CN115340970A - Culture medium additive for promoting growth of bacterial strain, culture medium containing additive and application of culture medium additive - Google Patents
Culture medium additive for promoting growth of bacterial strain, culture medium containing additive and application of culture medium additive Download PDFInfo
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- CN115340970A CN115340970A CN202211116137.4A CN202211116137A CN115340970A CN 115340970 A CN115340970 A CN 115340970A CN 202211116137 A CN202211116137 A CN 202211116137A CN 115340970 A CN115340970 A CN 115340970A
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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Abstract
The invention belongs to the technical field of microbial culture, and particularly relates to a culture medium additive for promoting growth of a bacterial strain, a culture medium containing the additive and an application of the culture medium additive, wherein the culture medium additive comprises the following components: resazurin, biotin, cobalamin, p-aminobenzoic acid, folic acid, pyridoxamine, thiamine, and riboflavin. By adding the additive, the improved GAM, MRS, PYG, BHI and other culture media have the advantages of simple preparation, rich nutrition, promotion of the growth rate of microorganisms, increase of the number of microorganisms, enhancement of the survival success rate of the cryopreserved strains and the like. More species can be enriched for unknown samples, thereby increasing the abundance of the isolated microorganisms; the growth rate can be significantly increased for microorganisms of known samples.
Description
Technical Field
The invention belongs to the technical field of microbial culture, and particularly relates to a culture medium additive for promoting growth of a bacterial strain, a culture medium containing the additive and application of the culture medium additive.
Background
Over the past decade, epidemiological and multinomial studies have shown that microbial composition has a significant impact on host health. For example, the gut microbiota is found to influence the development of the gastrointestinal tract, digestion and transformation of food, the immune response of the host, the energy balance of the host, and play a role in inflammatory bowel disease. The human intestinal microbiota is a diverse microbial environment, consisting mainly of anaerobic bacteria, containing up to 10 per gram of feces 12 And (4) cells.
At present, in an intestinal microorganism screening culture medium, research reports that GAM, MRS, PYG, BHI and other culture media have good effects are reported, and a certain amount of microorganisms can be cultured to a certain extent. The GAM culture medium can revive and freeze the strains more and can culture a certain amount of microorganisms abundantly. The MRS culture medium for the lactobacillus separation culture in food can enrich more lactobacillus. PYG medium is often used as a enrichment medium for bifidobacteria, while BHI medium is used for the cultivation of various fastidious (fastidious) pathogenic microorganisms. Although these media allow the culture of a certain number of strains, they have certain drawbacks. For example, GAM medium is rich in components compared with other media, can culture a plurality of live bacteria, and still lacks trace elements necessary for the growth of a plurality of microorganisms. Many strains grow more slowly on MRS medium, PYG medium, BHI medium, etc.
Disclosure of Invention
The invention aims to solve the defects of the prior art and provide a culture medium additive for promoting the growth of a strain, a culture medium containing the additive and application of the culture medium additive.
The invention is realized by the following technical scheme:
a culture medium additive (m, microorganisms for short) for promoting the growth of a strain comprises the following components: resazurin (resazurin), biotin (biotin), cobalamin (cobalamin), p-aminobenzoic acid (p-aminobenzoic acid), folic acid (folic acid), pyridoxamine (pyridoxamine), thiamine (thiamine), and riboflavin (riboflavin).
Preferably, the medium additive comprises the following components: 0.05-0.5mg/L resazurin, 0.5-5 mug/L biotin, 0.5-5 mug/L cobalamin, 1.5-15 mug/L para aminobenzoic acid, 2.5-25 mug/L folic acid, 7.5-75 mug/L pyridoxamine, 25-250 mug/L thiamine, and 25-250 mug/L riboflavin.
Further, the medium additive comprises the following components: 0.1 mg/L resazurin, 1 mug/L biotin, 1 mug/L cobalamin, 3 mug/L para aminobenzoic acid, 5 mug/L folic acid, 15 mug/L pyridoxamine, 50 mug/L thiamine and 50 mug/L riboflavin.
The invention also protects the use of the above-mentioned medium additives for promoting the growth of unknown and/or known microorganisms.
A microbial culture medium comprises a basal culture medium and the culture medium additive.
Preferably, the basal medium is a medium disclosed in the industry.
Further, the culture media disclosed in the industry are commercially available or self-formulated according to published formulations, including solid, liquid, or semi-solid media.
