CN104762163A - Method for preparing biological yeast - Google Patents
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12G—WINE; PREPARATION THEREOF; ALCOHOLIC BEVERAGES; PREPARATION OF ALCOHOLIC BEVERAGES NOT PROVIDED FOR IN SUBCLASSES C12C OR C12H
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Abstract
The invention discloses a method for preparing biological yeast, and belongs to the field of a wine making technology. The method comprises the following steps: producing a bacteria seed liquid, a yeast seed liquid and a mould solid seeds respectively, inoculating raw materials into the bacteria seed liquid, yeast seed liquid and mould solid seeds according to a ratio. On the basis of the traditional technology of yeast for making hard liquor, with the combination of a pure culture biological technology and a traditional raw material fermentation technology, the yeast overturning process is eliminated, the production period is shortened, and due to the fact that the yeast storage period which is as long as three months is eliminated, the production efficiency of the yeast for making hard liquor is improved, and the method has a profound historical significance for the promotion of the scientific and technological progress of the liquor industry.
Description
Technical field
The present invention relates to a kind of method preparing biological song, belong to brewing technical field.
Background technology
Daqu traditional zymotic is preparation technology go through 4 months, consumes a large amount of manpower and materials, and extraneous fungus strain is comparatively large with seasonal variation, and quality product is by various factors, wayward, causes Daqu quality product unstable.In recent years, along with the development of brewing technology, in the preparation technology of Daqu, realize mechanize from raw material pulverizing to buckling, and subsequent technique still rests on traditional-handwork operant level.Modern biotechnology combines with traditional yeast-making technology by this research, introduce totally enclosed, full automatic solid fermentation apparatus, which reduces unnecessary living contaminants, can not by the restriction in season, and temperature requiredly can be regulated flexibly in enzyme each period according to function yeast Different growth phases and producing, realize mechanization production completely biological bent, for the mechanize process of liquor industry opens up new brilliant work.
Summary of the invention
The object of the invention is to: provide a kind of and prepare biological bent method, improve traditional Daqu production technique, utilize modern biotechnology, automatic and mechanical is produced biological bent, realize continuous controlledization of producing, enhance productivity, promote Liquor-making Enterprises & and to make wine overall scientific and technical innovation dynamics.
The inventive method mainly comprises the following steps:
(1) bacterium seed liquor is prepared: bacillus pumilus, solution starch forest Zymomonas mobilis, Lactococcus lactis are connect a ring respectively in seed culture medium, carries out activation culture, obtain mixed bacteria liquid;
(2) yeast starter liquid is prepared: Hansenula anomala, Pi Texun pichia spp, Candida valida are connect a ring respectively in seed culture medium, carries out activation culture, obtain mixed bacteria liquid;
(3) prepare mould solid state bacterial: the physiological saline getting the spore of volume branch Mucor, access Daqu raw material quiescent culture, then, is seeded to kind of bent tank 32-36 DEG C and cultivates 72 ± 8h, obtain mould solid state bacterial;
(4) preparation is biological bent: wheat and barley are with mass ratio (8-10): 1 mixes, add water the wet feed of moistening one-tenth water content 47.5 ± 2.5% (w/w), with the inoculum size access mould solid state bacterial of the inoculum size access yeast seed liquor of the inoculum size of 1 ± 0.5% (w/w) access bacterium seed liquor, 2 ± 0.5% (w/w), 3 ± 0.5% (w/w) in wet feed, mixing, ulking thickness 25 ± 5cm, fermentation initial stage insulation 30 ± 1 DEG C, cultivated through 6-8 days, make bent heart top fire temperature reach 60 ± 2 DEG C, push up fiery 3-5 days.
In one embodiment of the invention, described bacillus pumilus is bacillus pumilus (Bacilluspumilus) CGMCC No 1.1168.
In one embodiment of the invention, described solution starch forest Zymomonas mobilis separates starch forest Zymomonas mobilis (Silvimonasamylolytica) CGMCC No 1.8860.
In one embodiment of the invention, described Lactococcus lactis is Lactococcus lactis (Lactococcus lactis) CGMCCNo 1.2470.
In one embodiment of the invention, described Hansenula anomala is Hansenula anomala (Hansenula anomala) CGMCC No 2.764.
In one embodiment of the invention, described Pi Texun pichia spp is Pi Texun pichia spp (Pichiapetersonii) CGMCC No 2.1473.
In one embodiment of the invention, described Candida valida is Candida valida (Candida robusta) CGMCC No 2.865.
