CN107523605A - 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 - Google Patents
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 Download PDFInfo
- Publication number
- CN107523605A CN107523605A CN201710911269.9A CN201710911269A CN107523605A CN 107523605 A CN107523605 A CN 107523605A CN 201710911269 A CN201710911269 A CN 201710911269A CN 107523605 A CN107523605 A CN 107523605A
- Authority
- CN
- China
- Prior art keywords
- culture medium
- lactobacillus
- lactobacillus rhamnosus
- lactobacillus acidophilus
- layer
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Classifications
-
- 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
-
- 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
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Analytical Chemistry (AREA)
- Toxicology (AREA)
- Immunology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
It is used to carry out the culture medium of counting and its application simultaneously to Lactobacillus rhamnosus and lactobacillus acidophilus the invention provides a kind of, the culture medium is by being obtained after Lincomycin Hydrochloride modified MRS agar, and the culture medium after the initiative modified MRS agar by Lincomycin Hydrochloride of the present invention is used to count while Lactobacillus rhamnosus and lactobacillus acidophilus.The present invention still further provides the method that Lactobacillus rhamnosus and lactobacillus acidophilus are carried out while counted using above-mentioned culture medium, above-mentioned culture medium is taken first and is placed solidifies to obtain basal layer, biased sample to be measured and above-mentioned culture medium are then added on basal layer and is placed and solidifies to obtain intermediate layer, then above-mentioned culture medium is added on the intermediate layer and is placed solidifies to obtain top layer, above-mentioned basal layer, intermediate layer and top layer form Sandwich Plates, after Sandwich Plates finally are carried out into Anaerobic culturel, Lactobacillus rhamnosus and lactobacillus acidophilus are counted respectively.The above method reduces experimental ware, experiment reagent and cost of human resources.
Description
Technical field
The invention belongs to strain counting technology field, and in particular to one kind is used for Lactobacillus rhamnosus and lactobacillus acidophilus
Carry out culture medium and its application counted simultaneously.
Background technology
Lactic acid bacteria has improvement stomach function, adjustment gut flora, suppresses growth of pathogenic bacteria, improves protein and vitamin generation
Thank, prevent constipation, alleviate lactose intolerance, be antitumor, strengthen immune system and reduce cholesterol and other effects, therefore by increasingly
More is added in food and health products.Lactobacillus acidophilus, Bifidobacterium, Lactobacillus rhamnosus and streptococcus thermophilus etc. belong to
Lactic acid bacteria, in order to ensure the physiological hygiene function that above-mentioned lactic acid bacteria is brought into normal play in human body, food tissue and health products tissue
It is required to be identified the content of different lactic acid bacteria culturers in lactic acid bacteria product, therefore, studies easy-to-use lactic acid bacteria meter
Counting method, for strengthening the monitoring of lactic acid bacteria product, improving product quality and protecting the interests of consumer and health to be respectively provided with pole
Its important meaning.
《People's Republic of China (PRC) light industry standard QB/T 4575-2013》In disclose lactobacillus acidophilus, rhamnose breast
The method of counting of the lactic acid bacterias such as bacillus, Bifidobacterium and streptococcus thermophilus, the method for counting make by using selective medium
Target bacteria growing, suppress non-targeted bacteria growing, to reach the purpose of selectivity counting.For example, for lactobacillus acidophilus and other
The selective enumeration method of the product mix of lactic acid bacteria, the MRS culture mediums being modified using Clindamycin Hydrochloride and Ciprofloxacin Hydrochloride are entered
Row Anaerobic culturel is to suppress other lactobacter growths in addition to lactobacillus acidophilus, and lactobacillus acidophilus growth is good on this culture medium
It is good, it thus can realize the selectivity counting of lactobacillus acidophilus；For the product mix of Lactobacillus rhamnosus and other lactic acid bacterias
Selective enumeration method, the MRS culture mediums for using vancomycin to be modified carry out Anaerobic culturel to suppress its in addition to Lactobacillus rhamnosus
His lactobacter growth, and Lactobacillus rhamnosus well-grown on this culture medium, it thus can realize the selectivity of Lactobacillus rhamnosus
Count；For Bifidobacterium and the selective enumeration method of the product mix of other lactic acid bacterias, it is modified using mupirocin lithium salt solution
MRS culture mediums carry out Anaerobic culturel to suppress other lactobacter growths in addition to Bifidobacterium, and bifid bar on this culture medium
Bacteria growing is good, thus can realize the selectivity counting of Bifidobacterium.
However, the above method can only be counted for single culture, and the lactic acid bacteria bacterium contained in lactic acid bacteria product
Kind species is more, and when being counted using the above method, the selectivity counting of each lactic acid bacteria culturers is required for individually being detected
Experiment, each test experience is required for preparing suitable selective medium, not only consumes substantial amounts of experiment equipment and (such as tests
Vessel, experiment reagent), and the cost of human resources is also higher.Therefore, two can be realized simultaneously by needing research and development one-time detection experiment badly
The method of counting of kind or two or more lactic acid bacteria culturers, to reach the consumption for reducing experimental ware, experiment reagent etc., save simultaneously
Human cost, improve the purpose of operating efficiency.
