Bifidobacterium viable cell number detection medium, detection method and application thereof

Abstract

The application belongs to the technical field of microorganism detection, and particularly relates to a bifidobacterium viable count detection culture medium, a detection method and application. The application provides a culture medium for detecting bifidobacterium, wherein the culture medium comprises MRS culture medium and an antibiotic solution; the antibiotic solution comprises streptomycin, kanamycin and sodium propionate. The culture medium can ensure normal growth of bifidobacterium, has strong inhibition on lactobacillus, can effectively realize accurate detection of bifidobacterium, and avoids interference of lactobacillus.

Description

Technical Field

[0001] This invention belongs to the field of microbial detection technology, specifically relating to the culture medium, detection method and application for detecting the number of viable Bifidobacteria. Background Technology

[0002] Bifidobacteria are among the most common microorganisms in the gut microbiota. As one of the most important strains of probiotics, their content in the gut can be as high as 10. 11 CFU / g contents. Bifidobacteria grow and colonize throughout a person's entire lifespan, with a particularly high relative proportion found in young adults and long-lived elderly individuals. Studies have shown that Bifidobacteria grow optimally between 37°C and 41°C, and can also grow in the human gut at temperatures above 45°C. Therefore, Bifidobacteria can withstand high temperatures and have good environmental tolerance. Bifidobacteria are widely used in food production and storage because they can grow and reproduce well in food.

[0003] Scientific research has shown that Bifidobacteria have a variety of probiotic functions, such as: improving intestinal diseases caused by immune system disorders, such as inflammatory bowel disease, ulcerative colitis, Crohn's disease, and pouchitis; adhering to intestinal epithelial cells and secreting antimicrobial peptides, while increasing the thickness of the mucus layer and inhibiting pathogens from invading the intestine; scavenging free radicals, increasing the activity of host antioxidant enzymes, and reducing the content of malondialdehyde in serum and liver, thereby alleviating oxidative damage to the body and delaying aging; improving diseases caused by intestinal flora imbalance, such as constipation and diarrhea; secreting β-galactosidase to alleviate lactose intolerance; inhibiting the growth of putrefactive bacteria and decomposing carcinogens to achieve anti-cancer effects (such as colon cancer); lowering serum cholesterol and preventing atherosclerosis; reducing inflammatory responses caused by tumor necrosis factor-α and lipopolysaccharide; and increasing serum calcium ion concentration and lowering alanine aminotransferase levels in patients with type 2 diabetes.

[0004] In industrial production, products typically contain several different species of lactic acid bacteria. However, there is currently no accurate and specific method for detecting the number of lactic acid bacteria, especially Bifidobacteria, in products containing a mixture of several species. For the detection of Lactobacillus and Bifidobacteria, there is a national standard method GB4789.35-2016. However, the modified MRS agar medium containing mupirocin lithium salt and cysteine ​​hydrochloride used in GB4789.35-2016 cannot accurately detect the number of Bifidobacteria because Lactobacillus can also grow on this medium, making the Bifidobacteria detection value inaccurate. On the other hand, the Bifidobacterium medium (BBL medium) used in GB4789.34-2016 also supports the growth of Lactobacillus, and the detection result includes both Lactobacillus and Bifidobacteria, not just Bifidobacteria.

[0005] Given the importance of Bifidobacteria, developing an accurate method for detecting them is of great significance. Summary of the Invention

[0006] To address the aforementioned problems, this invention provides a culture medium for detecting Bifidobacteria, which can selectively proliferate Bifidobacteria and is effectively used for the detection of Bifidobacteria.

[0007] On one hand, the present invention provides a culture medium for detecting Bifidobacteria, the culture medium comprising an antibiotic solution; the antibiotic solution comprising streptomycin, kanamycin and sodium propionate.

[0008] Specifically, the culture medium also includes MRS culture medium.

[0009] More specifically, the formulation of the MRS culture medium is as follows: peptone 10.0-15.0 g / L, beef meal 5.0-8.0 g / L, yeast powder 4.0-7.0 g / L, glucose 15-20.0 g / L, Tween 80 1.0-2.0 mL / L, K2HPO4·7H2O 1.0-2.0 g / L, CH3COONa·3H2O 3.0-5.0 g / L, triammonium citrate 1.0-2.0 g / L, MgSO4·7H2O 0.1-0.2 g / L, MnSO4·4H2O 0.05-0.1 g / L, and agar powder 15.0-20.0 g / L.

