Production process for producing spores by variable-temperature liquid fermentation of feeding bacillus licheniformis and application of spores

Abstract

The invention provides a production process for producing spores through variable-temperature liquid fermentation of bacillus licheniformis for feeding and application of the production process, a two-stage variable-temperature liquid fermentation process of bacillus licheniformis is adopted, process conditions are optimized, and the fermentation time is shortened by rapidly forming spores, so that the production efficiency is improved, and the production cost is reduced. The cost is greatly reduced, the yield of bacillus licheniformis spores is remarkably increased, popularization and application of bacillus licheniformis in the breeding industry are facilitated, and good economic benefits, social benefits and ecological benefits can be obtained.

Description

Technical Field

[0001] The present invention belongs to the technical field of microbial fermentation, and more particularly, relates to a production process and use of variable-temperature liquid fermentation of Bacillus licheniformis for spore production for feeding purposes. Background Art

[0002] Live microbial agents for feeding can maintain and adjust the microecological balance in the animal intestine, directly or indirectly interfere with, eliminate, and antagonize pathogenic bacteria, thereby improving the disease resistance and animal production level. Due to their lack of drug resistance and residue, and their role in improving animal production performance and preventing diseases, they can overcome some adverse consequences caused by long-term and large-scale use of antibiotics, and are considered potential, natural, and environmentally and health-friendly alternatives to antibiotics. The spores of Bacillus licheniformis can pass through the gastrointestinal environment smoothly without any treatment and effectively act on the animal intestine. Moreover, it is a facultative aerobic microorganism and can grow and reproduce under anaerobic conditions. This unique physiological and biochemical characteristic is considered to be the most ideal live bacterial agent for promoting growth to replace antibiotics, and has broad development and application prospects.

[0003] In the production of Bacillus licheniformis fermentation for spore production, generally soybean meal, corn flour or glucose, inorganic salts, etc. are used. Using these raw materials that are easily available and inexpensive to produce spores, the fermentation cycle is relatively long, and the energy consumption such as ventilation and stirring, and the labor cost in production are correspondingly high. If peptone, yeast extract or corn steep liquor and other relatively expensive substances are used to replace soybean meal and corn in the fermentation, the spore production cycle of the fermentation can be shortened relatively. However, because these substances used as culture media are many times more expensive than ordinary agricultural and sideline products such as soybean meal and corn flour in the market price, using these substances to produce Bacillus licheniformis spores, the cost is acceptable for the pharmaceutical industry, but relatively high for the aquaculture industry as a feed additive. The relatively high production cost is one of the reasons hindering the production and promotion and application of Bacillus licheniformis in the aquaculture industry.

[0004] Chinese Patent Application Publication No. CN110172429 A discloses a culture medium and a culture method for improving the antibacterial activity of biocontrol Bacillus spores against plant pathogenic fungi. Bacillus licheniformis is used for variable-temperature fermentation culture for 36 h. However, the variable-temperature fermentation culture refers to that the fermentation temperature is 32 °C from 0 to 24 h, and the fermentation temperature rises to 37 °C from 25 to 36 h, which is low-temperature fermentation in the first stage and high-temperature fermentation in the second stage.

[0005] The Chinese invention patent with the publication number of CN114921504 B discloses a method for producing microbial oil and carotenoids by variable-temperature fermentation. Using a three-stage variable-temperature fermentation process with a total fermentation time of 120 h. On the one hand, its fermentation time is too long, reducing the fermentation efficiency and increasing the fermentation cost; on the other hand, the technical problem it solves is to increase the oil and carotenoid contents in Rhodosporidium toruloides through the three-stage variable-temperature fermentation process.

[0006] Therefore, in production, there is an urgent need to develop a production process for sporulation of Bacillus licheniformis for feed by variable-temperature liquid fermentation, which can shorten the fermentation time by quickly forming spores, greatly reducing the cost while significantly increasing the yield of Bacillus licheniformis spores, and is beneficial to the promotion and application of Bacillus licheniformis in the aquaculture industry. Summary of the Invention

[0007] To solve the problems existing in the prior art, the present invention provides a production process and its use for sporulation of Bacillus licheniformis for feed by variable-temperature liquid fermentation. It adopts a two-stage variable-temperature liquid fermentation process of Bacillus licheniformis, optimizes the process conditions, shortens the fermentation time by quickly forming spores, greatly reduces the cost while significantly increasing the yield of Bacillus licheniformis spores, is beneficial to the promotion and application of Bacillus licheniformis in the aquaculture industry, and will obtain good economic, social and ecological benefits.

[0008] To achieve the above purpose, the present invention is implemented by the following scheme:

[0009] On the one hand, the present invention provides a production process for sporulation of Bacillus licheniformis for feed by liquid fermentation. The production process includes a two-stage variable-temperature liquid fermentation process of Bacillus licheniformis, wherein the first stage is high-temperature fermentation and the second stage is low-temperature fermentation; the high temperature is above 40 °C, and the low temperature is not more than 32 °C.

[0010] Furthermore, in the two-stage variable-temperature liquid fermentation process, the first stage is high-temperature aerobic fermentation and the second stage is low-temperature anaerobic fermentation.

[0011] In some embodiments, it was unexpectedly found in experiments that high temperature can promote the growth and reproduction of Bacillus licheniformis, significantly shortening the growth period of seeds and the time from the logarithmic growth phase to the stationary phase. When fermented to the stationary phase after the end of the logarithmic growth phase, the number of vegetative bodies basically no longer increases. However, subsequent low-temperature anaerobic fermentation can significantly increase the formation rate of vegetative bodies transforming into spores. The possible reasons are as follows: Under high-temperature and aerobic fermentation conditions, the enzyme activities of various biological enzymes in microorganisms increase, the physiological metabolism of microbial cells accelerates, the biosynthesis of the organism accelerates, and the growth and reproduction of microorganisms are accelerated. Under the subsequent fermentation conditions, the sudden change from high temperature to low temperature and from high dissolved oxygen to anaerobic conditions causes a strong stress response. Such conditions that are not conducive to growth and reproduction can promote the large-scale transformation of Bacillus licheniformis from vegetative bodies into spores, rapidly forming spores, thereby shortening the fermentation time. At the same time, the significant increase in the number of spores also significantly improves the antibacterial activity of the fermentation broth against pathogenic bacteria such as Staphylococcus aureus.

[0012] In some embodiments, the present invention screened the oxygen environment in the front and back stages of the variable-temperature fermentation process. The experimental results showed that when preferably adopting a two-stage variable-temperature fermentation process: high-temperature aerobic fermentation in the front stage and low-temperature anaerobic fermentation in the back stage, the number of spores of Bacillus licheniformis produced was significantly increased. At the same time, the fermentation time was shortened, and the production energy consumption of ventilation and stirring was greatly reduced, which was beneficial to reducing production costs. At the same time, the antibacterial activity of the fermentation broth against Staphylococcus aureus was also significantly improved.