The content of the invention
The technical problems to be solved by the invention are to overcome the method for counting of lactic acid bacteria product in the prior art can only pin
Single culture is counted, the defects of causing the cost of experimental ware, experiment reagent and human resources higher, and then provide one
The method that Lactobacillus rhamnosus and lactobacillus acidophilus count simultaneously in kind biased sample, so as to reduce experimental ware, experiment reagent
Cost, raising operating efficiency with human resources are allowed to be advantageous to counting of the enterprise to lactic acid bacteria culturers.
The present invention solve the technical scheme that uses of above-mentioned technical problem for：
It is a kind of to be used to carry out Lactobacillus rhamnosus and lactobacillus acidophilus the culture medium that counts simultaneously, the culture medium be by
Obtained after Lincomycin Hydrochloride modified MRS agar.
Above-mentioned culture medium is to be prepared via a method which：
(1) MRS agar is melted and be incubated at 45 DEG C~55 DEG C；
(2) the Clindamycin Hydrochloride solution that concentration is 0.005~0.01mg/mL is added into step (1)；Wherein, it is described
The volume ratio of MRS agar and the Clindamycin Hydrochloride solution is 100：(0.5~1.2).
The Clindamycin Hydrochloride solution concentration is 0.01mg/mL.
The volume ratio of the MRS agar and the Clindamycin Hydrochloride solution is 100：1.
A kind of application of above-mentioned culture medium in Lactobacillus rhamnosus and lactobacillus acidophilus count simultaneously.
A kind of method for Lactobacillus rhamnosus and lactobacillus acidophilus count simultaneously using above-mentioned culture medium, including such as
Lower step,
The preparation of basal layer：Take the culture medium and place and solidify to obtain basal layer；
The preparation in intermediate layer：Biased sample to be measured is added on the basal layer and the culture medium is well mixed and placed
Solidify to obtain intermediate layer；
The preparation on top layer：The culture medium is added on the intermediate layer and is placed and solidifies to obtain top layer；
The basal layer, intermediate layer and top layer form Sandwich Plates；
Anaerobic culturel is carried out to the Sandwich Plates, then the Lactobacillus rhamnosus and lactobacillus acidophilus carried out respectively
Count；
Wherein, the biased sample to be measured comprises at least Lactobacillus rhamnosus and/or lactobacillus acidophilus.
The biased sample to be measured also includes Bifidobacterium or streptococcus thermophilus.
The temperature of the Anaerobic culturel is 35 DEG C~37 DEG C, and the time is 48h~72h.
The temperature of the Anaerobic culturel is 36 DEG C, time 72h.
The volume ratio for respectively constituting the culture medium on the basal layer, intermediate layer and top layer is (5~10)：(10~
15)：(5~10).
The above-mentioned technical proposal of the present invention has the following advantages that：
(1) culture medium of the present invention for being used to carry out Lactobacillus rhamnosus and lactobacillus acidophilus counting simultaneously, should
Culture medium is by being obtained after Lincomycin Hydrochloride modified MRS agar, and the present invention is initiative by Lincomycin Hydrochloride modified MRS fine jade
Culture medium after fat is used to count while Lactobacillus rhamnosus and lactobacillus acidophilus.
(2) it is of the present invention that what is counted simultaneously is carried out to Lactobacillus rhamnosus and lactobacillus acidophilus using above-mentioned culture medium
Method, take MRS agar mediums that Lincomycin Hydrochloride is improved first and place and solidify to obtain basal layer, then on basal layer plus
Enter the MRS agar mediums of biased sample and Lincomycin Hydrochloride improvement to be measured and place and solidify to obtain intermediate layer, then in centre
The MRS agar mediums of Lincomycin Hydrochloride improvement are added on layer and are placed and solidify to obtain top layer, above-mentioned basal layer, intermediate layer and table
Layer forms Sandwich Plates, and after Sandwich Plates finally are carried out into Anaerobic culturel, Lactobacillus rhamnosus and lactobacillus acidophilus are entered respectively
Row counts.On the one hand, the initiative MRS agar mediums using Lincomycin Hydrochloride improvement of the above method are realized to be measured
Lactobacillus rhamnosus and counted in biased sample while lactobacillus acidophilus, because Lactobacillus rhamnosus is after above method culture
Sandwich Plates in bacterial plaque form and bacterial plaque diameter be more than 2mm, Sandwich Plates of the lactobacillus acidophilus after above method culture
In in bacterial plaque form and bacterial plaque diameter be less than the Sandwich Plates of 1mm, Bifidobacterium and streptococcus thermophilus after above method culture
Middle no bacterial plaque form does not grow completely, therefore can realize sandlwood in biased sample by the size of bacterial plaque diameter in once experiment
Count, namely realized in one-time detection experiment to the same of two kinds of lactic acid bacteria culturers while sugared lactobacillus and lactobacillus acidophilus
When count, thus reduce experimental ware, experiment reagent and cost of human resources, improve operating efficiency, be advantageous to enterprise pair
The quick counter of different strain lactic acid bacteria；On the other hand, using with basal layer, intermediate layer and the Sandwich Plates on top layer to be measured
Biased sample, which carries out culture, can be effectively prevented from Lactobacillus rhamnosus and lactobacillus acidophilus in the most bottom surface of ordinary flat and most
Top surface forms special-shaped bacterial plaque, and the diameter of special-shaped bacterial plaque is different from normal bacterial plaque, such as lactobacillus acidophilus is in ordinary flat
Most bottom surface and most top surface can form diameter and be more than 2mm bacterial plaque, therefore can not be distinguished as Lactobacillus rhamnosus or acidophilus breast bar
Bacterium, count results are formed and disturbed, the accuracy of counting is thus drastically increased using Sandwich Plates technology；In addition, using
Lincomycin Hydrochloride improvement MRS agar mediums to biased sample to be measured carry out strain counting when, Lactobacillus rhamnosus and
Mutually noiseless between the flora that lactobacillus acidophilus grows on the MRS agar mediums that Lincomycin Hydrochloride is improved, this also enters
One step improves the accuracy of strain counting.
