CN106047779B - A kind of pit mud functional microorganism not producing pit mud stink substances PC - Google Patents

A kind of pit mud functional microorganism not producing pit mud stink substances PC Download PDF

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CN106047779B
CN106047779B CN201610687369.3A CN201610687369A CN106047779B CN 106047779 B CN106047779 B CN 106047779B CN 201610687369 A CN201610687369 A CN 201610687369A CN 106047779 B CN106047779 B CN 106047779B
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pit mud
jgn4
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杜海
徐岩
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Jiangnan University
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Abstract

The invention discloses the pit mud functional microorganisms that one kind does not produce pit mud stink substances PC, belong to microorganisms technical field.The present invention has obtained three plants and has not produced or the pit mud functional microorganism of low yield pit mud stink substances PC, after functional microorganism of the invention is additionally added to pit mud, it is able to suppress the growth of the bacterial strain belonged to, and the microorganism structure that other belong to is influenced less, obtained pit mud is able to maintain that the normal fermentation of white wine but can reduce PC content in white wine.The present invention also provides the control strategies of smell substance PC in fermented food.

Description

A kind of pit mud functional microorganism not producing pit mud stink substances PC
Technical field
The present invention relates to the pit mud functional microorganisms that one kind does not produce pit mud stink substances PC, belong to microorganisms technical field.
Background technique
White wine is one of world-renowned six big Spirits.Luzhou-flavor liquo occupies importantly in China liquor consumption market Position, yield and occupation rate of market account for 70% or so of entire liquor industry.The most characteristic production technology of Luzhou-flavor liquo it It ferments first is that being stored using mud.Pit provides anaerobic environment as fermented grain round, and pit mud therein provides a large amount of brewing With bacterium, generate many to the contributive flavor substance of wine body.Wherein, the generation of Luzhou-flavor liquo main body aroma-producing substance ethyl hexanoate It is closely related with cellar mud microorganisms.However, pit mud not only contributes to a large amount of beneficial flavor substance, some bad flavors Matter such as pit mud is smelly also to enter wine body.Although the smelly puzzlement liquor industry of pit mud is for many years, the control for lacking such peculiar smell is effectively controlled Measure.
After the volatile component in pit mud detects in Xu Yan team, using modern times separation and flavor investigative technique, confirmation is produced The smelly compound of raw pit mud is 4- methylphenol (p-cresol, PC).Pass through yeast, fermented grain and cellar in quantitative analysis brewing process PC content in mud specifies that PC is mainly derived from pit mud, and PC content is related with the position in pit in pit mud.Fermented grain is certainly The ability that body produces PC is weaker.Yellow water is contacted with fermented grain, and PC is made to enter fermented grain;Fermented grain is distilled after fermentation, and PC enters with fraction In former wine.
Cellar mud microorganisms flora largely influences Luzhou-flavor liquo quality and flavor characteristic.Therefore, it is necessary to On the basis of the microbial physiology biochemical characteristic for clearly producing PC, screen similar to its ecological niche do not produce or the bacterial strain of low yield PC. And by strengthening these bacterial strains in pit mud in order to the reduction PC content from source and guaranteeing liquor flavor quality.
Summary of the invention
To solve the above-mentioned problems, the present invention is screened from white spirit pit mud has obtained three plants and has not produced or low yield pit mud stink The pit mud functional microorganism of Substance P C does not produce Lactobacillus acidipiscis JGn2 of PC respectively, does not produce PC's The Clostridium butyricum JGn6 of Clostridium sporogenes JGn4, low yield PC.
The Lactobacillus acidipiscis JGn2 has been preserved on May 10th, 2016 Chinese common micro- Biological inoculum preservation administrative center, deposit number are CGMCC NO.12432.
The Clostridium sporogenes JGn4 is preserved in China General Microbiological on May 10th, 2016 Culture presevation administrative center, deposit number are CGMCC NO.12433.
The Clostridium butyricum JGn6 is preserved in China General Microbiological on May 10th, 2016 Culture presevation administrative center, deposit number are CGMCC NO.12434.
