CN112813134A - Chromogenic medium, preparation method and strain detection method - Google Patents
Chromogenic medium, preparation method and strain detection method Download PDFInfo
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Abstract
The invention relates to the technical field of microorganisms, and provides a chromogenic culture medium which comprises the following components: 8-12 parts of maltose, 4-6 parts of yeast powder, 6-14 parts of peptone, 0.4-0.8 part of L-cysteine hydrochloride, 4-6 parts of sodium acetate, 2-4 parts of dipotassium hydrogen phosphate, 2-4 parts of triammonium citrate, 0.2-0.5 part of magnesium sulfate, 0.02-0.06 part of manganese sulfate, 13-17 parts of agar, 1-2 parts of tween and 900-1000 parts of distilled water. Through the technical scheme, the problem that other small amounts of unidentified strains cannot be mixed in the leavening agent consisting of the lactobacillus bulgaricus and the streptococcus thermophilus for detection in the prior art is solved.
Description
Technical Field
The invention relates to the technical field of microorganisms, in particular to a chromogenic medium, a preparation method and a strain detection method.
Background
The common yoghurt in the market is a milk product which is prepared by taking fresh milk as a raw material, sterilizing the fresh milk by a bus, adding a starter (a starter) into the milk, fermenting the milk, and cooling and filling the fermented milk.
The selection of the strains plays an important role in the quality of the leavening agent, proper strains can be selected according to the variety of the produced fermented dairy products, comprehensive selection is carried out on the characteristics of the optimal temperature, the acid production capacity, whether viscous substances are produced or not of the strains, and the symbiosis among the strains is also considered so as to enable the strains to be beneficial to each other in production and propagation. The yogurt is produced by adopting freeze-dried strains which are mainly a mixed starter of lactobacillus bulgaricus and streptococcus thermophilus, wherein the streptococcus thermophilus produces acid, and the lactobacillus bulgaricus produces acid and flavor.
The quality of the leavening agent is strictly controlled, and the requirements on the microorganism of the leavening agent are as follows: detecting the viable count of the lactobacillus according to GB 4789.35; mould and yeast testing, following GB 4789.15; enterobacteriaceae assays, following SN/T0738; staphylococcus aureus detection, following GB 4789.10; listeria monocytogenes, following GB 4789.30;
the method prevents a small amount of unidentified strains from being mixed into a leavening agent only comprising lactobacillus bulgaricus and streptococcus thermophilus, but in the conventional detection process, non-lactic acid bacteria and mildew fermentation detection is carried out on the product, so that a detection method for pollution of other non-local lactic acid bacteria is lacked, and a standard detection method is not available at present. For example, if a starter composed of only lactobacillus bulgaricus and streptococcus thermophilus is mixed with one or more of lactobacillus plantarum, lactobacillus casei, lactobacillus paracasei, lactobacillus rhamnosus, lactobacillus acidophilus buchneri and the like which should not be present, the detection cannot be performed by the detection method, which is a technical problem which has been difficult to solve for a long time.
There are many media for culturing lactic acid bacteria, but in the case of both Lactobacillus bulgaricus and Streptococcus thermophilus, there are cases where difficulty in growth is encountered, and therefore a suitable medium should be selected. In general, lactic acid bacteria are tolerant to low pH values but are very sensitive to other adverse conditions, e.g. some samples cannot be frozen before inoculation and the selection of a suitable medium is also a difficult problem to solve.
The chromogenic culture medium utilizes the specificity of bacteria, combines a specific enzyme substrate by a selective culture method, allows the target bacteria to grow and present a color or other colony characteristics which are obviously different from the colonies of other growing bacteria, and thereby identifies the target bacteria. The most important advantage of the chromogenic culture is that the specimen is directly used, and the separation culture and the identification of bacteria are combined together, so that the effect of direct identification while separation is achieved. To date, there have been many chromogenic cultures such as Candida chromogenic medium, group B Streptococcus chromogenic medium, Staphylococcus aureus chromogenic medium, etc., but Lactobacillus bulgaricus and Streptococcus thermophilus chromogenic medium have not been found.
