CN110734873B - Method for increasing number of bacillus coagulans spores and application - Google Patents
Method for increasing number of bacillus coagulans spores and application Download PDFInfo
- Publication number
- CN110734873B CN110734873B CN201910958273.XA CN201910958273A CN110734873B CN 110734873 B CN110734873 B CN 110734873B CN 201910958273 A CN201910958273 A CN 201910958273A CN 110734873 B CN110734873 B CN 110734873B
- Authority
- CN
- China
- Prior art keywords
- bacillus coagulans
- fermentation
- spores
- culture
- glucose
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/135—Bacteria or derivatives thereof, e.g. probiotics
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Biotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Virology (AREA)
- Medicinal Chemistry (AREA)
- Biomedical Technology (AREA)
- Polymers & Plastics (AREA)
- Microbiology (AREA)
- Food Science & Technology (AREA)
- Nutrition Science (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Mycology (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention discloses a method for increasing the number of Bacillus coagulans spores and application thereof, wherein the strain is Bacillus coagulans (Bacillus coagulans)13002, and the preservation number is CGMCC NO. 7431. The specific method comprises the following steps: (1) activating and culturing bacillus coagulans 13002 in a seed culture medium to obtain a seed culture solution; (2) the activated seed culture solution is connected to a fermentation culture medium, the number of spores of the culture solution is increased by changing nutrient substances in the fermentation culture medium and adjusting control parameters of the fermentation process, harmful metabolites are not produced, the oxidation resistance of an organism is improved, the occurrence of lipid peroxidation is reduced, and the number of spores can reach 3.048 multiplied by 109CFU/mL. Meanwhile, the process is simple, the conditions are mild, special equipment is not needed, the production cost is reduced, and the production can be expanded.
Description
Technical Field
The invention relates to the field of microbial fermentation engineering, in particular to bacillus coagulans 13002 and a method for increasing the number of spores thereof.
Background
Bacillus coagulans (gram-positive bacteria) belongs to the genus Bacillus, and has rod-like shape, terminal spore, and no flagellum. The metabolic type is facultative anaerobic, aerobic conditions can produce more biomass than anaerobic conditions. The optimal growth temperature is 40-50 ℃, the optimal growth pH value is generally 6.6-7.0, and the lactic acid can be produced by fermentation culture. Meanwhile, the bacillus coagulans is a potential stock in the field of probiotics, has stress resistance such as high temperature resistance, cholate resistance, acid resistance and the like, can adapt to the hypoxic intestinal environment, and has certain inhibition capability on part of antibiotics and certain harmful bacteria.
In recent years, bacillus coagulans has become one of the hot spots of domestic and foreign research, and the research focuses on how to obtain bacillus coagulans cells cultured at high density by using a cheap and easily available culture medium. The research on bacillus coagulans in China is just at the beginning. In 2016, 5 and 30 months, the national health committee announced Bacillus coagulans (Bacillus coagulousns) in the list of strains available for food, further expanding the research of Bacillus coagulans to the application in the food field.
The spores are dormant bodies which are extremely low in water content and extremely strong in stress resistance and are formed in cells when essential nutrients of bacterial nutrients are about to be exhausted. Because of the strong stress resistance of the spores, the activity of the microbial inoculum can be ensured and the product quality can be improved in the industrial mechanical production and high-temperature drying process. Because of the advantages of spores, the product forms for bacillus coagulans in the industry are being transferred from vegetative somatic forms like the spore forms.
The current research on bacillus coagulans mainly focuses on obtaining more vegetative somatic cells through a high-density culture technology, but the attention on the number of spores is less, and the optimization of the number of spores on a culture medium and a culture mode is not performed, so that the research on the spore form culture turning to low cost in the industry is lacked.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provides the bacillus coagulans 13002 and a method for improving the number of spores thereof, wherein the obtained bacillus coagulans does not produce harmful metabolites, has high oxidation resistance, and the number of spores can reach 3.048 multiplied by 109CFU/mL. The invention not only accords with the research trend of the strain, but also is the strainThe deep research and the practical application of the seeds provide a theoretical basis.
The purpose of the invention is realized by the following technical scheme:
a method for increasing the number of spores of Bacillus coagulans, comprising the following steps:
(1) preparing a seed culture solution: the method comprises the following steps of (1) enabling bacillus coagulans bacterial suspension in logarithmic growth phase to be in a volume ratio of 2-10: inoculating 100 percent of the seed culture medium to carry out activated culture; the seed culture medium is as follows: calculated by mass ratio, 0.5-2.5% of glucose, 0.5-1.5% of bacteriological peptone, 1-3% of yeast extract powder, 0.5-1.5% of sodium chloride and the balance of water, wherein the pH value is 6.0-7.5;
(2) the volume ratio of the components is 2-10: 100, inoculating the seed culture solution into a fermentation culture medium, performing fermentation culture for 48-60h at 40-50 ℃ and the rotating speed of a shaking table of 150-250 rpm, and collecting the fermentation culture solution; the formula of the fermentation medium is as follows: calculated according to the mass ratio, 0.5-2.5% of glucose, 0.5-1.5% of bacteriological peptone, 1-3% of yeast extract powder, 0.5-1.5% of sodium chloride, 0.01-0.03% of manganese sulfate, 0.1-0.5% of dipotassium hydrogen phosphate and the balance of bran water extract, wherein the concentration of the extract is 5-15%, and the pH value is 6.0-7.5;
the strain is Bacillus coagulans (Bacillus sp.)13002, is preserved in China general microbiological culture Collection center (CGMCC) in 2013, 4 and 8 months, and has the preservation number of CGMCC NO.7431 for short.
