CN109337836A - A kind of lactic acid bacteria isolation medium and preparation method thereof and lactic acid bacterial screening method - Google Patents
A kind of lactic acid bacteria isolation medium and preparation method thereof and lactic acid bacterial screening method Download PDFInfo
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- CN109337836A CN109337836A CN201811259118.0A CN201811259118A CN109337836A CN 109337836 A CN109337836 A CN 109337836A CN 201811259118 A CN201811259118 A CN 201811259118A CN 109337836 A CN109337836 A CN 109337836A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/02—Separating microorganisms from their culture media
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- 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
- C12N1/14—Fungi; Culture media therefor
- C12N1/16—Yeasts; Culture media therefor
Abstract
The present invention relates to lactic acid bacteria isolation medium in a kind of lactic acid bacteria isolation medium in Liquor Making Technology field more particularly to a kind of fermented grain and preparation method thereof and lactic acid bacterial screening methods.Lactic acid bacteria isolation medium in a kind of fermented grain is disclosed, contains yeast -25~100mL/L of fermented grain leaching liquor in the formula of the culture medium.By carrying out centrifugal treating to yeast in culture medium-fermented grain leaching liquor, culture medium background colour can be overcome to deepen, disadvantage more than impurity, the culture plate of production can carry out bacterium colony identification and screening with High Throughput Screening Assay, can meet lactobacter growth separation and the requirement of high flux screening in fermented grain simultaneously.Compared with manually selecting, working efficiency can be significantly improved.
Description
Technical field
The present invention relates to newborn in a kind of lactic acid bacteria isolation medium in Liquor Making Technology field more particularly to a kind of fermented grain
Sour bacterium isolation medium and preparation method thereof and lactic acid bacterial screening method.
Background technique
Liquor production is open solid ferment process, and fermented grain microbiologic population composition is sufficiently complex.It is movable through making
Long-term domestication, microorganism in fermented grain fermented grain peracid, nutriment multiplicity environment in be able to preferably grow and be metabolized.
Lactic acid bacteria is the general name for capableing of a quasi-microorganism of fermenting carbohydrate substance generation lactic acid, is prevalent in liquor fermentation
In the process, there is important influence to white wine quality, furthers investigate the Acid production metabolism mechanism of different type lactic acid bacteria to wine brewing skill
Art research is of great significance.
Lactic acid bacteria separation method common at present is that MRS basal medium is directly separated, the lactic acid in solid ferment process
Bacterium pass through long-term domestication, it is preferably to be grown in fermented grain environment, thus for the separation of lactic acid bacteria in solid state fermentation fermented grain and
Speech, common MRS are separately cultured that based selective is low, some micro substances needed for lacking fermented grain lactobacter growth.Therefore can divide
From kinds of lactobacillus and negligible amounts, and interference of the separating effect vulnerable to microorganisms such as yeast.
It is dry because also needing to eliminate as much as background when carrying out lactic acid bacteria separation using High Throughput Screening Assay since bacterium colony is smaller
It disturbs, reduces the influence that culture medium impurity and background color identify instrument bacterium colony.
So far, the separation and screening about lactic acid bacteria in Maotai-flavor fermented grain, there has been no add yeast-in the medium
Fermented grain leaching liquor, and the report that unbonded High Throughput Screening Assay separates lactic acid bacteria and screens.
Summary of the invention
One of the objects of the present invention is to provide a kind of lactic acid bacteria isolation mediums.
It is a further object of the present invention to provide a kind of lactic acid bacteria isolation mediums that can be used in high flux screening.
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 leaching liquor, adds in different bacteria culture medias, but separating effect does not protrude.The leaching
Extract is not added with production yeast, and does not carry out high-temperature boiling.
High Throughput Screening Assay is that one kind is identified and selected microorganism single colonie technology by instrument Imaging fast.In white wine
High Throughput Screening Assay is introduced in microbe research, compared with manually selecting, can significantly improve working efficiency.But it is
Guarantee that instrument can effectively identify the single colonie in plate, the plate for carrying out high flux screening should meet culture medium background color not
It can require too deeply, without too many impurity interference etc..Because of yeast-fermented grain leaching liquor complicated component, pigment content is higher, and directly addition is big
Song-fermented grain leaching liquor can deepen culture medium background colour, and bring more impurity into.Therefore, it is while meets lactic acid bacteria point in fermented grain
From and high flux screening requirement, yeast-fermented grain leaching liquor preparation method and dosage need to be advanced optimized.