In one embodiment of the invention, described volume branch Mucor is volume branch Mucor (Mucor circinelloides) CGMCC No3.2208.
One embodiment of the present invention adopt following steps:
(1) bacterium seed liquor is prepared: bacillus pumilus, solution starch forest Zymomonas mobilis, Lactococcus lactis are connect a ring respectively in seed culture medium, and carry out activation culture, the cell concentration of gained bacterium liquid is 10
6-10
7individual/mL; Described seed culture medium preferably beef cream protein culture medium; Described activation culture condition optimization 35-37 DEG C, 100-120rpm.
(2) prepare yeast starter liquid: the Hansenula anomala of preservation, Pi Texun pichia spp, Candida valida are connect a ring respectively in seed culture medium, carries out activation culture, the cell concentration of gained bacterium liquid is 10
5-10
6individual/mL; The preferred wort of described seed culture medium or the bent juice of rice; Described activation culture condition optimization 28-30 DEG C, 100-120rpm.
(3) mould solid state bacterial is prepared: the physiological saline getting the spore of volume branch Mucor, spore content about 10
5-10
6individual/mL, get 1 ± 0.5mL access and 30-35g water content is housed is adjusted in the 500mL shaking flask of the Daqu raw material of 45%-50%, 32-36 DEG C of static gas wave refrigerator, incubation time is 72 ± 8h; Then, be seeded to kind of bent tank 32-36 DEG C and cultivate 72 ± 8h, obtain mould solid state bacterial.
(4) preparation is biological bent: wheat and barley are with mass ratio (8-10): 1 mixes, add water the wet feed of moistening one-tenth water content 47.5 ± 2.5% (w/w), with the inoculum size of 1 ± 0.5% (w/w) access bacterium seed liquor in wet feed, the inoculum size access yeast seed liquor of 2 ± 0.5% (w/w), the inoculum size access mould solid state bacterial of 3 ± 0.5% (w/w), mixing, ulking thickness 25 ± 5cm, fermentation initial stage insulation 30 ± 1 DEG C, cultivated through 6-8 days, bent heart top fire temperature is made to reach 60 ± 2 DEG C, push up fiery 3-5 days, whole koji time shorten to 15 ± 1 day, final biological song.
The present invention also provides a kind of method applying the bent brewing spirit of described biology: after the biological bent pulverizing of gained, for brew house, with the 10%-15% (w/w) that song amount is charging capacity, grain unstrained spirits, than 1 ︰ (4-5), carries out wine brewing by " six-distribution method " technique and produces.
The present invention has the following advantages:
1, apply modern purebred cultivation biotechnology to combine with traditional raw material fermentation technique, eliminate and turn over bent operation, shorten the production cycle, and eliminate and reach the trimestral Daqu shelf lives, improve biological bent production efficiency.2, this production technique is simple, and processing ease, mechanize, automatic production, save manpower.3, this production technique avoids the impact of environment on quality product, and human factor impact is also less, all can produce throughout the year, and indices all reaches or is better than traditional Daqu.4, biological song is applied in pond, cellar for storing things and finds, although biological bent not through storing, but cellar for storing things pond temperature variation meets completely " front slow, in very, delay to fall afterwards " optimum regime, Jiao Chi goes out the wine body framework ingredient of wine and judges result and also demonstrate biological song and have good application prospect simultaneously.5, biological bent application, not only greatly improves the quality of former wine, and for function yeast in wine brewing Tiny ecosystem domestication and be enriched with vital role, thus promote former wine quality further.
Accompanying drawing explanation
Fig. 1 enters Jiao Jiao pond temperature curve, and biological bent 1-3 is that biology prepared by embodiment 1-3 is bent respectively
Embodiment
Further illustrate technical solution of the present invention below in conjunction with specific embodiment, these embodiments can not be interpreted as it is restriction to technical scheme.
