CN109234179A - Yeast isolation culture medium and preparation method thereof in a kind of fermented grain - Google Patents
Yeast isolation culture medium and preparation method thereof in a kind of fermented grain Download PDFInfo
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- CN109234179A CN109234179A CN201811266282.4A CN201811266282A CN109234179A CN 109234179 A CN109234179 A CN 109234179A CN 201811266282 A CN201811266282 A CN 201811266282A CN 109234179 A CN109234179 A CN 109234179A
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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- C12N1/02—Separating microorganisms from their culture media
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Abstract
The present invention relates to yeast isolation culture mediums in a kind of yeast isolation culture medium of microbial fermentation engineering technical field more particularly to a kind of fermented grain and preparation method thereof.Yeast isolation culture medium in a kind of fermented grain is disclosed, the formula of the culture medium includes following component: yeast powder 5-10g/L, peptone 10-20g/L, glucose 5-20g/L, agar 15-22g/L, yeast-fermented grain leaching liquor 100-800mL/L, surplus is water.For different times fermented grain fermentation character, suitably adjusting medium pH, be conducive to each yeast-like fungi in system separation brewing process.There is culture medium of the present invention composition to enrich, with strong points, the wide advantage of screening range.
Description
Technical field
The present invention relates to a kind of yeast isolation culture medium of microbial fermentation engineering technical field more particularly to a kind of wine
Yeast isolation culture medium and preparation method thereof in unstrained spirits.
Background technique
Saccharomycete be widely present in koji-making, accumulation, in cellar, be important micro- life during liquor production in air, the hall that dries in the air
Object.It is divided by function and produces wine function bacterium and produce fragrant function bacterium, growth metabolism situation has the yield of white wine with flavor important
It influences.To study the mechanism of action of the different saccharomycete in liquor production, need that saccharomycete is carried out to be completely separating screening first.
The culture medium of existing separation saccharomycete includes yeast extract powder peptone dextrose culture-medium (YPD culture medium), potato
Glucose agar medium (PDA culture medium), WL nutrient agar (WL culture medium), rose bengal medium etc., wherein
YPD culture medium and WL culture medium frequency of use are higher, and separating effect is more prominent.So far, point about saccharomycete in fermented grain
From there has been no add yeast-fermented grain leaching liquor report in the medium.
Summary of the invention
One of the objects of the present invention is to provide a kind of yeast isolation culture mediums.
It is a further object of the present invention to provide a kind of yeast isolation culture mediums with strong points.
It is a further object of the present invention to provide the culture mediums that one kind can separate saccharomycete in different times fermented grain.
It is a further object of the present invention to provide contained yeast-fermented grain leaching liquor preparation sides in a kind of above-mentioned culture medium
Method.
It is a further object of the present invention to provide a kind of preparation methods of above-mentioned culture medium.
Yeast-fermented grain leaching liquor is added in the medium, can preferably be simulated brewing microorganism growing environment, be promoted fermented grain
The separation of microorganism.It is reported that researcher once attempted to go out cellar fermented grain and boil when bacterium separates in Luzhou-flavor fermented grain
Boiled-out water oscillation is prepared into yeast-fermented grain leaching liquor, adds in different bacteria culture medias, but separating effect is not
It is prominent.The yeast-fermented grain leaching liquor is not added with production yeast, and does not carry out high-temperature boiling.
In order to solve the above technical problems, the technical solution adopted in the present invention content is specific as follows:
It is a kind of for separating the culture medium of saccharomycete in fermented grain, the formula of the culture medium includes following component: yeast extract
5-10 g/L, peptone 10-20g/L, glucose 5-20g/L, agar 15-22g/L, yeast-fermented grain leaching liquor 100-800mL/
L, surplus are water.
As preferred embodiment, the formula of the culture medium includes following component: yeast extract 5-10g/L, peptone
10- 15g/L, glucose 10-20g/L, agar 18-20g/L, yeast-fermented grain leaching liquor 200-700mL/L, surplus is water.
It include following component as one kind more preferably embodiment, formula of the culture medium: yeast extract 5-
7g/L, peptone 10-15g/L, glucose 15-20g/L, agar 18-20g/L, yeast-fermented grain leaching liquor 500-700 mL/L.
