CN103571763B - One strain self can synthesize yeast saccharomyces cerevisiae and the application thereof of terpene substances - Google Patents
One strain self can synthesize yeast saccharomyces cerevisiae and the application thereof of terpene substances Download PDFInfo
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Abstract
The invention discloses yeast saccharomyces cerevisiae and application thereof that a strain self can synthesize terpene substances, belong to field of fermentation engineering.The present invention screen from white wine wine unstrained spirits obtain a strain can the yeast saccharomyces cerevisiae of the multiple terpene owner of self de novo synthesis, this bacterial strain is preserved in China General Microbiological center on September 4th, 2013, deposit number is CGMCC No.8130, this bacterial strain is cultivated through liquid or solid state fermentation, can produce linalool, α-terpinol, geraniol, myrcenol, nerolidol, α-do not have red medicine alcohol, farnesol, α-farnesene, β-farnesene, not have red medicine alkene 10 kinds of terpene substances.In wine brewing process, add this yeast strain, the local flavor of white wine can be improved and improve its nutritive value.
Description
Technical field
The present invention relates to yeast saccharomyces cerevisiae and application thereof that a strain self can synthesize terpene substances, belong to fermentation engineering field.
Background technology
Terpene substances is the of paramount importance compounds in grape wine, has the fragrance of the pleasant such as fruit perfume (or spice), the fragrance of a flower; In addition, terpene its there is higher physiologically active and very high pharmacological action, there is nourishing function.Terpene substances generally has very low fragrance threshold value, it is also confirmed to be the important micro-flavour substances of China white wine, but except containing compared with except the terpene substances of horn of plenty in medicine aromatic white spirit, the content of terpene substances all very low (10 μ about g/L) in the white wine of other odor types, therefore, improve the content of terpene substances in white wine, not only can improve the quality of white wine from local flavor, more can increase the nutritive value of white wine.
The terpene substances kind that the yeast saccharomyces cerevisiae that can synthesize terpene substances of report can synthesize both at home and abroad is at present limited to linalool, α-terpinol, geraniol, farnesol, Geraniol these 5 kinds, and be by yeast saccharomyces cerevisiae de novo synthesis about this several terpene substances, still need the raw material/intermediate utilizing certain special could synthesize still without final conclusion.In grape wine, terpene substances is considered to major part and comes from grape fruit, additional beta-glucosidase in brewing process, is that to improve in grape wine that terpene substances is topmost be also a kind of method admitted most.And terpene substances in white wine is little, its content neither be very stable, and main problem is exactly that the precursor of terpene substances in brewed spirit raw material is few, and Glycosylase has Substratspezifitaet, also only have a few, additional Glycosylase also not necessarily can improve terpene substances content.By directly adding these terpene substances in white wine, no doubt can improve the content of terpene substances in wine, but industrial terpene substances security and consumption can not be protected, the terpene substances cost that plant origin extracts is very high again.Therefore, find the way of production of terpene substances and regulated and controled, just can reach the effect improving terpene substances output.
The invention provides a kind of method increasing terpene substances content in white wine, the key of the method is the yeast saccharomyces cerevisiae of a strain self synthesis terpene substances, this is domestic first to the relevant report self can synthesizing terpene substances yeast saccharomyces cerevisiae, this bacterial strain is also the bacterial strain that self can produce terpene alcohols material that screening obtains in brewed spirit process first, possesses the efficient function producing terpene substances.
Summary of the invention
First technical problem that the present invention will solve is to provide the yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) that a strain self can synthesize terpene substances, this bacterial strain screening is in certain scent type wine distiller's yeast, China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on September 4th, 2013, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.8130.
Obtain the 26S rDNA sequence of obtained strains, in GenBank, carry out Blast contrast determine its kind, the bacterial strain the highest with its homologous sequence is Saccharomyces cerevisiae, and similarity is 99%.
Described bacterial strain can produce linalool, α-terpinol, geraniol, myrcenol, nerolidol, α-do not have red medicine alcohol, farnesol, α-farnesene, β-farnesene, not have red medicine alkene 10 kinds of terpene substances.
