Method for optimizing fermentation of haemophilus parasuis through response surface

Abstract

The invention discloses a response surface optimization haemophilus parasuis fermentation method, through a single factor experiment and a Box-Behnken response surface optimization test, by taking the maximum viable count of haemophilus parasuis 12 type as an evaluation index, the optimized haemophilus parasuis 12 type fermentation medium comprises the following components: yeast extract = 0.5919998%, NAD = 0.0952694%, magnesium sulfate = 0.211466 g / L, and bovine serum concentration = 9.91374%. The haemophilus parasuis used in the invention is a rare pathogenic bacterium and belongs to a gram-negative bacterium. The strain mainly infects pigs, causes serious respiratory system diseases and seriously affects stable development of the pig breeding industry; the HPS fermentation process is optimized and researched, so that the yield of the haemophilus parasuis is increased, and meanwhile, the application value and the economic benefit of the haemophilus parasuis as a vaccine product subsequently can be increased.

Description

Technical Field

[0001] The invention belongs to the technical field of biological fermentation, and particularly relates to a method for optimizing fermentation of Haemophilus parasuis using a response surface methodology. Background Art

[0002] Haemophilus parasuis (HPS) is a rare pathogenic bacterium belonging to the Gram-negative bacteria. The bacterium mainly infects pigs, causing serious respiratory diseases, which seriously affects the stable development of the pig farming industry. HPS has many characteristics, such as sensitivity to environmental changes, large demand for oxygen, and is also very sensitive to temperature, pH and other conditions. The inappropriateness of these conditions will affect the growth and reproduction of HPS, thereby affecting its productivity. Fermentation technology is one of the key factors in the production of HPS products, involving many aspects, such as culture medium composition, different strains, fermentation conditions, etc. These factors directly affect cell growth and metabolism. Optimizing the fermentation process can effectively increase production, reduce production costs, and improve product quality and stability. In actual operation, the optimization of the fermentation process can be achieved by adjusting the ratio of substances in the culture medium, optimizing the screening of strains, and improving the production environment. The production of HPS products requires continuous exploration and research of fermentation technology to meet market demand and improve economic benefits.

[0003] The basis of HPS fermentation is a reasonable medium formula. Different medium components can affect cell growth, metabolism and product formation. HPS medium usually includes carbon source, nitrogen source, inorganic salt, growth factor and other additives. The medium optimization of Haemophilus parasuis (HPS) refers to the optimization of the proportion and concentration of each component on the basis of fermentation to achieve higher yield and better product quality. The specific details are fermentation basis and medium optimization: select suitable bacterial strains and culture medium, and optimize according to the characteristics of HPS, such as adding suitable carbon, nitrogen source and inorganic salt. Nitrogen source is necessary for HPS growth and metabolism. Commonly used nitrogen sources include yeast powder, broth paste, peptone, etc. Studies have shown that adding appropriate amounts of peptone and yeast powder can increase the growth rate and yield of HPS. For the HPS fermentation process, experiments are required to determine the optimal stirring speed, and factors such as the type of reactor and gas distribution oxygen should be considered comprehensively. By optimizing the components and concentrations of the culture medium, the growth rate and yield of HPS can be effectively improved, and the product quality and stability can also be improved.

[0004] There are many methods to optimize fermentation parameters, such as optimization method, single factor method, orthogonal test method. Usually, in multi-parameter optimization experiments, the best data point cannot be intuitively judged only by the optimization method experimental results or single factor experimental results. Therefore, it is necessary to combine the orthogonal experimental method and determine the trend of each data point by means of fitting equations. Response surface method is a mathematical modeling method, which belongs to the orthogonal experimental method. This method fits the function of the data points in the experiment and uses graphical technology to display the fitted function so that the best test point can be selected intuitively. Summary of the invention

[0005] The purpose of the present invention is to provide a method for optimizing the fermentation process of Haemophilus parasuis by response surface.

