CN110791545B - Culture medium for detecting bifidobacteria and detection method - Google Patents
Culture medium for detecting bifidobacteria and detection method Download PDFInfo
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Abstract
The invention relates to a culture medium for detecting bifidobacteria and a detection method, wherein the culture medium for detecting the bifidobacteria is prepared by adding estrogen into a lactic acid bacteria culture medium, and the concentration of the estrogen in the culture medium is 1-10 ppm. According to the invention, the estrogen with a certain concentration is added into the lactic acid bacteria culture medium, so that the proliferation of other lactic acid bacteria is inhibited and the proliferation of bifidobacteria is promoted, and therefore, the detection of the bifidobacteria is facilitated.
Description
Technical Field
The invention belongs to the technical field of microbial detection, and particularly relates to a culture medium for detecting bifidobacteria and a detection method, in particular to a culture medium for detecting bifidobacteria in compound lactic acid bacteria 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, heredity, 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 lower the pH, so that E.coli, Salmonella, etc., are inhibited from growing at low pH in bacteria sensitive to acidic conditions. While the activity of intestinal lactobacilli (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.
The fermented food contains abundant lactic acid bacteria, and the fermented sugar can generate a large amount of lactic acid, so that the fermented food has positive influence on the digestion and immunity related physiological functions of a human body. Common food lactic acid bacteria include several genera of lactobacillus, bifidobacterium, streptococcus thermophilus, etc., and they are often mixed as required to prepare functional foods or nutraceuticals. However, there is currently no very accurate and specific method for the detection of such combinations of lactic acid bacteria, in particular bifidobacteria. For the detection of lactobacillus and bifidobacterium, a national standard method GB4789.35-2016 is adopted, and for the total number detection of bifidobacterium, the mupirocin lithium salt and cysteine hydrochloride modified MRS agar culture medium used in GB4789.35-2016 cannot accurately detect the number of bifidobacterium, because lactobacillus can also grow on the culture medium, so that the detection value of bifidobacterium is inaccurate. In contrast, in the case of the bifidobacterium culture medium (BBL medium) used in GB4789.34-2016, lactobacillus was able to grow on the BBL medium, and the results of the detection included lactobacillus and bifidobacterium, but were not representative of only bifidobacterium.
Bifidobacteria are an important beneficial intestinal microorganism. The bifidobacterium is used as a physiological beneficial bacterium, and has various important physiological functions of biological barrier, nutrition, anti-tumor, immunity enhancement, gastrointestinal tract function improvement, aging resistance and the like on human health. Therefore, the method has very practical significance for detecting the bifidobacteria.
The prior application CN107805656A provides a culture medium for detecting bifidobacterium and a detection method. However, the culture medium has the characteristic of complex formula. The present invention is directed to the above-mentioned specific aspects of the present invention, and provides a culture medium capable of selectively proliferating bifidobacteria and its use for detecting bifidobacteria.
Disclosure of Invention
In order to detect bifidobacteria in the compound lactic acid bacteria, 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 bifidobacteria, which is characterized in that estrogen is added into a lactobacillus culture medium, wherein the concentration of the estrogen in the culture medium is 1-10 ppm. According to the invention, the estrogen with a certain concentration is added into the lactic acid bacteria culture medium, so that the proliferation of other lactic acid bacteria is inhibited and the proliferation of bifidobacteria is promoted, and therefore, the detection of the bifidobacteria is facilitated. Although the mechanism of this selective promotion of bifidobacteria is not clear, it is presumed that the receptors activated by estrogen are present in bifidobacteria and the receptors inhibited by estrogen are present in other lactic acid bacteria.
In a preferred embodiment of the present invention, the concentration of estrogen in the medium is 1 to 4 ppm.
In another preferred embodiment of the present invention, the estrogen includes, but is not limited to, one or more of estradiol benzoate, estradiol valerate, ethinyl estradiol ether, gestagen; estradiol benzoate and/or ethinyl estradiol are preferred.
In a preferred embodiment of the invention, the estrogen is a combination of estradiol benzoate and ethinyl estradiol ether. Preferably, the ratio of the estradiol benzoate to the ethinyl estradiol is 1-3: 1; further preferably 2: 1. When estradiol benzoate and ethinyl estradiol are mixed in a certain ratio, the growth of bifidobacteria has a remarkable synergistic effect compared with that of the individual estradiol benzoate and the ethinyl estradiol.