It is of the invention not produce or the bacterial strain of low yield PC is to screen to obtain from Luzhou-flavor liquor pit mud.
It is of the invention not produce or the bacterial strain of low yield PC has the property that
(1) it does not produce or low yield PC;
(2) acid producing ability is strong;Lactobacillus acidipiscis JGn2 can produce the acetic acid and capric acid of higher concentration; Clostridium sporogenes JGn4 can produce the 4- methvl-pentanoic acid of high level;Clostridium butyricum JGn6 can produce the butyric acid of high level.
(3) after being additionally added to pit mud, it is able to suppress the growth of the bacterial strain belonged to, and to the microorganism structure shadow that other belong to It rings less, obtained pit mud is able to maintain that the normal fermentation of white wine.
A second object of the present invention is to provide it is a kind of control mud stink substances PC composite bacteria agent, the composite bacteria agent with Lactobacillus acidipiscis JGn2, Clostridium sporogenes JGn4 and Clostridium Butyricum JGn6 is major microorganisms.
In one embodiment of the invention, in the composite bacteria agent, Lactobacillus acidipiscis JGn2, Clostridium sporogenes JGn4, Clostridium butyricum JGn6 viable count ratio be (1~ 10): (1~10): (1~10).
In one embodiment of the invention, in the composite bacteria agent, Lactobacillus acidipiscis JGn2, Clostridium sporogenes JGn4, Clostridium butyricum JGn6 viable count ratio be 1:1: 1。
In one embodiment of the invention, the composite bacteria agent is to prepare Lactobacillus acidipiscis The seed liquor of JGn2, Clostridium sporogenes JGn4, Clostridium butyricum JGn6, then according to Volume ratio (1~2): (1~2): the ratio of (1~2) is mixture prepared by seed liquor.
In one embodiment of the invention, in the composite bacteria agent also containing it is any can be applied to fermented food or The bacterial strain, such as bacillus licheniformis, saccharomyces cerevisiae, bacillus subtilis of any kind etc. of person's fermented food preparation.
In one embodiment of the invention, any load that can be used for fermented food is also contained in the composite bacteria agent Body.
Third object of the present invention is to provide the control method of smell substance PC in fermented food a kind of, the method is Composite bacteria agent of the invention or JGn2, JGn4, JGn6 of the invention individually or are in any proportion mixed, are inoculated into In the production process of fermented food, by not producing or the bacterial strain competitiveness of low yield PC substitutes in primary ferment food production system The bacterial strain of PC is produced, and then controls the smell substance PC in fermented food.
In one embodiment of the invention, the fermented food is white wine.
In one embodiment of the invention, the method is by the bacterium solution of composite bacteria agent or of the invention JGn2, JGn4, JGn6 are individually or the mixed bacterium suspension of the bacterium solution and pit mud that mix in any proportion cultivates pit mud in culture medium, And it is inoculated with 1 time during the cultivation process or multiple composite bacteria agent or JGn2, JGn4, JGn6 (individually or in any proportion mixing) Bacterial strain bacterium solution is strengthened, and pit mud mixes bacteria microorganism after being strengthened, and is then further cultured for the mixed bacteria microorganism of pit mud after strengthening and obtains Strengthen pit mud, recycles and strengthen pit mud progress brewed spirit.
In one embodiment of the invention, the control method is by Lactobacillus acidipiscis JGn2, Clostridium sporogenes JGn4, Clostridium butyricum JGn6 prepare bacterial strain bacterium solution simultaneously, Bacterium suspension is mixed with pit mud simultaneously to be cultivated.
In one embodiment of the invention, in the control method, Lactobacillus acidipiscis JGn2, Clostridium sporogenes JGn4, Clostridium butyricum JGn6 are according to inoculum concentration volume ratio (1~2): (1~2): the ratio inoculation of (1~2) prepares bacterial strain bacterium solution.
In one embodiment of the invention, the culture medium is caproic acid bacterium culture medium.
