Probiotic fermentation culture medium and design method

Abstract

The invention relates to a probiotic fermentation culture medium and a design method. Soybean meal and corn flour are used for replacing peptone to serve as a new culture medium of a nitrogen source and a carbon source, and the specific proportion is researched; key factors and influence degrees of influence results are confirmed, interaction effects among important influence factors are analyzed, a prediction model between the factors and response values is established, and the factor level when the response values are most appropriate is obtained; the method comprises the following steps: carrying out experimental optimization on the optimal matrix component proportion of a probiotic strain fermentation culture medium by utilizing Minitab software and adopting a Plackett-Burman design and a Box-Behnken center combinatorial design in a response surface analysis method; the production efficiency of the strain is improved, the economic cost of fermentation is reduced, and a foundation is laid for large-scale fermentation of probiotic liquid.

Description

Technical Field

[0001] The invention belongs to the technical field of probiotics and relates to a probiotic fermentation culture medium and a design method thereof. Background Art

[0002] Probiotic powder is an important type of probiotic product, widely used in food, health care products and medicine. The general process of probiotic powder production includes strain culture: first select high-quality probiotic strains, perform centrifugation, extraction and other treatments, and then carry out liquid culture in a suitable culture medium to allow the strain to multiply in large quantities; fermentation: add the strain to a fermentation medium containing nutrients, and ferment under suitable temperature, pH and other conditions; separation and extraction: after the fermentation is completed, separate the bacteria from the culture medium by centrifugation or filtration to obtain the bacteria; emulsification: after the bacteria are separated, add a protective agent and mix. Drying: convert the mixed substance into a solid powder form. Common drying methods include spray drying, vacuum freeze drying, etc. Probiotic liquid fermentation is a crucial step before the production of probiotic powder, and it is particularly important in the entire research, and the fermentation medium has an important influence on the fermentation effect and cost.

[0003] According to the composition and source, microbial culture media can be divided into natural culture media, synthetic culture media and semi-synthetic culture media. Natural culture media: contain natural organic matter with unclear or unstable chemical composition; synthetic culture media: a type of culture media composed entirely of known chemical reagents; semi-synthetic culture media: combine the characteristics of natural culture media and synthetic culture media, and use part of natural organic matter as carbon source, nitrogen source and growth factor. At present, probiotic fermentation culture media are mainly semi-synthetic culture media, for example, peptone is used as the main nitrogen source, carbon source and growth factor, but the cost of reagents such as peptone is relatively high, which is not conducive to large-scale production. Therefore, the development of low-cost and efficient production culture media is one of the current research focuses in the field of probiotic fermentation. However, there are few reports on the design methods of effective low-cost probiotic fermentation culture media components.

[0004] In summary, the development of new low-cost, efficient production culture media and corresponding design methods is of great significance in the field of probiotic fermentation. Summary of the invention

[0005] In view of the deficiencies of the prior art and actual needs, the present invention provides a probiotic fermentation medium and a design method to promote the efficient, low-cost, large-scale production of probiotics.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for designing a probiotic fermentation medium, the method comprising the following steps:

[0008] (S1) Plackett-Burman experiment was used to investigate factors A, B, C, D, E, F and G. Factor A was soybean flour, factor B was corn flour, factor C was NH 4 NO 3 , D factor is NaCl, E factor is K 2 HPO 4 ·3H 2 O, F factor is MgSO 4 7H 2 O and G factors are glycerol. For each factor, the lowest level is "-1" and the highest level is "1". The number of viable bacteria is used as the response value. The factors are ranked according to the degree of influence on the response value to screen the factors with significant influence on the response value.

