CN110819579A

CN110819579A - Preparation method of solid bacillus subtilis microbial inoculum

Info

Publication number: CN110819579A
Application number: CN201911347786.3A
Authority: CN
Inventors: 毛相朝; 孙建安; 赵甜宇
Original assignee: Ocean University of China
Current assignee: Ocean University of China
Priority date: 2019-12-24
Filing date: 2019-12-24
Publication date: 2020-02-21

Abstract

The invention belongs to the technical field of microbial agents, and discloses a preparation method of a solid bacillus subtilis microbial agent. The preparation method of the solid bacillus subtilis preparation comprises the following steps: (1) preparing a seed solution, namely inoculating a bacillus subtilis strain to an LB culture medium for culture to obtain the seed solution; (2) preparing bacterial sludge, namely inoculating the seed solution into a culture medium according to the inoculation amount of 7-12% for culture, and centrifuging after the culture is finished to obtain the bacterial sludge; (3) preparing a bacterial suspension, mixing the bacterial sludge obtained in the step (2) with 5-15% of glycerol by mass concentration in a proportion of 1: 6-8 to prepare bacterial suspension; (4) and (5) drying by air blowing. According to the method, liquid state fermentation culture is carried out on bacillus subtilis strains to obtain a culture medium with a high spore number, the culture condition is obtained, and then the solid state microbial agent with simple operation and low cost is prepared through a forced air drying process under the protection condition of a carrier and a protective agent.

Description

Preparation method of solid bacillus subtilis microbial inoculum

Technical Field

The invention relates to the technical field of microbial agents, and particularly relates to a preparation method of a solid bacillus subtilis microbial agent.

Background

The bacillus subtilis is a gram-positive rod-shaped bacterium capable of forming endogenous adversity-resistant spores, and can inhibit the growth of harmful bacteria, regulate the balance of intestinal flora, improve the intestinal micro-ecological environment, improve the activity of digestive enzymes, promote the absorption of nutrient substances and improve the immunity. Meanwhile, the bacillus subtilis is a plant rhizosphere growth-promoting bacterium, can be used as a biological fertilizer, and can improve the resistance of crops, promote the growth of the crops, improve the soil and improve the quality of the crops.

In recent years, bacillus subtilis is widely applied to feeds and fertilizers due to the advantages of no toxicity, no harm, strong stress resistance and the like, and can be used as a substitute of antibiotics, chemical fertilizers and pesticides. However, in the preparation process of the microbial inoculum at home and abroad, peptone, yeast powder and the like are mostly adopted for strain fermentation culture, the defects of complex components and high cost exist, the adopted drying technologies are mostly vacuum freeze drying, spray drying and the like, the vacuum freeze drying has wide application range and high survival rate of the microbial inoculum, but equipment is expensive, long time consumption and complex operation, the spray drying technology is simple to operate, continuous production is easy, but heat consumption is large, investment cost is high, and high temperature is not beneficial to survival of the microbial inoculum. Therefore, how to reduce energy consumption cost, shorten preparation period and improve the survival rate of the microbial inoculum in the preparation process of the microbial inoculum has important theoretical value and practical significance for efficient preparation and wide popularization and application of the bacillus subtilis microbial inoculum.

Disclosure of Invention

The invention provides a preparation method of a solid bacillus subtilis microbial inoculum in order to overcome the defects of the background technology. The method utilizes bacillus subtilis strains to carry out liquid fermentation culture to obtain a culture medium with high spore number and culture conditions, and then prepares a solid microbial agent with simple operation and low cost through a forced air drying process under the protection condition of a carrier and a protective agent.

In order to achieve the aim of the invention, the preparation method of the solid bacillus subtilis preparation comprises the following steps:

(1) preparing a seed solution, namely inoculating a bacillus subtilis strain to an LB culture medium for culture to obtain the seed solution;

(2) preparing bacterial sludge, namely inoculating the seed liquid into a culture medium according to the inoculation amount of 7-12% for culture, and centrifuging after the culture is finished to obtain bacterial sludge, wherein the culture medium is an LB culture medium containing one or more of corn starch, bean pulp and glucose;

(3) preparing a bacterial suspension, mixing the bacterial sludge obtained in the step (2) with 5-15% of glycerol by mass concentration in a proportion of 1: 6-8 to prepare bacterial suspension;

(4) and (3) drying by blowing, taking zeolite powder, corn starch, bran powder or shrimp head powder as a drying carrier, and mixing the carrier and the bacterial suspension in a ratio of 2: 3-4, and then blowing and drying for 6-10h at the temperature of 45-55 ℃.