(3) it is of the present invention that what is counted simultaneously is carried out to Lactobacillus rhamnosus and lactobacillus acidophilus using above-mentioned culture medium
Method, it is (5~10) by the volume ratio control of formation base layer, intermediate layer and the culture medium on top layer：(10~15)：(5~10)
Within the scope of, be advantageous to being uniformly distributed for strain in biased sample to be measured, be easy to count.
Brief description of the drawings
Fig. 1 is the life to different strain in biased sample to be measured after Sandwich Plates progress Anaerobic culturel that embodiment 1 provides
Long bacterial plaque figure；Wherein, bacterial plaque diameter is Lactobacillus rhamnosus more than 2mm；Bacterial plaque diameter is lactobacillus acidophilus less than 1mm；
Bifidobacterium and streptococcus thermophilus do not grow completely without bacterial plaque form.
Embodiment
Technical scheme will be clearly and completely described below, it is clear that described embodiment is this hair
Bright part of the embodiment, rather than whole embodiments.Based on the embodiment in the present invention, those of ordinary skill in the art are not having
There is the every other embodiment made and obtained under the premise of creative work, belong to the scope of protection of the invention.In addition, below
As long as it is mutual not form conflict can each other for involved technical characteristic in described different embodiments of the present invention
With reference to.
Embodiment 1
What the present embodiment provided carries out what is counted simultaneously to Lactobacillus rhamnosus in biased sample to be measured and lactobacillus acidophilus
Method, comprise the following steps,
The preparation of culture medium：MRS agar is melted and carries out water-bath insulation at 46 DEG C, is then maintained to 100mL temperature
The Clindamycin Hydrochloride solution that 1mL concentration is 0.005mg/mL is added in 46 DEG C of MRS agar, you can obtain culture medium；
(1) the MRS agar mediums that the 8mL Lincomycin Hydrochlorides for taking temperature to be maintained at 46 DEG C are improved are poured into aseptic flat board
And place solidify Sandwich Plates basal layer；
(2) the standard strain of Lactobacillus rhamnosus, lactobacillus acidophilus, Bifidobacterium and streptococcus thermophilus is increased into bacterium respectively
48h, take each 1mL of above-mentioned four kinds of enrichment liquids to be used as biased sample after mixing respectively, and biased sample is subjected to 10 times of gradient dilutions
To 10-6Gradient concentration, then take and above-mentioned be diluted to 10-6The dilution 1mL of gradient concentration, as dilution to be measured；In basal layer
The above-mentioned dilution to be measured of upper addition 1mL and temperature are maintained at the MRS agar mediums of 46 DEG C of 12mL Lincomycin Hydrochlorides improvement
After well mixed, place solidify Sandwich Plates intermediate layer；
(3) addition temperature is maintained at the MRS agar mediums of 46 DEG C of 7mL Lincomycin Hydrochlorides improvement simultaneously on the intermediate layer
Place solidify Sandwich Plates top layer；
(4) by above-mentioned Sandwich Plates in 36 DEG C of Anaerobic culturel 48h, then respectively to Lactobacillus rhamnosus and lactobacillus acidophilus
Counted；
Above-mentioned biased sample to be measured is made up of Lactobacillus rhamnosus, lactobacillus acidophilus, Bifidobacterium and streptococcus thermophilus.