Disclosure of Invention
The invention provides a chromogenic medium, a preparation method and a strain detection method, and solves the problem that in the prior art, a small amount of unidentified strains cannot be mixed in a leavening agent consisting of lactobacillus bulgaricus and streptococcus thermophilus for detection.
The technical scheme of the invention is as follows:
a chromogenic culture medium comprises the following components in parts by weight: 8-12 parts of maltose, 4-6 parts of yeast powder, 6-14 parts of peptone, 0.4-0.8 part of L-cysteine hydrochloride, 4-6 parts of sodium acetate, 2-4 parts of dipotassium hydrogen phosphate, 2-4 parts of triammonium citrate, 0.2-0.5 part of magnesium sulfate, 0.02-0.06 part of manganese sulfate, 13-17 parts of agar, 1-2 parts of tween and 900-1000 parts of distilled water.
As a further technical scheme, the chromogenic medium further comprises 0.01-0.015 part of vancomycin.
As a further technical scheme, the chromogenic medium further comprises 0.003-0.004 part of bromocresol purple ethanol solution.
A preparation method of a chromogenic medium comprises the following steps:
s1, weighing the raw materials according to the formula for later use;
s2, dissolving maltose, yeast powder, peptone, L-cysteine hydrochloride, sodium acetate, dipotassium hydrogen phosphate, triammonium citrate, magnesium sulfate, manganese sulfate, agar and tween in distilled water, heating and boiling until completely dissolving, sterilizing under high pressure, and cooling to 50 ℃ to obtain an S2 culture medium;
s3, adding vancomycin into the S2 culture medium to obtain an S3 culture medium for later use;
and S4, adding the bromocresol purple ethanol solution into the S3 culture medium, uniformly mixing, and pouring a flat plate to obtain a chromogenic culture medium for later use.
As a further technical scheme, in the step S2, the autoclaving temperature is 120-125 ℃, the time is 15-20 min, and the temperature is cooled to 50 ℃ for standby.
A detection method using a chromogenic medium comprises the following steps:
a1, adding the bacterial powder to be detected into sterile normal saline to prepare bacterial powder diluent;
a2, adding the bacterial powder diluent in the step A1 to the surface of the chromogenic medium for coating;
a3, observing the formation of sterile colonies in anaerobic culture.
As a further technical scheme, the bacterial powder to be detected comprises at least 1 microorganism of microorganisms belonging to streptococcus thermophilus, microorganisms belonging to lactobacillus bulgaricus, microorganisms belonging to lactobacillus plantarum, microorganisms belonging to lactobacillus casei, microorganisms belonging to lactobacillus paracasei, microorganisms belonging to lactobacillus rhamnosus, microorganisms belonging to lactobacillus acidophilus, microorganisms belonging to lactobacillus buchneri and microorganisms belonging to alkaline environment or ammonia gas produced by metabolism.
As a further technical scheme, the bacterial powder to be detected is one of the following:
a test bacterium containing a microorganism belonging to Streptococcus thermophilus and a microorganism belonging to Lactobacillus bulgaricus;
a test bacterium containing a microorganism belonging to Streptococcus thermophilus and a microorganism belonging to Lactobacillus bulgaricus and also a microorganism which is metabolized to produce ammonia gas or an alkaline environment.
The invention has the beneficial effects that:
1. the invention provides a chromogenic medium which can inhibit the growth of lactobacillus bulgaricus and streptococcus thermophilus on the medium, and the discoloration of other types of lactic acid bacteria or ammonia bacteria generated by metabolism after the growth of the lactic acid bacteria or the ammonia bacteria on the medium, thereby enhancing the maintenance of the product quality.
2. In the preparation process of the culture medium, vancomycin and bromocresol purple dissolving liquid are added, so that growth of lactobacillus bulgaricus and streptococcus thermophilus on the culture medium can be inhibited only in bacteria to be detected containing microorganisms of streptococcus thermophilus and lactobacillus bulgaricus; other lactic acid producing bacteria used in a composition containing Streptococcus thermophilus and microorganisms other than Lactobacillus bulgaricus, or strains which metabolically produce ammonia gas, develop discoloration upon growth on this medium, and a medium useful as a selective medium for the aforementioned assay.
