CN102586153A - Method for carrying out high-density culture on bacillus subtilis - Google Patents

Abstract

The invention belongs to the field of microbial fermentation, and particularly relates to a method for carrying out high-density culture on bacillus subtilis, which aims to improve the yield of the bacillus subtilis. According to the method for carrying out high-density culture on the bacillus subtilis, provided by the invention, the bacillus subtilis is adopted as strains, and is activated to be vaccinated into a fermentation culture medium so as to be cultured. Through optimization, the cell biomass of the bacillus subtilis can reach 4.32*109cfu/mL, which is 31.48 times higher than the cell biomass of the bacillus subtilis before optimization (1.33*108cfu/mL). Through research on culture medium formula and culture conditions, the method lays a foundation for industrial fermentation production and further development and utilization of the bacillus subtilis.

Description

A kind of method for cultivating Bacillus subtilis at high density

technical field

The invention belongs to the field of microbial fermentation, and more specifically relates to a method for cultivating Bacillus subtilis at high density.

technical background

As feed additives, antibiotics play a positive role in the prevention of livestock and poultry epidemics, but the widespread use of antibiotics has brought about a series of problems such as environmental pollution, residual toxicity, drug resistance of bacteria, and threats to human and animal safety. In addition, antibiotics will inhibit and kill the beneficial flora in the intestinal tract, resulting in dysbiosis of the normal flora in the intestinal tract, leading to the occurrence of epidemic diseases and double infection, which is harmful to the health of humans and animals. In 1974, Parker proposed a new concept of "probiotics" as opposed to "antibiotics". Probiotics, derived from the Greek word "beneficial to life", refer to the general term of live beneficial microorganisms that can colonize in the intestinal tract of the host and produce definite health effects. Live bacterial preparations and their metabolites in a healthy state.

In recent years, many studies have found that Bacillus has a significant antagonistic effect on animal and human pathogens, which has attracted the close attention of the majority of microbiology and immunology workers. The spores of Bacillus have the advantages of heat resistance, anti-drying, and easy transportation and use. In Western countries, Bacillus has been applied to human functional food and animal feed additives. Bacillus subtilis ( Bacillus subtilis ) is a kind of Bacillus, a Gram-positive aerobic bacterium, and it is one of the two types of bacillus allowed by the Ministry of Agriculture of China to be used as a feed additive. Because of its non-toxicity, no residue, and no pollution, At the same time, it has strong protease, lipase, amylase and other activities, which play an important role in improving the microenvironment of animal digestive tract, promoting the digestion and absorption of animal nutrition, improving the conversion rate of animal feed and enhancing the immunity of livestock and poultry. More and more developed into feed microecological preparations, widely used in animal husbandry, feed and other industries.

At present, the research on Bacillus subtilis mainly focuses on the antibacterial effect, the separation, purification and characterization of antibacterial substances, the enzyme production characteristics of Bacillus subtilis and the application effect in soybean meal, etc. There are few reports on the fermentation technology.

Contents of the invention

The invention provides a method for cultivating Bacillus subtilis at high density, so as to increase the cell yield of Bacillus subtilis.

The method for high-density cultivation of Bacillus subtilis provided by the present invention is specifically as follows: Bacillus subtilis is used as a strain, activated and then inoculated into a fermentation medium for cultivation.

The fermentation medium is: soluble starch 12.5-17.5g/L, peptone 20-25 g/L, NaCl 1.5-2.75 g/L, Na ₂ HPO ₄ 0.25-0.75g/L, MgSO ₄ 0.25-0.75g/L, pH6.0-8.0; culture conditions: culture temperature 35-45 ℃, rotation speed 150-200r/min, liquid volume 60-100mL/250mL; inoculum volume 5-10% (v/v), culture time 16-18h .

More preferably, the fermentation medium is: soluble starch 15g/L, peptone 22.5g/L, NaCl 2.5g/L, Na ₂ HPO ₄ 0.5g/L, MgSO ₄ 0.5g/L, pH7.0; culture temperature 37 ℃, rotation speed 200r/min, liquid volume 80mL/250mL; inoculum volume 5% (v/v), culture time 16h.

The activation uses beef extract peptone medium: beef extract 3g, peptone 10g, sodium chloride 5g, agar 15g-20g, distilled water 1000mL, pH7.0-7.2, 37°C for 24h activation. ``

After optimizing the above conditions, the cell biomass of Bacillus subtilis can reach 4.32×10 ⁹ cfu/mL, which is 31.48 times higher than that before optimization (1.33×10 ⁸ cfu/mL).

