CN102373160A - Screening and application of high glucose, high alcohol and high acid-resistant saccharomyces cerevisiae ATCC9763 - Google Patents
Screening and application of high glucose, high alcohol and high acid-resistant saccharomyces cerevisiae ATCC9763 Download PDFInfo
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Abstract
The invention discloses screening and application of high glucose, high alcohol and high acid-resistant saccharomyces cerevisiae ATCC9763. The application is as follows: 1, according to the characteristics of high glucose resistance, high alcohol resistance, quick growth and the like of the saccharomyces cerevisiae, the saccharomyces cerevisiae can be applied to fermentation and production of wine; 2, according to the characteristics of high gas yield, quick growth and the like of the saccharomyces cerevisiae, the saccharomyces cerevisiae can be applied to fermentation and production of sour dough; 3, according to the characteristics of high acid resistance, aerobic/anaerobic growing capability and the like of the saccharomyces cerevisiae, the saccharomyces cerevisiae can be applied to fermentation and production of animal feeds; and 4, according to the characteristics of quick growth, aerobic/anaerobic growing capability and the like of the saccharomyces cerevisiae, the saccharomyces cerevisiae can be taken as a gene engineering strain. The invention has the advantages: the saccharomyces cerevisiae derived from farmhouse self-made wine has the characteristics of high acid resistance, high glucose resistance, high gas yield, quick growth and the like, and can be applied to production of wine, fermentation of sour dough and fermentation of animal feeds; and moreover, the saccharomyces cerevisiae grows quickly, can grow under the aerobic/anaerobic condition, and can be taken as a good engineering candidate strain.
Description
Technical field
The present invention relates to screening and the application of the anti-high sugar of a strain, high alcohol and peracid yeast saccharomyces cerevisiae ATCC9763, relate in particular to this yeast, sour flour dough fermentation, the application in animal-feed production and the genetically engineered at wine fermentation.
Background technology
Yeast saccharomyces cerevisiae (
Saccharomyces cerevisiae), claim bread yeast or budding yeast again, be and mankind's relation primary yeast the most widely.It not only is applied to fields such as bread, steamed bun, wine brewing; In molecule and the cytobiology, also be used as the eucaryon model animals in modern times, its effect is equivalent to the model animals intestinal bacteria of protokaryon.
On Pericarpium Vitis viniferae, carpopodium and carpopodium, growth has a large amount of unartificial yeasts, and after grape was broken, squeezes, yeast got in the Sucus Vitis viniferae, sugar contained in the Sucus Vitis viniferae is fermented, degrades, thereby produce pure and sweet wine.Farmers''s soil system wine of collection of the present invention also has good fruital and aroma that yeast produces except that having the fruital of grape own, taste fresh is salubrious.
Summary of the invention
The purpose of this invention is to provide screening and the application of the anti-high sugar of a strain, high alcohol and peracid yeast saccharomyces cerevisiae ATCC9763, characteristics such as this yeast saccharomyces cerevisiae has heatproof, ethanol-tolerant degree, acidproof, anti-sugar, oxytolerant, and gas deliverability is strong, and it is quick to grow.
The screening of the anti-high sugar of one strain, high alcohol and peracid yeast saccharomyces cerevisiae ATCC9763; Utilize YPD culture medium flat plate counting process and DGGE method analysis wine stoste flora quantity and composition and sift out 7 strain bacterium; Analyze and sequencing result in conjunction with DGGE, confirm that yeast saccharomyces cerevisiae ATCC9763 wherein is a dominant bacteria in the wine.
In the YPD substratum, add the glucose and the alcohol of different concns respectively, measure its OD behind 24 h
600Value detects different concns sugar and the alcohol influence to its growth.
The YPD substratum is adjusted to different pH values, measures its OD behind 24 h
600Value detects the influence of different pH values to its growth.
Culture bacteria is placed differing temps, measure its OD behind 24 h
600Value detects the influence of differing temps to its growth.
