CN111172059A - Fermentation method for increasing number of bacillus coagulans spores - Google Patents
Fermentation method for increasing number of bacillus coagulans spores Download PDFInfo
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Abstract
The invention discloses a fermentation method for increasing the number of bacillus coagulans spores, and relates to the technical field of bacterial culture. Inoculating bacillus coagulans into a fermentation culture medium, and respectively performing first feeding and second feeding on the fermentation culture medium after fermentation; wherein, the components of the culture medium of the fermentation tank comprise: carbon source, nitrogen source, magnesium salt, manganese salt, sodium salt, potassium salt, lycopene, calcium carbonate and bran. Adding a feeding carbon source and a feeding nitrogen source during first feeding; during the second feeding, calcium carbonate, bran, corncob and amino acid are added. According to the application, lycopene, calcium carbonate and bran are added as components of the culture medium, so that strain degeneration can be inhibited, and bacillus coagulans is promoted to produce spores. And then, two times of material supplement is carried out, so that the components required by the culture medium in the fermentation process can be supplemented, the generation amount of spores is increased, and the total thallus number, the spore rate, the spore number and the storage capacity of bacillus coagulans in subsequent products of fermentation are improved.
Description
Technical Field
The invention relates to the technical field of bacterial culture, and particularly relates to a fermentation method for increasing the number of bacillus coagulans spores.
Background
The bacillus coagulans is a gram-positive bacterium which can form spores and produce lactic acid. The thallus is rod-shaped, two ends are blunt and round, and single or short chain can generate spores which are produced at the end. It has the functions of maintaining intestinal microecological balance, raising digestive function and health level of lactic acid bacteria, resisting acid, choline, heat, dryness and other adverse resistance of bacillus.
In recent years, the use of microecologics in cultivation is gradually increased, and bacillus coagulans is gradually paid more attention in recent years due to the characteristics of lactic acid bacteria and bacillus coagulans, and becomes a key point of domestic and overseas research. A few domestic manufacturers try to generate bacillus coagulans products, but the spore rate is generally low, so that the quality of related products is generally not high, and therefore, the process for improving the spore rate and the spore number is particularly important.
In view of this, the invention is particularly proposed.
Disclosure of Invention
The invention aims to provide a fermentation method for increasing the number of bacillus coagulans spores.
The invention is realized by the following steps:
in a first aspect, embodiments provide a fermentation method for increasing the number of bacillus coagulans spores, which includes inoculating bacillus coagulans into a fermentation medium, and performing a first feeding and a second feeding into the fermentation medium after fermentation respectively;
wherein the components of the culture medium of the fermenter comprise: 4-4.8g/L of carbon source, 9.5-11.4g/L of nitrogen source, 0.1-0.12g/L of magnesium salt, 0.05-0.06g/L of manganese salt, 2-2.4g/L of sodium salt, 2-2.4g/L of potassium salt, 0.5-0.6g/L of lycopene, 0.5-0.6g/L of calcium carbonate and 10-12g/L of bran;
during the first feeding, 1-1.2g/L of feeding carbon source and 1-1.2g/L of feeding nitrogen source are added into the fermentation medium; and during the second feeding, 0.5-0.6g/L of calcium carbonate, 10-12g/L of bran, 0.5-0.6g/L of corncob and 0.35-0.46g/L of amino acid are added into the fermentation medium.
In alternative embodiments, the supplemental carbon source comprises one or more of glucose, corn starch, potato starch, tapioca starch, rice mash, sucrose; preferably, the feed carbon source is glucose;
optionally, the feeding nitrogen source comprises one or more of peptone, yeast extract powder, corn steep liquor dry powder, bean cake powder, peanut cake powder and fish meal; preferably, the fermentation medium is added with 1-1.2g/L of the feeding nitrogen source which comprises 0.5-0.6g/L of peptone and 0.5-0.6g/L of yeast extract powder;
optionally, 0.35-0.46g/L of the amino acids added to the fermentation medium include 0.3-0.4g/L methionine and 0.05-0.06g/L isoleucine.
In alternative embodiments, the bacillus coagulans performs the first feeding 10-12h after the start of fermentation in the fermentation medium;
preferably, the second feeding is carried out 18-20h after the beginning of the fermentation of the Bacillus coagulans in the fermentation medium.