The remarkable advantage of the present invention: Bacillus subtilis is a kind of probiotics with very application value and market prospect, but there are still some problems in the actual application process. The effect of Bacillus subtilis under natural conditions is often unsatisfactory. Therefore, it is necessary to improve it to make breakthroughs in processing technology, dosage form and storage technology. The invention lays a foundation for the industrialized fermentation production and further development and utilization of the bacillus subtilis through the research on the medium formula and the culture conditions.

Description of drawings

Fig. 1 The growth curve of Bacillus subtilis;

Fig. 2 The influence of different carbon sources on the growth of Bacillus subtilis;

Figure 3 The effect of soluble starch addition on the growth of Bacillus subtilis;

Fig. 4 The influence of different nitrogen sources on the growth of Bacillus subtilis;

Figure 5 The effect of peptone addition on the growth of Bacillus subtilis;

Fig. 6 The influence of different inorganic salt species on the growth of Bacillus subtilis;

Fig. 7 The influence of medium initial pH value on the growth of Bacillus subtilis;

Fig. 8 The influence of culture temperature on the growth of Bacillus subtilis;

Fig. 9 The influence of rotating speed on the growth of Bacillus subtilis;

Fig. 10 The influence of medium loading volume on the growth of Bacillus subtilis;

Figure 11 Effect of inoculum size on the growth of Bacillus subtilis.

Detailed ways

The Bacillus subtilis used in the following examples is the Bacillus subtilis E bacterial strain (references: Zhao Ying et al., Optimization of process conditions for the production of fulvic acid by fermentation of bagasse by Bacillus subtilis. Journal of Fuzhou University (Natural Science Edition), 2010, 38 (2 ):290-296), further describe the present invention, but the present invention is not limited thereto.

Example 1

1. Determination of Growth Curve of Bacillus subtilis

After activating Bacillus subtilis, inoculate it into beef extract-peptone liquid medium (beef extract 3g, peptone 10g, sodium chloride 5g, distilled water 1000mL, pH7.0-7.2) at an inoculum size of 5% (v/v), The volume of liquid is 100mL/250mL, cultured at 30°C and 200r/min, and the OD ₆₀₀ value and the number of colonies at different culture times are measured by spectrophotometry and plate colony counting method respectively, with the culture time as the abscissa and the OD The ₆₀₀ value and the number of colonies are plotted as the ordinate to the growth curve (Figure 1). According to the growth curve, the optimal culture time of Bacillus subtilis was 16h.

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2. Optimization of medium formula for Bacillus subtilis

The effect of different carbon sources, nitrogen sources and inorganic salts on the biomass of Bacillus subtilis cells was studied with beef extract peptone liquid medium as the base medium. Culture conditions: inoculum size 5% (v/v), liquid volume 100mL/250mL, culture at 30°C, 200r/min for 16h.

2.1 Effects of carbon source types on the biomass of Bacillus subtilis cells

Using 1% (expressing g/100mL, hereinafter similar) sucrose, cyclodextrin, maltose, soluble starch, sodium acetate, and glucose as carbon sources to replace the beef extract in the beef extract peptone liquid medium, investigate their effects on subtilis The effect of Bacillus cell biomass is plotted with the type of carbon source as the abscissa and the OD ₆₀₀ increase as the ordinate. Figure 2 shows that when soluble starch is used as carbon source, the OD ₆₀₀ growth of Bacillus subtilis is the largest, which is 0.798 (measured by 10-fold dilution of the bacterial suspension); while sodium acetate and glucose are used as carbon sources, the OD ₆₀₀ growth is higher than that of Small. Considering the comprehensive cell biomass and medium cost, it was determined to use soluble starch as the carbon source.

2.2 The effect of carbon source addition on the biomass of Bacillus subtilis cells

To compare the effects of different additions of soluble starch on the growth of Bacillus subtilis, plot the carbon source addition as the abscissa and the OD ₆₀₀ growth as the ordinate, and measure the OD ₆₀₀ after diluting the bacterial suspension 10 times (Figure 3). The results showed that with the increase of soluble starch, the growth of OD ₆₀₀ also increased continuously; when the carbon source was added at 1.5%, the OD ₆₀₀ growth of Bacillus subtilis reached the highest, which was 0.544; then the soluble starch continued to increase The amount of addition, OD ₆₀₀ growth decreased. Therefore, it was determined that the optimum addition amount of Bacillus subtilis soluble starch was 1.5%.

