CN112980742A - Bacillus coagulans culture medium and fermentation method thereof - Google Patents
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Abstract
The invention provides a culture medium of bacillus coagulans, which is characterized by comprising 5-10g/L glucose, 10-30% (v/v) tomato juice, 10-30% (v/v) carrot juice, 5-10g/L fructo-oligosaccharide, 10-20g/L isomalto-oligosaccharide, 5-10g/L galacto-oligosaccharide and 5-10g/L LD-tagatose. The invention takes glucose, tomato juice, carrot juice, fructo-oligosaccharide, isomaltooligosaccharide, galacto-oligosaccharide and D-tagatose as culture substrates, and solves the problems of poor taste, low spore yield and short life span when common culture media are used for fermentation. Tomato juice, carrot juice, fructo-oligosaccharide, isomaltooligosaccharide, galacto-oligosaccharide and D-tagatose can make the fermented taste better. Fructo-oligosaccharide, isomaltooligosaccharide, galacto-oligosaccharide and D-tagatose have the effect of prolonging the life of the bacillus coagulans. In addition, the temperature change of the fermentation by adopting a two-temperature fermentation method (47 ℃ and 37 ℃) improves the spore yield and prolongs the life cycle of the bacillus coagulans.
Description
Technical Field
The invention relates to the field of fermentation, in particular to a bacillus coagulans culture medium and a fermentation method thereof.
Background
Bacillus coagulans can produce both spores and lactic acid and is a facultative anaerobe. The spore has the advantages of strong stress resistance, high temperature resistance, acid and alkali resistance and the like. The bacillus coagulans can pass through the strong acid environment of gastric juice without being damaged after being sporulated, then smoothly reaches the intestinal tract, produces acid in the intestinal tract, and reduces the pH value of the intestinal tract, so that harmful bacteria in the intestinal tract are killed, the proliferation of probiotics such as bifidobacteria in the intestinal tract can be promoted, and the micro-ecological environment of the host intestinal tract is improved. The bacillus coagulans can be used for food, health products, livestock and aquatic feeds and the like. Several techniques for producing Bacillus coagulans by fermentation through adjustment of the medium formulation have been developed in the market, including the use of yeast powder, peptone, corn flour, soybean flour, and the like. In addition, only one temperature (40 ℃) fermentation is generally used in the market to obtain spore-forming Bacillus coagulans.
In the prior art, yeast powder, peptone, corn flour, soybean flour and the like are used as culture medium components, so that the taste is seriously influenced, and the prior art is not suitable for beverage production. Because the culture medium is lack of oligosaccharide and rare sugar, the survival period of the bacillus coagulans cannot be prolonged, so that the thallus loses activity quickly during short-term storage. The method for obtaining the bacillus coagulans capable of producing spores by adopting fermentation at a temperature (40 ℃) has the spore production rate of about 80 percent generally, is not very high, so that the fermented bacillus cannot resist the strong acid environment of gastric juice and cannot smoothly reach the small intestine, and the life cycle of the bacillus coagulans is reduced due to low spore rate.
Disclosure of Invention
The invention aims to overcome the defects in the prior art, and provides a bacillus coagulans culture medium and a fermentation method thereof, which aim to solve the problems of poor taste, low spore yield and short life span when a common culture medium is used for fermentation.
In order to solve the technical problems, the invention adopts the technical scheme that: a culture medium of bacillus coagulans is characterized by comprising 5-10g/L glucose, 10-30% (v/v) tomato juice, 10-30% (v/v) carrot juice, 5-10g/L fructo-oligosaccharide, 10-20g/L isomalto-oligosaccharide, 5-10g/L galacto-oligosaccharide and 5-10g/L LD-tagatose.
Further, in the culture medium of the bacillus coagulans, the content of tomato juice is the same as the content of carrot juice; the culture medium of the bacillus coagulans comprises a seed culture medium and a fermentation culture medium, and the seed culture medium and the fermentation culture medium have the same composition.
Further, the culture medium of the bacillus coagulans comprises 5g/L glucose, 15% (v/v) tomato juice, 15% (v/v) carrot juice, 10g/L fructo-oligosaccharide, 20g/L isomalto-oligosaccharide, 10g/L galacto-oligosaccharide and 5 g/LD-tagatose.