[0013] Further, in the high-temperature aerobic fermentation in the front stage, the following steps are included:

[0014] (1) Inoculate the slant seeds of Bacillus licheniformis into a triangular flask seed culture medium and culture.

[0015] (2) Inoculate the triangular flask seeds into the first-stage seed tank culture medium and culture.

[0016] (3) Inoculate the first-stage seed tank seeds into the culture medium of the main fermentation tank, perform high-temperature aerobic culture for a certain period of time, and then enter the subsequent low-temperature anaerobic fermentation.

[0017] Further, in the high-temperature aerobic fermentation in the front stage, in step (2), the inoculation amount of the first-stage seed tank is 0.1-0.3%, and the culture time is 6-8 h; in step (3), the inoculation amount of the main fermentation tank is 3-5%, and the culture time is 16-20 h.

[0018] In some embodiments, the present invention screened the inoculation amount of the first-stage seed tank in the previous high-temperature aerobic fermentation. The experimental results showed that: when the inoculation amount of the first-stage seed tank in step (2) was preferably 0.3%, the spore yield of Bacillus licheniformis reached the highest at this time, the fermentation time was the shortest, and the antibacterial activity against Staphylococcus aureus could also reach 93%; in terms of production, a larger inoculation amount was beneficial to the improvement of spore yield, but due to process conditions limitations in production, the volume of the Erlenmeyer flask was about 300-500 ml per piece, and the volume was limited. Therefore, the filling amount of the seed liquid was limited to 50-80 ml per piece. Therefore, the inoculation amount was limited by process conditions, and the maximum inoculation amount within the preferred optional range was 0.3%.

[0019] In some embodiments, the present invention screened the culture time in the first-stage seed tank in the previous high-temperature aerobic fermentation. The experimental results showed that: when the culture time in the first-stage seed tank was not less than 6 h, its spore yield decreased slightly with the increase of culture time, but the yield was still extremely high. However, it was found in the experiment that when the culture time reached 6 hours, the bacterial cells were the strongest at this time, and the vitality and quantity of the seeds both reached the highest. At the same time, the antibacterial activity of the fermentation broth against Staphylococcus aureus was significantly enhanced, and the antibacterial rate reached 93%. When the culture time reached 8 h, the vitality and strength of the Bacillus spore seeds both decreased; when the culture time was 5 h or shorter, its spore yield decreased significantly. Therefore, when the culture time was preferably 6 h, the vitality and quantity of the seeds both reached the highest at this time, so as to achieve the highest spore yield, and the bacterial cells were the strongest at this time. At the same time, the antibacterial activity against Staphylococcus aureus was significantly enhanced, and the antibacterial rate could reach 93%.

[0020] In some embodiments, the present invention screened the inoculation amount of the main fermentation tank seeds in the previous high-temperature aerobic fermentation. The experimental results showed that: when the different inoculation amounts in the main fermentation tank decreased during the previous high-temperature aerobic fermentation, the spore yield decreased significantly, the fermentation time increased, and the antibacterial activity of the fermentation broth against Staphylococcus aureus decreased significantly, but compared with the existing fermentation process, there was still a significant improvement; when the inoculation amount in the main fermentation tank increased to 5%, its final spore yield decreased slightly but remained basically unchanged, and the difference from the result under the optimal conditions was not large, but the production cost increased significantly. Therefore, considering the dual factors of ensuring the effect and controlling the cost, when the inoculation amount of the main fermentation tank seeds was preferably 4%, the spore yield of Bacillus licheniformis reached the highest at this time, the fermentation time was the shortest, and the antibacterial activity against Staphylococcus aureus reached the highest.

[0021] In some embodiments, in the previous-stage high-temperature aerobic fermentation of the present invention, the culture time in the main fermentation tank was screened. The experimental results showed that when the culture time in the main fermentation tank did not exceed 20 h, the spore yield increased significantly with the increase of the culture time. At the same time, the antibacterial activity of the fermentation broth against Staphylococcus aureus was significantly improved, and the antibacterial rate reached 95%. However, when the culture time exceeded 20 h, with the increase of the culture time, the spore yield basically did not increase, and the fermentation time increased slightly, while the antibacterial effect of the spores remained basically unchanged. With the increase of the fermentation time, the energy consumption for ventilation and stirring was extremely high. Therefore, considering both cost and effect, the preferred culture time in the main fermentation tank was 20 h.

[0022] Further, in the previous-stage high-temperature aerobic fermentation, the temperature was 45 °C; in the subsequent-stage low-temperature anaerobic fermentation, the temperature was 30 - 32 °C.

[0023] In some embodiments, in the present invention, the optimal temperatures of the high temperature in the previous stage and the low temperature in the subsequent stage in the two-stage variable-temperature fermentation process were screened. The experimental results showed that when the high-temperature fermentation temperature in the previous stage was 45 °C and the low-temperature fermentation temperature in the subsequent stage was 30 - 32 °C in the preferred two-stage variable-temperature fermentation process, the spore yield increased significantly, the fermentation time was significantly shortened, and at the same time, the antibacterial activity of the fermentation broth against Staphylococcus aureus reached the highest.

[0024] Further, in the fermentation of Bacillus licheniformis, the raw material was soybean meal.

[0025] Further, in the fermentation of Bacillus licheniformis, the fermentation raw material was pretreated by enzymatic hydrolysis with neutral protease. By weight, the ratio of soybean meal powder to water was 1:6.

[0026] Further, in the previous-stage high-temperature aerobic fermentation, the culture medium was prepared by 25 - 35 g / L of enzymatically hydrolyzed soybean meal powder (calculated by the amount before enzymatic hydrolysis), 6.0 - 8.0 g / L of disodium hydrogen phosphate, and 4.0 - 8.0 g / L of ammonium chloride, sterilized at 121 °C for 20 min.

[0027] Preferably, the contents of the components of the culture medium were: 25 g / L of soybean meal powder, 8.0 g / L of disodium hydrogen phosphate, and 8.0 g / L of ammonium chloride.

[0028] In some embodiments, the present invention screened the content of the culture medium components in the previous high-temperature aerobic fermentation. The experimental results showed that when the content of soybean meal powder did not exceed 35 g / L, the spore yield increased significantly with the increase of the content of soybean meal powder. At the same time, the fermentation time was continuously shortened, and the antibacterial activity of the fermentation broth against Staphylococcus aureus was significantly increased, with the antibacterial rate reaching 94%. However, when the content of soybean meal powder exceeded 40 g / L, with the increase of the content of soybean meal powder, the spore yield remained basically unchanged, while the fermentation time increased, and the antibacterial activity of the fermentation broth against Staphylococcus aureus also basically did not change. This may be because the content of soybean meal powder has an obvious impact on the growth of Bacillus licheniformis spores and the final antibacterial activity. Within a certain content range, with the increase of the content of soybean meal powder, the spore formation significantly increases, and the antibacterial activity against Staphylococcus aureus also significantly enhances. Therefore, in the present invention, it is preferred that in the previous high-temperature aerobic fermentation step, the content of the culture medium components is 25 - 35 g / L of enzymatically hydrolyzed soybean meal powder (calculated based on the amount before enzymatic hydrolysis), 6.0 - 8.0 g / L of disodium hydrogen phosphate, and 4.0 - 8.0 g / L of ammonium chloride, which is prepared by sterilization at 121°C for 20 min. And it is preferred that the content of each component of the culture medium is 35 g / L of soybean meal powder, 7.0 g / L of disodium hydrogen phosphate, and 4.0 g / L of ammonium chloride.