In an alternative embodiment, the stirring speed in the fermentation medium is adjusted to 150-200r/min when the bacillus coagulans is inoculated to the fermentation medium;
preferably, when the bacillus coagulans is inoculated to the fermentation medium, the amount of gas introduced into the fermentation medium is adjusted, and the ratio of the volume of liquid in the fermentation medium to the volume of gas introduced is 1: 1-1.2;
in an alternative embodiment, the stirring speed in the fermentation medium is adjusted to 280-320r/min at 14-16h after inoculation of the bacillus coagulans into the fermentation medium;
preferably, the amount of gas introduced into the fermentation medium is adjusted 14-16h after the bacillus coagulans is inoculated into the fermentation medium, and the ratio of the volume of liquid in the fermentation medium to the volume of gas introduced is 1: 2-2.4.
In an alternative embodiment, before inoculating the bacillus coagulans into the fermentation medium, the method further comprises the steps of performing rejuvenation activation on the bacillus coagulans, then inoculating the bacillus coagulans into a shake flask culture medium for culture and serving as a seed solution, and inoculating the seed solution into the fermentation medium after expanding the seed solution;
preferably, the shake flask culture medium comprises: 8-12g/L of peptone, 4-6g/L of yeast powder, 16-24g/L of glucose, 4-6g/L of sodium acetate, 1.6-2.4g/L of diammonium citrate and 1.6-2.4g/L of dipotassium phosphate;
preferably, the inoculation amount of the seed liquid after propagation inoculated into the fermentation medium is 0.5-1%;
preferably, the temperature of the fermentation of the Bacillus coagulans in the fermentation medium is 39-41 ℃.
In alternative embodiments, the pH of the fermentation medium is 7.0-7.2.
In an alternative embodiment, the Bacillus coagulans ends fermentation 40-48h after starting fermentation within the fermentation medium.
In alternative embodiments, the carbon source comprises one or more of glucose, corn starch, potato starch, tapioca starch, rice mash, sucrose; preferably, the carbon source is glucose;
optionally, the nitrogen source comprises one or more of peptone, yeast extract powder, corn steep liquor dry powder, bean cake powder, peanut cake powder and fish meal; preferably, 9.5-11.4g/L of the nitrogen source in the fermentation medium comprises 4.5-5.4g/L of peptone, 4.5-5.4g/L of yeast extract powder and 0.5-0.6g/L of corn steep liquor dry powder;
optionally, the magnesium salt comprises magnesium sulfate;
optionally, the manganese salt comprises manganese sulfate;
alternatively, the sodium salt comprises sodium chloride;
optionally, the potassium salt comprises one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and potassium chloride. In an alternative embodiment, the total number of cells of the fermented Bacillus coagulans reaches 8.64X 109CFU/mL-9.07×109CFU/mL, the number of spores reached 7.66X 109CFU/mL-8.15×109CFU/mL, the spore rate reaches 88.66% -89.86%.
The invention has the following beneficial effects:
this application is through improving the component of fermentation medium in the fermentation cylinder, adds lycopene, calcium carbonate and bran as the component of fermentation medium, not only can protect bacillus coagulans cell membrane system, can also promote the effect that bacillus coagulans produced the spore simultaneously to the three is mutually in coordination, and is synergistic each other.
The lycopene has an anti-oxidation effect, can protect a bacillus coagulans cell membrane system, can block a process that a strain undergoes gene mutation under an external condition, and can effectively inhibit the strain degeneration condition.
Calcium carbonate can neutralize bacteria to produce acid and absorb toxic metabolites, and solid matters such as calcium carbonate and the like are added to play a role in promoting bacillus coagulans to produce spores; the bran can provide various substances such as necessary carbon source, nitrogen source and the like for the growth of the bacillus coagulans, and can play a role in promoting the bacillus coagulans to produce spores in the later fermentation period.