Embodiment 2
What the present embodiment provided carries out what is counted simultaneously to Lactobacillus rhamnosus in biased sample to be measured and lactobacillus acidophilus
Method, comprise the following steps,
Mix lyophilized bacterium powders of the bacterium powder sample A by Lactobacillus rhamnosus, lactobacillus acidophilus, Bifidobacterium and streptococcus thermophilus
It is well mixed to form；
The preparation of culture medium：MRS agar is melted and carries out water-bath insulation at 45 DEG C, is then maintained to 100mL temperature
The Clindamycin Hydrochloride solution that 1mL concentration is 0.01mg/mL is added in 45 DEG C of MRS agar, you can obtain culture medium；
(1) the MRS agar mediums that the 10mL Lincomycin Hydrochlorides for taking temperature to be maintained at 45 DEG C are improved pour into aseptic flat board
In and place solidify Sandwich Plates basal layer；
(2) the biased sample A of Lactobacillus rhamnosus, lactobacillus acidophilus, Bifidobacterium and streptococcus thermophilus is carried out 10 times
Gradient dilution is to 10-6Gradient concentration and 10-7Gradient concentration, the corresponding basal layer of each gradient concentration, on two basal layers
It is separately added into 1mL above-mentioned 10-6、10-7Gradient dilution liquid, 45 DEG C then are maintained to being separately added into temperature on two basal layers again
The improvement of 10mL Lincomycin Hydrochlorides MRS agar mediums, being both needed on the basis of each can be mould by gradient dilution liquid and hydrochloric acid woods
Element improvement MRS agar mediums be well mixed and place solidify Sandwich Plates intermediate layer；
(3) the MRS fine jades that 45 DEG C of 10mL Lincomycin Hydrochlorides are improved are maintained to addition temperature on two intermediate layers respectively
Fat culture medium and placing solidify Sandwich Plates top layer；
(4) by above-mentioned two Sandwich Plates in 35 DEG C of Anaerobic culturel 72h, then respectively to Lactobacillus rhamnosus and acidophilus breast
Bacillus is counted；
Clump count is chosen between 30CFU~300CFU, the Sandwich Plates of no sprawling colony growth count total plate count, bacterium
Fall the calculating of sum in accordance with the following methods：
(1) if 10-6Gradient dilution liquid and 10-7There was only the clump count on a dilution factor Sandwich Plates in gradient dilution liquid
Between 30CFU~300CFU, then the calculating of total plate count is in accordance with the following methods：Two Sandwich Plates clump counts of calculating are averaged
Value, then average value is multiplied by corresponding extension rate, as total plate count result；
(2) if above-mentioned 10-6、10-7The Sandwich Plates clump count of gradient dilution liquid is between 30CFU~300CFU, then bacterium
The calculating for falling sum calculates according to the following equation：
In formula：N=∑ C/ (n1+0.1n2)d
N --- clump count in sample；
∑ C --- flat board (flat board of the clump count containing optimum range) clump count sum；
n1--- the first dilution factor (low extension rate) flat board number；
n2--- the second dilution factor (highly diluted multiple) flat board number；
D --- dilution gfactor (the first dilution factor)；
Embodiment 3
What the present embodiment provided carries out what is counted simultaneously to Lactobacillus rhamnosus in biased sample to be measured and lactobacillus acidophilus
Method, comprise the following steps,
Mix lyophilized bacterium powders of the bacterium powder sample B by Lactobacillus rhamnosus, lactobacillus acidophilus, Bifidobacterium and streptococcus thermophilus
It is well mixed to form；
The preparation of culture medium：MRS agar is melted and carries out water-bath insulation at 55 DEG C, is then maintained to 100mL temperature
The Clindamycin Hydrochloride solution that 1.2mL concentration is 0.006mg/mL is added in 55 DEG C of MRS agar, you can obtain culture medium；
(1) the MRS agar mediums that the 5mL Lincomycin Hydrochlorides for taking temperature to be maintained at 55 DEG C are improved are poured into aseptic flat board
And place solidify Sandwich Plates basal layer；
(2) the biased sample B of Lactobacillus rhamnosus, lactobacillus acidophilus, Bifidobacterium and streptococcus thermophilus is carried out 10 times
Gradient dilution is to 10-7Gradient concentration and 10-8Gradient concentration, the corresponding basal layer of each gradient concentration, on two basal layers
It is separately added into 1mL above-mentioned 10-7、10-8Gradient dilution liquid, 55 DEG C then are maintained to being separately added into temperature on two basal layers again
The improvement of 15mL Lincomycin Hydrochlorides MRS agar mediums, being both needed on the basis of each can be mould by gradient dilution liquid and hydrochloric acid woods
Element improvement MRS agar mediums be well mixed and place solidify Sandwich Plates intermediate layer；
(3) it is maintained at the MRS agar that 55 DEG C of 5mL Lincomycin Hydrochlorides are improved to addition temperature on two intermediate layers respectively
Culture medium and placing solidify Sandwich Plates top layer；
(4) by above-mentioned two Sandwich Plates in 37 DEG C of Anaerobic culturel 48h, then respectively to Lactobacillus rhamnosus and acidophilus breast
Bacillus is counted；
Clump count is chosen between 30CFU~300CFU, the Sandwich Plates of no sprawling colony growth count total plate count, bacterium
Fall the calculating of sum in accordance with the following methods：
(1) if 10-7Gradient dilution liquid and 10-8There was only the clump count on a dilution factor Sandwich Plates in gradient dilution liquid
Between 30CFU~300CFU, then the calculating of total plate count is in accordance with the following methods：Two Sandwich Plates clump counts of calculating are averaged
Value, then average value is multiplied by corresponding extension rate, as total plate count result；
(2) if above-mentioned 10-7、10-8The Sandwich Plates clump count of gradient dilution liquid is between 30CFU~300CFU, then bacterium
The calculating for falling sum calculates according to the following equation：
In formula：N=∑ C/ (n1+0.1n2)d
N --- clump count in sample；
∑ C --- flat board (flat board of the clump count containing optimum range) clump count sum；