In one embodiment of the invention, the method is specifically: by Lactobacillus acidipiscis JGn2, Clostridium sporogenes JGn4, Clostridium butyricum JGn6 are according to inoculum concentration volume ratio (1~2): (1~2): the ratio inoculation of (1~2) prepares bacterial strain bacterium solution, and bacterium solution and pit mud are then mixed bacterium suspension in caproic acid bacteria Culture medium culture pit mud 1 day, and strengthened again every 3 days inoculation do not produce/low yield PC bacterium solution connects twice, pit mud is mixed after being strengthened Bacteria microorganism;Then, pit mud mixes bacteria microorganism 9 days after fermented and cultured is strengthened in new caproic acid bacterium culture medium, cellar of being strengthened Mud recycles and strengthens pit mud progress brewed spirit.
Fourth object of the present invention is to provide a kind of microbial bacterial agent, and the microbial bacterial agent contains of the present invention Do not produce or the bacterial strain of low yield PC in any one or it is a variety of.
In one embodiment of the invention, the microbial bacterial agent contains of the invention do not produce or the bacterial strain of low yield PC Thallus living cells, of the invention not producing or the present invention of the dry mycelium of the bacterial strain of low yield PC, immobilization of obtaining of freeze-drying Do not produce or the cell of the bacterial strain of low yield PC, of the invention do not produce or the liquid bacterial agent of the bacterial strain of low yield PC, of the invention do not produce Or the solid fungicide of the bacterial strain of low yield PC, or in the form of other are any existing for of the invention do not produce or the bacterial strain of low yield PC.
In one embodiment of the invention, also it can be applied to fermented food containing any in the microbial bacterial agent Or the bacterial strain, such as bacillus licheniformis, saccharomyces cerevisiae, bacillus subtilis of any kind etc. of fermented food preparation.
In one embodiment of the invention, any load that can be used for fermented food is also contained in the microbial bacterial agent Body.
It is not produced or the application of the bacterial strain of low yield PC fifth object of the present invention is to provide described, 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 not produced of the invention or the addition of the bacterial strain of low yield PC To during white wine, grape wine, yellow rice wine or brewing fruit wine.
In one embodiment of the invention, the application is not produced of the invention or low yield PC is added to cellar In mud.
Beneficial effects of the present invention:
The present invention has obtained three plants and has not produced or the pit mud functional microorganism of low yield pit mud stink substances PC, function of the invention After energy microorganism is additionally added to pit mud, it is able to suppress the growth of the bacterial strain belonged to, and the microorganism structure that other belong to is influenced Less, the pit mud obtained is able to maintain that the normal fermentation of white wine but can reduce PC content in white wine.The present invention also provides fermentations The control strategy of smell substance PC in food.
Biomaterial preservation
Lactobacillus acidipiscis JGn2, taxology are named as Lactobacillus acidipiscis, China General Microbiological culture presevation administrative center, deposit number CGMCC are preserved on May 10th, 2016 NO.12432, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
Clostridium sporogenes JGn4, taxology are named as clostridium sporogenes Clostridium Sporogenes is preserved in China General Microbiological culture presevation administrative center on May 10th, 2016, and deposit number is CGMCC NO.12433, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
Clostridium butyricum JGn6, taxology are named as clostridium butyricum Clostridium butyricum, China General Microbiological culture presevation administrative center, deposit number CGMCC are preserved on May 10th, 2016 NO.12434, preservation address are Yard 1, BeiChen xi Road, Chaoyang District, Beijing City 3.
Detailed description of the invention
Fig. 1: pit mud stable structure flora.
Specific embodiment
Culture medium:
Reinforced clostridial medium (gL-1): peptone 10, beef extract 10, yeast extract 3, glucose 5, sodium chloride 5, acetic acid Sodium 3, magnesium sulfate 0.2, ammonium sulfate 0.5, dipotassium hydrogen phosphate 1, potassium dihydrogen phosphate 0.5).
