CN113234791A

CN113234791A - Count detection method of bacillus coagulans

Info

Publication number: CN113234791A
Application number: CN202110709630.6A
Authority: CN
Inventors: 刘笑尘; 李鑫
Original assignee: Changsha Heguang Biotechnology Co ltd
Current assignee: Changsha Heguang Biotechnology Co ltd
Priority date: 2021-06-25
Filing date: 2021-06-25
Publication date: 2021-08-10
Anticipated expiration: 2041-06-25
Also published as: CN113234791B

Abstract

The invention relates to a bacillus coagulans counting and detecting method, which comprises the following steps: (S1) performing the following operations of the sample to be tested in an aseptic operation: diluting, thermally activating, ultrasonically dispersing and homogenizing to obtain a sample diluent; (S2) carrying out continuous gradient dilution on the sample diluent until the estimated bacteria content of the sample diluent is 10-300CFU/mL, and then finishing the dilution; (S3) after culturing the sample of the appropriate dilution gradient in BC medium at pH 5.0-5.5, the colony count of Bacillus coagulans is performed. The method can be widely applied to detection of different bacillus coagulans strains, and has the advantages of accurate counting result, small average error and detection period of only 26-30 hours to obtain the result.

Description

Count detection method of bacillus coagulans

Technical Field

The invention relates to the technical field of microbial detection, in particular to a method for detecting bacillus coagulans nutriments and spores in bacillus coagulans liquid or powder.

Background

Bacillus coagulans (Bacillus coagulans) is the only Bacillus lactobacillus which is currently licensed by governments of various countries to be added into food and feed in the world. The main beneficial function of the feed additive is that lactic acid is produced in the intestinal metabolism process, and the feed additive has very important effects on promoting digestion and absorption and maintaining the balance of intestinal flora. Meanwhile, because the spores have excellent stress resistance such as high temperature resistance, acid and alkali resistance, cholate resistance and the like, the application range of probiotics in the food industry is greatly expanded, bacillus coagulans is concerned by numerous food and feed enterprises in recent years, and bacillus coagulans products are sold on the market by adding a lot of bacillus coagulans.

As compared with other traditional probiotics, the research and application time of the bacillus coagulans is short, no unified detection method is published in national standards, and the detection method of each unit is only suitable for self-researched strains. The bacterial count detection is systematic work, polysaccharide and glycoprotein secreted by bacillus coagulans in the metabolic process can cause strong adhesion state among thalli, and the bacillus coagulans is difficult to disperse by a common treatment mode. Therefore, any one of the pretreatment mode, the dispersion mode, the culture medium components and the culture conditions in the bacillus coagulans counting detection may cause the counting result to be inaccurate, the average error to be too high and the like.

Patent CN202010644874.6 discloses a detection method of bacillus coagulans powder spores, relating to the technical field of microorganism detection and comprising the following steps: (1) adding a bacillus coagulans powder sample to be detected into sterile physiological saline containing Tween 80 and provided with glass beads, and homogenizing on a constant-temperature oscillator to prepare a sample homogeneous solution; (2) drawing the homogenized sample solution for water bath heating, taking out after heating, and cooling to room temperature to obtain a sample solution to be detected; (3) and (3) carrying out gradient dilution on the sample solution to be detected by 10 times, taking the sample solution to be detected with proper dilution gradient, culturing, observing and detecting. This patent adds the tween 80 of certain concentration through the normal saline for the sample homogeneity dilution and carries out the improvement test, under the active condition of not influencing sample growth, has played the effect of effectual dispersion sample bacterial powder spore. However, the patent has the following disadvantages: (1) after the water bath heating is carried out and the homogenization is carried out, dispersed bacteria are easy to agglomerate again after settling in the standing and heating environment, and the counting result is deviated. (2) The bacteria powder is dispersed in a triangular flask by shaking the normal saline containing the Tween 80, so that the bacteria powder is suitable for dispersing the bacteria with low bonding degree, and the problem of the bacterial dispersity under the strong bonding state cannot be thoroughly solved. (3) It is well known that the growth of bacteria requires suitable media components and culture conditions, and this patent does not provide media components and culture conditions that are widely applicable to each bacillus coagulans strain, with a narrow range of applicability. (4) The patent needs to culture results after 48 +/-2 hours, has long detection period and is not beneficial to actual production. Therefore, in order to solve the above problems, a method which has a small error in result and can be widely applied to detection of different bacillus coagulans strains is needed.

