CN115369148A - A method for counting lactic acid bacteria in microecological products - Google Patents

Abstract

The invention belongs to the technical field of microbial detection, and particularly provides a method for counting lactic acid bacteria in a microecological product, which comprises the following steps: (1) Preparing a culture medium, adjusting the pH value to 5.3-5.5, and adding natamycin into the culture medium; (2) Adding sterile normal saline into the microecological preparation, and uniformly mixing to prepare bacterial suspension; (3) Sequentially carrying out 10-time gradient dilution on the bacterial suspension by using sterile normal saline to obtain diluents with different dilution times; (4) After the diluted solutions were coated on a culture medium, respectively, and cultured, the number of lactic acid bacteria colonies was counted. The counting method of the lactic acid bacteria in the microecological product provided by the invention is simple and convenient to operate, can be used for widely and effectively inhibiting the growth of various enzyme bacteria and saccharomycetes and inhibiting the generation of mycotoxin, can eliminate the influence of the bacteria such as saccharomycetes and bacillus subtilis under the condition of not influencing the lactic acid bacteria, can be used for accurately counting the lactic acid bacteria, and can be used for improving the counting accuracy of the lactic acid bacteria in the composite microecological preparation.

Description

A method for counting lactic acid bacteria in microecological products

technical field

The invention belongs to the technical field of microorganism detection, and in particular relates to a method for counting lactic acid bacteria in microecological products.

Background technique

Microecological products are a class of beneficial or harmless microorganisms that can change the composition of the gastrointestinal microbiota, make beneficial or harmless microorganisms dominate the population, inhibit the proliferation of pathogenic or harmful microorganisms through competition, and regulate the microecology of the gastrointestinal tract Balanced product. Composite microecological products are products composed of different strains, such as lactic acid bacteria, bacillus, yeast and so on. Enterococcus faecium and Pediococcus can produce lactic acid and belong to the lactic acid bacteria category. They are probiotics and play an important role in maintaining the ecological balance of animal intestinal flora. They are often used in compound microecological products. One of the indicators for evaluating the quality of micro-ecological products is the number of viable bacteria in the product. The plate colony counting method is a commonly used microbial counting method. The separation and counting of lactic acid bacteria in existing complex bacteria mostly uses MRS medium, but some fungi, such as Yeast can also grow on the MRS, thereby interfering with the isolation and counting of lactic acid bacteria. Therefore, the present invention provides a new method for counting lactic acid bacteria, which can reduce the interference of other components in the compound microecological preparation on the counting of lactic acid bacteria.

Contents of the invention

The purpose of the present invention is to overcome the problem in the prior art that fungi such as yeast can interfere with the counting of lactic acid bacteria in probiotics.

For this reason, the invention provides a kind of enumeration method of lactic acid bacteria in microecological product, comprises the following steps:

(1) Prepare the medium and adjust the pH to 5.3-5.5, and add natamycin to the medium;

(2) Take the probiotics, add sterile saline, and mix to make a bacterial suspension;

(3) The bacterial suspension is sequentially diluted 10 times with sterile physiological saline to obtain dilutions of different dilution multiples;

(4) After the diluted solution was applied to the culture medium and cultured, the colonies of lactic acid bacteria were counted.

Specifically, each liter of medium in the above step (1) includes 10.0g peptone, 10.0g beef extract powder, 5.0g yeast extract powder, 20g glucose, 2.0g ammonium citrate, 3.0g sodium acetate, 2.0g potassium dihydrogen phosphate , 0.1g magnesium sulfate, 0.05g manganese sulfate, 1.0g Tween 80, 25g agar, and the balance is water.

Specifically, hydrochloric acid is used in the above step (1) to adjust the pH to 5.3-5.5.

Specifically, the natamycin in the above step (1) is a 0.01-0.03 g/ml natamycin solution.

Specifically, the preparation method of the above-mentioned natamycin solution is as follows: weigh the natamycin, add it into the sterilization buffer under aseptic conditions, oscillate and mix to obtain the natamycin solution.

