CN113957118B - Detection method for viable count of bacillus coagulans - Google Patents
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Abstract
The invention discloses a detection method of viable count of bacillus coagulans, and relates to the field of microbial detection. The method comprises the following steps: 1) Sample pretreatment: carrying out ultrasonic treatment on a bacillus coagulans sample, and oscillating the bacillus coagulans sample by a shaking table to obtain a bacterial liquid; 2) And (3) viable count detection: mixing the bacterial liquid and the detection culture medium uniformly, pouring the mixture into a water agar plate to form a double-layer plate, culturing at constant temperature, and counting viable bacteria, wherein the detection culture medium comprises peptone, antibacterial peptide, sorbitol, maltose, a naphthalenesulfonate formaldehyde condensate and light calcium carbonate. The method combines the basic method for probiotic viable bacteria detection, comprehensively optimizes the components of the detection culture medium, the pretreatment condition of the sample and the culture mode of the double-plate, effectively avoids the low-solubility bacillus coagulans from being polluted by the mixed bacteria, provides a powerful growth environment for the bacillus coagulans, has simple detection steps, and has the characteristics of good parallelism of counting results, good reproducibility, short culture period, accurate quantification, small influence of human factors and the like.
Description
Technical Field
The invention relates to the field of microbial detection, in particular to a method for detecting the viable count of bacillus coagulans.
Background
The bacillus coagulans is widely used at present as a probiotic replacing antibiotics, has the advantages of capability of producing organic acid, strong stress resistance, pathogenic bacteria inhibition and the like, is an excellent product replacing lactic acid bacteria, and effectively avoids the defects of poor stress resistance, short storage life and the like of the lactic acid bacteria. 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 lactobacillus and bacillus and is also the focus of research in the animal husbandry industry.
The viable count of probiotics is one of the indexes for evaluating the product quality at present, and an ideal viable count method has the characteristics of good parallelism of count results, good reproducibility and the like, but because most of bacillus coagulans are obtained by liquid submerged fermentation and spray drying, and because various culture mediums and metabolites such as saccharides, small peptides and the like exist in the fermentation process, the bacillus coagulans and the bacillus coagulans are adsorbed and crosslinked together after high-temperature spray drying, the density of bacteria is too high, and the bacteria are tightly combined together to form a biological membrane which is not easy to separate, so that the viable count method of the existing probiotics is used, and the detection difference of the viable count of the bacillus coagulans is large due to different treatment modes during detection. In addition, because the bacillus coagulans belongs to facultative anaerobes and has a growth speed lower than that of other bacillus, the low-concentration bacillus coagulans is easily polluted by other bacillus, so that the bacillus coagulans cannot be detected.
Therefore, it is urgently needed to develop a viable bacteria number detection scheme with accurate quantification and good reproducibility for bacillus coagulans.
Disclosure of Invention
The invention provides a method for detecting the viable count of bacillus coagulans, which aims to solve the technical problem that the existing method for detecting the viable count of bacillus coagulans is poor in reproducibility and accuracy.
In order to solve the technical problems, the invention aims to provide a method for detecting the viable count of bacillus coagulans, which comprises the following steps:
1) Sample pretreatment: adding bacillus coagulans particles or a crude sample into sterile physiological saline, carrying out ultrasonic treatment, adding tween 80 and sterilized glass beads, oscillating in a shaking table, and diluting to obtain a bacterial liquid;
2) And (3) viable count detection: uniformly mixing the bacterial liquid and a detection culture medium, pouring the mixture into a water agar plate, standing the mixture for solidification to form a double-layer plate, culturing the double-layer plate at constant temperature, and counting viable bacteria;
wherein the detection culture medium comprises the following raw materials in parts by weight: 8-12 parts of peptone, 0.05-0.2 part of antibacterial peptide, 1-5 parts of sorbitol, 10-30 parts of maltose, 0.5-1.5 parts of naphthalenesulfonate formaldehyde condensate and 0.5-2 parts of light calcium carbonate.