[0029] On the other hand, the present invention provides the use of a two-stage variable-temperature fermentation process for preparing a preparation for increasing the spore yield of Bacillus licheniformis. In the two-stage variable-temperature fermentation process, the previous stage is high-temperature aerobic fermentation, and the latter stage is low-temperature anaerobic fermentation.

[0030] On yet another aspect, the present invention provides a feed microbial viable preparation, wherein the microorganism is Bacillus licheniformis; and the feed microbial viable preparation is produced by the production process described in any one of the above technical solutions.

[0031] The beneficial effects of the present invention are as follows:

[0032] 1. The two-stage variable-temperature liquid fermentation process of Bacillus licheniformis provided by the present invention optimizes the process conditions. First, it rapidly propagates in large quantities under higher temperature conditions, and then promotes spore formation under lower temperature and anaerobic conditions. By rapidly forming spores, the fermentation time is shortened, the energy consumption of aeration and stirring and the labor cost are greatly reduced. At the same time, the spore yield of Bacillus licheniformis is significantly increased, the production cost per unit product is significantly reduced, the application advantage of the Bacillus licheniformis product of the present invention is improved, which is conducive to the popularization and application of Bacillus licheniformis in the aquaculture industry, and good economic, social and ecological benefits will be obtained.

[0033] 2. The present invention uses soybean meal, a cheap and easily available agricultural and sideline product, as the main raw material. The soybean meal raw material is first enzymatically hydrolyzed to produce a large amount of small peptides and amino acids, which play a role in replacing beef extract and peptone. In this way, the small molecular organic nitrogen source Bacillus licheniformis is easier to use. After being cultured at 45°C, its growth and reproduction speed is accelerated, and the number of spores of Bacillus licheniformis is significantly increased, from the original liquid fermentation spore number of about 4 billion per milliliter to the present invention The number of spores per milliliter is stabilized at more than 5 billion.

[0034] 3. The two-stage variable temperature liquid fermentation process of Bacillus licheniformis provided by the present invention significantly improves the spore yield, and the increase in spore yield significantly improves the antibacterial activity of the fermentation liquid against animal pathogenic bacteria such as Staphylococcus aureus, thereby significantly improving the ability of Bacillus licheniformis to eliminate pathogens in the intestines of animals when used as a live bacterial agent, further improving the disease resistance of the animals themselves, reducing the use of drugs, further improving the digestion and absorption of nutrients by animals, and increasing daily weight gain, which will greatly improve the production level of farmed animals.

[0035] 4. The present invention provides a production process for the production of spores by variable temperature liquid fermentation of Bacillus licheniformis for feed, which provides new ideas and technical support for the processing of feed additives in the breeding industry and has good application prospects. BRIEF DESCRIPTION OF THE DRAWINGS DETAILED DESCRIPTION

[0036] The present invention will be further described in detail below in conjunction with the examples. It should be pointed out that the examples described below are intended to facilitate the understanding of the present invention and do not have any limiting effect on the present invention.

[0037] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0038] Unless otherwise specified, the materials, reagents, equipment, etc. used in the following examples can be obtained from commercial sources.

[0039] The Bacillus licheniformis strain 56374 used in the present invention was introduced by the inventor from the China Industrial Microbiological Culture Collection Center (CICC), and the public can also obtain it through other public channels. Since it does not belong to the innovative content of the present invention, it will not be introduced in detail.

[0040] In the present invention, the number of spores is different from the number of bacteria. The number of bacteria generally refers to the number of vegetative bodies. Under different fermentation culture conditions, not all vegetative bodies can be transformed into spores. The process with the main purpose of forming spores is different from the process and culture medium with other vegetative bodies or antibacterial substances as the purpose.

[0041] In the present invention, except for the culture medium, the fermentation broth contains spores and metabolites of Bacillus licheniformis, and generally, the antibacterial activity mainly refers to the bacteriostatic effect of the metabolites.

[0042] In the actual production of the current fermentation industry, the number of spores of Bacillus licheniformis obtained by conventional fermentation processes is 3 - 4 billion / mL, the triangular flask seed culture takes 12 - 16 h, the fermentation time in the seed tank is 12 - 14 h, and the fermentation time in the fermentation tank is 36 - 48 h.

[0043] Example 1 A production process for variable - temperature liquid fermentation of Bacillus licheniformis for spore production in feed

[0044] The production process provided in this example is the optimal process, and the steps are as follows:

[0045] I. Treatment of the fermentation raw material soybean meal:

[0046] Mix soybean meal powder with water at a weight ratio of 1:6, add it to a heat - preservation storage tank, heat it to 45 - 50 °C, add neutral protease, add neutral protease at a dosage of 1000 international units per kilogram of soybean meal, keep warm for 4 - 6 h, and stir 2 - 3 times during this period for later use.

[0047] II. Front - stage high - temperature aerobic fermentation:

[0048] Inoculate the strain Bacillus licheniformis 56374 into the triangular flask seed culture medium, and culture it on a shaker at 45 °C and 180 - 200 rpm for 7 h to obtain triangular flask seeds; inoculate the triangular flask seeds into the culture medium of the first - stage seed tank at an inoculation amount of 0.3%, and culture it at 45 °C, 180 - 200 rpm, and an aeration rate of 1:0.5 - 0.6 for 6 h to obtain first - stage seed tank seeds; inoculate the first - stage seed tank seeds into the culture medium of the main fermentation tank at an inoculation amount of 4%, and culture it under the same conditions for 20 h, and then immediately enter the next - stage low - temperature fermentation; the culture media of the triangular flask, the first - stage seed tank, and the main fermentation tank are all prepared by adding 35 g / L of enzymatically hydrolyzed soybean meal powder (calculated based on the amount before enzymatic hydrolysis), 7.0 g / L of disodium hydrogen phosphate, and 4.0 g / L of ammonium chloride, and sterilizing at 121 °C for 20 min.

[0049] III. Back - stage low - temperature anaerobic fermentation:

[0050] Cool down the high - temperature fermentation broth in the above - mentioned step II to 30 - 32 °C (since there is a difference of 1 °C in the temperature control of the fermentation tank in production, it is sufficient to control within the said temperature range), and culture it under static conditions at 30 - 32 °C for 10 - 12 h. When the spore rate of Bacillus licheniformis reaches more than 85% by microscopic examination, stop the fermentation; then adsorb and dry or spray - dry the fermentation product into a finished product.