Preferably, the bran water extract is prepared by the following steps: adding bran into water, boiling for 0.5-1.5 h at 70-80 ℃, and filtering. The 5-10% bran water is: adding 50-100 g of bran into 1L of distilled water, boiling for 0.5-1.5 h at 70-80 ℃, filtering with 4 layers of gauze to obtain 5-10% bran water, cooling, and storing in a refrigerator at 4 ℃ for later use.
Preferably, the fermentation medium contains 1.5% of glucose, 1% of bacteriological peptone, 2% of yeast extract powder, 1% of sodium chloride, 0.02% of manganese sulfate, 0.3% of dipotassium hydrogen phosphate and the balance of 10% of bran water extract.
Preferably, the activated culture conditions in step (1) are: the temperature is 40-50 ℃, the rotating speed of a shaking table is 150-250 rpm, and the culture time is 18-24 h.
Preferably, the pH of the fermentation medium is 7.0-7.5.
Preferably, the inoculation amount of the seed culture solution in the step (2) is 6-7%.
Preferably, the temperature of the fermentation culture conditions in the step (2) is 45 ℃, the rotation speed of a shaking table is 200rpm, and the fermentation time is 52 h.
The bacillus coagulans prepared by the method is applied to preparation of probiotic health products.
Compared with the prior art, the invention has the following advantages and beneficial effects:
(1) the working leaven of the invention is cultured by two culture mediums in sequence, so that the bacillus coagulans is activated as much as possible and is in a synchronous growth state.
(2) The monopotassium phosphate, manganese sulfate and bran water adopted by the fermentation medium can ensure growth factors required by spore formation and promote the large-scale formation of spores.
(3) The fermentation temperature of the bacillus coagulans 13002 is 40-50 ℃, so that the probability of mixed bacteria pollution can be reduced.
(4) The bacillus coagulans does not produce harmful metabolites, and is high in oxidation resistance proved by animal experiments.
The invention optimizes the fermentation culture medium, improves the number of spores in the fermentation process of the bacillus coagulans by changing nutrient substances in the fermentation culture medium and adjusting control parameters of the fermentation process, does not produce harmful metabolites, is beneficial to improving the oxidation resistance of organisms and reducing the occurrence of lipid peroxidation, and the number of the spores can reach 3.048 multiplied by 109CFU/mL. Meanwhile, the process is simple, the conditions are mild, special equipment is not needed, the production cost is reduced, and the production can be expanded.
Drawings
FIG. 1 is a plot of the spore number of Bacillus coagulans in seed medium and modified fermentation medium of example 1.
FIG. 2 is a graph of the interaction effect of the Box-Behnken optimization experiment fitting response surface in example 1; (a) a is a contour diagram of interaction influence of glucose and B, namely bran water concentration, (B) A is a contour diagram of interaction influence of glucose and B, namely bran water concentration, on a 3D curved surface, (C) A is a contour diagram of interaction influence of glucose and C, namely dipotassium hydrogen phosphate, (D) A is a contour diagram of interaction influence of glucose and C, namely dipotassium hydrogen phosphate, on a 3D curved surface, (e) B is a contour diagram of interaction influence of bran water concentration and C, namely dipotassium hydrogen phosphate, and (f) B is a contour diagram of interaction influence of bran water concentration and C, namely dipotassium hydrogen phosphate on a 3D curved surface.
FIG. 3 is a graph showing the results of detection of nitroreductase in example 2; the right tube is a blank group without inoculated bacteria, the middle tube is an experimental group added with bacillus coagulans 13002, and the left tube is an experimental group added with a quality control strain escherichia coli ATCC 25922.
FIG. 4 is a graph showing the results of the assay for hemolytic activity in example 2; (a) streaking bacillus coagulans 13002; (b) inoculating bacillus coagulans 13002 by puncture; (c) streaking and inoculating a quality control strain escherichia coli; (d) and (3) performing puncture inoculation on the quality control strain escherichia coli.
FIG. 5 is a graph showing the results of the indole test in example 2; the left test tube is blank test, the middle test tube is experimental group added with bacillus coagulans 13002, and the right test tube is experimental group added with quality control strain escherichia coli.
FIG. 6 is a graph showing the results of the gelatin dissolution experiment in example 2; the upper test tube is a blank test group, and the lower test tube is a test group added with bacillus coagulans 13002.
Detailed Description
The present invention will be further described in detail with reference to the following examples for better understanding, but the scope of the present invention as claimed is not limited to the scope shown in the examples.
Example 1: research on increasing spore number of bacillus coagulans
In the first step, bacillus coagulans suspension in logarithmic growth phase is mixed according to the volume ratio of 3: 100 percent of the culture medium is inoculated into a seed culture medium for activation culture. The seed culture medium is as follows: calculated according to the mass ratio, 1.5 parts of glucose, 1.0 part of bacteriological peptone, 2 parts of yeast extract powder and 1 part of sodium chloride are added with distilled water to reach 100 parts of constant volume, and the pH value is 6.8.