Culture bacteria is placed aerobic and anaerobic condition respectively, every at a distance from 2 its OD of h sampling and measuring
600Value is drawn the similarities and differences that its growth curve also compares its OD value under aerobic and anaerobic condition.
Be that yeast saccharomyces cerevisiae ATCC9763 is applied to the wine brew: this yeast saccharomyces cerevisiae can be under the environment of sugared concentration 0.5-6.5% raised growth, can tolerate the alcoholic strength of 0-29%, all can survive under the need/anaerobic condition, therefore can be applicable to Production of Wine industry.
Yeast saccharomyces cerevisiae ATCC9763 is applied to sour flour dough fermentation: this yeast can be under pH 2.5-8.5 raised growth, acidproof ability is strong, can resist the acidic substance that milk-acid bacteria produces in the sour flour dough; Growth rapidly can reach logarithm stationary phase in 22 h, produce CO
2Ability is strong, can be applicable to sour flour dough fermentation industry.
Yeast saccharomyces cerevisiae ATCC9763 is applied to animal-feed production: this yeast growth is rapid, can reach logarithm stationary phase in 22 h, can be animal-feed more protein is provided, and vitamin B group, nucleic acid and mineral substance also can produce some nourishing function active substances simultaneously; The acidproof ability of this yeast is strong, can unite probiotic bacterium such as milk-acid bacteria is used together, improves the feed local flavor, for animal provides prebiotic substance; In addition, all can survive under this yeast need/anaerobic condition, with the feed submerged fermentation favourable condition is provided for producing.
Yeast saccharomyces cerevisiae ATCC9763 is applicable as engineering strain: this yeast growth is rapid, but all a large amount growths under the need/anaerobism cultivate easily, can be used as model animals, research yeast bio information science with perfect heterologous gene and in yeast, have complementary functions.
The present invention uses as follows:
1, yeast saccharomyces cerevisiae ATCC9763 is applied to the wine brew:
This yeast saccharomyces cerevisiae can be under the environment of sugared concentration 0.5-6.5% raised growth, can tolerate the alcoholic strength of 0-29%, all can survive under the need/anaerobic condition, therefore can be applicable to Production of Wine industry.
2, yeast saccharomyces cerevisiae ATCC9763 is applied to the sour flour dough fermentation:
This yeast can be under pH 2.5-8.5 raised growth, acidproof ability is strong, can resist the acidic substance that milk-acid bacteria produces in the sour flour dough; Growth rapidly can reach logarithm stationary phase in 22 h, produce CO
2Ability is strong, can be applicable to sour flour dough fermentation industry.
3, yeast saccharomyces cerevisiae ATCC9763 is applied to animal-feed production:
This yeast growth is rapid, can reach logarithm stationary phase in 22 h, can be animal-feed more protein is provided, and vitamin B group, nucleic acid and mineral substance also can produce some nourishing function active substances simultaneously; The acidproof ability of this yeast is strong, can unite probiotic bacterium such as milk-acid bacteria is used together, improves the feed local flavor, for animal provides prebiotic substance; In addition, all can survive under this yeast need/anaerobic condition, with the feed submerged fermentation favourable condition is provided for producing.
4, yeast saccharomyces cerevisiae ATCC9763 is applicable as engineering strain:
This yeast growth is rapid, but all a large amount growths under the need/anaerobism cultivate easily, can be used as model animals, research yeast bio information science with perfect heterologous gene and in yeast, have complementary functions.
Advantage of the present invention is: derive from farmers' and make yeast saccharomyces cerevisiae ATCC9763 vinous by oneself; Heatproof, ethanol-tolerant degree, acidproof, anti-sugar, oxytolerant; Gas deliverability is strong, and growth can be applicable in sour flour dough fermentation, wine making, animal-feed production, the engineering bacteria structure fast.
The present invention identifies with Physiology and biochemistry and molecular biology method the bacterial strain that screening obtains, and determines that it is yeast saccharomyces cerevisiae ATCC9763, afterwards it has been carried out a series of anti-sugar; Acidproof; Ethanol-tolerant and oxytolerant experiment confirm that it ferments at sour flour dough, the potential value in wine brewing and the animal-feed.
The optimum breeding temperature of wine yeast is 22～30 ℃, and when temperature surpasses 35 ℃, yeast is state of paralysis, stops growing fully and ferments in time more than 40 ℃; Contain a certain amount of oxygen in the fermentation initial stage Sucus Vitis viniferae, help obtaining strong yeast, guarantee carrying out smoothly of fermentation,, should control the existence of oxygen, guarantee under anaerobic to ferment and ageing in the fermentation middle and later periods; Different yeast utilizes ability different to sugar, and the yeast that fermentation rate is high is suitable for brewageing sack, and sweet wine is brewageed in low being suitable for of fermentation rate.In addition, total acidity will suit in the fermenting process, with pH meter, should be controlled at 3～5 scope.
Fodder yeast usually with candiyeast or Kluyveromyces fragilis through cultivate, drying processes, be not have the Powdered or granular product that fermenting power, cell are dead state.It contains materials such as rich in protein (about 30～40%), vitamin B group, amino acid, is widely used as the protein supplements of animal-feed; It can promote growing of animal, shortens feeding period, increases meat amount and egg amount, and improvement meat and raising lean ratio improve the glossiness of fur, and can strengthen the resistance against diseases of poult poultry.
In addition, because of yeast saccharomyces cerevisiae be all Eukaryotic animal and plant cell and have a lot of identical structures, cultivate easily again, be used as the Eukaryotic model animals of research, also be to be understood one of maximum biology at present by people.Important protein matter its homologue that much all in yeast, comes to light earlier in the human body comprises albumen, signal protein and the protein processive enzyme of relevant cell cycle.
The yeast saccharomyces cerevisiae ATCC9763 of the present invention's screening has heatproof (25-40 ℃), ethanol-tolerant degree (0-29%), acidproof (pH 2.5-8.5), anti-sugar (0.5-6.5%), oxytolerant; Gas deliverability is strong; Characteristics such as it is quick to grow are for sour flour dough fermentation, wine making, animal-feed production, engineering bacteria structure provide good candidate strain.
Description of drawings
Fig. 1 is a yeast saccharomyces cerevisiae bacterium colony form.
Fig. 2 is the yeast saccharomyces cerevisiae thalli morphology.
Fig. 3 DGGE analytical results.
Fig. 4 sequenced genes base sequence.
Fig. 5 is the tolerance experiment of yeast saccharomyces cerevisiae to glucose.
Fig. 6 is the tolerance experiment of yeast saccharomyces cerevisiae to acid.
Fig. 7 is the tolerance experiment of yeast saccharomyces cerevisiae to temperature.
Fig. 8 is the tolerance experiment of yeast saccharomyces cerevisiae to alcohol.
Fig. 9 is the growth curve of yeast saccharomyces cerevisiae under need/anaerobic condition.
Figure 10 is a yeast saccharomyces cerevisiae aerogenesis situation.
Embodiment 1:
1. wine stoste doubling dilution is got 100 μ L and is coated YPD or BHI agar plate, is cultured to 24-36 hour under 37 ℃, anaerobism or the aerobic condition, chooses the colony count scope and carries out live bacterial count and observe its colonial morphology at the flat board of 30-300; Simultaneously, the thalline of selecting different colonial morphologies carries out next step checking.
2. will select the 7 strain bacterium that obtain and carry out gramstaining, observe its thalli morphology down in oily mirror.
3. every kind of bacterium liquid is got 1 mL and extracted genomic dna more than: [pH 8.0 for 500 mM NaCl, 50 mM Tris-HCl for the lysis buffer of adding 700 L in 2 mL centrifuge tubes; 50 mM EDTA, 4% SDS], 300 L phenol/chloroforms/primary isoamyl alcohol (25:24:1; Promega) and 0.4 g granulated glass sphere (0.35 g/0.1 mm, 0.05 g/0.5 mm), mixing; And centrifuge tube placed on the vortex vibrator vibration 10 min, behind centrifugal 5 min of 20000 * g, in supernatant, add 150 L, 10 M ammonium acetates; DNA uses twice phenol/chloroform/isoamyl alcohol extracting and a chloroform extraction, isopropanol precipitating, 70% washing with alcohol immediately; After air-dry, add 100 L TE damping fluids (10 mM Tris-HCl, 1 mM EDTA; PH 8.0) the dissolving DNA deposition, add 2 lRNase (10 mg/ml) and hatch 15 min at 37 ℃.
4. amplification 18S rDNA primer is FF390 (F) (5 '-CGATAACGAACGAGACCT-3 '), FR1 (R)+(5 '-CCCCCGCCGCGCGCGGCGGGCGGGGCGGGGGCACGGGCCGAICCATTCAA
TCGGTAIT-3 ') amplification system (50 L: comprise 100 ng dna profilings, 1 * EX Taq buffer, 200 μ M dNTP; Each primer of 200 nM; 1.5 mM MgCl2,670 ng/ μ L BSA, 1.25 U EX Taq DNA polysaccharases) with reference to methods such as Muyzer.Reaction conditions: 94 ℃, 5 min; 94 ℃ of 30 s; 56 ℃, 30 s; 72 ℃, 1 min, 30 circulations; 72 ℃, 7 min.
5. use 8% polyacrylamide gel, 35%～65% [100% denatured gradient comprises 40% (v/v) methane amide and 7 M urea] deformation gradient.Electrophoresis adopts DCode Universal Mutation Detection System (denaturing gradient gel electrophoresis appearance, Bio-Rad Laboratories, Hercules; CA; USA), at first prerunning 10 min under 220 V voltages, electrophoresis 16 h under the fixed voltage of 85 V subsequently.Electrophoresis carries out cma staining after finishing.
6. experimental result is shown in Fig. 1-4: can see that from DGGE glue bacterial strain 1-4 is identical bacterium and in wine, preponderates; Its respective strap is cut the glue order-checking obtain its corresponding base sequence (Fig. 4), after the BLAST comparison, determine that it is yeast saccharomyces cerevisiae ATCC9763.This yeast saccharomyces cerevisiae bacterium colony form similar with bacterium (Fig. 1), but thicker, be creamy white, surface wettability, thickness, easy quilt is provoked; Thalli morphology is unicellular (Fig. 2), generally is oval, circle, cylindrical or lemon illiteracy shape, and the daughter cell that forms that sprouts does not separate with parent cell as yet, has grown sprouting again, forms the cell of bunchiness.
Embodiment 2:
1. yeast saccharomyces cerevisiae ATCC9763 is inoculated into by 1% amount that to contain glucose concn be that 37 ℃ of aerobic conditions are cultivated 24 h down, in OD among 0.5,1.5,2.5,3.5,4.5,5.5,6.5% the liquid YPD
600Under measure its light absorption value.
2. experimental result is as shown in Figure 5: raise with sugared concentration, its thalline quantity rises earlier and afterwards descends.Sugar concentration is that 5.5% o'clock this bacteria growing is in the best state, its OD
600=3.89; Sugar concentration raises, and its growth is suppressed, when sugared concentration is 6.5%, and OD
600=3.44.
Embodiment 3:
1. yeast saccharomyces cerevisiae ATCC9763 is inoculated among the pH value different liquid YPD (2.5,3.5,4.5,5.5,6.5,7.5,8.5) by 1% amount, 37 ℃ of aerobic conditions are cultivated 24 h down, in OD
600Under measure its light absorption value.
2. experimental result is as shown in Figure 6: raise with pH, its thalline quantity aggregate performance is to rise earlier afterwards to descend.When pH=2.5, thalline minimum number OD
600=0.66; During pH=6.5, thalline quantity reaches the highest, its OD
600=1.60; Afterwards, thalline quantity raises with pH and reduces.
Embodiment 4:
1. yeast saccharomyces cerevisiae ATCC9763 is inoculated among the liquid YPD by 1% amount, cultivates 24 h, OD down in 25,30,37,42 ℃ of aerobic conditions
600Under measure its light absorption value.
2. experimental result is as shown in Figure 7: raise with temperature, its thalline quantity aggregate performance is to rise earlier afterwards to descend.In the time of 25 ℃, thalline minimum number, OD
600=2.19; When temperature was 30 ℃, thalline quantity reached the highest, its OD
600=2.93; Afterwards, thalline quantity begins to descend its OD in the time of 40 ℃
600=1.88.
Embodiment 5:
1. yeast saccharomyces cerevisiae ATCC9763 being inoculated into spirituosity concentration by 1% amount is among 0,2,4,5,6,8,9,10,12,13,14,16,17,18,21,25,29% the liquid YPD; 37 ℃ of aerobic conditions are cultivated 24 h down, in OD
600Under measure its light absorption value.
2. experimental result is as shown in Figure 8: raise with alcoholic strength, its thalline quantity is totally on a declining curve, when alcoholic strength is higher than 8%, and the few and poor growth of thalline quantity, its OD
600=0.036.However, we can very clearly see that with the naked eye great amount of bubbles is attached on the test tube wall, show that indirectly yeast saccharomyces cerevisiae ATCC9763 can be survived and aerogenesis (Figure 10) under the 0-29% alcoholic strength.
Embodiment 6:
1. yeast saccharomyces cerevisiae ATCC9763 is inoculated among the liquid YPD by 1% amount, 37 ℃, under need/anaerobic condition, adopts OD
600Assay method is measured the growth curve of different time (4,6,8,10,12,14,16,18,20,22,24 h) yeast saccharomyces cerevisiae ATCC9763.
2. experimental result is as shown in Figure 9: yeast saccharomyces cerevisiae ATCC9763 difference is little under the 0-8 h, aerobic and anaerobic condition; Afterwards, under the aerobic condition yeast saccharomyces cerevisiae ATCC9763 biomass apparently higher than anaerobic condition, 20 h, aerophil and anerobes all reach the highest increment, aerobic condition OD
600=3.28, anaerobic condition OD
600=2.80; Afterwards, thalline quantity descends to some extent.
Claims (9)
1. the screening of the anti-high sugar of a strain, high alcohol and peracid yeast saccharomyces cerevisiae ATCC9763; It is characterized in that utilizing YPD culture medium flat plate counting process and DGGE method analysis wine stoste flora quantity and composition and sift out 7 strain bacterium; Analyze and sequencing result in conjunction with DGGE, confirm that yeast saccharomyces cerevisiae ATCC9763 wherein is a dominant bacteria in the wine.
2. the screening of the anti-high sugar of a strain according to claim 1, high alcohol and peracid yeast saccharomyces cerevisiae ATCC9763 is characterized in that, in the YPD substratum, adds the glucose and the alcohol of different concns respectively, measures its OD behind 24 h
600Value detects different concns sugar and the alcohol influence to its growth.
3. the screening of the anti-high sugar of a strain according to claim 1, high alcohol and peracid yeast saccharomyces cerevisiae ATCC9763 is characterized in that, the YPD substratum is adjusted to different pH values, measures its OD behind 24 h
600Value detects the influence of different pH values to its growth.
4. the screening of the anti-high sugar of a strain according to claim 1, high alcohol and peracid yeast saccharomyces cerevisiae ATCC9763 is characterized in that, culture bacteria is placed differing temps, measures its OD behind 24 h
600Value detects the influence of differing temps to its growth.
5. the screening of the anti-high sugar of a strain according to claim 1, high alcohol and peracid yeast saccharomyces cerevisiae ATCC9763 is characterized in that culture bacteria is placed aerobic and anaerobic condition respectively, and is every at a distance from 2 its OD of h sampling and measuring
600Value is drawn the similarities and differences that its growth curve also compares its OD value under aerobic and anaerobic condition.
6. the application of the anti-high sugar of the described strain of claim 1, high alcohol and peracid yeast saccharomyces cerevisiae ATCC9763; It is characterized in that it being that yeast saccharomyces cerevisiae ATCC9763 is applied to the wine brew: this yeast saccharomyces cerevisiae can be under the environment of sugared concentration 0.5-6.5% raised growth; Can tolerate the alcoholic strength of 0-29%; All can survive under the need/anaerobic condition, therefore can be applicable to Production of Wine industry.
7. the application of the anti-high sugar of the described strain of claim 1, high alcohol and peracid yeast saccharomyces cerevisiae ATCC9763; It is characterized in that yeast saccharomyces cerevisiae ATCC9763 is applied to sour flour dough fermentation: this yeast can be under pH 2.5-8.5 raised growth; Acidproof ability is strong, can resist the acidic substance that milk-acid bacteria produces in the sour flour dough; Growth rapidly can reach logarithm stationary phase in 22 h, produce CO
2Ability is strong, can be applicable to sour flour dough fermentation industry.
8. the application of the anti-high sugar of the described strain of claim 1, high alcohol and peracid yeast saccharomyces cerevisiae ATCC9763; It is characterized in that yeast saccharomyces cerevisiae ATCC9763 is applied to animal-feed production: this yeast growth is rapid; Can reach logarithm stationary phase in 22 h; Can be animal-feed more protein is provided, vitamin B group, nucleic acid and mineral substance also can produce some nourishing function active substances simultaneously; The acidproof ability of this yeast is strong, can unite probiotic bacterium such as milk-acid bacteria is used together, improves the feed local flavor, for animal provides prebiotic substance; In addition, all can survive under this yeast need/anaerobic condition, with the feed submerged fermentation favourable condition is provided for producing.
9. the application of the anti-high sugar of the described strain of claim 1, high alcohol and peracid yeast saccharomyces cerevisiae ATCC9763; It is characterized in that yeast saccharomyces cerevisiae ATCC9763 is applicable as engineering strain: this yeast growth is rapid; But all a large amount growths under the need/anaerobism; Cultivate easily, can be used as model animals, research yeast bio information science with perfect heterologous gene and in yeast, have complementary functions.
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| CN105238705A (en) * | 2015-09-30 | 2016-01-13 | 福建省农业科学院农业工程技术研究所 | Saccharomyces cerevisiae strain and application thereof |
| CN106414710A (en) * | 2015-05-22 | 2017-02-15 | 艾丝碧西有限公司 | Novel indigenous natural yeast and lactobacillus isolated from traditional korean malt for baking bread |
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| CN105238705A (en) * | 2015-09-30 | 2016-01-13 | 福建省农业科学院农业工程技术研究所 | Saccharomyces cerevisiae strain and application thereof |
| CN105238705B (en) * | 2015-09-30 | 2018-12-18 | 福建省农业科学院农业工程技术研究所 | A kind of saccharomyces cerevisiae bacteria strain |
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| CN112300950A (en) * | 2019-08-02 | 2021-02-02 | 内蒙古优然牧业有限责任公司 | Acid-resistant saccharomyces cerevisiae and application thereof |
| CN112300950B (en) * | 2019-08-02 | 2024-01-12 | 内蒙古优然牧业有限责任公司 | Acid-resistant saccharomyces cerevisiae and application thereof |
| CN112094763A (en) * | 2020-09-02 | 2020-12-18 | 青岛普罗百世生物科技有限公司 | Saccharomyces cerevisiae and application thereof in fattening pig feed |
| KR102593399B1 (en) * | 2022-12-19 | 2023-10-24 | 주은바이오주식회사농업회사법인 | Microbial fertilizer additive composition using citrus juice and method for preparing the same |
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