CN106635848A - Schizosaccharomyces pombe capable of simultaneously degrading arginine and urea - Google Patents
Schizosaccharomyces pombe capable of simultaneously degrading arginine and urea Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
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- C12H—PASTEURISATION, STERILISATION, PRESERVATION, PURIFICATION, CLARIFICATION OR AGEING OF ALCOHOLIC BEVERAGES; METHODS FOR ALTERING THE ALCOHOL CONTENT OF FERMENTED SOLUTIONS OR ALCOHOLIC BEVERAGES
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Abstract
The invention discloses achizosaccharomyces pombe capable of simultaneously degrading arginine and urea, and belongs to the technical field of microorganisms. The achizosaccharomyces pombe S.pombe C-11 provided by the invention can achieve a degrading capacity on the arginine in sorghum juice to 1.01mmol/L or above, with the generation of relatively little urine (0.54mmol/L), and the achizosaccharomyces pombe can achieve a degrading capacity on the urine to 2.03mmol/L or above. The achizosaccharomyces pombe provided by the invention is applicable to a Baijiu fermentation process; and by properly increasing the content of the strain in the fermentation process, the achizosaccharomyces pombe is conducive to decrease of an EC content in the fermentation process.
Description
Technical field
The present invention relates to the schizosaccharomyces pombe bacterium of a plant of degrade arginine and carbamide, belongs to microbial technique neck simultaneously
Domain.
Background technology
Urethanes (Ethyl Carbamate, EC) are a kind of 2A classes carcinogens, it is considered to be food relaying is yellow bent
Another important pollutant after syphilis element.Many researchers detect urethanes in many Wines and food
Presence.Since then, the content of urethanes is received significant attention in fermented food.Have been reported have detected different flavor into
The content of EC in product Chinese liquor, as a result shows that EC average contents are about 100 μ gL in Chinese liquor-1, less than 150 μ gL-1International limit
Amount standard.Wherein, EC contents are relatively low in delicate fragrance type, Maotai-flavor, medicated incense type and special aromatic white spirit, and Luzhou-flavor, sesame-flavor, phoenix
EC contents are higher in the Chinese liquor such as odor type.
Bakeing with fermented food, Jing often adds carbamide as the nitrogen source of yeast in fermentation liquid.Have been reported display
Carbamide can generate EC with ethanol synthesis；The main source for finding EC in Sucus Vitis viniferae sweat simultaneously is by arginine decomposition
The carbamide for coming.Japanese researchers construct the saccharomyces sake of arginase gene disappearance by genetic engineering means, using the ferment
Mother's fermentation finds the accumulation without carbamide, and EC contents significantly decline.Model essay comes, Xu Yan etc. it has also been found that in fermented grain carbamide and
The variation tendency of EC is similar to, therebetween with certain mutual relation.EC in Wine may be resulted from before distillation, distillation
When and distillation after each stage, at present, generally believe distillation before, that is, fermentation stage formed EC be mainly derived from carbamide.
Therefore, the schizosaccharomyces pombe bacterium of can degrade arginine and carbamide is obtained, for using schizosaccharomyces pombe as master
The production for wanting the fermented food of functional microorganism is just particularly important, for food safety has significant contribution.
The content of the invention
In order to solve the above problems, the invention provides the schizosaccharomyces pombe bacterium of one plant of degraded arginine and carbamide
(Schizosaccharomyces pombe)。
The schizosaccharomyces pombe bacterium (Schizosaccharomyces pombe), is preserved on June 28th, 2016
China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number be CGMCC NO.12715, preservation address
For Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
The schizosaccharomyces pombe bacterium CGMCC NO.12715 are screened from Chinese liquor fermented grain and obtained.
