CN109468259B - Culture medium for promoting spore generation - Google Patents
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- CN109468259B CN109468259B CN201811241570.4A CN201811241570A CN109468259B CN 109468259 B CN109468259 B CN 109468259B CN 201811241570 A CN201811241570 A CN 201811241570A CN 109468259 B CN109468259 B CN 109468259B
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Abstract
The invention relates to a culture medium for promoting spore generation, which comprises the following components in parts by weight: corn flour: 3-5, peptone: 0.3-0.5, soybean meal powder: 2-3, magnesium salt: 0.01-0.03, chloride: 0.001-0.01, potassium salt: 0.01-0.03, calcium salt: 0.01-0.05, manganese salt: 0.003-0.005. The medium is used as a liquid fermentation medium. Compared with the prior art, the culture medium can effectively shorten the spore generation time, is easy to operate, and obviously improves the spore yield.
Description
Technical Field
The invention relates to a culture medium, in particular to a culture medium for promoting sporulation.
Background
The bacillus subtilis is widely applied to the field of microbial fermentation as a probiotic. The bacillus subtilis can promote the growth of beneficial anaerobic bacteria in an animal body, generate organic acids such as lactic acid and the like, reduce the pH value of an intestinal tract and indirectly inhibit the growth of other pathogenic bacteria; can improve immunoglobulin and antibody level, enhance cellular immunity and humoral immunity, and improve population immunity; synthesizing enzymes such as alpha-amylase, protease, lipase, cellulase and the like, playing a role together with digestive enzymes in animal bodies in the digestive tract, balancing the composition of flora in the intestinal tract and improving the health of the intestinal tract.
In industrial production, the fermentation of bacillus subtilis mainly has solid fermentation and liquid fermentation, solid fermentation is comparatively stable relatively, but has certain restriction to the bacterial volume, liquid fermentation can make the bacterial volume promote greatly, form high bacterial volume, but high bacterial volume does not represent high spore, the fungus that does not form the spore can be dead mostly in spray drying in liquid fermentation, and if the spore rate is low in the fungus powder, the fungus that does not form the spore can die in the short time, greatly reduced the content of fungus in the fungus powder and guaranteed its stability. Therefore, the proportion of the spores in the liquid fermentation is guaranteed, and the method has great significance.
Chinese patent CN201510174012.0 discloses a preparation method of a paenibacillus bacteriocin preparation, which comprises the following steps: (1) inoculating Paenibacillus (Paenibacillus sp.) into a culture medium for culturing the Paenibacillus, performing shake fermentation culture to obtain a fermentation broth, and centrifuging the obtained fermentation broth to obtain a supernatant; (2) mixing the obtained supernatant with ethanol, wherein the volume ratio of the ethanol to the supernatant is 2: 1-4: and 1, standing the obtained mixed solution at the temperature of 2-10 ℃ for 12-36 hours, centrifuging the mixed solution, collecting precipitates, and removing the solvent to obtain the product. The formulation of the medium is preferably: 1-3% of nitrogen source, 1-10% of carbon source, 0.01-0.03% of L-cystine, 0.01-0.03% of sodium sulfate, 0.1-0.3% of Na2HPO4 & 12H2O 0.1, 1-3% of sodium acetate, 0.05-0.15% of sodium chloride, 0.1-0.3% of sodium bicarbonate and the balance of water.
Chinese patent CN201810095745.9 discloses a preparation method of bacillus amyloliquefaciens G5 spore powder, which comprises the following steps of: corn starch, glucose, bean pulp, calcium carbonate, ammonium chloride, dipotassium hydrogen phosphate, magnesium sulfate and manganese sulfate; secondly, culturing spore fermentation liquor; preparing spore bacteria powder, and adding ammonium sulfate and diatomite into the spore fermentation liquor; then carrying out plate-frame filtration; obtaining the spore powder of the bacillus amyloliquefaciens G5. Therefore, in the preparation method of the bacillus amyloliquefaciens G5 spore powder, the raw materials of the spore production culture medium are common, convenient and easily available, and the production cost is reduced; the obtained spore fermentation liquor has a large number of spores, and the spore rate is more than or equal to 98 percent; ammonium sulfate and diatomite are used as flocculating agents, the preparation time of the spore powder is shortened, the number of effective viable bacteria in the obtained spore powder is large, and the yield of the effective viable bacteria is more than or equal to 95%. The spore production culture medium comprises the following components: 10-40 g/L of corn starch, 0.5-3 g/L of glucose, 5-25 g/L of soybean meal, 0.1-1.5 g/L of calcium carbonate, 0.1-1.0 g/L of ammonium chloride, 0.1-1.0 g/L of dipotassium hydrogen phosphate, 0.01-0.7 g/L of magnesium sulfate and 0.01-0.7 g/L of manganese sulfate.