Further, a fermentation method of bacillus coagulans is characterized by comprising the following steps:
s1, preparing a seed culture medium and a fermentation culture medium, wherein the seed culture medium and the fermentation culture medium comprise 5-10g/L glucose, 10-30% (v/v) tomato juice, 10-30% (v/v) carrot juice, 5-10g/L fructo-oligosaccharide, 10-20g/L isomalto-oligosaccharide, 5-10g/L galacto-oligosaccharide and 5-10g/L LD-tagatose;
s2, culturing: sequentially inoculating bacillus coagulans into a seed culture medium and a fermentation culture medium for fermentation;
and S3, calculating the spore rate by microscopic examination after the fermentation is finished, and measuring the viable bacteria concentration after the storage for 3 months at 4 ℃.
Further, the bacillus coagulans is inoculated into the seed culture medium in the S2 under the culture conditions that: the inoculation amount of the bacillus coagulans is 2-6% (v/v), the culture temperature is 45-50 ℃, the initial culture pH value is 6.6-7.0, the rotation speed of the seed culture medium is 180-250r/min, and the culture time is 6-12 h.
Further, in the step S2, Bacillus coagulans is inoculated into a fermentation medium for fermentation culture, wherein the fermentation culture comprises early-stage high-temperature fermentation and later-stage low-temperature fermentation;
early-stage high-temperature fermentation: inoculating the bacillus coagulans cultured in the seed culture medium into a fermentation culture medium, wherein the inoculation amount is 2-6% (v/v), the initial pH value is set to be 6.6-7.0, the temperature is set to be 45-50 ℃, the preferred temperature is 47 ℃, and oxygen is fully supplied for fermentation for 36-48 h;
and (3) low-temperature fermentation at the later stage: setting pH value at 6.6-7.0, temperature at 37-42 deg.C, preferably 37 deg.C, and fermenting for 8-12 h.
Compared with the prior art, the invention has the beneficial effects that: glucose, tomato juice, carrot juice, fructo-oligosaccharide, isomaltooligosaccharide, galacto-oligosaccharide and D-tagatose are used as culture substrates, so that the problems of poor taste, low spore yield and short life span when a common culture medium is used for fermentation are solved. Tomato juice, carrot juice, fructo-oligosaccharide, isomaltooligosaccharide, galacto-oligosaccharide and D-tagatose can make the fermented taste better. Fructo-oligosaccharide, isomaltooligosaccharide, galacto-oligosaccharide and D-tagatose have the effect of prolonging the life of the bacillus coagulans. In addition, the temperature change of the fermentation method adopting the two-temperature fermentation method (namely 45-50 ℃ and 37-42 ℃) improves the spore yield and prolongs the life cycle of the bacillus coagulans.
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The disclosure of the present invention is illustrated with reference to the accompanying drawings. It is to be understood that the drawings are designed solely for the purposes of illustration and not as a definition of the limits of the invention. In the drawings, like reference numerals are used to refer to like parts. Wherein:
FIG. 1 is a process diagram of a fermentation method of Bacillus coagulans.
FIG. 2 is a comparison graph of spore yield of the present invention compared with the prior art.
In FIG. 2, A is Bacillus coagulans cultured by the prior art one-temperature fermentation method (40 ℃); b is Bacillus coagulans cultured by the two-temperature fermentation method (47 ℃ and 37 ℃) of the invention, and light-colored parts indicated by arrows are spores.
Detailed Description
It is easily understood that according to the technical solution of the present invention, a person skilled in the art can propose various alternative structures and implementation ways without changing the spirit of the present invention. Therefore, the following detailed description and the accompanying drawings are merely illustrative of the technical aspects of the present invention, and should not be construed as all of the present invention or as limitations or limitations on the technical aspects of the present invention.
