and Tween-80 are used to replace the growth factors in the original culture medium. The optimal growth factor is screened out based on the number of viable bacteria, and the optimal addition amount of the screened growth factor is selected according to the mass fraction of 0.02%, 0.04%, 0.06%, 0.08%, and 0.10%.

[0057] Results and analysis: Figure 5As shown, different types of growth factors can stimulate the growth of strains to a certain extent. Tween-80, as a surfactant, can effectively stimulate the growth and reproduction of strains, and the effect is significantly higher than that of several other substances. Adding 0.8g / L Tween-80 has the best effect on the proliferation of strains.

[0058] 2. Response Surface Optimization of Culture Medium Formulation

[0059] (I) Plackett-Burman test

[0060] The Plackett-Burman test was designed using Design-expert software. On the basis of the results of the single-factor test, glucose, soluble starch, corn syrup powder, yeast extract, diammonium hydrogen citrate, sodium acetate trihydrate, and Tween-80 were used as influencing factors, and the number of viable bacteria was used as the response value. The specific design and response range are shown in Table 1. According to the results of the Plackett-Burman test, the three factors that have the greatest impact on the number of viable Lactobacillus buchneri were selected.

[0061] Table 1 Plackett-Burman experimental design and factor levels

[0062]

[0063] Experimental results: From the single factor experiment, it can be seen that when the culture medium contains 3% glucose, 2% soluble starch, 3% yeast extract, 3% corn steep liquor powder, 0.7% sodium acetate trihydrate, 0.3% diammonium hydrogen citrate and 0.08% Tween-80 by mass fraction, the number of viable Lactobacillus buchneri reaches the highest. Therefore, the upper and lower levels are selected as the experimental levels. The design and results are shown in Table 2.

[0064] Table 2 Plackett-Burman test results

[0065]

[0066] The test results were analyzed by variance analysis, and the Lenth method was used to identify significant effects in the Plackett-Burman test results, and the half-normal probability effect diagram of the standardized effect value of each factor was obtained (see Figure 6 ) and a Pareto diagram of the standardized effects of the factors (see Figure 7 ).Depend on Figure 6 It can be seen that the standardized effect points of factors A, D, and F are far from the fitting line, so they are significant influencing factors (P < 0.05), that is, the significant factors affecting the activity of Lactobacillus buchneri are glucose, corn syrup powder, and diammonium hydrogen citrate, and the standardized effect points of other factors are small; Figure 7The magnitude and importance of the effect were further determined, and the t values ​​of factors A, D, and F all exceeded the significant values, among which factors A and D were shown to be extremely significant.

[0067] As shown in Table 3, the P value of the model is 0.0048<0.05, indicating that it is significant, that is, the model is reliable and fits well in the entire regression area being studied; the multiple correlation coefficient R2=0.9748, indicating that the correlation is good; the difference between the correction coefficient and the prediction coefficient is less than 0.2, indicating that the model design is reasonable; the coefficient of variation of the model is 3.85%, and the precision reaches 16.1312, indicating that the credibility and accuracy of the experiment are high.

[0068] By performing multiple regression fitting on the data, the regression equation is obtained:

[0069] Y=9.30+0.9142A+0.1992B+0.2692C+0.6408D+0.0375E+0.5342F-0.0708G

[0070] Table 3 Plackett-Burman test factors, levels and significance analysis

[0071]

[0072] (II) Steepest climbing test

[0073] The climbing direction and step length were determined by the effect values ​​of the three factors that contributed most to the number of live Lactobacillus buchneri in the Plackett-Burman test. The specific design is shown in Table 4.

[0074] Table 4 Steepest climbing test design

[0075] Test No. Glucose (g / L) Corn syrup powder (g / L) Diammonium hydrogen citrate (g / L) 1 20 19 3 2 30 26 4 3 40 33 5 4 50 40 6 5 60 47 7

[0076] From the regression equation obtained above, it can be seen that the partial regression coefficient of factor A is 0.9142, indicating that the effect of factor A on the viable count of Lactobacillus buchneri is a positive effect, that is, as the concentration of factor A increases, the viable count shows an upward trend; similarly, factors D and F also have positive effects. Therefore, when designing the center point of the response surface experiment, three significant factors are selected to climb upward. Since the most significant factor is A, A is used as the climbing unit, that is, the step length of A is 10g / L, the step length of D is 7.009g / L≈7g / L, and the step length of F is 1.169g / L≈1g / L.

[0077] The other components of the culture medium in the ramp test were all added at the optimal amount in the single factor test, and the experimental design and results are shown in Table 5. As can be seen from Table 5, the number of viable bacteria in treatment 2 reached the maximum value, so treatment 3 was taken as the center point of the response surface experiment.