In the present invention, the addition of yeast-fermented grain leaching liquor is particularly important for screening, and is generally used for the culture medium of yeast isolation, often
For universal culture medium, it is not added with particular matter, can not preferably simulate the original growing environment of saccharomycete, increases separating difficulty.
In order to advanced optimize medium component, in common yeast isolation culture medium, appropriate yeast-fermented grain leaching is added
Extract is conducive to the separation of microorganism in fermented grain.
Preferably, inventor targetedly adjusts medium pH, favorably also according to fermented grain fermenting characteristic
Saccharomycete in separation different times fermented grain.
As an alternative embodiment, the pH of the culture medium is 3.0-6.0.In the present invention, as a kind of optional
Embodiment, the culture medium is used to separate saccharomycete in fermented grain sample.
For fermented grain sample be selected from what period and which batch be not particularly limited, as fermented grain sample can for accumulation
Fermented grain enters to store fermented grain, or fermented grain in storing.
Preferably, in culture medium prescription, also contain chloramphenicol 0.1-1.0g/L.By to yeast isolation
Chloramphenicol is added in culture medium, inhibits the growth of bacterium, improves yeast isolation effect.
Alternatively, above-mentioned yeast-fermented grain leaching liquor the preparation method comprises the following steps:
(1) fermented grain and yeast are weighed, is mixed；
(2) boiling pure water is added into fermented grain-yeast mixture；
(3) it is spare to obtain filtrate for cooled and filtered.
As preferred embodiment, in step (2), boiling time 5-30min.
As more preferably embodiment, in step (2), boiling time 10-15min.
In some embodiments, yeast-fermented grain leaching liquor culture medium of processing is boiled in addition, does not boil processing with being added
Yeast-fermented grain leaching liquor culture medium compare, can more promote the growth of saccharomycete.
As optional embodiment, in step (1), the mass ratio of yeast and fermented grain is 1:1-1:20.
As a kind of more preferably embodiment, in step (1), the mass ratio of yeast and fermented grain is 1:3-1:10.
As an alternative embodiment, the mass ratio of the mixture and water is 1:2-1:20 in step (2).
As a preferred embodiment, the mass ratio of the mixture and water is 1:4-1:10.
As preferred embodiment, in step (3), it is filtered after boiling using gauze.
On the other hand, the present invention also provides a kind of preparation method of above-mentioned yeast isolation culture medium, steps are as follows:
(1) each component is weighed according to formula, by yeast powder, peptone, glucose, agar powder, yeast-fermented grain leaching liquor adds
Enter in container, adjusts pH, surplus water polishing, sterilizing with acid solution；
(2) chloramphenicol is dissolved in ethyl alcohol, shaken up, prepare chloramphenicol mother liquor；
(3) mixture after step (1) sterilizing is cooled to 50-60 DEG C, is mixed with the chloramphenicol mother liquor of step (2).
As a preferred option, in step (1), the acid solution used is that hydrochloric acid solution, glacial acetic acid solution or lactic acid are molten
Liquid.
It is further preferred that 121 DEG C carry out sterilizing 15-20min to culture medium in step (1).
In addition, the present invention additionally provides a kind of method for separating saccharomycete simultaneously, wherein this method includes dilute using plate
Fermented grain is made a series of dilution bacterium solutions and is coated in above-mentioned culture medium by interpretation of the law cultivates, to reach the work of yeast isolation
With.Remaining condition of culture and method are then identical as the condition of culture of saccharomycete in the prior art or method.
There are also on the one hand, the present invention provides the applications of saccharomycete of the above-mentioned culture medium in separation fermented grain.
Beneficial effects of the present invention:
(1) present invention adds yeast-fermented grain leaching liquor in the isolation medium of saccharomycete for the first time, which can be more preferable
Simulation make microorganism growing environment, promote fermented grain microorganism separation.Inventor during the experiment, once attempts fermented grain
It is prepared into yeast-fermented grain leaching liquor with boiled-out water oscillation is boiled, for separating saccharomycete, but separating effect does not protrude.This
Outside, inventor also attempts the separation that the yeast without high-temperature boiling-fermented grain leachate is used for saccharomycete, and separating effect is not also dashed forward
Out, therefore, in the present invention, it is necessary that high-temperature boiling and yeast and wine, which pay for common yeast-fermented grain leaching liquor,.