The schizosaccharomyces pombe bacterium CGMCC NO.12715 have the property that：
(1) the arginic degradation capability during schizosaccharomyces pombe bacterium of the invention is to Sorghum vulgare Pers. juice reaches 1.01mmolL-1And
Only generate less carbamide (0.54mmolL-1)；
(2) schizosaccharomyces pombe bacterium pair of the invention, to the degradation capability of the carbamide in Sorghum vulgare Pers. juice 2.03mmol is up to
L-1；
(3) various flavor substances are produced；
(4) in for alcoholic beverage sweat, by properly increasing its content during the fermentation, can effectively reduce
Arginine and urea content, are conducive to the reduction of EC contents in sweat.
Second object of the present invention is to provide the microbial bacterial agent containing the CGMCC NO.12715 bacterial strains.
In one embodiment of the invention, the microbial bacterial agent contain CGMCC NO.12715 thalline work it is thin
CGMCC NO.12715 dry myceliums, immobilized CGMCC NO.12715 cells, CGMCC that born of the same parents, lyophilization are obtained
The liquid bacterial agent of NO.12715, the solid fungicide of CGMCC NO.12715, or the CGMCC existed in other any forms
NO.12715 bacterial strains.
In one embodiment of the invention, in the microbial bacterial agent also containing it is any can apply to food or
The bacterial strain of any kind prepared by food, such as Bacillus licheniformis, saccharomyces cerevisiae, bacillus subtilises etc..
In one embodiment of the invention, it is CGMCC deposit number also to be contained in the microbial bacterial agent
The Lactobacillus plantarum and deposit number of NO.12717 is the Lactobacillus fermenti of CGMCC NO.12716.
In one embodiment of the invention, the microbial bacterial agent be by deposit number be CGMCC NO.12715
Schizosaccharomyces pombe, deposit number are the Lactobacillus fermenti of CGMCC NO.12716, deposit number for CGMCC NO.12717's
After Lactobacillus plantarum is activated respectively, according to volume ratio 1:1:1 is mixed to get liquid bacterial agent.
In one embodiment of the invention, also containing the carrier that can be arbitrarily used for food in the microbial bacterial agent.
Third object of the present invention is to provide application of the microbial bacterial agent in food technology field, especially applies
In fermented food technical field.
Fourth object of the present invention is to provide the CGMCC NO.12715 bacterial strains or described contains CGMCC
The application of the microbial bacterial agent of NO.12715, is to be applied to food technology field, especially fermented food technical field.
In one embodiment of the invention, the application, is to be applied to the aspects such as brewed wine, Spirit, such as in vain
Wine, wine, yellow wine, fruit wine aspect.
In one embodiment of the invention, the application, is by the CGMCC NO.12715 bacterial strains or described
Microbial bacterial agent containing CGMCC NO.12715 is added to during Chinese liquor, wine, yellow wine or brewing fruit wine.
5th purpose of the present invention is to provide a kind of preparation method of alcoholic beverages, and methods described is in alcoholic beverage
In sweat extra inoculation containing deposit number for the foxtail millet wine fragmentation yeast-like fungi of CGMCC NO.12715 microbial bacterial agent to
In ferment substrate.
In one embodiment of the invention, the microbial bacterial agent is with foxtail millet wine fragmentation yeast-like fungi as unique microorganism；Institute
The method of stating is that CGMCC NO.12715 bacterial strains are additionally inoculated with liquor fermentation fermented grain to 107CFU/g fermented grains.
In one embodiment of the invention, the microbial bacterial agent be by deposit number be CGMCC NO.12715
Schizosaccharomyces pombe, deposit number are the Lactobacillus fermenti of CGMCC NO.12716, deposit number for CGMCC NO.12717's
After Lactobacillus plantarum is activated respectively, according to volume ratio 1:1:1 is mixed to get liquid mixing microbial inoculum.
In one embodiment of the invention, methods described is the extra microbe inoculation microbial inoculum in liquor fermentation fermented grain
It is 10 to microbial bacteria agent content7CFU/g fermented grains.
In one embodiment of the invention, methods described is microorganism formulation according to total inoculum concentration 107CFU/g fermented grains
In being seeded to solid-state fermentation culture medium, and the Daqu (massive raw stater for alcholic liquor) of 10wt% is inoculated with, places standing for fermentation 6d in 30 DEG C of constant incubators.
