CN109880786B - Method for promoting sporulation of bacillus coagulans - Google Patents
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Abstract
The invention relates to a method for promoting spore formation of bacillus coagulans, belonging to the technical field of biological fermentation. Compared with the prior art, the supplement of the carbon source in the method can promote the increase of the number of viable bacteria, improve the density of the bacteria and accelerate the formation of spores, and the addition of the calcium carbonate can effectively shorten the formation time of the spores, improve the number of the spores and simultaneously increase the tolerance of the spores.
Description
Technical Field
The invention belongs to the technical field of biological fermentation, and particularly relates to a method for promoting spore formation of bacillus coagulans.
Background
Bacillus coagulans (Bacillus coagulons) is a gram-positive bacterium (G +). The bacterium can produce lactic acid and also can produce dormant sporophytes with strong tolerance; therefore, the bacillus subtilis has the advantages of both lactic acid bacteria and bacillus. Bacillus coagulans can form a large number of metabolites during growth, such as Coagulin (Coagulin), L-lactic acid, and lactosporin. Has the beneficial characteristics of increasing the number of intestinal probiotics, treating diarrhea, enhancing immunity, improving irritable bowel syndrome, reducing cholesterol, inhibiting the growth of pathogenic bacteria and the like; the compound microbial inoculum has broad-spectrum antibacterial property, can replace antibiotics to effectively inhibit the propagation of harmful bacteria in aquatic products and livestock breeding, and improves the micro-ecological environment of host intestinal tracts; meanwhile, some enzymes and nutrient substances can be generated, the absorption of the host to the ingested food nutrition is promoted, and the growth, development and metabolism of the host are accelerated.
As a kind of lactic acid bacillus, the bacillus coagulans is widely used in food, medicine, health care and livestock and aquatic product industries, and the corresponding products are in endless and the demand is increasing. Because the spores have the advantages of strong stress resistance, high temperature resistance, acid and alkali resistance, high cold storage germination survival rate and the like, the product form of the bacillus coagulans is being transferred from the form of vegetative cells to the form of spores in the market. Although many researches on high-density culture and sporulation-promoting culture of bacillus coagulans at home and abroad in recent years are optimized from a bacillus coagulans culture medium and a culture mode, few researches on both high-density culture and improvement of spore yield are reported. At present, regarding the problems of unstable spore formation rate and product quality, complex culture medium components and the like commonly existing in the industrial production of bacillus, the spore yield is improved and the fermentation time is shortened by changing the temperature, dissolved oxygen and stirring speed in the fermentation process, but the method has the disadvantages of complicated operation, large energy consumption and increased production cost in the actual operation.
Disclosure of Invention
In order to overcome the defects in the prior art, the invention aims to provide a method for promoting the sporulation of bacillus coagulans. The method is characterized in that in the process of bacillus coagulans culture and fermentation, glucose and calcium carbonate are sequentially added at intervals, the addition of a carbon source can promote the increase of the number of live bacteria, improve the density of bacteria and accelerate the formation of spores, and the addition of calcium carbonate can effectively shorten the formation time of the spores, improve the number of the spores and increase the tolerance of the spores.
In order to achieve the purpose, the invention adopts the specific scheme that:
a method for promoting the formation of bacillus coagulans spores comprises the step of sequentially and intermittently feeding a carbon source and calcium carbonate into a culture medium in the early logarithmic growth stage of bacillus coagulans fermentation culture.
As a further optimization of the above scheme, the carbon source is glucose.
As a further optimization of the above scheme, the method specifically comprises the following steps:
the method comprises the following steps: inoculating the original strain of the bacillus coagulans into a first-stage culture medium according to the inoculation amount with the volume ratio of 1: 15-25 for activated culture, wherein the culture conditions are as follows: the rotating speed of a shaking table is 200-220 rpm, the temperature is 35-38 ℃, the time is 20-22 hours, and the first-level strain is obtained after the culture is finished; the primary culture medium comprises the following components in percentage by mass: 1.6-1.8% of glucose, 1.0-1.4% of peptone, 0.6-1.0% of yeast powder, 0.4-0.6% of magnesium sulfate and the balance of water; the pH value is 7.8-8.2;
step two, inoculating the primary strain obtained in the step one into a secondary culture medium according to the inoculation amount with the volume ratio of 1:30 for fermentation culture, wherein the culture conditions are as follows: the rotating speed of a shaking table is 200-240 rpm, the temperature is 35-38 ℃, and the time is 40-50 h; when the culture is carried out for 8-10 h, supplementing a glucose solution at one time, wherein the supplementing amount of the glucose solution is 1.2-1.6% of the total volume of the secondary culture medium, and the concentration of the glucose solution is 1.0 g/mL; continuing culturing and fermenting for 14-18 h, and supplementing calcium carbonate solid with the final concentration of 40-80 g/L at one time;
the secondary culture medium comprises the following components in percentage by mass: 1.2-1.6% of glucose, 1.2-1.6% of peptone, 0.8-1.2% of yeast powder, 0.4-0.6% of magnesium sulfate and the balance of water; the pH value is 7.8-8.2.