[0051] 4. Calculation of antibacterial rate of fermentation broth:

[0052] Detection method of antibacterial effect:

[0053] 1) Blank group: LB agar plate colony counting method: Staphylococcus aureus (ATCC 25922) cultured in LB liquid medium was diluted to 5×10 3 CFU / mL, take 0.1mL of bacterial solution, wait for the LB agar medium to cool to about 50℃, mix the bacterial solution with 10mL of culture medium, mix well and pour into the plate (do 3 replicates for each treatment). Culture at 37℃ for 24h, record the number of colonies on the culture medium, and record it as Q1;

[0054] 2) Sample group: Centrifuge the fermented Bacillus licheniformis at 10000r / min for 10min, remove the bacterial precipitate, take 1mL of supernatant and 0.1mL of Staphylococcus aureus dilution, and mix the supernatant and Staphylococcus aureus dilution with 9mL of LB agar medium when the LB agar medium cools to about 50℃, mix well and pour on the plate (do 3 replicates for each treatment). Culture at 37℃ for 24h, record the number of colonies on the medium, and record it as Q2;

[0055] 3) Antibacterial rate % = (Q1-Q2) / Q1×100%;

[0056] The specific process conditions and fermentation results are shown in Table 1:

[0057] Table 1 Optimal production process of spore production by variable temperature liquid fermentation of feed Bacillus licheniformis

[0058]

[0059] As can be seen from Table 1, the two-stage variable temperature liquid fermentation process of Bacillus licheniformis is adopted in this embodiment, the process conditions are optimized, the optimal fermentation temperature, culture time and culture medium are selected, and the fermentation time is shortened by rapidly forming spores, so that the cost is greatly reduced while the yield of Bacillus licheniformis spores is significantly increased, which is beneficial to the promotion and application of Bacillus licheniformis in the breeding industry; on the other hand, while the spore fermentation level is improved, the antibacterial activity against pathogenic bacteria such as Staphylococcus aureus is also significantly improved, and the antibacterial rate can reach 95%, thereby significantly improving the ability of Bacillus licheniformis to remove pathogens in the intestines of animals when used as a live bacterial agent, further improving the disease resistance of animals themselves, reducing the use of antibacterial drugs, further improving the digestion and absorption of nutrients by animals, and increasing daily weight gain, which will greatly improve the production level and economic benefits of farmed animals.

[0060] Therefore, it can be seen that the production process provided in this embodiment is the optimal process. Compared with the existing fermentation process, the spore yield of Bacillus licheniformis produced is significantly increased, reaching 6.68 billion / mL. At the same time, compared with the existing process that requires at least 50h or even longer fermentation time, the fermentation time in this embodiment is significantly shortened. Starting from the shake flask seeds, it only takes about 33h, significantly improving the fermentation efficiency (the fermentation time marked in the existing literature usually only refers to the fermentation time in the main fermenter).

[0061] Example 2 A production process for variable-temperature liquid fermentation and spore production of Bacillus licheniformis for feed (different process conditions)

[0062] This embodiment provides production processes under different conditions, and the steps are as follows:

[0063] In this embodiment, the high-temperature aerobic fermentation in the first stage of step two is as follows:

[0064] The Bacillus licheniformis strain 56374 is inoculated into the shake flask seed medium and cultured on a shaker at 45°C and 180 - 200 rpm for 8 h to become shake flask seeds; the shake flask seeds are inoculated into the medium of the first-stage seed tank at an inoculation amount of 0.2% and cultured for 7 h under the conditions of 45°C, 180 - 200 rpm, and an aeration rate of 1:0.5 - 0.6 to become the first-stage seed tank seeds; the first-stage seed tank seeds are inoculated into the medium of the main fermenter at an inoculation amount of 3% and cultured for 18 h under the same conditions, and then immediately enter the next low-temperature fermentation; the media of the shake flask, the first-stage seed tank, and the main fermenter are all prepared by adding 30 g / L of enzymatically hydrolyzed soybean meal powder (calculated based on the amount before enzymatic hydrolysis), 6.0 g / L of disodium hydrogen phosphate, and 6.0 g / L of ammonium chloride, and sterilizing at 121°C for 20 min.

[0065] The remaining steps are the same as those in Example 1.

[0066] The specific process conditions and fermentation results are shown in Table 2:

[0067] Table 2 Optimal production process for variable-temperature liquid fermentation and spore production of Bacillus licheniformis for feed

[0068]

[0069] As can be seen from Table 2, in this embodiment, the two-stage variable-temperature liquid fermentation process of Bacillus licheniformis is still adopted, but the process conditions are changed. It is found that when the culture time of the shake flask seeds, the inoculation amount and culture time of the first-stage seed tank, the inoculation amount and culture time of the main fermentation tank, and the content of the culture medium components are changed, the number of spores of Bacillus licheniformis finally produced is 5.83 billion / mL. The fermentation time starting from the shake flask seeds is still 33h. Compared with the optimal process conditions, the spore yield has decreased, but it is still significantly higher than the number of spores produced by the existing fermentation process. Moreover, the antibacterial rate of the fermentation broth against Staphylococcus aureus can also reach 86%. Therefore, it can be known that compared with the existing process, the process conditions provided in this embodiment can also significantly increase the spore yield, shorten the fermentation time, and increase the antibacterial rate against Staphylococcus aureus. However, in the preferred production process and optimal process conditions of Example 1, the number of spores of Bacillus licheniformis produced can reach the highest, the fermentation efficiency is significantly improved, and at the same time, the antibacterial rate of the fermentation broth against Staphylococcus aureus can also reach the highest.

[0070] Example 3 A production process for variable-temperature liquid fermentation of Bacillus licheniformis for feeding (different process conditions)

[0071] This embodiment provides a production process under different conditions, and the steps are as follows:

[0072] In this embodiment, the high-temperature aerobic fermentation in the first stage of step (2) is as follows:

[0073] The Bacillus licheniformis strain 56374 is inoculated into the shake flask seed medium and cultured on a shaker at 45°C and 180 - 200 rpm for 9 h to become shake flask seeds; the shake flask seeds are inoculated into the medium of the first-stage seed tank at an inoculation amount of 0.1% and cultured at 45°C, 180 - 200 rpm, and an aeration rate of 1:0.5 - 0.6 for 8 h to become the first-stage seed tank seeds; the first-stage seed tank seeds are inoculated into the medium of the main fermentation tank at an inoculation amount of 5% and cultured under the same conditions for 16 h, and then immediately enter the next low-temperature fermentation; the media of the shake flask, the first-stage seed tank, and the main fermentation tank are all prepared by adding 25 g / L of enzymatically treated soybean meal powder (calculated based on the amount before enzymatic hydrolysis), 8.0 g / L of disodium hydrogen phosphate, and 8.0 g / L of ammonium chloride, and sterilizing at 121°C for 20 min.

[0074] The remaining steps are the same as those in Example 1.