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 lactic acid bacteria in fermented grain, the formula of the culture medium includes following component: peptone
(8-12g/L), beef extract (6.4-9.6g/L), yeast powder (3.2-4.8g/L), glucose (16-24g/L), dipotassium hydrogen phosphate
(1.6-2.4g/L), diammonium hydrogen citrate (1.6-2.4g/L), sodium acetate (4-6g/L), magnesium sulfate (0.16-0.24g/L), sulphur
Sour manganese (0.034-0.048g/L), agar (10-20g/L), tween (0.8-1.2g/L), yeast-fermented grain leaching liquor 25.0~
100.0 mL/L, wherein solvent is water；
As preferred experiment method, the formula of the culture medium includes following component: peptone (9-11g/L), beef
Cream (7.2-8.8g/L), yeast powder (3.6-4.4g/L), glucose (18-22g/L), dipotassium hydrogen phosphate (1.8-2.2g/L), lemon
Lemon acid hydrogen diammonium (1.8-2.2g/L), sodium acetate (4.5-5.5g/L), magnesium sulfate (0.18-0.22g/L), manganese sulfate (0.036-
0.044g/L), agar (12-16g/L), tween (0.9-1.1g/L), yeast-fermented grain leaching liquor (25~75mL/L), wherein molten
Agent is water.
Embodiment more preferably, peptone 10.0g/L, beef extract 8.0g/L, yeast powder 4.0g/L, glucose
20.0g/L, dipotassium hydrogen phosphate 2.0g/L, diammonium hydrogen citrate 2.0g/L, sodium acetate 5.0g/L, magnesium sulfate 0.2g/L, manganese sulfate
0.04g/L, agar 14.0g/L, Tween 80 1.0g/L, yeast-fermented grain leaching liquor 50-75mL/L, solvent is water.
As a kind of embodiment that can choose, yeast is selected from liquor production process in the yeast-fermented grain leaching liquor
Used in fermented as wheat made of yeast.
As a preferred embodiment, yeast was produced selected from Maotai-flavor liquor in the yeast-fermented grain leaching liquor
High-temperature daqu used in journey.
As a kind of embodiment that can choose, fermented grain is selected from liquor production in the yeast-fermented grain leaching liquor
The grain after boiling ferments in the process.
As a preferred embodiment, fermented grain was produced selected from Maotai-flavor liquor in the yeast-fermented grain leaching liquor
Sorghum in journey after boiling is fermented.
As further preferred embodiment, fermented grain is produced selected from Maotai-flavor liquor in the yeast-fermented grain leaching liquor
The fermentation in pits fermented grain of different rounds in the process.
As further preferred embodiment, fermented grain is produced selected from Maotai-flavor liquor in the yeast-fermented grain leaching liquor
The fermentation in pits fermented grain of 1-3 round in the process.
As a kind of highly preferred embodiment, fermented grain is raw selected from Maotai-flavor liquor in the yeast-fermented grain leaching liquor
The fermentation in pits fermented grain of 3rd round during production.
As preferred experiment method, in culture medium prescription, also contain amphotericin B 6-8mg/L.By to culture medium
The middle amphotericin B that is added can inhibit the growth of fungi in sample, reduce the interference that miscellaneous bacteria separates lactic acid bacteria, improve lactic acid
The effect of bacterium separation.
In order to advanced optimize medium component, lactic acid bacteria is separately cultured in based formulas, adjusts culture medium using acid solution
PH is 4.0-5.5.For conventional MRS medium pH 6.2 or so, this pH is to have been carried out according to fermented grain acidity adjusted, is conducive to wine
The growth of unstrained spirits lactic acid bacteria.
As a preferred embodiment, the acid solution is lactic acid solution.
Inventor is found through experiments that, relative to yeast-fermented grain leaching liquor lactic acid bacteria isolation medium is not added, containing big
Song-fermented grain leaching liquor culture medium is more suitable for the growth of lactic acid bacteria.
In some embodiments, inventors have found that when preparing yeast-fermented grain leaching liquor, if yeast: the mass ratio of fermented grain
It is excessively high that extract is easy to cause to boil paste, and extract color is deep, when culture medium as lactic acid bacteria coated plate, it is unfavorable for lactic acid bacteria
The observation of bacterium colony.If yeast: the mass ratio of fermented grain is low, can not provide distinctive nutriment in enough fermented grains, unfavorable
The growth and separation of lactic acid bacteria in fermented grain.
As a preferred option, 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 yeast-fermented grain mixture；
(3) it is spare to obtain filtrate for cooled and filtered；
(4) it takes out and thaws after freeze overnight, take supernatant spare after centrifugation.
As a preferred embodiment, in step (2), boiling time 10-30min.
As a kind of more preferably embodiment, the boiling time is 15min.
As a preferred embodiment, being filtered in step (3) using 4 layers of gauze.
Compared with filtrate is without freeze overnight, filtrate is handled by freeze overnight, enables to not readily dissolve in filtrate
Ingredient dissolution it is more complete.Defrosting centrifugation is carried out, has the function that preferably to remove color removal of impurities, so that yeast-fermented grain extraction
Liquid background colour is lower.For making lactic acid bacteria culture plate, it is more advantageous to Q-Pix high throughput screening system and bacterium colony is identified
And screening.
