CN109943494B - Culture medium and preparation method thereof - Google Patents
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Abstract
The invention provides a culture medium, which comprises: a bacteriostatic agent comprising salt and anthocyanins. The culture medium does not contain antibiotics, has the function of inhibiting mixed bacteria by adding the anthocyanin instead of the antibiotics, skillfully utilizes the function of the anthocyanin as an indicator when the anthocyanin changes color along with acid production of lactic acid bacteria, avoids using indicators such as resazurin, bromocresol purple, bromocresol green, litmus, MTT, TTC and the like, has the promotion effect on the proliferation of bifidobacterium by purple potato starch, and can conveniently and safely separate the bifidobacterium from excrement.
Description
Technical Field
The invention relates to the field of biology, in particular to a culture medium and a preparation method thereof.
Background
Probiotics are viable microorganisms with a probiotic effect that can colonize the intestinal tract through the digestive tract, and bifidobacteria are now widely accepted as probiotics. In the food field, the Ministry of health lists bifidobacteria in the List of strains for food, the List of strains for health food and the List of strains for infant food, and most of the bifidobacteria are contained in various probiotic fermented beverages, probiotic yogurt, probiotic milk powder and milk tablets in the market.
Scientific research finds that more than 100 kinds of bacteria exist in the digestive tract of a healthy human body, the number of the bacteria reaches more than 100 million, and the imbalance of the species and the proportion of the microbial flora in the intestinal tract of the human body can bring about a plurality of health problems. The bifidobacteria exist in human intestinal tracts and form a reciprocal coexistence relationship with a host in a long-term evolution process. The quantity of bifidobacteria in intestinal tracts of infants is high, the incidence rate of intestinal infection of infants can be effectively reduced, but the bifidobacteria changes obviously with the increase of human age, and a large number of researches prove that disorder of intestinal flora can cause various diseases, and the quantity of probiotics in the intestinal flora is sufficient, so that some diseases can be prevented and relieved. For example, Bifidobacterium in intestinal tract has bidirectional regulating effect in preventing diarrhea and relieving constipation, and can be used for regulating intestinal dysfunction. In addition, the intestinal bifidobacteria can inhibit the growth of other harmful bacteria, metabolize lactose to relieve lactose intolerance symptoms, generate nutrients such as vitamins, amino acids and the like necessary for human bodies, improve the utilization rate of calcium, phosphorus and iron and promote the absorption of vitamin D. Clinical tests prove that the bifidobacteria supplement has various health-care functions of enhancing the immunity of the organism, reducing blood pressure and blood fat, resisting inflammation, allergy, tumor and the like. Clinically, live bifidobacterium preparations have been widely used for the treatment of gastrointestinal diseases such as diarrhea and constipation.
Bifidobacteria have been found to date to have over 30 subtypes, most isolated from infant fecal samples. However, since bifidobacterium is a strict anaerobic bacterium and has strict requirements on growth environment and nutritional conditions, it is very difficult to separate bifidobacterium from a fecal sample with a complex flora, the separation efficiency is low, and the target bacterium is easily lost. Therefore, when bifidobacteria are isolated, it is common to add specific antibiotics to the medium to inhibit the growth of some other intestinal bacteria (e.g., escherichia coli, enterococcus, etc.) in order to ensure the nutrition of bifidobacteria. At present, most of culture media for separating the bifidobacteria contain one or more antibiotics such as mupirocin, sodium propionate, neomycin sulfate, paromomycin, thienamycin, thiothiclomycin and the like, the drug resistance of strains is easily enhanced in the separation process, and antibiotic resistance genes carried by the bifidobacteria are likely to be transferred to pathogenic bacteria in host intestinal tracts, so that hidden troubles are brought to the health of hosts. Bifidobacteria have been widely used in the food industry and in the field of medical hygiene as a popular probiotic. The safety problem of the exogenous bifidobacteria is particularly important when the exogenous bifidobacteria enter the intestinal tract of a human body to exert the probiotic effect.