[0075] The specific process conditions and fermentation results are shown in Table 3:

[0076] Table 3 The optimal production process for variable-temperature liquid fermentation of Bacillus licheniformis for feeding

[0077]

[0078]

[0079] As can be seen from Table 3, in this embodiment, the two-stage temperature-variable liquid fermentation process of Bacillus licheniformis is still adopted, but the process conditions are changed. It is found that when the culture time of the Erlenmeyer flask seeds, the inoculation amount and culture time of the first-stage seed tank, the inoculation amount and culture time of the main fermentation tank, and the content of the culture medium components are changed, the number of spores of Bacillus licheniformis finally produced is 5.47 billion / mL. The fermentation time starting from the Erlenmeyer flask seeds is still 33 h. Compared with the optimal process conditions, the spore yield has decreased, but it is still significantly higher than the number of spores produced by the existing fermentation process. Moreover, the antibacterial rate of the fermentation broth against Staphylococcus aureus can also reach 80%. Therefore, it can be known that compared with the existing process, the process conditions provided in this embodiment can also significantly increase the spore yield, shorten the fermentation time, and increase the antibacterial rate against Staphylococcus aureus. However, in the preferred Example 1, the production process and the optimal process conditions can achieve the highest number of spores of Bacillus licheniformis, significantly improve the fermentation efficiency, and at the same time, the antibacterial rate of the fermentation broth against Staphylococcus aureus can also reach the highest.

[0080] Screening of the temperatures of the front and back stages of the two-stage temperature-variable fermentation process in Example 4

[0081] To obtain the best effect in Example 1, the temperatures of the front and back stages of the temperature-variable fermentation process were screened in this embodiment, and there are the following methods (the remaining unmentioned operation processes and conditions are all the best conditions in Example 1 and are the same; the fermentation time is calculated according to the most suitable time during cultivation under different changed conditions and is not fixed as the best cultivation time in Example 1):

[0082] 1. Adopt the two-stage temperature-variable fermentation process: (1) High-temperature fermentation in the front stage (45 °C); (2) Low-temperature fermentation in the back stage (30 - 32 °C);

[0083] 2. Adopt the two-stage temperature-variable fermentation process: (1) Low-temperature fermentation in the front stage (30 - 32 °C); (2) High-temperature fermentation in the back stage (45 °C);

[0084] 3. Soybean meal raw materials, without being processed by the process of the present invention, but fermented according to the existing fermentation process. The existing fermentation process includes: First, using soybean meal as the main raw material, adding corn flour or starch or sucrose, and then adding inorganic nitrogen, mineral salts, etc., culturing at 37°C, preparing seeds in a triangular flask shaker, then inoculating into a seed tank, and finally inoculating into a main fermentation tank, culturing until the spore formation rate reaches more than 85%, and stopping fermentation; Second, using beef extract and peptone as the main raw materials, adding inorganic salts, mineral salts, etc., culturing at 37°C, with the same process as above, culturing until the spore formation rate reaches more than 85%, and stopping fermentation (using beef extract, peptone, sucrose, etc. as raw materials, the fermentation time is relatively short, but the number of spores formed is slightly less).

[0085] The specific results are shown in Table 4 as follows:

[0086] Table 4 Effects of different temperatures in the front and back stages of the variable-temperature fermentation process on spore yield, fermentation time, and antibacterial activity against Staphylococcus aureus

[0087]

[0088]

[0089] It can be seen from Table 4 that compared with the existing fermentation process, when the best process in Example 1 is adopted, that is, the two-stage variable-temperature fermentation process: high-temperature fermentation in the front stage (45°C) and low-temperature fermentation in the back stage (30 - 32°C), the number of spores in the liquid fermentation of Bacillus licheniformis can reach 6.67 billion per milliliter, and the fermentation time is only 33 h. At the same time, the antibacterial rate of the fermentation broth against Staphylococcus aureus is significantly increased, reaching 91%, and the antibacterial activity is significantly enhanced; when the temperatures in the front and back stages are changed, that is, low-temperature fermentation in the front stage (30 - 32°C) and high-temperature fermentation in the back stage (45°C), the inappropriate temperature at this time makes all Bacillus licheniformis vegetative cells, and almost no transformation into spores occurs.

[0090] In the present invention, it is unexpectedly found that high temperature can promote the growth and reproduction of Bacillus licheniformis, significantly shorten the growth period of seeds and the time from the logarithmic growth phase to the stationary phase. After fermenting to the stationary phase at the end of the logarithmic growth phase, the number of vegetative cells basically no longer increases, and the subsequent low-temperature anaerobic fermentation can significantly increase the spore formation rate of vegetative cells. The principle may be: Under the conditions of high temperature and aerobic fermentation, the enzyme activity of various biological enzymes in microorganisms increases, the physiological metabolism of microbial cells accelerates, the biosynthesis of the organism accelerates, and the growth and reproduction of microorganisms are accelerated; under the later fermentation conditions, the sudden change from high temperature to low temperature and from high dissolved oxygen to anaerobic causes a strong stress reaction, and this condition that is not suitable for growth and reproduction can promote the large-scale transformation of Bacillus licheniformis from vegetative cells into spores, quickly forming spores and thus shortening the fermentation time.

[0091] Therefore, the present invention determines the fermentation process and sequence. When preferably adopting a two-stage temperature-variable fermentation process: the first stage is high-temperature fermentation (45°C), and the second stage is low-temperature fermentation (30 - 32°C), the spore count of Bacillus licheniformis produced is significantly increased. At the same time, the fermentation time is shortened, and the energy consumption and labor cost of aeration and stirring are greatly reduced. Thus, the application advantage of the Bacillus licheniformis product of the present invention is improved. At the same time, the antibacterial activity against Staphylococcus aureus is also significantly enhanced, and the antibacterial rate reaches 91%.

[0092] Example 5 Screening of the oxygen environment in the front and back stages of the two-stage temperature-variable fermentation process

[0093] To obtain the best effect in Example 1, the oxygen environment in the front and back stages of the temperature-variable fermentation process was screened in this example, and there are the following methods (the remaining operation processes and conditions not mentioned are the best conditions in Example 1 and are the same; the fermentation time is calculated according to the most suitable time during cultivation under different changed conditions and is not fixed as the best cultivation time in Example 1):

[0094] 1. Adopt a two-stage temperature-variable fermentation process: (1) The first stage is high-temperature aerobic fermentation; (2) The second stage is low-temperature anaerobic fermentation;

[0095] 2. Adopt a two-stage temperature-variable fermentation process: (1) The first stage is high-temperature anaerobic fermentation; (2) The second stage is low-temperature aerobic fermentation;

[0096] 3. The soybean meal raw material is not processed by the process of the present invention but is fermented according to the existing fermentation process, and the existing fermentation process is specifically as described in Example 4.