(2) culture medium of the invention solves fermented grain yeast isolation effect relative to conventional yeast isolation culture medium
The bad problem of fruit, preferably simulates Yeast Growth environment, is more advantageous to the system separation of saccharomycete in fermented grain sample,
It is applicable in the with strong points of yeast isolation, and for the fermented grain for process different phase of making wine, there is wide excellent of screening range
Point.
The above description is only an overview of the technical scheme of the present invention, in order to better understand the technical means of the present invention,
And it can be implemented in accordance with the contents of the specification, and in order to allow above and other objects, features and advantages of the invention can
It is clearer and more comprehensible, special below to lift preferred embodiment, detailed description are as follows.
Detailed description of the invention
Fig. 1 is that yeast-fermented grain extracts the isolated influence of growth of the liquid hold-up difference to saccharomycete in culture medium.
Fig. 2 is influence of the medium pH difference to Yeast Growth separation in accumulation fermented grain sample.
Specific embodiment
Technical solution of the present invention is further illustrated below by way of specific embodiment, and specific embodiment does not represent to this hair
The limitation of bright protection scope.Other people according to the present invention theory made it is some it is nonessential modification and adjustment still fall within this hair
Bright protection scope.
Term " fermented grains of different times " used in the present invention refers to the fermented grain of any stage during entirely making wine
Sample.
" pH is natural " refers to that medium pH is not adjusted in the present invention.
" pH is modulated " refers to that medium pH addition acid solution is adjusted in the present invention.
" accumulation fermented grain " carries out the fermented grain sample of heap fermentation for one's parents of drying in the air in the present invention.
Wherein " enter to store fermented grain " to refer to that heap fermentation terminates the fermented grain sample for preparing to be transferred in pit.
" fermented grain in storing " is the fermented grain sample that anaerobic fermentation is carried out in pit.
Wherein " fermented grain is stored out " refer to that fermentation in pits terminates to prepare to open the fermented grain sample that cellar carries out boiling.
Yeast used in yeast-fermented grain leaching liquor in the embodiment of the present invention is the leavening of liquor production, and sugar
Agent, moreover it is possible to which certain flavor substance, including low temperature yeast, daqu of middle temperature, high-temperature daqu are provided for white wine.The yeast used comes
Yeast made of being fermented used in the liquor production process as wheat, specially used in Maotai-flavor liquor production process
High-temperature daqu.
Fermented grain used in yeast-fermented grain leaching liquor is that digested post-fermentation is good during liquor production in invention of the present invention
Grain, be the important carrier of wine brewing process, certain flavor substance can be also provided for white wine, including sorghum, corn, wheat,
Rice, glutinous rice, rice husk, wheat bran.Fermented grain used in yeast-fermented grain leaching liquor is selected from Maotai-flavor liquor production process in embodiment
The fermentation in pits fermented grain of difference round made of the middle sorghum fermentation after boiling, specially third round fermentation in pits fermented grain.
1 yeast isolation culture medium prescription of embodiment and preparation method thereof
The component and content of 1 culture medium of table
| Component | Culture medium 1 | Culture medium 2 | Culture medium 3 | Culture medium 4 | Culture medium 5 |
| Yeast extract | 5g | 5g | 5g | 7g | 7g |
| Peptone | 10g | 10g | 10g | 10g | 15g |
| Glucose | 10g | 10g | 15g | 20g | 20g |
| Agar | 18g | 18g | 20g | 20g | 20g |
| Leaching liquor | 100mL | 150mL | 200mL | 200mL | 200mL |
Note: medium pH is natural.
1, leaching liquor the preparation method comprises the following steps:
(1) yeast and fermented grain are weighed and is mixed according to the ratio of 1:5；
(2) yeast-fermented grain mixture and water are added into boiling pure water 10-15min according to the ratio of mass ratio 1:7；
(3) after cooling, using filtered through gauze, it is spare to obtain filtrate.
2, yeast isolation culture medium preparation method:
(1) each component is weighed by the formula of table 1, container is added in yeast powder, peptone, glucose, agar powder and leaching liquor
In, with water polishing to 1L, mix, 121 DEG C of sterilizing 15-20min；
(2) chloramphenicol is dissolved in 100% ethyl alcohol, shaken up, the chloramphenicol mother liquor that preparation concentration is 60g/L；
(3) mixture after step (1) sterilizing being cooled to 50-60 DEG C, the chloramphenicol mother liquor that step (2) are added mixes,
Chloramphenicol concentration in culture medium is set to be diluted to 0.2g/L.
