CN107475159B - Bacillus subtilis and its application in Sauce flavor white wine - Google Patents
Bacillus subtilis and its application in Sauce flavor white wine Download PDFInfo
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Abstract
The invention discloses a kind of bacillus subtilis for brewed spirit, application of the microbial inoculum and combinations thereof in fermentation white wine.Bacillus subtilis provided by the invention has the ability for generating the liquor flavors ingredient such as Sauce flavor and high yield propionic acid, while not only having had proteinase activity high, but also the characteristic strong with high temperature resistant, acidproof and resistance to ethyl alcohol ability.The bacterial strain can be used for fermenting and producing white wine, improves white wine propionic acid content, improves distillation yield and liquor flavor.
Description
Technical field
The invention belongs to brewed spirit fields, relate in particular to a kind of bacillus subtilis and the bacillus subtilis
Application of the bacterium in liquor production.
Background technique
In the techniques such as koji-making, accumulation and fermentation in sauce incense liquor production process can enriched bacillus, they are
Form basis and the key of Maotai-flavor liquor flavor components.Wherein bacillus cereus, pantothenic acid branch bacillus, huge gemma bar
Bacterium, bacillus subtilis and bacillus licheniformis these fifth types bacterial strain be the fragrant function bacterium of the main production of Maotai-flavor liquor (Zhong Shuxia,
Equal modern food science and technology, 2017,33 (4): 89-95).Bacillus is most in Maotai-flavor yeast finished product song, the kind of bacillus
Class and quantity will directly determine bent superiority and inferiority, and then influence the style and features of fermented grain attenuation degree and wine.Paste flavor fermented grain has heap
Accumulated temperature degree is high, pH value is low and the characteristics of containing 5% or so ethyl alcohol, therefore, the gemma with high temperature resistant, resistance to high ethano concentration and acidity
Bacillus will have apparent survival advantage.
Bacillus subtilis (Bacillus subtilis) is the important mushroom for producing amylase and protease, is also simultaneously
One of the main producing strains of flavor substance in Maotai-flavor liquor.In white wine many fragrance matters derive from protein, protein and
The effect of its enzyme is most important to the flavor effect of white wine (Nie Huifang, waits brewing science and technology, 2015,12:41-44).Lin Qun etc.
(brewing science and technology, 2013,11:30-32) isolates and purifies to obtain 1 bacillus subtilis from liquor production yeast and fermented grain,
Using sorghum as fermentation substrate, the GC-MS analysis qualitative detection for producing Flavor substance from strain fermentation goes out more than 30 kinds of fermentating metabolisms and produces
Object, including Pyrazine, acids, esters, alcohols, ketone and aromatic compound etc..3-hydroxy-2-butanone and ammonia are microbial fermentation production
The precursor substance of Tetramethylpyrazine, the bacterial strain that high proteinase yield is added in brewing system being capable of precursor in increase system
Ammonia, to improve the content of Tetramethylpyrazine in white wine (Xu Yan waits brewing science and technology 2011,7:37-40).Bacillus makes
With can effectively improve total acid in wine base, total ester and each main flavor content (Zeng Tingting, waits brewing science and technology, 2012,7:
32-34)。
Organic acid is the important taste compound of white wine and the precursor of flavor component.Their molecular weight is bigger, mouth
Taste is softer.The showing as hearing in fragrant flavor feature of propionic acid has tart flavour, and import is soft, and (Shen Yifang, liquor production technology are complete with micro-puckery
Book, 2013, p768).It is organic containing 6 kinds of acetic acid, propionic acid, isobutyric acid, butyric acid, isovaleric acid and lactic acid etc. in the white spirit wine of Hengshui
Acid.Isolated 69 plants of acid-producing bacterias in its yeast, fermented grain, wherein 1 plant of representative acetic acid bacteria, 4 plants of lactic acid bacteria, propionic acid
2 plants of bacterium (Sun Pengfei waits China to make, 2016,35 (3): 36-40).Lactic acid and the excessively high wind for influencing white wine of content of ethyl lactate
Taste or even distillation yield.Bacterium acidi propionici can generate propionic acid using lactic acid as carbon source through fermentation, have the effect of " increasing the third drop cream ", base liquor
Style protrusion, (Xin Xiuming waits food industry scientific and technological, 2012,6,240-243) good in taste.Propionic acid and its derivative are the world
Upper generally acknowledged Atoxic bacteriostat, has good anticorrosive mildewproof to act on, and to people and animals in terms of cereal warehousing and fresh feed preservation
Substantially harmless, it is also nutritious to animal, thus as good food preservative, cereal and feed addictive, herbicide
(Xu Hong waits food and fermentation industries, 1997,23 (6): 62-65) is widely applied by people.