Second technical problem that the present invention will solve is to provide a kind of method utilizing described bacterial strain to produce terpenol:
(1) seed culture medium: YNB6.7g/L, glucose 20g/L, pH7.0,0.22 μm of membrane filtration is degerming;
(2) fermention medium: KH
2pO
41g/L, (NH
4)
2sO
45g/L, MgSO
40.5g/L, NaCl0.1g/L, CaCl
20.1g/L, glucose 20g/L, pH7.0,0.22 μm of membrane filtration is degerming;
(3) seed culture method: 250mL taper bottled substratum 100mL, shaking speed 150rpm, cultivates 24 ~ 48h for 30 DEG C;
(4) fermentation culture method: with the inoculum size of about 1% (v/v) access fermention medium, 250ml taper bottled substratum 100mL, shaking speed 150rpm, cultivate 48 ~ 72h for 30 DEG C.
The 3rd technical problem that the present invention will solve is to provide the application of described bacterial strain at field of food, is in wine brewing process, to inoculate this yeast to improve the local flavor of wine, to improve the content of terpene alcohols material in white wine.
Described inoculation both can be liquid microbial inoculum mode, also can be liquid microbial inoculum mode.
Described liquid microbial inoculum preparation method: by the liquid nutrient medium (raw material 1 ~ 5%, v/v) after seed liquor inoculation (inoculum size 2-10%) to sterilizing, 30 DEG C, 150rpm shake flask fermentation 24 ~ 48h; Described raw material is Chinese sorghum, barley, wheat, pea, corn, wheat bran, glutinous rice or highland barley juice.
Described solid-state microbial inoculum preparation method: by the raw material substratum after seed liquor inoculation (inoculum size 2-10%) to sterilizing (1 ~ 5%, w/w), solid state fermentation 48 ~ 120h in 30 DEG C of environment; Described raw material substratum is Chinese sorghum, barley, wheat, pea, corn, wheat bran, glutinous rice or highland barley.
Described yeast is added with the form of solid-state inoculation or liquid inoculation in brewed spirit process, linalool in white wine, α-terpinol, geraniol, myrcenol, nerolidol, α-do not have red medicine alcohol, farnesol, α-farnesene, β-farnesene can be increased, do not have the concentration of red medicine alkene 10 kinds of terpene substances, the local flavor of wine can be improved and improve the nutritive value of wine.
The present invention provides the yeast saccharomyces cerevisiae that a strain self can synthesize terpene substances first, this bacterial strain screens and obtains from certain scent type wine unstrained spirits, liquid and solid state fermentation shows, do not need particular precursor material, this bacterial strain just can produce multiple terpene substances, comprises linalool, α-terpinol, geraniol, myrcenol, nerolidol, α-do not have red medicine alcohol, farnesol, α-farnesene, β-farnesene, does not have red medicine alkene 10 kinds of terpene substances.In wine brewing process, access this bacterium can improve wine local flavor, improve wine nutritive value, realize the lifting of China white wine quality, the technical progress of China white wine and development are had great importance.
Accompanying drawing explanation
Fig. 1 bacterial classification systematic evolution tree (CGMCC8130)
Embodiment
The research of terpenoid substance is produced in the liquid state fermentation of embodiment 1 yeast
Be inoculated in YNB fermention medium by described yeast saccharomyces cerevisiae CGMCC No.8130,150rpm, 30 DEG C of liquid state fermentation 48h, measured the content of terpenoid substance by HS-SPME-GC-MS, its concrete grammar is as follows:
1. preparation of samples: add the CaCl of 1% in fermented liquid
2, after dissolving at 4 DEG C with the centrifugal 10min of the rotating speed of 8000rpm, get after supernatant at the membrane filtration with 0.22 μm.
2. gas chromatography-mass spectrography detects: in 15mL ml headspace bottle, add 8mL settled solution, 3g sodium-chlor, the 4-methyl-2-amylalcohol adding 10 μ L, as interior mark, carries out headspace solid-phase microextraction.The condition of headspace solid-phase microextraction is: three-phase (Car/DVB/PDMS) extracting head, 60 DEG C of preheating 5 ~ 10min, extraction absorption 30 ~ 50min, GC desorb 5 ~ 10min.
3. qualitative, quantitative: calculate retention index RI by the mass spectrum of non-principal component, compares with the retention index of the standard substance of the corresponding conventional ingredient brewageed in product (as white wine, red wine etc.), the volatile component detected with this qualitative confirmation; Test substance after qualitative is done typical curve relative to interior mark, after GC-MS detects, utilizes test substance and internal standard substance matter relative peak area to go out the content of test substance than the corresponding typical curve Equation for Calculating of substitution: the peak area of often kind of test substance adopts Selective ion mode method integration (SIM).