[0009] (S2) Conduct a rapid climbing experiment on the factors selected by the Plackett-Burman experiment to obtain the addition level of each factor;

[0010] (S3) According to the Box-Behnken central composite experimental design principle, the factors selected by the Plackett-Burman experiment and the addition levels of the factors obtained by the rapid climbing experiment, a response surface analysis experiment is designed, a response surface analysis is performed, regression coefficients and variance analysis are generated, a quadratic polynomial regression equation is established, partial derivatives of the regression equation are calculated to predict the optimal experimental points of the factors, and the addition levels of the factors are determined, thereby determining the composition of the optimized culture medium;

[0011] (S4) With other factors and culture conditions unchanged, the probiotics are inoculated into the optimized culture medium for culture, the number of viable bacteria in the fermentation broth is detected, the difference between the number of viable bacteria in the fermentation broth after the culture medium is optimized and the predicted experimental results is compared, and the optimization effect of the culture medium components is analyzed.

[0012] In the present invention, a new design optimization strategy for probiotic fermentation medium was developed, and the multi-factor soybean powder, corn powder, NH 4 NO 3 , NaCl, K 2 HPO 4 ·3H 2 O、MgSO 4 7H 2 O and the effects of glycerol on the number of live bacteria, and the development and design of using relatively cheap natural raw materials such as soybean powder and corn powder as main nitrogen sources, carbon sources and growth factors to achieve efficient and low-cost probiotic fermentation, which is conducive to promoting the large-scale production of probiotic products.

[0013] It can be understood that the soybean powder and corn powder in the present invention refer to soybean powder and corn powder that can be used in the field of microbial fermentation. Specifically, the soybean powder is commercially available food-grade soybean powder, which is obtained by grinding soybeans after washing, drying and baking, and has a protein content of more than 40%. The corn powder is commercially available food-grade corn powder, which is obtained by washing, drying, shelling and grinding corn, and has a starch content of more than 65%.

[0014] Preferably, in step (S1), the minimum level "-1" of factor A is 10 g / L and the maximum level "1" is 15 g / L, the minimum level "-1" of factor B is 10 g / L and the maximum level "1" is 15 g / L, the minimum level "-1" of factor C is 2 g / L and the maximum level "1" is 3 g / L, the minimum level "-1" of factor D is 1 g / L and the maximum level "1" is 1.5 g / L, the minimum level "-1" of factor E is 1 g / L and the maximum level "1" is 1.5 g / L, the minimum level "-1" of factor F is 1 g / L and the maximum level "1" is 1.5 g / L, and the minimum level "-1" of factor G is 3 mL / L and the maximum level "1" is 4.5 mL / L.

[0015] Preferably, in step (S1), the factors that have a significant impact on the response value are screened as factor A, factor B and factor C.

[0016] Preferably, the regression equation is: Y=9.611+0.085X 1 +0.006X 2 +0.190X 3 -0.074X 1 *X 1 -0.1667X 2 *X 2 -0.098X 3 *X 3 +0.003X 1 *X 2 +0.020X 1 *X 3 -0.039X 2 *X 3 , where Y is the number of viable bacteria, X is 1 is the content coefficient of soybean powder in the culture medium, X 2 is the content coefficient of corn flour in the culture medium, X 3 NH 4 NO 3 Content coefficient, “*” represents the mathematical algorithm “multiplication”.

[0017] In the present invention, the designed prediction regression equation can accurately predict the response value, indicating that it can well predict the actual fermentation situation and ensure the effectiveness of the designed culture medium.

[0018] It can be understood that the present invention can be designed for testing any probiotics, and the probiotics can include any one or a combination of at least two of Lactobacillus probiotics, Bifidobacterium probiotics, Streptococcus probiotics, Enterococcus probiotics, Lactococcus probiotics, Pediococcus probiotics, Staphylococcus probiotics, Bacillus probiotics or Leuconostoc probiotics.

