WO2022083007A1 - Fermentation method for producing high-content bacillus coagulans spore probiotics by means of using cocoa powder and use thereof - Google Patents

Abstract

Provided are a fermentation method for producing high-content Bacillus coagulans spore probiotics by means of using cocoa powder and the use thereof. By means of adding an appropriate amount of cocoa powder to Bacillus coagulans for solid fermentation, spore formation can be effectively accelerated, the germination rate of Bacillus coagulans is increased, and at the same time, the acid resistance and cholate resistance of the spores of Bacillus coagulans are improved. In addition, the content of flavonoid aglycones in the cocoa powder can also be increased in the fermentation process.

Description

A kind of fermentation method using cocoa powder to produce high-content Bacillus coagulans spore probiotics and its application technical field

The invention belongs to the technical field of microbial fermentation, and in particular relates to a fermentation method for producing high-content Bacillus coagulans spore probiotics by using cocoa powder and its application.

Background technique

The information disclosed in this Background section is only for enhancement of understanding of the general background of the invention and should not necessarily be taken as an acknowledgement or any form of suggestion that this information forms the prior art already known to a person of ordinary skill in the art.

Microecological preparations have the effect of regulating the intestinal dysfunction of animals, maintaining the balance of intestinal flora and improving immunity, and their beneficial effects have been recognized. In addition to the above advantages, Bacillus coagulans probiotics also have the characteristics of lactic acid production, strong stress resistance, high temperature and high pressure resistance, and easy storage. At present, there have been many reports on the liquid fermentation of Bacillus coagulans, and the fermentation level is about 5 × 10 ⁸ cfu/ml. After further processing such as adsorption, drying and pulverization, the number of viable bacteria in the product is lower.

Compared with liquid fermentation, solid fermentation equipment is simple, energy consumption is low, cost is low, and the fermentation level is not high. At present, there are few reports on solid fermentation of Bacillus coagulans, and the tolerance of common Bacillus coagulans is poor. In order to ensure that the microecological preparations are not easily deactivated during long-term storage, thermal stability, acid resistance, bile salt resistance, etc. must be considered during preparation. a series of questions. Rapid and mass production of spores is the key to the preparation of Bacillus coagulans formulations. The inventors found that the yield of liquid fermentation is high, but Bacillus coagulans is not easy to survive, and the solid fermentation period is long. Therefore, in order to better exert the probiotic effect of Bacillus coagulans, it is necessary to optimize its sporulation conditions.

SUMMARY OF THE INVENTION

In order to overcome the above-mentioned technical problems, the present invention provides a fermentation method for producing high-content Bacillus coagulans spore probiotics by using cocoa powder and its application. Fermentation can effectively accelerate the formation of spores and improve the germination rate of Bacillus coagulans. At the same time, the fermentation process can also increase the content of flavonoid aglycones in cocoa powder, further improving the prebiotic effect of the prepared products, so it has good practical application value.

For realizing the above-mentioned technical purpose, the technical scheme adopted in the present invention is as follows:

The first aspect of the present invention provides the use of cocoa powder in the fermentation of Bacillus coagulans.

The fermentation is solid fermentation.

More specifically, the application includes at least:

1) increase the spore-forming content of Bacillus coagulans;

2) Increase the content of flavonoid aglycone in cocoa powder.

The second aspect of the present invention provides a fermentation method for producing high-content Bacillus coagulans spore probiotics by using cocoa powder, the method comprising: inoculating the activated Bacillus coagulans into a liquid medium for expanded culture, waiting for When Bacillus coagulans enters the initial stage of logarithmic growth, the liquid Bacillus coagulans is inoculated into the solid medium, and at the same time, cocoa powder is added to the solid medium for solid fermentation.

The third aspect of the present invention provides the application of the above fermentation method in the preparation of Bacillus coagulans preparations. The Bacillus coagulans preparation can be widely used in food, medicine, health care and animal husbandry and aquaculture industries. At the same time, because it is rich in nutrients such as flavonoid aglycone, its nutritional and health care value is further improved.

