CN110669817B - Culture medium for detecting lactobacillus and detection method - Google Patents
Culture medium for detecting lactobacillus and detection method Download PDFInfo
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- CN110669817B CN110669817B CN201911158085.5A CN201911158085A CN110669817B CN 110669817 B CN110669817 B CN 110669817B CN 201911158085 A CN201911158085 A CN 201911158085A CN 110669817 B CN110669817 B CN 110669817B
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- culture medium
- lactobacillus
- estrogen receptor
- receptor inhibitor
- raloxifene
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/06—Quantitative determination
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/045—Culture media therefor
Abstract
The invention relates to a culture medium for detecting lactobacillus and a detection method, wherein the culture medium for detecting the lactobacillus is prepared by adding an estrogen receptor inhibitor into a lactobacillus culture medium, and the concentration of the estrogen receptor inhibitor in the culture medium is 10-100 ppm. According to the invention, the estrogen receptor inhibitor with a certain concentration is added into the lactobacillus culture medium, so that the proliferation of other lactobacillus is inhibited and the proliferation of lactobacillus is promoted, and therefore, the detection of lactobacillus is facilitated.
Description
Technical Field
The invention belongs to the technical field of microbial detection, particularly relates to a culture medium for detecting lactobacillus and a detection method, and particularly relates to a culture medium for detecting lactobacillus in compound lactobacillus and a detection method.
Background
Lactic Acid Bacteria (LAB) are a general term for a group of bacteria that can utilize fermentable carbohydrates to produce large amounts of lactic acid. The bacteria are widely distributed in nature, have abundant species diversity, at least comprise 18 genera, and more than 200. Except for a very small number, most of them are flora which are indispensable in human body and have important physiological functions, and are widely present in intestinal tracts of human body.
Lactic acid bacteria are not only ideal materials for research classification, biochemistry, genetics, molecular biology and genetic engineering (theoretically having important academic values), but also have extremely high application values in important fields closely related to human life, such as industry, agriculture and animal husbandry, food and medicine. The bacterial colony of the lactobacillus on the solid culture medium is small and grows slowly. Can grow quickly in a liquid fermentation culture medium, can obtain thalli with higher purity after centrifugal washing, and has aerobic and anaerobic properties.
Research shows that oligosaccharide can promote proliferation of lactic acid bacteria, and has various ways for promoting proliferation of beneficial bacteria and inhibiting functions of pathogenic microorganisms: one is a barrier effect, described as competitive inhibition of intestinal mucosal epithelium; the other is the inhibition of the metabolite on pathogenic bacteria. Stewart et al (1993), Tomilka et al (1992) have demonstrated that Isomaltooligosaccharides (IMO) promote proliferation of bifidobacteria, lactic acid bacteria, and that low-energy fatty acids produced by metabolism of these bacteria can lower the pH, so that E.coli, Salmonella, etc. are inhibited from growing at low pH in bacteria sensitive to acidic conditions. The activity of intestinal lactobacillus (such as bifidobacterium) lectin is obviously higher than that of pathogenic bacteria (such as escherichia coli and salmonella), which is probably one of the reasons why the normal intestinal flora has obvious competitive advantages in competitive exclusion.
Lactobacillus has an important role in maintaining the health of the human and higher animal organisms. In the aspect of nutrition physiology, the lactobacillus can improve the digestion and absorption of protein, lactose, calcium and other nutrient substances, and can generate a plurality of vitamins for the digestion and absorption of organisms; can inhibit the reproduction of putrefying bacteria and pathogenic bacteria in intestinal tract, reduce the content of blood ammonia and cholesterol in blood, and maintain the balance of flora in intestinal tract. The lactobacillus has the immunoregulation function, such as the obvious functions of promoting cell division, promoting antibody generation, activating macrophage, inducing interferon generation and the like, and improving the disease resistance of organisms. The lactobacillus has activating effect on macrophage, and can enhance cytotoxicity of macrophage. Furthermore, the lactobacillus can induce human Peripheral Blood Mononuclear Cells (PBMC) to produce IL-12, IL-18 and gamma-interferon, thereby inducing Th1 type cellular immune response. In addition, lactic acid bacteria can inhibit hydroxymethylglutarate coenzyme A reductase and reduce the absorption of cholesterol in the intestinal tract, thereby reducing the cholesterol content in the body. Many studies have also demonstrated that lactic acid bacteria have the effect of lowering serum cholesterol and triglycerides. The lactobacillus has high affinity to gastric epithelial cells, can generate organic acids such as lactic acid, acetic acid and the like by competing with pathogenic bacteria for adhesion sites, is distributed on gastrointestinal mucosa in a large amount, antagonizes the pathogenic bacteria by mechanisms such as barrier action and the like on the adhesion of the pathogenic bacteria, generates substances such as organic acid cell-like proteins and the like, influences related metabolism generated by bacterial toxins and plays a role in protecting gastrointestinal tracts. Therefore, the identification and detection of lactobacillus is very important.