2.3 Effects of nitrogen source types on the biomass of Bacillus subtilis cells

On the basis of carbon source optimization, 1% peptone, ammonium dihydrogen phosphate, ammonium chloride, ammonium nitrate, ammonium sulfate, and sodium nitrate were used as nitrogen sources to investigate their effects on the biomass of Bacillus subtilis cells. The source species is plotted on the abscissa, and the increase in OD ₆₀₀ is plotted on the ordinate (Figure 4). The results showed that when Bacillus subtilis used peptone as the nitrogen source, the OD ₆₀₀ growth was the highest, which was 1.475; while when the inorganic nitrogen source was used, the cell biomass was relatively low, so peptone was selected as the nitrogen source of the medium.

2.4 The effect of nitrogen source addition on the biomass of Bacillus subtilis cells

Take the amount of peptone added as the abscissa and the increase in OD ₆₀₀ as the ordinate, and measure the OD ₆₀₀ after diluting the bacterial suspension 10 times (Figure 5). The results showed that with the increase of peptone addition, the OD ₆₀₀ growth rate also increased; when the peptone addition amount was 2.25%, the OD ₆₀₀ growth rate of Bacillus subtilis reached the highest, which was 0.718; then continue to increase the peptone addition amount , the growth rate of OD ₆₀₀ decreased. Therefore, it was determined that the optimum peptone addition amount of Bacillus subtilis was 2.25%.

2.5 Effects of inorganic salt types on the biomass of Bacillus subtilis cells

Based on the optimization of carbon source and nitrogen source, 0.5% potassium dihydrogen phosphate, disodium hydrogen phosphate, potassium chloride, sodium molybdate, manganese sulfate, magnesium sulfate, calcium chloride and ferrous sulfate were used as inorganic salts Add it to the medium, take the type of inorganic salt as the abscissa, and the OD ₆₀₀ increase as the ordinate, dilute the bacterial suspension 10 times and measure the OD ₆₀₀ (Figure 6) to determine the best inorganic salt. The results showed that when NaCl, Na ₂ HPO ₄ , MgSO ₄ were used as inorganic salts, the OD ₆₀₀ of Bacillus subtilis increased significantly, which were 0.751, 0.872, and 0.758, respectively. Therefore, NaCl, Na ₂ HPO ₄ , and MgSO ₄ were selected as The best inorganic salt for Bacillus subtilis culture. On the basis of determining the best type of inorganic salts, the optimal addition amount of inorganic salts and their interaction should be studied in depth.

2.6 The effect of inorganic salt addition on the biomass of Bacillus subtilis cells

The types and concentrations of inorganic salts required for the growth of different microorganisms are different. The above experimental results show that NaCl, Na ₂ HPO ₄ and MgSO ₄ have obvious effects on the growth of Bacillus subtilis, so the orthogonal test L ₉ (3 ⁴ ) was designed with these three salts as the factors to optimize the formulation of inorganic salts . Through the single factor test, it is preliminarily determined that the optimal levels of the above inorganic salts are NaCl 0%-0.25%, Na ₂ HPO ₄ 0%-0.1%, MgSO ₄ 0%-0.1%.

Table 1 Factors and levels of Bacillus subtilis inorganic salt optimization orthogonal test

Table 2 Orthogonal test results of Bacillus subtilis inorganic salt optimization

It can be seen from Table 2 that the primary and secondary sequence of inorganic salts affecting the growth of Bacillus subtilis is C>A>B, and the optimal combination is C ₂ A ₃ B ₂ ; however, it can be seen from the test data in the orthogonal table that the increase in OD ₆₀₀ The highest combination is C ₂ A ₃ B ₃ . The verification test of the inorganic salt orthogonal test was further carried out. Under these two combination conditions, the bacterial suspension was diluted 10 times and the OD ₆₀₀ was measured. The OD ₆₀₀ increase of the combination C ₂ A ₃ B ₂ was 0.867, and the combination C ₂ A The OD ₆₀₀ increase of ₃ B ₃ is 0.813, so the best combination is determined to be C ₂ A ₃ B ₂ , that is, MgSO ₄ 0.05%, NaCl 0.25%, and Na ₂ HPO ₄ 0.05%.