An embodiment according to the present invention is shown in connection with fig. 1. A fermentation method of bacillus coagulans is characterized by comprising the following steps:
s1, preparing a seed culture medium and a fermentation culture medium, wherein the seed culture medium and the fermentation culture medium comprise 5-10g/L glucose, 10-30% vol tomato juice, 10-30% vol carrot juice, 5-10g/L fructo-oligosaccharide, 10-20g/L isomalto-oligosaccharide, 5-10g/L galacto-oligosaccharide and 5-10g/L LD-tagatose;
s2, culturing: sequentially inoculating bacillus coagulans into a seed culture medium and a fermentation culture medium for fermentation; the inoculation amount of the bacillus coagulans is 2-6% vol, the culture temperature is 45-50 ℃, the initial culture pH value is 6.6-7.0, the rotation speed of a seed culture medium is 180-250r/min, and the culture time is 6-12 h; the fermentation culture comprises early-stage high-temperature fermentation and later-stage low-temperature fermentation; early-stage high-temperature fermentation: inoculating the bacillus coagulans cultured in the seed culture medium into a fermentation culture medium, wherein the inoculation amount is 2-6% vol, the initial pH value is set to be 6.6-7.0, the temperature is set to be 45-50 ℃, and oxygen is fully supplied for fermentation for 36-48 h; and (3) low-temperature fermentation at the later stage: setting pH value at 6.6-7.0 and temperature at 37-42 deg.C, and fermenting for 8-12 h.
And S3, calculating the spore rate by microscopic examination after the fermentation is finished, and measuring the viable bacteria concentration after the storage for 3 months at 4 ℃.
The culture medium of the bacillus coagulans is characterized by comprising 5-10g/L glucose, 10-30% vol tomato juice, 10-30% vol carrot juice, 5-10g/L fructo-oligosaccharide, 10-20g/L isomalto-oligosaccharide, 5-10g/L galacto-oligosaccharide and 5-10g/L LD-tagatose. In the culture medium of the bacillus coagulans, the content of tomato juice is the same as the content of carrot juice; the culture medium of the bacillus coagulans comprises a seed culture medium and a fermentation culture medium, and the seed culture medium and the fermentation culture medium have the same composition.
The following examples and comparative examples are specifically described.
Example 1: a bacillus coagulans culture medium and a fermentation method thereof are disclosed:
(1) preparing a seed culture medium: the formula is as follows:
5g/L glucose, 25% (v/v) tomato juice, 25% (v/v) carrot juice, 5g/L fructo-oligosaccharide, 10g/L isomalto-oligosaccharide, 5g/L galacto-oligosaccharide and 5g/L D-tagatose;
(2) seed culture:
the culture conditions are as follows: the inoculation amount is 2 percent (v/v), the temperature is 47 ℃, the initial pH value is 6.6, the liquid loading amount is 200ml (1000ml triangular flask), the rotating speed is 180r/min, and the fermentation time is 12 h;
(3) preparing a fermentation medium: the formula is as follows:
5g/L glucose, 25% (v/v) tomato juice, 25% (v/v) carrot juice, 5g/L fructo-oligosaccharide, 10g/L isomalto-oligosaccharide, 5g/L galacto-oligosaccharide and 5g/L D-tagatose;
(4) and (3) sterilization: mixing the fermentation culture medium in 50L fermentation tank, and sterilizing at 115 deg.C for 20 min;
(5) inoculation: after the fermentation medium is cooled to 47 ℃, 1L of seed liquid is inoculated into a 50L fermentation tank;
(6) fermentation culture:
early-stage high-temperature fermentation: the inoculation amount is 2 percent (v/v), oxygen is fully supplied, the temperature is 47 ℃, the initial pH value is 7.0, and the fermentation time is 48 hours;
and (3) low-temperature fermentation at the later stage: supplying oxygen sufficiently, at 40 deg.C, pH 6.6, and fermenting for 12 hr;
adding glucose 2 times during fermentation, and controlling the final concentration not higher than 20% (v/v);
(7) calculating the spore yield by microscopic examination after the fermentation is finished;
(8) viable bacteria concentration was measured after 3 months of storage at 4 ℃.