3. The components of the culture medium are mutually cooperated, and the growth of lactobacillus bulgaricus and streptococcus thermophilus can be inhibited by adding vancomycin, while the growth of lactobacillus plantarum, lactobacillus casei, lactobacillus paracasei, lactobacillus rhamnosus, lactobacillus acidophilus and lactobacillus buchneri is inconsistent.
4. When the morphology of colonies and the shape of bacteria are similar, the identification of the species requires professional knowledge. Therefore, a culture method is required to be used for mixing trace lactobacillus casei and the like into a fermenting agent mainly comprising streptococcus thermophilus and lactobacillus bulgaricus, carrying out metabolic analysis and optimizing a culture medium; the optimal addition amount of vancomycin is selected according to the resistance of the lactobacillus to antibiotics, and the vancomycin is added into the optimized culture for culture, so that streptococcus thermophilus and lactobacillus bulgaricus are just inhibited, and the mixed lactobacillus plantarum, lactobacillus casei, lactobacillus paracasei, lactobacillus rhamnosus, lactobacillus acidophilus and lactobacillus buchneri can grow. And then through the color development principle of bromocresol purple, the culture medium and the bacterial colony display different colors for judgment through acid or alkaline color taking generated after metabolism of different microorganisms.
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The present invention will be described in further detail with reference to the accompanying drawings and specific embodiments.
FIG. 1 is a photograph of a culture medium test of a first bacterial powder to be tested;
FIG. 2 is a photo of the detection of the culture medium of the second bacterial powder to be detected;
FIG. 3 is a photograph of the medium detection of the third bacterial powder to be tested.
Detailed Description
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 of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any inventive step, are intended to be within the scope of the present invention.
Example 1
S1, medium material composition: 10g/L of maltose, 5g/L of yeast powder, 10g/L, L g/L of peptone-cysteine hydrochloride 0.6g/L, 5g/L of sodium acetate, 3g/L of dipotassium phosphate, 3g/L of ammonium citrate tribasic, 0.3g/L of magnesium sulfate, 0.04g/L of manganese sulfate, 15g/L of agar, 801 g/L of tween and the balance of distilled water;
s2, dissolving maltose, yeast powder, peptone, L-cysteine hydrochloride, sodium acetate, dipotassium hydrogen phosphate, triammonium citrate, magnesium sulfate, manganese sulfate, agar and Tween 80 in distilled water, heating and boiling until the components are completely dissolved, sterilizing at 121 ℃ under high pressure for 10min, and cooling to 50 ℃ to obtain an S2 culture medium;
s3, adding vancomycin into the S2 culture medium, wherein the addition amount of the vancomycin is 1 vancomycin added into each 100ml of sterile culture medium, and the content of each vancomycin is 1mg to obtain an S3 culture medium for later use;
s4, adding the bromocresol purple ethanol solution into the S3 culture medium, wherein a color-changing component, namely bromocresol purple enables the culture medium to change color in environments with different acid and alkali, adding 300uL of 1.5% bromocresol purple (w/v, ethanol is dissolved) into each 100ml of the culture medium, uniformly mixing, and pouring a flat plate to obtain a color development culture medium for later use;
s5, weighing 1g of first bacterial powder to be tested in a sterile weighing vessel, adding 9g of sterile normal saline, and oscillating to prepare 1:10 bacterial powder diluent; and (3) sucking 1ml of sample diluent, adding the sample diluent into a culture medium agar plate respectively in the inoculation amount of 0.3ml, 0.3ml and 0.4ml, performing surface coating by using a coating rod, and observing the formation of sterile colonies after anaerobic culture at 36 +/-1 ℃ for 48 +/-2 hours.
FIG. 1 is a photograph of a medium test of the first test powder, the color of the plate is light green, only the microbial composition of Streptococcus thermophilus and Lactobacillus bulgaricus is contained, and no colony of any other bacterial species is grown.