2.7 Effect of the initial pH value of the medium on the biomass of Bacillus subtilis cells

On the basis of carbon source, nitrogen source and inorganic salt optimization, adjust the initial pH of the fermentation medium of Bacillus subtilis to be 4.0, 5.0, 6.0, 7.0, 8.0, 9.0, 10.0 respectively, and the initial pH value of the medium is the abscissa, Taking the increase in OD ₆₀₀ as the ordinate, the OD ₆₀₀ was measured after the bacterial suspension was diluted 10 times (Figure 7). The results showed that with the increase of the initial pH value of the medium, the OD ₆₀₀ increase also increased; when the pH value was 7, the OD ₆₀₀ increase of Bacillus subtilis reached the highest value, which was 0.928; continue to increase the medium pH value, OD ₆₀₀ growth decreased, therefore, the initial pH value of the optimum medium for Bacillus subtilis was determined to be 7.0.

3. Optimizing the Culture Conditions of Bacillus subtilis

On the basis of optimization of fermentation medium (soluble starch 15g/L, peptone 22.5g/L, NaCl 2.5g/L, Na ₂ HPO ₄ 0.5g/L, MgSO ₄ 0.5g/L, pH 7.0), further The culture conditions of Bacillus subtilis were optimized.

3.1 Effect of culture temperature on the biomass of Bacillus subtilis cells

Bacillus subtilis was inoculated into the optimized fermentation medium with an inoculum of 5% (v/v), and placed at 25°C, 30°C, 35°C, 40°C, 45°C, 50°C respectively, at 200r/ Cultivate for 16 hours under the condition of 1 min, take the culture time as the abscissa, and the OD ₆₀₀ increase as the ordinate, and measure the OD ₆₀₀ after diluting the bacterial suspension 10 times (Figure 8). The results showed that when the temperature was 45℃, the OD ₆₀₀ value of Bacillus subtilis was the highest, which was 0.716. Therefore, 45°C was determined to be the optimum temperature for culturing Bacillus subtilis.

3.2 Effect of dissolved oxygen on the biomass of Bacillus subtilis cells

3.2.1 The effect of rotational speed on the biomass of Bacillus subtilis cells

Inoculate Bacillus subtilis into the optimized fermentation medium at an inoculum of 5% (v/v), at 100r/min, 130r/min, 170r/min, 200r/min, 230r/min, 260r/min Incubate at 45°C for 16 hours at a constant speed. Taking the rotational speed as the abscissa and the OD ₆₀₀ increase as the ordinate, the OD ₆₀₀ was measured after the bacterial suspension was diluted 10 times (Figure 9). The results showed that with the increase of the rotational speed, the OD ₆₀₀ growth of Bacillus subtilis increased, and when the rotational speed was 170r/min, the OD ₆₀₀ growth reached the maximum, which was 0.985. When the speed continued to increase to 200r/min, the OD ₆₀₀ growth began to decline. Therefore, 170r/min was chosen as the optimum speed for the cultivation of Bacillus subtilis.

3.2.2 The effect of medium filling volume on the biomass of Bacillus subtilis cells

5% (v/v) inoculum was inoculated into the optimized fermentation medium, and six different cultures of 40mL/250mL, 60mL/250mL, 80mL/250mL, 100mL/250mL, 120mL/250mL and 140mL/250mL were selected respectively The volume of the base solution, cultured at 45°C and 170r/min for 16h. Take the volume of the culture medium as the abscissa, and the OD ₆₀₀ increase as the ordinate, and measure the OD ₆₀₀ after diluting the bacterial suspension 10 times (Figure 10). The results showed that when the filling volume was 80mL/250mL, the OD ₆₀₀ increase was the highest, which was 0.947. Therefore, 80mL/250mL was selected as the initial filling volume of the medium.

3.3 Orthogonal test of culture conditions

Design an orthogonal experiment L ₂₇ (3 ¹³ ) to optimize the culture conditions of Bacillus subtilis to investigate the influence of four factors, temperature, initial pH of the medium, rotational speed, and liquid volume, on the growth of the strain, and optimize the results according to the single factor experiment See Table 3, Table 4 and Table 5 for the selection of factor levels, orthogonal test design and analysis.

Table 3 Bacillus subtilis culture condition optimization factor level table

Table 4 Orthogonal test results of optimization of Bacillus subtilis culture conditions

Table 5 Analysis of variance table

It can be seen from Table 5 that temperature and rotation speed have significant effects on the growth of Bacillus subtilis. According to the analysis in Table 4, the optimal experimental combination is A ₁ B ₂ C ₃ D ₁ , and in the 27 experiments, the combination with the highest increase in OD ₆₀₀ is A ₁ B ₃ C ₃ D ₂ , due to factors B and D Factors, namely, pH value and liquid volume, are not significantly affected. At the same time, considering that more liquid volume can save production costs, the optimal combination that is conducive to the growth of bacteria is selected as A ₁ B ₂ C ₃ D ₂ . Further carry out the verification test of the orthogonal test of Bacillus subtilis culture conditions, that is, select two conditions of A ₁ B ₃ C ₃ D ₂ and A ₁ B ₂ C ₃ D ₂ to cultivate Bacillus subtilis, and measure its concentration after the bacterial suspension is diluted 10 times. _OD600 value. Under the conditions of the optimal combination A ₁ B ₂ C ₃ D ₂ , the OD ₆₀₀ increase of the bacterial suspension was 0.735, and under the condition of the combination A ₁ B ₃ C ₃ D ₂ , the OD ₆₀₀ of the bacterial suspension increased Therefore, the optimal combination of Bacillus subtilis culture conditions is determined to be A ₁ B ₂ C ₃ D ₂ through verification tests, that is, the temperature is 37°C, the initial pH of the medium is 7.0, the rotation speed is 200r/min, and the liquid volume is 80mL/ 250mL.

3.4 Effect of inoculum size on the biomass of Bacillus subtilis cells

On the basis of the optimization of medium components and culture conditions, the activated Bacillus subtilis was added with 1% (v/v), 3% (v/v), 5% (v/v), 10% (v /v), 15% (v/v), and 20% (v/v) inoculums were inoculated into the fermentation medium, with the inoculation amount as the abscissa and the OD ₆₀₀ growth as the ordinate, the bacterial suspension was diluted 10 OD ₆₀₀ was measured after multiple times (Fig. 11). The results showed that the inoculum amount had little effect on the growth of the strain. When the inoculum amount of Bacillus subtilis was 5%, the OD ₆₀₀ increased the most, which was 0.428. Therefore, it was determined that the optimal inoculum volume of Bacillus subtilis was 5% (v/v).

3. summary

The optimized medium formula and culture conditions of Bacillus subtilis were: soluble starch 15g/L, peptone 22.5g/L, NaCl 2.5g/L, Na ₂ HPO ₄ 0.5g/L, MgSO ₄ 0.5g/L, pH7 .0; culture temperature 37°C, rotation speed 200r/min, liquid volume 80mL/250mL; inoculum volume 5% (v/v), culture time 16h.

Implementation: Optimizing Experiments

Medium formula before optimization: beef extract 3g, peptone 10g, sodium chloride 5g, distilled water 1000mL, pH7.0-7.2;

The culture conditions before optimization: culture temperature 30°C, rotation speed 200r/min, liquid volume 100mL/250mL, inoculum volume 5% (v/v), culture time 24h.

The optimized medium formula and culture conditions were used for the fermentation production of Bacillus subtilis. The OD ₆₀₀ was measured after the bacterial suspension was diluted 25 times. The OD ₆₀₀ increase before optimization was 0.111, and the OD ₆₀₀ increase after optimization was ^0.526 ; The former (1.33×10 ⁸ cfu/mL) increased by 31.48 times.

Claims (4)

1. A method for high-density cultivation of Bacillus subtilis, characterized in that: Bacillus subtilis ( Bacillus subtilis ) is used as bacterial classification, inoculated into fermentation medium after activation and cultivated; fermentation medium is: soluble starch 12.5-17.5 g/L, peptone 20-25 g/L, NaCl 1.5-2.75 g/L, Na ₂ HPO ₄ 0.25-0.75g/L, MgSO ₄ 0.25-0.75g/L, pH6.0-8.0; culture conditions: culture The temperature is 35-45°C, the rotation speed is 150-200r/min, the liquid volume is 60-100mL/250mL; the inoculum volume is 5-10% (v/v), and the incubation time is 16-18h. 2. the method for high-density culture Bacillus subtilis according to claim 1, is characterized in that: described fermentation medium is: soluble starch 15g/L, peptone 22.5g/L, NaCl 2.5g/L, Na ₂ HPO ₄ 0.5g/L, MgSO ₄ 0.5g/L, pH 7.0. 3. The method for high-density culture of Bacillus subtilis according to claim 1, characterized in that: the culture conditions are: culture temperature 37°C, rotation speed 200r/min, liquid filling volume 80mL/250mL; inoculum volume 5% (v/v), culture time 16h. 4. the method for high-density culture Bacillus subtilis according to claim 1, is characterized in that: described activation adopts beef extract peptone medium: beef extract 3g, peptone 10g, sodium chloride 5g, agar 15g-20g, distilled water 1000mL, pH7.0-7.2, activated at 37°C for 24h.

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