CN111893072A - Preparation method of bacillus coagulans powder - Google Patents
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Abstract
The invention provides a preparation method of bacillus coagulans bacterial powder. Wherein the number of viable bacillus coagulans in the fermentation liquid can reach 8.0 x 109More than CFU/mL, more than 90% of spore rate, and 1.0 x 10 viable count of spray-dried powder11CFU/g is higher than the standard. The bacillus coagulans preparation prepared by the invention has high viable count and spore rate, can resist high temperature and acid, effectively inhibits the growth of harmful bacteria, improves the intestinal micro-ecological environment, and promotes the digestion and absorption of livestock and poultry on feed.
Description
Technical Field
The invention relates to a preparation method of bacillus coagulans powder, belongs to the technical field of microbial fermentation, and particularly relates to a preparation technology of bacillus coagulans liquid fermented to the powder.
Background
In the breeding industry, with the widespread use of antibiotics and chemical bactericides, a series of disadvantages are gradually brought, such as the generation of drug resistance of pathogenic bacteria, drug residues, damage to normal flora of intestinal tracts, damage to environment and the like. Therefore, it is very important to find a product capable of replacing antibiotics, and the technology for preparing the product capable of replacing antibiotics plays an important role in the development of the future breeding industry.
The probiotic preparation is used as one of substitute antibiotics, and has the advantages of no toxicity, no harm, environmental protection, improvement of intestinal health, inhibition of pathogenic bacteria growth and the like. The lactobacillus mainly comprises lactobacillus and bacillus, wherein the lactobacillus can produce organic acid, has good inhibition effect on pathogenic bacteria, can colonize intestinal tracts, but has poor stress resistance, easy inactivation and short shelf life. Although bacillus is stronger in stress resistance, it is inferior in function to lactic acid bacteria.
Bacillus coagulans (Bacillus coagulans) is a Bacillus which has the function of lactobacillus and can produce lactic acid, is used for regulating the balance of intestinal flora, promoting the development of intestinal tracts and enhancing the functions of the intestinal tracts and is used as facultative anaerobe. And can effectively inhibit the growth of harmful bacteria and improve the intestinal micro-ecological environment. The bacillus coagulans has high temperature and high pressure resistance and high gastric acid and bile resistance, so that the bacillus coagulans integrates the advantages of lactic acid bacteria and bacillus.
In domestic fermentation culture research on bacillus coagulans, most researchers culture the bacillus coagulans at 35-37 ℃, and the spore rate is about 70 percent and rarely reaches more than 90 percent. According to the purchased bacillus coagulans, experiments prove that the optimum growth temperature is 45-50 ℃, and the spore rate can reach more than 90% after the bacillus coagulans is cultured for 36-48h in the temperature range.
Trehalose is a non-reducing sugar composed of two glucose molecules with 1, 1-glycosidic bonds, and has acid, heat, freezing, radiation, and protein denaturation preventing effects. When the biological cells are in severe environments such as dry, high temperature and high pressure, the trehalose has good non-specific protection effect on biological and biological macromolecules. Scientists find that desert plant selaginella tamariscina almost withers when in drought and can be revived in a strange way when in water, alpine plant revived grass can resist ice, snow and severe cold, and some insects do not freeze or do not die under the conditions of high cold, high temperature, dry water loss and the like, namely the life strange trace created by trehalose in the bodies of the insects. In addition, sodium alginate is used as a coating protection material, the coating protection material has the capabilities of concentrating a solution, forming gel and forming a film, and after the coating protection material is uniformly mixed with a bacterial liquid, a tiny balloon is formed by spray drying, so that the bacterial coating protection material plays a role in coating and protecting bacteria. Meanwhile, the problems of moisture absorption, stickiness and the like caused by exposure of the skimmed milk powder to the air are solved. The bacterial liquid spray drying process is the thallus coating process.
Disclosure of Invention
The invention aims to provide a preparation method of bacillus coagulans powder, and the production process well solves the problems of high cost, low spore rate, mixed bacteria pollution in the fermentation process, short shelf life and the like; trehalose and sodium alginate are used as protective agents, the trehalose plays a role of life sugar and plays a role of protecting thalli, the sodium alginate plays a role of coating, the bacterial powder can be isolated from the external environment, meanwhile, the phenomena of moisture absorption, stickiness and the like caused by exposure of skimmed milk powder to the air are well solved, and the bacterial liquid drying process is a thallus coating process.
