CN106635848B - The schizosaccharomyces pombe bacterium of one plant of degrade simultaneously arginine and urea - Google Patents
The schizosaccharomyces pombe bacterium of one plant of degrade simultaneously arginine and urea Download PDFInfo
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- C—CHEMISTRY; METALLURGY
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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 the schizosaccharomyces pombe bacterium of one plant of degrade simultaneously arginine and urea, belong to microorganisms technical field.Schizosaccharomyces pombe bacterium S.pombe C-11 of the invention reaches 1.01mmolL to the arginic degradation capability in sorghum juice‑1And only generate less urea (0.54mmolL‑1), 2.03mmolL is up to the degradation capability of urea‑1；Schizosaccharomyces pombe bacterium of the invention, can be used for during liquor fermentation, by properly increasing its content during the fermentation, can be conducive to the reduction of EC content in fermentation process.
Description
Technical field
The present invention relates to the schizosaccharomyces pombe bacterium of one plant of degrade simultaneously arginine and urea, belong to microbial technique neck
Domain.
Background technique
Urethanes (Ethyl Carbamate, EC) is a kind of 2A class carcinogenic substance, it is considered to be food relaying is yellow bent
Another important pollutant after mould toxin.Many researchers detect urethanes in many Wines and food
Presence.Since then, the content of urethanes receives significant attention in fermented food.Have been reported have detected different flavor at
The content of EC in product white wine, EC average content is about 100 μ gL in white wine as the result is shown-1, it is lower than 150 μ gL-1International limit
Amount standard.Wherein, EC content is lower in delicate fragrance type, Maotai-flavor, medicinal-flavor and special aromatic white spirit, and Luzhou-flavor, sesame-flavor, phoenix
EC content is higher in the white wine such as odor type.
It is baking in fermented food, nitrogen source of the urea as yeast is often added in fermentation liquid.Display has been reported
EC can be generated in urea and ethanol synthesis；Simultaneously find EC in grape juice fermentation process main source be by arginine decompose and
The urea come.Japanese researchers construct the saccharomyces sake of arginase gene missing by genetic engineering means, utilize the ferment
Mother's fermentation finds the accumulation of not urea, and EC content sharp fall.Model essay comes, Xu Yan etc. it has also been found that in fermented grain urea and
The variation tendency of EC is similar, has certain correlation therebetween.There may be before distillation, distillation by EC in Wine
When and distillation after each stage, currently, before generally believing distillation, that is, the EC that fermentation stage is formed is mainly derived from urea.
Therefore, obtain the schizosaccharomyces pombe bacterium of arginine and urea of capable of degrading, for using schizosaccharomyces pombe bacterium as
The production of the fermented food of major function microorganism is just particularly important, and has significant contribution for food safety.
Summary of the invention
To solve the above-mentioned problems, the present invention provides the schizosaccharomyces pombe bacterium of one plant of degrade arginine and urea
(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 are CGMCC NO.12715, preservation address
For Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
The schizosaccharomyces pombe bacterium CGMCC NO.12715 is to screen to obtain from white wine fermented grain.
The schizosaccharomyces pombe bacterium CGMCC NO.12715 has the property that
(1) schizosaccharomyces pombe bacterium of the invention reaches 1.01mmolL to the arginic degradation capability in sorghum juice-1And
Only generate less urea (0.54mmolL-1)；
(2) schizosaccharomyces pombe bacterium pair of the invention is up to 2.03mmol to the degradation capability of the urea in sorghum juice
L-1；
(3) a variety of flavor substances are produced；
(4) for by properly increasing its content during the fermentation, can be effectively reduced in alcoholic beverage fermentation process
Arginine and urea content are conducive to the reduction of EC content in fermentation process.
A second object of the present invention is to provide the microbial bacterial agents for containing the CGMCC NO.12715 bacterial strain.
In one embodiment of the invention, the microbial bacterial agent contain CGMCC NO.12715 thallus work it is thin
Born of the same parents, CGMCC NO.12715 cell, the CGMCC for being freeze-dried obtained CGMCC NO.12715 dry mycelium, immobilization
The solid fungicide of the liquid bacterial agent of NO.12715, CGMCC NO.12715, or in the form of other are any existing for CGMCC
NO.12715 bacterial strain.
In one embodiment of the invention, in the microbial bacterial agent also containing it is any can be applied to food or
The bacterial strain, such as bacillus licheniformis, saccharomyces cerevisiae, bacillus subtilis of any kind etc. of food preparation.
In one embodiment of the invention, deposit number is also contained in the microbial bacterial agent is CGMCC
The lactobacillus plantarum and deposit number of NO.12717 is the lactobacillus fermenti of CGMCC NO.12716.