By adopting the scheme, the basic method for detecting the viable bacteria of the probiotics is comprehensively optimized aiming at the components of a bacillus coagulans sample detection culture medium, the pretreatment condition of the sample, the culture mode of a double-plate and the like, the low-concentration antibacterial peptide can effectively prevent the pollution of mixed bacteria and has no harm to the bacillus coagulans, the bacillus coagulans with lower solubility is effectively prevented from being polluted by the mixed bacteria, maltose and sorbitol are used as carbon sources, the two carbon sources have good growth promotion effect on the bacillus coagulans, the naphthalenesulfonate formaldehyde condensate can better scatter hydrophobic particles, the bacillus coagulans is better released, and the light calcium carbonate can neutralize organic acid generated in the growth process of the bacillus coagulans, so that the pH value of the culture medium is balanced, and a powerful growth environment is provided for the bacillus coagulans.
Preferably, the detection culture medium further comprises the following raw materials in parts by weight: 3-15 parts of beef extract, 2-10 parts of yeast powder, 0.1-0.5 part of dipotassium phosphate trihydrate, 0.2-1 part of magnesium sulfate heptahydrate, 0.05-0.5 part of manganese sulfate tetrahydrate and 8-10 parts of agar powder.
Preferably, the molecular weight of the antibacterial peptide is 1000-4000.
Preferably, in the step 2), the volume ratio of the bacterial liquid to the detection culture medium is 1:15.
preferably, the detection medium is a semi-solid medium.
Generally, by adopting the scheme, the detection culture medium is similar to a semi-solid culture medium, which is beneficial to distinguishing the growth of the bacillus coagulans, and the bacillus coagulans belongs to facultative anaerobes and is more beneficial to the anaerobic growth of thalli.
Preferably, the pH of the detection medium is 7.0 to 7.2.
Preferably, the preparation method of the detection medium comprises the following steps:
s101, weighing required amounts of peptone, sorbitol, beef extract, yeast powder and maltose, and dissolving with distilled water to obtain a salt solution A;
s102, weighing required amounts of dipotassium phosphate trihydrate, magnesium sulfate heptahydrate and manganese sulfate tetrahydrate, and adding distilled water to dissolve to obtain a salt solution B;
s103, adding the salt solution A into the salt solution B, fully and uniformly stirring, adding the required agar powder, heating to dissolve the agar powder, adding the required light calcium carbonate, fixing the volume of distilled water to the required volume, and sterilizing to obtain a sterile culture medium;
s104, respectively dissolving the antibacterial peptide and the naphthalene sulfonate formaldehyde condensate with distilled water according to required amounts, and performing sterile filtration to obtain sterile mixed solution;
and S105, adding the sterile mixed solution into a sterile culture medium, uniformly mixing, and preserving heat for later use.
Preferably, the water agar plate comprises 2wt% agar powder and the balance water.
Preferably, in the step 1), the power of the ultrasonic treatment is 240W-480W, the ultrasonic time is 5min-8min, and the shaking time of the shaking table is 20min-40min.
Preferably, in the step 2), the temperature for constant-temperature culture is 37-50 ℃ and the time is 24-48 h.
By adopting the scheme, the culture temperature, the ultrasonic and the oscillation time parameters in the detection stage are optimally controlled, the detection reproducibility of the viable count of the bacillus coagulans is better, and the detection result is accurate.
Compared with the prior art, the embodiment of the invention has the following beneficial effects:
the method combines a basic method for probiotic viable bacteria detection, and is comprehensively optimized for components of a bacillus coagulans sample detection culture medium, pretreatment conditions of the sample, a double-plate culture mode and the like, the low-concentration antibacterial peptide can effectively prevent the contamination of mixed bacteria, the bacillus coagulans with lower solubility is effectively prevented from being contaminated by the mixed bacteria, and a powerful growth environment is provided for the bacillus coagulans.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
In view of the problems existing in the detection of the bacillus coagulans at present, a special detection method of the bacillus coagulans, a special pretreatment mode and a basic method for detecting the viable bacteria of probiotics are researched, comprehensive optimization is carried out on components of a detection culture medium, pretreatment conditions, a culture mode and the like of a bacillus coagulans sample, and the method for detecting the viable bacteria number in a special bacillus coagulans product is provided.