Chinese patent (CN 102703358A) discloses a kind of preparation method of bacillus high-temperature daqu.Chinese patent
(CN 103865833A) discloses a kind of preparation method of thermophilic bacillus subtilis microbial agent.Chinese patent (CN
It 101445786A) discloses a plant height and produces the bacillus subtilis of Tetramethylpyrazine and its side of fermentation producing tetramethylpyrazine
Method.Chinese patent (CN 104745503A) discloses a kind of bacillus subtilis and its application in light flavor type white wine.China
Patent (CN 104388284A) discloses the method for improving ethyl propionate content in special aromatic white spirit.
On the one hand amino acid content can be improved in high proteinase yield live strain during liquor fermentation, to increase in white wine
Flavor substance, the yield and quality of white wine on the other hand also can be improved.The separation screening of high proteinase yield bacterium, facilitates sauce
The research of high proteinase yield microorganism in fragrant yeast is of great significance to the paste flavor yeast and sauce incense liquor of producing excellent.
The bacillus subtilis strain of fermentation white wine reported at present only relates to produce the heat resistance aspect of Tetramethylpyrazine and bacterial strain,
The disadvantages of there are bacterial strains to have a single function, comprehensive performance is insufficient.There has been no having both, protease production is high, Sauce flavor is prominent at present
Out, tolerance is good, produces the report that propionic acid etc. liquor flavors physical capacity strong bacillus subtilis and its applied in white wine.Change
Kind white wine quality and flavor, meet the market requirement always white wine manufacturer problems faced.
Inventive technique content:
It is an object of the present invention to provide a bacillus subtilis strain, which produces that Sauce flavor ability is good, egg
White enzymatic activity is high, it is strong to produce propionic acid ability, has stronger tolerance, can be used for fermenting and producing white wine.
It is a further object to provide the microbial inoculums containing bacillus subtilis.
It is a further object to provide the methods of fermentation of bacillus subtilis white wine.
It is also another object of the present invention to provide the methods of preparation bacillus subtilis solid fungicide.
It is also another object of the present invention to provide a kind of application of bacillus subtilis and microbial inoculum in fermentation white wine.
The invention discloses one plant of new bacillus subtilises, which is characterized in that the bacterial strain is bacillus subtilis
S10Bacillus subtilis S10 is preserved in specified preservation mechanism, State Intellectual Property Office-Chinese Typical Representative culture and protects
Hiding center, deposit number are CCTCC NO:M2017466, and preservation date is on September 1st, 2017.China typical culture collection
Center abbreviation CCTCC is located at Wuhan City, Hubei Province Wuhan University in the school, postcode 430072, phone: 027-68752319,
Email:cctcc@whu.edu.cn。
Bacillus subtilis S10 bacterial strain is to obtain from distillery, Hubei Province fermented grain through separation screening.The bacterial strain just like
Lower feature: bacillus subtilis S10 fermentation in producing protease culture medium produces basic protein enzyme activity up to 241.69 (U/mL).The bacterium
Strain has stronger tolerance to ethyl alcohol, organic acid and high temperature.The bacterial strain can ferment sorghum saccharified liquid produce propionic acid content reach
305.4mg/L produces 3-hydroxy-2-butanone content and reaches 254.5mg/L.The bacterial strain solid state fermentation sorghum can generate pure Sauce flavor, and addition should
The white spirit original wine paste flavor of bacterial strain brewing is prominent, in good taste.
Bacillus subtilis S10 plant shape state is characterized in: bacterium colony is creamy white, and surfacing is coarse, relatively dry, and edge is not
Neatly, cell is rod-short, produces gemma.
S10 plants of biochemical characters of bacillus subtilis are: using glucose, sucrose, mannitol, D- xylose, starch.It can be
It is grown in beef-protein medium containing 7%NaCl.