n1--- the first dilution factor (low extension rate) flat board number；
n2--- the second dilution factor (highly diluted multiple) flat board number；
D --- dilution gfactor (the first dilution factor)；
Embodiment 4
What the present embodiment provided carries out what is counted simultaneously to Lactobacillus rhamnosus in biased sample to be measured and lactobacillus acidophilus
Method, comprise the following steps,
The preparation of culture medium：MRS agar is melted and carries out water-bath insulation at 47 DEG C, is then maintained to 100mL temperature
The Clindamycin Hydrochloride solution that 0.5mL concentration is 0.01mg/mL is added in 47 DEG C of MRS agar, you can obtain culture medium；
(1) the MRS agar mediums that the 8mL Lincomycin Hydrochlorides for taking temperature to be maintained at 47 DEG C are improved are poured into aseptic flat board
And place solidify Sandwich Plates basal layer；
(2) the standard strain of Lactobacillus rhamnosus and lactobacillus acidophilus is increased into bacterium 48h respectively, takes above two to increase bacterium respectively
Each 1mL of liquid is used as biased sample after mixing, and biased sample is carried out into 10 times of gradient dilutions to 10-6Gradient concentration, then take
State and be diluted to 10-6The dilution 1mL of gradient concentration, as dilution to be measured；The above-mentioned dilutions to be measured of 1mL are added on basal layer
With temperature be maintained at 47 DEG C of 12mL Lincomycin Hydrochlorides improvement MRS agar mediums it is well mixed after, place solidification and prepare
The intermediate layer of Sandwich Plates；
(3) addition temperature is maintained at the MRS agar mediums of 47 DEG C of 7mL Lincomycin Hydrochlorides improvement simultaneously on the intermediate layer
Place the top layer that solidification prepares Sandwich Plates；
(4) by above-mentioned Sandwich Plates in 35 DEG C of Anaerobic culturel 48h, then respectively to Lactobacillus rhamnosus and lactobacillus acidophilus
Counted；
Above-mentioned biased sample to be measured is made up of Lactobacillus rhamnosus, lactobacillus acidophilus.
Embodiment 5
What the present embodiment provided carries out what is counted simultaneously to Lactobacillus rhamnosus in biased sample to be measured and lactobacillus acidophilus
Method, comprise the following steps,
The preparation of culture medium：MRS agar is melted and carries out water-bath insulation at 50 DEG C, is then maintained to 100mL temperature
The Clindamycin Hydrochloride solution that 0.8mL concentration is 0.009mg/mL is added in 50 DEG C of MRS agar, you can obtain culture medium；
(1) the MRS agar mediums that the 8mL Lincomycin Hydrochlorides for taking temperature to be maintained at 50 DEG C are improved are poured into aseptic flat board
And place solidify Sandwich Plates basal layer；
(2) the standard strain of Lactobacillus rhamnosus, lactobacillus acidophilus and Bifidobacterium is increased into bacterium 48h respectively, taken respectively
State and biased sample is used as after each 1mL of three kinds of enrichment liquids is mixed, and biased sample is subjected to 10 times of gradient dilutions to 10-6Gradient is dense
Degree, then take and above-mentioned be diluted to 10-6The dilution 1mL of gradient concentration, as dilution to be measured；Added on basal layer on 1mL
State dilution to be measured and the MRS agar mediums of 12mL Lincomycin Hydrochlorides improvement that temperature is maintained at 50 DEG C it is well mixed after,
Place solidify Sandwich Plates intermediate layer；
(3) addition temperature is maintained at the MRS agar mediums of 50 DEG C of 7mL Lincomycin Hydrochlorides improvement simultaneously on the intermediate layer
Place solidify Sandwich Plates top layer；
(4) by above-mentioned Sandwich Plates in 36 DEG C of Anaerobic culturel 72h, then respectively to Lactobacillus rhamnosus and lactobacillus acidophilus
Counted；
Above-mentioned biased sample to be measured is made up of Lactobacillus rhamnosus, lactobacillus acidophilus and Bifidobacterium.
Experimental example 1
After the standard strain of Lactobacillus rhamnosus, lactobacillus acidophilus, Bifidobacterium and streptococcus thermophilus is increased into bacterium 48h respectively
4 kinds of enrichment liquids are obtained, and 4 kinds of enrichment liquids are subjected to 10 times of gradient dilutions to 10-6Gradient concentration, then take respectively above-mentioned four kinds it is dilute
Release to 10-6Each 0.25mL of bacterium solution of gradient concentration, as four kinds of dilutions to be measured, use《National Standard of the People's Republic of China
GB 4789.35-2016》In method above-mentioned four kinds of dilutions to be measured are counted, as first group of count results；
The MRS agar mediums of Lincomycin Hydrochloride improvement are prepared using the method in embodiment 1, equally using embodiment
Method in 1 prepares the basal layer of Sandwich Plates；Equally use Lactobacillus rhamnosus, lactobacillus acidophilus, bifid bar in embodiment 1
Bacterium and the standard strain 48h enrichment liquids of streptococcus thermophilus, and enrichment liquid is subjected to 10 times of gradient dilutions to 10-6Gradient concentration, and
Above-mentioned four kinds are taken to be diluted to 10 respectively afterwards-6Each 0.25mL of bacterium solution of gradient concentration, as four kinds of dilutions to be measured, on basal layer
It is separately added into above-mentioned four kinds of dilutions to be measured and temperature is maintained at the MRS agar cultures that 46 DEG C of 12mL Lincomycin Hydrochlorides are improved
Base simultaneously places the intermediate layer that solidification prepares Sandwich Plates, and the table of Sandwich Plates is then prepared using method same as Example 1
Layer and progress Anaerobic culturel, Anaerobic culturel counts after terminating to above-mentioned four kinds of dilutions to be measured, as second group of counting
As a result；
Lactobacillus rhamnosus and acidophilus breast bar in the biased sample provided using the method for counting of the present invention embodiment 1
Bacterium is counted, as the 3rd group of count results；
Above-mentioned first group of count results, second group of count results and the 3rd group of count results are as shown in table 1.