[0019] Preferably, the Lactobacillus probiotics include Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus casei, Lactobacillus paracasei, Lactobacillus reuteri, Lactobacillus acidophilus, Lactobacillus delbrueckii, Lactobacillus salivarius, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus jensenii, Lactobacillus sakei, Lactobacillus crispatus, Lactobacillus salivarius, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus jensenii, Lactobacillus sakei, Lactobacillus crispatus, Lactobacillus salivarius, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus johnsonii, Lactobacillus jensenii, Lactobacillus salivarius ... crispatus or Propionibacterium freudenreichii, or a combination of at least two thereof.

[0020] Preferably, the Bifidobacterium probiotic includes any one or a combination of at least two of Bifidobacterium animalis, Bifidobacterium longum subsp. longum, Bifidobacterium breve, Bifidobacterium adolescentis, Bifidobacterium infantis subsp. infanttis or Bifidobacterium bifidum.

[0021] Preferably, the Streptococcus probiotic includes Streptococcus salivarius subsp. thermophilus.

[0022] Preferably, the Enterococcus probiotics include Enterococcus faecalis and / or Enterococcus faecium.

[0023] Preferably, the Lactococcus probiotic includes Lactococcus lactis.

[0024] Preferably, the Pediococcus probiotics include Pediococcus acidilactici and / or Pediococcus pentosaceus.

[0025] Preferably, the Staphylococcus probiotics include any one of Mammaliicoccus vitulinus, Staphylococcus xylosus or Staphylococcus carnosus, or a combination of at least two thereof.

[0026] Preferably, the Bacillus probiotics include Bacillus coagulans and / or Bacillus licheniformis.

[0027] Preferably, the Leuconostoc probiotic includes Leuconostoc mesenteroides.

[0028] Preferably, the culturing in step (S4) uses an oscillation parameter of 150 to 250 r / min, a temperature of 26 to 32° C., and a culturing time of 18 to 72 h.

[0029] In a second aspect, the present invention provides an application of the probiotic fermentation medium design method described in the first aspect in culturing probiotics, wherein the application comprises designing a medium using the probiotic fermentation medium design method described in the first aspect, and culturing probiotics using the designed medium.

[0030] The present invention develops a culture medium design strategy suitable for probiotic fermentation, which can be tested and designed for any probiotics, and explores culture media that can achieve efficient and low-cost fermentation, thereby facilitating large-scale production.

[0031] In a third aspect, the present invention provides a probiotic fermentation medium, wherein the probiotic fermentation medium is designed by the probiotic fermentation medium design method described in the first aspect.

[0032] Preferably, the probiotic fermentation medium contains soybean powder, corn powder, NH 4 NO 3 , NaCl, K 2 HPO 4 ·3H 2 O、MgSO 4 7H 2 O and glycerol.

[0033] Preferably, the probiotic fermentation medium contains 23.5 g / L soybean powder, 19.5 g / L corn powder, 6 g / L NH 4 NO 3 , 1.5g / L NaCl, 1.5g / L K 2 HPO 4 ·3H 2 O, 1.5 g / L MgSO 4 7H 2 O and 3mL / L glycerol.

[0034] It can be understood that, based on the optimal concentration ratio formula designed by the present invention, the concentration of each component is appropriately adjusted, and the culture medium that can achieve similar culture effects should be within the protection scope of the present invention. For example, the probiotic fermentation culture medium contains 23-24 g / L soybean powder, 19-20 g / L corn flour, 5.5-6.5 g / L NH 4 NO 3 , 1~2g / L NaCl, 1~2g / L K 2 HPO 4 ·3H 2 O, 1-2 g / L MgSO 4 7H2 O and 2.5-3.5 mL / L glycerol.