[0006] To achieve the above object, the present invention adopts the following technical solutions: A method for optimizing Haemophilus parasuis fermentation using a response surface method comprises the following steps: 1) After inactivating bovine serum in a water bath at 56°C for 30 min, add the NAD solution filtered through a 0.22 μm filter membrane into a sterilized TSB liquid medium and mix well. Inoculate the glycerol stock solution of Haemophilus parasuis type 12 into the above TSB liquid medium and culture at 37°C and 180 r / min shaking for 10 h for preliminary activation to obtain a bacterial solution. 2) Add inactivated bovine serum and NAD solution filtered through a 0.22 μm filter membrane to TSA solid medium, take 30 μL of the bacterial solution obtained in step 1) and inoculate it into TSA solid medium for three-zone streaking and culture at 37°C for 9 hours, pick a single colony and inoculate it into TSB medium for secondary activation to obtain seed solution; 3) The seed liquid obtained in step 2) was inoculated into TSB liquid culture medium, the culture temperature was controlled at 37°C, the shaking speed was 180 r / min, the yeast extract was 0.591998%, NAD was 0.0952694%, magnesium sulfate was 0.211466 g / L, bovine serum concentration was 9.91374%, and fermentation was carried out for 9 hours to obtain fermentation liquid.

[0007] Furthermore, the yeast extract = 0.591998%, NAD = 0.0952694%, magnesium sulfate = 0.211466 g / L, bovine serum concentration = 9.91374% were optimized by response surface methodology, and the specific steps are: 1) Single factor experiment: The seed liquid was inoculated into TSB liquid medium, and the fermentation time was 9 hours. The yeast extract content, NAD content, magnesium sulfate content, and bovine serum content were selected as variables for single factor experiments. The maximum viable count of Haemophilus parasuis type 12 was used as the determination index to screen out the parameter range of yeast extract content, NAD content, magnesium sulfate content, and bovine serum content; 2) Response surface method optimization: According to the parameter range screened in step 1), the fermentation time was fixed at 9h, the yeast extract content, NAD content, magnesium sulfate content, and bovine serum content were taken as the four factors to be investigated, and the maximum number of viable bacteria of Haemophilus parasuis type 12 was taken as the response value. The Box-Behnken principle was used to design a 4-factor 3-level test table for response surface experiment, and the multiple regression equation was simulated; 3) Solving the multiple regression equation in step 2) yielded the optimal fermentation medium composition: yeast extract = 0.591998%, NAD = 0.0952694%, magnesium sulfate = 0.211466 g / L, bovine serum concentration = 9.91374%.

[0008] Furthermore, the parameter ranges of the yeast extract content, NAD content, magnesium sulfate content, and bovine serum content are: yeast extract content is 0.05% to 7%, NAD content is 0.016% to 0.2%, magnesium sulfate content is 0 g / L to 0.7 g / L, and bovine serum content is 5% to 12%.

[0009] Furthermore, the multiple regression equation is: Y=-1051.03+205.72A+9274.33B-4982.50C+610.67D-532.50AB+355.00AC+510.00AD -3875.00BC-16100.00BD+7500.00CD-12.89A²-2901.67B²+2285.83C²-25226.67D²; Wherein, Y is the maximum viable count of Haemophilus parasuis type 12, A is the bovine serum content, B is the magnesium sulfate content, C is the yeast extract content, and D is the NAD content.

[0010] The Haemophilus parasuis is Haemophilus parasuis type 12, and the strain is preserved and provided by Zhaofenghua Biotechnology (Fuzhou) Co., Ltd.

[0011] The beneficial effects of the present invention are: 1) The Haemophilus parasuis used in the present invention is an important pathogen in pig reproduction and production, and its metabolites have important medical and industrial application values. Basic research on the fermentation of HPS is an important link in exploring the potential of HPS metabolites and improving production efficiency.

[0012] 2) The present invention uses response surface methodology to optimize and improve the maximum viable count of Haemophilus parasuis type 12. Compared with the fermentation broth obtained under the initial fermentation conditions and the optimal level combination conditions of the single-factor experiment, the yield of viable count of Haemophilus parasuis type 12 in the fermentation broth obtained by optimizing the optimal fermentation medium composition obtained by the response surface methodology is significantly improved.

[0013] 3) The response surface optimization used in the present invention to increase the number of live bacteria of Haemophilus parasuis type 12 also provides an excellent method for the development of livestock vaccines, which can effectively solve the problems of low live bacteria concentration and low live bacteria concentration in the fermentation broth, and has good application potential in the field of biomedicine. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The figure shows the results of the single factor optimization experiment of yeast extract content in the culture medium. The statistical analysis of the maximum bacterial concentration and the maximum viable count of Haemophilus parasuis type 12 when the yeast extract content in the culture medium is 0.05%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, and 0.7%.

