CN104560748A - Yeast-enriched identification medium for inhibiting mycetes and preparation method of yeast-enriched identification medium - Google Patents
Yeast-enriched identification medium for inhibiting mycetes and preparation method of yeast-enriched identification medium Download PDFInfo
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- CN104560748A CN104560748A CN201410846362.2A CN201410846362A CN104560748A CN 104560748 A CN104560748 A CN 104560748A CN 201410846362 A CN201410846362 A CN 201410846362A CN 104560748 A CN104560748 A CN 104560748A
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- 240000004808 Saccharomyces cerevisiae Species 0.000 title claims abstract description 55
- 238000002360 preparation method Methods 0.000 title claims abstract description 6
- 230000002401 inhibitory effect Effects 0.000 title abstract 2
- JXKPEJDQGNYQSM-UHFFFAOYSA-M sodium propionate Chemical compound [Na+].CCC([O-])=O JXKPEJDQGNYQSM-UHFFFAOYSA-M 0.000 claims abstract description 12
- 235000010334 sodium propionate Nutrition 0.000 claims abstract description 12
- 229960003212 sodium propionate Drugs 0.000 claims abstract description 10
- 239000004324 sodium propionate Substances 0.000 claims abstract description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 7
- 229920001817 Agar Polymers 0.000 claims abstract description 6
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 6
- 239000001888 Peptone Substances 0.000 claims abstract description 6
- 108010080698 Peptones Proteins 0.000 claims abstract description 6
- 239000008272 agar Substances 0.000 claims abstract description 6
- 239000008103 glucose Substances 0.000 claims abstract description 6
- 235000019319 peptone Nutrition 0.000 claims abstract description 6
- 239000000843 powder Substances 0.000 claims abstract description 6
- 230000001954 sterilising effect Effects 0.000 claims abstract 4
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 claims abstract 2
- 239000007788 liquid Substances 0.000 claims description 24
- 238000003860 storage Methods 0.000 claims description 23
- 241000233866 Fungi Species 0.000 claims description 12
- 230000005764 inhibitory process Effects 0.000 claims description 11
- 238000012797 qualification Methods 0.000 claims description 9
- 238000000034 method Methods 0.000 claims description 7
- XEEYBQQBJWHFJM-UHFFFAOYSA-N Iron Chemical compound [Fe] XEEYBQQBJWHFJM-UHFFFAOYSA-N 0.000 claims description 6
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 claims description 6
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 claims description 3
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 claims description 3
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 3
- 229910019142 PO4 Inorganic materials 0.000 claims description 3
- ZLMJMSJWJFRBEC-UHFFFAOYSA-N Potassium Chemical compound [K] ZLMJMSJWJFRBEC-UHFFFAOYSA-N 0.000 claims description 3
- WCUXLLCKKVVCTQ-UHFFFAOYSA-M Potassium chloride Chemical compound [Cl-].[K+] WCUXLLCKKVVCTQ-UHFFFAOYSA-M 0.000 claims description 3
- 239000001110 calcium chloride Substances 0.000 claims description 3
- 229910001628 calcium chloride Inorganic materials 0.000 claims description 3
- AIUDWMLXCFRVDR-UHFFFAOYSA-N dimethyl 2-(3-ethyl-3-methylpentyl)propanedioate Chemical class CCC(C)(CC)CCC(C(=O)OC)C(=O)OC AIUDWMLXCFRVDR-UHFFFAOYSA-N 0.000 claims description 3
- 229910052742 iron Inorganic materials 0.000 claims description 3
- 229910052943 magnesium sulfate Inorganic materials 0.000 claims description 3
- 235000019341 magnesium sulphate Nutrition 0.000 claims description 3
- SQQMAOCOWKFBNP-UHFFFAOYSA-L manganese(II) sulfate Chemical compound [Mn+2].[O-]S([O-])(=O)=O SQQMAOCOWKFBNP-UHFFFAOYSA-L 0.000 claims description 3
- 229940066779 peptones Drugs 0.000 claims description 3
- NBIIXXVUZAFLBC-UHFFFAOYSA-K phosphate Chemical compound [O-]P([O-])([O-])=O NBIIXXVUZAFLBC-UHFFFAOYSA-K 0.000 claims description 3
- 239000010452 phosphate Substances 0.000 claims description 3
- 239000011591 potassium Substances 0.000 claims description 3
- 229910052700 potassium Inorganic materials 0.000 claims description 3
- 229940049547 paraxin Drugs 0.000 claims 1
- 241000894006 Bacteria Species 0.000 abstract description 14
- 239000002609 medium Substances 0.000 abstract description 6
- 230000000694 effects Effects 0.000 abstract description 3
- 239000011550 stock solution Substances 0.000 abstract 7