[0035] Compared with the prior art, the present invention has at least the following beneficial effects:

[0036] The present invention provides a design optimization method for probiotic fermentation medium components, using soybean powder, using corn flour to replace peptone as a nitrogen source and carbon source, and studying its specific ratio, confirming the key factors and influence degree of the results, understanding the interaction effect between important influencing factors, establishing a prediction model between factors and response values, and obtaining the factor level when the response value is most appropriate. Fermentation medium optimization is a multi-factor multi-level experimental design. It is difficult to obtain a better medium formula only through orthogonal experiments or uniform designs. The present invention uses Minitab software to establish a mathematical model including the first-order terms, square terms and first-order interaction terms between any two factors of each factor, and the data is processed by a computer. The implicit rules in the data are intuitively expressed by a stereogram and described by a mathematical model, thereby revealing the deep-level rules, and designing the experimental optimization of the most suitable component ratio of the probiotic strain fermentation medium; improving the production efficiency of the strain and reducing the economic cost of its fermentation, laying the foundation for the large-scale fermentation of the probiotic bacterial liquid. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Main effect diagram for the Plackett-Burman design experiment of the present invention;

[0038] Figure 2 The response surface analysis diagram of the Box-Behnken central combination design experiment of the present invention is as follows;

[0039] Figure 3 It is a contour map of the Box-Behnken central combination design experiment of the present invention. DETAILED DESCRIPTION

[0040] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0041] If no specific techniques or conditions are specified in the examples, the techniques or conditions described in the literature in the field or the product instructions are used. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0042] In the specific embodiment of the present invention, the strain of Lactobacillus plantarum is used as an example for testing, and the method for detecting the number of viable bacteria in the fermentation broth is carried out with reference to GB 4789.35 National Food Safety Standard Food Microbiology Test Lactic Acid Bacteria Test.

[0043] The main process of live bacteria detection includes: taking 25mL of bacterial solution and adding it to a sterile conical flask containing 225mL of diluent, shaking it thoroughly, and diluting it in a 10-fold series gradient. Select an appropriate dilution gradient, take 1mL of diluent and add it to a sterile plate, add 15mL to 20mL of melted MRS agar medium to each plate, and put it into an incubator after the MRS agar medium solidifies, at 36℃±1℃, culture for 48h to 72h, and count after the culture is completed.

[0044] In the present invention, corn flour was purchased from Shanghai Jinshan Cereals, Oils and Foods Co., Ltd., and soybean flour was purchased from Shanghai Jinshan Cereals, Oils and Foods Co., Ltd.

[0045] Example 1

[0046] The present invention provides a method for optimizing components of a probiotic fermentation medium, comprising the following steps:

[0047] S1, 1 mL of the probiotic strain was inoculated into 9 mL of MRS liquid medium;

[0048] S2, placing the culture medium in a shaker for shaking culture;

[0049] S3, take 5 mL of bacterial solution in the logarithmic growth phase after 20 h of culture and inoculate it into a conical flask containing 45 mL of MRS liquid culture medium;

[0050] S4, placing the flask in a shaker for shaking culture;

[0051] S5, using Plackett-Burman design, examines the seven factors (A, B, C, D, E, F, G), and selects the factors that have a greater impact on the number of viable bacteria. For each factor, the lowest level is "-1" and the highest level is "1", and the number of viable bacteria is used as the response value. The experimental factors and levels are shown in the table below:

[0052] S6, conduct a rapid climbing experiment design for the important influencing factors selected by the Plackett-Burman experiment, and then use the highest point of the rapid climbing experiment as the center point of the subsequent Box-Behnken central combination experimental design.

[0053] S7, according to the Box-Behnken central composite experimental design principle, the three important influencing factors determined by the Plackett-Burman experimental design and the addition level of the important factors determined by the steepest climbing experiment, designed a response surface analysis of 3 factors and 3 levels with a total of 15 experimental points. Import the experimental results into the Minitab program data table, select "Statistics" click "DOE" select "Response Surface Analysis", import factors and response values, select "Encoded", generate regression coefficients and variance analysis, and establish a quadratic polynomial regression equation. In order to predict the response value, the partial derivative of the regression equation is calculated to predict the optimal experimental point (X 1 , X 2 , X 3 ), at which point the levels of the three factors were determined, thereby determining the composition of the optimized culture medium.