The application provides a detection method of viable count of bacillus coagulans, which comprises the following steps: s1, preparing a detection culture medium; s2, preprocessing a sample; and S3, detecting the viable count.
In one embodiment, the detection medium in S1 comprises 8-12 g/L of peptone, 0.05-0.2 g/L of antimicrobial peptide, 1-5 g/L of sorbitol, 3-15 g/L of beef extract, 2-10 g/L of yeast powder, 10-30 g/L of maltose, 0.1-0.5 g/L of dipotassium hydrogen phosphate trihydrate, 0.2-1 g/L of magnesium sulfate heptahydrate, 0.05-0.5 g/L of manganese sulfate tetrahydrate, 0.5-1.5 g/L of naphthalenesulfonate formaldehyde condensate, 8-10 g/L of agar powder and 0.5-2 g/L of light calcium carbonate, distilled water is added to be fixed to 1L, and the pH is 7.0-7.2.
In one embodiment, the antimicrobial peptide is selected to have a molecular weight of from 1000 to 4000.
In one embodiment, the antibacterial peptide is sigma (CAS NO:116229-36-8, molecular weight 1483.89, purity ≥ 95%).
The low-concentration antibacterial peptide can effectively prevent the pollution of mixed bacteria, does not harm bacillus coagulans, and is easily polluted by bacillus with high growth speed, such as bacillus subtilis, bacillus licheniformis and the like, once polluted, even if the pollution quantity is extremely small, no method is provided for detecting the bacillus coagulans accurately, so that the antibacterial peptide with the limited concentration can be added, and the pollution of the mixed bacteria can be effectively avoided.
Simultaneously, maltose and sorbitol are used as carbon sources, the two carbon sources have a good growth promoting effect on bacillus coagulans, and are more favorable for long-acting growth of strains compared with common glucose, because the glucose enables the bacillus coagulans to rapidly produce a large amount of acid, the pH value of a growth system is rapidly reduced, and the glucose can bring a substrate inhibition effect, so that the growth of the bacillus coagulans is inhibited, and the sorbitol and the maltose are used as the carbon sources in the application, so that the growth of the bacillus coagulans is promoted.
In addition, the naphthalene sulfonate formaldehyde condensate used as a dispersant is a strong anionic surfactant, so that hydrophobic particles can be better broken up, and bacteria can be better released; the precipitated calcium carbonate can neutralize organic acid generated in the growth process of the bacillus coagulans, so that the pH value of the culture medium is balanced, and a powerful growth environment is provided for the bacillus coagulans; the detection culture medium is similar to a semi-solid culture medium, so that the identification of the growth of the bacillus coagulans is facilitated, and the bacillus coagulans belongs to facultative anaerobes and is more favorable for the anaerobic growth of thalli.
In one embodiment, the configuration process of the detection medium in S1 comprises the following steps:
s101, weighing required amounts of peptone, sorbitol, beef extract, yeast powder and maltose, dissolving in a proper amount of distilled water, and fully dissolving to obtain a salt solution A;
s102, weighing required amounts of dipotassium phosphate trihydrate, magnesium sulfate heptahydrate and manganese sulfate tetrahydrate, and respectively adding a small amount of water to dissolve to obtain a salt solution B;
s103, adding the salt solution A into the salt solution B, fully and uniformly stirring, adding the required agar powder into the uniformly stirred solution, heating and boiling to fully dissolve the agar, then adding the required light calcium carbonate, adding the distilled water to a required volume, and sterilizing at 115 ℃ for 20min to obtain a sterile culture medium for later use;
s104, respectively dissolving the antibacterial peptide and the naphthalenesulfonate formaldehyde condensate into distilled water according to required amounts, and sterilizing the solution through a 0.22um filter membrane under an aseptic condition to prepare an aseptic mixed solution for later use;
and S105, adding the sterile mixed solution into a cooled sterile culture medium at 50 ℃, uniformly mixing, and preserving heat for later use.