In the second step, the seed culture solution is mixed with a mixture of 6: inoculating 100% of the extract into fermentation medium, fermenting at 45 deg.C and shaking table rotation speed of 200rpm for 52 hr, collecting fermentation liquid with spore number greater than 3 × 109CFU/mL. The formula of the fermentation medium is as follows: calculated according to the mass ratio, 1.5 parts of glucose, 1 part of bacteriological peptone, 2 parts of yeast extract powder, 1 part of sodium chloride, 0.02 part of manganese sulfate and 0.3 part of dipotassium hydrogen phosphate are added with 10% bran water to fix the volume to 100 parts, and the pH value is 7.0.
Through intensive study on the culture conditions of the Bacillus coagulans 13002, the logarithmic value of the fermentation time to the spore number of the Bacillus coagulans is shown in FIG. 1, and as can be seen from FIG. 1, the spore number reaches the highest value after 52h, and the spore number is 3.29 multiplied by 10 at the moment9CFU/mL。
The formula and culture conditions of the fermentation medium in the second step are obtained through the following single-factor optimization experiment, orthogonal experiment, Plackett-Burmen experiment and Box-Behnken optimization experiment, and specifically are as follows:
1. fermentation medium single factor optimization
1.1 Effect of different carbon sources
Culturing Bacillus coagulans for 60h by using basal culture media with different carbon sources at the temperature of 45 ℃ and the speed of 160r/min, and measuring the number of spores. The basic culture medium comprises: according to the mass ratio, 1.0 part of bacteriological peptone and 2.0 parts of yeast extract powder are added with 1.0 part of different carbon sources, the volume is fixed to 100 parts by using distilled water, and the pH is adjusted to 6.8. As can be seen from Table 1, when the carbon source is glucose or bran water, the number of spores is large, and both carbon sources are preferable for increasing the number of spores of Bacillus coagulans 13002.
TABLE 1 Effect of different carbon sources on the number of Bacillus coagulans spores
1.2 Effect of different Nitrogen sources
Culturing Bacillus coagulans for 60h at 45 deg.C and 160r/min with basic culture medium containing different nitrogen sources, and measuring spore number. The basic culture medium comprises: calculated according to the mass ratio, 1.0 part of glucose is added with 2.0 parts of different nitrogen sources, 10 percent of bran water is used for fixing the volume to 100 parts, and the pH value is adjusted to 6.8. As can be seen from Table 2, when the carbon source is yeast extract powder or peptone, the number of spores is large, and these two nitrogen sources are preferable for increasing the number of spores of Bacillus coagulans 13002.
TABLE 2 Effect of different nitrogen sources on the number of spores of Bacillus coagulans
1.3 Effect of different inorganic salts
Culturing Bacillus coagulans with different inorganic salt basal culture media at 45 deg.C and 160r/min for 60h, and determining spore number. The basic culture medium comprises: calculated according to the mass ratio, 1.0 part of glucose, 1.0 part of bacteriological peptone and 2.0 parts of yeast extract powder are added with 0.5 part of different inorganic salts, 10 percent of bran water is used for fixing the volume to 100 parts, and the pH is adjusted to 6.8. As can be seen from Table 3, when the inorganic salt is sodium chloride, manganese sulfate or dipotassium hydrogen phosphate, the number of spores is large, and these three inorganic salts are preferable for increasing the number of spores of Bacillus coagulans 13002.
TABLE 3 Effect of different inorganic salts on the number of spores of Bacillus coagulans
2. Orthogonal experimental optimization
In the above single-factor analysis, it was confirmed that seven single factors, i.e., glucose, bran water, bacteriological peptone, yeast extract powder, sodium chloride, manganese sulfate, and dipotassium hydrogen phosphate, were suitable for increasing the number of spores of Bacillus coagulans 13002. Therefore, the orthogonal experiment is adopted to carry out preliminary optimization on the single factors.
2.1 optimization of carbon and Nitrogen sources
Culturing Bacillus coagulans with culture medium of different carbon sources and nitrogen sources at 45 deg.C and 160r/min for 60h, and determining spore number. The formulation of the medium and the number of spores obtained are shown in Table 4.
TABLE 4 carbon and nitrogen source optimization experiment table
The combination of the media components analyzed for the test results (as shown in Table 5) to give the highest spore count was 1.0 part glucose, 10% bran water concentration, 2.0 parts yeast extract powder, and 1.0 part bacteriological peptone.
TABLE 5 analysis table of carbon source and nitrogen source optimization results
2.2 optimization of inorganic salts
Culturing Bacillus coagulans for 60h at 45 deg.C and 160r/min with basal medium containing different inorganic salts, and determining spore number. The basic culture medium comprises the following formula: glucose 1.0 part, yeast extract powder 2.0 parts, bacteriological peptone 1.0 part, inorganic salt addition amount shown in table 6, volume fixed to 100 parts with 10% bran water, pH adjusted to 6.8.
Table 6 inorganic salt optimization experimental table
The combination of inorganic salts in the medium having the highest number of spores was analyzed on the test results (as shown in Table 7) to be 0.6 part of sodium chloride, 0.03 part of manganese sulfate, and 0.2 part of dipotassium hydrogen phosphate.