The invention provides a preparation method of bacillus coagulans powder, which is characterized in that bacillus coagulans liquid is fermented and expanded and then subjected to thallus concentration and enrichment, and the obtained thallus concentrated solution is uniformly mixed with a protective agent and then subjected to spray drying to prepare a dry powder preparation.
Specifically, the preparation method of the bacillus coagulans powder comprises the following steps:
first, strain activation and seed liquid preparation
Taking out the frozen tube from a refrigerator at the temperature of minus 80 ℃, streaking and inoculating the frozen tube on a test tube slant culture medium, and carrying out anaerobic culture at the temperature of 45-50 ℃ for 18-24 h; inoculating the activated bacillus coagulans into a seed liquid culture medium, and culturing for 18-24h under the conditions of liquid loading capacity of 500mL/1000L, 45-50 ℃ and 200 rpm/min;
the test tube slant culture medium and the seed liquid culture medium are MRS culture media, wherein 20g/L of agar powder is added to the test tube slant culture medium, no agar powder is added to the seed liquid culture medium, the culture medium is sterilized for 20min at 121 ℃, and the culture medium is cooled for standby;
second, preparation of fermentation broth
Inoculating the Bacillus coagulans seed liquid into a primary fermentation liquid according to the inoculation amount of 5 percent, wherein the liquid filling amount is 50L/100L, 45-50 ℃, 200rpm/min, and an aeration rate of 1.5m3Culturing for 18-24h under the condition of/h; transferring the primary fermentation liquid into secondary fermentation liquid, wherein the liquid loading amount is 1000L/1500L, 45-50 deg.C, 200rpm/min, and the ventilation amount is 2m3Culturing for 36-48h under the condition of/h;
the first-stage fermentation liquid and the second-stage fermentation liquid culture medium are prepared as follows: 30-50g/L of glucose, 10-15g/L of yeast extract, 10-20g/L of peptone, 2-5g/L of dipotassium phosphate, 0.05-0.1g/L of manganese sulfate, 2-5g/L of ammonium chloride and 15-20g/L of calcium carbonate, sterilizing at 121 ℃ for 20min, and cooling to 45-50 ℃ with water for later use;
thirdly, concentrating and coating the fermentation liquor
Treating the fermentation liquor by 10 nm-specification ceramic membrane filtration equipment to obtain a thallus concentrated solution, wherein the solid content of the thallus concentrated solution is 10-30%; adding 5-10% skimmed milk powder, 1-5% trehalose as protective agent and 0.2-0.5% sodium alginate as coating agent, dissolving and mixing;
the fourth step, spray drying
Centrifugal spray drying equipment is adopted, the conditions are set to be that the air inlet temperature is 110-150 ℃, the air outlet temperature is 80-100 ℃, and the dry powder obtained under the conditions is the bacterial powder.
Wherein, the viable count of the bacillus coagulans after the fermentation culture in the second step can reach 8.0 x 109CFU/mL or more and the spore rate of 90% or more.
The viable count of the bacterial powder obtained in the fourth step is 1.0 x 1011Above CFU/g, and the water content is 5-9%.
After the technical scheme is adopted, the technical scheme of the invention has at least the following advantages:
1. the bacillus coagulans is cultured at 45-50 ℃, and when the bacillus coagulans is cultured by high-temperature fermentation, the spore rate is improved, and other mixed bacteria are inhibited or killed, so that no pollution is caused in the fermentation process.
2. The ceramic membrane is adopted to filter and enrich the thalli, most of water is removed, the purposes of improving the concentration of the thalli, shortening the drying time and saving time and energy consumption cost are achieved.
3. The drying process of the concentrated bacterial liquid in the spray drying equipment is the thallus coating process, thereby reducing the coating step.