As a further optimization of the scheme, the primary strain activated in the step one is inoculated in a secondary culture medium, and is subjected to at least one amplification culture, and then the fermentation culture in the step two is carried out; the culture conditions of the amplification culture are as follows: the volume ratio of the inoculation amount is 1:30, the rotating speed of a shaking table is 200-240 rpm, the temperature is 35-38 ℃, and the time is 16-20 h.
Has the advantages that:
1. according to the invention, glucose and calcium carbonate are fed on the basis of a basic culture medium, so that necessary conditions are provided for improving the yield of spores in industrial production of bacillus coagulans, shortening the product period, improving the spore resistance and prolonging the shelf life of spore products, and after glucose is added, the viable count is obviously increased by about 10 times; after calcium carbonate is added, the maturation time of spores is obviously shortened, and the yield of spores is stabilized at 108CFU/mL, significantly improved spore productivity of Bacillus coagulans.
2. The invention promotes spore formation by a two-step method, firstly, the supplement of a carbon source can promote the increase of the number of live bacteria, improve the density of the bacteria and accelerate the formation of spores, and in the initial growth stage of the bacillus coagulans, the method of adding glucose in fed-batch and fed-batch manner is adopted in the application to improve the number of the live bacteria to 1.0 multiplied by 109CFU/mL. The addition of calcium carbonate can effectively shorten the spore formation time, improve the spore number and increase the tolerance of spores. The shortening of the spore formation time in industrial production can effectively reduce the energy consumption and the production cost in production, and the components of the culture medium in the application are simple and easy to obtain, thereby obviously reducing the production cost.
3. According to research and comparison, the bacillus coagulans does not form spores under the condition that calcium carbonate is not added when the bacillus coagulans is cultured for 16 hours; while spores were observed at the 16 th hour of the culture after addition of calcium carbonate at the 18 th hour of the culture. The addition amount of the calcium carbonate is 40-80 g/L, and the addition of the calcium carbonate can quickly neutralize lactic acid formed in the fermentation process of the bacillus coagulans, so that bacteria quickly enter an environment suitable for spore formation, and the spore formation time is shortened; and the calcium carbonate is a cheap and easily-obtained production raw material, and is easier to separate from the product in industrial production compared with other solid raw materials.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.
Example 1
A method for promoting sporulation of Bacillus coagulans comprises the following specific steps:
1) inoculating original bacillus coagulans strains into a first-stage culture medium for activation, wherein the volume ratio of the inoculated strains is 1:15, the liquid loading amount is 30mL/250mL, the culture conditions are that the rotating speed of a shaking table is 200rpm, the culture temperature is 36 ℃, the culture time is 22 hours, and the first-stage strains are obtained after the culture. The primary culture medium comprises the following components in percentage by mass: 1.8% of glucose, 1.2% of peptone, 0.6% of yeast powder, 0.3% of magnesium sulfate and the balance of water; the pH was 7.8.
2) Inoculating the activated bacillus coagulans primary strain in the step 1) into a secondary liquid culture medium for amplification culture, wherein the volume ratio of the inoculum size is 1:30mL, and the liquid loading amount is 30mL/250 mL; the culture conditions are that the rotating speed of a shaking table is 230rpm/min, the culture temperature is 37 ℃, the culture time is 16 hours, and the second-level strains are obtained after the culture is finished.
3) Inoculating the bacillus coagulans secondary strain treated in the step 2) into a secondary culture medium for fermentation culture, wherein the volume ratio of the inoculation amount is 1:30mL, and the liquid loading amount is 30mL/250 mL; the culture conditions are that the rotating speed of a shaking table is 230rpm/min and the culture temperature is 37 ℃; when the culture is carried out for 8 hours, adding a sterile glucose solution into the culture medium, wherein the adding amount of the glucose solution is 1.5 percent of the volume of the culture medium, and the concentration of the glucose solution is 1.0 g/mL; continuing to culture for 16 h, adding sterile calcium carbonate solid to ensure that the final concentration of calcium carbonate in the culture medium is 60 g/L; the culture period is 40-45 hours, the culture solution is collected after the culture is finished to obtain a bacillus coagulans liquid product with high spore rate, and the viable count is detected by adopting a dilution coating flat plate method to obtain 4.5 multiplied by 109Heating in 80 deg.C water bath for 10min to inactivate vegetative somatic cells, diluting and spreading to obtain spore with spore number of 3.2 × 108 CFU/mL。
The secondary culture medium comprises the following components in percentage by mass: 1.2-1.6% of glucose, 1.2-1.6% of peptone, 0.8-1.2% of yeast powder, 0.4-0.6% of magnesium sulfate and the balance of water; the pH value is 7.8-8.2.