Example 2: a bacillus coagulans culture medium and a fermentation method thereof are disclosed:
(1) preparing a seed culture medium: the formula is as follows:
5g/L glucose, 15% (v/v) tomato juice, 15% (v/v) carrot juice, 10g/L fructo-oligosaccharide, 20g/L isomalto-oligosaccharide, 10g/L galacto-oligosaccharide and 5g/L D-tagatose;
(2) seed culture:
the culture conditions are as follows: the inoculation amount is 5% (v/v), the temperature is 45 ℃, the initial pH value is 7.0, the liquid loading amount is 200ml (1000ml triangular flask), the rotating speed is 220r/min, and the fermentation time is 8 h;
(3) preparing a fermentation medium: the formula is as follows:
5g/L glucose, 15% (v/v) tomato juice, 15% (v/v) carrot juice, 10g/L fructo-oligosaccharide, 20g/L isomalto-oligosaccharide, 10g/L galacto-oligosaccharide and 5g/L D-tagatose;
(4) and (3) sterilization: mixing the fermentation culture medium in 50L fermentation tank, and sterilizing at 115 deg.C for 20 min;
(5) inoculation: after the fermentation medium is cooled to 47 ℃, inoculating 2.5L of seed liquid into a 50L fermentation tank;
(6) fermentation culture:
early-stage high-temperature fermentation: the inoculation amount is 5% (v/v), oxygen is fully supplied, the temperature is 47 ℃, the initial pH value is 7.0, and the fermentation time is 36 h;
and (3) low-temperature fermentation at the later stage: supplying oxygen sufficiently, wherein the temperature is 37 ℃, the pH value is 7.0, and the fermentation time is 12 h;
adding glucose 2 times during fermentation, and controlling the final concentration not higher than 20% (v/v);
(9) calculating the spore yield by microscopic examination after the fermentation is finished;
(10) viable bacteria concentration was measured after 3 months of storage at 4 ℃.
Example 3: a bacillus coagulans culture medium and a fermentation method thereof are disclosed:
(1) preparing a seed culture medium: the formula is as follows:
5g/L glucose, 25% (v/v) tomato juice, 25% (v/v) carrot juice, 5g/L fructo-oligosaccharide, 10g/L isomalto-oligosaccharide, 5g/L galacto-oligosaccharide and 10g/L D-tagatose;
(2) seed culture:
the culture conditions are as follows: the inoculation amount is 6% (v/v), the temperature is 47 ℃, the initial pH value is 7, the liquid loading amount is 200ml (1000ml triangular flask), the rotating speed is 250r/min, and the fermentation time is 6 h;
(3) preparing a fermentation medium: the formula is as follows:
5g/L glucose, 25% (v/v) tomato juice, 25% (v/v) carrot juice, 5g/L fructo-oligosaccharide, 10g/L isomalto-oligosaccharide, 5g/L galacto-oligosaccharide and 10g/L D-tagatose;
(4) and (3) sterilization: mixing the fermentation culture medium in 50L fermentation tank, and sterilizing at 115 deg.C for 20 min;
(5) inoculation: after the fermentation medium is cooled to 45 ℃, inoculating 3L of seed liquid into a 50L fermentation tank;
(6) fermentation culture:
early-stage fermentation: the inoculation amount is 6% (v/v), oxygen is fully supplied, the temperature is 45 ℃, the initial pH value is 7.0, and the fermentation time is 36 h;
and (3) later-stage fermentation: supplying oxygen sufficiently, at 40 deg.C, pH 7.0, and fermenting for 8 hr;
adding glucose 2 times during fermentation, and controlling the final concentration not higher than 20% (v/v);
(11) calculating the spore yield by microscopic examination after the fermentation is finished;
(12) viable bacteria concentration was measured after 3 months of storage at 4 ℃.
The comparative experiment was carried out using conventional methods:
(1) preparing a seed culture medium: the formula is as follows:
20g/L of glucose, 10g/L of yeast powder and 20g/L of peptone;
(2) seed culture:
the culture conditions are as follows: the inoculation amount is 1% (v/v), the temperature is 40 ℃, the initial pH value is 7, the liquid loading amount is 200ml (1000ml triangular flask), the rotating speed is 220r/min, and the fermentation time is 24 h;
(3) preparing a fermentation medium: the formula is as follows:
20g/L of glucose, 10g/L of yeast powder and 20g/L of peptone;
(4) and (3) sterilization: mixing the fermentation culture medium in 50L fermentation tank, and sterilizing at 115 deg.C for 20 min;
(5) inoculation: after the fermentation medium is cooled to 40 ℃, 0.5L of seed liquid is inoculated into a 50L fermentation tank;
(6) fermentation culture:
the inoculation amount is 1% (v/v), oxygen is fully supplied, the temperature is 40 ℃, the initial pH value is 7.0, and the fermentation time is 48 h;
adding glucose 2 times during fermentation, and controlling the final concentration not higher than 10% (v/v);
(7) calculating the spore yield by microscopic examination after the fermentation is finished;
(8) viable bacteria concentration was measured after 3 months of storage at 4 ℃.