In some embodiments, yeast-fermented grain leaching liquor culture medium of processing is boiled in addition, does not boil with addition big
Song-fermented grain leaching liquor culture medium is compared, and the growth of lactic acid bacteria can be more promoted.
As preferred embodiment, in step (1), the mass ratio of yeast and fermented grain is 1:1~1:5；
As a kind of more preferably embodiment, yeast described in step (1) and the mass ratio of fermented grain are 1:2-1:5.
As a kind of embodiment still more preferably, the mass ratio of the yeast and fermented grain is 1:3-1:5.
As a kind of highly preferred embodiment, the mass ratio of the yeast and fermented grain is 1:5.
As preferred embodiment, in step (2), the mass ratio of yeast-fermented grain solid mixture and pure water be 1:4~
1:6。
As a kind of more preferably embodiment, in step (2), the yeast-fermented grain solid mixture and pure water
Ratio is 1:4-1:5.
As preferred embodiment, in step (4), the filtrate centrifugal condition be at 4 DEG C 10000-13000rpm from
Heart 5-15min.Above-mentioned lactic acid bacteria isolation medium can be prepared via a method which to obtain:
(1) by peptone, beef extract, yeast powder, glucose, dipotassium hydrogen phosphate, diammonium hydrogen citrate, sodium acetate, sulfuric acid
Magnesium, manganese sulfate, agar, Tween 80, yeast-fermented grain leaching liquor is added to the container, and after adding water constant volume, adjusts pH using acid solution,
Sterilizing；
(2) amphotericin B is dissolved in dimethyl sulfoxide, filtration sterilization must store liquid；
(3) mixture after step (1) sterilizing is cooled to 60 DEG C hereinafter, the amphotericin B solution that step (2) are added is mixed
It is even；
As an alternative embodiment, carrying out 15~20min of sterilizing to culture medium at 121 DEG C in step (1).
There are also on the one hand, the present invention provides culture medium the combination high pass measuring appratus in separating fermented grain lactic acid bacterias
Using.
Wherein, the lactobacteria-containing fermented grain is selected from the fermented grain during brewed spirit after boiling is fermented.
As a preferred embodiment, the wine of the fermented grain different rounds in Maotai-flavor liquor brewing process
Unstrained spirits.
As a preferred embodiment, the fermented grain is in Maotai-flavor liquor production process through the first~tri- round
Fermented grain after fermentation.
As a kind of more preferably embodiment, the fermented grain is in Maotai-flavor liquor production process through third round
Fermented grain after fermentation.
The present invention additionally provides a kind of method of separating lactic acid bacterium simultaneously, and wherein this method includes using plate dilution method will
Lactic bacteria strain to be separated is placed in above-mentioned lactic acid bacteria isolation medium and is cultivated, to reach the work of lactic bacteria strain separation
With.Remaining condition of culture and method are then identical as the condition of culture of lactic acid bacteria in the prior art or method.
In addition, the present invention also provides a kind of lactic acid bacteria progress using Q-Pix high throughput screening system to separating above
The method of screening.Selecting for traditional single colonie is mostly manually selected, not only time-consuming, but also vulnerable to pollution, high flux screening
System not only greatly improves screening efficiency, but also can avoid living contaminants well by batch operation, but its imaging system
System requires culture medium color too deep, and cannot have excessive impurity interference bacterium colony identification.
Specially cultured plate and 96 orifice plates containing culture medium are taken off and are placed in the screening instrument to sterilize in advance,
Flat-plate bacterial colony is identified by Q-Pix imaging system, and selection needs choosing colony, is selected automatically after determining by instrument.
Beneficial effects of the present invention:
(1) present invention adds yeast-fermented grain leaching liquor in the isolation medium of lactic acid bacteria for the first time, which can be more preferable
Simulation make microorganism growing environment, promote fermented grain microorganism separation.Inventor during the experiment, to rich fragrance wine
It when bacterium is separated in unstrained spirits, once attempted that cellar fermented grain will be gone out, and was prepared into leaching liquor with boiled-out water oscillation is boiled, adds not
In same bacteria culture media, it to be used for separation of bacterial.Also nutritional ingredient rich in fermented grain, but separating effect does not protrude.
In addition, inventor, which has also been attempted, will be used for yeast-fermented grain leaching liquor without high-temperature boiling the separation of lactic acid bacteria, it is also difficult to effectively
Isolate lactic acid bacteria, therefore, in the present invention, high-temperature boiling and yeast and the common leaching liquor of fermented grain are necessary.
(2) culture medium of the invention can overcome culture medium with compared with directly adding yeast-fermented grain leaching liquor culture medium
Background colour is deepened, the disadvantage more than impurity, can meet lactic acid bacteria separation and the requirement of high flux screening in fermented grain simultaneously.The training of production
Feeding plate can carry out bacterium colony identification and screening with High Throughput Screening Assay.Compared with manually selecting, work can be significantly improved
Make efficiency.