Purple sweet potato, also called purple sweet potato, is an annual herbaceous plant of Ipomoea of Convolvulaceae, is rich in vitamins, minerals, carbohydrates, anthocyanins and the like, and has the advantages of simple cultivation, wide adaptability, high yield and the like. At present, the method is mainly applied to the research and development of food, medicine and food. The purple sweet potato starch is prepared by selecting fresh high-quality purple sweet potatoes and carrying out processes of peeling, separation, extraction, drying and the like, reserves carbohydrates, vitamins, mineral substances, anthocyanin and the like in the purple sweet potatoes, and has a promoting effect on the proliferation of bifidobacteria. The purple sweet potato anthocyanin is a natural pigment, is reddish in color under an acidic condition and is blue in color under an alkaline condition, and is also an ideal indicator. The freeze-dried purple sweet potato anthocyanin is prepared by a freeze-drying process, is not subjected to long-time high-temperature treatment, and is high in active ingredients and ideal in efficacy.
Disclosure of Invention
The present application is based on the discovery and recognition by the inventors of the following facts and problems:
the inventor finds that the purple sweet potato starch has a promoting effect on the proliferation of bifidobacteria, and the freeze-dried purple sweet potato anthocyanin can replace antibiotics to play a role in inhibiting mixed bacteria, and simultaneously plays a role in an indicator along with the color change generated by lactic acid bacteria during acid production. Based on the discovery of the problems, the inventor provides a culture medium which does not contain antibiotics, the freeze-dried purple sweet potato anthocyanin can be added to replace the antibiotics to inhibit mixed bacteria, meanwhile, the anthocyanin is skillfully utilized to change the color along with acid production of lactic acid bacteria to serve as an indicator, and calcium carbonate or indicators (resazurin, bromcresol purple, bromcresol green, litmus, MTT, TTC and the like) are prevented from being added; and the purple sweet potato starch has a promoting effect on the proliferation of the bifidobacteria, so that the bifidobacteria can be separated from the fecal sample more conveniently and more safely by using the culture medium.
In a first aspect of the invention, a culture medium is provided. According to an embodiment of the invention, the medium comprises: a bacteriostatic agent comprising salt and anthocyanins. The inventor finds that the anthocyanin can replace antibiotics to play a role in inhibiting mixed bacteria, and simultaneously skillfully utilizes the color change of the anthocyanin along with acid production of lactic acid bacteria to play a role in an indicator, so that indicators such as resazurin, bromocresol purple, bromocresol green, litmus, MTT, TTC and the like are avoided. The culture medium according to the embodiment of the invention can separate and obtain the bifidobacteria from the fecal sample more conveniently and more safely.
According to an embodiment of the present invention, the above-mentioned culture medium may further comprise at least one of the following technical features:
according to the embodiment of the invention, the anthocyanin is freeze-dried purple sweet potato anthocyanin. The inventor finds that anthocyanins from different sources have different inhibition degrees on intestinal bacteria, wherein the freeze-dried purple sweet potato anthocyanins have better inhibition effects on staphylococcus aureus, escherichia coli, enterococcus, streptococcus and other aerobic bacteria.
The salt has no special requirement as long as the salt has the bacteriostatic effect. According to a particular embodiment of the invention, the salt comprises lithium chloride. The lithium chloride and the anthocyanin play a bacteriostatic role together.
According to the embodiment of the invention, the starch is purple sweet potato starch. The inventor finds that the purple sweet potato starch has a promoting effect on the proliferation of bifidobacteria.
According to an embodiment of the invention, the medium comprises: 10-20 parts of purple sweet potato starch; 1-4 parts of freeze-dried purple sweet potato anthocyanin; 5-15 parts of tryptone; 5-10 parts of casein phosphopeptide; 5-15 parts by weight of soybean oligosaccharide; 5 parts by weight of sodium chloride; 3 parts of lithium chloride; 15 parts of agar. The inventor finds that the freeze-dried purple sweet potato anthocyanin can inhibit the growth of other bacteria and does not inhibit the growth of bifidobacteria under the condition of the weight parts, the purple sweet potato starch has a promoting effect on the proliferation of the bifidobacteria, and the anthocyanin in the weight parts can play a role of an indicator along with the color change of acid production of lactic acid bacteria, so that calcium carbonate or indicators (resazurin, bromocresol purple, bromocresol green, litmus, MTT, TTC and the like) are avoided.
According to the embodiment of the invention, the culture medium further comprises tween 80, and the content of the tween 80 is 1 part by weight.