The beneficial technical effects of the above one or more technical solutions are:

The above technical scheme provides that solid fermentation is carried out by adding a suitable content of cocoa powder to Bacillus coagulans, which can effectively accelerate the formation of spores, improve the germination rate of Bacillus coagulans, and simultaneously improve the acid resistance and bile salt resistance of the spores; and the fermentation process can also improve. The content of flavonoid aglycone in the cocoa powder further improves the probiotic effect of the prepared product, so that the prepared Bacillus coagulans preparation can be widely used in food, medicine, health care, animal husbandry and aquatic products and other industries, so it has good practical application value.

Detailed ways

It should be noted that the following detailed description is exemplary and intended to provide further explanation of the invention. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

It should be noted that the terminology used herein is for the purpose of describing specific embodiments only, and is not intended to limit the exemplary embodiments according to the present invention. As used herein, unless the context clearly dictates otherwise, the singular is intended to include the plural as well, furthermore, it is to be understood that when the terms "comprising" and/or "including" are used in this specification, it indicates that There are features, steps, operations, devices, components and/or combinations thereof. It should be understood that the protection scope of the present invention is not limited to the following specific specific embodiments; it should also be understood that the terms used in the examples of the present invention are for describing specific specific embodiments, rather than for limiting the protection scope of the present invention.

As mentioned above, liquid fermentation has high yield, but Bacillus coagulans is not easy to survive, and the solid fermentation cycle is long. Therefore, in order to better exert the probiotic effect of Bacillus coagulans, it is necessary to optimize its sporulation conditions.

In view of this, in a specific embodiment of the present invention, the application of cocoa powder in the fermentation of Bacillus coagulans is provided.

The fermentation is solid fermentation.

In another specific embodiment of the present invention, the application at least includes:

1) increase the spore-forming content of Bacillus coagulans;

2) Increase the content of flavonoid aglycone in cocoa powder.

In yet another specific embodiment of the present invention, there is provided a fermentation method for producing high-content Bacillus coagulans spores using cocoa powder, the method comprising: inoculating the activated Bacillus coagulans into a liquid medium for expanded culture, waiting for When Bacillus coagulans enters the initial stage of logarithmic growth, the liquid Bacillus coagulans is inoculated into the solid medium, and at the same time, cocoa powder is added to the solid medium for solid fermentation.

Wherein, in the step of "inoculating the activated Bacillus coagulans into the liquid medium", the inoculation amount is controlled to be 0.5-5% (w/w), preferably 2%.

The liquid medium is composed of the following components by mass percentage: 1.2-1.8% (preferably 1.5%) of glucose, 1.0-1.4% (preferably 1.2%) of peptone, 0.5-1.0% (preferably 0.5%) of yeast powder ), K ₂ HPO ₄ 0.05-0.2% (preferably 0.1%), the balance is water, the pH is weakly alkaline, preferably 7.5-8.0; further, the liquid medium is composed of: glucose 1.5%, Peptone 1.2%, yeast powder 0.5%, K ₂ HPO ₄ 0.1%, the balance is water, and the pH is 7.5.

The liquid fermentation culture conditions include: culturing at 35-45° C. for 16-20 hours, and the rotational speed is controlled at 180-220 rpm; preferably, culturing at 40° C. for 18 hours, and the rotational speed is controlled at 200 rpm; by controlling the liquid medium and the culture conditions, the coagulated spores are Bacilli accumulate nutrients and multiply rapidly, thus entering the logarithmic growth phase faster.

In another specific embodiment of the present invention, when Bacillus coagulans enters the initial stage of logarithmic growth, the amount of spores in the fermentation broth can reach 6×10 ⁸ cfu/ml.

In another specific embodiment of the present invention, the solid medium includes a medium substrate and additives, wherein the medium substrate is a mixture of wheat bran and bran powder, and the additives include carbon sources, nitrogen sources and salts .

In another specific embodiment of the present invention, in the mixture of wheat bran and bran powder, the mass ratio of wheat bran and bran powder is 1-3:1, preferably 1:1.

The carbon source is molasses, the nitrogen source is peptone, and the salt is K ₂ HPO ₄ .

In another specific embodiment of the present invention, the added amount of the carbon source is 0.1-0.3% (w/w, preferably 0.2%) of the medium substrate, and the added amount of the nitrogen source is 0.1-0.3% of the medium substrate % (w/w, preferably 0.2%), the added amount of the salts is 0.1-0.3% (w/w, preferably 0.2%) of the medium substrate.