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
Influence of Fig. 1 culture medium of the present invention to lactobacter growth and screening effect.
The influence that Fig. 2 leaching liquor centrifugal treating screens lactic acid bacteria.
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.
The lactic acid bacteria that embodiment uses in the present invention be confirm through Morphological Identification, and brewed spirit process can be in wine
The lactic acid bacteria grown in unstrained spirits.
Lactic acid bacteria in the embodiment of the present invention from fermented grain be the fermented grain after boiling is fermented during liquor production,
Fermented grain specially in Maotai-flavor liquor production process after the fermentation of third round.
In the embodiment of the present invention yeast used in yeast-fermented grain leaching liquor used in the liquor production process by
Yeast, specially high-temperature daqu used in Maotai-flavor liquor production process made of wheat fermentation.
The method that high flux screening instrument Q-Pix is identified and selected in the present invention are as follows: by cultured plate and contain
There are 96 orifice plates of culture medium to take off to be placed in the screening instrument to sterilize in advance, flat-plate bacterial colony, choosing are identified by Q-Pix imaging system
Taking needs choosing colony, is selected automatically after determining by instrument.
1 lactic acid bacteria of embodiment is separately cultured based formulas and preparation method thereof
The component and content of 1 culture medium of table
Yeast-fermented grain leaching liquor the preparation method comprises the following steps:
(1) according to yeast shown in table 1-fermented grain ratio and content, fermented grain and yeast are weighed, is mixed；
(2) 10~20min of boiling pure water is added into yeast-fermented grain mixture；
(3) it after cooling, is filtered using 4 layers of gauze, it is spare to obtain filtrate；
(4) it will take out and thaw after -20 DEG C of freeze overnights of above-mentioned filtrate, at 4 DEG C after 12000rpm centrifugation 10min, take supernatant
Liquid is spare.
Lactic acid bacteria isolation medium preparation method:
(1) each component is weighed by the formula of table 1, it will be by peptone, beef extract, yeast powder, glucose, dipotassium hydrogen phosphate, lemon
Lemon acid hydrogen diammonium, sodium acetate, magnesium sulfate, manganese sulfate, agar, Tween 80 and yeast-fermented grain leaching liquor are added to the container, and cream is added
Acid solution adjusts pH to 5.5,121 DEG C of sterilizing 15-20min；
(2) amphotericin B is dissolved in dimethyl sulfoxide to shake up, filtration sterilization, it is spare that 16g/L mother liquor is made；
(3) mixture after step (1) sterilizing is cooled to 60 DEG C hereinafter, the amphotericin B mother liquor with step (2) is mixed
It is even.
The culture medium of the present invention of embodiment 2 evaluates lactobacter growth and screening effect
It is imitated to evaluate influence of the culture medium of the present invention to fermented grain lactobacter growth situation and screened to fermented grain lactic acid bacteria
Fermented grain bacterium solution is diluted to 10 using plate dilution method by the evaluation of fruit, the present invention-2~10-3, each group that is coated in embodiment 1
Anaerobic culturel is carried out on culture medium, incubation time is 5-7 days.Selecting diluted concentration is 10-2Lactobacter growth plate compares,
Growing state is as shown in table 2, Fig. 1.
These are inoculated with lactic acid bacteria culture plate to be identified and selected using high flux screening instrument Q-Pix.Detection
The results are shown in Table 2.
Wherein, the depth rank for indicating background color with 1,2,3,4,5,6,7,8, wherein color depth is ordered as 1 < 2 < 3
< 4 < 5 < 6 < 7 < 8, i.e., 1 to 8, which represent background color, successively increases.
Influence of the culture medium of the present invention of table 2 to lactobacter growth in fermented grain
Experimental result: the results are shown in Table 2, through Morphological Identification, culture medium 1-4 and control group 1 (common MRS culture medium)
There is lactobacter growth in culture medium.
Contrast groups are not plus leaching liquor is so background color is most shallow, but lactic acid bacterium number is minimum, is unable to reach experiment and wants
It asks, and there is a small amount of miscellaneous bacteria to generate in culture medium.
Because joined leaching liquor in culture medium 1-4, culture medium quantity is significantly more than the culture medium in control group 1.Explanation
Leaching liquor dosage is conducive to the growth of lactic acid bacteria in 25-100ml/L.And in current culture medium background color,
High flux screening instrument Q-Pix can also be identified and choosing colony.
So culture medium 1-4 can preferably realize the screening of lactic acid bacteria, but quantity slightly has difference.Wherein, culture medium 2
The growth population of lactic acid bacteria is most, and background color meets instrument identification and screening requires, and effect is best.
The influence of yeast and the different ratio of fermented grain to lactobacter growth and screening effect in 3 culture medium of embodiment
It is in order to test influence of the different ratio to lactobacter growth of yeast and fermented grain, fermented grain lactic acid bacteria is dilute by plate
Interpretation of the law is coated on the yeast containing ratio shown in table 3: being cultivated on the culture medium of fermented grain extract, remaining group of each group culture medium
Divide with content and other situations with the culture medium 2 in embodiment 1.The results are shown in Table 3.Wherein incubation time and lactic acid bacteria dilution
Concentration is same as Example 2.
These are inoculated with lactic acid bacteria culture plate to be identified and selected using high flux screening instrument Q-Pix, is detected
As a result as shown in Figure 2.
Wherein, the depth rank for indicating background color with 1,2,3,4,5,6,7,8, wherein color depth is ordered as 1 < 2 < 3
< 4 < 5 < 6 < 7 < 8, i.e., 1 to 8, which represent background color, successively increases.
The influence of 3 yeast of table and the different ratio of fermented grain to fermented grain sample lactobacter growth
Experimental result: had centainly according to result yeast and the different ratio of fermented grain to the growth of fermented grain lactic acid bacteria
Influence, when yeast and fermented grain ratio are 1:5, fermented grain lactobacter growth situation is best, and background color depth can expire
The requirement of sufficient instrument identification and screening, effect are best.
When ratio is 1:6, although instrument can identify bacterium colony, since yeast ratio is reduced, the nutriment provided
It reduces, lactobacter growth negligible amounts are unable to satisfy requirement of experiment.
These results suggest that the proportion of yeast and fermented grain in yeast-fermented grain leaching liquor can influence the growth of lactic acid bacteria,
And background color depth, to influence the separating effect of lactic acid bacteria.
Influence of the different proportion of yeast-fermented grain mixture and water to lactobacter growth in 4 culture medium of embodiment
In order to test in yeast fermented grain leaching liquor preparation method, the different ratio of yeast-fermented grain mixture and water is to lactic acid
The influence of bacterium growth, is coated on the yeast containing ratio shown in table 4-fermented grain extract by plate dilution method for fermented grain lactic acid bacteria
It is cultivated on the culture medium of liquid.In addition to yeast-fermented grain extract: other than the ratio of water is different, remaining component of each group culture medium
With content with the culture medium 2 in embodiment 1.Wherein incubation time and lactic acid bacteria diluted concentration are same as Example 2.As a result such as table
Shown in 4.
4 yeast of table-fermented grain mixture: influence of the different proportion of water to fermented grain sample lactobacter growth
Experimental result: as shown in table 4, when the ratio of yeast-fermented grain mixture and water is respectively 1:4,1:5,1:6, system
It can identify bacterium colony, but lactic acid bacterium number is different, and increase with the ratio of dampening, lactic acid bacterium number is reduced.And work as yeast-
When the ratio of fermented grain mixture and water is 1:3, due to the water sorption of fermented grain and yeast, leaching liquor causes to filter in thick
Difficulty can not obtain leaching liquor, therefore the optimal proportion of yeast-fermented grain mixture and water is determined as 1:4.
Influence of the preparation method of 5 leaching liquor of embodiment to lactobacter growth and screening
One, the leaching liquor in culture medium boils the influence handled to lactobacter growth
Test 1 yeast-fermented grain extraction liquid and preparation method thereof
(1) according to shown in the culture medium 1 in embodiment 1, yeast: fermented grain mass ratio 5:1 weighs fermented grain and yeast, mixing；
(2) pure water 200mL is added into yeast-fermented grain mixture, mixes, boils 20min；
(3) it after cooling, is filtered using 4 layers of gauze, it is spare to obtain filtrate；
(4) it will take out and thaw after above-mentioned filtrate freeze overnight, at 4 DEG C after 12000rpm centrifugation 10min, take supernatant standby
With.
1 yeast of comparative example-fermented grain extracts liquid and preparation method thereof
(1) fermented grain and yeast with 1 phase homogenous quantities of test, mixing are weighed；
(2) pure water 200mL is added into yeast-fermented grain mixture, mixes；
(3) it is filtered, is filtered to get filtrate spare using 4 layers of gauze after cooling；
(4) it will take out and thaw after above-mentioned filtrate freeze overnight, at 4 DEG C after 12000rpm centrifugation 10min, take supernatant standby
With.
In order to test using the influence to lactobacter growth after yeast-fermented grain leaching liquor boils, the present invention is respectively using examination
Test 1 and the obtained leaching liquor of preparation method of comparative example 1 carry out making lactic acid bacterium culture medium, two groups of culture medium remaining components, content
And preparation method is identical as the culture medium 4 in embodiment 1, the culture medium prepared is respectively culture medium 5 and culture medium 6.
The lactic acid bacteria in fermented grain sample is coated on three kinds of culture mediums by plate dilution method.Lactobacter growth situation result such as table 5
It is shown.Wherein incubation time and lactic acid bacteria diluted concentration are same as Example 2.
5 yeast of table-fermented grain leaching liquor boils the influence handled to lactobacter growth
Experimental result: as shown in Table 5, the leaching liquor through boiling can isolate more fermented grain lactic acid bacterias, and dry without miscellaneous bacteria
Disturb, instrument can correctly identify lactic acid bacteria, therefore, yeast-fermented grain leaching liquor boil be conducive to the separation of saccharomycete of the present invention with
Screening.
Two, the influence that the leaching liquor centrifugal treating in culture medium screens lactic acid bacteria
Test 2 yeast-fermented grain extraction liquid and preparation method thereof
(1) according to shown in the culture medium 4 in embodiment 1, yeast: fermented grain mass ratio 5:1 weighs yeast and fermented grain, mixing；
(2) pure water 200mL is added into yeast-fermented grain mixture, mixes, boils 20min；
(3) it after cooling, is filtered using 4 layers of gauze, it is spare to obtain filtrate；
(4) it will take out and thaw after above-mentioned filtrate freeze overnight, at 4 DEG C after 12000rpm centrifugation 10min, take supernatant standby
With.
3 yeast of comparative example-fermented grain extracts liquid and preparation method thereof
(1) yeast and fermented grain with 2 phase homogenous quantities of test, mixing are weighed；
(2) the pure water 200mL with 2 same volumes of test is added into yeast-fermented grain mixture, mixes, boils 20min；
(3) it after cooling, is filtered using 4 layers of gauze, it is spare to obtain filtrate.
In order to test after yeast-fermented grain leaching liquor centrifugation using the influence to lactobacter growth, the present invention is respectively using examination
Test 2 and the obtained leaching liquor of preparation method of comparative example 2 carry out making lactic acid bacterium culture medium, two groups of culture medium remaining components, content
And preparation method is identical as the culture medium 3 in embodiment 1, the culture medium prepared is respectively culture medium 7 and culture medium 8.
The lactic acid bacteria in fermented grain sample is coated on two kinds of culture mediums by plate dilution method.The strain culturing plate that separation obtains is adopted
It is selected with high flux screening instrument Q-Pix.Specifically: cultured plate and 96 orifice plates containing culture medium are taken off lid and set
In the screening instrument to sterilize in advance, flat-plate bacterial colony is identified by Q-Pix imaging system, selection needs choosing colony, after determining by
Instrument is selected automatically.
Lactic acid bacteria the selection result is as shown in table 6 in fermented grain.Wherein incubation time and lactic acid bacteria diluted concentration and 2 phase of embodiment
Together.
The influence that 6 yeast of table-fermented grain leaching liquor centrifugal treating screens lactic acid bacteria
Experimental result: as shown in table 6 and Fig. 2, after chilled centrifugation, fermented grain lactic acid bacterium number is without significant difference.But not from
The culture medium of the heart, background color is deeper, and has impurity interference, is not able to satisfy the requirement of instrument identification and screening；And after being centrifuged
Culture medium background can effectively identify bacterium colony without significant impurity, the imaging system of high flux screening instrument.It is thus identified that extraction liquid cooling
Freeze the imaging identification that centrifugal treating is conducive to system.The above embodiment is only the preferred embodiment of the present invention, cannot be come with this
Limit the scope of protection of the invention, the variation for any unsubstantiality that those skilled in the art is done on the basis of the present invention
And replacement belongs to scope of the present invention.
Claims (10)
1. a kind of lactic acid bacteria isolation medium, which is characterized in that including following components:
Peptone 8-12g/L,
Beef extract 6.4-9.6g/L,
Yeast powder 3.2-4.8g/L,
Glucose 16-24g/L,
Dipotassium hydrogen phosphate 1.6-2.4g/L,
Diammonium hydrogen citrate 1.6-2.4g/L,
Sodium acetate 4-6g/L,
Magnesium sulfate 0.16-0.24g/L,
Manganese sulfate 0.032-0.048g/L,
Agar 10-20g/L,
Tween 80 0.8-1.2g/L,
Yeast-fermented grain leaching liquor 25-100mL/L,
Solvent is water.
2. lactic acid bacteria isolation medium according to claim 1, which is characterized in that the culture medium includes with the following group
Point:
Peptone 9-11g/L,
Beef extract 7.2-8.8g/L,
Yeast powder 3.6-4.4g/L,
Glucose 18-22g/L,
Dipotassium hydrogen phosphate 1.8-2.2g/L,
Diammonium hydrogen citrate 1.8-2.2g/L,
Sodium acetate 4.5-5.5g/L,
Magnesium sulfate 0.18-0.22g/L,
Manganese sulfate 0.036-0.044g/L,
Agar 12-16g/L,
Tween 80 0.9-1.1g/L,
Yeast-fermented grain leaching liquor 25-75mL/L,
Solvent is water；
Preferably, the culture medium contains following components:
Peptone 10.0g/L,
Beef extract 8.0g/L,
Yeast powder 4.0g/L,
Glucose 20.0g/L,
Dipotassium hydrogen phosphate 2.0g/L,
Diammonium hydrogen citrate 2.0g/L,
Sodium acetate 5.0g/L,
Magnesium sulfate 0.2g/L,
Manganese sulfate 0.04g/L,
Agar 14.0g/L,
Tween 80 1.0g/L,
Yeast-fermented grain leaching liquor 50-75mL/L,
Solvent is water；
Or preferably, in the yeast-fermented grain leaching liquor yeast be selected from liquor production process used in fermented by wheat
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. lactic acid bacteria isolation medium according to claim 1, which is characterized in that in the lactic acid bacteria isolation medium also
Contain 6-8mg/L amphotericin B.
4. lactic acid bacteria isolation medium according to claim 1, which is characterized in that the pH of the lactic acid bacteria isolation medium
For 4.0-5.5.
5. lactic acid bacteria isolation medium according to claim 1, which is characterized in that the yeast-fermented grain leaching liquor system
Preparation Method the following steps are included:
(1) yeast and fermented grain are weighed, is mixed,
(2) boiling pure water is added into yeast-fermented grain mixture,
(3) it is spare to obtain filtrate for cooled and filtered,
(4) it takes out and thaws after filtrate freeze overnight, take supernatant spare after centrifugation；
Preferably, in step (2), boiling time 10-30min；
More preferably, the boiling time is 15min；
Or it is preferred, in step (3), it is filtered using 4 layers of gauze.
6. lactic acid bacteria isolation medium according to claim 5, which is characterized in that in step (1), yeast and fermented grain quality
Than for 1:1-1:5；
Preferably 1:2-1:5；
More preferably 1:3-1:5；
It is preferably 1:5.
Or preferably, in step (2), the ratio of yeast-fermented grain solid mixture and pure water is 1:4-1:6；
It is further preferable that the ratio of the yeast-fermented grain solid mixture and pure water is 1:4-1:5.
7. lactic acid bacteria isolation medium according to claim 5, which is characterized in that in step (4), the centrifugation of the filtrate
Condition is that 10000-13000rpm is centrifuged 5-15min at 4 DEG C.
8. a kind of method for preparing the described in any item lactic acid bacteria isolation mediums of claim 1-7, which is characterized in that including with
Lower step:
(1) by peptone, beef extract, yeast powder, glucose, dipotassium hydrogen phosphate, diammonium hydrogen citrate, sodium acetate, magnesium sulfate, sulphur
Sour manganese, agar, Tween 80, yeast-fermented grain leaching liquor is added to the container, and after adding water constant volume, adjusts pH, sterilizing using acid solution；
(2) amphotericin B is dissolved in dimethyl sulfoxide, filtration sterilization must store liquid；
(3) mixture after step (1) sterilizing is cooled to 60 DEG C hereinafter, the amphotericin B solution that step (2) are added mixes；
Preferably, 15~20min of sterilizing is carried out to culture medium at 121 DEG C in step (1)；
Preferably, the acid solution is the lactic acid solution of mass fraction 85%.
9. culture medium as claimed in claim 1 to 7 is in the application for separating and screening lactic acid bacteria in fermented grain；
Preferably, the fermented grain is selected from the fermented grain after boiling is fermented during liquor production；
It is further preferred that fermented grain of the fermented grain in Maotai-flavor liquor production process after the fermentation of different rounds；
It is further preferable that fermented grain of the fermented grain in Maotai-flavor liquor production process after the fermentation of the first~tri- round.
10. most preferably, fermented grain of the fermented grain in Maotai-flavor liquor production process after the fermentation of third round.It utilizes
The method of any culture medium separating lactic acid bacterium of claim 1 to 7, which is characterized in that ferment is separated using plate dilution method
Female bacterium；
Preferably, the bacterial strain for separating acquisition is selected using Q-Pix high throughput screening system.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112063573A (en) * | 2020-08-27 | 2020-12-11 | 贵州茅台酒股份有限公司 | Formula and culture method of lactobacillus isolation medium |
| CN113293108A (en) * | 2021-05-08 | 2021-08-24 | 四川农业大学 | Preparation method of microbial agent for cleaning orchid pot soil, obtained biological agent and application |
| CN113913351A (en) * | 2021-11-29 | 2022-01-11 | 山西杏花村汾酒厂股份有限公司 | Method for screening lactic acid bacteria in fermented grains of fen-flavor liquor |
| CN114107434A (en) * | 2020-08-31 | 2022-03-01 | 贵州茅台酒股份有限公司 | Method for evaluating lactic acid production capacity of Daqu in high throughput |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060046292A1 (en) * | 2004-09-01 | 2006-03-02 | Pioneer Hi-Bred International, Inc. | Ferulate esterase producing strains and methods of using same |
| CN103834744A (en) * | 2014-03-27 | 2014-06-04 | 泸州品创科技有限公司 | Method for quantitatively analyzing clostridia, lactobacilli, bacilli and methanobacteria in pit mud |
| CN107488604A (en) * | 2016-06-12 | 2017-12-19 | 张家港市山牧新材料技术开发有限公司 | MRS culture mediums, its preparation method and Isolation and purification method are used in lactic acid bacteria separation |
| BR102016029488A2 (en) * | 2016-12-15 | 2018-08-07 | Universidade Federal Do Paraná | PROCESS AND EQUIPMENT FOR CONTROLLED COFFEE GRAIN FERMENTATION AND SPECIAL DRINKING |
| CN109234179A (en) * | 2018-10-29 | 2019-01-18 | 贵州茅台酒股份有限公司 | Yeast isolation culture medium and preparation method thereof in a kind of fermented grain |
| CN112063573A (en) * | 2020-08-27 | 2020-12-11 | 贵州茅台酒股份有限公司 | Formula and culture method of lactobacillus isolation medium |
| CN112111434A (en) * | 2020-10-14 | 2020-12-22 | 湖南鼎康酒业发展有限公司 | Excellent lactic acid bacteria, screening method and application of excellent lactic acid bacteria in preparation of Xiaoqu |
| US10912818B2 (en) * | 2013-02-05 | 2021-02-09 | Ludwig Stocker Hofpfisterei Gmbh | Use of microorganisms for the prevention and treatment of intestinal diseases |
-
2018
- 2018-10-26 CN CN201811259118.0A patent/CN109337836B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20060046292A1 (en) * | 2004-09-01 | 2006-03-02 | Pioneer Hi-Bred International, Inc. | Ferulate esterase producing strains and methods of using same |
| US10912818B2 (en) * | 2013-02-05 | 2021-02-09 | Ludwig Stocker Hofpfisterei Gmbh | Use of microorganisms for the prevention and treatment of intestinal diseases |
| CN103834744A (en) * | 2014-03-27 | 2014-06-04 | 泸州品创科技有限公司 | Method for quantitatively analyzing clostridia, lactobacilli, bacilli and methanobacteria in pit mud |
| CN107488604A (en) * | 2016-06-12 | 2017-12-19 | 张家港市山牧新材料技术开发有限公司 | MRS culture mediums, its preparation method and Isolation and purification method are used in lactic acid bacteria separation |
| BR102016029488A2 (en) * | 2016-12-15 | 2018-08-07 | Universidade Federal Do Paraná | PROCESS AND EQUIPMENT FOR CONTROLLED COFFEE GRAIN FERMENTATION AND SPECIAL DRINKING |
| CN109234179A (en) * | 2018-10-29 | 2019-01-18 | 贵州茅台酒股份有限公司 | Yeast isolation culture medium and preparation method thereof in a kind of fermented grain |
| CN112063573A (en) * | 2020-08-27 | 2020-12-11 | 贵州茅台酒股份有限公司 | Formula and culture method of lactobacillus isolation medium |
| CN112111434A (en) * | 2020-10-14 | 2020-12-22 | 湖南鼎康酒业发展有限公司 | Excellent lactic acid bacteria, screening method and application of excellent lactic acid bacteria in preparation of Xiaoqu |
Non-Patent Citations (3)
| Title |
|---|
| MATTHEW A. RENSCHLER等: "Using nitrous acid-modified de Man, Rogosa, and Sharpe medium to selectively isolate and culture lactic acid bacteria from dairy foods", 《JOURNAL OF DAIRY SCIENCE》 * |
| 窦晓等: "泸型酒酿造期间酒醅中可培养细菌的分离鉴定及其种群演替规律研究", 《食品工业科技》 * |
| 罗青春等: "浓香型白酒中优势乳酸菌和酵母菌间的相互关系", 《应用于环境生物学报》 * |
Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112063573A (en) * | 2020-08-27 | 2020-12-11 | 贵州茅台酒股份有限公司 | Formula and culture method of lactobacillus isolation medium |
| CN112063573B (en) * | 2020-08-27 | 2023-01-20 | 贵州茅台酒股份有限公司 | Lactic acid bacteria isolation medium formula and culture method |
| CN114107434A (en) * | 2020-08-31 | 2022-03-01 | 贵州茅台酒股份有限公司 | Method for evaluating lactic acid production capacity of Daqu in high throughput |
| CN113293108A (en) * | 2021-05-08 | 2021-08-24 | 四川农业大学 | Preparation method of microbial agent for cleaning orchid pot soil, obtained biological agent and application |
| CN113913351A (en) * | 2021-11-29 | 2022-01-11 | 山西杏花村汾酒厂股份有限公司 | Method for screening lactic acid bacteria in fermented grains of fen-flavor liquor |
| CN113913351B (en) * | 2021-11-29 | 2023-07-25 | 山西杏花村汾酒厂股份有限公司 | Screening method of lactic acid bacteria in fermented grains of fen-flavor liquor |
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