Further, the step (1) is inoculated on the LB culture medium in an inoculation amount of 0.8% -1.2%.

Further, the step (1) is carried out for 22-26h at 28-32 ℃ in a shaking flask at 180-220 rpm.

Further, the culture medium in the step (2) is LB culture medium containing a mixture of 2.3-2.8% by mass of corn starch, 2.8-3.2% by mass of soybean meal and 1.8-2.2% by mass of glucose.

Preferably, the medium in the step (2) is an LB medium containing a mixture of 2.5% by mass of corn starch, 3% by mass of soybean meal and 2% by mass of glucose.

Preferably, the seed solution in the step (2) is inoculated into a culture medium for culture according to the inoculation amount of 7.5% -8.5%.

Further, the step (2) is carried out for 22-26h at 28-32 ℃ in a shaking flask at 180-220 rpm.

Further, the centrifugation in the step (2) is performed at 4500 5500rpm at 3-5 ℃ for 10-20 min.

Preferably, the bacterial sludge in the step (3) is mixed with glycerol with the pH of 6.5-7.5 and the mass concentration of 4.5% -5.5% in a ratio of 1: redissolving at a ratio of 6.5-7.5 to obtain a bacterial suspension.

More preferably, the shrimp head powder is used as the drying carrier in the step (4).

Further preferably, the step (4) is carried out for 6h by blowing and drying at 50 ℃.

Compared with the prior art, the liquid fermentation medium has simple components, low price, short fermentation period, high cultured spore number, simple and convenient preparation method of the solid microbial agent, easy operation, low cost and suitability for industrial production, and the raw materials of the solid microbial agent, such as soybean meal, corn starch, shrimp heads and the like, are green and easy to obtain.

Drawings

FIG. 1 shows the influence of the addition amounts of corn starch (a), soybean meal (b) and glucose (c) on the number of spores in example 1 of the present invention;

FIG. 2 is a graph showing the effect of the carrier species on the survival rate of the cells in example 5 of the present invention, wherein Zeolite is zeolite powder, corn starch, wheat bran powder and shrimp head powder;

FIG. 3 is a graph showing the effect of the mixing ratio of the carrier and the bacterial suspension on the survival rate of the bacterial cells in example 6 of the present invention;

FIG. 4 is a graph showing the effect of protecting agent species on the survival rate of the cells in example 7 of the present invention, wherein Trehalo is Trehalose, skim milk powder, glycerol, soluble starch, chitosan and concentrate are comparative examples which are still reconstituted with physiological saline;

FIG. 5 shows the effect of protectant concentration on bacterial survival in example 8 of the present invention;

FIG. 6 shows the effect of the ratio of bacterial sludge to protective agent on the survival rate of bacterial cells in example 9 of the present invention.

Detailed Description

In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is described in further detail below with reference to the accompanying drawings and embodiments. Additional aspects and advantages of the invention will be set forth in part in the description which follows and, in part, will be obvious from the description, or may be learned by practice of the invention. It is to be understood that the following description is only illustrative of the present invention and is not to be construed as limiting the present invention.

The terms "comprises," "comprising," "includes," "including," "has," "having," "contains," "containing," or any other variation thereof, as used herein, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises a list of elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

When an amount, concentration, or other value or parameter is expressed as a range, preferred range, or as a range of upper preferable values and lower preferable values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or preferred value and any lower range limit or preferred value, regardless of whether ranges are separately disclosed. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted to include the ranges "1 to 4", "1 to 3", "1 to 2 and 4 to 5", "1 to 3 and 5", and the like. When a range of values is described herein, unless otherwise stated, the range is intended to include the endpoints thereof and all integers and fractions within the range.

The indefinite articles "a" and "an" preceding an element or component of the invention are not intended to limit the number requirement (i.e., the number of occurrences) of the element or component. Thus, "a" or "an" should be read to include one or at least one, and the singular form of an element or component also includes the plural unless the number clearly indicates the singular.

The technical features of the embodiments of the present invention may be combined with each other as long as they do not conflict with each other, and the reagents are all analytically pure.