In another specific embodiment of the present invention, the wheat bran, bran powder and cocoa powder are sieved and processed, and the sieve mesh number is 40-50 mesh, preferably 40 mesh. By further refining the particle size, on the one hand, nutrients are provided for the growth and reproduction of Bacillus coagulans, and on the other hand, the adhesion of Bacillus coagulans is facilitated.

In another specific embodiment of the present invention, the specific method of the solid fermentation is as follows: inoculate the above-mentioned Bacillus coagulans liquid bacterial liquid according to 10-30% (preferably 20%) of the quality of the solid medium, and simultaneously press 10% to 30% (preferably 20%) of the quality of the solid medium ~10% (preferably 3%) of cocoa powder is added, the water content is controlled to be 30-60% (preferably 50%), and the initial pH is adjusted to be 7.0-7.2.

In another specific embodiment of the present invention, the solid fermentation adopts variable temperature fermentation treatment, preferably, culture at 35-40°C for 10-15 hours, and then continue to culture at 40-45°C for 10-15 hours. Adopting the above-mentioned variable temperature fermentation treatment can promote spore formation on the one hand, and can also improve the yield of flavonoid aglycone on the other hand.

In another specific embodiment of the present invention, the application of the above fermentation method in the preparation of Bacillus coagulans preparation is provided. The Bacillus coagulans preparation can be widely used in food, medicine, health care and animal husbandry and aquaculture industries. At the same time, because it is rich in nutrients such as flavonoid aglycone, its nutritional and health care value is further improved.

The technical solutions of the present invention will be described below through specific embodiments. The raw materials used in the following examples are all commercially available, and the equipment used are all existing equipment.

Example 1

A fermentation method for producing high-content Bacillus coagulans spores by using cocoa powder, the method comprising: inoculating activated Bacillus coagulans into a liquid medium at 2% (w/w) for expanded culture, and waiting for the Bacillus coagulans to be expanded. When entering the initial stage of logarithmic growth, the liquid bacterial liquid of Bacillus coagulans is inoculated into the solid medium. At the same time, cocoa powder is added to the solid medium for solid fermentation.

Wherein, the liquid medium is composed of the following components by mass percentage: 1.5% glucose, 1.2% peptone, 0.5% yeast powder, 0.1% K ₂ HPO ₄ , the balance is water, and the pH is 7.5.

The liquid fermentation culture conditions include: culturing at 40° C. for 18 hours, and the rotational speed is controlled at 200 rpm; at this time, the amount of spores in the fermentation broth can reach 6×10 ⁸ cfu/ml.

The solid medium includes a medium substrate and an additive, wherein the medium substrate is a mixture of wheat bran and bran powder (mass ratio 1:1), and the additive is molasses, peptone and K ₂ HPO ₄ . The added amount of molasses is 0.2% of the medium substrate, the added amount of peptone is 0.2% of the medium substrate, and the added amount of K ₂ HPO ₄ is 0.2% of the medium substrate.

Wheat bran, bran powder and cocoa powder are sieved, and the sieve number is 40 meshes.

The specific method of the solid fermentation is as follows: inoculating the above-mentioned Bacillus coagulans liquid bacterial liquid according to 20% of the quality of the solid medium, adding cocoa powder according to 3% of the quality of the solid medium, controlling the water content to 50%, and adjusting the initial pH to 7.2 .

The solid fermentation is treated by variable temperature fermentation, specifically: culturing at 40° C. for 12 hours, and then continuing to culture at 45° C. for 12 hours.

Example 2

A fermentation method for producing high-content Bacillus coagulans spores by using cocoa powder, the method comprising: inoculating activated Bacillus coagulans into a liquid medium at 2% (w/w) for expanded culture, and waiting for the Bacillus coagulans to be expanded. When entering the initial stage of logarithmic growth, the liquid bacterial liquid of Bacillus coagulans is inoculated into the solid medium. At the same time, cocoa powder is added to the solid medium for solid fermentation.

Wherein, the liquid medium is composed of the following components by mass percentage: 1.5% glucose, 1.2% peptone, 0.5% yeast powder, 0.1% K ₂ HPO ₄ , the balance is water, and the pH is 7.5.