The primary culture medium and the secondary culture medium need to be sterilized before use, and the sterilization conditions are as follows: sterilizing at 115-121 ℃ for 20-30 min.
Example 2
A method for promoting sporulation of Bacillus coagulans comprises the following specific steps:
1) inoculating original bacillus coagulans strains into a first-stage culture medium for activation, wherein the volume ratio of the inoculated strains is 1:25, the liquid loading amount is 50mL/250mL, the culture conditions are that the rotating speed of a shaking table is 210rpm/min, the culture temperature is 37 ℃, the culture time is 20 hours, and the first-stage strains are obtained after the culture is finished. The primary culture medium comprises the following components in percentage by mass: glucose 1.6%, peptone 1.4%, yeast powder 1.0%, magnesium sulfate 0.4%, and water in balance, and pH 8.0.
2) Inoculating the first-level strain of the bacillus coagulans activated in the step 1) into a second-level culture medium, wherein the inoculation volume ratio is 1:25mL, and the liquid loading amount is 50mL/250 mL; the culture conditions are that the rotating speed of a shaking table is 220rpm/min, the culture temperature is 36.5 ℃, the culture time is 18 hours, and the second-level strains are obtained after the culture.
3) Inoculating the second-level strain of the bacillus coagulans treated in the step 2) into a second-level culture medium for fermentation culture, wherein the volume ratio of the inoculation amount is 1:50mL, and the liquid loading amount is 50mL/250 mL; the culture conditions are that the rotating speed of a shaking table is 240rpm/min and the culture temperature is 38 ℃; when the culture is carried out for 8 hours, adding a sterile glucose solution into the culture medium, wherein the adding amount of the glucose solution is 1.4 percent of the volume of the culture medium, and the concentration of the glucose solution is 1.0 g/mL; continuing to culture for 16 h, adding sterile calcium carbonate solid to ensure that the final concentration of calcium carbonate in the culture medium is 60 g/L; continuously culturing until the spore rate is maximum, collecting culture solution after culturing to obtain Bacillus coagulans liquid product with high spore rate, and detecting by dilution coating plate method to obtain viable count of 8.0 × 109Heating in 80 deg.C water bath for 10min to inactivate vegetative somatic cells, diluting and spreading to obtain spore with spore rate of 5.38 × 108 CFU/mL。
The secondary culture medium comprises the following components in percentage by mass: 1.2-1.6% of glucose, 1.2-1.6% of peptone, 0.8-1.2% of yeast powder, 0.4-0.6% of magnesium sulfate and the balance of water; the pH value is 7.8-8.2.
The primary culture medium and the secondary culture medium need to be sterilized before use, and the sterilization conditions are as follows: sterilizing at 115-121 ℃ for 20-30 min.
Example 3
A method for promoting sporulation of Bacillus coagulans comprises the following specific steps:
1) inoculating original strain into first-stage culture medium for activation, wherein the volume ratio of inoculum size is 1:25, the liquid loading amount is 50mL/250mL, the culture conditions are that the rotating speed of a shaking table is 210rpm/min, the culture temperature is 37.5 ℃, the culture time is 18 hours, and the first-stage strain is obtained after the culture is finished. The primary culture medium comprises the following components in percentage by mass: glucose 1.7%, peptone 1.3%, yeast powder 0.9%, magnesium sulfate 0.5%, and water in balance, and pH 8.2.
2) Inoculating the first-level strain of the bacillus coagulans activated in the step 1) into a second-level culture medium, wherein the inoculation volume ratio is 1:30mL, and the liquid loading amount is 30mL/250 mL; the culture conditions are that the rotating speed of a shaking table is 230rpm/min, the culture temperature is 36.5 ℃, the culture time is 16 hours, and the second-level strains are obtained after the culture is finished.