[0015] Figure 2 The results of the single factor optimization experiment of NAD content in the culture medium are shown in Figure 1. The statistical analysis of the maximum bacterial concentration and the maximum number of viable bacteria of Haemophilus parasuis type 12 when the NAD content in the culture medium is 0.016%, 0.05%, 0.075%, 0.1%, 0.125%, 0.15%, 0.175%, and 0.2%.

[0016] Figure 3 This is the result of the single factor optimization experiment of magnesium sulfate content in the culture medium. Statistical analysis of the maximum bacterial concentration and the maximum number of viable bacteria of Haemophilus parasuis type 12 when the magnesium sulfate content in the culture medium is 0g / L, 0.1g / L, 0.2g / L, 0.3g / L, 0.4g / L, 0.5g / L, 0.6g / L, and 0.7g / L.

[0017] Figure 4 This is the result of the single factor optimization experiment of bovine serum content in the culture medium. The statistical analysis of the maximum bacterial concentration and the maximum number of viable bacteria of Haemophilus parasuis type 12 when the bovine serum content in the culture medium is 5%, 6%, 7%, 8%, 9%, 10%, 11%, and 12%.

[0018] Figure 5 These are the contour map and response surface diagram of the interactive effect of bovine serum and magnesium sulfate on the number of viable bacteria.

[0019] Figure 6 These are the contour map and response surface diagram of the interactive effect of bovine serum and yeast extract on the number of viable cells.

[0020] Figure 7 These are the contour map and response surface diagram of the interactive effect of bovine serum and NAD on the number of viable bacteria.

[0021] Figure 8 These are the contour map and response surface diagram of the interactive effects of magnesium sulfate and yeast extract on the number of viable bacteria.

[0022] Fig. 9 These are the contour map and response surface diagram of the interactive effect of magnesium sulfate and NAD on the number of viable bacteria.

[0023] Fig.10 Contour map and response surface diagram of the interactive effect of yeast extract and NAD on the number of viable cells. DETAILED DESCRIPTION

[0024] In order to make the contents of the present invention easier to understand, the technical solution of the present invention is further explained below in combination with specific implementation methods. However, the following examples are merely examples of the present invention and do not represent the scope of protection of the rights limited by the present invention. The scope of protection of the present invention shall be subject to the claims.

[0025] The Haemophilus parasuis type 12 involved in the present invention is preserved and provided by Zhaofenghua Biotechnology (Fuzhou) Co., Ltd.; TSB liquid culture medium and TSA solid culture medium are used for strain preservation and activation; newborn calf serum, yeast extract, tryptone soy broth, tryptone soy agar, agar, magnesium sulfate, and NAD (nicotinamide adenine dinucleotide) are all purchased from Shanghai Bioengineering Company, and all reagents are analytically pure.

[0026] Example 1 Single factor optimization test to determine the optimal level interval of factors This experimental example adopts a single factor optimization test method, taking the number of viable bacteria of Haemophilus parasuis type 12 as an indicator, to determine the optimal level range of the four culture conditions of yeast extract content, NAD content, magnesium sulfate content, and bovine serum content. The specific implementation steps are as follows: (1) Preparation of seed solution: 5% bovine serum was inactivated in a water bath at 56°C for 30 min, and then added to sterilized TSB liquid medium together with 0.016% NAD solution filtered through a 0.22 μm filter membrane and mixed well. The glycerol stock of Haemophilus parasuis type 12 stored in a -80°C refrigerator was inoculated into the above TSB liquid medium, and cultured in a shaking incubator at 37°C and 180 r / min for 10 h for preliminary activation. 30 μL of the cultured bacterial solution was drawn into three zones on TSA solid medium added with 5% inactivated bovine serum and 0.016% NAD solution filtered through a 0.22 μm filter membrane. After culture in a 37°C incubator for 9 h, a single colony on the medium was picked and inoculated into a medium with the same components as those for preliminary activation for secondary activation. This was the seed solution. Wherein, the TSB liquid culture medium (g / L): 30g of tryptone soy broth dry powder, 0.5g of yeast extract, fixed to 1L with distilled water, natural pH.

[0027] Wherein, the TSA solid culture medium (g / L): 40g of tryptone soy agar dry powder, 0.5g of yeast extract, and 9g of agar; the volume is made up to 1L with distilled water, and the pH is natural.