Both chinese patent applications CN101538547A and CN107338206A disclose culture media for lactobacilli. However, the above culture medium has a complicated formulation, and it is difficult to achieve directional proliferation of lactobacillus. The present invention provides a culture medium capable of selectively proliferating lactobacillus in view of the above-mentioned specific features of the present invention, and an application thereof for detecting lactobacillus.
Disclosure of Invention
In order to detect lactobacillus from the compound lactobacillus, the invention provides a culture medium and a detection method thereof, and the invention adopts the following technical scheme.
The invention relates to a culture medium for detecting lactobacillus, which is characterized in that an estrogen receptor inhibitor is added into a lactobacillus culture medium, wherein the concentration of the estrogen receptor inhibitor in the culture medium is 10-100 ppm. According to the invention, the estrogen receptor inhibitor with a certain concentration is added into the lactobacillus culture medium, so that the proliferation of other lactobacillus is inhibited and the proliferation of lactobacillus is promoted, and therefore, the detection of lactobacillus is facilitated. Although the mechanism of such selective promotion of lactobacilli is not clear, it is presumed that selective proliferation of lactobacilli is facilitated by the addition of estrogen receptor inhibitors due to the presence of estrogen-inhibited receptors in lactobacilli and estrogen-activated receptors in other lactic acid bacteria.
In a preferred embodiment of the present invention, the concentration of the estrogen receptor inhibitor in the medium is 10 to 40 ppm.
In another preferred embodiment of the invention, the estrogen receptor inhibitor includes, but is not limited to, one or more of tamoxifen (Tamoxifene), fluvistron (Fulvestrant), Raloxifene (Raloxifene), Lasofoxifene (Lasofoxifene), Lasofoxifene tartrate (Lasofoxifene tartrate), alfixifene (Afimoxifene), indoxifene (Idoxifene); preferably fluviatron and/or raloxifene.
In another preferred embodiment of the invention, the estrogen receptor inhibitor is a combination of fluviatron and raloxifene; preferably, the ratio of the fluviasetron to the raloxifene is 1-3: 1; further preferably 2: 1. When the fluviasetron and the raloxifene are mixed according to a certain ratio, the fluviasetron and the raloxifene have a remarkable synergistic effect on the growth of lactobacillus compared with the fluviasetron and the raloxifene which are used independently.
For the purposes of the Fulvestrant used in the present invention, it is an estrogen (estrogen) receptor antagonist which binds to the estrogen receptor in a reversible manner.
Raloxifene used in the invention is white to yellowish white crystal or crystalline powder, has a melting point of 250-253 ℃, is easily soluble in chloroform or dimethylformamide, is easily soluble in acetonitrile, acetone or ethyl acetate, is difficult to dissolve in methanol, absolute ethyl alcohol or absolute ethyl ether, is difficult to dissolve in ethane, and is hardly soluble in water. In the present invention, the acetone solution is added to the culture medium.
In a preferred embodiment of the invention, the lactic acid bacteria medium is a basal medium, preferably a Columbia blood agar basal medium.
In another preferred embodiment of the present invention, the pH of the culture medium is 5 to 6. By controlling the pH of the medium within the above range, the growth of lactic acid bacteria is facilitated.
The invention also relates to a detection method of lactobacillus, which is characterized in that a sample to be detected is cultured by adopting the culture medium.