The culture medium related to the above two patents is mainly used for preparing bacterial preparation or bacterial powder, and has no special function of promoting the spore yield of bacillus.
Chinese patent CN102181389A discloses a method for breeding bacillus with high spore yield, which adopts a step-by-step heating post-culture method to breed the bacillus with high spore yield according to the heat stability characteristics of spores. The method comprises the following steps: activating bacillus, performing primary heating treatment, performing primary culture, performing secondary heating treatment, performing secondary culture, performing tertiary heating treatment, performing tertiary culture and collecting to obtain the bacillus with high spore yield. Although the method improves the spore yield of the bacillus, the method needs longer culture time, at least 3-5 days each time, at least 2 weeks for culturing once by activating and carrying out three-stage culture, prolongs the culture acclimatization time, needs heat treatment of different degrees after culture, needs careful temperature and time setting, and increases the control cost.
Chinese patent CN201810052769.6 discloses a strain domestication method for improving the spore yield of bacillus, which comprises the following steps: activating bacillus; and (3) carrying out subculture on the activated strains in sequence from a normal culture medium to a lean culture medium, and then increasing the concentration of nutrient substances to the normal culture medium for culture, wherein the number of the culture mediums is more than 3 according to the concentration, and the culture mediums are continuously subcultured for more than 3 generations. The above patent changes the living environment of the strain by changing the amount of the nutrient substances in the culture medium, and further improves the spore yield of the bacillus, but the above culture method is still too complicated, the required procedures are many, and the culture time and efficiency need to be improved.
Therefore, the establishment of the culture medium for promoting the generation of the spores can effectively shorten the time for forming the spores, greatly improve the formation rate of the spores and the total amount of the spores, and has important significance for industrial production.
Disclosure of Invention
The present invention aims to overcome the defects of the prior art and provide a culture medium for promoting sporulation.
The purpose of the invention can be realized by the following technical scheme:
a culture medium for promoting sporulation comprises the following components in parts by weight:
corn flour: 3-5, peptone: 0.3-0.5, soybean meal powder: 2-3, magnesium salt: 0.01-0.03, chloride: 0.001-0.01, potassium salt: 0.01-0.03, calcium salt: 0.01-0.05, manganese salt: 0.003-0.005.
Preferably, the culture medium comprises the following components in parts by weight:
corn flour: 3-5, peptone: 0.3-0.5, soybean meal powder: 2-3, glucose: 1-2, magnesium salt: 0.01-0.03, chloride: 0.001-0.01, potassium salt: 0.01-0.03, calcium salt: 0.01-0.05, manganese salt: 0.003-0.005.
Further preferably, the culture medium comprises the following components in parts by weight:
corn flour: 4.5, peptone: 0.3, soybean meal powder: 2, glucose: 1, magnesium salt: 0.015, chloride: 0.001, potassium salt: 0.02, calcium salt: 0.03, manganese salt: 0.004.
in the culture medium of the present invention, preferably, the magnesium salt is magnesium sulfate heptahydrate or magnesium sulfate.
In the culture medium of the present invention, preferably, the manganese salt is manganese sulfate or manganese sulfate monohydrate.
In the culture medium of the present invention, preferably, the chloride is potassium chloride or sodium chloride.
In the culture medium of the present invention, preferably, the calcium salt is calcium nitrate or calcium carbonate.
In the medium of the present invention, preferably, the potassium salt is potassium dihydrogen phosphate or dipotassium hydrogen phosphate.
Further preferably, the magnesium salt is magnesium sulfate heptahydrate, the manganese salt is manganese sulfate, the chloride is potassium chloride, the calcium salt is calcium nitrate, and the potassium salt is potassium dihydrogen phosphate.
The culture medium of the invention is used as a liquid fermentation culture medium.
When the culture medium is used as a liquid fermentation culture medium, every 1L of the liquid fermentation culture medium contains the following components:
corn flour: 3-5g, peptone: 0.3-0.5g, soybean meal powder: 2-3g, magnesium sulfate heptahydrate: 0.01-0.03g, potassium chloride: 0.001-0.01g, potassium dihydrogen phosphate: 0.01-0.03g, calcium nitrate: 0.01-0.05g, manganese sulfate: 0.003-0.005 g.