[0078] Table 5 Steepest climbing test design and results

[0079]

[0080]

[0081] Note: Δ is a step unit (III) Box-Behnken test

[0082] Based on the results of the steepest climbing test, the center point of the response surface test was determined. With glucose, yeast extract and lactose as influencing factors and viable count as the response value, a three-factor three-level Box-Behnken test was designed. The specific scheme is shown in Table 6.

[0083] Table 6 Box-Behnken test design

[0084]

[0085] According to the results of the steepest climbing test, the number of viable bacteria in treatment 2 was the highest. Based on this as the center point, a Box-Behnken test was conducted, and three factors, glucose, corn syrup powder, and diammonium hydrogen citrate, were selected as independent variables. A three-factor three-level test was designed, and the number of viable bacteria in the strain was used as the response value. The experimental design and results are shown in Table 7.

[0086] Table 7 Box-Behnken test design and results

[0087]

[0088]

[0089] The variance analysis of the response surface test results is shown in Table 8. It can be seen from Table 8 that the model is extremely significant (P < 0.01) and the lack of fit term is not significant (P > 0.05). The model correlation coefficient R2 is 0.9903, indicating that the model has a good fit; the correction coefficient is 0.9777, and the difference with the prediction coefficient 0.8741 is less than 0.2, indicating that the model design is reasonable and the model can be used to analyze and predict the viable count of Lactobacillus buchneri; the coefficient of variation of the model is 1.61%, and the precision reaches 23.8756, indicating that the reliability and accuracy of the test are high.

[0090] In addition, factors A, B, and C have significant effects on the number of viable bacteria; the interaction terms AB, AC, and BC are not significant; and the quadratic terms A2, B2, and C2 have significant effects on the number of viable bacteria. This shows that there is not a simple linear relationship between the factors and the number of viable bacteria, and the influence of each factor is in the order of A>C>B, that is, glucose>corn syrup powder>diammonium hydrogen citrate.

[0091] Table 8 Analysis of variance of Box-Behnken test results

[0092]

[0093]

[0094] The regression model is used to predict the number of viable bacteria under each fermentation condition. In order to observe the model prediction results, a correlation diagram between the model prediction value and the experimental value is drawn, such as Figure 8 As shown in the figure, the experimental values ​​of the samples are all near the 45° diagonal line, indicating that the predicted values ​​of the number of viable bacteria under each fermentation condition of the model are highly correlated with the experimental values, and the difference between the two is small. Fig. 9 ,As can be seen from the figure, most of the sample points fall within the ±2SD range of the model, ,which shows that the model has high prediction accuracy and ,can be used for high-density fermentation analysis of Lactobacillus buchneri.

[0095] By fitting the data through quadratic multiple regression, a quadratic polynomial equation was obtained: Y=12.51+0.6588A+0.1663B+0.33C+0.165AB+0.1325AC+0.1925BC-1.22A2-0.9275B2-1.16C2.

[0096] According to the above equation, the response surface analysis diagram was drawn using Design-expert. The results are as follows: Figures 10-12 The 3D graph shows the range of the test and the area where the maximum value is.

[0097] The model was analyzed and predicted using Design-expert, and the maximum response value was 1.2644×1010CFU / mL. At this time, the amount of glucose added was 32.8926g / L, the amount of corn syrup powder added was 26.9294g / L, and the amount of diammonium hydrogen citrate added was 5.1688g / L.

[0098] (IV) Model Verification

[0099] The optimal conditions optimized by the response surface model were used to carry out fermentation experiments, and the model prediction values ​​were compared with the experimental values ​​to verify the effectiveness of the model.

[0100] The carbon source, nitrogen source and other substances at the maximum response value predicted by the model were added to the culture medium in turn for testing and verified under the same conditions. The experiment was repeated 3 times and the final live count of Lactobacillus buchneri was 1.26±0.02×1010CFU / mL. The experimental value was very close to the predicted value, which shows that the model can accurately predict the actual fermentation situation, thereby proving the feasibility of optimizing the high-density fermentation of Lactobacillus buchneri by response surface.

[0101] (V) Fermentation process optimization

[0102] Lactobacillus buchneri was cultured in a 5L in vitro fermenter. The number of viable bacteria was used as the evaluation index to optimize the inoculation amount, fermentation temperature, initial pH and fermentation pH.