S10 plants of growth characteristics of bacillus subtilis are: streak inoculation on beef extract-peptone plate, respectively at 37 DEG C to 45
Equal well-grown is cultivated at DEG C.
S10 plants of 16S rDNA gene sequencings of bacillus subtilis are shown: S10 plants of bacillus subtilis and Bacillus
The homology of 10071 bacterial strain of subtilis CICC is greater than 99%.
The invention also discloses the microbial inoculums of S10 containing bacillus subtilis a kind of, which is bacillus subtilis
S10 plants of bacterium and its extracellular products and fermentation substrate.
Of the invention also discloses the application of bacillus subtilis S10 and its microbial bacterial agent in fermentation white wine.
Of the invention also discloses application of the bacillus subtilis S10 antimicrobial composition in fermentation white wine.
The reference strains used in the present invention are General Microbiological Culture, wherein bacillus subtilis CICC10071
It publishes in Chinese industrial Microbiological Culture Collection administrative center catalogue, in open state, scientific worker can be to the strain
Collection is asked for.Saccharomyces cerevisiae CCTCC AY92003 is common General Microbiological Culture, publishes and protects in Chinese Typical Representative culture
Hiding center catalogue, in open state, scientific worker can ask for China typical culture collection center.
Microbial inoculum disclosed by the invention is solid-state or liquid.Preferential, S10 gemma bacillus agent disclosed by the invention is solid
State.
The invention also discloses the methods of preparation solid-state microbial inoculum, include the following steps:
(1) S10 actication of culture: being inoculated in beef extract-peptone slant medium with one ring S10 strain of oese picking,
It sets in 37 DEG C of constant incubators and cultivates 20-24h；
(2) preparation of seed liquor: the activated S10 bacterial strain access of one ring of picking is equipped with the beef extract-peptone liquid of 100mL
In body culture medium, 37 DEG C, 180r/min shaking table culture it is spare for 24 hours；
(3) solid fermentation is preparation of culture medium: press wheat bran 40%, rice bran 30%, dregs of beans 30%, material: water=1:1, pH naturally,
It is uniformly mixed, is configured to solid fermentation culture medium, and be sub-packed in plastics square box resistant to high temperature, it is cooling after 121 DEG C of sterilizing 30min
For use；
(4) solid fungicide culture: preparatory cultured S10 seed liquor is accessed to solid fermentation culture by 5-10% inoculum concentration
It in base, is uniformly mixed in 30 DEG C of constant incubators of postposition and cultivates 2 days, every 12h stirring is primary；
(5) dry and crush: the S10 solid culture that above-mentioned fermentation finishes is placed in 55 DEG C of baking ovens to be dried to moisture content low
In 15%, after dry culture is crushed, sieved with 100 mesh sieve with Universalpulverizer, it is prepared into solid powdery microbial inoculum and is packed into
In the polybag of sealing, preservation in dry, shady place or fridge freshness retaining layer is placed；Wherein, S10 bacillus in microbial inoculum sample
Viable count reaches cfu/ grams of 40-300 hundred million.
(6) mixing is with packaging: after saccharomyces cerevisiae CCTCC AY92003 and S10 is mixed in viable count ratio=10:1 ratio
Wine brewing antimicrobial composition is made in sealing after polybag.
Advantages of the present invention:
1, bacillus subtilis S10 plants provided by the invention are one plant of new bacterial strains, the bacterial strain have very strong high temperature resistant,
The acidproof and growth ability of resistance to ethyl alcohol.The bacterial strain remains to well-grown at 45 DEG C, compared with 37 DEG C of cultivation temperatures, relative growth
Amount reaches 62.6%, higher than reference culture CICC1007 by 19.6%.The concentration of alcohol of S10 bacterial strain tolerable 6% or so, compared with standard
Bacterial strain alcohol-tolerant ability is strong.The acidic growth environment that the tolerable pH of S10 bacterial strain is 4.5 is strong compared with reference culture acid-fast ability.
2, bacillus subtilis S10 plants of fermentation sorghum Sauce flavors are prominent, and sensory evaluation effect is good.