The count results that every group of table 1
First group of count results is understood compared with second group of count results in table 1, (1) Lactobacillus rhamnosus uses《In
Magnificent people's republic's standard GB/T 4789.35-2016》Method of counting and second group of method of counting count detection result
Deviation r=0.019, far smaller than 0.15 deviation requirement, it can thus be appreciated that the hydrochloric acid woods used in method of counting of the present invention can be mould
The MRS agar mediums of element improvement do not interfere with the growth of Lactobacillus rhamnosus；
(2) lactobacillus acidophilus uses《National Standard of the People's Republic of China GB 4789.35-2016》Method of counting and
The count detection result error r=0.011 of two groups of method of counting, far smaller than 0.15 deviation requirement, it can thus be appreciated that of the invention
The MRS agar mediums of the Lincomycin Hydrochloride improvement used in method of counting do not interfere with the growth of lactobacillus acidophilus；
(3) MRS for the Lincomycin Hydrochloride improvement that Bifidobacterium and streptococcus thermophilus use in method of counting of the present invention
Do not grown on agar medium.
First group of count results is understood compared with the 3rd group of count results, (1) uses the method in the embodiment of the present invention 1
The count results of Lactobacillus rhamnosus and use in the mixing liquid of detection《National Standard of the People's Republic of China GB4789.35-
2016》In method to the deviation r=0.014 of the count results of Lactobacillus rhamnosus, far smaller than 0.15 deviation requirement, by
This shows that the method for counting accuracy of Lactobacillus rhamnosus in biased sample provided by the invention is higher, and sandlwood in biased sample
The growth of sugared lactobacillus will not be influenceed by lactobacillus acidophilus, Bifidobacterium and streptococcus thermophilus；
(2) count results of lactobacillus acidophilus and use in the mixing liquid of the method detection in the embodiment of the present invention 1 are used
《National Standard of the People's Republic of China GB4789.35-2016》In method to the deviation r=of the count results of lactobacillus acidophilus
0.006, far smaller than 0.15 deviation requirement, it is indicated above the counting side of lactobacillus acidophilus in biased sample provided by the invention
Method accuracy is higher, and the growth of lactobacillus acidophilus will not be by Lactobacillus rhamnosus, Bifidobacterium and thermophilic in biased sample
Streptococcic influence.
Experimental example 2
The mixing bacterium powder sample A of Lactobacillus rhamnosus, lactobacillus acidophilus, Bifidobacterium is subjected to 10 times of gradient dilutions extremely
10-6Gradient concentration and 10-7Gradient concentration, then take above-mentioned 10-6、10-7Each 1mL of dilution of gradient concentration, as dilution to be measured
Liquid, use《People's Republic of China (PRC) light industry standard QB/T4575-2013》In method above-mentioned dilution to be measured is distinguished
Carry out Lactobacillus rhamnosus, lactobacillus acidophilus counts；
Lactobacillus rhamnosus and acidophilus breast bar in the biased sample provided using the method for counting of the present invention embodiment 2
Bacterium is counted；
The count results of two methods are as shown in table 2.
The count results of the two methods of table 2
As shown in Table 2, using Lactobacillus rhamnosus in the mixing bacterium powder sample of the method detection in the embodiment of the present invention 2
Count results and use《People's Republic of China (PRC) light industry standard QB/T4575-2013》In method to Lactobacillus rhamnosus
Count results deviation r=0.041, far smaller than 0.15 deviation requirement, be indicated above biased sample provided by the invention
The method of counting accuracy of middle Lactobacillus rhamnosus is higher；The mixing bacterium powder sample detected using the method in the embodiment of the present invention 2
The count results of lactobacillus acidophilus and use in product《People's Republic of China (PRC) light industry standard QB/T 4575-2013》In
Method far smaller than 0.15 deviation requirement, is indicated above this hair to the deviation r=0.030 of the count results of lactobacillus acidophilus
The method of counting accuracy of lactobacillus acidophilus is higher in the biased sample of bright offer.