Still further, the culture medium disclosed in the industry is any one of PDA (potato dextrose agar) culture medium, LB (bacteriolysis broth) culture medium, LBs (lactobacillus selective) culture medium, TSA (tryptone soy agar) culture medium, TSB (tryptone soy broth) culture medium, RCM (clostridium fortified) culture medium, NB (nutrient broth) culture medium, GAM (gluf anaerobic) culture medium, MRS (mupirocin lithium salt-modified) culture medium, PYG (peptone yeast glucose) culture medium, BHI (brain heart infusion) culture medium.
Preferably, the microbial culture medium further comprises other substances that facilitate growth and/or observation of the microorganisms.
The invention also protects the application of the microorganism culture medium in promoting the growth of unknown and/or known microorganisms.
The invention has the beneficial effects that:
by adding the additive, the improved GAM, MRS, PYG, BHI and other culture media have the advantages of simple preparation, rich nutrition, promotion of the growth rate of microorganisms, increase of the number of microorganisms, enhancement of the survival success rate of the cryopreserved strains and the like. More species can be enriched for unknown samples, thereby increasing the abundance of the isolated microorganisms; the growth rate can be significantly increased for microorganisms of known samples.
Drawings
FIG. 1 is a graph of the effect of MRS (a), GAM (b), and mGAM (c) media on the number of microorganisms growing in fecal sample # 35;
FIG. 2 is a graph of the effect of MRS (a), GAM (b), and mGAM (c) media on the number of microorganisms growing in fecal sample # 37;
FIG. 3 is a graph showing the effect of YCFA (a) and mGAM (b) media on the number of microorganisms growing in fecal sample # 37;
FIG. 4 is a bacterial growth trend chart of Bifidobacterium longum subspecies infantis cultured in GAM and mGAM medium;
FIG. 5 is a graph showing the growth tendency of Bifidobacterium pseudocatenulatum cultured in GAM and mGAM medium;
FIG. 6 is a graph showing the growth tendency of Bacillus villageus in GAM and mGAM medium;
FIG. 7 is a graph showing the growth tendency of bacteria of Escherichia fergushensis cultured in GAM and mGAM medium;
FIG. 8 is a graph showing the growth tendency of Pediococcus acidilactici cultured in GAM and mGAM medium;
FIG. 9 is a graph showing the growth tendency of Candida albicans cultured in BHI and mBHI media;
FIG. 10 is a graph showing the growth tendency of Streptococcus salivarius cultured in BHI and mBHI media;
FIG. 11 is a bacterial growth trend chart of Lactobacillus rhamnosus cultured in BHI and mBHI culture media;
FIG. 12 is a graph of the growth tendency of Lactobacillus delbrueckii cultured in BHI and mBHI media;
FIG. 13 is a graph showing the bacterial growth trend of Weissella mesenteroides cultured in PYG, mPGG medium;
FIG. 14 is a graph showing the growth tendency of bacteria of Lactobacillus rhamnosus cultured in PYG, mPGG medium;
FIG. 15 is a graph showing the growth tendency of Lactobacillus delbrueckii cultured in PYG and mPGG medium.
Detailed Description
For a better understanding of the present invention, the present invention will be further described with reference to the following examples and the accompanying drawings, which are illustrative of the present invention and are not to be construed as limiting thereof.
Example 1:
respectively weighing 0.1 g of resazurin, 1 mg of biotin, 1 mg of cobalamin, 3 mg of p-aminobenzoic acid, 5mg of folic acid, 15 mg of pyridoxamine, 50 mg of thiamine and 50 mg of riboflavin, fully dissolving, mixing, diluting to a constant volume of 1L, adding 1mL of the mixture into 1L of a commercialized GAM medium (LA 4450, solebao), and preparing into an mGAM medium.
Taking a No. 35 fecal sample, performing gradient dilution, and taking a sample of No. 10 -7 The concentration of (A) is plated, and the plate is respectively coated in a commercial MRS culture medium (Solebao, M8540), a commercial GAM culture medium (Solebao, LA 4450) and an mGAM culture medium, and is cultured for 14 to 16 hours under the anaerobic condition at 37 ℃, and counting is carried out after bacterial colonies grow out, as shown in figure 1.
As can be seen from FIG. 1, the number of colonies of the growing microorganisms after the culture in the three media was 40, 108, 149, respectively, it can be seen that the effect of the mGAM medium added with the additive of the present invention is superior to that of the commercial MRS and GAM media.