[0054] S8, while keeping the other culture medium components and culture conditions unchanged, inoculate the strain into the optimized culture medium, place the culture medium in a shaker, perform shaking culture, and detect the number of viable bacteria in the fermentation broth. Compare the difference between the number of viable bacteria in the fermentation broth after the culture medium is optimized and the predicted experimental results to prove the optimization effect of the culture medium components.

[0055] The storage temperature of the probiotic strain before use in step S1 is -80°C.

[0056] In step S2, the shaking parameter of the shaker is 200 r / min, the culture temperature is 28° C., and the culture time is 18-24 h.

[0057] In step S4, the shaking parameter of the shaker is 200 r / min, the culture temperature is 28° C., and the culture time is 20 h.

[0058] The experimental factors and levels in step S5 are shown in Table 1 below.

[0059] Table 1

[0060]

[0061] Finally, in step S8, the shaking parameters of the shaker are 200 r / min, the temperature is 28° C., and the incubation time is 20 h.

[0062] Experimental results:

[0063] 1. Plackett-Burman Experimental Design Results

[0064] The results of the Plackett-Burman experiment (with viable count as the influencing value) were analyzed using Minitab software and are shown in Tables 2 and 3. From the experimental results, it can be seen that the influence of the seven influencing factors on the response value is as follows: NH 4 NO 3(C)>Soybean flour(A)>Corn flour(B)>K 2 HPO 4 ·3H 2 O(E)>MgSO 4 7H 2 O(F)>glycerol(G)>NaCl(D).

[0065] Table 2

[0066] A B C D E F G Y(log10 cfu / mL) 01 1 1 -1 1 1 -1 1 9.014 02 -1 1 1 -1 1 -1 -1 9.163 03 -1 -1 1 1 1 -1 1 8.916 04 -1 -1 -1 1 1 1 -1 8.752 05 1 1 1 -1 1 1 -1 9.681 06 1 -1 1 1 -1 1 -1 9.118 07 -1 1 -1 -1 -1 1 1 8.589 08 -1 1 1 1 -1 1 1 9.234 09 1 1 -1 1 -1 -1 -1 9.066 10 1 -1 -1 -1 1 1 1 8.957 11 -1 -1 -1 -1 -1 -1 -1 8.321 12 1 -1 1 -1 -1 -1 1 9.167

[0067] Table 3

[0068] effect coefficient Standard error of coefficient T P A 0.33800 0.16900 0.03932 4.30 0.013 B 0.25267 0.12633 0.03932 3.21 0.032 C 0.43000 0.21500 0.03932 5.47 0.005 D 0.03700 0.01850 0.03932 0.47 0.662 E 0.16467 0.08233 0.03932 2.09 0.104 F 0.11400 0.0570 0.03932 1.45 0.221 G -0.03733 -0.01867 0.03932 -0.47 0.660

[0069] From Table 3, we can see that among the main effects of these seven factors, glycerol showed a negative effect on the response value, while the other components showed a positive effect on the response value, among which soybean flour, corn flour and NH 4 NO 3 If there is a significant effect on the response value (confidence level>95%, P<0.05), it will be used as a significant factor for the next step of the experiment. Figure 1 As shown in the figure, the experimental results can be intuitively reflected in the main effect diagram.

[0070] 2. Rapid climbing test results

[0071] The optimal region of significant factors was determined by the rapid climbing experiment. As shown in Table 4, the number of viable bacteria was the highest under the condition of experiment No. 4. Therefore, condition No. 4 was used as the center point of the response surface design factor level.

[0072] Table 4

[0073]

[0074] 3. Determination of the optimal level of important factors

[0075] According to the Box-Behnken central composite design principle of response surface, with viable cell count concentration as the influencing value and three components affecting fermentation, a response surface analysis experiment with three factors and three levels with a total of 15 experimental points was designed. The factors and level values ​​of the response surface design are shown in Table 5, and the experimental results are shown in Table 6.