3, bacillus subtilis S10 plants of protease productions are high, and it is strong to produce Sauce flavor precursor substance 3-hydroxy-2-butanone ability.S10
The ability for producing protease and 3-hydroxy-2-butanone is higher than reference culture CICC1007 by 324.7% and 18.3% respectively.
4, the flavor substances ability such as bacillus subtilis S10 plants of production propionic acid is strong.S10 produces propionic acid, caproic acid ability respectively than mark
Quasi- bacterial strain CICC1007 high 60.7% and 29.8%.S10 plants of production health factor ɑ-linolenic acids and flavor substance bata-phenethyl alcohol simultaneously
Content than standard bacteria plant height.
5, the compound wine brewing microbial inoculum distillation yield for adding bacillus subtilis S10 is high, and fermentation liquor flavor sense organ is good, and mouthfeel is soft
With.
It is the i.e. bacillus subtilis S10 plants high temperature resistants with higher compared with reference culture CICC1007, resistance to ethyl alcohol, acidproof
Ability and the ability for producing liquor flavor component precursor substance 3-hydroxy-2-butanone, proteinase activity is high, and Sauce flavor is prominent, and has height
Produce the characteristic of propionic acid, fermentation white wine feature in good taste.
Detailed description of the invention
Fig. 1 is the systematic growth tree graph of bacillus subtilis S10 bacterial strain.
Specific embodiment
Below with reference to specific embodiment, the present invention is further explained.It should be appreciated that these embodiments are merely to illustrate
The present invention, and cannot limit the scope of the invention.
Embodiment
The separation and identification of 1 bacillus subtilis S10 bacterial strain of embodiment
The separation of 1.1 bacillus subtilis S10 bacterial strains: fermented grain sample is acquired from distillery, Hubei Province, weighs fermented grain sample
Product 10g is added in the triangular flask equipped with 90mL sterile water, and shaken well is placed in 80 DEG C of thermostat water baths and handles 20min, is shaken
After even dilution, tri- dilution gradients of 10-3,10-4,10-5 are taken to be applied on beef-protein medium, each dilution gradient
3 repetitions are done, sets and cultivates 36h in 37 DEG C of constant incubators, single colonie is obtained through further scribing line and isolates and purifies preservation for egg
White enzyme assay.By 25 bacillus for being isolated from fermented grain and 1 plant of standard bacillus subtilis CICC10071 bacterial strain through ox
It is seeded in beef extract-peptone fluid nutrient medium after the activation of meat extract plate, 37 DEG C, 170rpm shaking table culture 12h, by culture
Seed liquor is seeded in white enzyme fermentation culture medium of laying eggs according to 3% inoculum concentration, 37 DEG C, 170rpm culture 48h, centrifugation
(4000r, 10min), taking supernatant is sample to be tested, measures each bacterial strain fermentation liquor alkalinity egg according to national standard folin-phenol method
White enzyme activity, the 3 bottles of repetitions of each bacterial strain.Each Bacillus strain protease activity measurement result is as shown in table 1.Using the method through sieving
1 plant is obtained after choosing can generate the bacillus compared with high protein enzymatic activity, strain number S10, in white enzyme fermentation training of laying eggs
The alkaline protease activity supported in base reaches 241.69U/mL.
Above-mentioned Bacillus strain is seeded to 37 DEG C of glucose proteins peptone fluid nutrient medium, 170rpm culture for 24 hours respectively,
It is seeded in wheat solids culture medium according to 10% inoculative proportion, and blank group (not being inoculated with wheat solids culture medium) is set,
Cultivated according to 35 DEG C → 45 DEG C → 55 DEG C heatings, each temperature section respectively cultivates 2d, referring to color, fragrance, viscosity the fragrant sense organ of song
Evaluation criteria is filtered out with the bacillus for producing paste flavor function.The 3 bottles of repetitions of each bacterial strain, comprehensive score are to be repeated 3 times test
Comprehensive evaluation result.Wherein 6 bacillus and reference culture fermentation produce Sauce flavor Analyses Methods for Sensory Evaluation Results and are shown in Table 2.From table 2
It can be seen that the higher bacterial strain S10 production paste flavor ability of basic protein enzyme activity is obviously strong compared with other bacterial strains, show the production sauce of bacillus
Fragrant ability has certain correlation with its height for producing protease activity.