Terpene alcohols material composition situation in this Accharomyces cerevisiae fermented liquid is as shown in table 1, the total content of fermented liquid can reach 71.66ug/L, on composition, we can find linalool, α-terpinol, geraniol, myrcenol, nerolidol, α-do not have red medicine alcohol, farnesol, α-farnesene, β-farnesene, not have red medicine alkene 10 kinds of terpene substances, and its content is respectively 5.55 μ g/L, 27.17 μ g/L, 2.26 μ g/L, 6.21 μ g/L, 10.14 μ g/L, 4.00 μ g/L, 14.16 μ g/L, 8.34 μ g/L, 3.27 μ g/L, 1.56 μ g/L.
Table 1: terpene substances composition and content (μ g/L)
Embodiment 2 yeast saccharomyces cerevisiae CGMCC No.8130 is with the difference of business Saccharomyces cerevisiae S288C Produced by Solid-state Fermentation terpene substances and produce comparing of terpene substances with liquor production process
Laboratory simulation brewing process, adds this Accharomyces cerevisiae and business yeast saccharomyces cerevisiae respectively, and be that raw material carries out solid state fermentation with Chinese sorghum, concrete scheme is as follows:
1. the process of raw material: 200g is placed in large beaker, add the distilled water of 300mL, 100 DEG C of water bath processing 2h, midway moisturizing is to maintain certain moisture, add saccharifying enzyme 5mL(100U/mL afterwards) 60 DEG C of process 4h, packing (50g/ bag) 110 DEG C of sterilizing 30min in high-temperature resistance plastice bag.
2. inoculation and fermentation: the bacterial strain activated is accessed in sterilized Chinese sorghum by identical amount, solid state rheology 15 ~ 30 days at 30 DEG C.
The detection of terpene alcohol content: get the Chinese sorghum or wine unstrained spirits 5g that ferment in 50mL centrifuge tube, add the CaCl of 3%
2the aqueous solution, in 4 DEG C of environment, soak 12 ~ 24h, supersound process 2 ~ 5h, 8000rpm are centrifugal, and 10min gets supernatant liquor, after the membrane filtration of 0.22 μm, is measured the content of terpene substances by HS-SPME-GC-MS.Final result, as following table 2, shows that CGMCC No.8130 can produce than the more terpenes kind of S288C commercial yeast and concentration.
Table 2: terpene substances composition and content (μ g/Kg)
Note: nd, does not detect.
Embodiment 3 is added yeast saccharomyces cerevisiae CGMCC No.8130 and is improved terpene substances content in white wine, improves liquor flavor.
After yeast saccharomyces cerevisiae (CGMCC No.8130 and business are used) respectively activation, (final concentration is 10 for the inoculum size according to 1%
7cfu/kg) add to and mixed in the wine unstrained spirits of Daqu, then continue by traditional technology fermentation.
After wine sample is diluted to 10 °, measured the content of terpene substances by HS-SPME-GC-MS.Analytical results, as table 3, compared with Normal group, adds terpene substances kind and content showed increased in yeast saccharomyces cerevisiae CGMCC No.8130 group white wine, and difference is little compared with the control to add in business yeast saccharomyces cerevisiae group terpene substances kind and content in white wine.
Table 3: terpene substances composition and content (μ g/L)
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.
Claims (7)
1. a strain self can synthesize the yeast saccharomyces cerevisiae (Saccharomyces cerevisiae) of terpene substances, it is characterized in that, China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on September 4th, 2013, No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, Institute of Microorganism, Academia Sinica, deposit number is CGMCC No.8130.
2. utilize a method for yeast saccharomyces cerevisiae production terpene substances described in claim 1, it is characterized in that, comprise the following steps:
(1) seed culture: yeast saccharomyces cerevisiae being inoculated into seed culture medium liquid amount is in the Erlenmeyer flask of 40%, shaking speed 150rpm, cultivates 24 ~ 48h for 30 DEG C;
(2) fermentation culture: the bottled substratum of taper with the inoculum size of 1% ~ 5% (v/v), seed culture fluid being accessed fermention medium liquid amount 40%, shaking speed 150rpm, cultivates 48 ~ 72h for 30 DEG C;
Described seed culture medium: YNB6.7g/L, glucose 20g/L, pH7.0,0.22 μm of membrane filtration is degerming;
Described fermention medium: KH
2pO
41g/L, (NH
4)
2sO
45g/L, MgSO
40.5g/L, NaCl 0.1g/L, CaCl
20.1g/L, glucose 20g/L, pH7.0,0.22 μm of membrane filtration is degerming.