TABLE 7 analysis table of inorganic salt optimization results
3. Optimized design of fermentation medium formula
Media optimization was performed using Design Expert 8.0.6 software. When the above analysis was carried out in the single-factor and orthogonal experiments, seven factors of glucose, bran water, bacteriological peptone, yeast extract powder, sodium chloride, manganese sulfate and dipotassium phosphate were selected and analyzed, and it was found that when the medium contained 1.0 part of glucose, 2.0 parts of yeast extract powder, 1.0 part of bacteriological peptone, 0.6 part of sodium chloride, 0.03 part of manganese sulfate, 0.2 part of dipotassium phosphate and 10% of bran water, respectively, the obtained bacterial liquid had the highest number of spores. Therefore, these several concentrations were chosen as a benchmark for performing the Plackett-Burmen experiment, the steepest hill climbing experiment and the Box-Behnken optimization experiment.
3.1Plackett-Burmen experiment
TABLE 8 Plackett-Burmen Experimental results Table
Note: A-G respectively refer to glucose, water concentration of bran, bacteriological peptone, yeast extract powder, sodium chloride, manganese sulfate, and dipotassium hydrogen phosphate.
The data in the above table were analyzed using Design Expert 8.0.6 software, and the results are as follows:
TABLE 9 Plackett-Burmen Experimental analysis Table
Note: A-G respectively refer to glucose, water concentration of bran, bacteriological peptone, yeast extract powder, sodium chloride, manganese sulfate, and dipotassium hydrogen phosphate.
As can be seen from table 9, the contribution degrees of glucose, bran water concentration, and dipotassium phosphate were high in promoting the increase of the number of spores of bacillus coagulans 13002, and were 3.83%, 6.00%, and 16.04%, respectively, and the contribution values of the other factors were all less than 3%. The results show that the addition of glucose and dipotassium hydrogen sulfate or the change of the concentration of bran water in the seven promoting factors is more favorable for promoting the increase of the number of spores of bacillus coagulans.
According to the results of the Plackett-Burmen experiment, glucose, bran water concentration and dipotassium hydrogen phosphate are selected as research objects, and the steepest climbing experiment is carried out in the next step.
3.2 steepest climbing experiment
The results are shown in table 10, and by setting a steepest climbing experiment, the concentrations of glucose, bran water and dipotassium hydrogen phosphate in the fermentation medium were changed, and after the measurement, the response value with the highest spore number of the bacterial liquid was obtained by comparison, and as the central point of the response surface design factor, 1.5 parts of glucose, 7.5 parts of bran water and 0.3 part of dipotassium hydrogen phosphate were calculated by mass ratio.
TABLE 10 steepest climb test results
3.3Box-Behnken optimization experiment
TABLE 11 Box-Behnken Experimental results Table
The experimental data were designed for analysis using the central portfolio of Box-Behnken and then analyzed using Design Expert 8.0.6 software.
TABLE 12 Box-Behnken results analysis Table
As can be seen from table 12, p of the fitting model is 0.0008<0.05, coefficient of determination R2When the model is fitted well to the actual situation, 0.9515, the multivariate regression equation of the spore number Y to the independent variables glucose (a), bran water concentration (B), dipotassium hydrogen phosphate (C) is obtained from the above analysis:
Y＝2.43+0.062A+0.31B+0.36C-0.035AB-0.28AC-0.0001BC-0.36A2+0.23B2-0.43C2
this equation expresses that the linear relationship between the number of spores of bacillus coagulans 13002 and the 3 independent variables is significant, i.e. the method is reliable. The first order coefficient ratio of the regression equation is larger, the interaction coefficient of AC and AB in the interaction coefficient is larger, which shows that the interaction effect between the water concentration of the glucose and the bran and between the glucose and the dipotassium hydrogen phosphate is large, and the interaction coefficient of BC is smaller, which shows that the interaction effect between the water concentration of the bran and the potassium dihydrogen phosphate is small.
To examine the validity of the equation, a mathematical model of the number of spores of bacillus coagulans 13002 determined was subjected to analysis of variance, and partial regression coefficients of each factor were examined. The regression coefficient of B, C in the first term was highly significant, with p both less than 0.01, indicating that bran water concentration and dipotassium phosphate have a highly significant effect on the number of spores of bacillus coagulans 13002. The partial regression coefficient of A, C in the quadratic term reaches a very significant level (p)<0.01), the partial regression coefficient of B reaches a significant level (p)<0.05). The regression coefficient of the interaction term AC is more obvious than other factors, which shows that the interaction term of glucose and potassium dihydrogen phosphate has more obvious influence on the spore number of the bacillus coagulans 13002. Determining the coefficient R2The equation is stated to be good for experimental fitness as 0.9515.
In order to obtain the maximum spore count, the glucose, bran water concentration and potassium dihydrogen phosphate concentration in the medium need to be in the appropriate ratio. Thus, from FIG. 2 and analysis of variance, it is seen that the order of magnitude of the growth effect on Bacillus coagulans 13002 is AC > AB > BC.