1 B. amyloliquefaciens alkaline protease activity testing result of table
The different bacillus of table 2 produce paste flavor ability comparison result
The present inventor is by one plant of separation S10 plants new of bacillus subtilis (Bacillus subtilis S10) preservation
In preservation mechanism-China typical culture collection center that State Intellectual Property Office specifies, deposit number is CCTCC NO:
M2017466, preservation date are on September 1st, 2017.It is military to be located at Hubei Province by China typical culture collection center abbreviation CCTCC
Wuhan University, Chinese city in the school, postcode 430072, phone: 027-68752319, Email:cctcc@whu.edu.cn.
The identification of 1.2 S10 plants of bacillus subtilises: through observation of morphological characteristics, physiological and biochemical test and 16S rDNA base
Because the sequencing results identify S10.In authentication, this experimental selection bacillus subtilis Bacillus subtilis
CICC10071 bacterial strain is as control strain.Bacillus subtilis is common General Microbiological Culture, is published in Chinese industrial strain
In collection catalogue, in open state, scientific worker can ask for collection.
1.2.1 bacillus subtilis S10 plant shape state is characterized in: bacterium colony is creamy white, and surfacing is coarse, opaque, side
Edge is irregular, and cell is rod-short, produces gemma.
1.2.2 Phylogenetic identification is carried out by the analysis of 16S rDNA sequence:
The bacterium bacterial strain isolated and purified is inoculated into beef extract-peptone fluid nutrient medium, at 37 DEG C, 170rpm
Under conditions of shaken cultivation 16h, recycle bacterial genomes kit extract genomic DNA.
It takes kit to extract after obtaining DNA of bacteria, by PCR amplification 16S rDNA, configures the reaction system of 50 μ L, carefully
The 16S rDNA primer of bacterium is 27F (5'-GAGTTTGATCCTGGCTCAG-3') and 1492R (5'-
ACGGCTACCTTGTTACGACTT-3')。
PCR cycle program are as follows: 94 DEG C of initial denaturations 5min, 94 DEG C of 50s, 54 DEG C of 50s, 72 DEG C of 90s, 30 circulations；72 DEG C are prolonged
Stretch 10min.It expands obtained bacteria PCR product to send to the sequencing of Jin Weizhi Biotechnology Co., Ltd, the length of sequencing exists
1400bp or so, sequencing result manually proofread splicing using BioaEdit software sequences map, and spliced sequence is in NCBI core
Homologous sequence search comparison is carried out in sequence databank.According to homologous sequence search result, it is closer to choose test strain relationship
Type strain 16S rDNA sequence region sequence, take adjacent method phylogenetic tree construction with MEGA software.
Experiment obtains the gene order of 1042bp, and homologous sequence search is carried out in Genbank GenBank,
The homology of S10 and Bacillus subtilis CICC10071 are more than 99%, meet fixed same of Kuttzman&Robnett
Different strains are shown in the standard that difference is no more than 1% in kind.Fig. 1 is the phylogenetic tree done according to 16S rDNA sequence.
1.2.3: S10 plants of bacillus subtilis of bio-chemical characteristics measurement result: being shown in Table 3.According to 16S rDNA sequence
Analyze result and bio-chemical characteristics measurement result, reference literature report (east show pearl, Cai Miaoying common bacteria system identification hand
Beijing volume: Science Press, 2001,267-295.), it can determine whether that S10 bacterium is bacillus subtilis.
The bio-chemical characteristics result of S10 plants of 3 bacillus subtilis of table
| Measurement item | As a result | Measurement item | As a result |
| Catalase | + | Glucose | + |
| Anaerobic growth | ? | Xylose | + |
| Methyl red test | + | Mannitol | + |
| Nitrate reduction | + | Sucrose | + |
| 45 DEG C of growths | + | Glycerol | ? |
| PH 5.7 is grown | + | Decompose casein | + |
| 7%NaCl growth | + | Starch Hydrolysis | + |
| Utilize citrate | + | Glucose fermentation produces gas | ? |
2 bacillus subtilis S10 resistance capacity comparative analysis of embodiment
2.1 S10 plants of bacillus subtilises are compared with 1007 alcohol-tolerant ability of reference culture CICC
Containing concentration of alcohol (v/v) be respectively 2% by the access of the seed liquor of bacterial strain S10 and reference culture CICC1007,
4%, in 6%, 8% beef extract-peptone fluid nutrient medium, 37 DEG C are placed in, after 170rpm shaking flask culture for 24 hours, bacterium solution dilution 10
OD is measured with spectrophotometric after times600Value.As shown in table 4, in the culture medium for being added to 6% (v/v) ethyl alcohol, S10 still grow compared with
It is good, but CICC1007 bacterial strain is not grown then, shows that the ability of the resistance to ethyl alcohol of S10 is apparently higher than CICC1007 bacterial strain.