The liquid fermentation culture conditions include: culturing at 38° C. for 20 hours, and the rotational speed is controlled at 180 rpm; at this time, the amount of spores in the fermentation broth can reach 6×10 ⁸ cfu/ml.

The solid medium includes a medium substrate and an additive, wherein the medium substrate is a mixture of wheat bran and bran powder (mass ratio 2:1), and the additive is molasses, peptone and K ₂ HPO ₄ . The added amount of molasses is 0.2% of the medium substrate, the added amount of peptone is 0.2% of the medium substrate, and the added amount of K ₂ HPO ₄ is 0.1% of the medium substrate.

Wheat bran, bran powder and cocoa powder are sieved, and the sieve number is 40 meshes.

The specific method of the solid fermentation is as follows: inoculating the above-mentioned Bacillus coagulans liquid bacterial liquid according to 20% of the quality of the solid medium, adding cocoa powder according to 3% of the quality of the solid medium, controlling the water content to 40%, and adjusting the initial pH to 7.2 .

The solid fermentation is treated by variable temperature fermentation, specifically: culturing at 38° C. for 15 hours, and then continuing to culture at 43° C. for 12 hours.

Example 3

A fermentation method for producing high-content Bacillus coagulans spores by using cocoa powder, the method comprising: inoculating activated Bacillus coagulans into a liquid medium at 2% (w/w) for expanded culture, and waiting for the Bacillus coagulans to be expanded. When entering the initial stage of logarithmic growth, the liquid bacterial liquid of Bacillus coagulans is inoculated into the solid medium. At the same time, cocoa powder is added to the solid medium for solid fermentation.

Wherein, the liquid medium is composed of the following components by mass percentage: 1.5% glucose, 1.2% peptone, 0.5% yeast powder, 0.1% K ₂ HPO ₄ , the balance is water, and the pH is 7.5.

The liquid fermentation culture conditions include: culturing at 40° C. for 18 hours, and the rotational speed is controlled at 200 rpm; at this time, the amount of spores in the fermentation broth can reach 6×10 ⁸ cfu/ml.

The solid medium includes a medium substrate and an additive, wherein the medium substrate is a mixture of wheat bran and bran powder (mass ratio 1:1), and the additive is molasses, peptone and K ₂ HPO ₄ . The added amount of molasses is 0.2% of the medium substrate, the added amount of peptone is 0.2% of the medium substrate, and the added amount of K ₂ HPO ₄ is 0.2% of the medium substrate.

Wheat bran, bran powder and cocoa powder are sieved, and the sieve number is 40 meshes.

The specific method of the solid fermentation is as follows: inoculating the above-mentioned Bacillus coagulans liquid bacterial liquid according to 30% of the quality of the solid medium, adding cocoa powder according to 5% of the quality of the solid medium, controlling the water content to 50%, and adjusting the initial pH to 7.2 .

The solid fermentation is treated by variable temperature fermentation, specifically: culturing at 40°C for 15 hours, and then continuing to culture at 45°C for 10 hours.

Experimental example 1

A fermentation method for producing high-content Bacillus coagulans spores by using cocoa powder, the method comprising: inoculating activated Bacillus coagulans into a liquid medium at 2% (w/w) for expanded culture, and waiting for the Bacillus coagulans to be expanded. When entering the initial stage of logarithmic growth, the liquid bacterial liquid of Bacillus coagulans is inoculated into the solid medium. At the same time, cocoa powder is added to the solid medium for solid fermentation.

Wherein, the liquid medium is composed of the following components by mass percentage: 1.5% glucose, 1.2% peptone, 0.5% yeast powder, 0.1% K ₂ HPO ₄ , the balance is water, and the pH is 7.5.

The liquid fermentation culture conditions include: culturing at 40° C. for 18 hours, and the rotational speed is controlled at 200 rpm; at this time, the amount of spores in the fermentation broth can reach 6×10 ⁸ cfu/ml.

The solid medium includes a medium substrate and an additive, wherein the medium substrate is a mixture of wheat bran and bran powder (mass ratio 1:1), and the additive is molasses, peptone and K ₂ HPO ₄ . The added amount of molasses is 0.2% of the medium substrate, the added amount of peptone is 0.2% of the medium substrate, and the added amount of K ₂ HPO ₄ is 0.2% of the medium substrate.