Wherein, the preparation of solid-state fermentation culture medium：Claim 250g Sorghum vulgare Pers., 350mL distilled water is added, in 70-80 DEG C of incubator
Middle immersion 24h.Water is drained, 500g Sorghum vulgare Pers. is weighed in vacuum bag, 25mL distilled water is added；121 DEG C, sterilize 20min；Plus
Enzyme glycolysiss 1d.
Beneficial effects of the present invention：
The schizosaccharomyces pombe bacterium of the present invention, being at present can be while the best foxtail millet of arginine and carbamide and degradation effect of degrading
Wine Schizosaccharomyces.The present invention CGMCC NO.12715 to Sorghum vulgare Pers. juice in arginic degradation capability reach 1.01mmolL-1
And only generate less carbamide (0.54mmolL-1)；2.03mmolL is up to the degradation capability of the carbamide in Sorghum vulgare Pers. juice-1；
Produce various flavor substances；In for alcoholic beverage sweat, by properly increasing its content during the fermentation, can be effective
Arginine and urea content are reduced, is conducive to the reduction of EC contents in sweat.
Biomaterial preservation
Schizosaccharomyces pombe bacterium provided by the present invention, taxonomy is named as schizosaccharomyces pombe bacterium
Schizosaccharomyces pombe, are preserved in China Committee for Culture Collection of Microorganisms on June 28th, 2016
Common micro-organisms center, deposit number is CGMCC NO.12715, and preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number.
Lactobacillus fermenti provided by the present invention, taxonomy is named as Lactobacillus fermenti Lactobacillusfermentum,
China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on June 28th, 2016, deposit number is
CGMCC NO.12716, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
Lactobacillus plantarum provided by the present invention, taxonomy is named as Lactobacillus plantarum Lactobacillusplantarum,
China Committee for Culture Collection of Microorganisms's common micro-organisms center is preserved on June 28th, 2016, deposit number is
CGMCC NO.12717, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
Specific embodiment
EC assay reference literatures：Headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrum (GC-MS) are combined
Urethanes [J] in quantitative Spirit. food industry science and technology, 2012,33 (14):60-63.
The measure of urea content in fermented grain
Urea content determines reference literature：Jimenez-Martin E,Ruiz J,Perez-Palacios T,et
aL.Gas chromatography-mass spectrometry method for the determination of free
amino acids as their dimethyl-tert-butylsilyl(TBDMS)derivatives in animal
source food[J].Journal of Agricultural and Food Chemistry,2012,60(10):2456-
2463。
Embodiment 1：The schizosaccharomyces pombe of the present invention is to arginine, the degraded situation of carbamide
Yeast is that, by the flora that conversion of Arginine is carbamide, due to growing demand, yeast needs substantial amounts of nitrogen source.Environment
In arginine and carbamide all can absorb for yeast as nitrogen source, but yeast preferentially utilize arginine.Therefore, essence in environment
Propylhomoserin, carbamide and yeast etc. can all affect the content of carbamide in sweat, so as to affect the content of EC.And the quantity of yeast
With metabolic capacity be affect urea level key factor, study fermentation system in yeast by arginine metabolism into carbamide ability
It is necessary.
It is 300mg L according to arginine addition in arginine in fermented grain and carbamide testing result design metabolism-1It is (high
Itself contain certain arginine, extra addition 300mg L in fine strain of millet juice culture medium-1Afterwards total arginine content about 690mg/L, is close to
Arginine content in true fermented grain), carbamide addition is 200mg L-1.Fermentation carries out smart ammonia using Sorghum vulgare Pers. juice to experimental strain
Acid, the metabolism of carbamide.