Two times of material supplement is carried out, because the growth of the bacillus coagulans can be inhibited due to overhigh concentration of glucose, peptone and the like in the initial fermentation stage, and because the consumption of a carbon source and a nitrogen source is caused in the fermentation process, less glucose, peptone and the like can not provide the carbon source and the nitrogen source which are necessary for the growth of the bacillus coagulans in the logarithmic phase of the fermentation, therefore, the carbon source and the nitrogen source are mainly supplemented to promote the continuous growth of strains in the first time of material supplement, and calcium carbonate, bran and corncobs which can promote the bacillus coagulans to generate spores are added in the second time of material supplement, and simultaneously, sufficient organic nitrogen is supplemented to a culture medium, so that necessary nitrogen source and key substrates can be provided for the formation of the spores of the strains in the; and the stirring speed in the fermentation tank is increased before the second feeding so as to improve the oxygen content in the fermentation process and be beneficial to increasing the generation amount of spores.
In the application, the components of the fermentation medium are optimized, and through two times of feeding design and process improvement, the components required by the fermentation medium in the fermentation process can be supplemented, so that the yield of spores is increased, and the total cell number, the spore rate, the spore number and the storage capacity of bacillus coagulans in subsequent products of fermentation are greatly improved.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products available commercially.
The application provides a fermentation method for increasing the number of bacillus coagulans spores, which comprises the following steps:
s1, preparing a bacillus coagulans seed solution.
The bacillus coagulans is firstly rejuvenated and activated according to a conventional method, then inoculated into a shake flask culture medium and cultured for 40-48h at 39-41 ℃ and used as seed liquid, and the seed liquid is expanded and cultured. Among them, bacillus coagulans in the present application is commercially available and purchased from Qingdao root biotechnology group, Inc.
Preferably, the components of the shake flask medium comprise: 8-12g/L of peptone, 4-6g/L of yeast powder, 16-24g/L of glucose, 4-6g/L of sodium acetate, 1.6-2.4g/L of diammonium citrate and 1.6-2.4g/L of dipotassium phosphate.
S2, inoculating the bacillus coagulans into the fermentation medium.
Preparing a fermentation culture medium, transferring the fermentation culture medium into a fermentation tank, adjusting the pH value of the culture medium in the fermentation tank to 7.0-7.2, and sterilizing. Inoculating the seed liquid after the expanding culture into a fermentation culture medium in a fermentation tank, wherein the inoculation amount is 0.5-1%, and the fermentation temperature is 39-41 ℃; when the bacillus coagulans is inoculated to a fermentation medium, the stirring speed in the fermentation tank is adjusted to 150-200 r/min; simultaneously, adjust the gas volume that lets in the fermentation cylinder, the volume of the interior liquid of fermentation cylinder and the volume ratio of letting in gas are 1: 1-1.2.
Preferably, the components of the fermentation medium include: 4-4.8g/L of carbon source, 9.5-11.4g/L of nitrogen source, 0.1-0.12g/L of magnesium salt, 0.05-0.06g/L of manganese salt, 2-2.4g/L of sodium salt, 2-2.4g/L of potassium salt, 0.5-0.6g/L of lycopene, 0.5-0.6g/L of calcium carbonate and 10-12g/L of bran.
Preferably, the carbon source comprises one or more of glucose, corn starch, potato starch, tapioca starch, rice saccharification liquid and sucrose; preferably, the carbon source is glucose;
preferably, the nitrogen source comprises one or more of peptone, yeast extract powder, corn steep liquor dry powder, bean cake powder, peanut cake powder and fish meal; preferably, 9.5-11.4g/L of the nitrogen source in the fermentation medium comprises 4.5-5.4g/L of peptone, 4.5-5.4g/L of yeast extract powder and 0.5-0.6g/L of corn steep liquor dry powder. Alternatively, the magnesium salt comprises magnesium sulfate; the manganese salt comprises manganese sulfate; sodium salts include sodium chloride; the potassium salt comprises one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate and potassium chloride. The magnesium salt, the manganese salt, the sodium salt and the potassium salt are used as inorganic salts and can also be used as buffering agents to maintain osmotic pressure.