[0028] (2) Preparation of fermentation broth: Take 1% of the seed liquid as the inoculum and inoculate it into TSB liquid culture medium (basic culture medium containing 5% bovine serum and 0.016% NAD) with different yeast content, NAD content, MgSO4 content, and bovine serum content. Set the fermentation temperature to 37°C, the shaking speed to 180 r / min, and the fermentation time to 9 h. Collect the fermentation broth as the control group for later use.

[0029] Wherein, the TSB liquid culture medium (g / L): 30g of tryptone soy broth dry powder, 0.5g of yeast extract, fixed to 1L with distilled water, natural pH.

[0030] (3) Determination of Haemophilus parasuis type 12 bacterial concentration: Take 250 μL of the completely shaken fermentation liquid and use a multi-function microplate reader to measure its absorbance at a wavelength of 600 nm, which is the bacterial concentration of the corresponding fermentation liquid.

[0031] (4) Determination of viable count of Haemophilus parasuis type 12: Shake the collected fermentation liquid thoroughly and dilute it to 10% of the original liquid. -6 Times, take 100 μL of the diluted bacterial solution and spread it on dry TSA solid medium (basal medium containing 5% bovine serum and 0.016% NAD), culture it in a constant temperature incubator at 37°C and 180 r / min for 9 hours, and then observe and count the colonies on the TSA solid medium with the naked eye.

[0032] The TSA solid culture medium (g / L) comprises: 40 g of tryptone soy agar dry powder, 0.5 g of yeast extract, 9 g of agar, and the volume is adjusted to 1 L with distilled water, with natural pH.

[0033] (5) Single factor optimization experiment: Taking the number of viable bacteria of Haemophilus parasuis type 12 as the indicator and TSB medium as the basic medium, the effects of yeast extract content, NAD content, magnesium sulfate content, and bovine serum content on the production of viable bacteria of Haemophilus parasuis type 12 were evaluated. The fermentation conditions were culture temperature of 37°C, shake flask speed of 180 rpm, medium pH of 7, inoculation amount of 1% (v / v), and fermentation time of 9 h. On this basis, single factor experiments were conducted on the four factors of yeast extract content, NAD content, magnesium sulfate content, and bovine serum content, that is, under the premise of controlling other factors at the initial level, the level of a single factor was changed, and the number of viable bacteria of Haemophilus parasuis type 12 in the fermentation broth at different levels of a single factor was measured to obtain the optimal level range of the factor to be explored. The setting ranges of each factor were as follows: the yeast extract content of the culture medium was 0.05% (v / v), 0.1% (v / v), 0.2% (v / v), 0.3% (v / v), 0.4% (v / v), 0.5% (v / v), 0.6% (v / v), and 0.7% (v / v); the NAD content of the culture medium was 0.016% (v / v), 0.05% (v / v), 0.075% (v / v), 0.1% (v / v), 0.125% (v / v), and 0. .15% (v / v), 0.175% (v / v), 0.2% (v / v); the magnesium sulfate content of the culture medium was 0g / L, 0.1g / L, 0.2g / L, 0.3g / L, 0.4g / L, 0.5g / L, 0.6g / L, 0.7g / L; the bovine serum content of the culture medium was 5% (v / v), 6% (v / v), 7% (v / v), 8% (v / v), 9% (v / v), 10% (v / v), 11% (v / v), 12% (v / v). The results of the single factor optimization experiment are as follows: Figure 1-Figure 4As shown, the results showed that: with the increase of yeast extract content, the number of HPS live bacteria also showed an increasing trend, and it reached a peak when the yeast extract content reached 0.4%, and then the yeast extract content increased further and showed an overall downward trend. Therefore, the optimal range of yeast extract content in the culture medium was in the range of 0.4%-0.6%, and the three levels of yeast extract content 0.4%, 0.5%, and 0.6% were selected as the optimal factor levels; with the increase of NAD content, the number of HPS live bacteria also showed an increasing trend, and it reached a peak when the NAD content reached 0.075%, and then the NAD content increased further and showed an overall downward trend. Therefore, the optimal range of NAD content in the culture medium was in the range of 0.05%-0.1%, and the three levels of yeast extract content 0.05%, 0.075%, and 0.1% were selected as the optimal factor levels; with the increase of magnesium sulfate content in the culture medium, the number of HPS live bacteria also showed an increasing trend, and when the magnesium sulfate content reached 0.4 g / L, and then showed an overall downward trend with the increase of magnesium sulfate content in the culture medium. Therefore, the optimal range of magnesium sulfate content in the culture medium is in the range of 0.3-0.5 g / L, and 0.2 g / L, 0.3 g / L, and 0.4 g / L of magnesium sulfate content were selected as the optimal factor levels; with the increase of bovine serum content in the culture medium, the number of HPS viable bacteria also showed an increasing trend. When the bovine serum content reached 9%, the number of HPS viable bacteria reached its peak, and then showed an overall downward trend with the increase of bovine serum content in the culture medium. Therefore, the optimal range of bovine serum content in the culture medium is in the range of 8%-10%, and 8%, 9%, and 10% of bovine serum content were selected as the optimal factor levels.