Experiment and matched group setting：
Blank 1：Sorghum vulgare Pers. juice culture medium is inoculated with by 2% inoculum concentration, without arginine and carbamide；
Experimental group 1：Addition 300mg L-1Arginic Sorghum vulgare Pers. juice culture medium is inoculated with by 2% inoculum concentration；
Experimental group 2：Addition 200mg L-1The Sorghum vulgare Pers. juice culture medium of carbamide is inoculated with by 2% inoculum concentration；
Wherein, experimental strain is inoculated in culture medium by 2% inoculum concentration, 48～54h of quiescent culture at 30 DEG C.Determine and send out
Arginine, the content of carbamide and viable count in ferment starting point, the fermentation liquid of fermentation termination.
Sorghum vulgare Pers. juice culture medium：By Sorghum vulgare Pers.:Water=1:4(M:V), plus amylase cooking and liquefaction, 60 DEG C plus saccharifying enzyme glycolysiss, mistake
Filter centrifugation, adjusts pol to 10 ° of Bx.
Choose 4 plants relatively strong to arginine and/or urea metabolism ability sifted out in fermentation fermented grain of yeast
Saccharomyces cerevisiae C3、Zygosaccharomyces bailii C7、
Schizosaccharomycespombe C11 and Issatchenkia orientalis C16 carry out the generation of arginine and carbamide
Thank to capacity experimental.
As a result it is as shown in table 1.As can be seen from Table 1 the 4 saccharomycetes arginine that can degrade generates carbamide, but its energy
Power has have by force weak.Although the ability that S.cerevisiae C-3 degraded arginine generates carbamide is very strong, the degraded to carbamide
Ability is weaker, only 0.56mmolL-1.S.pombe C-11 reach 1.01mmolL to arginic degradation capability-1And only give birth to
Into less carbamide (0.54mmolL-1), 2.03mmolL is up to the degradation capability of carbamide-1, it is most strong in 4 plants of yeast
's.For this feature, if properly increasing its content during the fermentation, the drop of EC contents in sweat can be conducive to
It is low.
The saccharomycete of table 14 is to arginine and the metabolic capacity of carbamide
Schizosaccharomyces pombe bacterium Schizosaccharomycespombe C11, in being preserved on June 28th, 2016
State's Microbiological Culture Collection administration committee common micro-organisms center, deposit number is CGMCC NO.12715.
Additionally, the energy that the present invention is also obtained is while the Lactobacillus fermenti CGMCC of efficient degradation arginine and carbamide
NO.12716 (L. fermentum JSA30), Lactobacillus plantarum CGMCC NO.12717 (L.plantarum JD19), such as table 2
It is shown.Meanwhile, Lactobacillus plantarum CGMCC NO.12717 and Lactobacillus fermenti CGMCC NO.12716 can metabolism generation acid
The materials such as class, alcohols, ketone, phenols, for Chinese liquor important aroma compound is provided；CGMCC NO.12717 and CGMCC
NO.12716, enters stable phase after MRS culture medium, 30 DEG C of anaerobic fermentation 2d, and cell concentration is respectively up to OD600=3.5,
OD600=2.4, fermentation liquid pH is respectively 3.8,4.3；Lactic acid content respectively reaches 24g/L, 12g/L after fermentation 36h；Fermentation 3d
Yield of acetic acid respectively up to 5.7g/L, 6g/L.
Table 2CGMCC NO.12716 and CGMCC NO.12717 is to arginine and the metabolic condition of carbamide
Embodiment 2：Schizosaccharomyces pombe produces wind taste substance-measuring
Sorghum vulgare Pers. diffusion juice culture medium：Sorghum vulgare Pers. 200g → crush → add water the 800mL and μ L of alpha-amylase 200 → steaming and decocting 20min
→ the μ L of alpha-amylase 200 are added, α-amylase 1mL → 55 DEG C insulation 4h → filter to get filtrate is added after steaming and decocting 20min → cooling.
After by schizosaccharomyces pombe bacterium CGMCC NO.12715 bacterial strain seed liquor shake-flask culture 1d, by 107CFU/mL is inoculated with
Amount accesses the culture of Sorghum vulgare Pers. diffusion juice based on constant temperature quiescent culture 6d in 30 DEG C of incubators.And when detecting fermentation ends fermentation liquid wind
Taste material.