Example 2
S1, medium material composition: 8g/L of maltose, 4g/L of yeast powder, 6g/L, L g of peptone-cysteine hydrochloride 0.4g/L, 4g/L of sodium acetate, 2g/L of dipotassium phosphate, 2g/L of ammonium citrate tribasic, 0.2g/L of magnesium sulfate, 0.02g/L of manganese sulfate, 13g/L of agar, 802 g/L of tween and the balance of distilled water;
s2, dissolving maltose, yeast powder, peptone, L-cysteine hydrochloride, sodium acetate, dipotassium hydrogen phosphate, triammonium citrate, magnesium sulfate, manganese sulfate, agar and Tween 80 in distilled water, heating and boiling until the components are completely dissolved, sterilizing at 120 ℃ under high pressure for 10min, and cooling to 50 ℃ to obtain an S2 culture medium;
s3, adding vancomycin into the S2 culture medium, wherein the addition amount of the vancomycin is 1 vancomycin added into each 100ml of sterile culture medium, and the content of each vancomycin is 1mg to obtain an S3 culture medium for later use;
s4, adding the bromocresol purple ethanol solution into the S3 culture medium, wherein a color-changing component, namely bromocresol purple enables the culture medium to change color in environments with different acid and alkali, adding 300uL of 1.4% bromocresol purple (w/v, ethanol is dissolved) into each 100ml of the culture medium, uniformly mixing, and pouring a flat plate to obtain a color development culture medium for later use;
s5, weighing 1g of second to-be-tested bacterium powder in a sterile weighing vessel, adding 9g of sterile normal saline, and oscillating to prepare 1:10 bacterium powder diluent; and (3) sucking 1ml of sample diluent, adding the sample diluent into a culture medium agar plate respectively in the inoculation amount of 0.3ml, 0.3ml and 0.4ml, performing surface coating by using a coating rod, and observing the formation of sterile colonies after anaerobic culture at 36 +/-1 ℃ for 48 +/-2 hours.
FIG. 2 is a photograph showing the detection of the medium of the second test powder, in which the surrounding medium is changed from light green to light yellow, and which shows that the powder contains one or more of microorganisms belonging to Lactobacillus plantarum, microorganisms belonging to Lactobacillus casei, microorganisms belonging to Lactobacillus paracasei, microorganisms belonging to Lactobacillus rhamnosus, microorganisms belonging to Lactobacillus acidophilus, and microorganisms belonging to Lactobacillus buchneri, in addition to the microorganisms belonging to the Streptococcus thermophilus and microorganisms belonging to Lactobacillus bulgaricus.
Example 3
S1, medium material composition: 12g/L of maltose, 6g/L of yeast powder, 14g/L, L g of peptone-cysteine hydrochloride 0.8g/L, 6g/L of sodium acetate, 4g/L of dipotassium phosphate, 4g/L of ammonium citrate tribasic, 0.5g/L of magnesium sulfate, 0.06g/L of manganese sulfate, 17g/L of agar, 801 g/L of tween and the balance of distilled water;
s2, dissolving maltose, yeast powder, peptone, L-cysteine hydrochloride, sodium acetate, dipotassium hydrogen phosphate, triammonium citrate, magnesium sulfate, manganese sulfate, agar and Tween 80 in distilled water, heating and boiling until the components are completely dissolved, sterilizing at 121 ℃ under high pressure for 12min, and cooling to 50 ℃ to obtain an S2 culture medium;
s3, adding vancomycin into the S2 culture medium, wherein the addition amount of the vancomycin is 1 vancomycin added into each 100ml of sterile culture medium, and the content of each vancomycin is 1mg to obtain an S3 culture medium for later use;
s4, adding the bromocresol purple ethanol solution into the S3 culture medium, wherein a color-changing component, namely bromocresol purple enables the culture medium to change color in environments with different acid and alkali, adding 300uL of 1.4% bromocresol purple (w/v, ethanol is dissolved) into each 100ml of the culture medium, uniformly mixing, and pouring a flat plate to obtain a color development culture medium for later use;
s5, weighing 1g of second to-be-tested bacterium powder in a sterile weighing vessel, adding 9g of sterile normal saline, and oscillating to prepare 1:10 bacterium powder diluent; and (3) sucking 1ml of sample diluent, adding the sample diluent into a culture medium agar plate respectively in the inoculation amount of 0.3ml, 0.3ml and 0.4ml, performing surface coating by using a coating rod, and observing the formation of sterile colonies after anaerobic culture at 36 +/-1 ℃ for 48 +/-2 hours.