The method for measuring the number of spores in each embodiment of the present invention comprises: and (3) directly measuring the liquid bacterial liquid, and measuring after preparing the solid microbial inoculum into a leaching solution (2 g of a solid sample is added with 20mL of sterile water containing 7-10 glass beads and placed in a constant temperature oscillator with 200rpm to vibrate for 30min to obtain the leaching solution). Placing the bacterial liquid/leaching solution in a water bath at 80 ℃ for 15min, sucking 100 mu L of bacterial liquid/leaching solution, adding the bacterial liquid/leaching solution into 900 mu L of sterile physiological saline, uniformly mixing to prepare 1:10 sample uniform solution, sequentially carrying out 10-time serial dilution, respectively sucking 100 mu L of dilution solution with each dilution degree, injecting the dilution solution into a flat plate counting culture medium, uniformly mixing and paving the dilution solution by using a coating rod, inverting, culturing for 24h in a constant temperature incubator at 30 ℃, and selecting a culture dish with the colony number between 30 and 300 to count the colony number.

The water content is measured by a full-automatic water content measuring instrument.

The calculation formula of the product yield is as follows:

the survival rate is calculated as follows:

the calculation formula of the centrifugal recovery rate is as follows:

the preparation method of the solid bacillus subtilis preparation comprises the following steps:

(1) preparing a seed solution, namely inoculating a bacillus subtilis glycerol tube strain preserved at the temperature of-20 ℃ to an LB culture medium for culture to obtain the seed solution;

(2) preparing bacterial sludge, namely inoculating the seed liquid into a culture medium according to the inoculation amount of 6-8% for culture, and centrifuging after the culture is finished to obtain bacterial sludge, wherein the culture medium is an LB culture medium containing one or more of corn starch, bean pulp and glucose;

(3) preparing bacterial suspension, mixing bacterial mud obtained in the step (2) with physiological saline with the pH value of 7.0, and mixing the mixture in a ratio of 1:7 to prepare bacterial suspension;

(4) drying, mixing the carrier and the bacterial suspension, and drying at 50 ℃ for 6 h.

If not specifically stated, inoculating the LB culture medium with an inoculum size of 1% in the step (1); culturing for 24 hours in a shaking flask at the temperature of 30 ℃ and the speed of 200rpm in the step (1); culturing for 24 hours in a shaking flask at the temperature of 30 ℃ and the speed of 200rpm in the step (2); centrifuging at 5000rpm for 15min at 4 ℃ in the step (3); and (3) in the step (4), the drying mode is blast drying, the shrimp head powder is used as a drying carrier, and the ratio of the carrier to the bacterial suspension is 1: 2, and mixing.

Example 1

Inoculating a bacillus subtilis glycerol tube strain preserved at the temperature of minus 20 ℃ to an LB culture medium for culture to obtain a seed solution, inoculating the seed solution to the LB culture medium containing one of corn starch, soybean meal and glucose according to the inoculation amount of 7 percent for culture, culturing at the temperature of 30 ℃ and the rpm of 200 for 28h, and determining the number of spores. As shown in FIG. 2, with the increase of the mass contents of corn starch, soybean meal and glucose, the number of spores tends to increase first and then decrease, indicating that the concentration of the three substances is too high or too low and is not suitable for the formation of spores. At a corn starch content of 2%, the number of spores was the greatest (FIG. 1 a). The change of the content of the bean pulp has great influence on the number of spores, the difference of the number of spores is more obvious when the content is different, and the number of spores is the maximum when the content of the bean pulp is 3 percent and can reach 4.4 multiplied by 10⁹CFU/mL (FIG. 1 b). Neither glucose contents above or below 2.5% are suitable for maximum spore formation, and at a glucose content of 2.5%, the number of spores is the greatest (FIG. 1 c).

Example 2

Different from the example 1, the seed solution is inoculated into LB culture medium containing a mixture of corn starch, bean pulp and glucose according to the inoculation amount of 7% for culture, and the culture medium with the optimal ratio is determined by changing the addition amount of the corn starch, the bean pulp and the glucose. The number of spores is shown in Table 1.

TABLE 1 influence of varying amounts of corn starch, soybean meal, and glucose added on the number of spores

As can be seen from table 3, when the cells were cultured in LB medium containing a mixture of corn starch, soybean meal, and glucose, the number of spores was significantly larger than that of LB medium containing one of corn starch, soybean meal, and glucose, and the number of spores of bacillus subtilis was affected by each of the factors corn starch, soybean meal, and glucose. By analysis, the optimal scheme of the culture medium is LB culture medium containing a mixture of 2.5% of corn starch, 3% of soybean meal and 2% of glucose in mass fraction, and the spore number reaches 3.9X 10¹⁰CFU/mL。

Example 3

The optimal ratio of the culture medium in example 2 was selected, and on the basis of this, the culture temperature or the inoculation amount of the seed solution was changed, and when only the culture temperature was changed or only the inoculation amount was changed, the number of spores was as shown in Table 2.