S10 plants of 4 bacillus subtilis of table is compared with reference culture CICC1007 alcohol-tolerant ability
Note: "-" expression is not grown
2.2 S10 plants of bacillus subtilises are compared with reference culture CICC1007 acid-fast ability
Use acetic acid: lactic acid=1:1 adjusts LB liquid medium pH to 4.0,4.5,5.0,5.5 respectively.By bacterial strain S10 and
The seed liquor of reference culture CICC1007 be transferred to respectively pH be respectively 4.0,4.5,5.0,5.5 LB liquid medium in, set
In 37 DEG C, the culture of 170rpm shaking flask for 24 hours, dilution 10 times after survey OD600Value.As can be seen from Table 5, bacillus subtilis S10 exists
Still there is certain growth in the LB culture medium of pH4.5, and reference culture CICC1056 is not grown.The S10 plants of LB culture mediums in pH5.0
In grown preferably, and CICC1056 plant only have faint growths.Show that S10 acid-fast ability is apparently higher than CICC1007 bacterial strain.
S10 plants of 5 bacillus subtilis of table is compared with reference culture CICC1007 acid-fast ability
Note: "-" expression is not grown
2.3 bacillus subtilis S10 high temperature resistant growth ability measures
Two kinds of bacillus subtilis seed liquors of culture are seeded to the training of beef extract-peptone liquid respectively by same ratio
It supports in base, 170rpm shaking table culture for 24 hours, measures OD at 37 DEG C, 45 DEG C and 50 DEG C respectively600Value.Testing result is shown in Table 6, as a result
Show that S10 bacterial strain remains to normal growth at 45 DEG C, there is very strong heat-resisting quantity, compared with the increment that 37 DEG C are cultivated, 45
For relative growth yield at DEG C up to 62.6%, heat-resisting ability is better than reference culture CICC1007.
The high temperature resistant growth ability measurement result of 6 S10 of table and reference culture CICC1007
Embodiment 3, bacillus subtilis S10 bacterial strain pure-blood ferment flavor components compare detection
Bacillus subtilis S10 and reference culture CICC1007 are inoculated with sorghum saccharified liquid respectively, according to white wine of liquid
Fermentation condition carries out pure-blood ferment test, carries out gas chromatographic analysis to karusen distillation sample after fermentation, the results are shown in Table
7.As can be seen from Table 7, S10 plants produce 3-hydroxy-2-butanones, the ability of propionic acid and caproic acid respectively than CICC1007 plant height 18.3%,
60.7% and 29.8%.
Two bacillus are inoculated in respectively in wheat solids culture medium and are fermented, GC-MS points of tunning
It analyses the result shows that (table 8): liquor flavor substance isovaleric acid that S10 plant fermentations generate, bata-phenethyl alcohol, myristic acid and having healthy
The content of the alpha-linolenic acid of function is all remarkably higher than reference culture CICC1007.
S10 plants of 7 bacillus subtilis of table and CICC1007 pure-blood ferment result (mg/L)
The GC-MS of 8 liang of bacillus tunnings of table analyzes result (relative amount/%)
The preparation method of 4 S10 bacillus of embodiment wine brewing microbial inoculum and combinations thereof
It carries out in accordance with the following steps:
(1) S10 actication of culture: being inoculated in beef extract-peptone slant medium with one ring S10 strain of oese picking,
It sets in 37 DEG C of constant incubators and cultivates 20-24h.
(2) preparation of seed liquor: the activated S10 bacterial strain access of one ring of picking is equipped with 100mL containing 0.2% sodium sulfite
In beef extract-peptone fluid nutrient medium, 37 DEG C, 180r/min shaking table culture it is spare for 24 hours.