[0097] The specific results are shown in Table 5:

[0098] Table 5 Influence of different oxygen environments in the front and back stages of the fermentation process on spore yield, fermentation time, and antibacterial activity against Staphylococcus aureus

[0099]

[0100] As can be seen from Table 5, compared with the existing fermentation process, when adopting the best process in Example 1, the two-stage temperature-variable fermentation process: the first stage is high-temperature aerobic fermentation, and the second stage is low-temperature anaerobic fermentation, the spore count of the liquid fermentation of Bacillus licheniformis can reach 6.69 billion per milliliter, and the fermentation time is only 33 h. At the same time, the antibacterial activity of the fermentation broth against Staphylococcus aureus is significantly enhanced, and the antibacterial rate can reach 94%; when the oxygen environment in the front and back stages changes and the first stage is high-temperature anaerobic fermentation and the second stage is low-temperature aerobic fermentation, basically no spores are produced.

[0101] The principle may be as follows: Under anaerobic conditions in the first stage, the growth and metabolism of microorganisms are extremely slow, and the reproduction rate is extremely low. At this time, the fermentation success rate is extremely low. On the other hand, Bacillus licheniformis is a facultative aerobic microorganism and can only reproduce in large numbers under aerobic conditions. Therefore, under the conditions of high temperature and anaerobic in the first stage, the growth and reproduction of microorganisms are extremely few, and almost no spores are produced.

[0102] Therefore, the present invention determines the fermentation process. When the two-stage variable temperature fermentation process is preferably adopted: high-temperature aerobic fermentation in the first stage and low-temperature anaerobic fermentation in the second stage, the spore number of Bacillus licheniformis produced is significantly increased, the fermentation time is shortened, and at the same time, the antibacterial activity of the spores against Staphylococcus aureus is significantly improved, so that the antibacterial rate can reach 94%.

[0103] Screening of the high temperature in the first stage and the low temperature in the second stage in the two-stage variable temperature fermentation process of Example 6

[0104] To obtain the best effect in Example 1, the best temperatures of the high temperature in the first stage and the low temperature in the second stage in the two-stage variable temperature fermentation process were screened in this example. The first-stage temperatures were respectively selected as 40°C, 45°C, and 50°C, and the second-stage temperatures were 25°C, 30°C, and 35°C, and the existing fermentation process was used for fermentation. The changes in the final spore yield of Bacillus licheniformis, fermentation efficiency, and antibacterial activity against Staphylococcus aureus were observed (the remaining unmentioned operation processes and conditions were the best conditions in Example 1 and were the same; the existing fermentation process was specifically described in Example 4; the fermentation time was calculated according to the most suitable time during cultivation under different changed conditions and was not fixed as the best cultivation time in Example 1):

[0105] The specific results are shown in Table 6:

[0106] Table 6 Effects of different temperatures in the first and second stages of the fermentation process on spore yield, fermentation time, and antibacterial activity against Staphylococcus aureus

[0107]

[0108]

[0109] As can be seen from Table 6, 1) compared with the optimal conditions in Example 1, when only changing the temperature in the first stage or the second stage of the fermentation process, the spore yield decreased significantly, the fermentation time increased, and the antibacterial activity of the fermentation broth against Staphylococcus aureus also decreased significantly. However, compared with the existing fermentation process, there was still a significant improvement; 2) in the aerobic fermentation at high temperature in the first stage, choosing too high or too low a temperature would reduce the final spore yield, increase the fermentation time, and significantly reduce the antibacterial activity against Staphylococcus aureus. In the anaerobic fermentation at low temperature in the second stage, choosing a higher temperature would also significantly reduce the final spore yield, significantly increase the fermentation time, and significantly reduce the antibacterial activity. The principle may be as follows: on the one hand, in industrial fermentation, during microbial production, heat is generated during its growth, making it difficult to control low temperatures, and the cost of cooling at low temperatures in production is extremely high. Only by using groundwater for cooling in summer when maintaining a temperature close to the indoor environment at 30 - 32°C can the cost be significantly reduced. If the temperature needs to be maintained at about 25°C, refrigeration equipment needs to be used, and the cost will increase significantly at this time. Therefore, in the present invention, temperatures below 30°C can be not considered; while too high a temperature may not be suitable for the growth and reproduction of microorganisms in the early stage, so the final spore yield decreases, the fermentation time prolongs, and the antibacterial activity against Staphylococcus aureus also decreases significantly. Therefore, only when the high-temperature fermentation temperature in the first stage is 45°C and the low-temperature fermentation temperature in the second stage is 30°C can the final spore yield reach the highest, and at the same time, the fermentation efficiency can be significantly improved.

[0110] Therefore, in the preferred two-stage variable-temperature fermentation process of the present invention, the high-temperature fermentation temperature in the first stage is 45°C, and the low-temperature fermentation temperature in the second stage is 30°C.

[0111] In the aerobic fermentation step at high temperature in the first stage of Example 7, screening of the inoculum amount in the first-stage seed tank

[0112] To obtain the best effect in Example 1, the inoculum amount in the first-stage seed tank in the aerobic fermentation at high temperature in the first stage was screened in the following ways (the remaining operation processes and conditions not mentioned are the same as the optimal conditions in Example 1; the fermentation time is calculated according to the most suitable time during cultivation under different changed conditions and is not fixed as the optimal cultivation time in Example 1):

[0113] 1. The inoculum amount in the first-stage seed tank is 0.1%.

[0114] 2. The inoculum amount in the first-stage seed tank is 0.2%.

[0115] 3. The inoculum amount in the first-stage seed tank is 0.3% (the optimal inoculum amount in Example 1).

[0116] 4. The soybean meal raw material is fermented according to the existing fermentation process without being processed by the process of the present invention. The existing fermentation process is specifically as described in Example 4.

[0117] The specific results are shown in Table 7 as follows:

[0118] Table 7 Effects of different inoculation amounts in the first-stage seed tank on spore yield, fermentation time, and antibacterial activity against Staphylococcus aureus

[0119]

[0120] As can be seen from Table 7, 1) Compared with the existing fermentation process, when the optimal process in Example 1, the two-stage variable-temperature fermentation process, is adopted, only by changing the different inoculation amounts in the first-stage seed tank in the first-stage high-temperature aerobic fermentation step, the spore numbers of Bacillus licheniformis liquid fermentation are significantly increased, the fermentation time is significantly shortened at the same time, and its antibacterial activity against Staphylococcus aureus is also significantly enhanced compared with the existing fermentation process; 2) When the inoculation amount in the first-stage seed tank continuously increases, its final spore yield also continuously increases, the fermentation time gradually shortens, and the antibacterial activity against Staphylococcus aureus is also significantly enhanced, reaching 93%. This is because in production, due to process condition limitations, the volume of the Erlenmeyer flask is about 300-500 ml per piece, and the volume is limited. Therefore, the filling amount of the seed liquid is limited to 50-80 ml per piece. Therefore, the inoculation amount is limited by process conditions, and the maximum inoculation amount within the preferred optional range is 0.3%.

[0121] Therefore, the present invention selects the inoculation amount in the first-stage seed tank in the first-stage high-temperature aerobic fermentation step to be 0.1% - 0.3%, preferably the maximum inoculation amount of 0.3%.