[0076] Table 5

[0077]

[0078] Table 6

[0079] Experiment No. Corn flour Soybean powder <![CDATA[NH 4 NO 3 ]]> Y(log10 cfu / mL) 01 0 0 0 9.608 02 0 0 0 9.611 03 1 1 0 9.474 04 1 0 1 9.758 05 -1 1 0 9.231 06 1 -1 0 9.502 07 -1 0 -1 9.158 08 1 0 -1 9.223 09 0 1 1 9.471 10 -1 -1 0 9.274 11 0 0 0 9.615 12 -1 0 1 9.612 13 0 1 -1 9.281 14 0 -1 1 9.489 15 0 -1 -1 9.143

[0080] Taking the number of viable bacteria as the response value, Minitab software was used for regression fitting, and the regression equation Y = 9.611 + 0.085X 1 +0.006X 2 +0.190X 3 -0.074X 1 *X 1 -0.1667X 2 *X 2 -0.098X 3 *X 3 +0.003X 1 *X 2 +0.020X 1 *X 3 -0.039X 2 *X 3 , conduct normative analysis on the regression model, and combine the software to make response surface analysis diagrams and contour maps, such as Figure 2 and Figure 3 Each response surface represents the interaction between two independent variables. It can be seen from the response surface and the corresponding contour lines that the interaction between the two factors is quite obvious. After software analysis, the optimal experimental point (X 1 , X 2 , X 3 )=(0.696, -0.091, 1), and substitute it into the regression equation to get the predicted viable count concentration of 5.5×10 9 cfu / mL, according to the values ​​of the three factors, after conversion, the optimal values ​​of the three factors are (23.5, 19.5, 6), and the culture medium formula is 23.5g / L soybean powder, 19.5g / L corn flour, NH 4 NO 3 6g / L, NaCl 1.5g / L, K 2 HPO 4 ·3H 2 O 1.5g / L, MgSO 4 7H 2 O 1.5 g / L, glycerol 3 mL / L.

[0081] Example 2

[0082] This example conducts a verification experiment on the culture medium designed in Example 1.

[0083] In order to verify the degree of fit between the predicted values ​​of the model and the experimental values, a verification experiment was carried out under the optimal fermentation conditions, and the predicted medium formula was used for three repeated experiments. The specific experimental process included:

[0084] Take a 1mL frozen probiotic strain from a -80℃ freezer. After thawing, inoculate it into 9mL of MRS liquid culture medium and place it in a 28℃ shaker for 200r / min for 20h. After the culture is completed, take 5mL of the bacterial solution and transfer it to 45mL of optimized culture medium (the culture medium components are: soybean powder 23.5g / L, corn flour 19.5g / L, NH 4 NO 3 6g / L, NaCl 1.5g / L, K 2 HPO 4 ·3H 2 O 1.5g / L, MgSO 4 7H 2 O 1.5g / L, glycerol 3mL / L), placed in a 28℃ shaker, 200r / min, cultured for 20h. The average number of viable bacteria obtained in three experiments was 5.2×10 9 cfu / mL. Experimental value and model prediction value (5.5×10 9 cfu / mL) is relatively close, which shows that the model can well predict the actual fermentation situation.

[0085] Similarly, take a 1 mL frozen probiotic strain from a -80°C freezer. After thawing, inoculate it into 9 mL of MRS liquid culture medium and place it in a 28°C shaker for 200 r / min for 20 hours. After the culture is completed, take 5 mL of the bacterial solution and transfer it to 45 mL of culture medium (the culture medium components are: peptone 20 g / L, NH 4 NO 3 6g / L, NaCl 1.5g / L, K 2 HPO 4 ·3H 2 O 1.5g / L, MgSO 4 7H 2 O1.5g / L, glycerol 3mL / L), placed in a 28℃ shaker for 20h at 200r / min. The average number of viable bacteria obtained in three experiments was 5.2×10 9 cfu / mL. The experimental data showed that when the peptone in the culture medium was replaced by soybean powder and corn powder, the number of viable bacteria after fermentation did not change significantly. This shows that using soybean powder and corn powder to replace peptone can effectively reduce the fermentation cost.