Wheat bran, bran powder and cocoa powder are sieved, and the sieve number is 40 meshes.

The specific method of the solid fermentation is as follows: inoculating the above-mentioned Bacillus coagulans liquid bacterial liquid according to 20% of the quality of the solid medium, adding cocoa powder according to 3% of the quality of the solid medium, controlling the water content to 50%, and adjusting the initial pH to 7.2 .

The solid fermentation was cultured at 45°C for 24h.

Experimental example 2

A fermentation method for producing high-content Bacillus coagulans spores by using cocoa powder, the method comprising: inoculating activated Bacillus coagulans into a liquid medium at 2% (w/w) for expanded culture, and waiting for the Bacillus coagulans to be expanded. When entering the initial stage of logarithmic growth, the liquid bacterial liquid of Bacillus coagulans is inoculated into the solid medium. At the same time, cocoa powder is added to the solid medium for solid fermentation.

Wherein, the liquid medium is composed of the following components by mass percentage: 1.5% glucose, 1.2% peptone, 0.5% yeast powder, 0.1% K ₂ HPO ₄ , the balance is water, and the pH is 7.5.

The liquid fermentation culture conditions include: culturing at 40°C for 18 hours, and the rotational speed is controlled at 200 rpm; at this time, the amount of spores in the fermentation broth can reach 6×10 ⁸ cfu/ml.

The solid medium includes a medium substrate and an additive, wherein the medium substrate is wheat bran, and the additive is molasses, peptone and K ₂ HPO ₄ . The added amount of molasses is 0.2% of the medium substrate, the added amount of peptone is 0.2% of the medium substrate, and the added amount of K ₂ HPO ₄ is 0.2% of the medium substrate.

The wheat bran and cocoa powder are sieved, and the sieve number is 40 meshes.

The specific method of the solid fermentation is as follows: inoculating the above-mentioned Bacillus coagulans liquid bacterial liquid according to 20% of the quality of the solid medium, adding cocoa powder according to 3% of the quality of the solid medium, controlling the water content to 50%, and adjusting the initial pH to 7.2 .

The solid fermentation is treated by variable temperature fermentation, specifically: culturing at 40° C. for 12 hours, and then continuing to culture at 45° C. for 12 hours.

Experimental example 3

A fermentation method for producing high-content Bacillus coagulans spores by using cocoa powder, the method comprising: inoculating activated Bacillus coagulans into a liquid medium at 2% (w/w) for expanded culture, and waiting for the Bacillus coagulans to be expanded. When entering the initial stage of logarithmic growth, the liquid bacterial liquid of Bacillus coagulans is inoculated into the solid medium. At the same time, cocoa powder is added to the solid medium for solid fermentation.

Wherein, the liquid medium is composed of the following components by mass percentage: 1.5% glucose, 1.2% peptone, 0.5% yeast powder, 0.1% K ₂ HPO ₄ , the balance is water, and the pH is 7.5.

The liquid fermentation culture conditions include: culturing at 40° C. for 18 hours, and the rotational speed is controlled at 200 rpm; at this time, the amount of spores in the fermentation broth can reach 6×10 ⁸ cfu/ml.

The solid medium includes a medium substrate and an additive, wherein the medium substrate is a mixture of wheat bran and bran powder (mass ratio 1:1), and the additive is molasses, peptone and K ₂ HPO ₄ . The added amount of molasses is 0.2% of the medium substrate, the added amount of peptone is 0.2% of the medium substrate, and the added amount of K ₂ HPO ₄ is 0.2% of the medium substrate.

The wheat bran, bran powder and cocoa powder are sieved, and the sieve number is 60 meshes.

The specific method of the solid fermentation is as follows: inoculating the above-mentioned Bacillus coagulans liquid bacterial liquid according to 20% of the quality of the solid medium, adding cocoa powder according to 3% of the quality of the solid medium, controlling the water content to 50%, and adjusting the initial pH to 7.2 .

The solid fermentation is treated by variable temperature fermentation, specifically: culturing at 40° C. for 12 hours, and then continuing to culture at 45° C. for 12 hours.