[0122] Screening of the culture time in the first-stage seed tank in the first-stage high-temperature aerobic fermentation step of Example 8

[0123] To obtain the best effect in Example 1, the culture time of inoculating the Erlenmeyer flask seeds into the first-stage seed tank medium in the first-stage high-temperature aerobic fermentation was screened in the following ways (the remaining operation processes and conditions not mentioned are the best conditions in Example 1 and are the same; in this example, only the culture time in the first-stage seed tank is changed, and the fermentation time of the Erlenmeyer flask seeds and the fermentation tank is calculated according to the most suitable time under different changed conditions, and is not fixed as the best culture time in Example 1):

[0124] 1. The culture time is 8 h;

[0125] 2. The culture time is 7 h;

[0126] 3. The culture time is 6 h (the best culture time in Example 1);

[0127] 4. The culture time is 5 h;

[0128] 5. Soybean meal raw materials, without being processed by the process of the present invention, but fermented according to the existing fermentation process, and the existing fermentation process is specifically as described in Example 4.

[0129] The specific results are shown in Table 8:

[0130] Table 8 Influence of the culture time in the primary seed tank on the spore yield, fermentation time, and antibacterial activity against Staphylococcus aureus in the high-temperature aerobic fermentation step at the front stage

[0131]

[0132] It can be seen from Table 8 that: 1) Compared with the optimal conditions in Example 1, when only changing the culture time in the primary seed tank in the high-temperature aerobic fermentation at the front stage, it is found that too short or too long culture time will both reduce the spore yield, and the antibacterial activity of the fermentation broth against Staphylococcus aureus decreases. However, compared with the existing fermentation process, there is still a significant improvement; 2) When the culture time in the primary seed tank is not less than 6 h, although the spore yield decreases slightly with the increase of the culture time, the yield is still extremely high and basically remains unchanged. However, it is found in the experiment that when the culture time reaches 6 hours, the bacteria are the strongest at this time, and the vitality and quantity of the seeds both reach the highest. At the same time, the antibacterial activity of the fermentation broth against Staphylococcus aureus is significantly enhanced, and the antibacterial rate reaches 93%. When the culture time reaches 8 h, the vitality and strength of the Bacillus spore seeds both weaken; when the culture time is 5 h or shorter, the spore yield decreases significantly. In this example, the preferred seeds need to meet the following conditions: ① There are enough bacteria (vegetative bodies), and only after being cultured under the optimal conditions can they be transformed into spores in the largest number; ② The bacteria need to be strong enough, and old and weak bacteria are not suitable as seeds.

[0133] Therefore, in the present invention, the culture time in the primary seed tank in the high-temperature aerobic fermentation step at the front stage is selected to be 6 - 8 h, preferably 6 h. Under this condition, the vitality and quantity of the seeds both reach the highest, so as to obtain the highest spore yield. At this time, the bacteria are the strongest, and the antibacterial activity against Staphylococcus aureus is significantly enhanced, and the antibacterial rate can reach 93%.

[0134] Screening of the inoculation amount in the main fermentation tank in the high-temperature aerobic fermentation step in Example 9

[0135] To obtain the best effect in Example 1, the inoculation amount in the main fermentation tank in the high-temperature aerobic fermentation at the front stage was screened in this example. There are the following methods (the remaining operation processes and conditions not mentioned are all the optimal conditions in Example 1 and are the same; the fermentation time is calculated according to the most suitable time during cultivation under different changed conditions and is not fixed as the optimal cultivation time in Example 1):

[0136] 1. The inoculation amount in the main fermenter is 5%;

[0137] 2. The inoculation amount in the main fermenter is 3%;

[0138] 3. The inoculation amount in the main fermenter is 4%;

[0139] 4. The soybean meal raw material is not processed by the process of the present invention, but is fermented according to the existing fermentation process, and the existing fermentation process is specifically as described in Example 4.

[0140] The specific results are shown in Table 9:

[0141] Table 9 Effects of different inoculation amounts in the main fermenter on spore yield, fermentation time, and antibacterial activity against Staphylococcus aureus in the high-temperature aerobic fermentation step in the first stage

[0142]

[0143] As can be seen from Table 9, 1) Compared with the optimal conditions in Example 1, when the different inoculation amounts in the main fermenter decrease during the high-temperature aerobic fermentation in the first stage, the spore yield decreases significantly, the fermentation time increases, and the antibacterial activity of the fermentation broth against Staphylococcus aureus decreases significantly. However, compared with the existing fermentation process, there is still a significant improvement; 2) When the inoculation amount in the main fermenter increases to 5%, the final spore yield decreases slightly but remains basically unchanged, which is not much different from the results under the optimal conditions in Example 1, but the production cost increases significantly.

[0144] Therefore, considering both the effect and cost control, in this example, the inoculation amount of the seeds in the main fermenter in the high-temperature aerobic fermentation step in the first stage is selected to be 3% - 5%, preferably 4%.

[0145] Screening of the culture time in the main fermenter in the high-temperature aerobic fermentation in Example 10

[0146] To obtain the best effect in Example 1, the culture time in the main fermenter in the high-temperature aerobic fermentation in the first stage was screened in the following ways (the remaining operation processes and conditions not mentioned are the same as the optimal conditions in Example 1; only the culture time in the main fermenter is changed in this example, which has no effect on the fermentation time of the Erlenmeyer flask seeds and the seeds in the seed tank in the first two stages, so the fermentation time is still the best fermentation time in Example 1):

[0147] 1. The culture time is 16 h;

[0148] 2. The culture time is 18 h;

[0149] 3. The culture time is 20 h;

[0150] 4. The culture time is 22 h;

[0151] 5. Soybean meal raw material, without being processed by the process of the present invention, but fermented according to the existing fermentation process, and the existing fermentation process is specifically as described in Example 4.

[0152] The specific results are shown in Table 10:

[0153] Table 10 Effects of the culture time in the main fermenter on the spore yield, fermentation time, and antibacterial activity against Staphylococcus aureus in the previous high-temperature aerobic fermentation step

[0154]

[0155]

[0156] It can be seen from Table 10 that: 1) Compared with the optimal conditions in Example 1, when only reducing the culture time in the main fermenter during the previous high-temperature aerobic fermentation, the spore yield significantly decreases, and the antibacterial activity of the fermentation broth against Staphylococcus aureus significantly decreases. However, compared with the existing fermentation process, there is still a significant improvement; 2) It can be seen from the experimental results that when the culture time in the main fermenter does not exceed 20 h, its spore yield significantly increases with the increase of the culture time. At the same time, the antibacterial activity of the fermentation broth against Staphylococcus aureus significantly increases, and the antibacterial rate reaches 95%. When the culture time exceeds 20 h, with the increase of the culture time, the spore yield basically does not increase, while the fermentation time slightly increases, and the antibacterial effect of the spores basically remains unchanged. With the increase of the fermentation time, the energy consumption for ventilation and stirring is extremely large. Therefore, considering both cost and effect, in this example, the culture time in the main fermenter in step (3) is selected to be 16 h - 20 h, and the preferred culture time is 20 h.