In a second aspect of the invention, a method of preparing a culture medium is provided. According to an embodiment of the invention, the method comprises: 1) carrying out first mixing, pH adjustment and sterilization treatment on purple sweet potato starch, water, tryptone, casein phosphopeptide, tween 80, soybean oligosaccharide, sodium chloride, lithium chloride and agar to obtain mixed feed liquid A; 2) secondly, mixing the anthocyanin with water to obtain a solution B; 3) performing third mixing treatment on the mixed feed liquid A and the solution B to obtain the culture medium, wherein the anthocyanin is freeze-dried purple sweet potato anthocyanin, the amount of the freeze-dried purple sweet potato anthocyanin is 1-4 parts by weight, and the amount of the purple sweet potato starch is 10-20 parts by weight; the using amount of the tryptone is 5 to 15 weight parts; the using amount of the casein phosphopeptide is 5 to 10 weight parts; the dosage of the soybean oligosaccharide is 5 to 15 weight parts; the using amount of the sodium chloride is 5 parts by weight; the using amount of the lithium chloride is 3 parts by weight; the agar was used in an amount of 15 parts by weight, and tween 80 was used in an amount of 1mL based on 1000mL of the medium. The inventor finds that the freeze-dried purple sweet potato anthocyanin can inhibit the growth of other bacteria and does not inhibit the growth of bifidobacteria under the condition of the components in parts by weight, the purple sweet potato starch has a promoting effect on the proliferation of the bifidobacteria, and the freeze-dried purple sweet potato anthocyanin in parts by weight can play a role of an indicator along with the color change of acid production of lactic acid bacteria, so that calcium carbonate or indicators (resazurin, bromocresol purple, bromocresol green, litmus, MTT, TTC and the like) are avoided. The culture medium prepared by the method provided by the embodiment of the invention can be used for conveniently and safely separating the bifidobacteria from the fecal sample (namely, antibiotics are not required to be added, so that the drug resistance of the strain is easily enhanced in the separation process, and further, the antibiotic resistance gene carried by the bifidobacteria is prevented from being possibly transferred to pathogenic bacteria in the intestinal tract of a host).
According to an embodiment of the present invention, the method may further include at least one of the following technical features:
according to an embodiment of the present invention, the first mixing process is performed by: carrying out gelatinization treatment on the purple sweet potato starch; carrying out fourth mixing treatment on the gelatinized purple sweet potato starch, the tryptone, the casein phosphopeptide, the tween 80, the soybean oligosaccharide, the sodium chloride, the lithium chloride and the agar; and (5) carrying out constant volume treatment on the feed liquid after the fourth mixing treatment by using the water, and keeping the constant volume to 1000 ml. The starch is insoluble in water at normal temperature, and is easy to agglomerate and disperse unevenly when being directly mixed with other raw materials; the inventor finds that the purple sweet potato starch is gelatinized in advance and then mixed with tryptone, casein phosphopeptide, tween 80, soybean oligosaccharide, sodium chloride, lithium chloride and agar, so that the starch and other nutrient components can be dissolved and dispersed more uniformly.
According to the embodiment of the invention, the gelatinization treatment is realized by dissolving purple sweet potato starch in water with the temperature of 75-80 ℃ and stirring for dissolving and gelatinization. The inventor finds that the purple sweet potato starch is dissolved and gelatinized more completely at the temperature.
According to an embodiment of the present invention, the pH after the pH adjustment treatment is 6.5 to 7.0. The optimum pH value for the growth of the bifidobacterium is 6.5-7.0, and the inventor finds that when the pH value is less than 6.5, the color of a culture medium is reddish, and the differentiation is not obvious when colonies are selected; when the pH value is more than 7.0, the culture medium is dark and blue-purple, and the colony morphology is observed to be influenced.
According to an embodiment of the present invention, the sterilization treatment is performed at a temperature of 115 ℃ for 15 min. The inventors have found that autoclaving at the above temperatures for the above periods of time results in the greatest possible kill of undesirable species.
According to the embodiment of the invention, before the mixed material liquid A and the solution B are subjected to the third mixing treatment, the mixed material liquid A is cooled to 40-45 ℃ in advance. The inventors found that lowering the temperature to 40-45 ℃ and mixing with solution B did not destroy the anthocyanin structure.