TABLE 2 Effect of changing the cultivation temperature or the inoculum size on the number of spores

Example 4

The drying techniques commonly used at present include vacuum freeze drying, spray drying, forced air drying, microwave vacuum drying and the like. The time cost and the operation cost of vacuum freeze drying and microwave vacuum drying are higher, and the method is not suitable for commercial large-scale production. In the experiment, the culture medium with the best ratio in example 2 was selected, the inoculation amount was changed to 8%, and on the basis, two drying methods of spray drying and forced air drying were selected to prepare the microbial inoculum according to the method described in the embodiment of the present invention (i.e., the method described in example 1 above) for comparison. The results of the experiment are shown in Table 5.

TABLE 5 preparation of the microbial inoculum by different drying methods

As shown in Table 5, the survival rate and yield of spores were higher than those of zeolite powder when spray-dried using corn starch as an adjuvant, but the yield was only 30%. In the blast drying experiment, the zeolite powder is used as a carrier for drying, the survival rate and the yield of spores are higher than those of the corn starch used as the carrier and are far higher than those of the corn starch or the zeolite powder used as an auxiliary agent for spray drying, and solid matters are basically not lost. In consideration of higher heat energy and electric energy consumption of spray drying and lower yield, the method selects forced air drying to prepare the microbial inoculum.

Example 5

The product quality and the use effect of the microbial inoculum can be ensured only by selecting a proper matrix as a carrier for adsorbing the bacterial liquid. On the basis of selecting the forced air drying preparation microbial inoculum in the embodiment 4, in the embodiment, besides selecting zeolite powder or corn starch as a drying carrier, bran powder or shrimp head powder is also selected as the drying carrier, and as can be seen from fig. 2, when the shrimp head powder is used as the drying carrier, the survival rate is higher than that of other carriers and exceeds 100%, which may be because original vegetative cells are dormant due to severe environment in the drying process to form spores, or the shrimp heads may be used as a nutrient substance, so that the bacterial cells are favorable for re-proliferation in the drying process with low temperature. The shrimp waste contains valuable compounds such as chitin, protein, lipid, calcium salt and the like, but most shrimp byproducts cannot be utilized. The increasing amount of shrimp wastes causes serious environmental and economic problems, so that the shrimp wastes are used as microbial agent carriers to be utilized with high value, and the shrimp wastes have important significance.

Example 6

In example 5, the bacterium agent is prepared by forced air drying, and the ratio of the carrier to the bacterium suspension is changed on the basis of selecting shrimp head powder as a dry carrier, so that the survival rate of the bacterium is shown in figure 3. As can be seen from FIG. 3, the survival rate tended to increase first and then decrease as the amount of the carrier added decreased. Within a certain range, the increase of the addition amount of the bacterial suspension causes the increase of the combination of the thalli and the carrier, the survival rate is increased, and the survival rate is the highest when the carrier and the bacterial suspension are mixed in a ratio of 2: 3. When the addition amount of the bacterial suspension is increased to a certain degree, the carrier is supersaturated, and thalli cannot be combined with the carrier, so that the survival rate is reduced, and when the carrier and the bacterial suspension are mixed in a ratio of 1:3, a small amount of bacterial liquid cannot be adsorbed.

Example 7

In the drying process, the cells are dehydrated to cause the increase of osmotic pressure, the atrophy of cells and the denaturation of proteins, thereby causing the death of thalli. As shown in FIG. 4, trehalose, skimmed milk powder, glycerol, soluble starch, and chitosan were added to physiological saline as protective agents (all at 7 mass%), and the survival rate was higher than that of cells reconstituted with physiological saline directly, and the survival rate was highest when trehalose, skimmed milk powder, and glycerol were added as protective agents. The glycerol as an osmotic protective agent can enter cells to maintain the osmotic pressure, and meanwhile, the hydroxyl group of the glycerol can replace water molecules to form hydrogen bonds with proteins on cell membranes, so that the structure and the function of the proteins are maintained and protected. When the soluble starch and the chitosan are used as protective agents, the survival rate of the bacterial suspension is lower than that of bacterial suspension redissolved by normal saline, probably because the chitosan is poor in solubility after sterilization and cannot completely play a role, and the soluble starch is in a gel state after sterilization and cooling and is not beneficial to operation.