In one embodiment of the invention, it is CGMCC NO.12715 that the microbial bacterial agent, which is by deposit number,
Schizosaccharomyces pombe bacterium, deposit number are the lactobacillus fermenti of CGMCC NO.12716, deposit number is CGMCC NO.12717
Lactobacillus plantarum activate respectively after, be mixed to get liquid bacterial agent according to volume ratio 1:1:1.
In one embodiment of the invention, any carrier that can be used for food is also contained in the microbial bacterial agent.
Third object of the present invention is to provide the microbial bacterial agents in the application of food technology field, especially applies
In fermented food technical field.
Fourth object of the present invention is to provide the CGMCC NO.12715 bacterial strain 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 brewed wine, Spirit etc., such as white
In terms of wine, grape wine, yellow rice wine, fruit wine.
In one embodiment of the invention, the application is by the CGMCC NO.12715 bacterial strain or described
Microbial bacterial agent containing CGMCC NO.12715 is added to during white wine, grape wine, yellow rice wine or brewing fruit wine.
Fifth object of the present invention is to provide a kind of preparation method of alcoholic beverages, the method is in alcoholic beverage
The microbial bacterial agent containing the schizosaccharomyces pombe bacterium that deposit number is CGMCC NO.12715 is additionally inoculated in fermentation process to arrive
In fermentation substrate.
In one embodiment of the invention, the microbial bacterial agent is with schizosaccharomyces pombe bacterium for unique microorganism；
The method is that CGMCC NO.12715 bacterial strain is additionally inoculated in liquor fermentation fermented grain to 107CFU/g fermented grain.
In one embodiment of the invention, it is CGMCC NO.12715 that the microbial bacterial agent, which is by deposit number,
Schizosaccharomyces pombe bacterium, deposit number are the lactobacillus fermenti of CGMCC NO.12716, deposit number is CGMCC NO.12717
Lactobacillus plantarum activate respectively after, be mixed to get liquid mix bacterium agent according to volume ratio 1:1:1.
In one embodiment of the invention, the method is the additional microbe inoculation microbial inoculum in liquor fermentation fermented grain
It is 10 to microbial bacteria agent content7CFU/g fermented grain.
In one embodiment of the invention, the method is microorganism formulation according to total inoculum concentration 107CFU/g fermented grain
It is seeded in solid-state fermentation culture medium, and is inoculated with the yeast of 10wt%, place in 30 DEG C of constant incubators and be left to ferment 6d.
Wherein, the preparation of solid-state fermentation culture medium: claiming 250g sorghum, 350mL distilled water is added, in 70-80 DEG C of incubator
Middle immersion is for 24 hours.Water is drained, 500g sorghum is weighed in vacuum bag, adds 25mL distilled water；121 DEG C, sterilize 20min；Add
Enzymatic conversion 1d.
Beneficial effects of the present invention:
Schizosaccharomyces pombe bacterium of the invention is can degrade simultaneously at present arginine and urea and the best grain of degradation effect
Wine Schizosaccharomyces.CGMCC NO.12715 of the invention reaches 1.01mmolL to the arginic degradation capability in sorghum juice-1
And only generate less urea (0.54mmolL-1)；2.03mmolL is up to the degradation capability of the urea in sorghum juice-1；
Produce a variety of flavor substances；It, can be effective by properly increasing its content during the fermentation in alcoholic beverage fermentation process
Arginine and urea content are reduced, the reduction of EC content in fermentation process is conducive to.
Biomaterial preservation
Schizosaccharomyces pombe bacterium provided by the present invention, taxology are named as schizosaccharomyces pombe bacterium
Schizosaccharomyces pombe is preserved in China Committee for Culture Collection of Microorganisms on June 28th, 2016
Common micro-organisms center, deposit number are CGMCC NO.12715, and preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3
Number.
Specific embodiment
EC assay reference literature: headspace solid-phase microextraction (HS-SPME) and gas chromatography-mass spectrum (GC-MS) combination
Urethanes [J] food industry science and technology, 2012,33 (14): 60-63 in quantitative Spirit.
The measurement of urea content in fermented grain
Urea content measures 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 degradation situation of schizosaccharomyces pombe bacterium of the invention to arginine, urea
Yeast is the flora by conversion of Arginine for urea, and due to growing demand, yeast needs a large amount of nitrogen source.Environment
In arginine and urea all can be used as nitrogen source and be absorbed and utilized for yeast, but yeast preferentially utilizes arginine.Therefore, smart in environment
Propylhomoserin, urea and saccharomycete etc. can all influence the content of urea in fermentation process, to influence the content of EC.And the quantity of yeast
It is an important factor for influencing urea level with metabolic capability, yeast is by arginine metabolism at the ability of urea in research fermentation system
It is necessary to.