The innovative components in the application, namely lycopene, calcium carbonate and bran, can promote bacillus coagulans to produce spores. Specifically, lycopene has an anti-oxidation effect and can play a role in protecting a bacillus coagulans cell membrane system, and multiple experiments show that after bacillus coagulans is passaged, strain degeneration is obvious, and lycopene can block a process of gene mutation of a strain under an external condition, so that strain degeneration is effectively inhibited. Calcium carbonate can neutralize bacteria to produce acid and absorb toxic metabolites, and solid substances such as calcium carbonate and the like can play a role in promoting bacillus coagulans to produce spores according to experimental results; the bran can provide various substances such as necessary carbon source, nitrogen source and the like for the growth of the bacillus coagulans, and experiments show that the bran, like calcium carbonate, can play a role in promoting the bacillus coagulans to generate spores in the later fermentation stage.
In adding lycopene, calcium carbonate and bran to the culture medium jointly in this application, not only can protect bacillus coagulans cell membrane system, can also promote the effect that bacillus coagulans produced the spore simultaneously to the three is synergistic each other.
S3, feeding for the first time after fermentation.
Inoculating bacillus coagulans into a fermentation culture medium, and performing first feeding after 10-12h of starting fermentation; when feeding for the first time, adding 1-1.2g/L of feeding carbon source and 1-1.2g/L of feeding nitrogen source into the fermentation medium;
preferably, the supplementary carbon source comprises one or more of glucose, corn starch, potato starch, tapioca starch, rice saccharification liquid and sucrose; preferably, the carbon source is glucose;
preferably, the nitrogen source for feeding comprises one or more of peptone, yeast extract powder, corn steep liquor dry powder, bean cake powder, peanut cake powder and fish meal; preferably, 1-1.2g/L of the nitrogen source for feeding is added to the fermentation medium and comprises 0.5-0.6g/L of peptone and 0.5-0.6g/L of yeast extract powder.
In the application, when the first feeding is carried out, a carbon source and a nitrogen source are mainly supplemented, the growth of the bacillus coagulans can be inhibited due to overhigh concentrations of glucose, peptone and the like in the initial fermentation stage, and the carbon source and the nitrogen source which are necessary for the growth of the bacillus coagulans cannot be provided by less glucose, peptone and the like in the logarithmic phase of the fermentation due to the consumption of the carbon source and the nitrogen source in the fermentation process, so that the components for the first feeding are selected.
In the application, the time for first feeding is 10-12h, 10-12h is the logarithmic phase of strain growth, and the continuous growth of the strain can be promoted by supplementing a nitrogen source and a carbon source.
S4, performing second feeding.
Adjusting the stirring speed of the bacillus coagulans in the fermentation culture medium to 280-320r/min 14-16h after the bacillus coagulans starts to ferment in the fermentation culture medium; meanwhile, the amount of gas introduced into the fermentation medium is adjusted, and the ratio of the volume of liquid in the fermentation medium to the volume of introduced gas is 1: 2-2.4.
And (3) inoculating the bacillus coagulans into the fermentation medium, and performing secondary feeding 18-20 hours after fermentation is started. When feeding materials for the second time, adding 0.5-0.6g/L calcium carbonate, 10-12g/L bran, 0.5-0.6g/L corncob and 0.35-0.46g/L amino acid into the fermentation medium; preferably, 0.35-0.46g/L amino acids added to the fermentation medium include 0.3-0.4g/L methionine and 0.05-0.06g/L isoleucine.
And in this application before carrying out the second feed supplement, improved the stirring speed in the fermentation cylinder for material in the fermentation cylinder mixes more evenly, has improved the volume of letting in gas simultaneously, is about the twice of the volume of letting in gas for the first time, and then has promoted the oxygen content among the fermentation process, is favorable to increasing the yield of spore.
In addition, in the application, calcium carbonate, bran, corncobs and amino acids are selected as components of the second feeding, wherein the calcium carbonate, the bran and the corncobs can provide various substances such as necessary carbon sources and nitrogen sources for the growth of the bacillus coagulans, and experiments show that the bran, the corncobs and the calcium carbonate can play a role in promoting the bacillus coagulans to produce spores in the later fermentation period. The amino acid can be used as an organic nitrogen source in the later fermentation period, can provide a necessary nitrogen source for the formation of bacterial spores in the later fermentation period, and can be used as a key substrate.