Specifically, the amount of natamycin solution added in the above step (1) is 0.01-0.03% of the medium volume.

Specifically, the weight-to-volume ratio of the probiotics to the sterile saline in the above step (2) is (0.5-5):100.

Specifically, the dilution factor in the above step (2) is 10 ^-7 , 10 ^-8 and 10 ^-9 .

Specifically, the culture condition in the above step (3) is 35-39° C. for 24-48 hours.

Compared with the prior art, the present invention has the following advantages and beneficial effects:

The method for counting lactic acid bacteria in the micro-ecological product provided by the present invention is easy to operate, can widely and effectively inhibit the growth of various molds and yeasts, and can also inhibit the production of mycotoxins, and eliminate yeasts, yeasts, The influence of bacteria such as Bacillus subtilis, accurate counting of lactic acid bacteria can improve the counting accuracy of lactic acid bacteria in the compound microecological preparation.

Detailed ways

The technical solutions in the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are only some of the embodiments of the present invention, not all of them. Although representative embodiments of the present invention have been described in detail, those skilled in the art to which this invention pertains will appreciate that various modifications and changes can be made therein without departing from the scope of the invention. Accordingly, the scope of the present invention should not be limited to the embodiments, but should be defined by the appended claims and their equivalents.

The effect of the method for counting lactic acid bacteria in the micro-ecological product of the present invention is studied below through specific examples.

The bacterial activity of Enterococcus faecium used in the examples of the present invention is 3.3×10 ¹¹ CFU/g, that of Bacillus subtilis is 2×10 ¹¹ CFU/g, that of Bacillus coagulans is 9×10 ¹⁰ CFU/g, and that of lichen The activity of Bacillus is 2×10 ¹¹ CFU/g, the activity of Clostridium butyricum is 1×10 ¹⁰ CFU/g, the activity of Pediococcus is 3×10 ¹¹ CFU/g, and the activity of Saccharomyces is 1×10 ¹⁰ CFU/g.

Unless otherwise specified, the reagents used in the present invention are all analytically pure, and the water is all secondary water that meets the requirements in GB/T6682.

Example 1:

In this example, the growth effect of various fungi in the probiotics under different addition amounts of natamycin was studied, and the specific method is as follows.

(1) Preparation of physiological saline: Take 8.5g of sodium chloride in 1000mL of distilled water and autoclave at 121°C for 20min.

Preparation of natamycin solution: Weigh 0.5g of natamycin into a sterile Erlenmeyer flask, add 24.5mL of sterilizing buffer, shake well.

Prepare MRS medium: 10.0g peptone, 10.0g beef extract powder, 5.0g yeast extract powder, 20g glucose, 2.0g ammonium citrate, 3.0g sodium acetate, 2.0g potassium dihydrogen phosphate, 0.1g magnesium sulfate, 0.05g Add manganese sulfate, 1.0g Tween 80, and 25g agar into water to 1000mL, and adjust the pH to 5.4 with hydrochloric acid. Autoclave at 115°C for 30min.

Experimental group 1: Add 0.01% natamycin solution to the medium based on the volume of the medium, invert the plate, after solidification, place it upside down in an incubator at 37°C for 24 hours, and select the uncontaminated medium for later use.

Experimental group 2: Based on the medium volume, add 0.02% natamycin solution to the medium, invert the plate, after solidification, place it upside down in an incubator at 37°C for 24 hours, and select the uncontaminated medium for later use.

Experimental group 3: In terms of medium volume, add 0.03% natamycin solution to the medium, invert the plate, after solidification, place it upside down in an incubator at 37°C for 24 hours, and select the uncontaminated medium for later use.

Control group 1: no natamycin solution was added to the culture medium, and the plate was inverted, and after solidification, it was placed upside down in an incubator at 37°C for 24 hours, and the unstained culture medium was selected for use.