Embodiment 1 is produced biological bent according to following steps
(1) bacteria culture: 50mL beef extract-peptone liquid nutrient medium is positioned in 500mL triangular flask, from three bacterium function yeast bacillus pumilus Bacilluspumilus (CGMCC No 1.1168), solution starch forest Zymomonas mobilis Silvimonasamylolytica (CGMCC No 1.8860), Lactococcus lactis Lactococcus lactis (CGMCC No 1.2470) preservation inclined-plane, respectively scrape an articulating enters in beef extract-peptone liquid nutrient medium, pH value 7.0,37 DEG C of shaking tables are cultivated, shaking speed is 100rpm, and incubation time is 24h; Then, adopt beef extract-peptone liquid nutrient medium, pH value 7.0, adopt fermentor tank 37 DEG C to cultivate 24h, obtain bacterium seed liquor; Microscopy, every milliliter of thalline quantity remains on 10
7above, quantity is more, homogeneous, not containing other miscellaneous bacterias;
(2) yeast seeds: 50mL malt juice liquid medium is placed in 500mL triangular flask, from three yeast function yeast Hansenula anomala Hansenula anomala (CGMCC No 2.764), Pi Texun pichia spp Pichiapetersonii (CGMCC No 2.1473), Candida valida Candida robusta (CGMCC No 2.865) preservation inclined-plane, respectively scrape an articulating enters in malt juice liquid medium, 28 DEG C of shaking tables are cultivated, shaking speed is 100rpm, and incubation time is 24h; Then, adopt above-mentioned same medium, adjust ph 4.5, adopt fermentor tank 28 DEG C to cultivate 36h, obtain yeast seed liquor; Microscopy, every milliliter of thalline quantity remains on 10
6above, yeast is more healthy and stronger, size is even, without cavity in cell, does not infect other miscellaneous bacterias;
(3) mold species: get mould function yeast volume branch Mucor (Mucor circinelloides) CGMCC No 3.2208 slant preservation test tube, wash lower spore by stroke-physiological saline solution, use blood counting chamber to be adjusted to the order of magnitude 10
6individual/mL, gets that 30g/500mL is equipped with in 1mL access, water content is adjusted in 45% Daqu raw material, 32 DEG C of static gas wave refrigerator, and incubation time is 72h; Then, be seeded to kind of bent tank 32 DEG C of static gas wave refrigerator 72h, obtain mould solid state bacterial;
(4) biological bent: wheat mixes with mass ratio 8:1 with barley, add water moistening one-tenth water content 45.0% (w/w) wet feed, with the inoculum size access mould solid state bacterial of the inoculum size access yeast seed liquor of the inoculum size of 0.5% (w/w) access bacterium seed liquor, 1.5% (w/w), 2.5% (w/w) in wet feed, ulking thickness 20cm, the fermentation initial stage is incubated 29 DEG C, cultivated through 6 days, bent heart top fire temperature is made to reach 58 DEG C, top fire 3 days, whole koji time shorten to 14 day, final biological bent 1.
It is bent that embodiment 2 produces modern biotechnology according to following steps
(1) bacteria culture: 50mL beef extract-peptone liquid nutrient medium is positioned in 500mL triangular flask, from three bacterium function yeast B.pumilus (CGMCC 1.1168), S.amylolytica (CGMCC 1.8860), L.lactis (CGMCC 1.2470) preservation inclined-plane, respectively scrape an articulating enters in beef extract-peptone liquid nutrient medium, pH value 7.2,37 DEG C of shaking tables are cultivated, shaking speed is 100rpm, and incubation time is 24h; Then, adopt beef extract-peptone liquid nutrient medium, pH value 7.2, adopt fermentor tank 37 DEG C to cultivate 24h, obtain bacterium seed liquor; Microscopy, every milliliter of thalline quantity remains on 10
7above, quantity is more, homogeneous, not containing other miscellaneous bacterias;
(2) yeast seeds: the bent juice liquid nutrient medium of 50mL rice is placed in 500mL triangular flask, from three yeast function yeast H.anomala (CGMCC 2.764), P.petersonii (CGMCC 2.1473), C.robusta (CGMCC 2.865) preservation inclined-plane, respectively scrape an articulating enters in the bent juice liquid nutrient medium of rice, 29 DEG C of shaking tables are cultivated, shaking speed is 100rpm, and incubation time is 24h; Then, adopt above-mentioned same medium, adjust ph 4.7, adopt fermentor tank 29 DEG C to cultivate 36h, obtain yeast seed liquor; Microscopy, every milliliter of thalline quantity remains on 10
6above, yeast is more healthy and stronger, size is even, without cavity in cell, does not infect other miscellaneous bacterias;
(3) mold species: get a mould function yeast M.circinelloides (CGMCC 3.2208) slant preservation test tube, wash lower spore by stroke-physiological saline solution, uses blood counting chamber to be adjusted to the order of magnitude 10
6individual/mL, gets that 30g/500mL is equipped with in 1mL access, water content is adjusted in 47.5% Daqu raw material, 34 DEG C of static gas wave refrigerator, and incubation time is 72h; Then, be seeded to kind of bent tank 34 DEG C of static gas wave refrigerator 72h, obtain mould solid state bacterial;
(4) biological bent: wheat mixes with mass ratio 9:1 with barley, add water moistening one-tenth water content 47.5% (w/w) wet feed, with the inoculum size access mould solid state bacterial of the inoculum size access yeast seed liquor of the inoculum size of 1% (w/w) access bacterium seed liquor, 2% (w/w), 3% (w/w) in wet feed, ulking thickness 25cm, the fermentation initial stage is incubated 30 DEG C, cultivated through 7 days, bent heart top fire temperature is made to reach 60 DEG C, top fire 4 days, whole koji time shorten to 15 day, final biological bent 2.