Experiments show that solid matters such as calcium carbonate and the like can inhibit the growth of strains to a certain extent in the fermentation process, so that the final spore rate is high but the total cell number is low, and therefore, the solid matters are supplemented in the spore production period of the bacillus coagulans, and the influence of the solid matters on the proliferation of the bacillus coagulans is reduced.
In the application, the time of the second feeding is 18-20h after the fermentation is started, 18-20h is the period of the strains to generate spores, and substances for promoting spore production such as solid matters and the like are added to promote the spore generation under the condition of not influencing the increase of the total cell number in the initial stage of the strains.
S5, inoculating the bacillus coagulans to the fermentation medium, and finishing the fermentation when most spores fall off 40-48 hours after the fermentation is started.
In this application, optimize through the component to the fermentation medium, still optimize the time and the component of first time feed supplement and second time feed supplement simultaneously, improve the total thallus number and the spore quantity after having promoted the spore fermentation of condensing more than, from the production, reduced manufacturing cost, the loss of total thallus number when having reduced the pelletization, still improved the shelf life of after-treatment finished product simultaneously.
The features and properties of the present invention are described in further detail below with reference to examples.
Examples 1 to 5
Examples 1-5 provide a fermentation process for increasing the number of spores of bacillus coagulans, comprising the steps of:
s1, preparing a bacillus coagulans seed solution.
The bacillus coagulans is firstly rejuvenated and activated according to a conventional method, then inoculated into a shake flask culture medium and cultured for 48 hours at 40 ℃ and used as seed liquid, and the seed liquid is expanded and cultured.
The components of the shake flask culture medium comprise: 10g/L of peptone, 5g/L of yeast powder, 20g/L of glucose, 5g/L of sodium acetate, 2g/L of diammonium citrate, 2g/L of dipotassium phosphate and the balance of water.
S2, inoculating the bacillus coagulans into the fermentation medium.
The media of examples 1 to 5 were prepared according to Table 1, and the pH of the medium in the fermenter was adjusted to 7.0 to 7.2 to perform sterilization. Respectively inoculating the seed liquid after the expanding culture into a fermentation tank corresponding to the above embodiment 1-5 according to the inoculation amount of 1%, and fermenting at 40 ℃; when the bacillus coagulans is put in a fermentation tank, the stirring speed in the fermentation tank is adjusted to 200 r/min; meanwhile, the ratio of the amount of gas introduced into the fermentation tank to the volume of liquid in the fermentation tank is adjusted to be 1: 1.
s3, feeding for the first time after fermentation.
Feeding the bacillus coagulans for the first time 10 hours after the bacillus coagulans starts to ferment in the fermentation tank; the composition of the first feed medium is shown in table 1;
s4, performing second feeding.
Adjusting the stirring speed in the fermentation tank to 300r/min 14h after the bacillus coagulans starts to ferment in the fermentation tank; meanwhile, the ratio of the amount of gas introduced into the fermentation tank to the volume of liquid in the fermentation tank is adjusted to be 2: 1.
s5, finishing fermentation when most spores fall off 40-48h after the bacillus coagulans starts fermenting in the fermentation tank.
TABLE 1 fermentation formulations of examples 1-5, blank set (balance water)
Example 6
This example is substantially the same as example 3, except that:
in this example, the shake flask medium comprises: 8g/L of peptone, 6g/L of yeast powder, 24g/L of glucose, 4g/L of sodium acetate, 1.6g/L of diammonium citrate, 2.4g/L of dipotassium phosphate and the balance of water.
Example 7
This example is substantially the same as example 3, except that:
in this example, the shake flask medium comprises: peptone 12g/L, yeast powder 4g/L, glucose 16g/L, sodium acetate 6g/L, diammonium citrate 2.4g/L, dipotassium hydrogen phosphate 1.6g/L and the balance of water.
Example 8
This example is substantially the same as example 3, except that:
in this example, the carbon source and the feed carbon source are both sucrose; and the nitrogen source and the supplemented nitrogen source are both corn steep liquor dry powder and soybean cake powder, wherein the dosage of the corn steep liquor dry powder is consistent with that of the peptone, and the dosage of the soybean cake powder is consistent with that of the yeast extract powder.