(2) Prepare mixed bacterial suspension: weigh 1g Enterococcus faecium, 1g Pediococcus, 0.5g Bacillus subtilis, 0.25g Bacillus coagulans, 0.5g Bacillus licheniformis, 0.25g Clostridium butyricum, 0.5g yeast and mix , add 100mL sterile physiological saline, shake and mix at room temperature to make a bacterial suspension.

Prepare a single bacterial suspension: Weigh 2.5g Enterococcus faecium, 2.5g Pediococcus, 3g Bacillus subtilis, 1g Bacillus coagulans, 3g Bacillus licheniformis, 1g Clostridium butyricum and 1g yeast, respectively add 100mL In sterile normal saline, shake and mix at room temperature to make a single bacterial suspension.

(3) The bacterial suspension was sequentially diluted 10 times with sterile normal saline to obtain a 10 ^-9 dilution of mixed bacteria, a 10 ^-9 dilution of Enterococcus faecium, a 10 ^-8 dilution of Pediococcus, and a dilution of Bacillus subtilis 10 ^-9 dilution of Bacillus coagulans, 10 ^-9 dilution of Bacillus licheniformis, 10 ^-7 dilution of Clostridium butyricum, ^{10 -7 dilution} of yeast.

(4) Take 100 μL of the dilutions of each group, add them to the culture medium of each experimental group and control group, spread with a push rod, place them upside down in a constant temperature incubator at 37°C, and detect the colonies after 48 hours of cultivation. The test results are shown in Table 1.

Table 1 Microbial growth status under different natamycin additions

It can be seen from Table 1 that after adding natamycin to the medium, it can inhibit the growth of fungi such as yeast and bacillus without affecting the growth of lactic acid bacteria, which is convenient for subsequent accurate counting of lactic acid bacteria.

Example 2:

In this embodiment, the growth effect of fungi under different pH conditions is studied, and the specific method is as follows.

(1) Preparation of physiological saline: Take 8.5g of sodium chloride in 1000mL of distilled water and autoclave at 121°C for 20min.

Preparation of natamycin solution: Weigh 0.5g of natamycin into a sterile Erlenmeyer flask, add 24.5mL of sterilizing buffer, shake well.

Prepare MRS medium: 10.0g peptone, 10.0g beef extract powder, 5.0g yeast extract powder, 20g glucose, 2.0g ammonium citrate, 3.0g sodium acetate, 2.0g potassium dihydrogen phosphate, 0.1g magnesium sulfate, 0.05g Add manganese sulfate, 1.0g Tween 80, and 25g agar into water to 1000mL.

Experimental group 1: adjust the pH of the hydrochloric acid medium to 5.3, and sterilize under high pressure at 115°C for 30 minutes. Based on the volume of the medium, add 0.01% natamycin solution to the medium, invert the plate, and after solidification, place it upside down in an incubator at 37°C for 24 hours, and select the uncontaminated medium for later use.

Experimental group 2: hydrochloric acid was used to adjust the pH of the culture medium to 5.5, and it was sterilized under high pressure at 115° C. for 30 minutes. Based on the volume of the medium, add 0.01% natamycin solution to the medium, invert the plate, and after solidification, place it upside down in an incubator at 37°C for 24 hours, and select the uncontaminated medium for later use.

Control group 1: adjust the pH of the medium to 5.6 with hydrochloric acid, and sterilize under high pressure at 115° C. for 30 minutes. Based on the volume of the medium, add 0.01% natamycin solution to the medium, invert the plate, and after solidification, place it upside down in an incubator at 37°C for 24 hours, and select the uncontaminated medium for later use.

Control group 2: adjust the pH of the medium to 5.2 with hydrochloric acid, and sterilize under high pressure at 115° C. for 30 minutes. Based on the volume of the medium, add 0.01% natamycin solution to the medium, invert the plate, and after solidification, place it upside down in an incubator at 37°C for 24 hours, and select the uncontaminated medium for later use.