[0103] After optimizing the medium formulation, further improving the fermentation process can increase the growth density of the strain. Fig.13 As shown in (a), when the inoculation amount gradually increases from 1% to 5%, the number of viable bacteria shows a trend of first increasing and then decreasing. At 3% inoculation amount, the number of viable bacteria reaches a maximum value of 1.34×1010CFU / mL. When the inoculation amount is too low, the strain grows slowly in the early stage, the time to reach the stable period is delayed, and the activity is low. When the inoculation amount is too high, the strain grows too fast in the early stage, consumes nutrients and produces a large amount of metabolites, which ultimately leads to a low number of viable bacteria in the stable period. Fig.13 (b) It can be seen that there are significant differences in the number of viable bacteria at different fermentation temperatures. At a fermentation temperature of 37°C, the number of viable bacteria is the highest, reaching 1.26×1010 CFU / mL. Fig.13 (c) It can be seen that the initial pH has a significant effect on the number of viable bacteria. When the initial pH is set to 7.0, the number of viable bacteria is 1.28×1010CFU / mL, which is significantly higher than that of other groups. Lactic acid bacteria produce lactic acid during growth and metabolism. Excessive accumulation will affect the growth of the strain. Therefore, it is necessary to maintain a constant pH during fermentation. Fig.13 (d) It can be seen that when the pH is set to 6.5, the maximum number of viable bacteria can be 1.32×1010 CFU / mL, so the pH is controlled to be constant at 6.5 during fermentation.

[0104] The present invention takes industrialized production of Lactobacillus buchneri as the experimental target, uses relatively cheap culture medium raw materials, optimizes the key factors of the culture medium, and optimizes the culture process in combination with single-factor experiments. The above-mentioned optimized culture medium and culture process are used to ferment Lactobacillus buchneri, and the final viable count of Lactobacillus buchneri is 1.42×1010CFU / mL, which is 2.31 times higher than the initial basic culture medium 6.14×109CFU / mL.

[0105] This specific embodiment is only an explanation of the present invention, and it is not a limitation of the present invention. After reading this specification, those skilled in the art can make non-creative modifications to this embodiment as needed, but they are protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A high-density culture medium for Lactobacillus buchneri, characterized in that: The fermentation medium consists of the following components and proportions: glucose 28-35 g / L, corn steep liquor dry powder 20-30 g / L, yeast extract 20-30 g / L, sodium acetate trihydrate 5-9 g / L, diammonium hydrogen citrate 4-7 g / L, MgCl2 0.6-1 g / L, CaCO3 0.8-1 g / L, MnSO4 0.2-0.5 g / L, Tween-80 0.6-1 g / L, and the rest is water.

2. A high-density fermentation method of Lactobacillus buchneri, characterized in that: The Lactobacillus buchneri seed liquid is placed in a seed liquid culture medium at 37° C. and cultured in two stages, with the first stage culture for 20-24 hours and the second stage culture for 10-12 hours. The second stage seed liquid is inoculated into the sterilized fermentation medium of claim 1, and the initial pH is controlled to be 7.0, the fermentation pH is 6.5, the temperature is 37° C., and the rotation speed is 250 rpm. During the fermentation period, glucose is supplemented twice, and the culture is continued until the fermentation is completed. The total fermentation time is 40-44 hours.

3. A high-density fermentation method of Lactobacillus buchneri according to claim 2, characterized in that: Use a sterilized toothpick to pick up a single colony and inoculate it into the seed liquid culture medium, and culture it at 37℃ for 24 hours as the first-level seed liquid; take 2-5mL of the first-level seed liquid and inoculate it into fresh seed culture medium, and culture it at 37℃ for 12 hours as the second-level seed liquid.

4. The high-density fermentation method of a Lactobacillus buchneri according to claim 2 or 3, characterized in that: The seed liquid culture medium is MRS culture medium.

5. The high-density fermentation method of Lactobacillus buchneri according to claim 2, characterized in that: The Lactobacillus buchneri seed solution was inoculated into a 5 L fermentation tank culture medium at an inoculation rate of 3% (v / v).

6. The high-density fermentation method of Lactobacillus buchneri according to claim 5, characterized in that: The liquid volume of the fermentation tank culture medium is 70%-80% of the fermentation tank volume.

7. The high-density fermentation method of Lactobacillus buchneri according to claim 2, characterized in that: At the beginning of fermentation, nitrogen is used as the pressure-maintaining gas, and the tank pressure is 0.02-0.04MPa. After the inoculation is completed, nitrogen is continuously introduced for 20-30 minutes to remove the remaining oxygen in the tank.

8. The high-density fermentation method of Lactobacillus buchneri according to claim 2, characterized in that: The feeding time was 16h and 24h from the start of the fermentation timing, and 1% (w / w) glucose was supplemented respectively.

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