According to an embodiment of the invention, said solution B is previously sterilized by filtration through a filter. The inventors have found that the filtration sterilization with a membrane maximizes the removal of microorganisms from solution B.
According to the embodiment of the invention, the mixed material liquid A and the solution B are subjected to the third mixing treatment, and the volume ratio of the mixed material liquid A to the solution B is 19: 1.
According to an embodiment of the present invention, the solution B is prepared in advance as a solution having a concentration of 20, 40, 60, 80 mg/mL.
According to the embodiment of the invention, the concentration of the freeze-dried purple sweet potato anthocyanin in the culture medium is 1-4 mg/mL. The inventor finds that the freeze-dried purple sweet potato anthocyanin can inhibit the growth of other strains and cannot inhibit the growth of bifidobacteria under the concentration condition.
In a third aspect of the invention, a method of preparing a culture medium is provided. According to an embodiment of the invention, the method comprises: 1) stirring the purple sweet potato starch and 75-80 ℃ distilled water for dissolving and pasting; 2) adding tryptone, casein phosphopeptide, tween 80, soybean oligosaccharide, sodium chloride, lithium chloride and agar into the starch pasting liquid obtained in the step 1); 3) using distilled water to fix the volume of the mixed material liquid obtained in the step 2) to 1000 mL; 4) adjusting the pH value of the mixed material liquid obtained in the step 3) to 6.5-7.0; 5) sterilizing the mixed material liquid obtained in the step 4) for 15min at the temperature of 115 ℃; then cooling to 40-45 ℃ to obtain mixed feed liquid A; 6) dissolving the freeze-dried purple sweet potato anthocyanin in distilled water to obtain solutions with the concentrations of 20, 40, 60 and 80mg/mL, and performing filter membrane filtration sterilization to obtain a solution B; 7) carrying out fifth mixing treatment on the mixed feed liquid A and the solution B according to the volume ratio of 19:1 to prepare a solution with the concentration of 1-4 mg/mL, and standing and cooling to obtain the culture medium; the freeze-dried purple sweet potato anthocyanin accounts for 1-4 parts by weight, and the purple sweet potato starch accounts for 10-20 parts by weight; the using amount of the tryptone is 5 to 15 weight parts; the using amount of the casein phosphopeptide is 5 to 10 weight parts; the dosage of the soybean oligosaccharide is 5 to 15 weight parts; the using amount of the sodium chloride is 5 parts by weight; the using amount of the lithium chloride is 3 parts by weight; the dosage of the agar is 15 parts by weight; the amount of tween 80 was 1mL based on 1000mL of the medium. The inventor finds that the freeze-dried purple sweet potato anthocyanin can inhibit the growth of other bacteria and does not inhibit the growth of bifidobacteria under the condition of the components in parts by weight, the purple sweet potato starch has a promoting effect on the proliferation of the bifidobacteria, and the freeze-dried purple sweet potato starch in parts by weight can play a role of an indicator along with the color change of acid production of lactic acid bacteria, so that calcium carbonate or indicators (resazurin, bromocresol purple, bromocresol green, litmus, MTT, TTC and the like) are avoided. The culture medium prepared by the method provided by the embodiment of the invention can be used for separating and obtaining the bifidobacteria from the fecal sample.
In a fourth aspect of the invention, a method of isolating bifidobacterium enteric is presented. According to an embodiment of the present invention, a sample to be isolated is inoculated into the culture medium described above or a culture medium prepared by the method described above to perform separation and purification treatment, colonies obtained after separation and purification are subjected to aerotolerant screening to obtain anaerobic bacteria, and the anaerobic bacteria are inoculated into an enrichment culture medium to perform enrichment culture to obtain the bifidobacterium. The strain separation steps involved in the invention are as follows: the strain separation steps involved in the invention are as follows: 1) sample treatment: weighing 1g of a stool sample, dissolving the stool sample in 99mL of a buffered protein aqueous solution, and preparing 10 times of diluent by a tenfold dilution method; 2) coating culture: 0.1mL of sample diluent is coated on the agar plate of the invention and is inversely placed in an anaerobic box for culturing for 48 to 72 hours at 37 ℃; 3) separation and purification: selecting red and orange colonies, and streaking and purifying; 4) oxygen resistance test: culturing under aerobic or anaerobic condition, and selecting bacterial colony growing only under anaerobic condition; 5) enrichment culture: selecting a single colony, inoculating the single colony in a TPY liquid culture medium (the formula g/L of the TPY liquid culture medium is 10 parts of hydrolyzed casein, 5 parts of plant east, 2 parts of yeast powder, 5 parts of glucose, 2 parts of dipotassium phosphate, 0.5 part of magnesium chloride, 0.25 part of zinc sulfate, 0.15 part of calcium chloride, 1 part of Tween 80 and 0.5 part of cysteine) (the upper layer is isolated from the air by liquid paraffin), and culturing at the constant temperature of 37 ℃ for 24-48 h; 6) and (3) strain preservation: centrifuging the enriched culture solution to collect bacterial sludge, adding 70% glycerol for protection, and preserving at-80 deg.C. According to the method provided by the embodiment of the invention, the bifidobacteria can be separated and obtained from the fecal sample more conveniently and more safely.