Experimental example 4

A fermentation method for producing Bacillus coagulans spores, the method comprising: inoculating the activated Bacillus coagulans into a liquid medium at 2% (w/w) for expanded culture, and waiting for the Bacillus coagulans to enter the initial stage of logarithmic growth At the same time, the Bacillus coagulans liquid bacterial liquid was inoculated into the solid medium for solid fermentation.

Wherein, the liquid medium is composed of the following components by mass percentage: 1.5% glucose, 1.2% peptone, 0.5% yeast powder, 0.1% K ₂ HPO ₄ , the balance is water, and the pH is 7.5.

The liquid fermentation culture conditions include: culturing at 40° C. for 18 hours, and the rotational speed is controlled at 200 rpm; at this time, the amount of spores in the fermentation broth can reach 6×10 ⁸ cfu/ml.

The solid medium includes a medium substrate and an additive, wherein the medium substrate is a mixture of wheat bran and bran powder (mass ratio 1:1), and the additive is molasses, peptone and K ₂ HPO ₄ . The added amount of molasses is 0.2% of the medium substrate, the added amount of peptone is 0.2% of the medium substrate, and the added amount of K ₂ HPO ₄ is 0.2% of the medium substrate.

The wheat bran and bran powder are sieved and processed, and the sieve mesh number is 40 meshes.

The specific method of the solid fermentation is as follows: inoculate the above-mentioned Bacillus coagulans liquid bacterial liquid according to 20% of the quality of the solid medium, control the water content to 50%, and adjust the initial pH to 7.2.

The solid fermentation is treated by variable temperature fermentation, specifically: culturing at 40° C. for 12 hours, and then continuing to culture at 45° C. for 12 hours.

Effect verification

1. Determination of viable bacterial count and spore rate in fermentation products

Fermentation was carried out using the methods described in Example 1 and Experimental Examples 1-3 of the present invention. After fermentation, the content of viable bacteria in the product was determined by the dilution plate counting method. After crystal violet staining, the spore rate was observed by oil microscope, and quercetin was used as the flavonoid. The aglycone represents the component, and the quercetin content was determined by HPLC, and the determination results are shown in Table 1 below.

Table 1

sample	Viable count (cfu/ml)	Spore rate (%)
Example 1	8.2×10 9	98
Experimental example 1	2.5×10 9	91
Experimental example 2	1.8×10 9	89
Experimental example 3	3.4×10 9	86
Experimental example 4	1.9×10 9	80

It can be seen from Table 1 that the fermentation method adopted in Example 1 of the present invention has the highest number of viable bacteria in the final fermentation product, and its spore rate is also significantly higher than that of each experimental example group.

2. Determination of flavonoid aglycone components in fermentation products

Fermentation treatment was carried out using the methods described in Example 1 and Experimental Examples 1-3 of the present invention, and quercetin was used as a representative component of flavonoid aglycone, and the content of quercetin was measured by HPLC. The measurement results are shown in Table 2 below.

Table 2

sample	Quercetin content (mg/L)
Example 1	2.25
Experimental example 1	1.02

Experimental example 2	0.82
Experimental example 3	1.36
Experimental example 4	-

It can be seen from Table 2 that the quercetin content in Example 1 is much higher than other experimental examples, indicating that the fermentation method of the present application can effectively improve the flavonoid aglycone content in the cocoa powder.

3. Effects of bile salts and pH on Bacillus coagulans spores obtained by fermentation

Since Bacillus coagulans is used as a probiotic, it needs to enter the gastrointestinal tract environment of animals (such as humans), and the gastric acid pH of the general human body is 2.0-3.0, and it is a bile salt environment, so the spores of Bacillus coagulans in the gastrointestinal tract Survival and proliferation must adapt to strong acid and bile salt environments. Therefore, the 2h survival rate of Bacillus coagulans spores in strong acid environment and bile salt environment in Example 1 and Experimental Examples 1-4 was investigated, and the experimental results are shown in Table 3 and Table 4.

table 3

sample	pH 2.0
Example 1	56.72%
Experimental example 1	50.35%
Experimental example 2	51.89%
Experimental example 3	53.56%
Experimental example 4	50.78%

Table 4

sample	0.3% (bile salt concentration)
Example 1	98.56%
Experimental example 1	96.78%
Experimental example 2	97.24%
Experimental example 3	98.51%
Experimental example 4	88.26%

As can be seen from Table 3 and Table 4, the fermentation method of the present application can effectively improve the Bacillus coagulans spores obtained by fermentation have good adaptability to strong acid environment and bile salt environment, thereby helping it to play a role when used as probiotics. Probiotic properties.