Analyze with headspace solid-phase microextraction technology (HS-SPME) and gas chromatography-mass spectrum (GC-MS) method.Take respectively
Supernatant and distillate after 8mL centrifugations, in being put into the headspace sampling bottle equipped with 3g sodium salts.With 4- methyl -2- amylalcohols as internal standard.
Bottle cap is screwed, ml headspace bottle is placed in into 50 DEG C of constant temperature extracting 45min.Laggard GC-MS analyses are extracted, GC-MS analysis conditions are shown in text
Offer.As a result it is as shown in table 3.
The main flavor of table 3
Embodiment 3：The solid state analogue fermentation of CGMCC NO.12715
The preparation of solid-state fermentation culture medium：Claim 250g Sorghum vulgare Pers., add 350mL distilled water, soak in 70-80 DEG C of incubator
24h.Water is drained, 500g Sorghum vulgare Pers. is weighed in vacuum bag, 25mL distilled water is added.121 DEG C, sterilize 20min.Plus enzyme glycolysiss
1d。
Matched group：Additionally there are addition 300mg L-1Arginic solid-state fermentation culture medium in be inoculated with 10wt% it is big
Song, standing for fermentation 6d in 30 DEG C of constant incubators.
Experimental group 1：By the seed liquor of schizosaccharomyces pombe bacterium CGMCC NO.12715 with final concentration 107CFU/g fermented grains
Inoculum concentration is seeded to and additionally have addition 300mg L-1Arginic solid-state fermentation culture medium in, and be inoculated with the Daqu (massive raw stater for alcholic liquor) of 10wt%,
Place standing for fermentation 6d in 30 DEG C of constant incubators.
Experimental group 2：By deposit number be the schizosaccharomyces pombe of CGMCC NO.12715, deposit number be CGMCC
After the Lactobacillus fermenti of NO.12716, deposit number are activated respectively for the Lactobacillus plantarum of CGMCC NO.12717, according to volume
Than 1:1:1 is mixed to get liquid mixing microbial inoculum.The cell concentration in liquid mixing microbial inoculum is determined, then with 107CFU/g fermented grains
Inoculum concentration is seeded to and additionally have addition 300mg L-1Arginic solid-state fermentation culture medium in, and be inoculated with the Daqu (massive raw stater for alcholic liquor) of 10wt%,
Place standing for fermentation 6d in 30 DEG C of constant incubators.
Experimental group 3：Compared with experimental group 1, only by the 300mg L of extra addition-1Arginine is substituted for extra addition
200mg L-1Carbamide.
Experimental group 4：Compared with experimental group 2, only by the 300mg L of extra addition-1Arginine is substituted for extra addition
200mg L-1Carbamide.
Experimental group 3 parallel, results averageds each with matched group, as shown in table 4-5.As shown in Table 4, by inoculation
The mixed microorganism microbial inoculum forced fermentation of CGMCC NO.12715 or the present invention, can effectively degrade arginine and carbamide.Together
When, the product special flavour that experimental group is obtained is more preferably excellent, especially experimental group 2.
The fermentation results of table 4
Wherein, Cit be citrulline, Arg be arginine, Orn is ornithine.Δ represent fermentation after respective substance content-
Content before fermentation, such as Δ Cit represent the citrulline that citrulline content is deducted in initial medium in fermentation 6d after fermentation things
Content.
The fermentation results of table 5
It is understood that for those of ordinary skills, with technology according to the present invention scheme and its can send out
Bright design in addition equivalent or change, and all these changes or replace the guarantor that should all belong to appended claims of the invention
Shield scope.
Claims (10)
1. one plant of foxtail millet wine fragmentation yeast-like fungi, it is characterised in that the foxtail millet wine fragmentation yeast-like fungi was preserved in China on 06 28th, 2016
Microbiological Culture Collection administration committee common micro-organisms center, deposit number is CGMCC NO.12715, and preservation address is north
The institute 3 of Jing Shi Chaoyang Districts North Star West Road 1.