[0034] (6) Data processing: Each experiment was repeated three times and plotted using Origin 2021. Data are presented as mean ± SE. IBM SPSS Statistics 22 was used for one-way analysis of variance (ANOVA) analysis, and the LSD method was used for post hoc multiple comparisons.

[0035] Example 2 Optimization of response surface methodology to increase the number of viable Haemophilus parasuis Based on the single factor optimization experiment, this experimental example uses the Box-Behnken Design principle to complete the response surface experimental design of four factors and three levels of yeast extract content, NAD content, magnesium sulfate content, and bovine serum content. The number of viable bacteria of Haemophilus parasuis type 12 is used as the response value, and the regression equation between the response value and each factor is established. After regression analysis and verification, the response surface diagram of the regression equation is drawn. The optimal fermentation conditions of Haemophilus parasuis type 12 are predicted by the generated response surface optimization model, and the accuracy and effectiveness of the response surface optimization model are verified by the model verification experiment. The steps are as follows: (1) Preparation of seed solution: 5% bovine serum was inactivated in a water bath at 56°C for 30 min, and then mixed with 0.016% NADNAD solution filtered through a 0.22 μm filter membrane in sterile TSB liquid medium. Haemophilus parasuis type 12 stored in a -80°C refrigerator was inoculated into the above TSB liquid medium, and cultured in a shaking incubator at 37°C and 180 r / min for 10 h for preliminary activation. 30 μL of the cultured bacterial liquid was drawn into three zones on a TSA solid medium to which an appropriate amount of inactivated bovine serum and an appropriate amount of NAD solution filtered through a sterile 0.22 μm filter membrane were added. After 9 h of culture, a single colony on the medium was picked and inoculated into a medium with the same components as those for preliminary activation for secondary activation. This was the seed solution. Wherein, the TSB liquid culture medium (g / L): 30g of tryptone soy broth dry powder, 0.5g of yeast extract, fixed to 1L with distilled water, natural pH.

[0036] The TSA solid culture medium (g / L) comprises: 40 g of tryptone soy agar dry powder, 0.5 g of yeast extract, 9 g of agar, and the volume is adjusted to 1 L with distilled water, with natural pH.

[0037] (2) Box-Behenken test: On the basis of the single factor optimization experiment, 1% seed liquid was fixed as the inoculation amount, 37°C was the fermentation temperature, 180r / min was the shaking flask speed, and 9h was the fermentation time. The Box-Behnken Design principle was used to complete the response surface experimental design of four factors and three levels of yeast extract content, NAD content, magnesium sulfate content, and bovine serum content. The specific levels are shown in Table 1. The number of viable bacteria was taken as the response value. The experimental design scheme and experimental results are shown in Table 2.

[0038] Table 1 Table 2 After fermentation is completed, the fermentation liquid is collected for later use.

[0039] (3) Determination of HPS bacterial concentration in fermentation broth: Take 250 μL of completely shaken fermentation broth and measure its absorbance at a wavelength of 600 nm using a multifunctional microplate reader, which is the bacterial concentration of the corresponding fermentation broth.

[0040] (4) Determination of HPS viable bacteria count in fermentation broth: Shake the collected fermentation broth thoroughly and dilute it to 10% of the original solution. -6times, take 100 μL of the diluted bacterial solution and spread it on dry TSA solid medium. After culturing in a constant temperature incubator at 37°C and 180 r / min for 9 hours, observe and count the colonies on the TSA solid medium with the naked eye.

[0041] The TSA solid culture medium (g / L) comprises: 40 g of tryptone soy agar dry powder, 0.5 g of yeast extract, 9 g of agar, and the volume is adjusted to 1 L with distilled water, with natural pH.