[0157] Screening of the culture medium in the previous high-temperature aerobic fermentation in Example 11

[0158] To obtain the best effect in Example 1, the content of the components of the culture medium in the previous high-temperature aerobic fermentation was screened in the following ways (the remaining operation processes and conditions not mentioned are the same as the optimal conditions in Example 1; the fermentation time is calculated according to the most suitable time during cultivation under different changed conditions and is not fixed as the optimal culture time in Example 1):

[0159] 1. Culture medium: Prepared from 35 g / L of enzymatically hydrolyzed soybean meal powder (calculated based on the amount before enzymatic hydrolysis), 7.0 g / L of disodium hydrogen phosphate, and 4.0 g / L of ammonium chloride, sterilized at 121 °C for 20 min.

[0160] 2. Culture medium: Prepared by 30 g / L of enzymatically treated soybean meal powder (calculated based on the amount before enzymatic treatment), 6.0 g / L of disodium hydrogen phosphate, and 6.0 g / L of ammonium chloride, sterilized at 121 °C for 20 min;

[0161] 3. Culture medium: Prepared by 25 g / L of enzymatically treated soybean meal powder (calculated based on the amount before enzymatic treatment), 8.0 g / L of disodium hydrogen phosphate, and 8.0 g / L of ammonium chloride, sterilized at 121 °C for 20 min;

[0162] 4. Culture medium: Prepared by 40 g / L of enzymatically treated soybean meal powder (calculated based on the amount before enzymatic treatment), 7.0 g / L of disodium hydrogen phosphate, and 4.0 g / L of ammonium chloride, sterilized at 121 °C for 20 min;

[0163] 5. The soybean meal raw material is not processed by the process of the present invention, but is fermented according to the existing fermentation process, and the existing fermentation process is specifically as described in Example 4.

[0164] The specific results are shown in Table 11:

[0165] Table 11 Effects of the content of culture medium components on the spore yield, fermentation time, and antibacterial activity against Staphylococcus aureus in the previous high-temperature aerobic fermentation step

[0166]

[0167] As can be seen from Table 11, 1) Compared with the optimal conditions in Example 1, when only changing the content of each component of the culture medium in the previous high-temperature aerobic fermentation, the spore yield decreased significantly, the fermentation time increased, and the antibacterial activity of the fermentation broth against Staphylococcus aureus decreased significantly. However, compared with the existing fermentation process, there was still a significant improvement; 2) From the experimental results, it can be seen that when the content of soybean meal powder does not exceed 35 g / L, its spore yield increases significantly with the increase of the content of soybean meal powder, the fermentation time is continuously shortened, and the antibacterial activity of the fermentation broth against Staphylococcus aureus increases significantly, and the antibacterial rate reaches 94%. When the content of soybean meal powder exceeds 40 g / L, with the increase of the content of soybean meal powder, the spore yield basically remains unchanged, the fermentation time increases, and the antibacterial activity of the fermentation broth against Staphylococcus aureus also basically does not change. This may be because the content of soybean meal powder has an obvious impact on the growth of Bacillus licheniformis spores and the final antibacterial activity. Within a certain content range, with the increase of the content of soybean meal powder, the formation of spores increases significantly, and the antibacterial activity against Staphylococcus aureus also increases significantly.

[0168] Therefore, in the present invention, preferably in the high-temperature aerobic fermentation step in the front stage, the content of the culture medium components is 25-35 g / L of enzymatically hydrolyzed soybean meal powder (calculated based on the amount before enzymatic hydrolysis), 6.0-8.0 g / L of disodium hydrogen phosphate, and 4.0-8.0 g / L of ammonium chloride, which is prepared by sterilization at 121 °C for 20 min. And on the premise of ensuring the same effect and lower cost, the content of each component of the culture medium is preferably 35 g / L of soybean meal powder, 7.0 g / L of disodium hydrogen phosphate, and 4.0 g / L of ammonium chloride.

[0169] Although the present invention has been disclosed above with preferred embodiments, it is not intended to limit the present invention. Any person familiar with this technology can make various modifications and decorations without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention should be defined by the claims.

Claims

1. A production process for liquid fermentation of Bacillus licheniformis to produce spores, characterized in that, the production process includes a two-stage variable-temperature liquid fermentation process of Bacillus licheniformis, wherein the first stage is high-temperature fermentation and the second stage is low-temperature fermentation; the high temperature is above 40°C, and the low temperature is not more than 32°C.

2. The production process according to claim 1, characterized in that, the first stage is high-temperature aerobic fermentation and the second stage is low-temperature anaerobic fermentation.

3. The production process according to claim 2, characterized in that, in the first-stage high-temperature aerobic fermentation, the following steps are included: (1) Inoculate the slant seeds of Bacillus licheniformis into the triangular flask seed medium and culture; (2) Inoculate the triangular flask seeds into the first-stage seed tank medium and culture; (3) Inoculate the first-stage seed tank seeds into the medium of the main fermentation tank, perform high-temperature aerobic culture for a certain period of time, and then enter the second-stage low-temperature anaerobic fermentation.

4. The production process according to claim 3, characterized in that, in the first-stage high-temperature aerobic fermentation, in step (2), the inoculation amount of the first-stage seed tank is 0.1-0.3%, and the culture time is 6-8h; in step (3), the inoculation amount of the seeds in the main fermentation tank is 3-5%, and the culture time is 16-20h.

5. The production process according to claim 4, characterized in that, in the first-stage high-temperature aerobic fermentation, the temperature is 45°C; in the second-stage low-temperature anaerobic fermentation, the temperature is 30-32°C.

6. The production process according to claim 5, characterized in that, in the fermentation of Bacillus licheniformis, the raw material is soybean meal.

7. The production process according to claim 6, characterized in that, in the fermentation of Bacillus licheniformis, the fermentation raw material is pretreated by neutral protease hydrolysis. By weight, the ratio of soybean meal powder to water is 1:

6.

8. The production process according to claim 7, characterized in that, in the first-stage high-temperature aerobic fermentation, the medium is prepared by 25-35 g / L of enzymatically hydrolyzed soybean meal powder (calculated by the amount before enzymolysis), 6.0-8.0 g / L of disodium hydrogen phosphate, and 4.0-8.0 g / L of ammonium chloride, sterilized at 121°C for 20 min.

9. The use of a two-stage variable-temperature fermentation process for preparing a preparation for increasing the spore yield of Bacillus licheniformis, characterized in that, in the two-stage variable-temperature fermentation process, the first stage is high-temperature aerobic fermentation and the second stage is low-temperature anaerobic fermentation.

10. A feed microbial viable bacteria preparation, characterized in that, the microorganism is Bacillus licheniformis; the feed microbial viable bacteria preparation is produced by the production process according to any one of claims 1-8.

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