Example 9
This example is substantially the same as example 3, except that:
in this embodiment, the stirring rate in the fermenter was always maintained at 200r/min, and the ratio of the amount of gas introduced into the fermenter to the volume of liquid in the fermenter was always adjusted to 1: 1.
comparative example 1
The composition "lycopene" in the medium of example 3 was replaced by "ascorbic acid".
Comparative example 2
The medium of example 3 was replaced by the component "calcium carbonate" with "calcium phosphate".
Comparative example 3
The composition "bran" in the medium of example 3 was replaced by "rice bran".
Comparative example 4
The components "lycopene, calcium carbonate and bran" in the medium of example 3 were replaced with "ascorbic acid, calcium phosphate and rice bran".
Comparative example 5
The medium of example 3 was modified to include the components "lycopene 0.8g/L, calcium carbonate 0.8g/L and bran 1.5 g/L".
Comparative example 6
The second feeding operation in example 3 was omitted.
Comparative example 7
The second feed of example 3 was performed by replacing the amounts of calcium carbonate, bran, corncob, methionine and isoleucine by 0.55g/L, 0.8g/L, 15g/L, 12g/L and 15g/L, 0.8g/L and 0.6g/L, respectively.
Blank group
Blank medium referring to table 1, the blank was fed only once conventionally and contained no lycopene, calcium carbonate and bran.
The above examples 1 to 9, comparative examples 1 to 7 and blank groups were fermented according to the above-mentioned methods, and after completion of the fermentation, samples were taken and the total cell count and spore count were measured by a plate dilution and coating counting method. See table 2 for experimental results.
Note: and (3) calculating the number of spores, namely sucking 10ml of sample into a test tube, placing the test tube in a water bath kettle at 90 ℃ for 10 minutes in a water bath manner, and detecting the number of bacteria by using a plate dilution coating counting method to obtain the number of spores. The ratio of spores was 100% to the number of spores/total cell count.
TABLE 2 Total cell number, spore number and spore ratio at the end of fermentation
As can be seen from the above table, when other carbon sources, supplemented carbon sources, nitrogen sources and supplemented nitrogen sources are selected, the cell number and spore number are obviously reduced compared with those in example 3, which shows that the addition of refined materials such as glucose, yeast extract powder, peptone and the like has a great gain effect on the total cell number, and meanwhile, the lack of refined materials such as glucose and the like can also cause the stagnation of the growth of bacillus coagulans at the initial fermentation stage; in example 9, the ratio of the amount of gas introduced into the fermenter to the volume of the liquid in the fermenter was always adjusted to 1: 1, the dissolved oxygen is insufficient in the later stage of fermentation, so that the spore formation process of bacillus coagulans is inhibited, the thalli are autolyzed, and the number of thalli and spores is obviously reduced; in addition, as can be seen from comparative examples 1-5, lycopene and bran have certain gain effect on the increase of the cell number in the fermentation process; the calcium carbonate and the bran can greatly improve the spore rate in the later fermentation period; as can be seen from comparative example 6, the elimination of the second feeding resulted in a substantial reduction in the spore rate of clotted spores; as can be seen from comparative example 7, the number of spores of Bacillus coagulans was not increased or decreased slightly by further increasing the amount of the second feeding, which indicates that the effect of the second feeding selected by the present application is not good enough when the amount of the second feeding exceeds the range of the present application. Overall, the best results are obtained in example 3.
To sum up, this application is through improving the component of culture medium in the fermentation cylinder, adds lycopene, calcium carbonate and bran as the component of culture medium, and wherein, lycopene has anti-oxidant effect, can play the effect of protection bacillus coagulans cell membrane system to lycopene can also block the process that the bacterial takes place gene mutation under external conditions, effectively inhibits the bacterial degeneration condition. Calcium carbonate can neutralize bacteria to produce acid and absorb toxic metabolites, and solid matters such as calcium carbonate and the like are added to play a role in promoting bacillus coagulans to produce spores; the bran can provide various substances such as necessary carbon source, nitrogen source and the like for the growth of the bacillus coagulans, and can play a role in promoting the bacillus coagulans to produce spores in the later fermentation period.