It is bent that embodiment 3 produces modern biotechnology according to following steps
(1) bacteria culture: 50mL beef extract-peptone liquid nutrient medium is positioned in 500mL triangular flask, from three bacterium function yeast B.pumilus (CGMCC 1.1168), S.amylolytica (CGMCC 1.8860), L.lactis (CGMCC 1.2470) preservation inclined-plane, respectively scrape an articulating enters in beef extract-peptone liquid nutrient medium, pH value 7.4,37 DEG C of shaking tables are cultivated, shaking speed is 100rpm, and incubation time is 24h; Then, adopt beef extract-peptone liquid nutrient medium, pH value 7.4, adopt fermentor tank 37 DEG C to cultivate 24h, obtain bacterium seed liquor; Microscopy, every milliliter of thalline quantity remains on 10
7above, quantity is more, homogeneous, not containing other miscellaneous bacterias;
(2) yeast seeds: 50mL malt juice liquid medium is placed in 500mL triangular flask, from three yeast function yeast H.anomala (CGMCC 2.764), P.petersonii (CGMCC 2.1473), C.robusta (CGMCC 2.865) preservation inclined-plane, respectively scrape an articulating enters in malt juice liquid medium, 30 DEG C of shaking tables are cultivated, shaking speed is 100rpm, and incubation time is 24h; Then, adopt above-mentioned same medium, adjust ph 5.0, adopt fermentor tank 30 DEG C to cultivate 36h, obtain yeast seed liquor; Microscopy, every milliliter of thalline quantity remains on 10
6above, yeast is more healthy and stronger, size is even, without cavity in cell, does not infect other miscellaneous bacterias;
(3) mold species: get a mould function yeast M.circinelloides (CGMCC 3.2208) slant preservation test tube, wash lower spore by stroke-physiological saline solution, uses blood counting chamber to be adjusted to the order of magnitude 10
6individual/mL, get 1mL access and 30g/500mL is housed, water content is adjusted in 50% Daqu raw material, 36 DEG C of static gas wave refrigerator, and incubation time is 72h; Then, be seeded to kind of bent tank 36 DEG C of static gas wave refrigerator 72h, obtain mould solid state bacterial;
(4) biological bent: wheat mixes with mass ratio 10:1 with barley, add water moistening one-tenth water content 50.0% (w/w) wet feed, with the inoculum size access mould solid state bacterial of the inoculum size access yeast seed liquor of the inoculum size of 1.5% (w/w) access bacterium seed liquor, 2.5% (w/w), 3.5% (w/w) in wet feed, ulking thickness 30cm, the fermentation initial stage is incubated 31 DEG C, cultivated through 8 days, bent heart top fire temperature is made to reach 62 DEG C, top fire 5 days, whole koji time shorten to 16 day, final biological bent 3.
By the use standard of biological bent traditionally wrapped starter, test at pilot plant, adopt " six-distribution method " technique, take Chinese sorghum as alcoholic raw material, high-quality barley, wheat, pea mix, add biological bent, with the 10%-15% (w/w) that song amount is charging capacity, grain unstrained spirits is than 1 ︰ (4-5), and mud cellar for storing things solid state fermentation, adopts continuous grain batching, mixed-steaming and mixed-heating, amount quality picking wine, poor unstrained spirits goes out rice steamer and to splash spreading for cooling after proportioning water, adds song and enters cellar for storing things.Through the test of 6 months by a definite date, result shows to use biological bent pond, cellar for storing things temperature to meet the processing requirement (shown in Fig. 1) of " front slow in delay to fall very afterwards ", produce ethyl hexanoate content in top grade wine and one-level wine and be greater than 300mg/100mL and 200mg/100mL (shown in table 2) respectively, wine body framework ingredient is suitable with the Jiao Chi of the traditional Daqu of use, and judge through the room of sampling wine, according to the Comment on Standard that company formulates, adopt and secretly comment method, by look, fragrant, taste and style are given a mark and write comment, with color and luster 10 points, fragrance 25 points, taste 50 points and style 15 are divided into centesimal system, by total score divided rank, top grade 90 and more than, one-level 80 and more than, secondary 70 and more than.Judge result display wine body of adopting and all reach top grade and primary standard (shown in table 3), demonstrate modern biotechnology song and there is good application prospect.