When the culture medium is used, the fermentation process conditions are as follows: temperature: ventilating at 36-38 deg.c: 3-5m3H, pH: 7.0-7.4, rotation speed: 200-300rpm, preferably the temperature: aeration at 37 ℃: 3-5m3H, pH: 7.2, rotation speed: 200 rpm.
When the culture medium is used, the following method is adopted:
A. preparation of Bacillus seed liquid
Activating bacillus preserved on the inclined plane, then inoculating the bacillus to a shake flask culture medium, and carrying out shake culture at 36-38 ℃ and 200-;
B. fermentation of bacterial species
Fermenting the Bacillus seed liquid obtained in step A in a fermenter using the culture of claim 1 as a liquid medium, wherein the Bacillus has a spore content of 94.74-97.86% and a spore content of 1.73-1.95X 1010CFU/mL。
In the invention, the bacillus is bacillus or coccus capable of forming bacillus, including bacillus, lactobacillus and sporosarcina, preferably bacillus subtilis and bacillus licheniformis.
The components of the culture medium are mainly adjusted according to the salt components, and the potassium chloride is utilized, and the calcium nitrate, the monopotassium phosphate, the magnesium sulfate heptahydrate, the manganese sulfate and the like are added, so that the thalli can be in a proper growth environment, the generation of spores is effectively promoted, the time is shortened, and the yield of the spores is improved.
Wherein MgSO4.7H2Mg of O2+Is an important enzyme activator for EMP, TCA pathway and lysine production, Mg2+With MnSO4Mn in (1)2+Can effectively promote the generation of spores; KCl balances osmotic pressure and plays a role in regulating osmotic pressure in a culture medium; KH (Perkin Elmer)2PO4K in (1)+Is a cofactor of certain enzymes (fructokinase, phosphopyruvate transphosphatase, etc.), maintains potential difference and osmotic pressure, and plays an important role in buffering pH value; ca (NO)3)2Ca in (1)2+Is an activator of various enzymes and is more related to the permeability of cell membranes; and NO3-Can supply inorganic nitrogen source, can effectively shorten the time for forming the spores of the bacillus subtilis, and simultaneously greatly improves the formation rate of the spores and the total amount of the spores. The synergistic effect of the components greatly saves the electric power and manpower saved by shortening the fermentation time in the industrial production process, so that the fermentation efficiency is obviously improved.
Compared with the prior art, the culture medium can effectively shorten the spore generation time, greatly improve the spore formation rate and the total amount of spores, is easy to operate, and obviously improves the spore yield.
Detailed Description
The present invention will be described in detail with reference to specific examples.
Comparative example
1. Preparation of Bacillus subtilis seed solution
The bacillus subtilis preserved on the inclined plane is activated and then inoculated into 100mL shake flask culture medium, and is cultured for 24h at 37 ℃ and 200rpm in a shaking way.
The formulation of the fermentation medium used in the comparative example was: peptone: 10g/L, 5g/L of yeast powder, 5g/L of sodium chloride and 2g/L of glucose.
2. Fermentation of bacterial species
Fermenting with a 50L fermentation tank, and fermenting the strain with conventional fermentation process. The fermentation process conditions are as follows: temperature: aeration at 37 ℃: 3-5m3H, pH: 7.2, rotation speed: 200 rpm.
Examples 1 to 5
1. Preparation of Bacillus subtilis seed solution
Inoculating the bacillus subtilis preserved on the inclined plane to a shake flask culture medium, performing shake culture at 37 ℃ and 200rpm for 22-24h, streaking to an LB (Langmuir Blodgett) plate for culture at 37 ℃, separating a single colony, and repeatedly activating for 2-3 times. Inoculating activated Bacillus subtilis to 100mL shake flask culture medium, and shake culturing at 37 deg.C and 200rpm for 24h to obtain seed solution.
The formulations of the fermentation media used in examples 1-5 are shown in Table 1.
TABLE 1 culture Medium formulations in examples 1-5
2. Fermentation of bacterial species
Fermenting with a 50L fermentation tank, and fermenting the strain with conventional fermentation process. The fermentation process conditions are as follows: temperature: aeration at 37 ℃: 3-5m3H, pH: 7.2, rotation speed: 200 rpm.