According to an embodiment of the present invention, the method further comprises at least one of the following additional technical features:
according to an embodiment of the present invention, the sample to be separated is subjected to a dilution process in advance.
According to an embodiment of the invention, the concentration of the diluted treated sample is 1 x 10-3～1*10-6g/mL。
It should be noted that the strain identification method of the present invention comprises the following steps:
1. morphological identification: gram staining is carried out on the strain, the characteristics of the color, the shape, the arrangement condition and the like of the thallus are observed by an oil lens, the thallus cell is a rod-shaped rod, one end or two ends of the thallus cell are thick or split, and X, Y, V type of the thallus cell is preliminarily identified as the bifidobacterium;
2. identification of a full-automatic microbiological analysis system: selecting a single colony, streaking the single colony on a Columbia blood plate, and carrying out anaerobic culture at 37 ℃ for 48 h; dipping a fresh colony by using an aseptic cotton swab, and grinding the colony in a 0.45% NaCl solution to prepare 2.7-3.0 McF bacterial suspension; selecting an anaerobe identification card, and testing on a computer; and analyzing the identification report.
Detailed Description
The following describes embodiments of the present invention in detail. The following examples are illustrative and are intended to be illustrative of the invention and are not to be construed as limiting the invention.
Example 1:
(1) the formula of the separation culture medium comprises (per liter):
10g of purple sweet potato starch, namely 10g,
1g of freeze-dried purple sweet potato anthocyanin,
15g of tryptone, namely 15g of tryptone,
5g of casein phosphopeptide,
the volume of Tween 801 mL is,
15g of soybean oligosaccharide,
5g of sodium chloride, namely 5g of sodium chloride,
3g of lithium chloride, namely 3g of lithium chloride,
15g of agar, namely 15g of agar,
distilled water was added to 1000 mL.
(2) The preparation method of the culture medium comprises the following steps:
1. weighing purple sweet potato starch according to the formula, dissolving in 75-80 ℃ distilled water, stirring for dissolving and pasting,
2. adding tryptone, casein phosphopeptide, tween 80, soybean oligosaccharide, sodium chloride, lithium chloride and agar into the starch pasting solution,
3. distilled water is added to the volume of 1000mL,
4. adjusting the pH value to 6.5-7.0,
4. sterilizing the culture medium at 115 deg.C under high pressure for 15min, cooling to 40-45 deg.C,
5. dissolving the freeze-dried purple sweet potato anthocyanin in distilled water to prepare a high-concentration solution of 20mg/mL, filtering and sterilizing by using a filter membrane,
6. transferring the mixture into a standby culture medium according to the ratio of 1:19 to prepare a culture medium solution with the final concentration of 1mg/mL,
7. pour the plate, stand and cool for use.
And (3) separating results: the colony on the separating plate has different colors, including blue, red and orange, and the colony is selected to purify and separate to obtain bifidobacterium.
Example 2:
(1) the formula of the separation culture medium comprises (per liter):
20g of purple sweet potato starch, namely,
2g of freeze-dried purple sweet potato anthocyanin,
10g of tryptone,
8g of casein phosphopeptide,
5g of soybean oligosaccharide,
5g of sodium chloride, namely 5g of sodium chloride,
3g of lithium chloride, namely 3g of lithium chloride,
15g of agar, namely 15g of agar,
distilled water was added to 1000 mL.