(3) solid fermentation is preparation of culture medium: press wheat bran 40%, rice bran 30%, dregs of beans 30%, material: water=1:1, pH naturally,
It is uniformly mixed, is configured to solid fermentation culture medium, and be sub-packed in plastics square box resistant to high temperature, it is cooling after 121 DEG C of sterilizing 30min
For use.
(4) solid fungicide culture: preparatory cultured S10 seed liquor is accessed to solid fermentation culture by 5-10% inoculum concentration
It in base, is uniformly mixed in 30 DEG C of constant incubators of postposition and cultivates 2 days, every 12h stirring is primary.
(5) dry and crush: the S10 solid culture that above-mentioned fermentation finishes is placed in 55 DEG C of baking ovens to be dried to moisture content low
In 15%, after dry culture is crushed, sieved with 100 mesh sieve with Universalpulverizer, it is prepared into solid powdery microbial inoculum and is packed into
In the polybag of sealing, preservation in dry, shady place or fridge freshness retaining layer is placed.Sampling surveys number method using dilution plate and detects sample
The viable count of S10 bacillus reaches cfu/ grams of 40-300 hundred million in product.
(6) mix and packaging: will containing S10 plant solid fungicide fill polybag after sealing after the wine brewing containing single culture is made
Microbial inoculum；Wine is made in sealing after polybag after saccharomyces cerevisiae CCTCC AY92003 and S10 is mixed in viable count ratio=10:1 ratio
Wine antimicrobial composition.
Application of 5 S10 of the embodiment wine brewing microbial inoculum in liquid liquor fermentation
Saccharomyces cerevisiae AY92003 and bacillus subtilis S10 wine brewing microbial inoculum is pressed into 1:0,10:1,1:1,1:10 and 0 respectively:
1 bacterium number carries out liquid liquor fermentation than combined inoculation sorghum saccharified liquid.Gas-chromatography point is carried out to fermentation liquid after fermentation
Analysis, the results are shown in Table 9.As can be seen from the table, when saccharomyces cerevisiae AY92003 is mixed with bacillus subtilis S10 bacterium number by 10:1
When fermentation, in fermentation liquid ethyl alcohol, ethyl acetate, vinegar drone, propionic acid, caproic acid equal size highest.Show to add one in saccharomyces cerevisiae
After the S10 wine brewing microbial inoculum of certainty ratio, there is apparent facilitation for improving yeast of white wine and liquor flavor content of material.
9 saccharomyces cerevisiae AY92003 of table and S10 different proportion mixed fermentation interpretation of result (mg/L)
6 S10 of embodiment wine brewing combination microbial inoculum is improving the application in yeast of white wine and wine quality
6.1 test method
By, according to 200kg/ group stacking, control group is inoculated with high temperature after the fermented grain spreading for cooling after the distillation of certain Liquor-making Enterprises & brew house
Yeast ratio is the 15% of inventory, and two experimental group inoculation high-temperature daqu ratios are 10% (reduction yeast use of inventory
33.3%) amount, while being separately added into S10 wine brewing antimicrobial composition (S10 group) of the preparation of embodiment 6 and with bacillus subtilis mark
The CICC1007 of quasi- bacterial strain substitution S10 makes wine antimicrobial composition (CICC1007 group), and inoculative proportion is the 0.5% of inventory
(percent by volume) substitutes 33.3% high-temperature daqu.It is carried out high temperature stacking fermentation 3 days after inoculation, windrow temperature is up to after 45 DEG C
Pit entry fermentation 30d, sampling distillation.Three Duplicate Samples of each processing.To ingredients such as alcohols, acids, esters in the sample of distillation
Content carries out gas chromatographic analysis.Key instrument: gas chromatographicanalyzer.
6.2 vinosity chromatography results and sensory evaluation
As can be seen from Table 10: in the experimental group fermented grain of the wine brewing antimicrobial composition containing S10 alcoholic strength up to 5.5%, than
CICC1007 group improves 12.2%.Ethyl acetate in S10 group, propionic acid, caproic acid, bata-phenethyl alcohol and 3-hydroxy-2-butanone yield respectively compared with
CICC1007 group improves 21.2%, 34.8%, 12.0%, 15.5% and 19.0%.To the Sauce flavor for improving former wine and change
Kind mouthfeel effect is obvious.Show that addition wine brewing antimicrobial composition containing S10 is for improving former wine distillation yield and wine quality in fermented grain
There is apparent facilitation, and high-temperature daqu dosage 33.3% can be reduced.