(2) Preparation of mixed bacterial suspension: Weigh 0.03g Enterococcus faecium, 0.04g Bacillus subtilis, 0.09g Bacillus coagulans, 0.03g Bacillus licheniformis, 0.6g Clostridium butyricum, 0.6g saccharomyces, mix, add 100mL of sterile saline was shaken and mixed at room temperature to obtain a mixed bacterial suspension.

Preparation of single bacterial suspension: Weigh 2.5g of Enterococcus faecium, 3g of Bacillus subtilis, 1g of Bacillus coagulans, 3g of Bacillus licheniformis, 1g of Clostridium butyricum, and 1g of yeast, and add them to 100mL sterile saline , shake and mix at room temperature to make a single bacteria suspension.

(4) Bacterial suspensions were serially diluted 10 times with sterile normal saline to obtain ^10-7 dilutions of mixed bacteria, ^10-9 dilutions of Enterococcus faecium, ^10-9 dilutions of Bacillus subtilis, and Bacillus coagulans Bacillus 10 ^-8 dilution, Bacillus licheniformis 10 ^-9 dilution, Clostridium butyricum 10 ^-7 dilution, yeast 10 ^-7 dilution.

(5) Take 100 μL of the dilutions of each group, add them to the culture medium of each experimental group and control group, spread with a push rod, place them upside down in a constant temperature incubator at 37°C, and detect the colonies after 48 hours of cultivation. The test results are shown in Table 2.

Table 2 Microbial growth status under different medium pH

It can be seen from Table 1 that when the pH of the MRS medium is higher than 5.5, Bacillus subtilis and Enterococcus faecium grow, but other bacteria do not grow; when the pH of the MRS medium does not exceed 5.5, only Enterococcus faecium grows. It can be seen that only the MRS medium with a pH not exceeding 5.5 is suitable for the isolation and counting of Enterococcus faecium in the complex bacteria containing Bacillus subtilis. However, too low a pH value is not conducive to the growth of Enterococcus faecium, so the optimal pH of the culture medium suitable for counting Enterococcus faecium in complex bacteria is 5.3-5.5.

Example 3:

In this example, the effect of natamycin on the growth inhibition of various fungi in Chinese medicine after being treated in different ways is studied, and the specific method is as follows.

(1) Preparation of physiological saline: Take 8.5g of sodium chloride in 1000mL of distilled water and autoclave at 121°C for 20min.

Preparation of natamycin solution: Weigh 0.5g of natamycin into a sterile Erlenmeyer flask, add 24.5mL of sterilizing buffer, shake well.

Preparation of sterilized natamycin solution: Weigh 0.5g of natamycin into a sterile Erlenmeyer flask, add 24.5mL of sterilizing buffer. Shake well and autoclave at 121°C for 20 minutes.

Prepare MRS medium: 10.0g peptone, 10.0g beef extract powder, 5.0g yeast extract powder, 20g glucose, 2.0g ammonium citrate, 3.0g sodium acetate, 2.0g potassium dihydrogen phosphate, 0.1g magnesium sulfate, 0.05g Add manganese sulfate, 1.0g Tween 80, and 25g agar into water to 1000mL, and adjust the pH to 5.5 with hydrochloric acid. Autoclave at 115°C for 30min.

Experimental group 1: Add 0.01% natamycin solution to the medium based on the volume of the medium, invert the plate, after solidification, place it upside down in an incubator at 37°C for 24 hours, and select the uncontaminated medium for later use.

Control group 1: Add 0.01% sterilized natamycin solution to the culture medium based on the volume of the culture medium, invert the plate, after solidification, place it upside down in an incubator at 37°C for 24 hours, and select the uncontaminated culture medium spare.

Control group 2: no natamycin solution was added to the culture medium, and the plate was inverted, and after solidification, it was placed upside down in an incubator at 37°C for 24 hours, and the unstained culture medium was selected for use.

(2) Prepare mixed bacterial suspension: weigh 0.03g Enterococcus faecium, 0.04g Bacillus subtilis, 0.09g Bacillus coagulans, 0.04g Bacillus licheniformis, 0.6g Clostridium butyricum, 0.2g Pediococcus, 0.6g Yeasts were mixed, and 100 mL of sterile saline was added, shaken and mixed evenly at room temperature, to obtain a mixed bacterial suspension.

Prepare a single bacterial suspension: Weigh 2.5g Enterococcus faecium, 2.5g Pediococcus, 3g Bacillus subtilis, 1g Bacillus coagulans, 3g Bacillus licheniformis, 1g Clostridium butyricum and 1g yeast, respectively add 100mL In sterile normal saline, shake and mix at room temperature to make a single bacterial suspension.

(3) The bacterial suspension was sequentially diluted 10 times with sterile normal saline to obtain a 10 ^-9 dilution of mixed bacteria, a 10 ^-9 dilution of Enterococcus faecium, a 10 ^-8 dilution of Pediococcus, and a dilution of Bacillus subtilis 10 ^-9 dilution of Bacillus coagulans, 10 ^-9 dilution of Bacillus licheniformis, 10 ^-7 dilution of Clostridium butyricum, ^{10 -7 dilution} of yeast.

(4) Take 100 μL of the dilutions of each group, add them to the medium of the experimental group 1 and the control group 1-2 respectively, spread it with a push rod, place it upside down in a constant temperature incubator at 37°C, and detect the colonies after 48 hours of cultivation . The test results are shown in Table 3.

Table 3 Microbial growth status under different natamycin treatments

It can be seen from Table 3 that natamycin has no antibacterial effect after high temperature and high pressure treatment.

Example 4:

This embodiment provides a method for counting lactic acid bacteria in probiotics, comprising the following steps:

(1) Preparation of physiological saline: Take 8.5g of sodium chloride in 1000mL of distilled water and autoclave at 121°C for 20min.

Preparation of natamycin solution: Weigh 0.5g of natamycin into a sterile Erlenmeyer flask, add 24.5mL of sterilizing buffer, shake well.

Prepare MRS medium: 10.0g peptone, 10.0g beef extract powder, 5.0g yeast extract powder, 20g glucose, 2.0g ammonium citrate, 3.0g sodium acetate, 2.0g potassium dihydrogen phosphate, 0.1g magnesium sulfate, 0.05g Add manganese sulfate, 1.0g Tween 80, and 25g agar into water to 1000mL, and adjust the pH to 5.4 with hydrochloric acid. Autoclave at 115°C for 30min.

In terms of medium volume, add 0.03% natamycin solution to the medium, invert the plate, and after solidification, place it upside down in a 37°C incubator for 24 hours, and select the uncontaminated medium for later use.

(2) Preparation of mixed bacterial suspension: Weigh 2g of Enterococcus faecium, 0.5g of Bacillus subtilis, 0.25g of Bacillus coagulans, 0.5g of Bacillus licheniformis, 0.25g of Clostridium butyricum, and 0.5g of saccharomyces, and add 100mL of Bacterial saline was shaken and mixed at room temperature to make a bacterial suspension.

(3) The bacterial suspension was serially diluted 10 times with sterile physiological saline to obtain a mixed bacterial dilution with a dilution factor of 10 ⁻⁹ .

(4) Take 100 μL of the diluted solution and add it to the medium that has been cultured in empty space, spread it with a push rod, place it upside down in a constant temperature incubator at 37°C, and detect the colonies after 48 hours of cultivation.

Through the plate counting method, the Enterococcus faecium in the mixed bacteria was measured to be 163 billion.

Example 5:

This embodiment provides a method for counting lactic acid bacteria in probiotics, comprising the following steps:

(1) Preparation of physiological saline: Take 8.5g of sodium chloride in 1000mL of distilled water and autoclave at 121°C for 20min.

Preparation of natamycin solution: Weigh 0.5g of natamycin into a sterile Erlenmeyer flask, add 24.5mL of sterilizing buffer, shake well.

Prepare MRS medium: 10.0g peptone, 10.0g beef extract powder, 5.0g yeast extract powder, 20g glucose, 2.0g ammonium citrate, 3.0g sodium acetate, 2.0g potassium dihydrogen phosphate, 0.1g magnesium sulfate, 0.05g Add manganese sulfate, 1.0g Tween 80, and 25g agar into water to 1000mL, and adjust the pH to 5.4 with hydrochloric acid. Autoclave at 115°C for 30min.

In terms of medium volume, add 0.03% natamycin solution to the medium, invert the plate, and after solidification, place it upside down in a 37°C incubator for 24 hours, and select the uncontaminated medium for later use.

(2) Preparation of mixed bacterial suspension: Weigh 0.2g of Enterococcus faecium, 0.5g of Bacillus subtilis, 0.5g of Bacillus coagulans, 0.5g of Bacillus licheniformis, 0.5g of Clostridium butyricum, and 1.8g of saccharomyces, add 100mL Sterile physiological saline was shaken and mixed at room temperature to make a bacterial suspension.

(3) The bacterial suspension was serially diluted 10 times with sterile physiological saline to obtain a mixed bacterial dilution with a dilution factor of 10 ⁻⁸ .

(4) Take 100 μL of the diluted solution and add it to the medium that has been cultured in empty space, spread it with a push rod, place it upside down in a constant temperature incubator at 37°C, and detect the colonies after 48 hours of cultivation.

By plate counting method, the Enterococcus faecium in the mixed bacteria was measured to be 16.2 billion.

The above examples are only illustrations of the present invention, and do not constitute a limitation to the protection scope of the present invention. All designs that are the same as or similar to the present invention fall within the protection scope of the present invention.

Claims (9)

1. a method for enumerating lactic acid bacteria in micro-ecological products, is characterized in that, comprises the following steps: (1) Prepare the medium and adjust the pH to 5.3-5.5, and add natamycin to the medium; (2) Take the probiotics, add sterile saline, and mix to make a bacterial suspension; (3) The bacterial suspension is sequentially diluted 10 times with sterile saline to obtain dilutions of different dilution multiples; (4) After the diluted solution was applied to the culture medium and cultured, the colonies of lactic acid bacteria were counted. 2. the enumeration method of lactic acid bacteria in the microecological product as claimed in claim 1 is characterized in that: in described step (1), every liter of medium comprises 10.0g peptone, 10.0g beef extract powder, 5.0g yeast extract powder, 20g glucose, 2.0g ammonium citrate, 3.0g sodium acetate, 2.0g potassium dihydrogen phosphate, 0.1g magnesium sulfate, 0.05g manganese sulfate, 1.0g Tween 80, 25g agar, and the balance is water. 3. The method for counting lactic acid bacteria in micro-ecological products according to claim 1, characterized in that: in the step (1), hydrochloric acid is used to adjust the pH to 5.3-5.5. 4. The counting method of lactic acid bacteria in the micro-ecological product as claimed in claim 1, characterized in that: in the step (1), natamycin is a natamycin solution of 0.01-0.03g/ml. 5. the enumeration method of lactic acid bacteria in the microecological product as claimed in claim 4 is characterized in that, the preparation method of described natamycin solution is: take by weighing natamycin, add sterilization buffer under aseptic condition solution, shake and mix to obtain natamycin solution. 6. The method for counting lactic acid bacteria in micro-ecological products according to claim 4, characterized in that: the added amount of natamycin solution in the step (1) is 0.01-0.03% of the medium volume. 7. the enumeration method of lactic acid bacteria in the probiotics product as claimed in claim 1 is characterized in that: the weight-to-volume ratio of probiotics and sterile saline is (0.5-5):100 in the described step (2). The method for counting lactic acid bacteria in micro-ecological products according to claim 7, characterized in that: the dilution factor in the step (2) is 10 ⁻⁷ , 10 ⁻⁸ and 10 ⁻⁹ . 9. The method for counting lactic acid bacteria in micro-ecological products according to claim 1, characterized in that: the culture condition in the step (3) is 35-39° C. for 24-48 hours.