And then, two times of material supplement are carried out, because the growth of the bacillus coagulans can be inhibited due to overhigh concentration of glucose, peptone and the like in the initial fermentation stage, and less glucose, peptone and the like in the logarithmic phase of the fermentation can not provide the carbon source and the nitrogen source which are necessary for the growth of the bacillus coagulans in the fermentation process due to the consumption of the carbon source and the nitrogen source, the carbon source and the nitrogen source are mainly supplemented to promote the continuous growth of the strains in the first time of material supplement, and calcium carbonate, bran and corncobs which can promote the bacillus coagulans to generate spores are added in the second time of material supplement, and simultaneously, sufficient organic nitrogen is supplemented to the culture medium, so that necessary nitrogen source and key substrates can be provided for the formation of the spores of the strains in. The method can supplement the components required by the culture medium in the fermentation process through two times of material supplement, and is further favorable for increasing the generation amount of spores. By adopting the fermentation process of the experiment, the total number of the bacillus coagulans after fermentation can reach 8.64 multiplied by 109CFU/mL-9.07×109CFU/mL, the number of spores reached 7.66X 109CFU/mL-8.15×109CFU/mL, the spore rate reaches 88.66% -89.86%, and the total thallus number, spore rate, spore number and storage capacity of the bacillus coagulans in subsequent products of fermentation are greatly improved.
The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention, and various modifications and changes may be made by those skilled in the art. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (10)
1. A fermentation method for increasing the number of bacillus coagulans spores is characterized by comprising the steps of inoculating bacillus coagulans into a fermentation culture medium, and respectively feeding materials into the fermentation culture medium for the first time and feeding materials into the fermentation culture medium for the second time after fermentation;
wherein the components of the fermentation medium comprise: 4-4.8g/L of carbon source, 9.5-11.4g/L of nitrogen source, 0.1-0.12g/L of magnesium salt, 0.05-0.06g/L of manganese salt, 2-2.4g/L of sodium salt, 2-2.4g/L of potassium salt, 0.5-0.6g/L of lycopene, 0.5-0.6g/L of calcium carbonate and 10-12g/L of bran;
during the first feeding, 1-1.2g/L of feeding carbon source and 1-1.2g/L of feeding nitrogen source are added into the fermentation medium; and during the second feeding, 0.5-0.6g/L of calcium carbonate, 10-12g/L of bran, 0.5-0.6g/L of corncob and 0.35-0.46g/L of amino acid are added into the fermentation medium.
2. The fermentation method for increasing the number of bacillus coagulans spores, according to claim 1, wherein the supplementary carbon source comprises one or more of glucose, corn starch, potato starch, tapioca starch, rice saccharification liquid and sucrose; preferably, the feed carbon source is glucose;
optionally, the feeding nitrogen source comprises one or more of peptone, yeast extract powder, corn steep liquor dry powder, bean cake powder, peanut cake powder and fish meal; preferably, 1 to 1.2g/L of the nitrogen source for feeding to the fermentation medium comprises 0.5 to 0.6g/L of peptone and 0.5 to 0.6g/L of yeast extract powder;
optionally, 0.35-0.46g/L of the amino acids added to the fermentation medium include 0.3-0.4g/L methionine and 0.05-0.06g/L isoleucine.
3. The method of claim 1, wherein the first feeding of the bacillus coagulans is performed 10-12h after the beginning of the fermentation of the bacillus coagulans in the fermentation medium;
preferably, the second feeding is carried out 18-20h after the beginning of the fermentation of the Bacillus coagulans in the fermentation medium.
4. The fermentation method for increasing the number of bacillus coagulans spores as set forth in claim 1, wherein the stirring speed of the fermentation medium is adjusted to 150-200r/min when the bacillus coagulans is inoculated into the fermentation medium;
preferably, when the bacillus coagulans is inoculated to the fermentation medium, the amount of gas introduced into the fermentation medium is adjusted, and the ratio of the volume of liquid in the fermentation medium to the volume of gas introduced is 1: 1-1.2.