FIG. 3 is a photograph showing the detection of the medium of the third test powder containing, in addition to microorganisms belonging to Streptococcus thermophilus and Lactobacillus bulgaricus, one or more of microorganisms capable of metabolizing and decomposing proteins to produce ammonia, such as Bacillus subtilis, etc., which form colonies that change the surrounding medium from light green to deep blue purple.
The present invention is not limited to the above preferred embodiments, and any modifications, equivalent substitutions, improvements, etc. within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (8)
1. The chromogenic culture medium is characterized by comprising the following components in parts by weight: 8-12 parts of maltose, 4-6 parts of yeast powder, 6-14 parts of peptone, 0.4-0.8 part of L-cysteine hydrochloride, 4-6 parts of sodium acetate, 2-4 parts of dipotassium hydrogen phosphate, 2-4 parts of triammonium citrate, 0.2-0.5 part of magnesium sulfate, 0.02-0.06 part of manganese sulfate, 13-17 parts of agar, 1-2 parts of tween and 900-1000 parts of distilled water.
2. The chromogenic medium according to claim 1, further comprising 0.01 to 0.015 parts of vancomycin.
3. The chromogenic medium according to claim 2, further comprising 0.003 to 0.004 part of bromocresol purple ethanol solution.
4. A preparation method of a chromogenic medium is characterized by comprising the following steps:
s1, weighing the raw materials according to the formula of claim 3 for later use;
s2, dissolving maltose, yeast powder, peptone, L-cysteine hydrochloride, sodium acetate, dipotassium hydrogen phosphate, triammonium citrate, magnesium sulfate, manganese sulfate, agar and tween in distilled water, heating and boiling until the components are completely dissolved, sterilizing at high pressure, and cooling to obtain an S2 culture medium;
s3, adding vancomycin into the S2 culture medium to obtain an S3 culture medium for later use;
and S4, adding the bromocresol purple ethanol solution into the S3 culture medium, uniformly mixing, and pouring a flat plate to obtain a chromogenic culture medium for later use.
5. The method for preparing a chromogenic medium according to claim 4, wherein in step S2, the autoclaving temperature is 120-125 ℃ for 15-20 min, and the temperature is cooled to 50 ℃ for further use.
6. A detection method using a chromogenic medium, comprising the steps of:
a1, adding the bacterial powder to be detected into sterile normal saline to prepare bacterial powder diluent;
a2, adding the bacterial powder diluent in the step A1 to the surface of the chromogenic medium for coating;
a3, observing the formation of sterile colonies in anaerobic culture.
7. The detection method according to claim 6, wherein the bacterial powder to be detected comprises at least 1 microorganism selected from the group consisting of a microorganism belonging to Streptococcus thermophilus, a microorganism belonging to Lactobacillus bulgaricus, a microorganism belonging to Lactobacillus plantarum, a microorganism belonging to Lactobacillus casei, a microorganism belonging to Lactobacillus paracasei, a microorganism belonging to Lactobacillus rhamnosus, a microorganism belonging to Lactobacillus acidophilus, a microorganism belonging to Lactobacillus buchneri, and a microorganism belonging to the metabolic generation of ammonia gas or an alkaline environment.
8. The detection method according to claim 6, wherein the bacterial powder to be detected is one of the following:
a test bacterium containing a microorganism belonging to Streptococcus thermophilus and a microorganism belonging to Lactobacillus bulgaricus;
a test bacterium containing a microorganism belonging to Streptococcus thermophilus and a microorganism belonging to Lactobacillus bulgaricus and also a microorganism which is metabolized to produce ammonia gas or an alkaline environment.
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