In addition, when being counted using the inventive method, due to folder of the Lactobacillus rhamnosus after the inventive method culture
It is more than 2mm in bacterial plaque form and bacterial plaque diameter in layer flat board, in Sandwich Plates of the lactobacillus acidophilus after the inventive method culture
It is less than in bacterial plaque form and bacterial plaque diameter in the Sandwich Plates of 1mm, Bifidobacterium and streptococcus thermophilus after above method culture
Do not grown completely without bacterial plaque form, therefore rhamnose in biased sample can be realized by the size of bacterial plaque diameter in once experiment
While counting, namely realized in one-time detection experiment to two kinds of lactic acid bacteria culturers while lactobacillus and lactobacillus acidophilus
Count, thus reduce experimental ware, experiment reagent and cost of human resources, improve operating efficiency, be advantageous to enterprise's realization
To the quick counter of different strain lactic acid bacteria in biased sample；At the same time, using with basal layer, intermediate layer and the folder on top layer
Layer flat board, which carries out culture to biased sample to be measured, can be effectively prevented from Lactobacillus rhamnosus and lactobacillus acidophilus in Sandwich Plates
Most bottom surface and most top surface form special-shaped bacterial plaque, the diameter of special-shaped bacterial plaque is different from normal bacterial plaque, therefore can not be distinguished as
Lactobacillus rhamnosus or lactobacillus acidophilus, the accuracy of counting is thus drastically increased using Sandwich Plates；Furthermore use
Lincomycin Hydrochloride improvement MRS agar mediums to biased sample to be measured carry out strain counting when, Lactobacillus rhamnosus and
Mutually noiseless between the flora that lactobacillus acidophilus grows on the MRS agar mediums that Lincomycin Hydrochloride is improved, this also enters
One step improves the accuracy of strain counting.
Experimental example 3
The mixing bacterium powder sample B of Lactobacillus rhamnosus, lactobacillus acidophilus, Bifidobacterium and streptococcus thermophilus is carried out 10 times
Gradient dilution is to 10-7Gradient concentration and 10-8Gradient concentration, then take above-mentioned 10-7、10-8Each 1mL of dilution of gradient concentration, make
For dilution to be measured, use《People's Republic of China (PRC) light industry standard QB/T 4575-2013》In method to above-mentioned to be measured
Dilution is counted；
Lactobacillus rhamnosus and acidophilus breast bar in the biased sample provided using the method for counting of the present invention embodiment 3
Bacterium is counted；
The count results of two methods are as shown in table 3.
The count results of the two methods of table 3
As shown in Table 3, using Lactobacillus rhamnosus in the mixing bacterium powder sample of the method detection in the embodiment of the present invention 3
Count results and use《People's Republic of China (PRC) light industry standard QB/T4575-2013》In method to Lactobacillus rhamnosus
Count results deviation r=0.005, far smaller than 0.15 deviation requirement, be indicated above biased sample provided by the invention
The method of counting accuracy of middle Lactobacillus rhamnosus is higher；It is thermophilic in the mixing liquid detected using the method in the embodiment of the present invention 3
The count results of Lactobacillus lactis and use《People's Republic of China (PRC) light industry standard QB/T 4575-2013》In method pair
The deviation r=0.032 of the count results of lactobacillus acidophilus, far smaller than 0.15 deviation requirement, is indicated above offer of the present invention
Biased sample in lactobacillus acidophilus method of counting accuracy it is higher.
Obviously, above-described embodiment is only intended to clearly illustrate example, and is not the restriction to embodiment.It is right
For those of ordinary skill in the art, can also make on the basis of the above description it is other it is various forms of change or
Change.There is no necessity and possibility to exhaust all the enbodiments.And the obvious change thus extended out or
Among changing still in the protection domain of the invention.
Claims (10)
1. a kind of culture medium for being used to carry out Lactobacillus rhamnosus and lactobacillus acidophilus counting simultaneously, it is characterised in that described
Culture medium is by being obtained after Lincomycin Hydrochloride modified MRS agar.
2. culture medium according to claim 1, it is characterised in that the culture medium is to be prepared via a method which：
(1) MRS agar is melted and be incubated at 45 DEG C~55 DEG C；
(2) the Clindamycin Hydrochloride solution that concentration is 0.005~0.01mg/mL is added into step (1)；
Wherein, the volume ratio of the MRS agar and the Clindamycin Hydrochloride solution is 100：(0.5~1.2).
3. culture medium according to claim 2, it is characterised in that the concentration of the Clindamycin Hydrochloride solution is
0.01mg/mL。
4. the culture medium according to Claims 2 or 3, it is characterised in that the MRS agar and the Clindamycin Hydrochloride are molten
The volume ratio of liquid is 100：1.
5. the culture medium described in a kind of any one of claim 1-4 to Lactobacillus rhamnosus and lactobacillus acidophilus count simultaneously
Application in number.
6. a kind of culture medium using described in claim any one of 1-4 is carried out simultaneously to Lactobacillus rhamnosus and lactobacillus acidophilus
The method of counting, comprises the following steps,
The preparation of basal layer：Take the culture medium and place and solidify to obtain basal layer；
The preparation in intermediate layer：Biased sample to be measured is added on the basal layer and the culture medium is well mixed and places solidification
Obtain intermediate layer；
The preparation on top layer：The culture medium is added on the intermediate layer and is placed and solidifies to obtain top layer；
The basal layer, intermediate layer and top layer form Sandwich Plates；
Anaerobic culturel is carried out to the Sandwich Plates, then the Lactobacillus rhamnosus and lactobacillus acidophilus counted respectively
Number；
Wherein, the biased sample to be measured comprises at least Lactobacillus rhamnosus and lactobacillus acidophilus.
7. according to the method for claim 6, it is characterised in that the biased sample to be measured also include Bifidobacterium and/or
Streptococcus thermophilus.
8. the method according to claim 6 or 7, it is characterised in that the temperature of the Anaerobic culturel is 35 DEG C~37 DEG C, when
Between be 48h~72h.
9. according to the method for claim 8, it is characterised in that the temperature of the Anaerobic culturel is 36 DEG C, time 72h.