Caproic acid bacterium culture medium (gL-1): yeast extract 5, peptone 5, sodium acetate 6, dipotassium hydrogen phosphate 0.4, magnesium sulfate 0.2, Ammonium sulfate 0.5, calcium carbonate 5, dipotassium hydrogen phosphate 1, potassium dihydrogen phosphate 0.5.Ethyl alcohol 20mL (is added) before inoculation.
Embodiment 1: the separation and identification of cellar mud microorganisms
(1) strain isolation: being suspended from 100mL 0.9%NaCl solution for the fresh pit mud of 5g, after solid medium coating, puts In 37 DEG C of anaerobic culture box stationary culture 3 days;Scribing line obtain single colonie after picking individual colonies in 12mL reinforced clostridial medium, Every piece of plate selects 3 in parallel, cultivates 3 days in 37 DEG C of anaerobic culture box.
(2) detection of strain culturing post-fermentation liquid flavor: fermentation liquid is through 8000rmin-1Supernatant is taken after centrifugation 10min 8mL measures it through head space micro-extraction-gas chromatography-mass spectrography (HS-SPME-GC-MS) technology after adding 3g sodium chloride to be saturated In volatile flavor.
GC-MS condition:
Extraction conditions: DVB/CAR/PBDS extracting head extracts 45min, and extraction temperature is 45 DEG C.
GC condition: 250 DEG C of injector temperature, carrier gas He, flow velocity 2mLmin-1, Splitless injecting samples, chromatographic column CP-Wax (60m×0.25mmi.d.×0.25μm,J&W Scientific).Temperature program when detection are as follows: 50 DEG C of constant temperature 2min, with 6 ℃·min-1Speed be warming up to 230 DEG C, keep 15min.
MS condition: EI ionization source, electron energy 70eV, 230 DEG C of ion source temperature, 35.00~350amu of scanning range.Matter Spectrum analysis database source is in NIST05a.L (Agilent company).
(3) bacterial strain 16S rRNA is identified: being obtained cell precipitation after fermentation liquid centrifugation, is extracted genome, measures conservative region 16S rRNA Sequence Identification kind, the primer sequence of PCR are 27F (5 '-AGAGTTTGATCCTGGCTCAG-3 ') and 1492R (5′-GGTTACCTTGTTACGACTT-3′)。
It separates to PC microorganism is produced in pit mud, is isolated by the judgement of preliminary colonial morphology to 34 plants of bacteriums, measurement It is compared after 16S rRNA gene order with Genbank database, determines kind, obtained several plants and do not produced or the bacterial strain of low yield PC, The results are shown in Table 1.
The separation and identification of 1 microorganism of table
Note: ND is indicated lower than detection limit;"-" expression does not compare
Wherein Lactobacillus acidipiscis JGn2 has been preserved on May 10th, 2016 Chinese common micro- Biological inoculum preservation administrative center, deposit number are CGMCC NO.12432.Clostridium sporogenes JGn4, China General Microbiological culture presevation administrative center is preserved on May 10th, 2016, deposit number is CGMCC NO.12433. Clostridium butyricum JGn6 is preserved in China General Microbiological culture presevation management on May 10th, 2016 Center, deposit number are CGMCC NO.12434.
Embodiment 2: the type and content measurement of the production flavor substance of pit mud functional microorganism
By the pure bacterium CGMCC NO.12432 of function isolated in pit mud, CGMCC NO.12433, CGMCC NO.12434 It is seeded to reinforced clostridial medium (gL respectively-1): peptone 10, beef extract 10, yeast extract 3, glucose 5, sodium chloride 5, vinegar Sour sodium 3, magnesium sulfate 0.2, ammonium sulfate 0.5, dipotassium hydrogen phosphate 1, potassium dihydrogen phosphate 0.5.The inspection of strain culturing post-fermentation liquid flavor Survey: fermentation liquid is through 8000rmin-1Supernatant 8mL is taken after centrifugation 10min, after adding 3g sodium chloride to be saturated, through head space micro-extraction-gas Phase chromatograph-mass spectrometer coupling (HS-SPME-GC-MS) technology measures volatile flavor therein.
Extraction conditions: DVB/CAR/PBDS extracting head extracts 45min, and extraction temperature is 45 DEG C.