3) Inoculating the bacillus coagulans secondary strain treated in the step 2) into a secondary culture medium, wherein the volume ratio of the inoculation amount is 1:30mL, and the liquid loading amount is 30mL/250 mL; the culture conditions are that the rotating speed of a shaking table is 200rpm/min and the culture temperature is 38 ℃; when the culture is carried out till the 8 th hour, adding 1.5 percent of glucose solution, wherein the adding amount of the glucose solution is 1.5 percent of the volume of the culture medium, and the concentration of the glucose solution is 1.0 g/mL; continuing to culture for 16 h, adding calcium carbonate solid to make the final concentration of calcium carbonate in the culture medium be 50 g/L; continuously culturing until the spore rate is maximum, collecting the culture solution after the culture to obtain a bacillus coagulans liquid product with high spore rate, and detecting by adopting a dilution coating plate method to obtain the viable count of 7.10 multiplied by 109Heating in 80 deg.C water bath for 10min to inactivate vegetative somatic cells, diluting and spreading to obtain spore with spore rate of 2.79 × 108 CFU/mL。
The secondary culture medium comprises the following components in percentage by mass: 1.2-1.6% of glucose, 1.2-1.6% of peptone, 0.8-1.2% of yeast powder, 0.4-0.6% of magnesium sulfate and the balance of water; the pH value is 7.8-8.2.
The primary culture medium and the secondary culture medium need to be sterilized before use, and the sterilization conditions are as follows: sterilizing at 115-121 ℃ for 20-30 min.
Example 4
A method for promoting sporulation of Bacillus coagulans comprises the following specific steps:
1) inoculating original strain into first-stage culture medium for activation, wherein the volume ratio of the inoculum size is 1:15, the liquid loading amount is 30mL/250mL, the culture conditions are that the rotating speed of a shaking table is 220rpm, the culture temperature is 36.5 ℃, the culture time is 18 hours, and the first-stage strain is obtained after the culture is finished. The primary culture medium comprises the following components in percentage by mass: 1.6% of glucose, 1.4% of peptone, 1.0% of yeast powder, 0.4% of magnesium sulfate and the balance of water.
2) Inoculating the activated bacillus coagulans primary strain in the step 1) into a secondary culture medium for amplification culture, wherein the volume ratio of the inoculum size is 1:30mL,the liquid loading amount is 30mL/250 mL; the culture conditions are that the rotating speed of a shaking table is 230rpm/min and the culture temperature is 37 ℃; adding a glucose solution into the culture medium when the culture is carried out for 8 hours, wherein the addition amount of the glucose solution is 1.5 percent of the volume of the culture medium, and the concentration of the glucose solution is 1.0 g/mL; continuing to culture for 16 h, adding calcium carbonate solid to make the final concentration of calcium carbonate in the culture medium be 40 g/L; culturing for 50h, collecting culture solution after culturing to obtain Bacillus coagulans liquid product with high spore rate, and detecting by dilution coating plate method to obtain viable count of 5.39 × 109Heating in 80 deg.C water bath for 10min to inactivate vegetative somatic cells, diluting and spreading to obtain spore with spore number of 3.04 × 108 CFU/mL。
The secondary culture medium comprises the following components in percentage by mass: 1.5% of glucose, 1.5% of peptone, 1.0% of yeast powder, 0.5% of magnesium sulfate and the balance of water.
Comparative example 5
As a control, comparative example 5 was conducted in the same manner as in example 4 except that: comparative example 5 in the fermentation culture to 16 hours when no calcium carbonate solid was added to the culture medium, after the culture was completed, the culture broth was collected to obtain a Bacillus coagulans liquid product, and the viable count was measured by a dilution spread plate method to obtain 6.12X 109Heating in a water bath at 80 ℃ for 10min to inactivate vegetative somatic cells in CFU/mL, and then detecting, wherein spores cannot be detected.
To further illustrate the properties of spores obtained using the methods of the present invention, spore tolerance was tested by the following tests.
Spores obtained by the method described in example 1 were taken as a test group; since calcium carbonate is sometimes added to the fermentation medium to promote cell proliferation in the prior art, spore formation (CaCO) was stimulated by calcium carbonate feeding using spore resistance formed by the spore formation medium as a control group3) The detection protocol was as follows:
first, the effect of temperature on the spore of Bacillus coagulans CGMCC 9951, the results are shown in Table 1 below.
Table 1: influence of temperature on spore of Bacillus coagulans CGMCC 9951
Secondly, the effect of bile salts on the spore of bacillus coagulans CGMCC 9951, and the results are shown in the following table 2.
Table 2: influence of bile salt on spore of Bacillus coagulans CGMCC 9951
Thirdly, the influence of pH on the spore of Bacillus coagulans CGMCC 9951, and the results are shown in the following table 3.