[0010] Preferably, the formulation of the MRS culture medium is as follows: peptone 10.0 g / L, beef meal 5.0 g / L, yeast powder 4.0 g / L, glucose 20.0 g / L, Tween 80 1.0 mL / L, K2HPO4·7H2O 2.0 g / L, CH3COONa·3H2O 5.0 g / L, triammonium citrate 2.0 g / L, MgSO4·7H2O 0.2 g / L, MnSO4·4H2O 0.05 g / L, and agar powder 15.0 g / L.

[0011] More specifically, the sterilization conditions for the MRS culture medium are: autoclaving at 121°C for 15-30 minutes.

[0012] Specifically, the antibiotic solution consists of: streptomycin 30-50 mg, kanamycin 45-65 mg, sodium propionate 0.1-0.2 g, and distilled water 50 mL.

[0013] In some embodiments, the antibiotic solution comprises: 30 mg streptomycin, 65 mg kanamycin, 0.1 g sodium propionate, and 50 mL distilled water.

[0014] In other embodiments, the antibiotic solution is composed of: 50 mg streptomycin, 45 mg kanamycin, 0.2 g sodium propionate, and 50 mL distilled water.

[0015] Specifically, the antibiotic solution is sterilized by filtration through a microporous membrane.

[0016] Preferably, the microporous filter membrane has a size of 0.22-0.45 μm.

[0017] More preferably, the microporous filter membrane has a size of 0.22 μm.

[0018] Specifically, the volume ratio of the antibiotic solution to the sterilized MRS culture medium is 1-3:19-50.

[0019] Preferably, the volume ratio of the antibiotic solution to the sterilized MRS culture medium is 1:19.

[0020] Specifically, the temperature of the sterilized MRS culture medium is 45-50℃.

[0021] More specifically, the temperature of the sterilized MRS culture medium can be any other temperature among 45°C, 46°C, 47°C, 48°C, 49°C, 50°C, or 45-50°C.

[0022] Preferably, the temperature of the sterilized MRS culture medium is 48°C.

[0023] In another aspect, the present invention provides a method for detecting Bifidobacteria, comprising the following steps:

[0024] S1. Dilute the sample with diluent;

[0025] S2. Inoculate the sample diluted in step S1 into the aforementioned culture medium for anaerobic culture.

[0026] S3, Colony Count.

[0027] Specifically, the diluent described in step S1 includes NaCl and tryptone.

[0028] More specifically, the amount of NaCl is 5-10g, and the amount of tryptone is 10-15g.

[0029] Preferably, the amount of NaCl is 8.5g and the amount of tryptone is 15g.

[0030] More specifically, the sterilization conditions for the diluent are: autoclaving at 121°C for 15-20 minutes.

[0031] Specifically, the anaerobic culture time in step S2 is 60-80 hours.

[0032] Preferably, the anaerobic culture time is 68-72 hours.

[0033] Specifically, in step S3, the colony counting is performed on plates with a colony count between 30 and 300.

[0034] Preferably, the colony counting in step S3 is performed on plates with a colony count between 50 and 200.

[0035] In another aspect, the present invention provides the application of the aforementioned culture medium or detection method in the detection of Bifidobacteria.

[0036] The technical advantages achieved by this invention are: Bifidobacteria can grow well on MRS medium supplemented with antibiotics, while Lactobacillus is significantly inhibited on this medium. It can effectively detect Bifidobacteria in complex cultures, and the detection method is accurate and reliable. Detailed Implementation

[0037] The present invention will be further described in detail below with reference to specific embodiments. The following embodiments are not intended to limit the present invention, but only to illustrate the present invention. Unless otherwise specified, the experimental methods used in the following embodiments are generally performed under conventional conditions. Unless otherwise specified, the materials and reagents used in the following embodiments are commercially available.

[0038] Example 1

[0039] 1.1 Instruments and Reagents

[0040] 1.1.1 Instruments and Equipment

[0041] Clean bench (manufacturer: Shanghai Hujing, model: SW-CJ-2FD);

[0042] Autoclave (Manufacturer: BOXUN, Model: YXQ-LB-75SII);

[0043] Anaerobic workstation (manufacturer: Jiangxue Technology, model: Basil).