5. The fermentation method for increasing the number of bacillus coagulans spores, according to claim 1, wherein the stirring speed in the fermentation medium is adjusted to 280-320r/min at 14-16h after the bacillus coagulans is inoculated into the fermentation medium;
preferably, the amount of gas introduced into the fermentation medium is adjusted 14-16h after the bacillus coagulans is inoculated into the fermentation medium, and the ratio of the volume of liquid in the fermentation medium to the volume of gas introduced is 1: 2-2.4.
6. The fermentation method for increasing the number of bacillus coagulans spores, according to any one of claims 1 to 5, further comprising the steps of performing rejuvenation activation on the bacillus coagulans before inoculating the bacillus coagulans into the fermentation medium, then inoculating the bacillus coagulans into a shake flask culture medium for culture and serving as a seed solution, and inoculating the seed solution into the fermentation medium after expanding the seed solution;
preferably, the shake flask culture medium comprises: 8-12g/L of peptone, 4-6g/L of yeast powder, 16-24g/L of glucose, 4-6g/L of sodium acetate, 1.6-2.4g/L of diammonium citrate and 1.6-2.4g/L of dipotassium phosphate;
preferably, the inoculation amount of the seed liquid after propagation inoculated into the fermentation medium is 0.5-1%;
preferably, the temperature of the fermentation of the Bacillus coagulans in the fermentation medium is 39-41 ℃.
7. The method of any one of claims 1-5, wherein the fermentation medium has a pH of 7.0-7.2.
8. The method for increasing the number of bacillus coagulans spores as set forth in any one of claims 1 to 5, wherein the bacillus coagulans finishes fermentation 40 to 48 hours after starting fermentation in the fermentation medium.
9. The fermentation method for increasing the number of bacillus coagulans spores, according to any one of claims 1 to 5, wherein the carbon source comprises one or more of glucose, corn starch, potato starch, tapioca starch, rice saccharification liquid, and sucrose; preferably, the carbon source is glucose;
optionally, the nitrogen source comprises one or more of peptone, yeast extract powder, corn steep liquor dry powder, bean cake powder, peanut cake powder and fish meal; preferably, 9.5-11.4g/L of the nitrogen source in the fermentation medium comprises 4.5-5.4g/L of peptone, 4.5-5.4g/L of yeast extract powder and 0.5-0.6g/L of corn steep liquor dry powder;
optionally, the magnesium salt comprises magnesium sulfate;
optionally, the manganese salt comprises manganese sulfate;
alternatively, the sodium salt comprises sodium chloride;
optionally, the potassium salt comprises one or more of dipotassium hydrogen phosphate, potassium dihydrogen phosphate, and potassium chloride.
10. The method of any one of claims 1-5, wherein the fermentation is followed by fermentation to increase the number of spores of Bacillus coagulansThe total number of the cells of the Bacillus coagulans after fermentation is 8.64 multiplied by 109CFU/mL-9.07×109CFU/mL, the number of spores reached 7.66X 109CFU/mL-8.15×109CFU/mL, the spore rate reaches 88.66% -89.86%.
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| CN111500508A (en) * | 2020-05-31 | 2020-08-07 | 郑州大学 | Liquid mixed fermentation method of clostridium butyricum and bacillus coagulans |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN111500508A (en) * | 2020-05-31 | 2020-08-07 | 郑州大学 | Liquid mixed fermentation method of clostridium butyricum and bacillus coagulans |
| CN111500508B (en) * | 2020-05-31 | 2021-06-15 | 郑州大学 | Liquid mixed fermentation method of clostridium butyricum and bacillus coagulans |
| CN112266888A (en) * | 2020-11-04 | 2021-01-26 | 河南柏裕植物免疫科技有限公司 | Method for improving biomass and spore production of growth-promoting bacteria of bacillus plant roots |
| CN112868932A (en) * | 2021-02-28 | 2021-06-01 | 国药三良(浙江)健康科技有限公司 | Pure meat cat food capable of improving digestion characteristic and relieving feces odor and preparation method thereof |
| CN112852680A (en) * | 2021-03-22 | 2021-05-28 | 四川润格生物科技有限公司 | Liquid fermentation method of bacillus coagulans with high spore number |
| CN113215056A (en) * | 2021-06-02 | 2021-08-06 | 亚太星原农牧科技海安有限公司 | Screening method of bacillus coagulans capable of easily forming spores |
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