2. a kind of microbial bacterial agent, it is characterised in that containing deposit number be CGMCC NO.12715 in the microbial bacterial agent
Foxtail millet wine fragmentation yeast-like fungi.
3. microbial bacterial agent according to claim 2, it is characterised in that in the microbial bacterial agent also containing it is any can
It is applied to the bacterial strain of any kind prepared by food or food.
4. microbial bacterial agent according to claim 3, it is characterised in that also contain deposit number in the microbial bacterial agent
For CGMCC NO.12717 Lactobacillus plantarum and deposit number for CGMCC NO.12716 Lactobacillus fermenti.
5. microbial bacterial agent according to claim 3, it is characterised in that the microbial bacterial agent is to be by deposit number
The schizosaccharomyces pombe of CGMCC NO.12715, deposit number are the Lactobacillus fermenti of CGMCC NO.12716, deposit number is
After the Lactobacillus plantarum of CGMCC NO.12717 is activated respectively, according to volume ratio 1:1:1 is mixed to get liquid bacterial agent.
6. a kind of preparation method of fermented food, it is characterised in that methods described has used the foxtail millet wine fragmentation described in claim 1
Yeast-like fungi or the arbitrary described microbial bacterial agent of claim 2～5.
7. preparation method according to claim 6, it is characterised in that the fermented food is brewed wine or Spirit.
8. a kind of preparation method of alcoholic beverages, it is characterised in that methods described is extra in the sweat of alcoholic beverage
Microbial bacterial agent of the inoculation containing the foxtail millet wine fragmentation yeast-like fungi that deposit number is CGMCC NO.12715 is in fermentation substrate.
9. method according to claim 8, it is characterised in that it by deposit number is CGMCC that the microbial bacterial agent is
The schizosaccharomyces pombe of NO.12715, deposit number are the Lactobacillus fermenti of CGMCC NO.12716, deposit number is CGMCC
After the Lactobacillus plantarum of NO.12717 is activated respectively, according to volume ratio 1:1:1 is mixed to get liquid bacterial agent.
10. according to the arbitrary described method of claim 8～9, it is characterised in that methods described is the volume in liquor fermentation fermented grain
Outer inoculation microbial bacterial agent to microbial bacteria agent content is 107CFU/g fermented grains.
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01320982A (en) * | 1988-06-23 | 1989-12-27 | Oozeki Syuzo Kk | Brewing method of refined japanese rice wine containing small amount of urea |
| CN1578839A (en) * | 2001-11-06 | 2005-02-09 | 英属哥伦比亚大学 | Modulating urea degradation in wine yeast |
-
2016
- 2016-10-19 CN CN201610911832.8A patent/CN106635848B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JPH01320982A (en) * | 1988-06-23 | 1989-12-27 | Oozeki Syuzo Kk | Brewing method of refined japanese rice wine containing small amount of urea |
| CN1578839A (en) * | 2001-11-06 | 2005-02-09 | 英属哥伦比亚大学 | Modulating urea degradation in wine yeast |
Non-Patent Citations (5)
| Title |
|---|
| DANMEI AN等: "Urea Excretion and Uptake by Wine Yeasts as Affected by Various Factors", 《AM.J.ENOL.VITIC》 * |
| SANTIAGO BENITO等: "New applications for Schizosaccharomyces pombe in the alcoholic", 《INTERNATIONAL JOURNAL OF FOOD SCIENCE AND TECHNOLOGY》 * |
| 付方圆: "葡萄酒中低产氨基甲酸乙酯和生物胺酿酒微生物特性的研究", 《中国优秀硕士学位论文全文数据库 工程科技Ⅰ辑》 * |
| 庞红勋: "本土葡萄酒酵母的选育及低醇葡萄酒的酿造", 《中国优秀硕士学位论文全文数据库（电子期刊） 工程科技Ⅰ辑》 * |
| 曹帅等: "白酒固态发酵过程中不同品种高粱原料对氨基甲酸乙酯形成的影响", 《食品与生物技术学报》 * |
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