- 239000007640 basal medium Substances 0.000 abstract 1
- 229960005091 chloramphenicol Drugs 0.000 abstract 1
- 238000011109 contamination Methods 0.000 abstract 1
- 239000004615 ingredient Substances 0.000 abstract 1
- 238000004659 sterilization and disinfection Methods 0.000 abstract 1
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 36
- 241000235648 Pichia Species 0.000 description 8
- 238000012216 screening Methods 0.000 description 4
- 239000001963 growth medium Substances 0.000 description 3
- 241000235036 Debaryomyces hansenii Species 0.000 description 2
- 241001149669 Hanseniaspora Species 0.000 description 2
- 241000235070 Saccharomyces Species 0.000 description 2
- 235000009754 Vitis X bourquina Nutrition 0.000 description 2
- 235000012333 Vitis X labruscana Nutrition 0.000 description 2
- 240000006365 Vitis vinifera Species 0.000 description 2
- 235000014787 Vitis vinifera Nutrition 0.000 description 2
- 235000021028 berry Nutrition 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 238000000855 fermentation Methods 0.000 description 2
- 230000004151 fermentation Effects 0.000 description 2
- 150000002500 ions Chemical class 0.000 description 2
- 244000005700 microbiome Species 0.000 description 2
- 238000000926 separation method Methods 0.000 description 2
- 230000002087 whitening effect Effects 0.000 description 2
- 241000186361 Actinobacteria <class> Species 0.000 description 1
- 240000000560 Citrus x paradisi Species 0.000 description 1
- 244000285963 Kluyveromyces fragilis Species 0.000 description 1
- 235000014663 Kluyveromyces fragilis Nutrition 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 239000003513 alkali Substances 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 239000003086 colorant Substances 0.000 description 1
- 230000000052 comparative effect Effects 0.000 description 1
- 238000012136 culture method Methods 0.000 description 1
- 238000012258 culturing Methods 0.000 description 1
- 238000013461 design Methods 0.000 description 1
- 239000006160 differential media Substances 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000011156 evaluation Methods 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 238000011835 investigation Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 230000000877 morphologic effect Effects 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 230000003716 rejuvenation Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 238000007423 screening assay Methods 0.000 description 1
- 239000011573 trace mineral Substances 0.000 description 1
- 235000013619 trace mineral Nutrition 0.000 description 1
- 238000011514 vinification Methods 0.000 description 1
Classifications
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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
-
- 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
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- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Mycology (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Engineering & Computer Science (AREA)
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- Tropical Medicine & Parasitology (AREA)
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Abstract
The invention provides a yeast-enriched identification medium for inhibiting mycetes and a preparation method of the yeast-enriched identification medium. The identification medium comprises the following ingredients by weight percent: 1+/-0.2% of yeast powder, 2+/-0.2% of peptone, 2+/-0.2% of glucose, 2+/-0.2% of agar, 4 +/-0.2% of a stock solution A, 0.1 +/-0.2% of a stock solution B, 2.2 +/-0.2% of sodium propionate and the balance of water, and the natural pH is 6.5 +/-0.2. The preparation method comprises the following steps: step A: preparing a basal medium, the stock solution A and the stock solution B according to the formula; step B: adding yeast powder, peptone, glucose, agar and water respectively according to the formula, uniformly mixing, adding the stock solution A and the stock solution B, then adding sodium propionate, so as to enable the natural pH of the medium to reach to 6.5 +/-0.2, sterilizing for 20 minutes at 121+/-0.2 DEG C, and after sterilization adding chloramphenicol and a stock solution C. By adopting the scheme, the mycete bacteria contamination in nature can be effectively inhibited, and the effects of yeast-enriched cultivation and preliminary identification are achieved.