3. Calculating the spore rate: after fermentation is finished, detecting the total bacterial load by adopting a flat coating dilution counting method; after 20min after passing through 80 ℃ water bath, plate coating, dilution and counting are carried out to detect the spore production. The spore rate is calculated by the following steps: spore productivity ═ 100% spore formation/total viable count.
Comparative example and fermentation media of examples 1 to 5 Bacillus subtilis was cultured, and the results are shown in Table 2.
TABLE 2 sporulation of Bacillus subtilis
From the results it follows: the fermentation culture medium can ensure that the spore rate of the bacillus subtilis reaches 94.74-97.86 percent and the spore amount reaches 1.79-1.85 multiplied by 10 under the condition of not influencing the total amount of viable bacteria10CFU/mL, spore formation time is shortened by 5-7h, namely total bacterial load is not influenced, and compared with the prior art, the spore formation efficiency is greatly improved by increasing the spore load and shortening the spore formation time.
Examples 6 to 8
1. Preparation of Bacillus seed liquid
Inoculating bacillus preserved on the inclined plane to a shake flask culture medium, performing shake culture at 37 ℃ and 200rpm for 24h, streaking to an LB (Luria Bertani) flat plate for culture at 37 ℃, separating a single colony, and repeatedly activating for 2 times. The activated bacillus is inoculated into 100mL shake flask culture medium, and shake-cultured for 24h at 37 ℃ and 200rpm to prepare seed liquid.
The bacilli cultured in examples 6-8 were Lactobacillus inulinus, Sporosarcina ureae, and Bacillus licheniformis, respectively.
2. Fermentation of bacterial species
Fermenting with 50L fermenter, and fermenting with the formula of fermentation medium (unit is g/L): corn flour: 4.5, peptone: 0.3, soybean meal powder: 2, glucose: 1, magnesium salt: 0.015, chloride: 0.001, potassium salt: 0.02, calcium salt: 0.03, manganese salt: 0.004, fermenting the strain by adopting a conventional fermentation process. The fermentation process conditions are as follows: temperature: aeration at 37 ℃: 4m3/h，pH：7Rotation speed: at 250 rpm.
3. Calculating the spore rate: after fermentation is finished, detecting the total bacterial load by adopting a flat coating dilution counting method; after 20min after passing through 80 ℃ water bath, plate coating, dilution and counting are carried out to detect the spore production. The spore rate is calculated by the following steps: spore productivity ═ 100% spore formation/total viable count.
The results of culturing Bacillus bacteria in the fermentation media of examples 6 to 8 are shown in Table 2.
TABLE 3 sporulation of Bacillus
From the results it follows: the fermentation culture medium can ensure that the spore amount of the bacillus reaches 1.73-1.95 multiplied by 10 under the condition of not influencing the total amount of the live bacteria10CFU/mL, the spore rate reaches 95.53-97.14%, the spore formation time is shortened by 6-7h, and the spore formation efficiency is greatly promoted.
The Bacillus is Bacillus or coccus capable of forming spore, including Bacillus, Lactobacillus and Sporosarcina, preferably Bacillus subtilis and Bacillus licheniformis.
The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.
Claims (1)
1. A method for using a culture medium for promoting sporulation,
the culture medium is a liquid fermentation culture medium and comprises the following components:
corn flour (g/L) 4.5
Peptone (g/L) 0.3
Soybean meal powder (g/L) 2
Glucose (g/L) 1
Magnesium sulfate heptahydrate (g/L) 0.015
Potassium chloride (g/L) 0.001
0.03 g/L of calcium nitrate
Dipotassium hydrogen phosphate (g/L) 0.02
Manganese sulfate (g/L) 0.004;
the method comprises the following steps:
A. preparation of Bacillus subtilis seed solution
Repeatedly activating the bacillus subtilis preserved on the inclined plane for 2-3 times, then inoculating the bacillus subtilis to a shake flask culture medium, and carrying out shake culture at 37 ℃ and 200rpm to obtain bacillus subtilis seed liquid;
B. fermentation of bacterial species
And fermenting the seed liquid by using the liquid fermentation medium and a fermentation tank, wherein the fermentation process conditions are as follows: temperature: aeration at 37 ℃: 3-5m3H, pH: 7.2, rotation speed: 200 rpm.
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| CN111394280A (en) * | 2020-03-26 | 2020-07-10 | 福建傲农生物科技集团股份有限公司 | Culture medium suitable for growth of bacillus licheniformis and application thereof |
| CN111349592B (en) * | 2020-05-11 | 2022-06-03 | 河南大学 | Spore production culture medium and preparation method of bacterial spores |
| 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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