GC condition: 250 DEG C of injector temperature, carrier gas He, flow velocity 2mLmin-1, Splitless injecting samples, chromatographic column CP-Wax (60m×0.25mmi.d.×0.25μm,J&W Scientific).Temperature program when detection are as follows: 50 DEG C of constant temperature 2min, with 6 ℃·min-1Speed be warming up to 230 DEG C, keep 15min.MS condition: EI ionization source, electron energy 70eV, ion source temperature 230 DEG C, 35.00~350amu of scanning range.Mass spectral analysis database source is in NIST05a.L (Agilent company).In conjunction with Mass spectrogram and retention time substance are qualitative, and carry out accurate quantitative analysis by the characteristic ion of various substances.
The results are shown in Table 2.
2 flavor substance content (unit mg/L) of table
The pit mud functional microorganism of above-mentioned three kinds of low yields/do not produce PC can produce more rich aroma substance, including 9 kinds Acid compounds, 6 kinds of alcohol compounds, 2 kinds of ketone compounds and 4 kinds of aldehyde compounds.Wherein, acids and alcohol compound be most Abundant, the two accounts for about the 45% of total type.Wherein, the less Lactobacillus of pit mud stink substances PC is produced Acidipiscis JGn2 can produce the acetic acid and capric acid of higher concentration.Clostridium butyricum JGn6 can produce higher contain The butyric acid of amount.Butyric acid is the precursor of caproic acid synthesis.The corresponding ethyl butyrate of above-mentioned acids and ethyl hexanoate are Luzhou-flavor liquo wind Main body aroma-producing substance in taste.Clostridium sporogenes JGn4 also contributes pyrazine substance more abundant, such object Matter imparting white wine is similar to bake fragrance.In addition, this three plants of functional microorganism common metabolic fragrance of a flower/alcohols of fruit perfume (or spice) class, esters The aldehyde material of substance and green grass fragrance.The fragrance and promotion organoleptic quality of horn of plenty white wine provide material base.
Embodiment 3: the physio-biochemical characteristics of pit mud functional microorganism
The physio-biochemical characteristics of pit mud functional microorganism are compared, the results are shown in Table 3.
The physio-biochemical characteristics of 3 bacterial strain of table
Note:aGemma position end raw (T) or proximal end are raw (ST);ND: uncertain;+: indicate microorganism can using the carbon source or The metabolin can be generated;: indicate that microorganism cannot utilize the carbon source or can generate the metabolin.
Embodiment 4: the application of bacterial strain
After CGMCC NO.12432, CGMCC NO.12433, CGMCC NO.12434 activation, respectively connect according to 0.1% Kind is measured to be inoculated in caproic acid bacterium culture medium with the mixed bacterium suspension of pit mud, is cultivated pit mud 1 day, and reinforcing was inoculated with not again every 3 days Production/low yield PC bacterium solution (i.e. the mixed bacteria liquid of CGMCC NO.12432, CGMCC NO.12433, CGMCC NO.12434, respectively 0.1%) it connects twice, pit mud mixes bacteria microorganism after being strengthened.Then, after fermented and cultured is strengthened in new caproic acid bacterium culture medium Pit mud mixes bacteria microorganism 9 days, the sample after being strengthened.
Sample carries out GC-MS detection and MiSeq genome 16S rDNA sequencing, pit mud after strengthening with observation respectively after fermentation The metabolism of microorganism flavor and Bacterial community situation of change.Control sample (sample before being transformed) does not add as of the invention The sample of CGMCC NO.12432, CGMCC NO.12433, CGMCC NO.12434 obtained according to the method described above.Table 4 is function It can bacterial strain reinforcing front and back PC content and microorganism structure change.The results show that do not produced using of the invention these/low yield PC Function stem strengthens pit mud, and PC content reduces 73.3%, and it is smelly to significantly reduce pit mud.Using obtain not produce PC micro- Biology building cellar mud microorganisms homeostasis system, it is smelly fundamentally to solve pit mud.Meanwhile as shown in Figure 1, using the above method pair After pit mud is strengthened, (hydrolytic bacteria group's fermentative microflora, mutual nutrition produce acetic acid flora and methanogen to the big pit mud stable state flora of the three of building Group) can use several kinds of carbon source, ultimately produce neutral products methane, the system can export simultaneously a variety of flavor substances (including C2-C10 short chain fatty acids), there is certain robustness (anti-interference ability).