In order to evaluate influence of the different formulations to Yeast Growth in culture medium of the present invention, the present invention uses plate dilution method
Fermented grain will be entered to store, a series of dilution bacterium solutions are made, and be respectively coated on culture medium shown in table 1 and cultivated, every group of dilution ladder
It is 10 there are three degree is equal-4、10-5、10-6(extension rate).
After cultivating 48h, it is 10 that 5 groups of culture mediums, which select dilution gradient,-5Plate compare, Yeast Growth situation is such as
Shown in table 2.
Influence of the culture medium prescription of the present invention of table 2 to Yeast Growth in fermented grain
| Yeast Growth situation | Culture medium 1 | Culture medium 2 | Culture medium 3 | Culture medium 4 | Culture medium 5 |
| Saccharomycete quantity (CFU/g) | 89×105 | 99×105 | 103×105 | 115×105 | 110×105 |
| Whether there is or not miscellaneous bacterias | A little | Nothing | Nothing | Nothing | Nothing |
Note: fermented grain is into storing fermented grain sample at this time.
Experimental result: saccharomycete has growth on above-mentioned 5 kinds of culture mediums in fermented grain, and quantity is slightly different, wherein cultivating
The quantity of base 1 and culture medium 2 is lower, and the saccharomycete quantity above culture medium 3,4,5 is higher, on the whole without significant difference, explanation
Culture medium in above-mentioned formula range could be used for the separation of saccharomycete in fermented grain.
Influence of the addition of 2 leaching liquor of embodiment to yeast isolation in fermented grain
In order to evaluate influence of the addition to Yeast Growth of leaching liquor in culture medium of the present invention, the present invention is dilute using plate
A series of dilution bacterium solutions are made in fermented grain by interpretation of the law, and are respectively coated on culture medium shown in table 3 and are cultivated, every group of dilution ladder
It is 10 there are three degree is equal-3、10-4、10-5(extension rate).
The component and content of 3 culture medium of table
| Component | Culture medium 6 | Culture medium 7 | Culture medium 8 | Contrast groups |
| Yeast extract | 5.0g | 5.0g | 5.0g | 5.0g |
| Peptone | 10.0g | 10.0g | 10.0g | 10.0g |
| Glucose | 10.0g | 10.0g | 10.0g | 10.0g |
| Agar | 20.0g | 20.0g | 20.0g | 20.0g |
| Leaching liquor | 700mL | 500mL | 200mL | 0 |
After cultivating 48h, selecting dilution gradient is 10-4Plate compare, Yeast Growth situation such as Fig. 1, table 4, table
Shown in 5.
Table 4 extracts influence of the liquid hold-up to Yeast Growth in fermented grain A
| Yeast Growth situation | Culture medium 6 | Culture medium 7 | Culture medium 8 | Contrast groups |
| Saccharomycete quantity (CFU/g) | 48×104 | 129×104 | 42×104 | 9×104 |
| Whether there is or not miscellaneous bacterias | A little | Nothing | Nothing | Nothing |
Note: fermented grain A is accumulation fermented grain sample.
Influence of the culture medium of the present invention of table 5 to Yeast Growth in fermented grain B
Note: fermented grain B is fermented grain sample in storing.
Above-mentioned four kinds of medium pHs are naturally, chloromycetin content is 0.2g/L.
Preparation method, substance and its content of leaching liquor, it is same as Example 1.
Yeast isolation culture medium is in addition to the ingredient and its content in table 3, other ingredients and its content and culture medium
Preparation method, it is same as Example 1.
Experimental result: saccharomycete has growth on above-mentioned 4 kinds of culture mediums in fermented grain A and fermented grain B, but quantity is different,
Saccharomycete quantity is above control group in the culture medium of middle addition leaching liquor, illustrates that certain nutriments in leaching liquor are conducive to
In fermented grain the growth of saccharomycete with separate.