The biological song of table 1 and the comparative analysis of traditional wrapped starter physical and chemical index
The biological bent pilot scale of table 2 goes out wine and compares (mg/100mL)
The biological bent wine that produces of table 3 judges result through the room of sampling wine
Although the present invention with preferred embodiment openly as above; but it is also not used to limit the present invention, any person skilled in the art, without departing from the spirit and scope of the present invention; all can do various changes and modification, what therefore protection scope of the present invention should define with claims is as the criterion.
Claims (6)
1. prepare a biological bent method, mainly comprise the following steps:
(1) bacterium seed liquor is prepared: bacillus pumilus, solution starch forest Zymomonas mobilis, Lactococcus lactis are connect a ring respectively in seed culture medium, carries out activation culture, obtain mixed bacteria liquid;
(2) yeast starter liquid is prepared: Hansenula anomala, Pi Texun pichia spp, Candida valida are connect a ring respectively in seed culture medium, carries out activation culture, obtain mixed bacteria liquid;
(3) prepare mould solid state bacterial: the physiological saline getting the spore of volume branch Mucor, access Daqu raw material quiescent culture, then, is seeded to kind of bent tank 32-36 DEG C and cultivates 72 ± 8h, obtain mould solid state bacterial;
(4) preparation is biological bent: wheat and barley are with mass ratio (8-10): 1 mixes, add water the wet feed of moistening one-tenth water content 47.5 ± 2.5% (w/w), with the inoculum size access mould solid state bacterial of the inoculum size access yeast seed liquor of the inoculum size of 1 ± 0.5% (w/w) access bacterium seed liquor, 2 ± 0.5% (w/w), 3 ± 0.5% (w/w) in wet feed, mixing, ulking thickness 25 ± 5cm, fermentation initial stage insulation 30 ± 1 DEG C, cultivated through 6-8 days, make bent heart top fire temperature reach 60 ± 2 DEG C, push up fiery 3-5 days.
2. method according to claim 1, it is characterized in that, described preparation bacterium seed liquor: bacillus pumilus, solution starch forest Zymomonas mobilis, Lactococcus lactis are connect a ring respectively in seed culture medium, and carry out activation culture, the cell concentration of gained bacterium liquid is 10
6-10
7individual/mL; Described seed culture medium is beef-protein medium; Described activation culture condition is 35-37 DEG C, 100-120rpm.
3. method according to claim 1, it is characterized in that, describedly prepare yeast starter liquid: Hansenula anomala, Pi Texun pichia spp, Candida valida are connect a ring respectively in seed culture medium, carries out activation culture, the cell concentration of gained bacterium liquid is 10
5-10
6individual/mL; Described seed culture medium is the bent juice of wort or rice; Described activation culture condition is 28-30 DEG C, 100-120rpm.
4. method according to claim 1, is characterized in that, described preparation mould solid state bacterial: the physiological saline getting the spore of volume branch Mucor, spore content about 10
5-10
6individual/mL, get 1 ± 0.5mL access and 30-35g water content is housed is adjusted in the 500mL shaking flask of the Daqu raw material of 45%-50%, 32-36 DEG C of static gas wave refrigerator, incubation time is 72 ± 8h; Then, be seeded to kind of bent tank 32-36 DEG C and cultivate 72 ± 8h, obtain mould solid state bacterial.
5. the biology prepared of method is bent according to claim 1.
6. biological bent application in brewed spirit described in claim 5.
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| CN111218406A (en) * | 2020-01-10 | 2020-06-02 | 浙江工业大学 | Mucor circinelloides MF-8 and application thereof in improving content of taxifolin in rhizoma smilacis glabrae |
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| CN111218406B (en) * | 2020-01-10 | 2022-03-15 | 浙江工业大学 | Mucor circinelloides MF-8 and application thereof in improving content of taxifolin in rhizoma smilacis glabrae |
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| Publication number | Publication date |
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| CN104762163B (en) | 2017-03-01 |
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