Each ingredient gas chromatographic analysis result in the fermentation fermented grain Spirit sample of table 10
Sensory evaluation comparison are as follows: control group wine sample score in 85-90, comment be sweet mellow, fragrance compared with harmony, wine body compared with
It is plentiful；For CICC1007 group wine sample score in 90-95, comment is that sweet mellow, fragrance is more mellow compared with harmony, wine body；S10 group wine sample
Score is in 95-100, and prominent, soft mellow, fragrance harmony, wine body are plentiful for paste flavor for comment.Show high-temperature daqu with the use of withered
Careless bacillus S10 microbial inoculum can be obviously improved vinosity, main due to white wine such as ethyl acetate, propionic acid, caproic acid and 3-hydroxy-2-butanones
The increase of flavor component content coordinates the fragrance in wine more, and mouthfeel is more preferably.
Claims (4)
1. one plant can generate Sauce flavor and bacillus subtilis of the high yield propionic acid for brewed spirit, which is characterized in that the bacterium
Strain is bacillus subtilis (Bacillus subtilis) S10, is preserved in China typical culture collection center, deposit number
For CCTCC NO:M2017466.
2. a kind of microbial bacterial agent containing the bacillus subtilis S10 described in claim 1, it is characterised in that its active constituent
For bacillus subtilis S10 thallus and its metabolite.
3. microbial bacterial agent according to claim 2, it is characterised in that the microbial inoculum is solid-state microbial inoculum.
4. preparing the preparation method of microbial inoculum as stated in claim 2, this method comprises the following steps:
(1) it S10 actication of culture: is inoculated in beef extract-peptone slant medium with one ring S10 strain of oese picking, sets 37
20-24h is cultivated in DEG C constant incubator；
(2) preparation of seed liquor: the activated S10 bacterial strain access of one ring of picking is trained equipped with the beef extract-peptone liquid of 100mL
Support base in, 37 DEG C, 180r/min shaking table culture it is spare for 24 hours；
(3) solid fermentation is preparation of culture medium: press wheat bran 40%, and rice bran 30%, dregs of beans 30%, material: water=1:1, pH is naturally, mixing
Uniformly, be configured to solid fermentation culture medium, and be sub-packed in plastics square box resistant to high temperature, after 121 DEG C of sterilizing 30min cool down to
With；
(4) solid fungicide culture: preparatory cultured S10 seed liquor is accessed to solid fermentation culture medium by 5-10% inoculum concentration
In, it is uniformly mixed in 30 DEG C of constant incubators of postposition and cultivates 2 days, every 12h stirring is primary；
(5) dry and crush: the S10 solid culture that above-mentioned fermentation finishes being placed in and is dried to moisture content in 55 DEG C of baking ovens and is lower than
15%, after dry culture is crushed, sieved with 100 mesh sieve with Universalpulverizer, it is prepared into solid powdery microbial inoculum and is packed into close
In the polybag of envelope, preservation in dry, shady place or fridge freshness retaining layer is placed；Wherein, in microbial inoculum sample S10 bacillus work
Bacterium number reaches cfu/ grams of 40-300 hundred million.
(6) it mixes and packaging: plastics after saccharomyces cerevisiae CCTCC AY92003 and S10 is mixed in viable count ratio=10:1 ratio
Wine brewing antimicrobial composition is made in sealing after bag.
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| CN109337775A (en) * | 2018-11-15 | 2019-02-15 | 安徽省领航动物保健品有限责任公司 | A kind of sweet osmanthus leaf of bamboo cordiale |
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| CN111172079B (en) * | 2020-02-18 | 2021-07-30 | 北京工商大学 | New strain of Ningqing bacillus and application thereof |
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| CN111518734A (en) * | 2020-06-11 | 2020-08-11 | 劲牌有限公司 | High-yield guaiacol bacillus subtilis and application thereof |
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