10. according to the method described in claim any one of 6-9, it is characterised in that respectively constitute the basal layer, intermediate layer and
The volume ratio of the culture medium on top layer is (5~10)：(10~15)：(5~10).
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710911269.9A CN107523605B (en) | 2017-09-29 | 2017-09-29 | Culture medium for simultaneously counting lactobacillus rhamnosus and lactobacillus acidophilus and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201710911269.9A CN107523605B (en) | 2017-09-29 | 2017-09-29 | Culture medium for simultaneously counting lactobacillus rhamnosus and lactobacillus acidophilus and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN107523605A true CN107523605A (en) | 2017-12-29 |
| CN107523605B CN107523605B (en) | 2021-02-19 |
Family
ID=60684149
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201710911269.9A Active CN107523605B (en) | 2017-09-29 | 2017-09-29 | Culture medium for simultaneously counting lactobacillus rhamnosus and lactobacillus acidophilus and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107523605B (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109136138A (en) * | 2018-11-18 | 2019-01-04 | 善恩康生物科技（苏州）有限公司 | The method and dedicated culture medium, corresponding method of counting of Bifidobacterium are cultivated in standard incubator |
| CN110669817A (en) * | 2019-11-22 | 2020-01-10 | 合生元（广州）健康产品有限公司 | Culture medium for detecting lactobacillus and detection method |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105349614A (en) * | 2015-12-17 | 2016-02-24 | 石家庄君乐宝乳业有限公司 | Lactobacillus plantarum specific culture medium and application thereof |
-
2017
- 2017-09-29 CN CN201710911269.9A patent/CN107523605B/en active Active
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105349614A (en) * | 2015-12-17 | 2016-02-24 | 石家庄君乐宝乳业有限公司 | Lactobacillus plantarum specific culture medium and application thereof |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109136138A (en) * | 2018-11-18 | 2019-01-04 | 善恩康生物科技（苏州）有限公司 | The method and dedicated culture medium, corresponding method of counting of Bifidobacterium are cultivated in standard incubator |
| CN110669817A (en) * | 2019-11-22 | 2020-01-10 | 合生元（广州）健康产品有限公司 | Culture medium for detecting lactobacillus and detection method |
| CN110669817B (en) * | 2019-11-22 | 2020-06-02 | 合生元（广州）健康产品有限公司 | Culture medium for detecting lactobacillus and detection method |
Also Published As
| Publication number | Publication date |
|---|---|
| CN107523605B (en) | 2021-02-19 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Adhikari et al. | Viability of microencapsulated bifidobacteria in set yogurt during refrigerated storage | |
| Lee et al. | A differential medium for lactic acid‐producing bacteria in a mixed culture | |
| KR101141781B1 (en) | Method for producing fermented milk using novel lactic acid bacteria | |
| CN110656060A (en) | Multi-linked lactobacillus composition and application thereof in female vaginal health | |
| CN110066750B (en) | Streptococcus thermophilus JMCC0024, and separation and purification method and application thereof | |
| CN115039885B (en) | Lactobacillus paracasei with function of inhibiting growth of Proteus mirabilis, and probiotic composition, fermentation liquor and application thereof | |
| CN111363704A (en) | Lactobacillus rhamnosus X253 beneficial to oral health, and separation and purification method and application thereof | |
| KR20110069796A (en) | Improvement of growth of bifidobacteria in fermented milk products | |
| MX2008009568A (en) | Novel lactic acid bacteria. | |
| CN109156686B (en) | Method for improving activity of probiotics in storage period of fermented fruit juice based on microencapsulation | |
| EP0111392B1 (en) | A culture containing viable cell mass of bifidobacteria and lactic acid bacteria, and a process for preparing said culture | |
| CN102014644A (en) | Method for manufacturing fermented milk | |
| CN110643673B (en) | Counting method of bacillus coagulans spores in feeding composite microecological product | |
| CN107523605A (en) | 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 | |
| Renschler et al. | Using nitrous acid-modified de Man, Rogosa, and Sharpe medium to selectively isolate and culture lactic acid bacteria from dairy foods | |
| Liu et al. | Screening of bifidobacteria with acquired tolerance to human gastrointestinal tract | |
| Miranda et al. | Enumeration of bifidobacteria using Petrifilm™ AC in pure cultures and in a fermented milk manufactured with a commercial culture of Streptococcus thermophilus | |
| CN105349614B (en) | Lactobacillus plantarum special media and its application | |
| CA2781516C (en) | Culture medium for specific growth, detection, and enumeration of bifidobacterium breve | |
| Wang et al. | Effect of probiotic Lactobacillus casei Zhang on fermentation characteristics of set yogurt | |
| Korbekandi et al. | Production and evaluation of a probiotic yogurt using Lactobacillus casei ssp. casei | |
| CN104946723B (en) | A kind of collective media and its application for lactic acid bacteria drug | |
| CN116286497A (en) | Anti-phage probiotics and application thereof in preparation of fermented dairy products | |
| Mahmoudi et al. | The influence of probiotic bacteria on the properties of Iranian white cheese | |
| CN110353044A (en) | A kind of micro- concentrated probiotics Yoghourt and preparation method thereof rich in bifidobacterium lactis |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |