CN111635918A - A kind of fermentation process and application of Bacillus coagulans high-yielding antibacterial polypeptide substance - Google Patents

Abstract

The invention discloses a fermentation process for high-yield antibacterial polypeptide substances of bacillus coagulans and application thereof, relating to the technical field of bacillus fermentation, wherein the fermentation process comprises the following steps: inoculating the bacillus coagulans seed liquid into a basic culture medium, and culturing for 20-30h at 40-45 ℃ under the conditions of 150-; meanwhile, amino acid substances are added in the middle logarithmic phase of the fermentation growth of the bacillus coagulans strain; adding alcohol substances in the stationary phase of the fermentation growth of the bacillus coagulans strain. The invention optimizes the basic culture medium, optimizes the carbon-nitrogen ratio, ensures that the bacterial strain grows in the most suitable culture medium environment, and metabolizes polypeptide substances. The content of the antibacterial polypeptide is increased from 1710 mu g/mL to 3940 mu g/mL, and the antibacterial active substance is mainly protein bacteriocin, is safe and nontoxic, and can be applied to the fields of food preservatives, preservatives or medical health care.

Description

A kind of fermentation process and application of Bacillus coagulans high-yielding antibacterial polypeptide substance

technical field

The invention relates to the technical field of Bacillus fermentation, in particular to a fermentation process of Bacillus coagulans high-yielding antibacterial polypeptide substances and its application.

Background technique

The metabolites of lactic acid bacteria include lactic acid, organic acids, unsaturated fatty acids and bacteriocins, which can inhibit the growth of some pathogenic microorganisms and have beneficial effects on the host. However, lactic acid bacteria have poor resistance to stress and are sensitive to acids and bile salts. Antibiotics have a good bacteriostatic effect on some pathogenic bacteria and can reduce the occurrence of certain diseases. However, due to the abuse of antibiotics, some microorganisms will produce Drug resistance, the formation of "super bacteria", more difficult to control.

Bacillus coagulans, also known as spore lactic acid bacteria, is a Gram-positive bacterium, facultative anaerobic, capable of producing spores, and has strong resistance to stress. The spore survival rate can reach 96.4% at a high temperature of 100 ° C for 10 minutes; at pH 2.0 Under acidic conditions, the survival rate of 6h was 48.2%; under the condition of 0.9% bile salt, the survival rate of 24h was 78.3%. It can also produce lactic acid, bacteriocin and other antibacterial substances. In 2016, it was included in the list of food strains that can be used by the state. However, the bacteriostatic substances produced by Bacillus coagulans are affected by the composition of the medium and the growth conditions, and their content is relatively low, and there is a lack of high bacteriostatic Bacillus coagulans-related mature products. It is very necessary to increase the yield of the bacteriostatic active substances of Bacillus coagulans, prepare corresponding products, and promote their wide application. The authorization announcement number is: CN106191178B, and the name is: a kind of invention patent of a method for Bacillus coagulans to produce bacteriostatic active substances. Optimal control of the process.

SUMMARY OF THE INVENTION

The invention provides a fermentation process of Bacillus coagulans high-yielding antibacterial polypeptide substances and application thereof, and solves the problem that the antibacterial metabolic yield of the existing Bacillus coagulans is generally low.

In order to solve the above-mentioned technical problems, the technical scheme of the present invention is:

A fermentation process of Bacillus coagulans high-yield antibacterial polypeptide substance, comprising the following steps:

Inoculate the Bacillus coagulans seed liquid into the basal medium, and culture at 40-45°C and 150-250rpm for 20-30h; during this time, add amino acids in the middle of the log phase of the fermentation growth of the Bacillus coagulans strain; Alcohols are added during the stationary phase of the fermentative growth of Bacillus coagulans strains.

Preferably, the basic medium comprises molasses 10-40g/L, yeast extract powder 10-30g/L, corn steep liquor dry powder 5-30g/L, beef extract powder 10-30g/L, sodium chloride 1-10g/L L, sodium acetate 2-10g/L, magnesium sulfate 0.1-1g/L, manganese sulfate 2-15mg/L and Tween 80 0.5-2g/L.