3. yeast saccharomyces cerevisiae described in claim 1 is in the application of field of food, it is characterized in that, inoculates described yeast and improve the local flavor of wine, improve the content of terpene substances in white wine in brewed spirit process.
4. application according to claim 3, is characterized in that, described terpene is linalool, α-terpinol, geraniol, myrcenol, nerolidol, α-and do not have red medicine alcohol, farnesol, α-farnesene, β-farnesene and do not have red medicine alkene.
5. application according to claim 3, is characterized in that, described inoculation is the liquid microbial inoculum of inoculation, and described liquid microbial inoculum seed liquor is inoculated in the liquid nutrient medium after sterilizing, 30 DEG C, 150rpm shake flask fermentation 24 ~ 48h.
6. application according to claim 5, is characterized in that, described liquid nutrient medium is Chinese sorghum, barley, wheat, pea, corn, wheat bran, glutinous rice or highland barley juice.
7. application according to claim 3, is characterized in that, described inoculation is the solid-state microbial inoculum of inoculation, and described solid-state microbial inoculum seed liquor is inoculated in the raw material substratum after sterilizing, solid state fermentation 48 ~ 120h in 30 DEG C of environment; Described raw material substratum is Chinese sorghum, barley, wheat, pea, corn, wheat bran, glutinous rice or highland barley.
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|---|---|---|---|---|
| ITUB20160092A1 (en) * | 2016-01-25 | 2017-07-25 | Univ Degli Studi Padova | YEAST STRAIN Saccharomyces cerevisiae DBVPG35P, ITS USE FOR THE FERMENTATIVE PRODUCTION OF FOOD, PARTICULARLY OF LISON AND LISON CLASSIC WINE, A RELATIVE PRODUCT AND A METHOD FOR THE SELECTION OF THE STRAINER |
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| CN106011178B (en) * | 2016-07-14 | 2019-02-26 | 珀莱雅化妆品股份有限公司 | A method of β-farnesene is prepared using sugared sorghum stalk and sugarcane produced by fermenting waste residues |
| CN112444580A (en) * | 2019-09-05 | 2021-03-05 | 中国食品发酵工业研究院有限公司 | Detection method for simultaneously and rapidly determining four specific functional aroma active ingredients in brewed rice wine |
| CN110951631B (en) * | 2019-12-31 | 2021-03-02 | 江南大学 | Hansenula polymorpha capable of producing geraniol and fermentation method thereof |
| CN111440733A (en) * | 2020-02-07 | 2020-07-24 | 天津大学 | Recombinant saccharomyces cerevisiae for producing terpineol, construction method and application |
| CN113774079A (en) * | 2021-08-13 | 2021-12-10 | 中国科学院天津工业生物技术研究所 | Recombinant saccharomyces cerevisiae and construction method and application thereof |
| CN114395513B (en) * | 2022-02-25 | 2023-08-25 | 江南大学 | Microbial agent for brewing fen-flavor liquor and application thereof |
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| WO2012116783A2 (en) * | 2011-02-28 | 2012-09-07 | Organobalance Gmbh | A yeast cell for the production of terpenes and uses thereof |
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Patent Citations (3)
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| CN102071153A (en) * | 2010-12-08 | 2011-05-25 | 江南大学 | Yeast engineering bacterium for producing d-limonene and construction method thereof |
| CN102071155A (en) * | 2010-12-09 | 2011-05-25 | 江南大学 | High-yield s-linalool genetic engineering strain and building method thereof |
| WO2012116783A2 (en) * | 2011-02-28 | 2012-09-07 | Organobalance Gmbh | A yeast cell for the production of terpenes and uses thereof |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
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| ITUB20160092A1 (en) * | 2016-01-25 | 2017-07-25 | Univ Degli Studi Padova | YEAST STRAIN Saccharomyces cerevisiae DBVPG35P, ITS USE FOR THE FERMENTATIVE PRODUCTION OF FOOD, PARTICULARLY OF LISON AND LISON CLASSIC WINE, A RELATIVE PRODUCT AND A METHOD FOR THE SELECTION OF THE STRAINER |
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Address after: 214122 Jiangsu city of Wuxi Province District Liangxi No. 898 South Road 7 layer beam Creek area of food science and Technology Park Patentee after: Jiangnan University Address before: No. 1800 road 214122 Jiangsu Lihu Binhu District City of Wuxi Province Patentee before: Jiangnan University |