[0042] (5) Data processing: Each experiment was repeated three times and plotted using Origin 2021. Data are mean ± SE. IBM SPSS Statistics 22 was used to perform one-way analysis of variance (ANOVA) to analyze statistical differences, and the LSD method was used for post hoc multiple comparisons. Different lowercase letters indicate significant differences between the two groups (P < 0.05).

[0043] (6) Model regression analysis: According to the experimental results, the regression equation between the response value and each factor is established as follows: Y=-1051.03+205.72A+9274.33B-4982.50C+610.67D-532.50AB+355.00AC+510.00AD-3875.00BC-16100.00BD+7500.00CD-12.89A²-2901.67B²+2285.83C²-25226.67D². Y represents the response value, that is, the number of viable bacteria. The regression variance analysis of the model is shown in Table 3. As can be seen from Table 3, the F value of the model is 15.52 and the P value is less than 0.0001, indicating that the model is extremely significant. The lack-of-fit term P=08985>0.05, indicating that the lack-of-fit term is not significant, the model is not overfitted, and the model can well explain the situation of each data point. Among the first-order terms, A and B are extremely significant, while C and D are not significant. According to the size of the F value, it can be obtained that the influence of each factor on the number of viable bacteria is magnesium sulfate>bovine serum>yeast extract>NAD. It is consistent with the results of the single-factor test. Among the interaction terms, AB is extremely significant, and AC, BC and BD are significant. The quadratic terms B² and C² are significant. The coefficient of determination of the model is R²=0.9395, and the degree of fit is high. The adjusted coefficient of determination of the model is R²Adj=0.8789, indicating that there are 12.11% of the data points that cannot be explained by this model. Therefore, the model can already well explain each data point in the experiment, and the model can be used as a prediction.

[0044] Table 3 Regression variance analysis of the model (7) Response surface analysis and determination of optimal culture conditions: After the reliability of the model is verified by regression analysis, the response surface diagram and contour map of the regression equation are drawn as shown in Figure 5-Figure 10As shown, Figure 5 It shows that when the magnesium sulfate content is greater than 0.3g / L, the contour lines in the direction of the magnesium sulfate content are denser, and the magnesium sulfate content plays a dominant role at this time; when the magnesium sulfate content is less than 0.3g / L, the contour lines in the direction of the bovine serum content are denser, and the bovine serum content plays a dominant role at this time. When the magnesium sulfate content is less than 0.35g / L, the number of live bacteria increases with the increase of bovine serum content; and when the magnesium sulfate content is constant, the number of live bacteria increases with the increase of bovine serum content; Figure 6 It shows that when the yeast extract content is greater than 0.5 g / L, the contour lines in the direction of the bovine serum content are denser, and the bovine serum content plays a dominant role at this time; when the yeast extract content is less than 0.5 g / L, the contour lines in the direction of the yeast extract content are denser, and the yeast extract content plays a dominant role at this time. When the bovine serum content is less than 9%, the number of viable bacteria decreases with the increase of the yeast extract content; and when the yeast extract content is constant, the number of viable bacteria increases with the increase of the bovine serum content. Figure 7 It shows that the contour density lines in the direction of bovine serum content are larger than those in the direction of NAD content. When the two factors of NAD content and bovine serum content interact, the magnesium sulfate content factor plays a dominant role, and at this time, the effect of NAD content on the number of viable bacteria is small; Figure 8 It shows that the iso-density line in the direction of magnesium sulfate content is larger than that in the direction of yeast extract content. When the two factors of yeast extract content and magnesium sulfate content interact, the magnesium sulfate content factor plays a dominant role, and at this time, the yeast extract content has little effect on the number of viable bacteria. Fig. 9 It shows that the iso-density line in the direction of magnesium sulfate content is larger than that in the direction of NAD content. When the two factors of NAD content and magnesium sulfate content interact, the magnesium sulfate content factor plays a dominant role, and at this time, NAD has little effect on the number of viable bacteria; Fig.10 The results showed that the iso-density line in the direction of yeast extract content was larger than that in the direction of NAD content, but the effects of NAD content and yeast extract content on the number of viable bacteria were relatively small. The optimal fermentation medium composition of Haemophilus parasuis type 12 obtained by model analysis was as follows: bovine serum concentration = 9.91374%, magnesium sulfate = 0.211466 g / L, yeast extract = 0.591998%, NAD = 0.0952694%. Under this condition, the maximum number of viable bacteria could reach 3×10 9 CFU / mL.