Finally, it should be noted that the above are only preferred embodiments of the present invention and are not intended to limit the present invention. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art will still Modifications may be made to the technical solutions described in the foregoing embodiments, or equivalent replacements may be made to some of them. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included within the protection scope of the present invention. Although the specific embodiments of the present invention are described above, they do not limit the protection scope of the present invention. Those skilled in the art should understand that on the basis of the technical solutions of the present invention, those skilled in the art can make Various modifications or deformations made are still within the protection scope of the present invention.

Claims (10)

1. The use of cocoa powder in the fermentation of Bacillus coagulans.
2. The application according to claim 1, wherein the fermentation is solid fermentation.
3. The application of claim 1, wherein the application at least includes:

   1) increase the spore-forming content of Bacillus coagulans;

   2) Increase the content of flavonoid aglycone in cocoa powder.
4. A fermentation method for producing high-content Bacillus coagulans spore probiotics by using cocoa powder, characterized in that the method comprises: inoculating the activated Bacillus coagulans into a liquid medium for expanded culture, and waiting for the Bacillus coagulans to enter the In the early stage of growth, the liquid bacterial liquid of Bacillus coagulans is inoculated into the solid medium at the same time, and cocoa powder is simultaneously added to the solid medium for solid fermentation.
5. The fermentation method according to claim 4, characterized in that, in the step of "inoculating the activated Bacillus coagulans into the liquid medium", the inoculation amount is controlled to be 0.5-5% (w/w), preferably 2%.
6. The fermentation method according to claim 4, wherein the liquid medium is composed of the following components by mass percentage: 1.2-1.8% (preferably 1.5%) of glucose, 1.0-1.4% (preferably 1.2%) of peptone ), yeast powder 0.5-1.0% (preferably 0.5%), K2HPO4 0.05-0.2% (preferably 0.1%), the balance is water, and pH is weakly alkaline, preferably 7.5-8.0; further, the said The liquid medium consists of: glucose 1.5%, peptone 1.2%, yeast powder 0.5%, K2HPO4 0.1%, the balance is water, and the pH is 7.5;

   The liquid fermentation culture conditions include: culturing at 35-45° C. for 16-20 hours, and the rotational speed is controlled at 180-220 rpm; preferably, the culture is at 40° C. for 18 hours, and the rotational speed is controlled at 200 rpm.
7. The fermentation method according to claim 4, wherein the solid medium comprises a medium substrate and an additive, wherein the medium substrate is a mixture of wheat bran and bran powder, and the additive includes a carbon source and a nitrogen source and salts;

   Preferably, in the mixture of wheat bran and bran powder, the mass ratio of wheat bran and bran powder is 1-3:1, preferably 1:1;

   Preferably, the carbon source is molasses, the nitrogen source is peptone, and the salt is K2HPO4;

   Preferably, the added amount of the carbon source is 0.1-0.3% (w/w, preferably 0.2%) of the medium substrate, and the added amount of the nitrogen source is 0.1-0.3% (w/w, preferably 0.1%) of the medium substrate 0.2%), the added amount of the salts is 0.1-0.3% (w/w, preferably 0.2%) of the medium substrate;

   Preferably, the wheat bran, bran powder and cocoa powder are sieved, and the sieve mesh number is 40-50 mesh, preferably 40 mesh.
8. The fermentation method according to claim 4, wherein the specific method of the solid fermentation is as follows: inoculate the above-mentioned Bacillus coagulans liquid bacterial liquid by 10-30% (preferably 20%) of the solid medium quality, while pressing the solid medium 1-10% (preferably 3%) of the mass is added to cocoa powder, the water content is controlled to be 30-60% (preferably 50%), and the initial pH is adjusted to 7.0-7.2.
9. The fermentation method according to claim 4, characterized in that the solid fermentation adopts variable temperature fermentation treatment; preferably, it is cultured at 35-40°C for 10-15 hours, and then cultured at 40-45°C for 10-15 hours.
10. Application of the fermentation method described in any one of claims 4-9 in the preparation of a Bacillus coagulans preparation.

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