And growing state of the saccharomycete in fermented grain A and fermented grain B on culture medium 7 is best, can remarkably promote ferment at this time
The growth of female bacterium with separate.But after leaching liquor additive amount is greater than 700mL, culture medium is not easy to solidify, and color is deeper, because
This, when extraction liquid hold-up is within 200 to 700mL, be more advantageous to the growth of saccharomycete with separate.
Influence of 3 medium pH of embodiment to yeast isolation in accumulation fermented grain sample
In order to evaluate the different influences to yeast isolation of medium pH of the present invention, the present invention will using plate dilution method
A series of dilution bacterium solutions are made in accumulation fermented grain, and are respectively coated on culture medium shown in table 6 and are cultivated, two groups of culture medium inoculateds
Bacterium solution dilution gradient have 3, be 10-4、10-5、10-6.Culture medium is in addition to pH is different, remaining component and content and preparation
Method is identical with the culture medium 8 in embodiment 2.
Note: wherein " pH is natural " refers to that medium pH is not added with acid solution and is adjusted.
" pH is modulated " refers to that medium pH addition acid solution (hydrochloric acid solution that concentration is 1mol/L) is adjusted, and makes to train
It supports base pH and is adjusted to 5.0.
Microorganism in heap fermentation process, net for catching fish or birds and enriched place, air, yeast, accumulates early period, fermented grain flora
Structure is more complex, accumulates the later period, and due to the domestication by certain time, Bacterial community gradually tends towards stability.
After cultivating 48h, selecting dilution gradient is 10-4Plate compare, Yeast Growth situation such as Fig. 2 and 6 institute of table
Show.
Influence of 6 medium pH of table to Yeast Growth in accumulation fermented grain C
| Yeast Growth situation | PH is natural | PH is modulated |
| Saccharomycete quantity (CFU/g) | 21×104 | 27×104 |
| Whether there is or not miscellaneous bacterias | Have | Nothing |
The experimental results showed that saccharomycete has growth on two kinds of culture mediums in accumulation fermented grain C, but the culture of pH is not adjusted
There are also a large amount of fungus growths on base, and the quantity of saccharomycete is slightly lower；And pH be adjusted to 5.0 after culture medium on without fungus growth,
And Yeast Growth quantity is more.
Therefore, saccharomycete in accumulation fermented grain sample can be efficiently separated, medium pH is also required to meet certain condition.
The influence of yeast and the different ratio of fermented grain to yeast isolation in 4 culture medium of embodiment
In order to test influence of the different ratio to yeast isolation of yeast and fermented grain, the present invention will using plate dilution method
A series of dilution bacterium solutions are made in fermented grain, and are respectively coated on culture medium shown in table 7 and are cultivated, the bacterium of 4 groups of culture medium inoculateds
Liquid dilution gradient has 3, is 10-2、10-3、10-4.Each group culture medium is different from fermented grain proportion in addition to yeast, remaining component and
Content and preparation method are identical as culture medium 8 in embodiment 2.
After cultivating 48h, it is 10 that each group culture medium, which selects dilution gradient,-2Plate compare, Yeast Growth situation is such as
Shown in table 7.
The influence of 7 yeast of table and the different ratio of fermented grain to fermented grain sample Yeast Growth in storing
Experimental result: saccharomycete has growth in above-mentioned culture medium in fermented grain, but when yeast and fermented grain matter in leaching liquor
When amount is than being 1:25, saccharomycete minimum number.And work as yeast: the ratio of fermented grain is greater than after 1:1, yeast and fermented grain mixture
It is easy to boil paste, the culture medium of preparation forms taupe, is unfavorable for cultivating the observation of bacterium colony in culture medium.Therefore, big in leaching liquor
It is bent with fermented grain mass ratio within the scope of 1:1 to 1:20, be more advantageous in fermented grain the growth of saccharomycete with separate.
The above embodiment is only the preferred embodiment of the present invention, and the scope of protection of the present invention is not limited thereto,
The variation and replacement for any unsubstantiality that those skilled in the art is done on the basis of the present invention belong to institute of the present invention
Claimed range.
Claims (10)
1. a kind of yeast isolation culture medium, which is characterized in that including following components:
Yeast extract 5-10g/L,
Peptone 10-20g/L,
Glucose 5-20g/L,
Agar 15-22g/L,
Yeast-fermented grain leaching liquor 100-800mL/L,
Surplus is water.