Preferably, the basic medium comprises molasses 40.00g/L, yeast extract powder 20g/L, corn steep liquor dry powder 10g/L, beef extract powder 20g/L, sodium chloride 5g/L, sodium acetate 5g/L, sulfuric acid Magnesium 0.58g/L, manganese sulfate 10mg/L and Tween 80 2g/L.

Preferably, the pH of the base medium is 7.0.

Preferably, the inoculation amount in step (1) is 2-7 mL of seed liquid/100 mL of basal medium for inoculation.

Preferably, the Bacillus coagulans seed solution is obtained by inoculating a single colony of Bacillus coagulans into PCA liquid medium at 40-45° C., 150-200 rpm, and culturing for 20-30 hours.

Preferably, the Bacillus coagulans is Bacillus coagulans BC99, the Bacillus coagulans BC99 is named Bacillus coagulans, and the deposit number is: CGMCC No.19487.

Preferably, the amino acid substance is one or more of serine, threonine, cysteine, phenylalanine, glycine or proline.

Preferably, the alcohol substance is one or more of ethanol, ethylene glycol or n-butanol.

An application of the fermentation process of the Bacillus coagulans high-yield antibacterial polypeptide substance, characterized in that: the antibacterial polypeptide substance of Bacillus coagulans prepared by the fermentation process of the Bacillus coagulans high-yield antibacterial polypeptide substance should be added to In food, health products, additives or preservatives.

By adopting the above technical scheme, the present invention optimizes the basal medium, optimizes the carbon and nitrogen ratio, ensures that the strain grows in the most suitable medium environment, and multi-metabolizes polypeptide substances; the main component monomers of the antibacterial polypeptide substances are: Amino acids, by adding specific amino acids as precursors for polypeptide synthesis, are added at specific times of bacterial growth to maximize the yield of polypeptide antibacterial substances; In the stable period, due to the exhaustion of nutrients, the bacteria will produce enzymes that decompose polypeptide substances and decompose them into amino acids for the growth of bacteria. The production of bacterial polypeptides did not decrease.

In the present invention, the content of antibacterial polypeptides produced by Bacillus coagulans is increased by 2 times through the optimization of the culture medium and the optimization of the fermentation process, and the antibacterial titer is measured by using golden yellow glucose as the indicator bacteria, and its inhibitory effect is the same as that of antibiotics. Equivalent, its antibacterial substances are mainly protein-based cytokines, after the determination of antibacterial titer, it is found that the antibacterial titer effect of the fermentation broth containing 3940μg/mL antibacterial polypeptide is equivalent to 0.6mg/mL of the same antibacterial titer. mL of ampicillin and 0.06 mg/mL of kanamycin sulfate have similar effects, safe, non-toxic and side effects, and do not produce drug resistance, and have good effects in the fields of food preservatives, food and health products.

Description of drawings

Fig. 1 is a graph showing the effect of different carbon sources on the antibacterial titer and antibacterial polypeptide content of Bacillus coagulans BC99;

Fig. 2 is the influence figure of molasses addition on the antibacterial titer and antibacterial polypeptide content of Bacillus coagulans BC99;

Figure 3 is a graph showing the effect of different nitrogen sources on the antibacterial titer and antibacterial polypeptide content of Bacillus coagulans BC99;

Fig. 4 is a graph showing the effect of Tween and ethylene glycol on the bacteriostatic titer and content of antibacterial polypeptides of Bacillus coagulans BC99;

Fig. 5 is a graph showing the effect of temperature on the bacteriostatic titer and content of antibacterial polypeptides of Bacillus coagulans BC99;

Fig. 6 is a graph showing the effect of adding amino acids on Bacillus coagulans BC99 antibacterial titer and antibacterial polypeptide content at different growth periods;

Fig. 7 is a graph showing the effect of adding alcohol substances on Bacillus coagulans BC99 antibacterial titer and antibacterial polypeptide content in different growth periods;

Figure 8 is a graph showing the effect of different pH values on the bacteriostatic ability of metabolites;

Figure 9 is a graph showing the effect of different temperatures on the bacteriostatic ability of metabolites;

Figure 10 is a graph of the effect of supernatant and bacterial cells on the bacteriostatic ability of metabolites;

Fig. 11 The effect of different enzymes on the bacteriostatic ability of bacteriostatic substances.

Detailed ways

The specific embodiments of the present invention will be further described below with reference to the accompanying drawings. It should be noted here that the descriptions of these embodiments are used to help the understanding of the present invention, but do not constitute a limitation of the present invention. In addition, the technical features involved in the various embodiments of the present invention described below can be combined with each other as long as they do not conflict with each other.

It should be noted that the experimental methods in the following examples are conventional methods unless otherwise specified; the materials and reagents used in the examples can be obtained from commercial sources unless otherwise specified. The quantitative tests in the following examples are all set to repeat the experiments three times, and the results are averaged.

Example 1

Optimal determination of basal medium

1. The influence of carbon source types

Preparation of control group basal medium: Dissolve 20.00 g of peptone, 20.00 g of glucose, 5 g of sodium chloride, 5 g/L of sodium acetate, 0.58 g of magnesium sulfate and 10 mg of manganese sulfate in 1 L of distilled water, adjust the pH to 7.0, and set aside;

Preparation of PCA medium: tryptone 5.0g, yeast extract powder 2.5g, glucose 1.0g dissolved in 1L distilled water, adjust pH to 7.0, set aside;

Preparation of the basal medium of the experimental group: lactose, sucrose, soluble starch, maltose, and molasses were used to replace the glucose in the basal medium of the control group.

Pick a single colony of Bacillus coagulans BC99 and inoculate it into PCA medium, cultivate at 42°C and 180 rpm for 24 hours to obtain Bacillus coagulans BC99 seed liquid, and then inoculate the Bacillus coagulans BC99 seed liquid into the basal medium of the control group and the basal culture of the experimental group respectively. culture medium at 42°C and 180rpm for 24h to obtain a fermentation broth. Among them, Bacillus coagulans BC99 preservation unit code: CGMCC-General Microbiology Center of China Microbial Culture Collection Management Committee; Address: No. 3, No. 1, Beichen West Road, Chaoyang District, Beijing; The detection result of Bacillus coagulans BC99 is: survival; Preservation date: March 18, 2020; Preservation number: CGMCC No.19487; Classification name: Bacillus coagulans.

The Oxford cup method was used for the fermentation broth, and Staphylococcus aureus was used as the indicator bacteria, and its antibacterial titer was measured:

Measurement of inhibition zone: the difference between the diameter of the outer inhibition zone and the diameter of the inner Oxford cup (8cm) is the final inhibition zone diameter, in mm.

Determination of polypeptide content: using the biuret method, take 1 mL of the centrifuged fermentation broth supernatant, accurately add 4 mL of biuret reagent, mix and shake well, react at room temperature for 30 minutes, and measure its absorbance at 540 nm. . Calculate the polypeptide content in the fermentation broth according to the standard curve.

Analysis of antibacterial activity: expressed in titer, unit AU/mL, as follows: the supernatant of the fermentation broth was diluted twice with sterile normal saline, and 100uL of each dilution gradient was sampled and added to the Oxford cup to detect the antibacterial activity. The highest dilution ratio at which the inhibition zone appears is defined as an active unit, and its reciprocal multiplied by the dilution ratio is the antibacterial activity titer (AU/mL) of the stock solution.

Among them, the average antibacterial titer of the basal medium in the control group was 2300AU/mL, and the content of antibacterial polypeptides was 1710 μg/mL; The results of titer and antibacterial polypeptide content are shown in Figure 1. It can be seen that the antibacterial titer and antibacterial polypeptide content of the basal medium of the experimental group of molasses are the best.

Continue to optimize the addition amount of molasses: the addition amount of molasses was tested at 10g/L, 20g/L, 30g/L, 40g/L, 50g/L, and the best antibacterial addition effect was investigated. The results are shown in the figure 2, it can be seen that the amount of molasses added has a good effect at 10-40g/L, but it reaches the best state at 40.00g/L.

2. The influence of nitrogen source types

Ammonium chloride, ammonium sulfate, yeast extract powder, soybean cake powder, corn steep liquor powder, soybean peptone, and beef extract powder were used to replace peptone, and the addition amount was 2g/100mL. The Oxford cup method was used, and Staphylococcus aureus was used as the indicator. Bacteria, the antibacterial titers and the content of antibacterial polypeptides were determined, and the results are shown in Figure 3. Among them, yeast extract powder, corn steep liquor dry powder, and beef extract powder had no significant difference in the antibacterial effect.

3. The effect of Tween and ethylene glycol

Tween 20, Tween 60, Tween 80 and ethylene glycol were added in an addition amount of 2 g/L, and the Oxford cup method was used to measure the antibacterial titer and the content of antibacterial polypeptides with Staphylococcus aureus as the indicator bacteria. The results are shown in Figure 4, and it can be seen that Tween and ethylene glycol have no significant difference in the bacteriostatic effect.

4. The influence of temperature

Cultures were carried out at 30°C, 37°C, 42°C, 45°C, and 50°C, and other conditions were the same. The effects of different temperatures on the bacteriostatic ability of Bacillus coagulans BC99 were investigated. At the end of the fermentation, the Oxford cup method was used to measure the antibacterial titer and antibacterial polypeptide content of the supernatant with Staphylococcus aureus as the indicator bacteria.

5. Nitrogen source orthogonal experiment

The above best medium ingredients are used: molasses 40.00g/L, Tween 80 2g/L, sodium chloride 5g/L, sodium acetate 5g/L, magnesium sulfate 0.58g/L, manganese sulfate 10mg/L as medium Ingredients, the nitrogen source: yeast extract powder, corn steep liquor dry powder, beef extract powder, carry out orthogonal test, and ferment at 42 ℃, as shown in Table 1:

Table 1 Orthogonal factors and levels of medium composition optimization

Level A Yeast Dip Powder (g/L) B corn steep liquor powder (g/L) C Beef Dipping Powder (g/L) 1 10 5 15 2 20 10 20 3 30 20 25

Using Staphylococcus aureus as indicator bacteria, the antibacterial titer and antibacterial polypeptide content were determined, and the results are shown in Table 2:

Table 2 Orthogonal test results of medium components

Orthogonal test results showed that the primary and secondary effects of nitrogen sources (yeast extract powder, corn steep liquor dry powder, beef extract powder) on Bacillus coagulans were C>A>B, and the optimal combination was C ₂ A ₂ B ₂ . The optimal medium composition obtained by single factor test and orthogonal test is: molasses 40.00g/L, yeast extract powder 20g/L, corn steep liquor dry powder 10g/L, beef extract powder 20g/L, Tween 80 2g/L L, sodium chloride 5g/L, sodium acetate 5g/L, magnesium sulfate 0.58g/L, manganese sulfate 10mg/L.

Example 2

Optimization of the fermentation process is determined

Taking the best proportioning obtained in Example 1 as the basal medium, amino acids (serine, threonine) were added in the early, middle and final stages of the growth logarithmic phase of fermentation, wherein the amino acids were sterilized separately. , the addition amount is 5-20g/L; after the fermentation, the Oxford cup method is used to measure the antibacterial titer and antibacterial polypeptide content of the supernatant with Staphylococcus aureus as the indicator bacteria. The results are shown in Figure 6, it can be seen that, When amino acids were added in the mid-log phase, the antibacterial titer was the largest after fermentation.

Taking the best proportioning obtained in Example 1 as the base medium, alcohols (ethanol, ethylene glycol, n-butanol in the middle of the logarithmic phase, the end of the logarithmic phase, and the initial stage of the stable phase of the fermentation growth were added respectively. One or more) substances, the concentration of which is 20-50%; after fermentation, the Oxford cup method is adopted, and Staphylococcus aureus is used as indicator bacteria to measure the bacteriostatic titer and the content of bacteriostatic polypeptides in the supernatant, and the results are shown in the figure As shown in Fig. 7, it can be seen that the antibacterial titer measured after fermentation is the largest when alcohol is added at the beginning of the stable period.

Taking the best proportioning obtained in Example 1 as the basal medium, the best fermentation process obtained in the present embodiment was fermented, and after the fermentation was finished, the Oxford Cup method was adopted, and Staphylococcus aureus was used as the indicator bacteria to measure the above. The antibacterial titer of the serum; the biuret method was used to determine the content of the polypeptide, and the maximum antibacterial titer produced by Bacillus coagulans was 5690AU/mL; the content of the antibacterial polypeptide was 3940μg/mL.