4. Trehalose is adopted as a protective agent before spray drying, the trehalose plays a role of life sugar and plays a role of protecting the thalli, and sodium alginate is used as a coating agent, so that the thalli can be guaranteed to be resistant to high temperature in the spray process, and the survival rate is improved. The microcapsule is formed after spray drying, so that the thallus is isolated from the air, and the stability and the quality guarantee period of the bacterial powder are increased. Meanwhile, the problems of moisture absorption, stickiness and the like caused by exposure of the skim milk powder in the air are solved.
Detailed Description
Preferred embodiments of the present invention will be described in more detail below. While the following describes preferred embodiments of the present invention, it should be understood that the present invention may be embodied in various forms and should not be limited by the embodiments set forth herein.
The bacillus coagulans adopted by the invention is a common strain and does not relate to biological preservation. In the following examples: the Bacillus coagulans (Bacillus coagulans) is purchased from Guangdong province microorganism strain preservation center, and has the following numbering: GDM 1.645.
Example 1: screening of optimum fermentation temperature of bacillus coagulans
Taking out a freezing tube of the bacillus coagulans from a refrigerator at the temperature of minus 80 ℃, inoculating MRS agar culture medium for anaerobic culture for 48h at the temperature of 37 ℃, selecting a few bacterial sludge to inoculate in a triangular flask liquid MRS culture medium of 100mL/250mL, and adopting a shaking table at the temperature of 37 ℃ and at the speed of 200rpm/min for 24h as bacillus coagulans seed liquid. Inoculating the bacillus coagulans seed solution into 500mL/1000mL triangular flask liquid MRS fermentation broth according to the inoculation amount of 5%, performing shake culture at 30 ℃, 37 ℃, 40 ℃, 45 ℃, 50 ℃, 55 ℃ and 200rpm/min for 48h, sampling every 24h, and detecting the viable count and the spore rate, wherein the obtained results are shown in Table 1.
Table 1:
as can be seen from the table data, the bacillus coagulans strain grows well at 45-50 ℃ and the spore rate is more than 90%.
Example 2: bacillus coagulans liquid fermentation at 45 DEG C
Strain activation and seed liquid preparation: taking out the frozen tube from a refrigerator at minus 80 ℃, streaking and inoculating the tube on a test tube MRS slant culture medium, and carrying out anaerobic culture at 45 ℃ for 24 h. The activated bacillus coagulans is inoculated in an MRS seed liquid culture medium, and cultured for 24h under the conditions of liquid loading capacity of 500mL/1000L, 45 ℃ and 200 rpm/min.
Preparing fermentation liquor: inoculating the Bacillus coagulans seed solution into the primary fermentation liquid according to the inoculation amount of 5%, wherein the liquid loading amount is 50L/100L, the temperature is 45 ℃, the rpm is 200/min, and the ventilation amount is 1.5m3Culturing for 24h under the condition of/h. Then transferring the primary fermentation liquid into secondary fermentation liquid, wherein the liquid loading amount is 1000L/1500L, the temperature is 45 ℃, the rpm is 200/min, and the ventilation volume is 2m3Culturing for 36h under the condition of/h. The number of viable bacillus coagulans after fermentation culture can reach 9.5 x 109CFU/mL, spore number 9.0 x 109CFU/mL, spore rate 94.7%.
The culture medium for the primary fermentation liquid and the secondary fermentation liquid in example 2 is prepared as follows: 50g/L glucose, 10g/L yeast extract, 20g/L peptone, 5g/L dipotassium phosphate, 0.1g/L manganese sulfate, 5g/L ammonium chloride and 20g/L calcium carbonate, sterilizing at 121 ℃ for 20min, and cooling to 45 ℃ with water for later use.
Example 3: liquid fermentation of bacillus coagulans at 50 DEG C
The experimental culture, the operation method and the culture medium of the fermentation broth are the same as those of example 2, except that the culture temperature used in example 3 is 50 ℃.
The viable count of the bacillus coagulans after 36 hours of fermentation culture can reach 9.1 x 109CFU/mL, number of spores 8.7 x 109CFU/mL, spore rate 95.6%.
Example 4: spray drying to prepare fungus powder
In example 2, the fermentation broth was concentrated by 10nm ceramic membrane filtration equipment to obtain a cell concentrate with 20% of solid content, and then 5% of skimmed milk powder, 1% of trehalose, and 0.2% of sea water were added according to the weight percentageSodium alginate, through fully stirring, mix. Centrifugal spray drying equipment is adopted, the spraying operation conditions are set to be that the air inlet temperature is 120 ℃, the air outlet temperature is 90 ℃, and dry powder obtained under the conditions is the bacterial powder. The number of viable bacteria in the obtained bacterial powder is 2.1 x 1011CFU/g, moisture 7%.
Example 5: spray drying to prepare fungus powder
In example 2, the fermentation broth is concentrated by a 10 nm-sized ceramic membrane filtration device to obtain a thallus concentrated solution with 20% of solid content, 10% of skimmed milk powder, 5% of trehalose and 0.5% of sodium alginate are added according to the weight percentage, and the mixture is fully stirred and uniformly mixed. Centrifugal spray drying equipment is adopted, the spraying operation conditions are set to be that the air inlet temperature is 120 ℃, the air outlet temperature is 90 ℃, and dry powder obtained under the conditions is the bacterial powder. The number of viable bacteria in the obtained bacterial powder is 2.8 x 1011CFU/g, water content 8%.
Example 6: spray drying to prepare fungus powder
The mixing procedure and the addition amount of the concentrated fermentation broth and the protective agent were the same as those of example 4. The spraying operation conditions are set as the air inlet temperature of 150 ℃ and the air outlet temperature of 90 ℃, and the dry powder obtained under the conditions is the bacterial powder. The number of viable bacteria in the obtained bacterial powder is 1.8 x 1011CFU/g, moisture 6.8%.
Comparative example 1: spray drying without coating
In example 2, the fermentation broth was concentrated by a ceramic membrane filtration apparatus to obtain a cell concentrate with a solid content of 20%, and the cell concentrate was directly spray-dried without adding any protective agent, wherein the spray conditions were set to 120 ℃ for inlet air and 90 ℃ for outlet air, and the dry powder obtained under these conditions was bacterial powder. The number of viable bacteria in the obtained bacterial powder is 0.9 x 1011CFU/g, moisture 6%.
Example 7: high temperature resistance test of bacillus coagulans dry powder
The bacterial powder obtained in the examples 4, 5 and 6 and the bacterial powder obtained in the comparative example 1 are respectively weighed in 25g to 225mL of sterile physiological saline, and then are respectively put into water bath with the temperature of 80 ℃, 90 ℃ and 100 ℃ for 10min, and the change of the viable count before and after the bacillus coagulans is detected. The results show that the bacillus coagulans powder obtained by the processes of examples 4 to 6 which were coated can tolerate higher temperatures than the bacillus coagulans powder which was not coated by the process of comparative example 1, as shown in table 2.
Table 2:
example 8: acid resistance test of Bacillus coagulans dry powder
The acid resistance of the bacillus coagulans dry powder is detected by adopting an artificial gastric acid simulation method, and the artificial gastric acid preparation method comprises the following steps: 0.2g of sodium chloride and 0.35g of pepsin are dissolved in a proper amount of distilled water, the pH value is adjusted to 2.0 by using 1mol/L hydrochloric acid, and the volume is determined to the scale by using a 100mL volumetric flask.
The test samples are prepared by adding 10% of the bacterial powder obtained in the examples 4, 5 and 6 and the bacterial powder obtained in the comparative example 1 into a sterile triangular flask simulating artificial gastric acid, carrying out constant-temperature water bath at 37 ℃ for 1h, and then detecting the number of viable bacteria in each group of samples. The survival rate was calculated by taking the number of viable bacteria at 0h as a control. The results show that the groups of examples 4-6 have stronger gastric acid tolerance after coating treatment, and the survival rate of the groups after 1h treatment at pH2.0 is more than 73 percent and is more than 30 percent higher than that of the groups without the coating treatment. Specifically, the results are shown in Table 3.
Table 3:
the preferred embodiments of the present invention have been described in detail, however, the present invention is not limited to the specific details of the above embodiments, and various simple modifications may be made to the technical solution of the present invention within the technical idea of the present invention, and these simple modifications are within the protective scope of the present invention.
It should be noted that the various features described in the above embodiments may be combined in any suitable manner without departing from the scope of the invention. The invention is not described in detail in order to avoid unnecessary repetition.
Claims (3)
1. A preparation method of bacillus coagulans powder is characterized by comprising the following steps:
first, strain activation and seed liquid preparation
Taking out the frozen tube from a refrigerator at the temperature of minus 80 ℃, streaking and inoculating the frozen tube on a test tube slant culture medium, and carrying out anaerobic culture at the temperature of 45-50 ℃ for 18-24 h; inoculating the activated bacillus coagulans into a seed liquid culture medium, and culturing for 18-24h under the conditions of liquid loading capacity of 500mL/1000L, 45-50 ℃ and 200 rpm/min;
the test tube slant culture medium and the seed liquid culture medium are MRS culture media, wherein 20g/L of agar powder is added to the test tube slant culture medium, no agar powder is added to the seed liquid culture medium, the culture medium is sterilized for 20min at 121 ℃, and the culture medium is cooled for standby;
second, preparation of fermentation broth
Inoculating the Bacillus coagulans seed solution into the primary fermentation liquid according to the inoculation amount of 5%, wherein the liquid loading amount is 50L/100L, 45-50 deg.C, 200rpm/min, and the ventilation amount is 1.5m3Culturing for 18-24h under the condition of/h; transferring the primary fermentation liquid into secondary fermentation liquid, wherein the liquid loading amount is 1000L/1500L, 45-50 deg.C, 200rpm/min, and the ventilation amount is 2m3Culturing for 36-48h under the condition of/h;
the first-stage fermentation liquid and the second-stage fermentation liquid culture medium are prepared as follows: 30-50g/L of glucose, 10-15g/L of yeast extract, 10-20g/L of peptone, 2-5g/L of dipotassium phosphate, 0.05-0.1g/L of manganese sulfate, 2-5g/L of ammonium chloride and 15-20g/L of calcium carbonate, sterilizing at 121 ℃ for 20min, and cooling to 45-50 ℃ with water for later use;
thirdly, concentrating and coating the fermentation liquor
Treating the fermentation liquor by 10 nm-specification ceramic membrane filtration equipment to obtain a thallus concentrated solution, wherein the solid content of the thallus concentrated solution is 10-30%; adding 5-10% skimmed milk powder, 1-5% trehalose as protective agent and 0.2-0.5% sodium alginate as coating agent, dissolving and mixing;
the fourth step, spray drying
Centrifugal spray drying equipment is adopted, the conditions are set to be that the air inlet temperature is 110-150 ℃, the air outlet temperature is 80-100 ℃, and the dry powder obtained under the conditions is the bacterial powder.
2. The method for preparing Bacillus coagulans powder according to claim 1, wherein the method comprises the following steps: in the second step, the number of viable bacillus coagulans after fermentation culture can reach 8.0 x 109CFU/mL or more and the spore rate of 90% or more.
3. The method for preparing Bacillus coagulans powder according to claim 1, wherein the method comprises the following steps: the viable count of the bacterial powder obtained in the fourth step is 1.0 x 1011Above CFU/g, and the water content is 5-9%.
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| CN112592870A (en) * | 2021-01-06 | 2021-04-02 | 北京波尔莱特饲料有限公司 | Bacillus coagulans G4, fermentation liquor thereof, preparation method and application |
| CN113846031A (en) * | 2021-09-28 | 2021-12-28 | 天津科技大学 | Preparation and application of mixed bacterium powder for inhibiting multiple pathogenic bacteria |
| CN113930370A (en) * | 2021-11-19 | 2022-01-14 | 广东容大生物股份有限公司 | Development and research of feed additive bacillus coagulans |
| CN113957118A (en) * | 2021-10-29 | 2022-01-21 | 播恩集团股份有限公司 | Detection method for viable count of bacillus coagulans |
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| CN113957118A (en) * | 2021-10-29 | 2022-01-21 | 播恩集团股份有限公司 | Detection method for viable count of bacillus coagulans |
| CN113930370A (en) * | 2021-11-19 | 2022-01-14 | 广东容大生物股份有限公司 | Development and research of feed additive bacillus coagulans |
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