[0086] Comparative Example 1

[0087] Compared with Example 2, the difference is that the soybean powder in the optimized culture medium is replaced by corn powder in equal amounts. The fermentation culture process is the same as that of Example 2 and will not be repeated here. The experimental results are shown in Table 7.

[0088] Comparative Example 2

[0089] Compared with Example 2, the difference is that the corn flour in the optimized culture medium is replaced by soybean flour in equal amounts. The fermentation culture process is the same as that of Example 2 and will not be repeated here. The experimental results are shown in Table 7.

[0090] Comparative Example 3

[0091] Compared with Example 2, the difference is that the corn flour in the optimized culture medium is replaced by corn flour in equal amounts. The fermentation culture process is the same as that of Example 2 and will not be repeated here. The experimental results are shown in Table 7.

[0092] Comparative Example 4

[0093] Compared with Example 2, the difference is that the soybean powder in the optimized culture medium is replaced by mung bean powder in equal amounts. The fermentation culture process is the same as that of Example 2 and will not be repeated here. The experimental results are shown in Table 7.

[0094] From the experimental results in Table 7, it can be found that when the optimized culture medium components are replaced by other substances (Comparative Examples 1 to 4), the number of viable bacteria in the fermentation broth is significantly reduced compared with Example 2. It can be seen that the optimized culture medium component ratio in the present invention cannot be changed arbitrarily.

[0095] Table 7

[0096] The number of viable bacteria in the fermentation broth (average value of 3 experiments) Comparative Example 1 <![CDATA[1.9×10 9 cfu / mL]]> Comparative Example 2 <![CDATA[3.2×10 9 cfu / mL]]> Comparative Example 3 <![CDATA[3.5×10 9 cfu / mL]]> Comparative Example 4 <![CDATA[2.8×10 9 cfu / mL]]>

[0097] In summary, the present invention can conveniently apply Plackett-Burman design and response surface analysis to carry out reasonable experimental arrangement and data analysis with the help of Minitab software, and use the software to make response surface analysis diagrams and contour maps, and obtain the optimal fermentation medium for probiotics: soybean powder 23.5g / L, corn flour 19.5g / L, NH 4 NO 3 6g / L, NaCl1.5g / L, K 2 HPO 4 ·3H 2 O 1.5g / L, MgSO 4 7H 2 O 1.5g / L, glycerol 3mL / L. Under this condition, the predicted results were verified and the number of viable bacteria in the fermentation broth was 5.2×10 9 cfu / mL, and the predicted value (5.5×10 9cfu / mL) is slightly different from the experimental value, indicating that the model can better predict the actual fermentation situation and is suitable for multi-factor optimization of fermentation conditions. The experimental results show that it is highly feasible to use soybean powder, corn powder and other low-cost raw materials to replace experimental reagents such as peptone; using the optimized culture medium to ferment probiotic strains not only greatly increases the concentration of viable bacteria, but also greatly reduces the economic cost, which is of great significance to the field of probiotic production.