Description
Technical field
The invention belongs to occurring in nature yeast culture screening method field, be specifically related to the enrichment culture and taxonomic identification substratum and preparation method thereof of yeast.
Background technology
Yeast is the eukaryotic microorganisms that a class is extensively present in physical environment.Yeast useful in a large number, particularly vineyard and grape berry surface is there is in physical environment.Simultaneously also there are other microorganisms a large amount of at occurring in nature, comprise bacterium and mould, actinomycetes.When doing the investigation of wild yeast resource classification, need kind, the quantity of wild yeast bacterium in true reflection environment.But yeast and mould belong to fungi together, and growing environment is identical, optimum growth temperature is all at 25-28 degree.Therefore cannot avoid the pollution of mould when culturing yeast bacterium, have a strong impact on yeast specie and observe and counting.Also needing separating for several times purifying when gathering nature wild yeast, wasting time and energy.
Summary of the invention
In order to solve above technical problem, the invention provides a kind of energy enrichment culture yeast, effective mould fungus inhibition, bacterium and actinomycetic yeast enrichment medium.Add trace element and developer simultaneously, can make not belong to yeast together and show different colours and morphological specificity, preliminary classification qualification is carried out to natural wild yeast.
A yeast enrichment qualification substratum for mould fungus inhibition, by weight percentage,
Comprise: 1 ± 0.2% yeast powder, 2 ± 0.2% peptones, 2 ± 0.2% glucose, 2 ± 0.2% agar, 4 ± 0.2% liquid storage A, 0.1 ± 0.2% liquid storage B and 2.2 ± 0.2% Sodium Propionates, surplus is water, and natural pH is 6.5 ± 0.2.
Preferably, described liquid storage A comprises: potassium primary phosphate, Repone K, calcium chloride and magnesium sulfate.
Preferably, described liquid storage B comprises: iron(ic) chloride and manganous sulfate.
The present invention adopts above technical scheme, and its advantage is, liquid storage A provides necessary ion elements for substratum, and liquid storage B provides necessary ion elements for substratum.Sodium Propionate can effective mould fungus inhibition, but does not affect Yeast Growth.Adopt this programme, effectively can suppress the pollution of occurring in nature mould bacterium, reach the effect of yeast enrichment cultivation and preliminary evaluation.
The present invention also provides a kind of yeast enrichment qualification culture method preparing mould fungus inhibition, comprises following step:
Steps A: add 1 ± 0.2% yeast powder, 2 ± 0.2% peptones, 2 ± 0.2% glucose, 2 ± 0.2% agar, water respectively by formula, mix, by formulated liquid storage A, liquid storage B;
Step B: add liquid storage A and liquid storage B, then add Sodium Propionate, makes substratum natural ph reach 6.5 ± 0.2, and 121 DEG C of sterilizings 20 minutes, add paraxin and liquid storage C after sterilizing.
The present invention adopts above technical scheme, and its advantage is, has added the Sodium Propionate of mould fungus inhibition in formula; Tradition needs separating for several times purifying to avoid the pollution of mould and bacterium; If there is mould, whole culture dish can be polluted.
Preferably, described liquid storage C adopts the tetrabromo-mcresolsulfonphthalein being dissolved in alcohol.
The present invention adopts above technical characteristic further, and its advantage is, liquid storage C is developer, and tetrabromo-mcresolsulfonphthalein color change interval PH is 3.8-5.4, and acid type is yellow, and alkali formula type is blue.Yeast is that self and surrounding environment pH-value change in process of growth, causes bacterium colony and colour-change around.
Beneficial effect of the present invention: enrichment culture and taxonomic identification organically combine by present method, can isolate each Yeasts from occurring in nature fast, simply and easily, effectively classify simultaneously.And saccharomycetic ecologicaI distribution and kind diversity can be observed from basal culture medium intuitively.There is provided technological method fast for the researcher being engaged in nature yeast resource exploration, the processing enterprise of simultaneously also screening barms for brewery or other needs from physical environment provides process for screening and identifying simply and easily.
Embodiment
Below preferably embodiment of the present invention is described in further detail:
Embodiment 1
Steps A: preparation basic medium and liquid storage A, liquid storage B;
Step B: conventional yeasts substratum (1% yeast powder, 2% peptone, 2% glucose, 2% agar, surplus are water), add liquid storage A and liquid storage B after mixing.Finally by adding Sodium Propionate 2.2%, substratum natural ph is made to reach 6.5.121 DEG C of sterilizings 20 minutes, add paraxin 1%, liquid storage C 0.1% after sterilizing.
Concrete formula is:
Liquid storage A: potassium primary phosphate 5.5g, Repone K 4.25g, calcium chloride 1.25g, magnesium sulfate 1.25g water are settled to 400ml.
Liquid storage B: iron(ic) chloride 0.25g, manganous sulfate 0.25g water is settled to 100ml.
Liquid storage C: 0.44g tetrabromo-mcresolsulfonphthalein being dissolved in 20ml concentration is in the alcohol of 50%.
Novel culture medium: by weight percentage: 1% yeast powder, 2% peptone, 2% glucose, 2% agar, 4% liquid storage A, 0.1% liquid storage B, natural pH are 6.5.
Comparative example:
As different from Example 1: by above-mentioned formulated substratum, the concentration that Sodium Propionate adds only is changed.
Embodiment 2: add 0.7% Sodium Propionate, natural pH value 6.27, other condition is identical with embodiment 1;
Embodiment 3: add 1.0% Sodium Propionate, natural pH value 6.31, other condition is identical with embodiment 1;
Embodiment 4: add 1.4% Sodium Propionate, natural pH value 6.35, other condition is identical with embodiment 1;
Embodiment 5: add 2.2% Sodium Propionate, natural pH value 6.50, other condition is identical with embodiment 1;
Inoculate mould, debaryomyces hansenii, pichia spp and yeast saccharomyces cerevisiae respectively, 25 DEG C of cultivations, after 6 days, embodiment 2,3 and 4 all has Molds and yeasts bacteria growing, and embodiment 5 only has Yeast Growth.
As can be seen here, when the weight ratio of Sodium Propionate is 2.2%, this substratum can the effective common barms of enrichment culture occurring in nature, the simultaneously pollution of mould fungus inhibition, can reach enrichment culture and carry out the object of preliminary classification.
Hansenula (
hanseniaspora) aobvious blue-greenish colour, surrounding media flavescence look, bacterium colony is smooth, and smooth surface is butteriness.
Pichia (
pichia) whitening look, surrounding media flavescence look, bacterium colony is smooth, and surface drying is coarse.
Yeast saccharomyces cerevisiae belongs to (
saccharomyces) whitening look, surrounding media flavescence look, bacterium colony conoid protuberance, smooth surface butteriness.
Can contrast from effect: first use if traditionally need yeast enrichment medium (YPD) to filter out yeast (3-5 days) again through separation and purification 1-3 time (needing within 3-5 days, cultivate) at every turn from occurring in nature, and then use differential medium to carry out taxonomic identification (needing 5-7 days).
The work of yeast collection and classification can be carried out by the present invention simultaneously, generally needs within 5-7 days, can isolate yeast from substratum, and directly observe out saccharomycetic kind from flat board.Greatly reduce and carry out the workload that yeast separation is purified to taxonomic identification in a traditional way.
Embodiment 6
The screening operation of Wild Saccharomyces cerevisiae bacterium
Grape berry destemming is broken, load in appropriate container and make it naturally trigger fermentation, after fermentation starting, get 1mL fermented liquid, be diluted to 10
-4or 10
-5, get diluent 0.1mL and coat the design's substratum, cultivate 5-7 days, observe for 25 DEG C.Flat board shows the yeast of three forms.A kind of colony characteristics shows as circle, smooth, butteriness, and smooth surface is moistening, band yellow-green colour, can be accredited as Hansenula (
hanseniaspora); A kind of colony characteristics shows as coniform, central protuberance, smooth surface, butteriness, white, can be accredited as yeast saccharomyces cerevisiae belong to (
saccharomyces); One primary yeast mark sheet is now white colony, and surface drying is Powdered, can be accredited as Pichia (
pichia).Experimental result shows, this grape fruit surface exists three kinds of yeast relevant to wine making, is respectively debaryomyces hansenii, yeast saccharomyces cerevisiae, pichia spp.
Picking list bacterium colony yeast from substratum, carries out physio-biochemical characteristics, strain excellent seed selection etc. that namely rejuvenation cultivation, preservation can be used for carrying out related yeasts bacterium and gos deep into scientific research.Basal culture medium substantially reduces working hour and the working strength of the classification of nature yeast screening assay, provides the method for the acquisition experiment material of convenient and efficient for being engaged in saccharomycetic researcher.
Above content is in conjunction with concrete preferred implementation further description made for the present invention, can not assert that specific embodiment of the invention is confined to these explanations.For general technical staff of the technical field of the invention, without departing from the inventive concept of the premise, some simple deduction or replace can also be made, all should be considered as belonging to protection scope of the present invention.
Claims (5)
1. the yeast enrichment qualification substratum of a mould fungus inhibition, it is characterized in that, by weight percentage, comprise: 1 ± 0.2% yeast powder, 2 ± 0.2% peptones, 2 ± 0.2% glucose, 2 ± 0.2% agar, 4 ± 0.2% liquid storage A, 0.1 ± 0.2% liquid storage B and 2.2 ± 0.2% Sodium Propionates, surplus is water, and natural pH is 6.5 ± 0.2.
2. the yeast enrichment qualification substratum of mould fungus inhibition as claimed in claim 1, it is characterized in that, described liquid storage A comprises: potassium primary phosphate, Repone K, calcium chloride and magnesium sulfate.
3. the yeast enrichment qualification substratum of mould fungus inhibition as claimed in claim 1, it is characterized in that, described liquid storage B comprises: iron(ic) chloride and manganous sulfate.
4. prepare a method for the yeast enrichment qualification substratum of mould fungus inhibition as claimed in claim 1, it is characterized in that, comprise following step:
Steps A: preparation basic medium and liquid storage A, liquid storage B;
Step B: add 1 ± 0.2% yeast powder, 2 ± 0.2% peptones, 2 ± 0.2% glucose, 2 ± 0.2% agar, water respectively by formula, liquid storage A and liquid storage B is added after mixing, add Sodium Propionate again, substratum natural ph is made to reach 6.5 ± 0.2,121 ± 0.2 DEG C of sterilizings 20 minutes, add paraxin and liquid storage C after sterilizing.
5. the yeast enrichment qualification substratum of mould fungus inhibition as claimed in claim 1, it is characterized in that, described liquid storage C adopts the tetrabromo-mcresolsulfonphthalein being dissolved in alcohol.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112280689A (en) * | 2020-10-18 | 2021-01-29 | 绿城农科检测技术有限公司 | Culture medium capable of promoting rapid growth of osmophilic yeast in honey and preparation method thereof |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101624573A (en) * | 2009-06-04 | 2010-01-13 | 新疆农业科学院微生物应用研究所 | Saccharomyces cerevisiae for brewing pomegranate fruit wine and pomegranate wine prepared by fermentation |
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101624573A (en) * | 2009-06-04 | 2010-01-13 | 新疆农业科学院微生物应用研究所 | Saccharomyces cerevisiae for brewing pomegranate fruit wine and pomegranate wine prepared by fermentation |
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| 刘姝 等: "优良山楂酒酿造酵母菌株的筛选与鉴定", 《食品研究与开发》 * |
| 王尤湘 等: "用于乳酸菌分离鉴定的几种培养基的筛选及应用", 《食品科学》 * |
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| 马乐好 等: "《微生物培养基实用手册》", 31 March 2006 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN112280689A (en) * | 2020-10-18 | 2021-01-29 | 绿城农科检测技术有限公司 | Culture medium capable of promoting rapid growth of osmophilic yeast in honey and preparation method thereof |
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