First order partial derivatives were calculated for the binary regression equation, and when the response value Y (number of spores) was the maximum value, the factor levels were 1.5 parts by mass of glucose (a), 10 parts by mass of bran water concentration (B), and 0.3 parts by mass of monopotassium phosphate (C) per 100 parts of the medium. At this time, the number of spores of Bacillus coagulans is theoretically predicted to be 2.97X 109CFU/mL。
The obtained culture medium formula is subjected to a verification test, and the actual spore numberMeasured as 3.048X 109CFU/mL。
4. Optimization of culture conditions
In the optimized fermentation medium, the culture conditions are optimized, and the optimal culture conditions are determined by changing the pH value of the fermentation medium, the inoculation quantity, the rotating speed and the temperature of the fermentation culture. The optimized fermentation medium formula is as follows: 1.5 parts of glucose, 1 part of bacteriological peptone, 2 parts of yeast extract powder, 1 part of sodium chloride, 0.02 part of manganese sulfate and 0.3 part of dipotassium hydrogen phosphate, and the volume is fixed to 100 parts by 10% bran water.
4.1 pH optimization of the Medium
The pH value of the fermentation medium is changed, and the number of spores is measured. As can be seen from Table 13, the number of spores was at most 2.82X 10 at pH 7.09CFU/mL, which is most favorable for the increase of the number of Bacillus coagulans spores.
TABLE 13 Effect of culture Medium pH on spore number
4.2 optimization of inoculum size in culture Medium
And changing the inoculation amount of the seed liquid of the fermentation medium, and measuring the number of spores. As can be seen from table 14, when the inoculation ratio is seed liquid: fermentation medium ═ 6: when the inoculation amount is 100 percent (namely 6 percent), the maximum number of spores can reach 2.97 multiplied by 109CFU/mL, which is most favorable for the increase of the number of Bacillus coagulans spores.
TABLE 14 Effect of inoculum size on spore number
4.3 optimization of culture temperature
The temperature of the fermentation culture was changed and the number of spores was measured. As can be seen from Table 15, the number of spores was as high as 3.21X 10 at a temperature of 45 deg.C9CFU/mL, which is most favorable for the increase of the number of Bacillus coagulans spores.
TABLE 15 Effect of temperature on spore count
4.4 optimization of the culture rotation speed
And changing the rotating speed in the fermentation culture process and measuring the number of spores. As can be seen from Table 16, the number of spores was as high as 2.88X 10 at a rotation speed of 200rpm9CFU/mL, which is most favorable for the increase of the number of Bacillus coagulans spores.
TABLE 16 Effect of rotational speed on spore count
The present invention will be described in more detail with reference to examples 2 and 3 for better illustration of the safety and oxidation resistance of the resulting bacterial suspension, but the scope of the present invention is not limited to the ranges shown in the examples.
Example 2 Bacillus coagulans safety study
In the first step, bacillus coagulans suspension in logarithmic growth phase is mixed according to the volume ratio of 2-10: 100 percent of the culture medium is inoculated into a seed culture medium for activation culture. The seed culture medium is as follows: according to the mass ratio, 0.5-2.5 parts of glucose, 0.5-1.5 parts of bacteriological peptone, 1-3 parts of yeast extract powder and 0.5-1.5 parts of sodium chloride are subjected to constant volume to 100 parts by using distilled water, the pH value is adjusted to 6.0-7.5, and the yeast extract powder can be used after being sterilized and cooled.
And the second step is that the volume ratio is 2-10: 100, inoculating the seed culture solution into a fermentation culture medium, performing fermentation culture for 48-60h at 40-50 ℃ and the rotation speed of a shaking table of 150-250 rpm, and collecting the fermentation culture solution. The formula of the fermentation medium is as follows: calculated according to the mass ratio, 0.5-2.5 parts of glucose, 0.5-1.5 parts of bacteriological peptone, 1-3 parts of yeast extract powder, 0.5-1.5 parts of sodium chloride, 0.01-0.03 part of manganese sulfate, 0.1-0.5 part of dipotassium hydrogen phosphate, 5-10% of bran water for constant volume to 100 parts, adjusting the pH value to 6.0-7.5, sterilizing and cooling for use.
The fermentation liquor obtained in the first step and the second step is used for safety evaluation research
1. Nitroreductase assay
Inoculating the bacillus coagulans 13002 subjected to fermentation culture into a nitroreductase detection culture medium according to the inoculation amount (v/v) of 3%, culturing at the constant temperature of 37 ℃ for 72h, sequentially adding an alpha-naphthylamine solution and a sulfanilic acid solution into a fermentation culture solution, and observing the change of the color of the culture medium. Coli ATCC25922 was a positive control strain. The nitroreductase detection medium without inoculated bacteria is a blank control. The nitroreductase detection culture medium comprises: peptone 10.0g, potassium nitrate 1.0g, distilled water 1L, pH 7.4, 121 deg.C and sterilizing for 15 min. If the culture medium turns red, the detection result of the nitroreductase is positive, otherwise, the detection result of the nitroreductase is negative. As a result, as shown in FIG. 3, the detection result of nitroreductase in Bacillus coagulans 13002 was negative, that is, no active nitroreductase was present in the bacterial solution, and nitrate could not be reduced to nitrite.