Disclosure of Invention

Aiming at the defects of the prior art, the invention aims at wide applicable range of bacillus coagulans strains, accurate counting result, small average error and short detection period, researches a diluent formula, a culture medium formula, culture conditions and a dispersion mode of bacteria in a strong bonding state, and provides a bacillus coagulans counting detection method. The method can be widely applied to detection of different bacillus coagulans strains, and has the advantages of accurate counting result, small average error and detection period of only 26-30 hours to obtain the result.

The technical scheme adopted by the invention for solving the technical problems is as follows:

a bacillus coagulans counting detection method comprises the following steps:

(S1) performing the following operations of the sample to be tested in an aseptic operation: diluting, thermally activating, ultrasonically dispersing and homogenizing to obtain a sample diluent;

(S2) carrying out continuous gradient dilution on the sample diluent until the estimated bacteria content of the sample diluent is 10-300CFU/mL, and then finishing the dilution;

(S3) after culturing the sample of the appropriate dilution gradient in BC medium at pH 5.0-5.5, the colony count of Bacillus coagulans is performed.

Further, in the step (S1), the sample to be tested is bacillus coagulans powder or bacillus coagulans liquid.

Further, the dilution in step (S1) is 10-1000 times, preferably 100-200 times. The dilution is carried out using a sterile diluent containing 0.5-2 wt% of a surfactant and 0.5-1.5 wt% of sodium chloride. Preferably, the sterile diluent contains 1.0-1.5 wt% surfactant and 0.9-1.2 wt% sodium chloride.

Preferably, the surfactant is a mixture of an anionic surfactant and a nonionic surfactant, and the anionic surfactant is selected from at least one of sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, ammonium dodecyl sulfate and sodium dodecyl polyoxyethylene ether sulfate; the HLB of the nonionic surfactant is 12-16, and is specifically selected from at least one of Tween 40, Tween 60, Tween 80, OP-10, polyoxyethylene alkylphenol, and polyoxyethylene monooleate.

More preferably, the mass ratio of the anionic surfactant to the nonionic surfactant is 4 to 6: 1.

further, the heat activation temperature of the step (S1) is 65-85 ℃ and the time is 2-15 min. Preferably, when the kit is used for detecting the bacillus coagulans liquid, the temperature is 65-70 ℃, and the time is 2-7 min; when the kit is used for detecting the bacillus coagulans powder, the temperature is 75-85 ℃, and the time is 7-15 min.

Further, the frequency of the ultrasonic transducer for ultrasonic dispersion in the step (S1) is 19-40KHz, the power is 50-200w, the period is 5-30S of ultrasound every 5-30S, and the total ultrasonic time is 60-120S; the beating frequency of the beating homogenizing sterile homogenizer is 3-12 times/second, and the beating time is 1-10 min; preferably, the beating frequency of the sterile homogenizer is 4-8 times/second, and the beating time length is 4-6 min.

Preferably, the dilution series in step (S2) is performed by serial dilution according to a multiple of 1:5-30, preferably according to a multiple of 1:10-20, until the estimated bacteria content in one dilution tube is 30-100CFU/mL, which is a suitable dilution for testing the colony number.

Preferably, the culturing in step (S3) is performed in a BC culture plate, and the BC culture medium comprises the following components in terms of weight-to-volume ratio of each component: 5-15g/L of tryptone, 4-10g/L of yeast extract, 4-10g/L of glucose, 4-8g/L of beef extract, 1-2g/L of sodium acetate, 0.5-2.0g/L of dipotassium phosphate, 0.1-0.7g/L of L-cysteine hydrochloride, 0.1-0.5g/L of anhydrous calcium chloride, 0.05-0.20g/L of manganese sulfate monohydrate and 15-30g/L of agar powder; preferably, each component comprises the following components in percentage by weight and volume: 10-12g/L of tryptone, 6-8g/L of yeast extract, 6-8g/L of glucose, 6-8g/L of beef extract, 1.2-1.5g/L of sodium acetate, 1-1.5g/L of dipotassium phosphate, 0.3-0.5g/L of L-cysteine hydrochloride, 0.2-0.3g/L of anhydrous calcium chloride, 0.1-0.15g/L of manganese sulfate monohydrate and 20-25g/L of agar powder.

Further, when preparing the culture medium, deionized water is used for fully dissolving all components except the agar powder, the volume is determined to be the corresponding volume, then the pH value is adjusted to 5.0-5.5 by using dilute acid, the dilute acid is dilute phosphoric acid or dilute hydrochloric acid, finally the agar powder is added, and the BC culture plate can be poured after sterilization. The culture conditions are that the culture temperature is 42-48 ℃ and the culture time is 20-30 h.

Preferably, the step (S3) of counting colonies adopts a two-step counting method, wherein the colonies are cultured for 20-24h, and then returned to the incubator for further culture, and newly grown colonies are counted again when the colonies are cultured for 26-30 h. To prevent closely spaced colonies from adhering during the culturing process, two or more adhered colonies were counted as one. The first counting is to count the number of bacillus coagulans colonies in each plate accurately when the bacillus coagulans is cultured for 20-24h, count the number of newly grown colonies when the bacillus coagulans is cultured for 26-30h, and count the first counting and the second counting as the final number of colonies of each plate.

When counting, the bacillus coagulans colony meets the following conditions: culturing for 20-30h to obtain Bacillus coagulans colony with diameter of 0.8-3.2mm, milky-white to yellowish color, neat and non-wrinkled edge, slight luster, and slight bulge, and its surface is frosted when observed with magnifier.

Further, the method for calculating the bacteria content of the sample in the step (S4) is: typically, the number of colonies on a single plate will have a gradient of 1 or 2 between 30 and 300, and a single plate will have a colony number between 30 and 300 and will be an authentic plate, and the bacillus coagulans content N of the sample is calculated by the following equation:

in the formula, X₁And X₂The number of the credible plates under the same gradient is obtained; sigma 1 and sigma 2 are the sum of the colony numbers of the credible plates under the corresponding gradients; d₁And D₂Is the dilution factor of the gradient; v is the volume of diluent used for plate coating in mL; k is toA constant in the average, K is 0.5 when neither Σ 1 nor Σ 2 is 0, and K is 1 when one of Σ 1 and Σ 2 is 0.

The invention has the following beneficial effects:

the method can detect the content of spores in bacillus coagulans powder and the content of vegetative thalli in bacillus coagulans liquid in the production process by performing water bath heat shock, the water bath heat shock can kill the vegetative thalli in a sample to be detected and can also perform heat shock on the spores of the bacillus coagulans, and the time for the bacillus coagulans subjected to heat shock to generate visible colonies is 4-6 hours earlier than that before the heat shock, so that the method is favorable for shortening the detection time in the actual scientific research and production processes, and has beneficial social value.

The sterile diluent uses the compounding of the anionic surfactant and the nonionic surfactant, and can play a better dispersing role and improve the detection accuracy compared with the single surfactant.

The invention combines ultrasonic treatment and homogenizer homogenization, and adds proper amount of dispersing emulsifier sodium dodecyl sulfate in the diluent, thus thoroughly solving the problems of tight cohesive mass and difficult dispersion between bacillus coagulans, having small counting detection result preparation and average error, and effectively solving the problem of poor result repeatability during the counting of bacillus coagulans.

The BC culture medium formula provided by the invention is specially developed for detecting the bacillus coagulans, and is matched with a more appropriate culture temperature, so that the growth cycle of bacillus coagulans bacterial colonies is further shortened by 14-16h compared with other methods, the culture and detection time is shortened to 26-30h, the result reporting time is effectively advanced, and the BC culture medium formula has remarkable effects of shortening the scientific research cycle and improving the production efficiency.

In the invention, the pH value of the BC culture medium is adjusted to 5.0-5.5, and Ca in the culture medium can be ensured in a proper acidic environment²⁺And Mn²⁺In a free state without influencing the growth and reproduction of the bacillus coagulans, and the Ca with proper concentration²⁺And Mn²⁺Is sporulation of Bacillus coagulans andcompared with the culture medium with the same formula and pH of 6.0-6.8, the colony number of the bacillus coagulans of the same sample is increased, which indicates that other detection methods have poor dispersion effect, and the culture medium is not special for the bacillus coagulans, so that the bacteria number is low during detection, and the method has very important significance for accurately detecting the content of the bacillus coagulans by scientific research and production workers.

Detailed Description

The invention is described below with reference to specific embodiments. Unless otherwise specified, the technical means used in the present invention are methods well known to those skilled in the art, and the raw materials or equipment used are obtained by conventional commercial methods. In addition, the embodiments should be considered illustrative, and not restrictive, of the scope of the invention, which is defined solely by the claims. It will be apparent to those skilled in the art that various changes or modifications can be made in the components and amounts of the materials used in these embodiments without departing from the spirit and scope of the invention.

The water used in the embodiment of the invention is deionized water; the experimental reagent is of reagent grade; all instruments, reagents and the like are commercially available in the conventional manner unless otherwise specified.

The experimental operations carried out in the examples of the present invention are all aseptic operations unless otherwise specified.

Example 1

Counting and detecting the content of spores in bacillus coagulans powder, wherein the bacillus coagulans powder is a product of a certain domestic company, and the content of the bacillus coagulans powder is 100 multiplied by 10 according to the package indication⁸CFU/g, abbreviated herein as sample No. 1.

1) And (5) preparing sterile diluent. Accurately weighing 4.5g of sodium chloride, 4g of sodium dodecyl sulfate and 1g of Tween 60, fully dissolving with deionized water, diluting to a constant volume of 500mL, placing in a 1L conical flask, sterilizing at 121 ℃ for 15min, and cooling to room temperature for later use.

2) And (4) preparing a BC flat plate. Accurately weighing 3g of tryptone, 1.8g of yeast extract, 1.8g of glucose, 1.8g of beef extract, 0.45g of sodium acetate, 0.3g of dipotassium phosphate, 0.09g of L-cysteine hydrochloride, 0.06g of anhydrous calcium chloride and 0.03g of manganese sulfate monohydrate, fully dissolving by using a small amount of hot deionized water, then fixing the volume to 300mL, adjusting the pH to 5.0 by using phosphoric acid, weighing agar powder and the culture medium, pouring the agar powder and the culture medium into a 500mL conical flask, taking out the agar powder after sterilizing for 15min at 115 ℃, subpackaging 20-30mL of the agar powder in each 90mm glass culture dish, and cooling for later use.

3) And (4) activating by heat. And (3) weighing 198mL of the sterile diluent in a sterile homogenizing bag, accurately weighing 2g of the No. 1 sample in the sterile homogenizing bag by using an analytical balance (the precision is 0.1mg), sealing, shaking up, carrying out water bath at 80 ℃ for 10min, and cooling to room temperature in cold water to obtain a sample pretreatment solution.

4) And (4) ultrasonic dispersion. And (3) placing the sterile homogenizing bag filled with the sample pretreatment liquid in an ultrasonic cleaning machine, setting the frequency of an ultrasonic transducer to be 30KHz, the power to be 100w, the period to be 10s of ultrasonic every 15s, and the total ultrasonic time to be 60 s.

5) Beating and homogenizing. And (3) placing the sample treatment solution subjected to ultrasonic dispersion in a sterile homogenizer, setting the beating frequency to be 6 times/second and the beating time to be 5min, and obtaining a 1:100 sample diluent.

6) And (4) performing gradient dilution. Taking 6 sterile glass test tubes of 25mL, subpackaging 9mL sterile diluents for each test tube, sequentially marking the test tubes as 3-8 from the first test tube, sucking 1000 mu L of the sample diluent into the first test tube, and uniformly mixing for 60s by using a vortex mixer to obtain 1: 1000 sample dilutions, and continuously repeating the 1:10 gradient dilution step until dilution in test tube No. 8 is complete, where the estimated bacteria content is around 10 CFU/mL.

7) And (4) coating a flat plate. Pipette 100. mu.L of the dilution from test tubes 6, 7, and 8 into the BC plate prepared in step 2), coat it uniformly with a glass coater, and then stand for 15min to allow the dilution to be completely absorbed, and coat it 3 times.

8) And (3) inverting the flat plate obtained in the step 7), putting the flat plate into a biochemical incubator, culturing at the culture temperature of 45 ℃ for 22h, counting colonies, putting the flat plate back into the incubator for continuous culture, and counting newly grown colonies when the flat plate is cultured for 28 h.

9) When the bacillus coagulans is cultured for 22-28h, the diameter of the bacterial colony is between 0.8-3.2mm, the bacterial colony is milky-white to light yellow, the edge is neat, has no wrinkles, is slightly glossy and slightly convex, and the number of the bacterial colonies with the typical bacillus coagulans bacterial colony morphology with a frosted surface is observed by using a magnifying glass, and the calculation result is shown in the following table 1:

TABLE 1

Test results were averaged over three tests in test tube No. 7. In the following examples, unless otherwise specified, the final test result was obtained as the average of three tests performed on test tube No. 7.

Example 2

The other conditions are the same as example 1, except that the amount of sodium dodecyl sulfate used in step 1) is 6g, the amount of tween 60 is 1.5g, and after the BC medium in step 2) is prepared to a constant volume, the pH is adjusted to 5.5 using phosphoric acid, and the detection results are shown in table 2 below:

TABLE 2

Example 3

The other conditions were the same as in example 1 except that in step 1), the amount of sodium laurylsulfate was 6g, the amount of tween 60 was 1g, and the results of the measurements are shown in table 3 below:

TABLE 3

Example 4

The other conditions were the same as in example 1 except that in step 1), 3g of sodium lauryl sulfate and 3g of Tween 60 were used. The results are shown in table 4 below:

TABLE 4

Example 5

The other conditions were the same as in example 1 except that in step 1), 5g of sodium lauryl sulfate was used and no Tween 60 was added. The results are shown in table 4 below:

TABLE 4

Example 6

The other conditions were the same as in example 1 except that 5g of Tween 60 was used in step 1) and sodium laurylsulfate was not added. The results are shown in table 6 below:

TABLE 6

Example 7

And (3) counting and detecting the content of the nutrient in the bacillus coagulans fermentation liquor, wherein the bacillus coagulans fermentation liquor is self-made in a laboratory, and the estimated content is 25-30 multiplied by 10⁸CFU/g, abbreviated herein as sample No. 2.

The other conditions are the same as example 1, except that 198mL of the sterile diluent is weighed in the sterile homogenizing bag in the step 3), 2mL of the No. 2 sample is accurately sucked in the sterile homogenizing bag by using a pipettor, the sample is sealed and shaken uniformly, and after water bath at 70 ℃ for 3min, the sample is cooled to room temperature in cold water to obtain a sample pretreatment solution. The results are shown in table 7 below:

TABLE 7

Comparative example 1

The other conditions were the same as in example 1, except that step 4) and step 5) were performed first, and then step 3) was performed, that is, the order of thermal activation, ultrasonic dispersion and beating homogenization was changed, and the results of the test of sample 1 were as shown in the following table: the results are shown in Table 8 below:

TABLE 8

Comparative example 2

The other conditions were the same as in example 1, except that 198mL of sterile diluent was measured in a sterile homogenizer bag, 2g of sample No. 1 was accurately measured in a sterile homogenizer bag using an analytical balance (precision 0.1mg), and after sealing and shaking up, the homogenizer bag was directly placed in an ultrasonic cleaning machine for ultrasonic treatment. I.e. without thermal activation in step 3). It was found that the colony counts could not be counted effectively at 22h and 28h, and that the absence of heat activation had the following effects: 1, spore germination efficiency is reduced, and colonies grow slowly, so that in the subsequent culture process, results can be obtained after 48 hours. 2, some of the spores that did not germinate soon could not be counted, resulting in a lower outcome.

Comparative example 3

The other conditions were the same as in example 1 except that the BC medium in step 2) was changed to MRS medium as follows: accurately weighing 3g of tryptone, 3g of beef extract, 1.5g of yeast extract, 1.5g of glucose, 1.5g of sodium acetate, 1.5g of diammonium citrate, 0.6g of dipotassium phosphate, 0.06g of tween-800.3, 0.06g of magnesium sulfate heptahydrate and 0.015g of manganese sulfate monohydrate, fully dissolving, fixing the volume to 300mL, adding 6g of agar powder, sterilizing at 115 ℃ for 15min, and performing water bath heat preservation at 50 ℃ for later use.

In the step 7), the flat plate coating is changed into flat plate pouring, and the method comprises the following steps: sucking 100 mu L of diluent from a No. 6 test tube, pouring 20-30mL of MRS culture medium reserved after the step 2) is changed into a sterile glass culture dish, slightly rotating the culture dish to fully and uniformly mix the diluent with the culture medium, and standing at room temperature until the culture medium is completely solidified; this procedure was repeated, and test tubes 7 and 8 were each poured for 3 replicates.

The results are shown in table 9 below:

TABLE 9

Comparative example 4

The other conditions are the same as example 1, except that after the BC medium in step 2) is prepared to a constant volume, the pH is adjusted to 6.5 by using phosphoric acid, and the detection results of sample 1 are shown in table 10 below:

watch 10

Comparative example 4 was confirmed to be three test tubes No. 6 and one test tube No. 7, the average of 4 results was 287, and the settlement result was 28.7X 10⁸CFU/g, rounded to give 29X 10⁸CFU/g。

By combining the above examples 1-3, it can be seen that the use of a certain proportion of compounded sodium dodecyl sulfate and tween 60 as surfactants results in good dispersion effect, and the relative standard deviation of the examples 1-3 is very low. The standard deviation of example 4 becomes large but still within an acceptable range. And the relative standard deviation is more than 10% by using a single surfactant.

Combining the above example 1 and comparative example 1, it can be seen that the relative standard deviation of example 1 under the same gradient of the same sample is significantly smaller than that of comparative example 1, and the detection result of comparative example 1 is 43 × 10⁸CFU/g is obviously lower than that of example 1, which shows that heat shock of the sample can cause dispersed thalli to re-agglomerate after sedimentation in the process of standing and heat shock, so that the reliability of the detection result is highA large reduction.

It can be seen from the above examples 1 and 3 that the colony count in comparative example 4 can be accurately performed only after 48 hours of culture, which is 20 hours longer than the detection time in example 1, and this shows that when a non-specific culture medium formula is used, anaerobic culture is adopted, and the temperature is low, the growth rate of bacillus coagulans is significantly reduced, and although the final results are similar, a large amount of time and cost are wasted, which is not favorable for the efficient performance of practical production and research and development work.

As can be seen from the above examples 1, 2 and 4, the counting result of comparative example 4 is only 24.58% of that of example 1, which indicates that Ca in the medium can be ensured by a suitable acidic environment²⁺And Mn²⁺In a free state without influencing the growth and reproduction of the bacillus coagulans, and the Ca with proper concentration²⁺And Mn²⁺Is the key for activating the spore germination of the bacillus coagulans, so the optimum pH value of the BC plate is 5.0-5.5.

Claims

1. A bacillus coagulans counting detection method comprises the following steps:

2. The method for detecting the count of Bacillus coagulans as claimed in claim 1, wherein the dilution in step (S1) is 10-1000 times, preferably 100-200 times.

3. The method for detecting the count of bacillus coagulans according to claim 1, wherein the dilution is performed using a sterile diluent containing 0.5-2 wt% of a surfactant and 0.5-1.5 wt% of sodium chloride; preferably, the sterile diluent contains 1.0-1.5 wt% surfactant and 0.9-1.2 wt% sodium chloride.

4. The method for detecting the count of bacillus coagulans according to claim 3, wherein the surfactant is a mixture of an anionic surfactant and a nonionic surfactant.

5. The method for detecting the count of Bacillus coagulans according to claim 4, wherein the anionic surfactant is at least one selected from the group consisting of sodium dodecyl sulfate, sodium dodecyl benzene sulfonate, ammonium dodecyl sulfate and sodium dodecyl polyoxyethylene ether sulfate; the HLB of the nonionic surfactant is 12-16, and is specifically selected from at least one of Tween 40, Tween 60, Tween 80, OP-10, polyoxyethylene alkylphenol, and polyoxyethylene monooleate.

6. The method for detecting the count of Bacillus coagulans according to claim 4, wherein the mass ratio of the anionic surfactant to the nonionic surfactant is 4-6: 1.

7. the method for detecting count of Bacillus coagulans according to claim 1, wherein the heat activation temperature in step (S1) is 65-85 deg.C for 2-15 min.

8. The method for detecting the count of bacillus coagulans according to claim 1, wherein the ultrasonic dispersion ultrasonic transducer of the step (S1) has a frequency of 19-40KHz, a power of 50-200w, a period of 5-30S ultrasonic every 5-30S, and a total ultrasonic time of 60-120S; the beating frequency of the beating homogenizing sterile homogenizer is 3-12 times/second, and the beating time is 1-10 min; preferably, the beating frequency of the sterile homogenizer is 4-8 times/second, and the beating time length is 4-6 min.

9. The method for detecting count of bacillus coagulans according to claim 1, wherein the culturing in step (S3) is performed in a BC culture plate, and the BC culture medium comprises the following components in terms of weight to volume ratio: 5-15g/L of tryptone, 4-10g/L of yeast extract, 4-10g/L of glucose, 4-8g/L of beef extract, 1-2g/L of sodium acetate, 0.5-2.0g/L of dipotassium phosphate, 0.1-0.7g/L of L-cysteine hydrochloride, 0.1-0.5g/L of anhydrous calcium chloride, 0.05-0.20g/L of manganese sulfate monohydrate and 15-30g/L of agar powder; preferably, each component comprises the following components in percentage by weight and volume: 10-12g/L of tryptone, 6-8g/L of yeast extract, 6-8g/L of glucose, 6-8g/L of beef extract, 1.2-1.5g/L of sodium acetate, 1-1.5g/L of dipotassium phosphate, 0.3-0.5g/L of L-cysteine hydrochloride, 0.2-0.3g/L of anhydrous calcium chloride, 0.1-0.15g/L of manganese sulfate monohydrate and 20-25g/L of agar powder.

10. The method for detecting the count of bacillus coagulans according to claim 1, wherein the step (S3) of counting colonies adopts a two-step counting method, wherein the colonies are cultured for 20-24h, and then returned to the incubator for further culture, and newly grown colonies are counted again when the colonies are cultured for 26-30h, and the final number of colonies per plate is obtained by the first counting and the second counting.