And (3) evaluating the spore yield result:
| group of | Ratio of spore to bud |
| Example 1 | 88％ |
| Example 2 | 95％ |
| Example 3 | 90％ |
| Comparative experiment | 82％ |
Experimental results show that the process can greatly improve the spore yield of the bacillus coagulans and has good taste.
Evaluation of viable count results after 3 months of storage at 4 ℃:
| group of | Viable count after 3 months |
| Example 1 | 3X107 |
| Example 2 | 8X107 |
| Example 3 | 5X107 |
| Comparative experiment | 7X105 |
Experimental results show that the technology can greatly improve the viable count after being stored for 3 months at 4 ℃. The technical scope of the present invention is not limited to the above description, and those skilled in the art can make various changes and modifications to the above-described embodiments without departing from the technical spirit of the present invention, and such changes and modifications should fall within the protective scope of the present invention.
Claims (6)
1. A culture medium for bacillus coagulans is characterized by comprising 5-10g/L glucose, 10-30% vol tomato juice, 10-30% vol carrot juice, 5-10g/L fructo-oligosaccharide, 10-20g/L isomalto-oligosaccharide, 5-10g/L galacto-oligosaccharide and 5-10g/L LD-tagatose.
2. The bacillus coagulans culture medium according to claim 1, wherein the bacillus coagulans culture medium contains the same amount of tomato juice as carrot juice; the culture medium of the bacillus coagulans comprises a seed culture medium and a fermentation culture medium, and the seed culture medium and the fermentation culture medium have the same composition.
3. The culture medium of bacillus coagulans according to claim 1, wherein the culture medium of bacillus coagulans comprises 5g/L glucose, 15% vol tomato juice, 15% vol carrot juice, 10g/L fructo-oligosaccharide, 20g/L isomalto-oligosaccharide, 10g/L galacto-oligosaccharide and 5 g/LD-tagatose.
4. A method for fermenting Bacillus coagulans based on the Bacillus coagulans culture medium as set forth in any one of claims 1 to 3, comprising the steps of:
s1, preparing a seed culture medium and a fermentation culture medium, wherein the seed culture medium and the fermentation culture medium comprise 5-10g/L glucose, 10-30% vol tomato juice, 10-30% vol carrot juice, 5-10g/L fructo-oligosaccharide, 10-20g/L isomalto-oligosaccharide, 5-10g/L galacto-oligosaccharide and 5-10g/L LD-tagatose;
s2, culturing: sequentially inoculating bacillus coagulans into a seed culture medium and a fermentation culture medium for fermentation;
and S3, calculating the spore rate by microscopic examination after the fermentation is finished, and measuring the viable bacteria concentration after the storage for 3 months at 4 ℃.
5. The method for fermenting Bacillus coagulans according to claim 4, wherein the Bacillus coagulans is inoculated into the seed culture medium in S2 under the culture conditions of: the inoculation amount of the bacillus coagulans is 2-6% vol, the culture temperature is 45-50 ℃, the initial culture pH value is 6.6-7.0, the rotation speed of the seed culture medium is 180-250r/min, and the culture time is 6-12 h.
6. The method for fermenting bacillus coagulans according to claim 5, wherein bacillus coagulans is inoculated into a fermentation medium in S2 for fermentation culture, and the fermentation culture comprises early-stage high-temperature fermentation and later-stage low-temperature fermentation;
early-stage high-temperature fermentation: inoculating the bacillus coagulans cultured in the seed culture medium into a fermentation culture medium, wherein the inoculation amount is 2-6% vol, the initial pH value is set to be 6.6-7.0, the temperature is set to be 45-50 ℃, the temperature is optimally 47 ℃, and oxygen is fully supplied for fermentation for 36-48 h;
and (3) low-temperature fermentation at the later stage: setting pH value at 6.6-7.0 and temperature at 37-42 deg.C, optimally at 37 deg.C, and fermenting for 8-12h with oxygen.
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