2. Detection of hemolytic Activity
The bacillus coagulans 13002 after fermentation culture is streaked and punctured on a Columbia blood agar plate by using a sterilized inoculating loop under the aseptic condition, and after inverted culture is carried out for 48 hours at 37 ℃, if a transparent loop is formed around a bacterial colony, the beta-hemolysis is realized; if a green circle appears, alpha-hemolysis is performed; if there is no hemolytic ring, it is γ -hemolysis, also called no hemolysis. Staphylococcus aureus ATCC 25923 was used as a positive reference strain. As a result, as shown in FIG. 4, a hemolytic cycle appeared around the colony of Staphylococcus aureus, which is a quality control strain, but no hemolytic cycle appeared around Bacillus coagulans 13002, i.e., Bacillus coagulans 13002 did not produce hemolytic toxins that can lyse erythrocytes, and thus it was safe.
3. Indole experiments
Inoculating the bacillus coagulans 13002 subjected to fermentation culture into a peptone water culture medium according to the inoculation amount (v/v) of 3%, culturing at the constant temperature of 37 ℃ for 72h, adding 1-2 mL of diethyl ether into the culture solution, standing for a moment, slowly adding 8 drops of indole reagent along the tube wall, observing the test result, and simultaneously performing a blank experiment. Coli ATCC25922 was a positive control strain. The peptone water culture medium comprises peptone 1.0g and sodium chloride 0.5g, and water with pH adjusted to 7.8 when added to 100 mL. If the liquid surface appears red, the liquid surface is positive, and if the liquid surface does not change color, the liquid surface is negative. As shown in FIG. 5, the medium of Escherichia coli ATCC25922 showed a red ring, and the indole test showed a positive result, whereas the medium inoculated with Bacillus coagulans 13002 showed no significant color change, and the result showed a negative result.
4. Gelatin dissolution test
Inoculating the bacillus coagulans 13002 subjected to fermentation culture into a gelatin culture medium according to the inoculation amount (v/v) of 3%, culturing at the constant temperature of 37 ℃ for 48h, taking out and placing in a refrigerator at the temperature of 4 ℃ to completely solidify gelatin, taking out after solidification and placing at room temperature, observing the melting speed of a inoculation tube and an aseptic control tube, wherein if the melting speed is obviously accelerated, a positive reaction is obtained, and otherwise, the negative reaction is obtained. The results are shown in FIG. 6, the melting rate of the gelatin in the inoculation tube is not obviously different from that in the sterile tube, and the results are negative.
Example 3: research on inoxidizability of bacillus coagulans
In the first step, bacillus coagulans suspension in logarithmic growth phase is mixed according to the volume ratio of 2-10: 100 percent of the culture medium is inoculated into a seed culture medium for activation culture. The seed culture medium is as follows: according to the mass ratio, 0.5-2.5 parts of glucose, 0.5-1.5 parts of bacteriological peptone, 1-3 parts of yeast extract powder and 0.5-1.5 parts of sodium chloride are subjected to constant volume to 100 parts by using distilled water, the pH value is adjusted to 6.0-7.5, and the yeast extract powder can be used after being sterilized and cooled.
And the second step is that the volume ratio is 2-10: 100, inoculating the seed culture solution into a fermentation culture medium, performing fermentation culture for 48-60h at 40-50 ℃ and the rotation speed of a shaking table of 150-250 rpm, and collecting the fermentation culture solution. The formula of the fermentation medium is as follows: calculated according to the mass ratio, 0.5-2.5 parts of glucose, 0.5-1.5 parts of bacteriological peptone, 1-3 parts of yeast extract powder, 0.5-1.5 parts of sodium chloride, 0.01-0.03 part of manganese sulfate, 0.1-0.5 part of dipotassium hydrogen phosphate, 5-10% of bran water for constant volume to 100 parts, adjusting the pH value to 6.0-7.5, sterilizing and cooling for use.
Animal experiment of the fermentation liquid for researching its oxidation resistance
1. Grouping and treatment of mice
The breeding temperature of the mice is 20-25 ℃, and the relative humidity is 45-55%. After 40 SPF-grade BALB/c male mice were pre-fed for one week, they were randomly divided into 4 groups of normal group, model group, positive group and Bacillus coagulans group, 10 mice per group, and the weights among the groups were not significantly different, as shown in Table 17.
Table 17 mouse grouping and treatment
Except the normal group, the other groups were injected with 50mg/kg of cyclophosphamide intraperitoneally for 3 days continuously to establish a liver injury mouse model. After modeling, each group of mice was gavaged in the manner shown in Table 17, once a day, at an injection rate of 1.0mL/100g for two weeks. Daily weight changes of the mice were recorded to adjust the gavage amount.
2. Detection of antioxidant index in mouse
In experiment 15d, the mice are fasted overnight, the mice are killed by dislocation of cervical vertebrae, the liver is taken, physiological saline is added to prepare 10% liver tissue homogenate, and the contents of SOD, CAT and MDA in the liver tissue are measured according to the kit specification. The superoxide dismutase (SOD) determination kit, the Catalase (CAT) determination kit and the Malondialdehyde (MDA) determination kit are all from Nanjing institute of bioengineering.
The results are shown in table 18, the liver SOD and CAT activities of the model group mice are both significantly lower than those of the normal group (p <0.05), and the content of the lipid peroxidation product MDA is significantly higher than that of the normal group (p <0.05), which indicates that the injection of cyclophosphamide causes the damage of the liver antioxidant system of the mice, and also indicates that the modeling of the liver-damaged mouse model is successful. The activities of SOD and CAT in the liver of the mouse with the bacillus coagulans group are obviously higher than those of the model group (p is less than 0.05), and the content of MDA is obviously lower than that of the model group (p is less than 0.05). And the effect of improving the activity of the antioxidant enzyme of the bacillus coagulans group mice is better than that of the positive group.
TABLE 18 results of in vivo antioxidant indices in mice
Acrolein, a metabolite of cyclophosphamide, can stimulate the organism to generate a large amount of active oxygen, so that the activities of SOD and CAT are reduced. In organisms, free radicals act on lipid to generate peroxidation, and the oxidation end product is Malondialdehyde (MDA), which can cause cross-linking polymerization of life macromolecules such as protein and nucleic acid, has cytotoxicity and can reflect the degree of lipid oxidation damage caused by cyclophosphamide. As can be seen from Table 18, Bacillus coagulans can significantly improve the SOD and CAT activities of liver-injured mice, and simultaneously reduce the content of MDA, indicating that Bacillus coagulans is helpful for improving the antioxidant capacity of organisms and reducing the occurrence of lipid peroxidation.
Claims (7)
1. A method for increasing the number of spores of Bacillus coagulans, comprising the steps of:
(1) preparing a seed culture solution: bacillus coagulans in logarithmic growth phase (Bacillus coagulans) The volume ratio of the bacterial suspension is 2-10: inoculating 100 percent of the seed culture medium to carry out activated culture; the seed culture medium is as follows: calculated by mass ratio, 0.5-2.5% of glucose, 0.5-1.5% of bacteriological peptone, 1-3% of yeast extract powder, 0.5-1.5% of sodium chloride and the balance of water, wherein the pH value is 6.0-7.5;
(2) the volume ratio of the components is 2-10: 100, inoculating the seed culture solution into a fermentation culture medium, performing fermentation culture for 48-60h at 40-50 ℃ and the rotating speed of a shaking table of 150-250 rpm, and collecting the fermentation culture solution; the formula of the fermentation medium is as follows: calculated according to the mass ratio, 0.5-2.5% of glucose, 0.5-1.5% of bacteriological peptone, 1-3% of yeast extract powder, 0.5-1.5% of sodium chloride, 0.01-0.03% of manganese sulfate, 0.1-0.5% of dipotassium hydrogen phosphate and the balance of bran water extract, wherein the concentration of the extract is 5-15%, and the pH value is 6.0-7.5;
the bacillus coagulans is bacillus coagulans (A), (B), (C)Bacillus coagulans)13002, deposited in China general microbiological culture Collection center (CGMCC) at 8.4.2013,the preservation number is CGMCC NO. 7431.
2. The method according to claim 1, characterized in that the bran water extract is prepared by: adding bran into water, boiling for 0.5-1.5 h at 70-80 ℃, and filtering.
3. The process according to claim 2, characterized in that the fermentation medium contains 1.5% glucose, 1% bacto peptone, 2% yeast extract powder, 1% sodium chloride, 0.02% manganese sulfate, 0.3% dipotassium hydrogen phosphate and the balance bran water extract, at a concentration of 10%.
4. The method according to claim 3, wherein the activation culture conditions in step (1) are: the temperature is 40-50 ℃, the rotating speed of a shaking table is 150-250 rpm, and the culture time is 18-24 h.
5. The method of claim 1 or 2 or 3 or 4, wherein the pH of the fermentation medium is 7.0 to 7.5.
6. The method according to claim 5, wherein the inoculation amount of the seed culture solution in the step (2) is 6-7%.
7. The method of claim 6, wherein the fermentation conditions of step (2) are at a temperature of 45 ℃, a shaker speed of 200rpm, and a fermentation time of 52 hours.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910958273.XA CN110734873B (en) | 2019-10-10 | 2019-10-10 | Method for increasing number of bacillus coagulans spores and application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201910958273.XA CN110734873B (en) | 2019-10-10 | 2019-10-10 | Method for increasing number of bacillus coagulans spores and application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN110734873A CN110734873A (en) | 2020-01-31 |
| CN110734873B true CN110734873B (en) | 2022-04-22 |
Family
ID=69268473
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201910958273.XA Active CN110734873B (en) | 2019-10-10 | 2019-10-10 | Method for increasing number of bacillus coagulans spores and application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN110734873B (en) |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW200932902A (en) * | 2008-01-21 | 2009-08-01 | Glory Biotech Co Ltd | Method for producing Bacillus coagulans with high bacterial count and products thereof |
| CN103160455A (en) * | 2013-03-27 | 2013-06-19 | 杭州保安康生物技术有限公司 | Preparation method of spore preparation of bacillus coagulans |
| CN104974966A (en) * | 2015-07-22 | 2015-10-14 | 江南大学 | Bacillus coagulans and high-density fermentation method thereof, and dry bacterium powder preparation method |
| CN105112326A (en) * | 2015-08-19 | 2015-12-02 | 华南理工大学 | Bacillus and high-density cultivation method for same |
| CN105154358A (en) * | 2015-08-19 | 2015-12-16 | 华南理工大学 | Bacillus and method for simultaneous saccharification and fermentation production of L-lactic acid with same |
| CN109880786A (en) * | 2019-03-15 | 2019-06-14 | 河南科技大学 | A method of promoting Bacillus coagulans spore formation |
| CN110403011A (en) * | 2019-07-17 | 2019-11-05 | 华南理工大学 | A kind of bacillus coagulans acidified milk and preparation method thereof |
| WO2019212928A1 (en) * | 2018-04-30 | 2019-11-07 | Muhammed Majeed | Modulation of immune function by bacillus coagulans |
| US10653729B2 (en) * | 2013-10-25 | 2020-05-19 | Nch Corporation | Delivery system and probiotic composition for animals and plants |
-
2019
- 2019-10-10 CN CN201910958273.XA patent/CN110734873B/en active Active
Patent Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| TW200932902A (en) * | 2008-01-21 | 2009-08-01 | Glory Biotech Co Ltd | Method for producing Bacillus coagulans with high bacterial count and products thereof |
| CN103160455A (en) * | 2013-03-27 | 2013-06-19 | 杭州保安康生物技术有限公司 | Preparation method of spore preparation of bacillus coagulans |
| US10653729B2 (en) * | 2013-10-25 | 2020-05-19 | Nch Corporation | Delivery system and probiotic composition for animals and plants |
| CN104974966A (en) * | 2015-07-22 | 2015-10-14 | 江南大学 | Bacillus coagulans and high-density fermentation method thereof, and dry bacterium powder preparation method |
| CN105112326A (en) * | 2015-08-19 | 2015-12-02 | 华南理工大学 | Bacillus and high-density cultivation method for same |
| CN105154358A (en) * | 2015-08-19 | 2015-12-16 | 华南理工大学 | Bacillus and method for simultaneous saccharification and fermentation production of L-lactic acid with same |
| WO2019212928A1 (en) * | 2018-04-30 | 2019-11-07 | Muhammed Majeed | Modulation of immune function by bacillus coagulans |
| CN109880786A (en) * | 2019-03-15 | 2019-06-14 | 河南科技大学 | A method of promoting Bacillus coagulans spore formation |
| CN110403011A (en) * | 2019-07-17 | 2019-11-05 | 华南理工大学 | A kind of bacillus coagulans acidified milk and preparation method thereof |
Non-Patent Citations (4)
| Title |
|---|
| Bacillus coagulans 13002 and fructo-oligosaccharides improve the immunity of mice with immunosuppression induced by cyclophosphamide through modulating intestinal-derived and fecal microbiota;Zhao shan等;《Food Research International》;20201016;109793 * |
| 凝结芽孢杆菌13002 的益生特性;金迅等;《食品工业科技》;20171128;摘要 * |
| 凝结芽孢杆菌13002 高密度培养;孙丽娜等;《食品工业科技》;20170713;第114页最后一段和115页第1段 * |
| 凝结芽孢杆菌13002产芽孢条件优化;涂家霖等;《食品工业科技》;20201013;88-96,102页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN110734873A (en) | 2020-01-31 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105567624B (en) | A kind of the Lactococcus lactis cream subspecies synergist and its application method of the lactic acid producing streptostacin that ferments | |
| CN106119152A (en) | The bacillus acidophilus of a kind of high-yield lactic acid rhzomorph and application thereof | |
| CN105112326A (en) | Bacillus and high-density cultivation method for same | |
| CN112251371B (en) | Lactococcus lactis, microecological preparation and application | |
| CN116286471B (en) | Application of tricholoma matsutake extract in improving number of live bacteria of lactobacillus reuteri high-density culture | |
| CN103283948A (en) | Bifidobacterium bifidum-oriented microecological preparation | |
| CN106978369A (en) | A kind of high-purity, resistance to storage at normal temperature VREF solid state fermentation culture process | |
| CN103289935A (en) | Compound strain microecological preparation and preparation method thereof | |
| CN102286411B (en) | Lactobacillus plantarum and application thereof in fermenting cabbage wrapper leaf | |
| CN102102095A (en) | Method for preparing lysozyme by fermenting marine streptomyces | |
| CN102669432B (en) | Compound probiotics preparation for geese and method for preparing same | |
| CN110734873B (en) | Method for increasing number of bacillus coagulans spores and application | |
| CN104745554B (en) | Bacillus produces the fermentation medium and fermentation process of protease and gemma | |
| CN111187744A (en) | High-density industrial fermentation medium for stratospheric bacillus and fermentation method thereof | |
| CN115181686A (en) | Bacillus subtilis culture preparation process not easy to be infected by mixed bacteria | |
| CN109722408A (en) | Promote the method for producing bacillus subtilis gemma | |
| CN108823135A (en) | A kind of fermentation medium and fermentation condition improving ocean brown bacillus number of viable and antibacterial activity | |
| CN112715890B (en) | Immobilized pickle starter and application thereof | |
| CN112175834B (en) | Application of lactobacillus plantarum in preservation of bacillus subtilis solid microbial inoculum and method for prolonging preservation period of bacillus subtilis | |
| CN104711208B (en) | A kind of lactic acid bacteria with high starch capacity of decomposition | |
| CN102816709A (en) | Method for preparing composite biological agent by double-bacterium co-culture | |
| CN111955742A (en) | Composite probiotics and preparation method thereof | |
| CN103404703A (en) | Stable and safe streptococcus thermophilus viable bacteria preparation and preparation method thereof | |
| CN104694441A (en) | Lactobacillus plantarum suitable for being made into freeze-dried powder | |
| CN103211102A (en) | Feed bionic acid processing and producing method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |