CN102604845A - Acid-proof ethanol-tolerance volatile-acid-producing yeast in Chinese white spirits and application thereof - Google Patents
Acid-proof ethanol-tolerance volatile-acid-producing yeast in Chinese white spirits and application thereof Download PDFInfo
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Abstract
The invention relates to acid-proof ethanol-tolerance volatile-acid-producing yeast in Chinese white spirits and application thereof, which belongs to the technical field of biological engineering; bacterial strains are issatchenkia orientalis CGMCC (China General Microbiological Culture Collection Center) No.4741; the bacterial strains are obtained through separation in a brewing procedure of white spirits factories in Guizhou Province of China; a sifting method comprises the following steps: taking fermenting primer samples, fermented grains, brewing raw materials or brewing environment matters; processing diluting, coating, acid producing, primary sifting in a culture medium, selecting bacterial colonies with marked acid production, shaking flask, cultivating the bacterial colonies so as to process secondary sifting; processing flask-shaking fermenting cultivation to the bacterial strains, measuring kinds and content of volatile-acid-producing through HS-SPME-GC-MS, sifting the bacterial strains so as to obtain the bacterial strains with strong volatile acid producing capacity; and the bacterial strains can be used for brewing industry such as brewed white spirits, distilled white sprits, compound white sprits and food industry.
Description
Technical field
The present invention relates to the yeast that acidproof in the China white wine, anti-alcoholic acid produces volatile acid, and the application of product volatile acid yeast in drinks and food brewery industry acidproof, alcohol performance.
Background technology
The acids composition is the important tastant of China white wine, plays to be fragrant, to help perfume (or spice), to reduce stimulation and to cushion the equilibrated effect.Volatile acid in the liquor, wherein acid such as acetate, butyric acid, caproic acid, sad, n-nonanoic acid, capric acid are especially remarkable to the local flavor contribution of liquor.The peculiar taste of wine in they and other fragrance matter are formed jointly simultaneously also is the precursor substance that generates Ester.An amount of acid helps to strengthen the mouthfeel and the aftertaste of liquor, can make the wine body plentiful, mellow, long times of aftertaste.What of volatile acid content have significant effects to the grade of wine, odor type etc.
The generation approach of acid in liquor, mainly from the mikrobe of fermentation generation volatile acid, they utilize hyles such as carbohydrate, accomplish the generation and the conversion of acid through complicated biochemical reaction.More to the living acetic bacterial research in the brewed spirit process at present, comprise caproic acid bacteria, butyric bacteria etc., but these bacteriums can only provide several kinds of limited acids in liquor production.But also there is some other important acid in the liquor, like isopropylformic acid, enanthic acid, sad, n-nonanoic acid, capric acid etc.At present, formation and the generation mikrobe for these important volatile acids is also unintelligible in the China white wine brewery industry.Therefore seek the mikrobe of synthetic these acids, and these mikrobes are applied to have great importance with the content that improves acid in the brewed spirit.
At present; Abroad to wine, the existing certain research of beer; Find that yeast is bigger to the local flavor contribution of wine, especially effect is bigger aspect the generation of volatilization acid, does not almost have but screen and use relevant research for the product volatile acid zymic in the China white wine brewing process.Therefore seek specific saccharomyces acidifaciens in the distinct Chinese characteristics brewed spirit process; Not only can be familiar with the generation source of important acid in the liquor; And these functions are produced the content that volatile acid yeast are applied to can effectively improve in the brewed spirit technology acids taste compound in the liquor, all have important role for the theoretical developments of distinct Chinese characteristics liquor and the lifting of brewed spirit industrial technology level.
The present invention screens the yeast that obtains and is from Chinese brewed spirit Daqu, wine unstrained spirits, brewing materials or brewages and screen gained the environment to have acidproof, anti-alcoholic acid performance, and product volatile acid ability is strong, is that function stem is used in good wine brewing.Bacterial strain can effectively be applied in the brewing process of China white wine, effectively regulates and control liquor acidity, optimizes the brewing wine production technique, reduces the brewing wine production cost, improves the liquor quality.
Summary of the invention
The technical problem that (1) will solve
The technical problem that the present invention will solve provides acidproof, anti-alcoholic acid and produces the volatile acid yeast strain.
Another technical problem to be solved by this invention has provided a kind of application of volatile acid yeast in the China white wine brewery industry of producing.
(2) technical scheme
Acidproof in a kind of China white wine, anti-alcoholic acid produces the yeast of volatile acid, its classification called after Issatchenkia orientalis (
Issatchenkia orientalis), this bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and its deposit number is CGMCC No.4741.
Described Issatchenkia orientalis CGMCC No.4741, its acidproof scope is: pH2.0～5.0; Anti-ethanol scope is: 0～15%.
Described Issatchenkia orientalis CGMCC No.4741, it produces acid is acetate, propionic acid, butyric acid, isopropylformic acid, caproic acid, sad, n-nonanoic acid, capric acid volatile acid, producing total volatile acid concentration is 400～600mg/L.
This produces the screening of volatile acid zymic:
Get the bent appearance of 0.5～5g Daqu, wine unstrained spirits or brewing materials and be dissolved in 15～150mL SPSS shaking table vibration 30～150 minutes, redilution, sour primary dcreening operation substratum is produced in coating; Select the tangible bacterium colony of flavescence on every side and produce significantly bacterial strain of acid, again with bacterial strain transfer respectively in acid with contain the alcoholic acid substratum, shake-flask culture carries out multiple sieve; Select yeast; Shake flask fermentation is cultivated, and measures through HS-SPME-GC-MS and produces sour kind and content, and screening obtains the strong bacterial strain of acid producing ability.
Producing volatile acid primary dcreening operation culture condition is: produce sour substratum (g/L): yeast powder 2.0, peptone 2.0, glucose 40.0, potassium primary phosphate 0.25, Repone K 0.4, calcium chloride 0.1, sal epsom 0.1, iron(ic)chloride 0.002, manganous sulfate 0.002, tetrabromo-mcresolsulfonphthalein 0.05, agar 20.0; Prepare with deionized water; Transfer pH5.0; Cultivated 2～5 days for 30 ℃, judge according to periphery of bacterial colonies variable color circle and produce acid ability.
The shake flask fermentation culture condition is: Chinese sorghum juice, wort or YPD fermention medium, and Chinese sorghum juice or malt extract medium production method are: 20～200g Chinese sorghum or Fructus Hordei Germinatus sample add the water of 1～4 times of volume after crushed; Boiling 1-5h; In the pasty state, the cooling back adds saccharifying enzyme 10～50 units/g sample, keeps 2～10h in 40～80 ℃; Filtration, centrifugal gained filtrating, pol is 10 ~ 15
oBx, pH are 3～5,25～35 ℃, 150rpm, shake-flask culture 24～120h.
Produce volatile acid kind and concentration determination: adopt the HS-SPME-GC-MS method to measure, add 5～10 mL clarified broths in the 15 mL head space bottles, 1～5 g NaCl is interior mark with 4-methyl-2-amylalcohol, carries out headspace solid-phase microextraction.The condition of headspace solid-phase microextraction is: three-phase (Car/DVB/PDMS) extracting head, 60 ℃ of preheating 5～10 min, extraction absorption 30～50 min, GC desorb 5～10 min.
Acidproof, the alcohol performance of bacterial strain: in the Chinese sorghum juice body substratum, 30 ℃, static cultivation 48h.Utilize ELIASA to measure bacteria concentration at the 600nm place, utilize sterilized water to regulate OD
600nm=1.0.Get 50 μ L bacterium liquid (OD
600nm=1.0) in pH 1～7, ethanol 0～20% gradient YPD substratum, cultivated 24 hours for 30 ℃, it is dense to utilize ELIASA to measure bacterium at the 600nm place then.
Issatchenkia orientalis CGMCC No.4741 is in the brewery industry and the Application in Food Industry of various brewing wines, liquor, assembled alcoholic drinks.To process the microbiobacterial agent of all forms such as liquid or solid with Issatchenkia orientalis CGMCC No.4741; Make an addition in all kinds of wine and the food brewing process and use; Can effectively increase its living weight in brewing wine; And sour kind and concentration in the effective coordination brewing wine, finally improve the quality of product.
(3) beneficial effect
It is from Chinese brewed spirit Daqu or brewing wine unstrained spirits, to screen gained that the present invention screens the yeast that obtains, and has acidproof, anti-alcoholic acid performance, and product volatile acid ability is strong, is good brewing functional bacterial strain.Bacterial strain can effectively be applied in the brewing process of China white wine, effectively regulates and control liquor acidity, optimizes the brewing wine production technique, reduces the brewing wine production cost, improves the liquor quality.
The biological material specimens preservation: acidproof in a kind of China white wine, anti-alcoholic acid produces the yeast of volatile acid, its classification called after Issatchenkia orientalis (
Issatchenkia orientalis), this bacterial strain has been preserved in Beijing China Committee for Culture Collection of Microorganisms of Institute of Microorganism, Academia Sinica common micro-organisms center, is called for short CGMCC, and its deposit number is CGMCC No.4741, preservation date on April 6th, 2011.
Description of drawings
Fig. 1: Issatchenkia orientalis (
Issatchenkia orientalis) CGMCC No.4741 cellular form electromicroscopic photograph.
Embodiment
Embodiment 1:
Produce the screening of volatile acid zymic: get 0.5g wine unstrained spirits and be dissolved in 20mL SPSS shaking table vibration 30 minutes; Redilution; Volatile acid primary dcreening operation substratum is produced in coating, selects the tangible bacterium colony of flavescence on every side and is significantly bacterial strain of product acid, bacterial strain is transferred respectively cultivate in producing sour culture media shaking vase again; Measure product volatile acid kind and content through HS-SPME-GC-MS, screening obtains to produce the strong bacterial strain of volatile acid ability.
Producing sour primary dcreening operation culture condition is: produce sour substratum (g/L): yeast powder 2.0, peptone 2.0, glucose 40.0, potassium primary phosphate 0.25, Repone K 0.4, calcium chloride 0.1, sal epsom 0.1, iron(ic)chloride 0.002, manganous sulfate 0.002, tetrabromo-mcresolsulfonphthalein 0.05, agar 20.0; Prepare with deionized water; Transfer pH5.0; Cultivated 2～5 days for 30 ℃, judge according to periphery of bacterial colonies variable color circle and produce acid ability.
The shake flask fermentation culture condition is: malt extract medium, and production method is: 20g Fructus Hordei Germinatus sample adds the water of 4 times of volumes after crushed; Boiling 5h, in the pasty state, the cooling back adds the saccharifying enzyme of 50 units/g; In 40 ℃ of maintenance 2h, filtration, centrifugal gained filtrating, pol is 10 ~ 15
oBx, pH are 5,28 ℃, 150rpm, shake-flask culture 48h.
Embodiment 2:
Produce volatile acid yeast and molecular biology identification thereof: the different product volatile acid yeast to obtaining carries out molecular biology identification, utilizes increase the respectively 26srDNA fragment of bacterial strain of the special classification primers designed of yeast, through detected through gel electrophoresis.The comparison of checking order subsequently, confirm institute sieve the zymic kind.Bacterial strain is preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, yeast kind and deposit number: Issatchenkia orientalis (
Issatchenkia orientalis) CGMCC No.4741.
Embodiment 3:
The acid resistance experiment of bacterial strain: get a ring yeast-inoculated in 3mL Chinese sorghum juice body substratum is housed, 30 ℃, static cultivation 48h.Utilize ELIASA to measure bacteria concentration at the 600nm place, utilize sterilized water to regulate OD
600nm=1.0.Get 50 μ L bacterium liquid (OD
600nm=1.0) in pH gradient YPD substratum, cultivated 24 hours for 30 ℃, it is dense to utilize ELIASA to measure bacterium at the 600nm place then, and the strain growth situation is as shown in table 1.
Table 1 bacterial strain acid resistance (OD
600Value)
Embodiment 4:
The alcohol resistance of bacterial strain can be tested: get a ring yeast-inoculated in 3mL YPD liquid nutrient medium is housed, and 30 ℃, static cultivation 48h.Utilize ELIASA to measure bacteria concentration at the 600nm place, utilize sterilized water to regulate OD
600nm=1.0.Get 50 μ L bacterium liquid (OD
600nm=1.0) in ethanol gradient YPD liquid nutrient medium, cultivated 24 hours for 30 ℃, it is dense to utilize ELIASA to measure bacterium at the 600nm place then, and the strain growth situation is as shown in table 2.
Anti-alcohol performance (the OD of table 2 bacterial strain
600Value)
Embodiment 5:
Yeast produces the volatile acid ability and measures: with wine and the used saccharomyces acidifaciens of beer is contrast, and yeast is carried out shake-flask culture, and fermentation culture conditions is: 100g Chinese sorghum sample after crushed; The water that adds 4 times of volumes, boiling 2～3h, in the pasty state; The cooling back adds the saccharifying enzyme of 30 units/g; Keep 2h, filtration, centrifugal gained filtrating, pol 10 in 60 ℃
oBx, 5.0,28 ℃ of pH, 150rpm cultivates 36h, and with total acid concentration in the titration measuring fermented liquid, GC/MS measures each volatile acid concentration in the fermented liquid.The result is as shown in table 3.
The different yeast of table 3 produce acid concentration
| The acid kind | Acid concentration (mg/l) |
| Acetate | 468.6 |
| Propionic acid | 8.6 |
| Butyric acid | 10.2 |
| Isopropylformic acid | 8.5 |
| Caproic acid | 2.7 |
| Enanthic acid | 2.2 |
| Sad | 4.2 |
| N-nonanoic acid | 3.5 |
| Capric acid | 1.9 |
| Total volatile acid | 510.4 |
Claims (6)
1. acidproof, anti-alcoholic acid produces the yeast of volatile acid in the China white wine, its classification called after Issatchenkia orientalis (
Issatchenkia orientalis), this bacterial strain has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center, and its deposit number is CGMCC No.4741.
2. Issatchenkia orientalis CGMCC No.4741 as claimed in claim 1 is characterized in that acidproof scope is: pH2.0～5.0; Anti-ethanol scope is: 0～15%.
3. Issatchenkia orientalis CGMCC No.4741 as claimed in claim 1 is characterized in that, produces acid and be acetate, propionic acid, butyric acid, isopropylformic acid, caproic acid, sad, n-nonanoic acid, capric acid volatile acid, and producing total volatile acid concentration is 400～600mg/L.
4. the cultural method of the described Issatchenkia orientalis CGMCC of claim 1 No.4741 is characterized in that the shake flask fermentation culture condition is: Chinese sorghum juice, wort or YPD fermention medium, and Chinese sorghum juice or malt extract medium production method are: 20～200g Chinese sorghum or Fructus Hordei Germinatus sample are after crushed; The water that adds 1～4 times of volume, boiling 1-5h, in the pasty state; The cooling back adds saccharifying enzyme 10～50 units/g sample; In 40～80 ℃ of maintenance 2～10h, filtration, centrifugal gained filtrating, pol is 10 ~ 15
oBx, pH are 3～5,25～35 ℃, 150rpm, shake-flask culture 24～120h.
5. the application of the described Issatchenkia orientalis CGMCC of claim 1 No.4741 is characterized in that brewery industry and Application in Food Industry at various brewing wines, liquor, assembled alcoholic drinks.
6. application as claimed in claim 5 is characterized in that processing with Issatchenkia orientalis CGMCC No.4741 making an addition to the microbiobacterial agent of liquid or solid in all kinds of wine and the food brewing process and use.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107177519A (en) * | 2017-06-20 | 2017-09-19 | 湖北白云边酒业股份有限公司 | Schizosaccharomyces pombe bacterium, its composition and application |
| CN113481113A (en) * | 2021-09-06 | 2021-10-08 | 中粮营养健康研究院有限公司 | Space breeding saccharomyces cerevisiae and application thereof in brewing wine |
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| CN102102084A (en) * | 2010-10-26 | 2011-06-22 | 湖北白云边酒业股份有限公司 | Issatchenkia orientalis and composition and application thereof |
| CN102242072A (en) * | 2011-05-12 | 2011-11-16 | 江南大学 | Strain produced by screening beta-damascenone in Chinese liquor brewing by utilizing flavor directional technology and application thereof |
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Patent Citations (4)
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| CN1793321A (en) * | 2005-11-22 | 2006-06-28 | 浙江杭州鑫富药业股份有限公司 | Microorganism of producing D-pantothenic acid enternal ester hydrolase and process for preparing D-pantothenic acid thereof |
| CN101029300A (en) * | 2007-02-06 | 2007-09-05 | 韶关学院 | Basylous yeast and its use in brewing myrica rubra |
| CN102102084A (en) * | 2010-10-26 | 2011-06-22 | 湖北白云边酒业股份有限公司 | Issatchenkia orientalis and composition and application thereof |
| CN102242072A (en) * | 2011-05-12 | 2011-11-16 | 江南大学 | Strain produced by screening beta-damascenone in Chinese liquor brewing by utilizing flavor directional technology and application thereof |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN107177519A (en) * | 2017-06-20 | 2017-09-19 | 湖北白云边酒业股份有限公司 | Schizosaccharomyces pombe bacterium, its composition and application |
| CN113481113A (en) * | 2021-09-06 | 2021-10-08 | 中粮营养健康研究院有限公司 | Space breeding saccharomyces cerevisiae and application thereof in brewing wine |
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Application publication date: 20120725 |