Example 8

The concentration of glycerol was varied based on the selection of glycerol as the protectant in example 7. As shown in FIG. 5, the survival rate of the cells can be improved by adding glycerol, the survival rate is highest and can reach about 140% when the concentration of the glycerol is 5%, the survival rate begins to decrease with the increase of the concentration of the glycerol, which may be related to the increase of osmotic pressure, and the product becomes sticky after adding 20% of the glycerol due to the overhigh concentration.

Example 9

In example 8, the ratio of the bacterial sludge to the protective agent was changed on the basis of 5% glycerol as the protective agent. As can be seen from FIG. 6, in a certain range, the smaller the ratio of the bacterial sludge to the protective agent, i.e., the greater the addition amount of the protective agent, the higher the survival rate, because the insufficient addition amount of the protective agent can make a large amount of bacterial cells be unprotected due to exposure, and the survival rate is affected. When the mass/volume mixing ratio of the bacterial sludge to the glycerol is 1:7, the survival rate is the best and can reach about 150%. As the ratio continues to increase, the survival rate decreases, probably because of the change in the concentration of the protectant around the cells due to the different mixing ratios of the bacterial sludge and the protectant, and too high a concentration affects the osmotic pressure and permeability of the Bacillus subtilis cells, resulting in poor survival rate.

Example 10

In example 9, the mass/volume mixing ratio of the bacterial sludge to the glycerol is 1:7, investigating the influence of the drying time on the viable count, wherein the moisture content of the solid fermentation sample is gradually reduced along with the increase of the drying time and is basically stable after 6 h. With the prolonging of the drying time and the reduction of the moisture content, the dry matter in unit mass increases, the number of the viable bacteria in unit mass increases, and after 6 hours, the number of the viable bacteria tends to be stable, and the drying time of 6-10 hours can be selected as the optimal time for preparing the microbial inoculum in consideration of the time cost.

It will be understood by those skilled in the art that the foregoing is only exemplary of the present invention, and is not intended to limit the invention, which is intended to cover any variations, equivalents, or improvements therein, which fall within the spirit and scope of the invention.

Claims

1. A preparation method of a solid-state bacillus subtilis preparation is characterized by comprising the following steps:

2. The method for preparing a solid-state Bacillus subtilis preparation according to claim 1, wherein the step (1) is carried out by inoculating the solid-state Bacillus subtilis preparation to LB medium in an inoculation amount of 0.8-1.2%.

3. The method for preparing a solid-state Bacillus subtilis preparation as claimed in claim 1, wherein the step (1) is carried out at 28-32 ℃ and 180-220rpm in a shake flask for 22-26 h.

4. The method for preparing a solid-state bacillus subtilis microbial inoculum according to claim 1, wherein the culture medium in the step (2) is an LB culture medium containing a mixture of 2.3-2.8% by mass of corn starch, 2.8-3.2% by mass of soybean meal and 1.8-2.2% by mass of glucose.

5. The method for preparing a solid-state Bacillus subtilis microbial inoculum according to claim 4, wherein the culture medium in the step (2) is LB culture medium containing a mixture of 2.5 mass percent of corn starch, 3 mass percent of soybean meal and 2 mass percent of glucose.

6. The method for preparing a solid-state Bacillus subtilis preparation as claimed in claim 1, wherein the seed solution obtained in step (2) is inoculated into a culture medium according to an inoculum size of 7.5-8.5%.

7. The method for preparing a solid-state Bacillus subtilis preparation as claimed in claim 1, wherein the step (2) is carried out at 28-32 ℃ and 180-220rpm in a shake flask for 22-26 h.

8. The method for preparing a solid-state Bacillus subtilis microbial inoculum according to claim 1, wherein the centrifugation in the step (2) is performed at 4500 5500rpm at 3-5 ℃ for 10-20 min.

9. The method for preparing a solid-state bacillus subtilis microbial inoculum according to claim 1, wherein the bacterial sludge in the step (3) is mixed with glycerol with the pH of 6.5-7.5 and the mass concentration of 4.5% -5.5% in a ratio of 1: redissolving at a ratio of 6.5-7.5 to obtain a bacterial suspension.

10. The method for preparing the solid-state bacillus subtilis preparation as claimed in claim 1, wherein in the step (4), shrimp head powder is used as a drying carrier; preferably, the step (4) is carried out for 6h by blowing and drying at 50 ℃.