Example 3

Antibacterial active substance properties

The Bacillus coagulans fermentation supernatant prepared by the best process obtained in Example 2 was basal medium with the best ratio drawn in Example 1, using 0.5mol/L HCl and 0.5mol/L The pH value of the supernatant of Bacillus coagulans was adjusted by NaOH, and the pH value gradient was: 2.0, 4.0, 6.0, 8.0, 10.0. After treatment at 37 °C for 1 h, the pH value was adjusted back to 6.0, and the Oxford cup bacteriostatic test was performed. Staphylococcus aureus was tested as an indicator bacteria, the size of the antibacterial titer (AU/mL) was determined, and the effect of different acidic conditions on its antibacterial ability was investigated. The results are shown in Figure 8.

The Bacillus coagulans fermentation supernatant prepared by the best process obtained in Example 2 was basal medium with the best ratio obtained in Example 1, at different temperature gradients (4°C, 25°C, respectively). , 37°C, 45°C, 80°C, 121°C), the antibacterial ability was determined after 30min treatment, and Staphylococcus aureus was used as the indicator bacteria for the test to determine the size of the antibacterial titer. The effect of bacteriostatic ability of bacillus, the results are shown in Figure 9.

The Bacillus coagulans fermented liquid supernatant prepared with the best process obtained in Example 2 is used as the base medium with the best ratio drawn in Example 1, and the comparison of bacteriostatic ability with its fermented bacterial liquid is carried out, The test was carried out with Staphylococcus aureus as the indicator bacteria, the size of the antibacterial titer was determined, and the effect of the bacteria of Bacillus coagulans on the antibacterial ability was investigated. The results are shown in Figure 10.

Taking the best proportioning obtained in Example 1 as the basal medium, the Bacillus coagulans fermentation broth supernatant prepared by the best process obtained in Example 2 was prepared with 0.5mol/L hydrochloric acid and 0.5mol/L Sodium hydroxide was used to adjust the pH of the supernatant to the optimum pH of pepsin, trypsin, proteinase K and catalase, and the fermentation supernatant of Bacillus coagulans was reacted with various enzymes in a water bath at 37°C. After 30 minutes, the pH was adjusted to 6.0 to study the antibacterial metabolites of Bacillus coagulans treated with different proteases, and the supernatant of Bacillus coagulans without enzyme treatment was used as the control group. By measuring the antibacterial titers of the control group and the test group, the antibacterial substances of Bacillus coagulans were investigated. The results are shown in Figure 11. The results show that the metabolites of Bacillus coagulans BC99 are protein bacteriocin substances.

Ampicillin and kanamycin sulfate were used for control experiments, and two antibiotics with a concentration of 50 mg/mL were selected as mother solutions, and were diluted to the original 10, 10 ^-2 , 10 ^-3 , 10 ^-4 , 10 ^-5 , 10 respectively. ^-6 times, with Staphylococcus aureus as the indicator bacteria to carry out the antibacterial experiment, with the best ratio obtained in Example 1 as the basal medium, the coagulated spores prepared with the best process obtained in Example 2 The size of the inhibition zone of the bacillus fermentation broth supernatant was compared. After the determination of the antibacterial titer, it was found that the inhibition zone effect of the fermentation broth containing 3940 μg/mL bacteriostatic polypeptide was comparable to that of 0.6 mg/mL ampicillin with the same inhibition zone size. Penicillin and 0.06mg/mL kanamycin sulfate have similar effects.

Staphylococcus aureus, Escherichia coli, Salmonella typhimurium, Bacillus subtilis, Shigella, Clostridium perfringens, and Saccharomyces cerevisiae were selected as indicator bacteria, and their bacterial concentrations were controlled at 10 ⁹ CFU/mL. The best ratio drawn in Example 1 is the base medium, and the Bacillus coagulans fermentation broth supernatant prepared by the best process obtained in Example 2 is subjected to a bacteriostatic spectrum test, and it is found that this strain of Bacillus coagulans is inhibited. The bacterial product has good bacteriostatic effect on Gram-positive bacteria, but no obvious bacteriostatic effect on fungal yeast.