4 function stem of table strengthens front and back PC content and microorganism structure change
The main acids for the pit mud for strengthening front and back is detected, the results are shown in Table 5.The results show that the cellar after strengthening The main organic acid caproic acid of mud has been increased to 4 times, and butyric acid content has been increased to 3.42 times.
The transformation of the table 5 front and back main organic acid concentration of pit mud compares
The pit mud that this method is obtained is used for brewed spirit, and the pit for comparing pit mud transformation front and back produces the main wind of wine Taste substance, as shown in table 6, ester content increase 50% or so, and acid content increases 44 times or so.
Wine main flavor concentration is produced in the pit of the transformation of table 6 front and back to compare
The pit mud strengthened through CGMCC NO.12432, CGMCC NO.12433 and CGMCC NO.12434, PC content reduce 73.3%, main organic acid concentration is significantly improved.Cellar mud microorganisms are constructed from steady using the obtained PC microorganism that do not produce It is smelly fundamentally to solve pit mud for state system.Obtained pit mud is used for the brewing of white wine, not only effectively reduces the content of PC, and And stability is good, difference is small between each batch, is able to maintain that being normally carried out for liquor fermentation, the flavor and taste of obtained white wine are bright Seem to improvement.
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 (8)

1. one plant of pit mud functional microorganism for not producing pit mud stink substances 4- methylphenol (PC), which is characterized in that the pit mud Functional microorganism is Clostridium sporogenes JGn4;Wherein, the Clostridium sporogenes JGn4, China General Microbiological culture presevation administrative center, deposit number CGMCC are preserved on May 10th, 2016 NO.12433。
2. a kind of microbial bacterial agent, which is characterized in that it is not producing for claim 1 that the microbial bacterial agent, which contains major microorganisms, The pit mud functional microorganism of pit mud stink substances 4- methylphenol.
3. microbial bacterial agent according to claim 2, which is characterized in that the microbial bacterial agent contains claim 1 The Clostridium sporogenes that the thallus living cells of Clostridium sporogenes JGn4, freeze-drying obtain Cell, the Clostridium of the dry mycelium of JGn4, the Clostridium sporogenes JGn4 bacterial strain of immobilization The solid fungicide of the liquid bacterial agent of sporogenes JGn4, Clostridium sporogenes JGn4 bacterial strain, or with it Clostridium sporogenes JGn4 existing for his any form.
4. microbial bacterial agent according to claim 2, which is characterized in that in the microbial bacterial agent also containing it is any can be with Bacterial strain applied to any kind prepared by fermented food or fermented food.
5. microbial bacterial agent according to claim 2, which is characterized in that can also be used containing any in the microbial bacterial agent In the carrier of fermented food.
6. microbial bacterial agent according to claim 2, which is characterized in that also contain in the microbial bacterial agent Lactobacillus acidipiscis JGn2 and/or Clostridium butyricum JGn6;
Wherein, the Lactobacillus acidipiscis JGn2 has been preserved on May 10th, 2016 Chinese common micro- Biological inoculum preservation administrative center, deposit number are CGMCC NO.12432;The Clostridium butyricum JGn6, China General Microbiological culture presevation administrative center, deposit number CGMCC are preserved on May 10th, 2016 NO.12434。
7. the application of the pit mud functional microorganism described in claim 1 for not producing pit mud stink substances 4- methylphenol, feature It is, the application, is produced applied to brewed wine, Spirit.
8. application according to claim 7, which is characterized in that the application is will not produce pit mud stink substances 4- methylbenzene The pit mud functional microorganism bacterial strain of phenol is added to during white wine, grape wine, yellow rice wine or brewing fruit wine.
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