Example 2:
respectively weighing 0.1 g of resazurin, 1 mg of biotin, 1 mg of cobalamin, 3 mg of p-aminobenzoic acid, 5mg of folic acid, 15 mg of pyridoxamine, 50 mg of thiamine and 50 mg of riboflavin, fully dissolving, mixing, diluting to a constant volume of 1L, adding 1mL of the mixture into 1L of a commercialized GAM medium (LA 4450, solebao), and preparing into an mGAM medium.
Taking No. 37 fecal sample, diluting with gradient, and taking 10 -7 The concentration of the bacterial strain is coated, the bacterial strain is respectively coated in a commercial MRS culture medium (Solebao, M8540), a commercial GAM culture medium (Solebao, LA 4450) and an mGAM culture medium, the culture is carried out for 14 to 16 hours under the anaerobic condition of 37 ℃, and counting is carried out after bacterial colonies grow out, as shown in figure 2.
As can be seen from FIG. 2, the number of colonies of the growing microorganisms after the culture in the three media was 37, 44, 93, respectively, it can be seen that the effect of the mGAM medium added with the additive of the present invention is superior to that of the commercial MRS and GAM media.
Example 3:
1 g of tyrosone, 0.25 g of yeast extract, 0.4 g of NaHCO were each weighed 3 0.1 g cysteine, 0.045 g K 2 HPO 4 ，0.045 g KH 2 PO 4 ，0.09 g NaCl，0.009 g MgSO 4 7H 2 O，0.009 g CaCl 2 0.1 mg resazurin, 1 mg hemin, 1 μ g biotin, 1 μ g cobalamin, 3 μ g p-aminobenzoic acid, 5 μ g folic acid and 15 μ g pyridoxamine, which are fully dissolved and then mixed, the volume is constant to 100 mL, thiamine and riboflavin are added after high-temperature sterilization, so that the final concentration of the thiamine and riboflavin is 0.05 μ g/mL, and a YCFA culture medium is prepared.
Respectively weighing 0.1 g of resazurin, 1 mg of biotin, 1 mg of cobalamin, 3 mg of p-aminobenzoic acid, 5mg of folic acid, 15 mg of pyridoxamine, 50 mg of thiamine and 50 mg of riboflavin, fully dissolving, mixing, diluting to a constant volume of 1L, adding 1mL of the mixture into 1L of a commercial GAM medium (Solebao, LA 4450), and preparing an mGAM medium.
Taking No. 37 fecal sample, diluting with gradient, and taking 10 -7 Coating the mixture in a YCFA culture medium and an mGAM culture medium respectively, culturing for 14 to 16 hours at 37 ℃ under an anaerobic condition, and counting after bacterial colonies grow out, wherein the concentration is shown in figure 3.
As can be seen from FIG. 3, the number of colonies of the growing microorganisms after culturing in the two media was 0 and 49, respectively, and it can be seen that the effect of the mGAM medium supplemented with the supplement of the present invention was superior to that of the YCFA medium.
Example 4:
respectively weighing 0.1 g of resazurin, 1 mg of biotin, 1 mg of cobalamin, 3 mg of p-aminobenzoic acid, 5mg of folic acid, 15 mg of pyridoxamine, 50 mg of thiamine and 50 mg of riboflavin, fully dissolving, mixing, diluting to a constant volume of 1L, adding 1mL of the mixture into 1L of a commercialized GAM medium (LA 4450, solebao), and preparing into an mGAM medium.
Placing Bifidobacterium longum subspecies of infant, bifidobacterium pseudoalboglobum, bacillus village, pegasseus fergusiensis, and Pediococcus acidilactici in commercial GAM culture medium (LA 4450) and commercial GAM culture medium containing the additive of the invention, and recording OD of the strain liquid at regular time 600 Values, as shown in fig. 4-8.
As can be seen from FIGS. 4 to 8, the growth rates of Bifidobacterium longum subspecies infantis, bifidobacterium pseudocatenulatum, bacillus village, escherichia fergushensis, pediococcus acidilactici in the commercial mGAM medium supplemented with the additive of the present invention were all superior to those in the commercial GAM medium.