Table 3 Bacteriostatic effect of Bacillus coagulans BC99 on different indicator bacteria

Note: The antibacterial titer of 5000-6000AU/mL is equivalent to the inhibition zone of 25-30mm; the antibacterial titer of 4000-5000AU/mL is equivalent to the inhibition zone of 20-25mm; the antibacterial titer of 3000-4000AU/mL is equivalent to the inhibition zone of 20-25mm. The bacterial circle is 15-20mm; the antibacterial titer is 2000-3000AU/mL, which is equivalent to the antibacterial circle 10-15mm

The embodiments of the present invention have been described in detail above with reference to the accompanying drawings, but the present invention is not limited to the described embodiments. For those skilled in the art, without departing from the principle and spirit of the present invention, various changes, modifications, substitutions and alterations to these embodiments still fall within the protection scope of the present invention.

Claims (10)

1. A fermentation process of high-yield antibacterial polypeptide substances of bacillus coagulans is characterized by comprising the following steps: the method comprises the following steps:

inoculating the bacillus coagulans seed liquid into a basic culture medium, and culturing for 20-30h at 40-45 ℃ under the conditions of 150-; meanwhile, amino acid substances are added in the middle logarithmic phase of the fermentation growth of the bacillus coagulans strain; adding alcohol substances in the stationary phase of the fermentation growth of the bacillus coagulans strain.

2. The fermentation process of high yield bacteriostatic polypeptide substance of bacillus coagulans according to claim 1, which is characterized in that: the basic culture medium comprises 10-40g/L of molasses, 10-30g/L of yeast extract powder, 5-30g/L of corn steep liquor dry powder, 10-30g/L of beef extract powder, 1-10g/L of sodium chloride, 2-10g/L of sodium acetate, 0.1-1g/L of magnesium sulfate, 2-15mg/L of manganese sulfate and 800.5-2 g/L of tween.

3. The fermentation process of high-yield bacteriostatic polypeptide substance of bacillus coagulans according to claim 1 or 2, which is characterized in that: the basic culture medium comprises 40.00g/L of molasses, 20g/L of yeast extract powder, 10g/L of corn steep liquor dry powder, 20g/L of beef extract powder, 5g/L of sodium chloride, 5g/L of sodium acetate, 0.58g/L of magnesium sulfate, 10mg/L of manganese sulfate and 802 g/L of tween.

4. The fermentation process of high-yield bacteriostatic polypeptide substance of bacillus coagulans according to claim 1 or 2, which is characterized in that: the pH of the basic culture medium is 7.0.

5. The fermentation process of high yield bacteriostatic polypeptide substance of bacillus coagulans according to claim 1, which is characterized in that: the inoculation amount in the step (1) is 2-7mL of seed liquid/100 mL of basal medium for inoculation.

6. The fermentation process of high yield bacteriostatic polypeptide substance of bacillus coagulans according to claim 1, which is characterized in that: the bacillus coagulans seed liquid is obtained by inoculating a single colony of bacillus coagulans to a PCA liquid culture medium, culturing at 40-45 ℃ and 150-200rpm for 20-30 h.

7. The fermentation process of high yield bacteriostatic polypeptide substance of bacillus coagulans according to claim 1, which is characterized in that: the Bacillus coagulans is Bacillus coagulans BC99, the Bacillus coagulans BC99 is named as Bacillus coagulans, and the preservation number is as follows: CGMCC No. 19487.

8. The fermentation process of high yield bacteriostatic polypeptide substance of bacillus coagulans according to claim 1, which is characterized in that: the amino acid substance is one or more of serine, threonine, cysteine, phenylalanine, glycine or proline.

9. The fermentation process of high yield bacteriostatic polypeptide substance of bacillus coagulans according to claim 1, which is characterized in that: the alcohol substance is one or more of ethanol, ethylene glycol or n-butanol.

10. Use of a process for the fermentation of a highly productive bacteriostatic polypeptide substance of bacillus coagulans according to any one of claims 1 to 9, wherein the process comprises the steps of: the bacteriostatic polypeptide substance of the bacillus coagulans prepared by the fermentation process of the bacillus coagulans high-yield bacteriostatic polypeptide substance is added into food, health-care products, additives or preservatives.

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