2. yeast isolation culture medium according to claim 1, which is characterized in that the yeast isolation culture medium packet
Containing following components:
Yeast extract 5-10g/L,
Peptone 10-15g/L,
Glucose 10-20g/L,
Agar 18-20g/L,
Yeast-fermented grain leaching liquor 200-700mL/L,
Surplus is water；
Preferably, the yeast isolation culture medium includes following components:
Yeast extract 5-7g/L,
Peptone 10-15g/L,
Glucose 15-20g/L,
Agar 18-20g/L
Yeast-fermented grain leaching liquor 500-700mL/L；
Or preferably, the yeast isolation culture medium also contains chloramphenicol 0.1-1.0g/L；
Or preferably, in the yeast-fermented grain leaching liquor yeast be selected from liquor production process used in fermented by wheat
Made of yeast；
It is further preferable that in the yeast-fermented grain leaching liquor yeast be selected from Maotai-flavor liquor production process used in high temperature it is big
It is bent；
Or preferably, in the yeast-fermented grain leaching liquor fermented grain be selected from liquor production during after boiling grain fermentation and
At；
It is further preferable that height of the fermented grain in Maotai-flavor liquor production process after boiling in the yeast-fermented grain leaching liquor
Fine strain of millet is fermented；
It is further preferred that fermented grain different rounds in Maotai-flavor liquor production process in the yeast-fermented grain leaching liquor
Fermentation in pits fermented grain；
It is further preferred that fermented grain 1-3 round in Maotai-flavor liquor production process in the yeast-fermented grain leaching liquor
Fermentation in pits fermented grain；
Most preferably, in the yeast-fermented grain leaching liquor in cellar of the fermented grain selected from the 3rd round in Maotai-flavor liquor production process
Fermentation fermented grain.
3. yeast isolation culture medium according to claim 1, which is characterized in that the culture medium is for separating fermented grain sample
Saccharomycete in product；
Preferably, the fermented grain sample is selected from any one period of wine brewing production process；
It is further preferable that the fermented grain sample is one or more in fermented grain in accumulating fermented grain, entering to store fermented grain and store.
4. yeast isolation culture medium according to claim 1, which is characterized in that the leaching liquor the preparation method comprises the following steps:
(1) fermented grain and yeast are weighed, is mixed,
(2) boiling pure water is added into fermented grain-yeast mixture,
(3) it is spare to obtain filtrate for cooled and filtered；
Preferably, in step (2), boiling time 5-30min；
It is further preferable that boiling time is 10-15min.
5. yeast isolation culture medium according to claim 4, which is characterized in that in step (1), the yeast and fermented grain
Mass ratio be 1:1-1:20；
Preferably, the mass ratio is 1:3-1:10.
6. yeast isolation culture medium according to claim 4, which is characterized in that in step (2), the mixture and water
Mass ratio be 1:2-1:20；
Preferably, the mass ratio is 1:4-1:10.
7. yeast isolation culture medium according to claim 1, which is characterized in that the pH of the culture medium is 3.0-6.0.
8. a kind of preparation method for preparing any one of claim 1-7 yeast isolation culture medium, which is characterized in that contain
Following steps:
(1) by yeast powder, peptone, glucose, agar powder, yeast-fermented grain leaching liquor is added to the container, adjusted with acid solution
PH, surplus water polishing, sterilizing；
(2) chloramphenicol is dissolved in ethyl alcohol, shaken up, prepare chloramphenicol mother liquor；
(3) mixture after step (1) sterilizing is cooled to 50-60 DEG C, is mixed with the chloramphenicol mother liquor of step (2).
Preferably, acid solution is hydrochloric acid solution, glacial acetic acid solution or lactic acid solution in step (1)；
Preferably, in step (1), sterilizing 15-20min is carried out to culture medium at 121 DEG C.
9. utilizing the method for the culture medium separation saccharomycete as claimed in claim 1 to 7, which is characterized in that the saccharomycete
Separation uses plate dilution method.
10. the application of saccharomycete of the culture medium as claimed in claim 1 to 7 in separation fermented grain.
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| CN109337836A (en) * | 2018-10-26 | 2019-02-15 | 贵州茅台酒股份有限公司 | A kind of lactic acid bacteria isolation medium and preparation method thereof and lactic acid bacterial screening method |
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