In one embodiment, the sample pretreatment in S2 comprises the following steps:
s201, grinding or crushing particles or crude samples, directly weighing other samples, adding 1g-10g of bacillus coagulans samples into a triangular flask of 0.5% sterile normal saline, performing ultrasonic treatment at 40 ℃ and power of 240W-480W for 5-8 minutes to obtain bacterial suspension;
s202, adding Tween 80 in an amount of 0.1% by mass into the bacterial suspension subjected to the S201 ultrasonic treatment, and shaking up. Adding a little sterilized glass beads with the diameter of 0.56-0.60cm; shaking the shaking table at the rotation rate of 200 at the temperature of 25 ℃ for 20-40 minutes at constant temperature, and then diluting the shaking table with sterile water to the bacterial liquid with the required detection gradient, so that the detection can be carried out.
3. And (3) detecting the viable count:
s301, preparation of 2wt% water agar plate: mixing 2wt% agar powder with water, sterilizing at 121 deg.C for 30min to obtain water agar, cooling, pouring 10-15ml into sterile plate, spreading, and blowing on ultra-clean bench for half an hour or standing at room temperature for more than 1 hr for complete solidification to obtain 2wt% water agar plate;
s302, according to bacterial liquid: the volume ratio of the detection culture medium is 1: and (2) adding the bacterial liquid treated in the step (2) into the detection culture medium in the step (1), wherein each plate contains 16mL of mixed liquid, uniformly mixing the mixed liquid on a vortex mixer to prevent foaming, pouring the mixed liquid into a 2wt% water agar plate, and standing for solidification to form a double-layer plate.
S303, placing the double-layer flat plate in a constant-temperature incubator, culturing at the temperature of 37-50 ℃ for 24-48 h, and counting viable bacteria.
The following description is given with reference to specific examples to illustrate the practical effects of the technical solutions of the present application.
Example one
A detection method of viable count of Bacillus coagulans comprises the following steps:
1. preparation of a detection culture medium:
s101, weighing required amounts of 10g/L peptone, 3g/L sorbitol, 10g/L beef extract, 5g/L yeast powder and 15g/L maltose, dissolving in appropriate amount of distilled water, and fully dissolving to obtain a salt solution A;
s102, weighing required amounts of 0.2g/L dipotassium phosphate trihydrate, 0.5g/L magnesium sulfate heptahydrate and 0.1g/L manganese sulfate tetrahydrate, and respectively adding a small amount of water to dissolve to obtain a salt solution B;
s103, adding the salt solution A into the salt solution B, fully and uniformly stirring, adding required 10g/L agar powder into the uniformly stirred solution, heating and boiling to fully dissolve agar, then adding required 1g/L light calcium carbonate, fixing the volume of distilled water to required volume, and sterilizing at 115 ℃ for 20min to obtain a sterile culture medium for later use;
s104, respectively dissolving 0.1g/L of antibacterial peptide and 1g/L of naphthalene sulfonate formaldehyde condensate in distilled water according to required amounts, and sterilizing through a 0.22um filter membrane under an aseptic condition to prepare an aseptic mixed solution for later use;
s105, adding the sterile mixed solution into a cooled sterile culture medium at 50 ℃, uniformly mixing, keeping the pH value of 7.0-7.2, and preserving the temperature for later use.
2. Sample pretreatment:
s201, grinding or crushing particles or crude samples, directly weighing other samples, taking 1g-10g of bacillus coagulans samples, adding the bacillus coagulans samples into a triangular flask of 0.5% sterile normal saline, controlling the temperature at 40 ℃, controlling the power at 240W, and carrying out ultrasonic treatment for 8 minutes to obtain bacterial suspension;
s202, adding Tween 80 in an amount of 0.1% by mass into the bacterial suspension subjected to the S201 ultrasonic treatment, and shaking up. Adding a little sterilized glass beads with the diameter of 0.56-0.60cm; and (4) shaking the shaking table at the rotation speed of 200 ℃ for 30 minutes at constant temperature, and then diluting the shaking table with sterile water to the bacterial liquid with the required detection gradient, so that the detection can be carried out.
3. And (3) detecting the viable count:
s301, preparing a water agar plate: the bottom layer is a water agar layer: mixing 2% agar powder with water, sterilizing at 121 deg.C for 30min to obtain water agar, cooling, pouring 10-15ml into sterile plate, spreading, and blowing on ultra-clean bench for half an hour or standing at room temperature for more than 1 hr for complete solidification to obtain water agar plate;
s302, according to the bacterial suspension: the volume ratio of the detection culture medium is 1: and (2) adding the bacterial liquid treated in the step (2) into the detection culture medium in the step (1), wherein each plate contains 16mL of mixed liquid, uniformly mixing the mixed liquid on a vortex mixer to prevent foaming, pouring the mixed liquid into a water agar plate, and standing the mixed liquid for solidification to form a double-layer plate.
S303, placing the double-layer flat plate in a constant-temperature incubator, culturing at the culture temperature of 45 ℃ for 36h, and counting viable bacteria.
Example two
1. Preparation of a detection culture medium:
s101, weighing required amounts of 8g/L peptone, 5g/L sorbitol, 15g/L beef extract, 10g/L yeast powder and 10g/L maltose, dissolving in appropriate amount of distilled water, and fully dissolving to obtain a salt solution A;
s102, weighing required amounts of 0.1g/L dipotassium phosphate trihydrate, 0.2g/L magnesium sulfate heptahydrate and 0.05g/L manganese sulfate tetrahydrate, and respectively adding a small amount of water to dissolve to obtain a salt solution B;
s103, adding the salt solution A into the salt solution B, fully and uniformly stirring, adding the required 9g/L agar powder into the uniformly stirred solution, heating and boiling to fully dissolve agar, then adding 0.5g/L light calcium carbonate of the required amount, fixing the volume of distilled water to the required amount, and sterilizing at 115 ℃ for 20min to obtain a sterile culture medium for later use;
s104, 0.05g/L antibacterial peptide and 0.5g/L naphthalenesulfonate formaldehyde condensate are respectively dissolved in distilled water according to required amounts, and sterilized by a 0.22um filter membrane under the aseptic condition to prepare an aseptic mixed solution for later use;
s105, adding the sterile mixed solution into a cooled sterile culture medium at 50 ℃, uniformly mixing, and keeping the temperature for later use, wherein the pH value is 7.0-7.2.
2. Sample pretreatment:
s201, grinding or crushing particles or coarse samples, directly weighing other samples, taking 1g-10g of a bacillus coagulans sample, adding the bacillus coagulans sample into a triangular flask containing 0.5% sterile normal saline, performing ultrasonic treatment at 40 ℃, and performing ultrasonic treatment at 300W for 7 minutes to prepare a bacterial suspension;
s202, adding Tween 80 in an amount of 0.1% by mass into the bacterial suspension subjected to the S201 ultrasonic treatment, and shaking up. Adding a little sterilized glass beads with the diameter of 0.56-0.60cm; and (4) shaking the shaking table at the rotation speed of 200 ℃ for 40 minutes at the constant temperature, and then diluting the shaking table with sterile water to the bacterial liquid with the required detection gradient, so that the detection can be carried out.
3. And (3) detecting the viable count:
s301, preparing a water agar plate: the bottom layer is a water agar layer: mixing 2% agar powder with water, sterilizing at 121 deg.C for 30min to obtain water agar, cooling, pouring 10-15ml into sterile plate, spreading, and blowing on ultra-clean bench for half an hour or standing at room temperature for more than 1 hr for complete solidification to obtain water agar plate;
s302, according to bacterial suspension: the volume ratio of the detection culture medium is 1: and (2) adding the bacterial liquid treated in the step (2) into the detection culture medium in the step (1), wherein each plate contains 16mL of mixed liquid, uniformly mixing the mixed liquid on a vortex mixer to prevent foaming, pouring the mixed liquid into a water agar plate, and standing the mixed liquid for solidification to form a double-layer plate.
S303, placing the double-layer flat plate in a constant-temperature incubator, culturing at the temperature of 50 ℃ for 24 hours, and counting viable bacteria.
EXAMPLE III
1. Preparation of a detection culture medium:
s101, weighing required amounts of peptone of 12g/L, sorbitol of 1g/L, beef extract of 3g/L, yeast powder of 2g/L and maltose of 30g/L, dissolving in appropriate amount of distilled water, and fully dissolving to obtain a salt solution A;
s102, weighing required amounts of 0.5g/L dipotassium phosphate trihydrate, 1g/L magnesium sulfate heptahydrate and 0.5g/L manganese sulfate tetrahydrate, and respectively adding a small amount of water to dissolve to obtain a salt solution B;
s103, adding the salt solution A into the salt solution B, fully and uniformly stirring, adding required 8g/L agar powder into the uniformly stirred solution, heating and boiling to fully dissolve agar, then adding required 2g/L light calcium carbonate, fixing the volume of distilled water to required volume, and sterilizing at 115 ℃ for 20min to obtain a sterile culture medium for later use;
s104, respectively dissolving 0.2g/L of antibacterial peptide and 1.5g/L of naphthalene sulfonate formaldehyde condensate in distilled water according to required amounts, and sterilizing through a 0.22um filter membrane under aseptic condition to prepare an aseptic mixed solution for later use;
s105, adding the sterile mixed solution into a cooled sterile culture medium at 50 ℃, uniformly mixing, keeping the pH value of 7.0-7.2, and preserving the temperature for later use.
2. Sample pretreatment:
s201, grinding or crushing particles or crude samples, directly weighing other samples, adding 1g-10g of bacillus coagulans samples into a triangular flask of 0.5% sterile normal saline, controlling the temperature at 40 ℃, controlling the power at 480W, and carrying out ultrasonic treatment for 5 minutes to obtain a bacterial suspension;
s202, adding Tween 80 in an amount of 0.1% by mass into the bacterial suspension subjected to the S201 ultrasonic treatment, and shaking up. Adding a little sterilized glass beads with the diameter of 0.56-0.60cm; and (4) shaking the shaking table at the rotation speed of 200 ℃ for 20 minutes at the constant temperature, and then diluting the shaking table with sterile water to the bacterial liquid with the required detection gradient, so that the detection can be carried out.
3. And (3) detecting the viable count:
s301, preparing a water agar plate: the bottom layer is a water agar layer: mixing 2% agar powder with water, sterilizing at 121 deg.C for 30min to obtain water agar, cooling, pouring 10-15ml into sterile plate, spreading, and blowing on ultra-clean bench for half an hour or standing at room temperature for more than 1 hr for complete solidification to obtain water agar plate;
s302, according to the bacterial suspension: the volume ratio of the detection culture medium is 1: and (2) adding the bacterial liquid treated in the step (2) into the detection culture medium in the step (1), wherein each plate contains 16mL of mixed liquid, uniformly mixing the mixed liquid on a vortex mixer to prevent foaming, pouring the mixed liquid into a water agar plate, and standing the mixed liquid for solidification to form a double-layer plate.
S303, placing the double-layer flat plate in a constant-temperature incubator, culturing at 37 ℃ for 48h, and counting viable bacteria.
Comparative example 1
The detection is carried out by adopting a method of local standard DB41T 1902-2019 feed additive-bacillus coagulans.
Comparative example 2
The bacillus coagulans is detected by adopting the standard of a certain fermentation enterprise in Shandong.
Comparative example 3
The detection is carried out by adopting a method of national standard GB/T26428-2010 Bacillus subtilis in microbial preparations for feeding.
Performance test
Samples of bacillus coagulans with different contents are collected on the market, and the samples are respectively as follows: coagulation 1 (100 x 10) 8 CFU/g), coagulation 2 (500 x 10), coagulation 8 CFU/g), coagulation 3 (1000 x 10), coagulation 8 CFU/g), coagulation 4 (8 x 10), coagulation 8 CFU/g), four samples were as per example 1, example 2, example 3, pairsThe tests of the proportion 1, the comparison example 2 and the comparison example 3 are carried out, the tests are carried out 3 times in parallel, and the test results are shown in the following table 1.
TABLE 1-detection results of viable count of examples 1 to 3 and comparative examples 1 to 3
According to the detection results of the examples 1 to 3 and the comparative examples 1 to 3 in the table 1, the parallelism of the viable count results obtained by the comparative examples 1 to 3 is very poor, the viable count results are high or low, the product quality cannot be accurately evaluated, and the detection results of the comparative examples are partially lower than the label value and are greatly influenced by other factors. The detection method has the advantages that the reproducibility is good, the stability of the parallel detection result is good, the viable count result is far higher than the label value, the low-concentration bacillus coagulans in the sample can be detected well, and the interference of other mixed bacteria is effectively avoided.
The above-mentioned embodiments are provided to further explain the objects, technical solutions and advantages of the present invention in detail, and it should be understood that the above-mentioned embodiments are only examples of the present invention and are not intended to limit the scope of the present invention. It should be understood that any modifications, equivalents, improvements and the like, which come within the spirit and principle of the invention, may occur to those skilled in the art and are intended to be included within the scope of the invention.
Claims (4)
1. A detection method of viable count of Bacillus coagulans is characterized by comprising the following steps:
1) Sample pretreatment: adding bacillus coagulans particles or a crude sample into sterile physiological saline, carrying out ultrasonic treatment, adding tween 80 and sterilized glass beads, oscillating in a shaking table, and diluting to obtain a bacterial liquid;
2) And (3) viable count detection: uniformly mixing a bacterial liquid and a detection culture medium, wherein the volume ratio of the bacterial liquid to the detection culture medium is 1:15, pouring a water agar plate which comprises 2wt% of agar powder and the balance of water, standing for solidification to form a double-layer plate, culturing the double-layer plate at constant temperature, and counting viable bacteria;
the pH value of the detection culture medium is 7.0-7.2, the detection culture medium is a semisolid culture medium, and the detection culture medium comprises the following raw materials in parts by weight: 8-12 parts of peptone, 0.05-0.2 part of antibacterial peptide, 1-5 parts of sorbitol, 10-30 parts of maltose, 0.5-1.5 parts of naphthalenesulfonate formaldehyde condensate, 0.5-2 parts of light calcium carbonate, 3-15 parts of beef extract, 2-10 parts of yeast powder, 0.1-0.5 part of dipotassium hydrogen phosphate trihydrate, 0.2-1 part of magnesium sulfate heptahydrate, 0.05-0.5 part of manganese sulfate tetrahydrate and 8-10 parts of agar powder; the antibacterial peptide is CAS NO of 116229-36-8 and sigma.
2. The method for detecting the viable count of bacillus coagulans according to claim 1, wherein the preparation method of the detection medium comprises the following steps:
s101, weighing required amounts of peptone, sorbitol, beef extract, yeast powder and maltose, and dissolving with distilled water to obtain a salt solution A;
s102, weighing required amounts of dipotassium phosphate trihydrate, magnesium sulfate heptahydrate and manganese sulfate tetrahydrate, and adding distilled water to dissolve to obtain a salt solution B;
s103, adding the salt solution A into the salt solution B, fully and uniformly stirring, adding the required agar powder, heating to dissolve the agar powder, adding the required light calcium carbonate, fixing the volume of distilled water to the required volume, and sterilizing to obtain a sterile culture medium;
s104, respectively dissolving the antibacterial peptide and the naphthalene sulfonate formaldehyde condensate with distilled water according to required amounts, and performing sterile filtration to obtain sterile mixed solution;
and S105, adding the sterile mixed solution into a sterile culture medium, uniformly mixing, and keeping the temperature for later use.
3. The method for detecting the viable count of bacillus coagulans according to claim 1, wherein in the step 1), the power of the ultrasonic treatment is 240W-480W, the ultrasonic time is 5min-8min, and the shaking time of the shaking table is 20min-40min.
4. The method for detecting the viable count of bacillus coagulans according to claim 1, wherein in the step 2), the temperature for constant-temperature culture is 37-50 ℃ and the time is 24-48 h.
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