It is 300mg L according to arginine additive amount in arginine in fermented grain and urea testing result design metabolism-1It is (high
Itself contain certain arginine in fine strain of millet juice culture medium, it is additional to add 300mg L-1Total arginine content about 690mg/L afterwards is approached
Arginine content in true fermented grain), urea additive amount is 200mg L-1.Fermentation carries out smart ammonia to experimental strain using sorghum juice
The metabolism of acid, urea.
Experiment and control group setting:
Blank 1: sorghum juice culture medium is inoculated with by 2% inoculum concentration, does not add arginine and urea；
Experimental group 1: addition 300mg L-1Arginic sorghum juice culture medium is inoculated with by 2% inoculum concentration；
Experimental group 2: addition 200mg L-1The sorghum juice culture medium of urea is inoculated with by 2% inoculum concentration；
Wherein, experimental strain is inoculated into culture medium by 2% inoculum concentration, 48~54h of stationary culture at 30 DEG C.Measurement hair
Ferment starting point, fermentation termination fermentation liquid in arginine, the content of urea and viable count.
Sorghum juice culture medium: by sorghum: water=1:4 (M:V) adds amylase cooking and liquefaction, 60 DEG C of sugaring enzymatic conversions, mistake
Filter centrifugation adjusts pol to 10 ° of Bx.
4 plants sifted out in fermentation fermented grain are chosen to arginine and/or the relatively stronger yeast of urea metabolism ability
Saccharomyces cerevisiae C3、Zygosaccharomyces bailii C7、Schizosaccharomyces
Pombe C11 and Issatchenkia orientalis C16 carries out the metabolic capability experiment of arginine and urea.
The results are shown in Table 1.The 4 saccharomycetes arginine that can degrade generates urea, but its energy as can be seen from Table 1
Power have by force have it is weak.Although S.cerevisiae C-3 degrades, the ability of arginine generation urea is very strong, the degradation to urea
Ability is weaker, only 0.56mmolL-1.S.pombe C-11 reaches 1.01mmolL to arginic degradation capability-1And it only gives birth to
At less urea (0.54mmolL-1), 2.03mmolL is up to the degradation capability of urea-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 content in fermentation process can be conducive to
It is low.
Metabolic capability of 14 saccharomycete of table to arginine and urea
Schizosaccharomyces pombe bacterium Schizosaccharomyces pombe C11, has been preserved on June 28th, 2016
State's Microbiological Culture Collection administration committee common micro-organisms center, deposit number are CGMCC NO.12715.
In addition, the lactobacillus fermenti CGMCC of energy while efficient degradation arginine and urea that the present invention also obtains
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, generation acid can be metabolized
The substances such as class, alcohols, ketone, phenols provide important aroma compound for white wine；CGMCC NO.12717 and CGMCC
NO.12716 enters stationary 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 are respectively 3.8,4.3；Lactic acid content respectively reaches 24g/L, 12g/L after fermentation 36h；Ferment 3d
Yield of acetic acid respectively up to 5.7g/L, 6g/L.
Metabolic condition of table 2CGMCC NO.12716 and the CGMCC NO.12717 to arginine and urea
Embodiment 2: schizosaccharomyces pombe bacterium produces wind taste substance-measuring
Sorghum diffusion juice culture medium: sorghum 200g → crushing → plus water 800mL and alpha-amylase 200 μ L → boiling
20min → addition alpha-amylase 200 μ L, boiling 20min → α-amylase 1mL → 55 DEG C are added after cooling and keep the temperature 4h → are filtered
Filtrate.
After schizosaccharomyces pombe bacterium CGMCC NO.12715 bacterial strain seed liquor shaking flask culture 1d, by 107CFU/mL inoculation
Amount access sorghum diffusion juice culture is based on constant temperature stationary culture 6d in 30 DEG C of incubators.And when detecting fermentation ends fermentation liquid wind
Taste substance.
It is analyzed with headspace solid-phase microextraction technology (HS-SPME) and gas chromatography-mass spectrum (GC-MS) method.It takes respectively
Supernatant and distillate after 8mL centrifugation, are put into the headspace sampling bottle equipped with 3g sodium salt.Using 4- methyl -2- amylalcohol as internal standard.
Bottle cap is screwed, ml headspace bottle is placed in 50 DEG C of constant temperature extracting 45min.Laggard GC-MS analysis is extracted, GC-MS analysis condition is shown in text
It offers.The results are shown in Table 3.
3 main flavor of table
The solid state analogue of embodiment 3:CGMCC NO.12715 ferments
The preparation of solid-state fermentation culture medium: claiming 250g sorghum, and 350mL distilled water is added, impregnates in 70-80 DEG C of incubator
24h.Water is drained, 500g sorghum is weighed in vacuum bag, adds 25mL distilled water.121 DEG C, sterilize 20min.Enzyme saccharification
1d。
Control group: additionally there is addition 300mg L-1Arginic solid-state fermentation culture medium in be inoculated with 10wt% it is big
Song is left to ferment 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 grain
Inoculum concentration, which is seeded to additionally, addition 300mg L-1Arginic solid-state fermentation culture medium in, and be inoculated with the yeast of 10wt%,
It places in 30 DEG C of constant incubators and is left to ferment 6d.