Example 5:
respectively weighing 0.05 g of resazurin, 0.5mg of biotin, 0.5mg of cobalamin, 1.5 mg of p-aminobenzoic acid, 2.5 mg of folic acid, 7.5 mg of pyridoxamine, 25 mg of thiamine and 25 mg of riboflavin, fully dissolving the components, mixing the components, diluting to a constant volume of 1L, adding 1mL of the mixture into 1L of commercial BHI culture medium (Solebao, LA 0360) to prepare the mBHI culture medium.
Putting Candida albicans, streptococcus salivarius, lactobacillus rhamnosus and Lactobacillus delbrueckii into a commercial BHI culture medium (Solebao, LA 0360) and an mBHI culture medium added with the additive, and recording OD of the bacterial liquid at regular time 600 Values, as shown in fig. 9-12.
As can be seen from FIGS. 9 to 12, the growth rates of Candida albicans, streptococcus salivarius, lactobacillus rhamnosus, and Lactobacillus delbrueckii in the commercial BHI medium added with the additive of the present invention were all superior to those of the commercial BHI medium.
Example 6:
respectively weighing 0.5 g of resazurin, 5mg of biotin, 5mg of cobalamin, 15 mg of p-aminobenzoic acid, 25 mg of folic acid, 75 mg of pyridoxamine, 250 mg of thiamine and 250 mg of riboflavin, fully dissolving the components, mixing the components, determining the volume to 1L, adding 1mL of the mixture into 1L of a commercial PYG medium (HB 0398, qingdao Haibo) to prepare the mPGG medium.
Respectively placing Weissella mesenteroides, lactobacillus rhamnosus and lactobacillus delbrueckii into a commercial PYG culture medium (Qingdao Haibo, HB 0398) and an mPGG culture medium added with the additive, and recording OD of a bacterial liquid at regular time 600 Values, as shown in FIGS. 13-15.
As can be seen from FIGS. 13-15, growth rates of Weissella mesenteroides, lactobacillus rhamnosus, and Lactobacillus delbrueckii in mPYG medium supplemented with the additives of the present invention were superior to those of the commercial PYG medium.
The above-mentioned embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various modifications and improvements made to the technical solution of the present invention by those skilled in the art without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.
Claims (10)
1. A culture medium additive for promoting the growth of a bacterial strain, which is characterized by comprising the following components: resazurin, biotin, cobalamin, p-aminobenzoic acid, folic acid, pyridoxamine, thiamine, and riboflavin.
2. The media supplement of claim 1, comprising the following components: 0.05-0.5mg/L resazurin, 0.5-5 mug/L biotin, 0.5-5 mug/L cobalamin, 1.5-15 mug/L p-aminobenzoic acid, 2.5-25 mug/L folic acid, 7.5-75 mug/L pyridoxamine, 25-250 mug/L thiamine and 25-250 mug/L riboflavin.
3. The media supplement of claim 1, comprising the following components: 0.1 mg/L resazurin, 1 mug/L biotin, 1 mug/L cobalamin, 3 mug/L para aminobenzoic acid, 5 mug/L folic acid, 15 mug/L pyridoxamine, 50 mug/L thiamine and 50 mug/L riboflavin.
4. Use of a medium additive according to any one of claims 1 to 3 for promoting the growth of an unknown and/or known microorganism.
5. A microbial culture medium, comprising: comprising a basal medium and a medium additive according to any one of claims 1 to 3.
6. The microbial culture medium of claim 5, wherein: the basic culture medium is a culture medium disclosed in the industry.
7. The microbial culture medium of claim 6, wherein: the culture media disclosed in the industry are commercially available or self-formulated according to published formulations, including solid, liquid, or semi-solid media.
8. The microbial culture medium of claim 6, wherein: the culture medium disclosed in the industry is any one of a PDA culture medium, an LB culture medium, an LBS culture medium, a TSA culture medium, a TSB culture medium, an RCM culture medium, an NB culture medium, a GAM culture medium, an MRS culture medium, a PYG culture medium and a BHI culture medium.
9. A microbial culture medium according to any one of claims 5 to 8, wherein: the microbial culture medium may also include other substances that facilitate the growth and/or observation of the microorganisms.
10. Use of a microbial culture medium according to any one of claims 5 to 9 for promoting the growth of an unknown and/or known microorganism.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN115948273A (en) * | 2022-06-21 | 2023-04-11 | 合肥瀚微生物科技有限公司 | Bifidobacterium bifidum for treating diabetes and related disorders |
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