(2) The preparation method of the culture medium comprises the following steps:
1. weighing purple sweet potato starch according to the formula, dissolving in 75-80 ℃ distilled water, stirring for dissolving and pasting,
2. adding tryptone, casein phosphopeptide, tween 80, soybean oligosaccharide, sodium chloride, lithium chloride and agar into the starch pasting solution,
3. distilled water is added to the volume of 1000mL,
4. adjusting the pH value to 6.5-7.0,
4. sterilizing the culture medium at 115 deg.C under high pressure for 15min, cooling to 40-45 deg.C,
5. dissolving the freeze-dried purple sweet potato anthocyanin in distilled water to prepare a high-concentration solution of 40mg/mL, filtering and sterilizing by using a filter membrane,
6. transferring the mixture into a standby culture medium according to the ratio of 1:19 to prepare a culture medium solution with the final concentration of 2mg/mL,
7. pour the plate, stand and cool for use.
And (3) separating results: the peripheral color of the bacterial colony on the separation flat plate is different, the bacterial colony has blue color, red color and orange color, the bacterial colony with red color and orange color is selected for purification, the number of the selected bacterial colony is reduced, and the workload is reduced.
Example 3:
(1) the formula of the separation culture medium comprises (per liter):
15g of purple sweet potato starch, namely,
3g of freeze-dried purple sweet potato anthocyanin,
8g of tryptone (E),
10g of casein phosphopeptide,
the volume of Tween 801 mL is,
10g of soybean oligosaccharide,
5g of sodium chloride, namely 5g of sodium chloride,
3g of lithium chloride, namely 3g of lithium chloride,
15g of agar, namely 15g of agar,
distilled water was added to 1000 mL.
(2) The preparation method of the culture medium comprises the following steps:
1. weighing purple sweet potato starch according to the formula, dissolving in 75-80 ℃ distilled water, stirring for dissolving and pasting,
2. adding tryptone, casein phosphopeptide, tween 80, soybean oligosaccharide, sodium chloride, lithium chloride and agar into the starch pasting solution,
3. distilled water is added to the volume of 1000mL,
4. adjusting the pH value to 6.5-7.0,
4. sterilizing the culture medium at 115 deg.C under high pressure for 15min, cooling to 40-45 deg.C,
5. dissolving the freeze-dried purple sweet potato anthocyanin in distilled water to prepare a high-concentration solution of 60mg/mL, filtering and sterilizing by using a filter membrane,
6. transferring the mixture into a standby culture medium according to the ratio of 1:19 to prepare a culture medium solution with the final concentration of 3mg/mL,
7. pour the plate, stand and cool for use.
And (3) separating results: the colony on the separating plate has different colors, including blue, red and orange, and the colony is selected to purify and separate to obtain bifidobacterium.
Example 4:
(1) the formula of the separation culture medium comprises (per liter):
15g of purple sweet potato starch, namely,
4g of freeze-dried purple sweet potato anthocyanin,
5g of tryptone,
10g of casein phosphopeptide,
10g of soybean oligosaccharide,
5g of sodium chloride, namely 5g of sodium chloride,
3g of lithium chloride, namely 3g of lithium chloride,
15g of agar, namely 15g of agar,
distilled water was added to 1000 mL.
(2) The preparation method of the culture medium comprises the following steps:
1. weighing purple sweet potato starch according to the formula, dissolving in 75-80 ℃ distilled water, stirring for dissolving and pasting,
2. adding tryptone, casein phosphopeptide, tween 80, soybean oligosaccharide, sodium chloride, lithium chloride and agar into the starch pasting solution,
3. distilled water is added to the volume of 1000mL,
4. adjusting the pH value to 6.5-7.0,
4. sterilizing the culture medium at 115 deg.C under high pressure for 15min, cooling to 40-45 deg.C,
5. dissolving the freeze-dried purple sweet potato anthocyanin in distilled water to prepare a high-concentration solution of 80mg/mL, filtering and sterilizing by using a filter membrane,
6. transferring the mixture into a standby culture medium according to the ratio of 1:19 to prepare a culture medium solution with the final concentration of 4mg/mL,
7. pour the plate, stand and cool for use.