Estradiol benzoate (Estradiol benzoate) used in the present invention is an estrogen drug. Can promote endometrial hyperplasia, enhance uterine smooth muscle contraction and promote mammary gland development and hyperplasia: the large dose inhibits prolactin release, acts against antiandrogens, and increases calcium deposition in bone.
For use in the present invention, ethinyl estradiol 3-cyclopentyl ether, also known as ethinyl estradiol, is a synthetic steroidal estrogen for the treatment and occasionally treatment of breast and prostate cancer.
Although the use of estrogen drugs for the treatment of gynecological diseases is widely used, the present invention innovatively finds the proliferative effect of estrogen on bifidobacteria.
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 method for detecting the bifidobacterium, which is characterized in that the culture medium is adopted to culture a sample to be detected.
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 comprises 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 counting the number of strains after culturing.
In another aspect, the present invention also relates to the use of the above-mentioned culture medium for detecting bifidobacteria for promoting the growth and proliferation of bifidobacteria.
Advantageous effects
According to the invention, the estrogen with a certain concentration is added into the lactic acid bacteria culture medium, so that the proliferation of other lactic acid bacteria is inhibited and the proliferation of bifidobacteria is promoted, and therefore, the detection of the bifidobacteria is facilitated.
Detailed Description
The invention discloses a culture medium for detecting bifidobacterium and a detection method. The skilled person can refer to the content and suitably modify the process parameters to realize the process. 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 of the techniques described herein, may be made and used 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
2.2MRS Medium
2.3 estradiol benzoate and ethinyl estradiol: 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 suction of sample inoculum (containing 1ml sterile pipette)Sterile phosphate buffer solution containing Bifidobacterium longum, Lactobacillus bulgaricus and Streptococcus thermophilus) 1ml, slowly pouring into a test tube containing 9ml sterile phosphate buffer solution along the tube wall, shaking the test tube to mix well, and making into 1:100 sample solution (containing 0.5 × 10 of Bifidobacterium longum, Lactobacillus bulgaricus and Streptococcus thermophilus)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: estradiol benzoate was added to the above autoclaved medium in an amount of 3ppm as a final concentration.
Preparation of improved medium 2: to the autoclaved medium was added ethinyl estradiol in an amount of 2ppm at the final concentration.
Preparation of modified medium 3: ethinyl estradiol was added to the autoclaved medium in an amount of 1ppm as well as estradiol benzoate in an amount of 2ppm at the final concentration.
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 experiment group 1 and experiment group 2 using the improved medium of the present invention, the growth of lactobacillus bulgaricus and streptococcus thermophilus was significantly inhibited, while the growth of bifidobacterium longum was promoted. For experimental group 3, although the growth of lactobacillus bulgaricus and streptococcus thermophilus was not substantially different from that of experimental group 1 and experimental group 2, the growth of bifidobacterium longum was significantly improved compared to that of experimental group 1 and experimental group 2, thereby demonstrating that the combination of estradiol benzoate and ethinylestradiol had a synergistic effect on the growth of bifidobacterium longum.
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 (8)
1. A culture medium for detecting bifidobacterium longum is characterized in that estrogen is added into a lactic acid bacteria culture medium, the concentration of the estrogen in the culture medium is 1-10 ppm, and the estrogen is selected from estradiol benzoate, ethinylestradiol ether or a combination of estradiol benzoate and ethinylestradiol ether.
2. The medium according to claim 1, wherein the concentration of estrogen in the medium is 1-4 ppm.
3. The medium of claim 1, wherein the estrogen is a combination of estradiol benzoate and ethinyl estradiol ether; the mass ratio of the estradiol benzoate to the ethinyl estradiol is 1-3: 1.
4. The culture medium of claim 1, wherein the lactic acid bacteria culture medium is a basal medium.
5. The culture medium according to claim 1, wherein the pH value of the culture medium is 5-6.
6. A method for detecting Bifidobacterium longum, characterized in that a sample to be tested is cultured in the medium according to any one of claims 1 to 5; the detection method is not for diagnostic purposes; the sample to be detected is a compound lactobacillus sample which consists of bifidobacterium longum, lactobacillus bulgaricus and streptococcus thermophilus.
7. The assay of claim 6 comprising the step of enumerating the strains after incubation.
8. Use of the culture medium according to any one of claims 1 to 5 for promoting growth and proliferation of Bifidobacterium longum.
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