[0098] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for designing a probiotic fermentation medium, characterized in that: The design method comprises the following steps: (S1) Plackett-Burman experiment was used to investigate factors A, B, C, D, E, F and G. Factor A was soybean powder, factor B was corn flour, factor C was NH4NO3, factor D was NaCl, factor E was K2HPO4·3H2O, factor F was MgSO4·7H2O, and factor G was glycerol. For each factor, the lowest level was "-1" and the highest level was "1". The number of viable bacteria was used as the response value. The factors were ranked according to the degree of influence on the response value, and the factors with significant influence on the response value were screened. (S2) Conduct a rapid climbing experiment on the factors selected by the Plackett-Burman experiment to obtain the addition level of each factor; (S3) According to the Box-Behnken central composite experimental design principle, the factors selected by the Plackett-Burman experiment and the addition levels of the factors obtained by the rapid climbing experiment, a response surface analysis experiment is designed, a response surface analysis is performed, regression coefficients and variance analysis are generated, a quadratic polynomial regression equation is established, partial derivatives of the regression equation are calculated to predict the optimal experimental points of the factors, and the addition levels of the factors are determined, thereby determining the composition of the optimized culture medium; (S4) With other factors and culture conditions unchanged, the probiotics are inoculated into the optimized culture medium for culture, the number of viable bacteria in the fermentation broth is detected, the difference between the number of viable bacteria in the fermentation broth after the culture medium is optimized and the predicted experimental results is compared, and the optimization effect of the culture medium components is analyzed.

2. The method for designing a probiotic fermentation medium according to claim 1, characterized in that: In step (S1), the minimum level "-1" of factor A is 10g / L and the maximum level "1" is 15g / L, the minimum level "-1" of factor B is 10g / L and the maximum level "1" is 15g / L, the minimum level "-1" of factor C is 2g / L and the maximum level "1" is 3g / L, the minimum level "-1" of factor D is 1g / L and the maximum level "1" is 1.5g / L, the minimum level "-1" of factor E is 1g / L and the maximum level "1" is 1.5g / L, the minimum level "-1" of factor F is 1g / L and the maximum level "1" is 1.5g / L, and the minimum level "-1" of factor G is 3mL / L and the maximum level "1" is 4.5mL / L.

3. The method for designing a probiotic fermentation medium according to claim 1 or 2, characterized in that: Step (S1) screens factors that have a significant impact on the response value as factor A, factor B and factor C.

4. The method for designing a probiotic fermentation medium according to any one of claims 1 to 3, characterized in that: The regression equation is: Y=9.611+0.085X1+0.006X2+0.190X3-0.074X1*X1-0.1667X2*X2-0.098X3*X3+0.003X1*X2+0.020X1*X3-0.039X2*X3, wherein Y is the number of viable bacteria, X1 is the content coefficient of soybean powder in the culture medium, X2 is the content coefficient of corn flour in the culture medium, and X3 is the content coefficient of NH4NO3 in the culture medium.

5. The method for designing a probiotic fermentation medium according to any one of claims 1 to 4, characterized in that: The probiotics include any one or a combination of at least two of Lactobacillus probiotics, Bifidobacterium probiotics, Streptococcus probiotics, Enterococcus probiotics, Lactococcus probiotics, Pediococcus probiotics, Staphylococcus probiotics, Bacillus probiotics or Leuconostoc probiotics.

6. The method for designing a probiotic fermentation medium according to any one of claims 1 to 5, characterized in that: The culturing in step (S4) uses an oscillation parameter of 150 to 250 r / min, a temperature of 26 to 32° C., and a culturing time of 18 to 72 h.

7. Application of the method for designing a probiotic fermentation medium according to any one of claims 1 to 6 in culturing probiotics, characterized in that: The application comprises designing a culture medium using the probiotic fermentation culture medium design method according to any one of claims 1 to 6, and culturing probiotics using the designed culture medium.

8. A probiotic fermentation medium, characterized in that: The probiotic fermentation medium is designed by the probiotic fermentation medium design method according to any one of claims 1 to 6.

9. The probiotic fermentation medium according to claim 8, characterized in that The probiotic fermentation medium contains soybean powder, corn powder, NH4NO3, NaCl, K2HPO4·3H2O, MgSO4·7H2O and glycerol.

10. The probiotic fermentation medium according to claim 9, characterized in that: The probiotic fermentation medium contains 23.5 g / L soybean powder, 19.5 g / L corn powder, 6 g / L NH4NO3, 1.5 g / L NaCl, 1.5 g / L K2HPO4·3H2O, 1.5 g / L MgSO4·7H2O and 3 mL / L glycerol.

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