In a preferred embodiment of the present invention, the sample to be tested is a composite lactobacillus sample.
In another preferred embodiment of the present invention, the complex lactic acid bacteria sample contains bifidobacterium, lactobacillus and streptococcus thermophilus; preferably Bifidobacterium longum, Lactobacillus bulgaricus and Streptococcus thermophilus.
In a preferred embodiment of the present invention, the detection method comprises the step of enumerating the strains after cultivation.
The invention also relates to the application of the culture medium for detecting the lactobacillus in promoting the growth and proliferation of the lactobacillus.
Advantageous effects
According to the invention, the estrogen receptor inhibitor with a certain concentration is added into the lactobacillus culture medium, so that the proliferation of other lactobacillus is inhibited and the proliferation of lactobacillus is promoted, and therefore, the detection of lactobacillus is facilitated.
Detailed Description
The invention discloses a culture medium for detecting lactobacillus and a detection method. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and products of the present invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications of the methods described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of the present invention without departing from the spirit and scope of the invention.
In order to further understand the present invention, 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 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.
Unless otherwise specified, the reagents involved in the examples of the present invention are all commercially available products, and all of them are commercially available.
Example 1
1. Apparatus and materials
Except for the conventional sterilization and culture equipment in the microbiological laboratory, other equipment and materials are as follows:
1.1 constant temperature incubator: 36 +/-1 ℃;
1.2 homogenizer and aseptic homogenizing bag, homogenizing cup or sterilizing milk bowl;
1.3 refrigerator: 2 to 5 ℃;
1.4 balance: the sensory quantity is 0.1 g;
1.5 sterile test tubes: 18mm × 180mm, 15mm × 100 mm;
1.6 sterile pipette: 1mL (with 0.01mL scale), 10mL (with 0.1mL scale)
1.7 spiral inoculator or L-shaped glass smearing rod
1.8 anaerobic incubator or anaerobic tank and matched consumables
1.9 vortex oscillator
2. Culture medium and reagent
2.1 Soy peptone
2.2 MRS Medium
2.3 Fluviasetron and raloxifene: chemical purity
2.4 dipotassium hydrogen phosphate: analytical purity
2.5 Potassium dihydrogen phosphate: analytical purity
2.6 phosphate buffer: 1g of soybean peptone, 1.21g of dipotassium hydrogenphosphate and 0.34g of potassium dihydrogenphosphate were dissolved in 1L of distilled water and sterilized at 121 ℃ for 10 minutes.
3. Detection program
3.1 sucking 1ml of sample bacterial liquid (sterile phosphate buffer solution containing Bifidobacterium longum, Lactobacillus bulgaricus and Streptococcus thermophilus) with 1ml of sterile pipette, slowly injecting into a test tube containing 9ml of sterile phosphate buffer solution along the tube wall, shaking the test tube to mix them uniformly, and making into 1:100 sample bacterial liquid (the contents of Bifidobacterium longum, Lactobacillus bulgaricus and Streptococcus thermophilus are all 0.5 × 10)5CFU/ml)。
3.2 preparation method of culture medium:
basal medium (control group) preparation: a colombian blood agar basal medium of 38 g was added to 950ml of distilled water, and the pH was adjusted to 5.1 with 6N hydrochloric acid, and autoclaved at 121 ℃ for 10 minutes.
Preparation of modified medium 1: to the autoclaved medium was added fluviatron at a final concentration of 30 ppm.
Preparation of improved medium 2: raloxifene was added to the autoclaved medium above in an amount to give a final concentration of 20 ppm.
Preparation of modified medium 3: to the autoclaved medium was added fluviatron at a final concentration of 20ppm and raloxifene at 10 ppm.
3.3 culturing of the compound lactobacillus: the compound lactobacillus is cultured by adopting the culture medium prepared by the 3.2 in a plate culture mode, each culture medium is provided with 3 plates, and all bacterial colonies on the plates are counted after anaerobic culture is carried out for 72h +/-2 h at 36 +/-1 ℃.
4. Colony counting
And selecting a plate with the colony number between 30 and 300CFU and no spread colony growth to count the total number of the colonies. The corresponding colony numbers were observed and recorded with a magnifying glass. Colony counts are expressed in colony-forming units (CFU).