And (3) separating results: the colony on the separating plate has different colors, including blue, red and orange, and the colony is selected to purify and separate to obtain bifidobacterium.
Example 5:
(1) the formula of the separation culture medium comprises (per liter):
10g of purple sweet potato starch, namely 10g,
2g of freeze-dried purple sweet potato anthocyanin,
15g of tryptone, namely 15g of tryptone,
5g of casein phosphopeptide,
the volume of Tween 801 mL is,
5g of soybean oligosaccharide,
5g of sodium chloride, namely 5g of sodium chloride,
3g of lithium chloride, namely 3g of lithium chloride,
15g of agar, namely 15g of agar,
distilled water was added to 1000 mL.
(2) The preparation method of the culture medium comprises the following steps: the same as in example 2.
And (3) separating results: the number of colonies on the separation plate is moderate, the peripheral colors of the colonies are different and have blue, red and orange colors, the colonies on the red and orange sides are selected for purification, and the bifidobacteria are obtained by separation.
Comparative example 1: (Freeze-dried purple sweet potato anthocyanin is not added)
The formula of the separation culture medium comprises (per liter):
10g of purple sweet potato starch, namely 10g,
15g of tryptone, namely 15g of tryptone,
5g of casein phosphopeptide,
the volume of Tween 801 mL is,
15g of soybean oligosaccharide,
5g of sodium chloride, namely 5g of sodium chloride,
3g of lithium chloride, namely 3g of lithium chloride,
15g of agar, namely 15g of agar,
distilled water was added to 1000 mL.
(2) The preparation method of the culture medium comprises the following steps: the same as in example 1.
And (3) separating results: the freeze-dried purple sweet potato anthocyanin is not added, the bacteriostatic action on intestinal bacteria is weak, the bacterial colony on a culture medium is various, and the bifidobacterium is obtained without separation.
Comparative example 2: (not adding purple potato starch)
The formula of the separation culture medium comprises (per liter):
3g of freeze-dried purple sweet potato anthocyanin,
10g of tryptone,
8g of casein phosphopeptide,
the volume of Tween 801 mL is,
10g of glucose is added into the mixture,
15g of soybean oligosaccharide,
5g of sodium chloride, namely 5g of sodium chloride,
40mg of mupirocin lithium salt,
15g of agar, namely 15g of agar,
distilled water was added to 1000 mL.
(2) The preparation method of the culture medium comprises the following steps: the same as in example 2.
And (3) separating results: colony is mostly white, circular, smooth colony on the culture medium, and the colour does not distinguish, and it is big to select colony quantity, and the work load is big.
Comparative example 3: (corn starch is used for replacing purple potato starch)
The formula of the separation culture medium comprises (per liter):
15g of corn starch, namely 15g of corn starch,
3g of freeze-dried purple sweet potato anthocyanin,
8g of tryptone (E),
10g of casein phosphopeptide,
the volume of Tween 801 mL is,
10g of soybean oligosaccharide,
5g of sodium chloride, namely 5g of sodium chloride,
15g of agar, namely 15g of agar,
distilled water was added to 1000 mL.
(2) The preparation method of the culture medium comprises the following steps: same as example 3
And (3) separating results: the purple sweet potato starch has a promoting effect on the proliferation of the bifidobacteria, the corn starch has no equivalent effect, the number of colonies on a culture medium is lower than that of the colonies in the embodiment 3, characteristic colonies are selected for identification, and the bifidobacteria are not obtained.
Comparative example 4: (excessive addition of lyophilized purple sweet potato anthocyanin)
The formula of the separation culture medium comprises (per liter):
15g of purple sweet potato starch, namely,
6g of freeze-dried purple sweet potato anthocyanin,
5g of tryptone,
10g of casein phosphopeptide,
10g of soybean oligosaccharide,
5g of sodium chloride, namely 5g of sodium chloride,
3g of lithium chloride, namely 3g of lithium chloride,
15g of agar, namely 15g of agar,
distilled water was added to 1000 mL.
(2) The preparation method of the culture medium comprises the following steps: the same as in example 4.
And (3) separating results: excessive purple sweet potato anthocyanin inhibits the growth of bifidobacteria, the number of colonies on a culture medium is lower than that in example 4, characteristic colonies are selected for identification, and no bifidobacteria are obtained.