Table 3: influence of pH on Bacillus coagulans CGMCC 9951 spore
Table 1 shows that although the heat resistance of spores formed by calcium carbonate stimulation is slightly worse than that of spores formed by a spore culture medium, the time of the conditioning and granulating process in feed granulation is very short, generally not more than 3min, and meanwhile, the granulating temperature generally not more than 100 ℃ is taken as the upper temperature limit, so the survival rate of the spores generated by using calcium carbonate in the feed granulation process is not greatly influenced. The test results are shown in Table 2, the survival rate of the calcium carbonate stimulating to form spores in 2 hours is as high as 99.79% under the condition of 0.3% of the concentration of the bile salt; the survival rate of the bacillus subtilis can reach 87.22% after being treated for 2 hours under the condition of 0.7% of the concentration of the bile salt, the survival rate of the bacillus subtilis is higher than that of the spores formed by a spore culture medium, and the survival and proliferation of the bacteria in the intestinal tract must be capable of tolerating 0.3% of the environment of the bile salt, so the spores generated by calcium carbonate stimulation can successfully survive and proliferate after entering the intestinal tract. Generally, the pH of gastric acid of animals is 2.0-3.0, and the retention time of food in stomach is 1-2h, so the pH is set to be 1.5, 2.0, 3.0 and 4.0, and the treatment time is 1h and 2 h. Table 3 shows that the survival rate of the calcium carbonate stimulated spore formation after 1 hour of treatment at pH 1.5 is only 7.14, which is slightly higher than that of the spore culture medium; however, the present patent finds that the survival rate of the spores is greater than 100% when the pH value is greater than or equal to 3, which indicates that the spores can germinate rapidly and efficiently and exert probiotic effect after being treated by the gastric juice with the pH value greater than or equal to 3. In conclusion, the acid-resistant and bile salt-resistant performances of the bacillus can be better exerted by stimulating the calcium carbonate to form spores.
It should be noted that the above-mentioned embodiments illustrate rather than limit the scope of the invention, which is defined by the appended claims. It will be apparent to those skilled in the art that certain insubstantial modifications and adaptations of the present invention can be made without departing from the spirit and scope of the invention.
Claims (2)
1. A method of promoting sporulation of bacillus coagulans, comprising: sequentially and intermittently feeding a carbon source and calcium carbonate into a culture medium at the initial logarithmic growth stage of the fermentation culture of the bacillus coagulans to promote the formation of bacillus coagulans spores; the carbon source is glucose;
the method for promoting the sporulation of the bacillus coagulans comprises the following steps:
the method comprises the following steps: inoculating the original strain of the bacillus coagulans into a first-stage culture medium according to the inoculation amount with the volume ratio of 1: 15-25 for activated culture, wherein the culture conditions are as follows: the rotating speed of a shaking table is 200-220 rpm, the temperature is 35-38 ℃, the time is 20-22 hours, and the first-level strain is obtained after the culture is finished; the primary culture medium comprises the following components in percentage by mass: 1.6-1.8% of glucose, 1.0-1.4% of peptone, 0.6-1.0% of yeast powder, 0.4-0.6% of magnesium sulfate and the balance of water; the pH value is 7.8-8.2;
step two, inoculating the primary strain obtained in the step one into a secondary culture medium according to the inoculation amount with the volume ratio of 1:30 for fermentation culture, wherein the culture conditions are as follows: the rotating speed of a shaking table is 200-240 rpm, the temperature is 35-38 ℃, and the time is 40-50 h; when the culture is carried out for 8-10 h, supplementing a glucose solution at one time, wherein the supplementing amount of the glucose solution is 1.2-1.6% of the total volume of the secondary culture medium, and the concentration of the glucose solution is 1.0 g/mL; continuing culturing and fermenting for 14-18 h, and supplementing calcium carbonate solid with the final concentration of 40-80 g/L at one time;
the secondary culture medium comprises the following components in percentage by mass: 1.2-1.6% of glucose, 1.2-1.6% of peptone, 0.8-1.2% of yeast powder, 0.4-0.6% of magnesium sulfate and the balance of water; the pH value is 7.8-8.2;
the bacillus coagulans is bacillus coagulans CGMCC 9951.
2. The method of claim 1, wherein the bacillus coagulans spore formation is promoted by: inoculating the first-stage strain activated in the first step into a second-stage culture medium, performing amplification culture at least once, and performing fermentation culture in the second step; the culture conditions of the amplification culture are as follows: the volume ratio of the inoculation amount is 1:30, the rotating speed of a shaking table is 200-240 rpm, the temperature is 35-38 ℃, and the time is 16-20 h.
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