Experimental group 2: by schizosaccharomyces pombe bacterium that deposit number is CGMCC NO.12715, deposit number CGMCC
After the lactobacillus fermenti of NO.12716, the lactobacillus plantarum that deposit number is CGMCC NO.12717 activate respectively, according to volume
Liquid mix bacterium agent is mixed to get than 1:1:1.The cell concentration in liquid mix bacterium agent is measured, then with 107CFU/g fermented grain
Inoculum concentration, which is seeded to additionally, addition 300mg L-1Arginic solid-state fermentation culture medium in, and be inoculated with the yeast of 10wt%,
It places in 30 DEG C of constant incubators and is left to ferment 6d.
Experimental group 3: compared with experimental group 1, the 300mg L that only will additionally add-1Arginine is substituted for additional addition
200mg L-1Urea.
Experimental group 4: compared with experimental group 2, the 300mg L that only will additionally add-1Arginine is substituted for additional addition
200mg L-1Urea.
Experimental group is parallel with each 3 of control group, and results are averaged, as shown in table 4-5.As shown in Table 4, pass through inoculation
CGMCC NO.12715 or mixed microorganism microbial inoculum forced fermentation of the invention, can effectively degrade arginine and urea.Together
When, the product special flavour that experimental group obtains is more preferably excellent, especially experimental group 2.
4 fermentation results of table
Wherein, Cit is citrulling, Arg is arginine, and Orn is ornithine.Δ represents the content-of respective substance after fermentation
Content before fermentation, such as Δ Cit represent citrulling content in fermentation 6d post-fermentation object and subtract the citrulling in initial medium
Content.
5 fermentation results of table
It, can according to the technique and scheme of the present invention and its hair it is understood that for those of ordinary skills
Bright design is subject to equivalent substitution or change, and all these changes or replacement all should belong to the guarantor of appended claims of the invention
Protect range.
Claims (10)
1. one plant of schizosaccharomyces pombe bacterium (Schizosaccharomyces pombe), which is characterized in that the grain wine fragmentation ferment
Female bacterium was preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center, deposit number on 06 28th, 2016
For CGMCC NO.12715, preservation address is Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
2. a kind of microbial bacterial agent, which is characterized in that containing deposit number in the microbial bacterial agent is CGMCC NO.12715
Schizosaccharomyces pombe bacterium.
3. microbial bacterial agent according to claim 2, which is characterized in that in the microbial bacterial agent also containing it is any can
Bacterial strain applied to any kind prepared by food or food.
4. microbial bacterial agent according to claim 3, which is characterized in that also contain deposit number in the microbial bacterial agent
It is CGMCC for the lactobacillus plantarum (Lactobacilus plantarum) and deposit number of CGMCC NO.12717
The lactobacillus fermenti (Lactobacilus fermentum) of NO.12716.
5. microbial bacterial agent according to claim 3, which is characterized in that the microbial bacterial agent is to be by deposit number
The schizosaccharomyces pombe bacterium of CGMCC NO.12715, lactobacillus fermenti, the deposit number that deposit number is CGMCC NO.12716
For CGMCC NO.12717 lactobacillus plantarum activate respectively after, be mixed to get liquid bacterial agent according to volume ratio 1:1:1.
6. a kind of preparation method of fermented food, which is characterized in that the method has used grain wine fragmentation described in claim 1
Saccharomycete or any microbial bacterial agent of claim 2~5.
7. preparation method according to claim 6, which is characterized in that the fermented food is brewed wine or Spirit.
8. a kind of preparation method of alcoholic beverages, which is characterized in that the method is additional in the fermentation process of alcoholic beverage
The microbial bacterial agent containing the schizosaccharomyces pombe bacterium that deposit number is CGMCC NO.12715 is inoculated with into fermentation substrate.
9. according to the method described in claim 8, it is characterized in that, it is CGMCC that the microbial bacterial agent, which is by deposit number,
The schizosaccharomyces pombe bacterium of NO.12715, deposit number are the lactobacillus fermenti of CGMCC NO.12716, deposit number CGMCC
After the lactobacillus plantarum of NO.12717 activates respectively, liquid bacterial agent is mixed to get according to volume ratio 1:1:1.
10. according to any method of claim 8~9, which is characterized in that the method is the volume in liquor fermentation fermented grain
Outer inoculation microbial bacterial agent to microbial bacteria agent content is 107CFU/g fermented grain.
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