Comparative example 5: (Freeze-dried blueberry anthocyanin is used for replacing freeze-dried purple sweet potato anthocyanin)
(1) The formula of the separation culture medium comprises (per liter):
10g of purple sweet potato starch, namely 10g,
2g of freeze-dried blueberry anthocyanin,
15g of tryptone, namely 15g of tryptone,
5g of casein phosphopeptide,
the volume of Tween 801 mL is,
5g of soybean oligosaccharide,
5g of sodium chloride, namely 5g of sodium chloride,
3g of lithium chloride, namely 3g of lithium chloride,
15g of agar, namely 15g of agar,
distilled water was added to 1000 mL.
(2) The preparation method of the culture medium comprises the following steps: the same as in example 2.
And (3) separating results: the number of colonies on the culture medium is larger than that in example 5, the inhibition effect on the mixed bacteria is inferior to that of freeze-dried purple sweet potato anthocyanin, characteristic colonies are selected for identification, and bifidobacteria are not obtained.
Comparative example 6: (the addition amount of the freeze-dried purple sweet potato anthocyanin is too small)
(1) The formula of the separation culture medium comprises (per liter):
10g of purple sweet potato starch, namely 10g,
0.5g of freeze-dried purple sweet potato anthocyanin,
15g of tryptone, namely 15g of tryptone,
5g of casein phosphopeptide,
the volume of Tween 801 mL is,
5g of soybean oligosaccharide,
5g of sodium chloride, namely 5g of sodium chloride,
3g of lithium chloride, namely 3g of lithium chloride,
15g of agar, namely 15g of agar,
distilled water was added to 1000 mL.
(2) The preparation method of the culture medium comprises the following steps: the same as in example 1.
And (3) separating results: the number of colonies on the culture medium is larger than that of the culture medium in example 1, the inhibition effect on the mixed bacteria is not obvious, the characteristic colonies are selected for identification, and the bifidobacteria are not obtained.
In the description herein, references to the description of the term "one embodiment," "some embodiments," "an example," "a specific example," or "some examples," etc., mean that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the invention. In this specification, the schematic representations of the terms used above are not necessarily intended to refer to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, various embodiments or examples and features of different embodiments or examples described in this specification can be combined and combined by one skilled in the art without contradiction.
Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention, and that variations, modifications, substitutions and alterations can be made to the above embodiments by those of ordinary skill in the art within the scope of the present invention.
Claims (3)
1. A method for separating intestinal bifidobacteria is characterized in that a sample to be separated is inoculated in a culture medium, the culture medium is cultured for 48-72h at 37 ℃, and red and orange colonies are selected and streaked for purification;
carrying out oxygen-resistant screening on the bacterial colony obtained after separation and purification so as to obtain anaerobic bacteria,
inoculating the anaerobic bacteria to an enrichment medium for enrichment culture so as to obtain the bifidobacteria;
the culture medium comprises:
10-20 parts by weight of purple sweet potato starch,
1-4 parts of freeze-dried purple sweet potato anthocyanin,
5-15 parts of tryptone,
5 to 10 parts by weight of casein phosphopeptide,
5 to 15 parts by weight of soybean oligosaccharide,
5 parts by weight of sodium chloride,
3 parts by weight of lithium chloride, namely,
15 parts by weight of agar-agar, wherein the agar is agar-agar gel,
and 801 parts of tween.
2. The method according to claim 1, wherein the sample to be separated is subjected to a dilution treatment in advance.
3. The method according to claim 2, wherein the concentration of the diluted sample is 1 x 10-3~1*10-6g/mL。
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| CN1880471A (en) * | 2005-06-14 | 2006-12-20 | 黑龙江省完达山乳业股份有限公司 | Method for detecting bifidobacteria in milk product |
| CN105779330A (en) * | 2016-03-04 | 2016-07-20 | 杭州清正生物科技股份有限公司 | Composite strain and beverage produced by fermenting composite strain |
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| CN1880471A (en) * | 2005-06-14 | 2006-12-20 | 黑龙江省完达山乳业股份有限公司 | Method for detecting bifidobacteria in milk product |
| CN105779330A (en) * | 2016-03-04 | 2016-07-20 | 杭州清正生物科技股份有限公司 | Composite strain and beverage produced by fermenting composite strain |
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