[0044] 1.1.2 Reagents:

[0045] Streptomycin (manufacturer: Hubei Qifei Pharmaceutical Chemical Co., Ltd., product number: QF0479);

[0046] Kanamycin (manufacturer: Jiangsu Rongshuo Pharmaceutical Co., Ltd., product number: 5-974648);

[0047] Sodium propionate (manufacturer: Shanghai Baishun Biotechnology Co., Ltd., product number: s100121-500g).

[0048] 1.2 Samples

[0049] Bifidobacterium: Bifidobacterium animalis subsp. lactis NJ241, preservation number CGMCCNo.20816, hereinafter referred to as Bifidobacterium.

[0050] Lactobacillus: Lactobacillus acidophilus LA-10A, preservation number CCTCC NO:M2019012, hereinafter referred to as Lactobacillus.

[0051] Note: All strains listed above are publicly available in the prior art and do not require preservation certification.

[0052] (1) Sample A: Under aseptic conditions, culture media (MRS medium) of Bifidobacterium and Lactobacillus at known concentrations were prepared, with each concentration being 1.0 × 10⁻⁶. 6 CFU / mL, total bacterial concentration 2.0 × 10⁻⁶ 6 cfu / mL.

[0053] (2) Sample B: A known concentration of Lactobacillus culture medium (MRS medium) was prepared under aseptic conditions. The concentration of Lactobacillus was 1.0 × 10⁻⁶. 6 cfu / mL.

[0054] (3) Sample C: Bifidobacterium culture medium (MRS medium) of known concentration was prepared under aseptic conditions. The concentration of Bifidobacterium was 1.0 × 10⁻⁶. 6 cfu / mL.

[0055] (4) Sample D: MRS culture medium only.

[0056] 1.3 Diluent

[0057] The diluent composition (g / L) is: NaCl 8.5g, tryptone 15g.

[0058] Preparation of diluent: Add the above components to 1000 mL of distilled water, heat to dissolve, dispense into containers, and autoclave at 121°C for 15 min.

[0059] 1.4 Culture medium

[0060] The composition of MRS medium (g / L) is as follows: peptone 10.0g, beef meal 5.0g, yeast powder 4.0g, glucose 20.0g, Tween 80 1.0mL, K2HPO4·7H2O 2.0g, CH3COONa·3H2O 5.0g, triammonium citrate 2.0g, MgSO4·7H2O 0.2g, MnSO4·4H2O 0.05g, and agar powder 15.0g.

[0061] Preparation of MRS culture medium: Add the above components to 950 mL of distilled water, heat to dissolve, adjust the pH to 6.2 ± 0.2, dispense, and autoclave at 121 °C for 15-20 min.

[0062] The antibiotic solution consists of: streptomycin 30mg, kanamycin 65mg, sodium propionate 0.1g, and distilled water 50mL.

[0063] Preparation of antibiotic solution: Weigh 30 mg of streptomycin, 65 mg of kanamycin and 0.1 g of sodium propionate and add them to 50 mL of sterile distilled water. Filter the solution through a 0.22 μm microporous membrane for sterilization.

[0064] Preparation of modified MRS medium: After sterilization, 950 mL of MRS medium was cooled to 48°C in a water bath, and 50 mL of filtered antibiotic solution was added to the medium.

[0065] 1.5 Detection Method

[0066] Based on the bacterial content in the sample, select 2-3 consecutive suitable dilutions. For each dilution, pipette 1 mL of the sample homogenate into a sterile Petri dish, and prepare two Petri dishes for each dilution (the samples of this invention are diluted sequentially from 1 to 10,000 times). After transferring the diluent into the Petri dish, spread approximately 15 mL of cooled modified MRS medium onto the dish and rotate the dish to mix thoroughly. Incubate anaerobically at 36°C for 72 hours, and count all colonies on the plate after incubation. The entire process from sample dilution to plate preparation should be completed within 15 minutes.

[0067] Dilution Procedure: Sample dilution is performed aseptically. Place 1 mL of thoroughly mixed sample into a test tube containing 9 mL of sterile diluent to create a 1:10 homogeneous dilution. Then, using a 1 mL sterile pipette, slowly pour 1 mL of the 1:10 dilution into the test tube containing 9 mL of sterile diluent, shaking well to create a 1:100 dilution. Using another 1 mL sterile pipette, repeat the above steps to create 10-fold serial dilutions. For each serial dilution, use a different 1 mL sterile pipette to prepare several appropriate homogeneous dilutions.

[0068] All AD samples were diluted sequentially according to the dilution ratios (1-10000 times) determined above, and then plated according to the above method.

[0069] Colony counting: Select plates with colony counts between 30 and 300 and count the suspected colonies. Count the number of Bifidobacteria in the plate, then multiply by the sample dilution factor to obtain the number of Bifidobacteria per milliliter of sample.

[0070] Example 2

[0071] The antibiotic solution consists of: streptomycin 50mg, kanamycin 45mg, sodium propionate 0.2g, and distilled water 50mL.

[0072] Everything else is the same as in Example 1.

[0073] Example effect data:

[0074] The diluted AD sample was spread onto MRS medium (modified MRS medium) containing antibiotic solution. The bacterial concentration results obtained from colony counting are shown in Table 1.

[0075] Table 1

[0076]

[0077] The diluted AD sample was plated on MRS medium, and the bacterial concentration in the sample was obtained by colony counting. The results are shown in Table 2.

[0078] Table 2

[0079] Sample A Sample B Sample C Sample D bacterial concentration <![CDATA[2.06×10 6 cfu / mL]]> <![CDATA[1.12×10 6 cfu / mL]]> <![CDATA[1.02×10 6 cfu / mL]]> 0 cfu / mL

[0080] As shown in Tables 1-2, Bifidobacteria can grow normally on the antibiotic culture medium of the present invention, and the number of Bifidobacteria detected by this culture medium is consistent with the actual number, indicating high detection accuracy. The MRS culture medium with added antibiotics of the present invention has a strong inhibitory effect on Lactobacillus, and the growth ability of Lactobacillus on the antibiotic culture medium of the present invention is almost zero.

[0081] Comparative Example

[0082] The comparative examples were set up according to the detection method of Example 1. The differences between the comparative examples and Example 1 are shown in Table 3:

[0083] Table 3

[0084] Comparative Example Difference from the antibiotic solution in Example 1 Comparative Example 1 Replace streptomycin with an equal amount of sodium propionate. Comparative Example 2 Replace sodium propionate with an equal amount of streptomycin. Comparative Example 3 No added sodium propionate and streptomycin

[0085] Results data:

[0086] The diluted AD sample was spread onto MRS medium supplemented with antibiotic solutions from comparative examples 1-3. The bacterial concentration in the sample was obtained by colony counting, and the results are shown in Table 4.

[0087] Table 4

[0088]

[0089]

[0090] The above results indicate that MRS culture medium with added comparative antibiotic solutions 1-3 had no effect on the growth of Bifidobacteria, but had a poor inhibitory effect on the growth of Lactobacillus, and was not suitable for detecting Bifidobacteria in Lactobacillus.

Claims

1. A culture medium for detecting Bifidobacteria, characterized in that, The culture medium consists of MRS medium and an antibiotic solution; the antibiotic solution contains streptomycin, kanamycin, and sodium propionate. The formulation of the MRS medium is as follows: peptone 10.0-15.0 g / L, beef meal 5.0-8.0 g / L, yeast powder 4.0-7.0 g / L, glucose 15-20.0 g / L, Tween 80 1.0-2.0 mL / L, K2HPO4·7H2O 1.0-2.0 g / L, CH3COONa·3H2O 3.0-5.0 g / L, triammonium citrate 1.0-2.0 g / L, MgSO4·7H2O 0.1-0.2 g / L, MnSO4·4H2O 0.05-0.1 g / L, and agar powder 15.0-20.0 g / L; The antibiotic solution is composed of: streptomycin 30-50 mg, kanamycin 45-65 mg, sodium propionate 0.1-0.2 g, and distilled water 50 mL; The volume ratio of the antibiotic solution to the sterilized MRS culture medium is 1-3:19-50.

2. The culture medium according to claim 1, characterized in that, The antibiotic solution is sterilized by filtration through a microporous membrane.

3. The culture medium according to claim 1, characterized in that, The temperature of the sterilized MRS medium is 45-50℃.

4. A method for detecting Bifidobacteria for non-disease diagnostic purposes, characterized in that, Includes the following steps: S1. Dilute the sample with diluent; S2. The sample diluted in step S1 is inoculated into the culture medium according to any one of claims 1-3 and cultured anaerobically. S3, Colony Count.

5. The detection method according to claim 4, characterized in that, The diluent described in step S1 includes NaCl and tryptone.

6. The use of the culture medium according to any one of claims 1-3 or the detection method according to any one of claims 4-5 in the detection of Bifidobacteria for non-disease diagnostic purposes.

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