5. Results and analysis
The counting results are shown in the table below.
From the above test results, the results of Bifidobacterium longum, Lactobacillus bulgaricus and Streptococcus thermophilus were not significantly different among the counting results obtained using the basal medium. In the experimental group 1 and the experimental group 2 using the improved medium of the present invention, the growth of bifidobacterium longum was significantly inhibited, the growth of streptococcus thermophilus was also inhibited to a certain extent, and the growth of lactobacillus bulgaricus was promoted.
For experimental group 3, by adding fluviasetron and raloxifene simultaneously, although the inhibitory effects on bifidobacterium longum and streptococcus thermophilus were not significantly different from those of experimental group 1 and experimental group 2, the promoting effect of experimental group 3 on lactobacillus bulgaricus was significantly enhanced, thereby demonstrating that the combination of fluviasetron and raloxifene had a synergistic effect on the proliferation of lactobacillus bulgaricus.
The foregoing describes preferred embodiments of the present invention, but is not intended to limit the invention thereto. Modifications and variations of the embodiments disclosed herein may be made by those skilled in the art without departing from the scope and spirit of the invention.
Claims (7)
1. A culture medium for detecting lactobacillus bulgaricus is characterized in that an estrogen receptor inhibitor is added into a lactobacillus culture medium, wherein the concentration of the estrogen receptor inhibitor in the culture medium is 10-100 ppm; the estrogen receptor inhibitor is selected from the group consisting of fluviatron, raloxifene or combinations thereof,
the lactobacillus culture medium is a Columbia blood agar basic culture medium.
2. The culture medium according to claim 1, wherein the concentration of the estrogen receptor inhibitor in the culture medium is 10-40 ppm.
3. The culture medium according to claim 1, wherein the estrogen receptor inhibitor is a combination of fluviasetron and raloxifene, and the ratio of the fluviasetron to the raloxifene is 1-3: 1.
4. The culture medium according to claim 1, wherein the pH value of the culture medium is 5-6.
5. A detection method of Lactobacillus bulgaricus is characterized in that a sample to be detected is cultured by using the culture medium according to any one of claims 1 to 4, the sample to be detected is a compound lactobacillus sample, the compound lactobacillus sample consists of Bifidobacterium longum, Lactobacillus bulgaricus and Streptococcus thermophilus, and the detection method is for non-diagnostic purposes.
6. The assay of claim 5 comprising the step of enumerating the strains after incubation.
7. Use of the culture medium according to any one of claims 1 to 4 for promoting growth and proliferation of Lactobacillus bulgaricus.
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| CN201911158085.5A CN110669817B (en) | 2019-11-22 | 2019-11-22 | Culture medium for detecting lactobacillus and detection method |
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Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104781385A (en) * | 2012-09-20 | 2015-07-15 | 丹尼斯科美国公司 | Microtiter plates for controlled release of culture components to cell cultures |
| CN107446863A (en) * | 2017-09-07 | 2017-12-08 | 国家粮食局科学研究院 | Application of the soya whey wastewater in high-activity lactic acid bacteria microbial inoculum is prepared |
| CN107523605A (en) * | 2017-09-29 | 2017-12-29 | 完美（广东）日用品有限公司 | It is a kind of to be used to carry out the culture medium of counting and its application simultaneously to Lactobacillus rhamnosus and lactobacillus acidophilus |
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- 2019-11-22 CN CN201911158085.5A patent/CN110669817B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104781385A (en) * | 2012-09-20 | 2015-07-15 | 丹尼斯科美国公司 | Microtiter plates for controlled release of culture components to cell cultures |
| CN107446863A (en) * | 2017-09-07 | 2017-12-08 | 国家粮食局科学研究院 | Application of the soya whey wastewater in high-activity lactic acid bacteria microbial inoculum is prepared |
| CN107523605A (en) * | 2017-09-29 | 2017-12-29 | 完美（广东）日用品有限公司 | It is a kind of to be used to carry out the culture medium of counting and its application simultaneously to Lactobacillus rhamnosus and lactobacillus acidophilus |
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