[0045] (9) Comparison of model validation experiment and optimization effect: To further test the reliability of the response surface model, the Design-Expert 12 software was used to activate, inoculate and ferment the predicted optimal experimental conditions. The number of viable bacteria in the control group (TSB basal medium containing 5% bovine serum and 0.016% NAD) was 1.2×10 8CFU / mL, the optimal culture medium composition predicted by the postharvest model (corrected bovine serum concentration = 9.9%, magnesium sulfate = 0.2 g / L, yeast extract = 0.6%, NAD = 0.1%) was set as the model validation experiment, and the number of viable bacteria obtained was 3×10 9 CFU / mL, which was 25 times higher than that of the control group; the results showed that the established response surface methodology was used to optimize the optimal process conditions suitable for the fermentation medium of Haemophilus parasuis. This showed that the model was reliable, and compared with the original fermentation conditions, the response surface optimization method could indeed increase the maximum number of viable Haemophilus parasuis type 12.

[0046] The above description is only a preferred embodiment of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention should fall within the scope of the present invention.

Claims

1. A method for optimizing the fermentation of Haemophilus parasuis using a response surface, characterized in that: The following steps are involved: 1) After inactivating bovine serum in a water bath at 56°C for 30 min, add the NAD solution filtered through a 0.22 μm filter membrane into a sterilized TSB liquid medium and mix well. Inoculate the glycerol stock solution of Haemophilus parasuis type 12 into the above TSB liquid medium and culture at 37°C and 180 r / min shaking for 10 h for preliminary activation to obtain a bacterial solution. 2) Add inactivated bovine serum and NAD solution filtered through a 0.22 μm filter membrane to TSA solid medium, take 30 μL of the bacterial solution obtained in step 1) and inoculate it into TSA solid medium for three-zone streaking and culture at 37°C for 8-10 hours, pick a single colony and inoculate it into TSB medium for secondary activation to obtain seed solution; 3) The seed liquid obtained in step 2) was inoculated into TSB liquid culture medium, the culture temperature was controlled at 37°C, the shaking speed was 180 r / min, the yeast extract was 0.591998%, NAD was 0.0952694%, magnesium sulfate was 0.211466 g / L, bovine serum concentration was 9.91374%, and fermentation was carried out for 8-10 hours to obtain fermentation liquid.

2. The method according to claim 1, characterized in that: The yeast extract = 0.591998%, NAD = 0.0952694%, magnesium sulfate = 0.211466 g / L, bovine serum concentration = 9.91374% were optimized by response surface methodology, and the specific steps are as follows: 1) Single factor experiment: The seed liquid was inoculated into TSB liquid medium, and the fermentation time was 8-10 hours. The yeast extract content, NAD content, magnesium sulfate content, and bovine serum content were selected as variables for single factor experiments. The maximum viable count of Haemophilus parasuis type 12 was used as the determination index to screen out the parameter range of yeast extract content, NAD content, magnesium sulfate content, and bovine serum content; 2) Response surface method optimization: According to the parameter range screened in step 1), the fermentation time was fixed at 8-10h, the yeast extract content, NAD content, magnesium sulfate content, and bovine serum content were taken as the four factors to be investigated, and the maximum number of viable bacteria of Haemophilus parasuis type 12 was taken as the response value. The Box-Behnken principle was used to design a 4-factor 3-level test table for response surface experiment, and the multiple regression equation was simulated; 3) Solving the multiple regression equation in step 2) yielded the optimal fermentation medium composition: yeast extract = 0.591998%, NAD = 0.0952694%, magnesium sulfate = 0.211466 g / L, bovine serum concentration = 9.91374%.

3. The method according to claim 2, characterized in that: The parameter ranges of the yeast extract content, NAD content, magnesium sulfate content, and bovine serum content are: yeast extract content is 0.05% to 7%, NAD content is 0.016% to 0.2%, magnesium sulfate content is 0g / L to 0.7g / L, and bovine serum content is 5% to 12%.

4. The method according to claim 2, characterized in that: The multiple regression equation is: Y=-1051.03+205.72A+9274.33B-4982.50C+610.67D-532.50AB+355.00AC+510.00AD -3875.00BC-16100.00BD+7500.00CD-12.89A²-2901.67B²+2285.83C²-25226.67D²; Wherein, Y is the maximum viable count of Haemophilus parasuis type 12, A is the bovine serum content, B is the magnesium sulfate content, C is the yeast extract content, and D is the NAD content.

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