CN109971657A - A kind of Rhizopus oryzae of high-yield glucoamylase and its application - Google Patents

A kind of Rhizopus oryzae of high-yield glucoamylase and its application Download PDF

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CN109971657A
CN109971657A CN201910393499.XA CN201910393499A CN109971657A CN 109971657 A CN109971657 A CN 109971657A CN 201910393499 A CN201910393499 A CN 201910393499A CN 109971657 A CN109971657 A CN 109971657A
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highland barley
rhizopus oryzae
yeast
culture medium
culture
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CN109971657B (en
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薛洁
张玉红
闫寅卓
耿晓杰
张晓蒙
文华英
金玮鋆
靳玉龙
张志薇
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Agricultural Products Development And Food Science Institute Tibet Academy Of Agricultural And Animal Husbandry Sciences
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Agricultural Products Development And Food Science Institute Tibet Academy Of Agricultural And Animal Husbandry Sciences
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    • C—CHEMISTRY; METALLURGY
    • 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
    • C12G3/00—Preparation of other alcoholic beverages
    • C12G3/02—Preparation of other alcoholic beverages by fermentation
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/14—Fungi; Culture media therefor
    • C12N1/145—Fungal isolates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/645—Fungi ; Processes using fungi
    • C12R2001/845—Rhizopus
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Abstract

The invention discloses a kind of Rhizopus oryzae of high-yield glucoamylase and its applications in the optimization of highland barley Chinese yeast yeast-making technology, belong to microorganism and fermented soy engineering field.The Rhizopus oryzae is isolated from Chinese different regions of Tibet highland barley Chinese yeast, its screening technique is to collect to crush highland barley Chinese yeast, mould is separated and purified using the method for culture medium, then pass through primary dcreening operation, secondary screening and fermenting experiment, bacterial strain carbohydrase, α-amylase and prolease activity are measured, the bacterial strain that fermenting property is good and saccharifying enzymic activity is relatively high is selected；The saccharification activity and quality stability of the highland barley Chinese yeast with Tibet region characteristic can be effectively improved using the bacterial strain.

Description

A kind of Rhizopus oryzae of high-yield glucoamylase and its application
Technical field
The invention belongs to microorganisms and fermented soy engineering field, and in particular to a kind of Rhizopus oryzae of high-yield glucoamylase And its screening technique, the Rhizopus oryzae of the high-yield glucoamylase have been saved in Guangdong Province's Culture Collection, deposit number is GDMCC NO.60628；The invention further relates to application of the Rhizopus oryzae in optimization highland barley Chinese yeast yeast-making technology.
Background technique
Traditional barley wine is that spy made of highland barley Chinese yeast diastatic fermentation is added using Tibet staple food crop highland barley as raw material The fermented wine of color distinctness, because it is the representative of Tibet spirits culture, has that mouthfeel is suitable for, alcoholic strength is low and the spies such as nutritive effect is high Point succession is so far.It is essential in the occasions such as to be Tibetan people receive guests in kith and kin's party, major holiday, welcome, wedding and funeral arrangements marriage Vintage wine.It is closely bound up with the production and living of local people.
Highland barley Chinese yeast is that the brewing fermentation agent of traditional natural vaccination ways production is used by the Tibetan people, rich in micro- Biology and its generate enzyme, take part in wine brewing during saccharification, liquefaction, alcoholic fermentation and flavor formation, quality good or not There is strong influence to distillation yield and wine body quality, therefore have the title of " bone that song is wine ".So micro- life in highland barley Chinese yeast Object has been largely fixed the quality and flavor of barley wine.Currently, due to geographical location, brewage process, starter-making materials and ring The influence of the factors such as border causes highland barley Chinese yeast unstable quality, and it is low distillation yield occur so as to cause barley wine, has bitterness etc. displeased The leading to the problem of of happy flavor substance, wine body is muddy, different regions taste difference is big and is not easy to store etc., and wine body quality is unstable.
Carbohydrase successively hydrolyzing alpha -1,4- grape the effect in highland barley Chinese yeast is since the non reducing end of starch Glycosidic bond generates glucose, is applied to microbial fermentation and generates in alcohol, provides raw material and nutriment for subsequent fermentation, is One of key enzyme in barley wine fermentation process.In addition, the mainly mould of saccharification is played in highland barley Chinese yeast, by the shallow lake in raw material Powder is converted to reduced sugar and grows utilization for other microorganisms.Saccharifying enzymic activity is one of the important indicator for evaluating distiller's yeast quality, Saccharifying enzymic activity is higher to be conducive to making full use of and be conducive to improve distillation yield to raw material.
So the mould that screening saccharifying enzymic activity is high, especially the screening good fungal strain of fermenting property, are to improve blueness Highland barley Chinese yeast saccharifying enzymic activity, the quality to produce the Chinese yeast and stabilization and promotion barley wine that are suitble to Tibet region barley wine brewing One of major measure.
Summary of the invention
It is an object of the present invention to being directed to the status of current Tibet region highland barley Chinese yeast unstable quality, one plant is provided The Rhizopus oryzae of high-yield glucoamylase can significantly improve and stablize the Rhizopus oryzae bacterial strain of highland barley Chinese yeast saccharifying enzymic activity, have for exploitation There is the highland barley Chinese yeast product of Tibet region characteristic to be of great significance.
It is another object of the present invention to provide the screening techniques of above-mentioned Rhizopus oryzae.
Another of the invention is that above-mentioned Rhizopus oryzae is used to make the highland barley Chinese yeast of Tibet region.
In order to achieve the above-mentioned object of the invention, the present invention provides the Rhizopus oryzae of one plant of high-yield glucoamylase, the taxology of the mould It is named as Rhizopus oryzae, biomaterial are as follows: Rhizopus oryzae YC666 was preserved on April 11st, 2019 Guangdong Province's Culture Collection, deposit number are GDMCC NO.60628.
High-yield glucoamylase Rhizopus oryzae provided by the invention is villus by the bacterium colony grown on 72h solid PDA medium It is shape, initially white, after become taupe, dry, big and loose, opaque, mycelia mutually winds, and easily provokes, and is paved with entire Plate；Rhizopus oryzae saccharifying enzymic activity provided by the invention is 1400~1500mg/gh, and liquefaction enzyme activity is 1.3~1.4g/ Gh, neutral protease vigor are 800~900 μ g/gmin, and acid protease activity is 60~100 μ g/gmin.
The present invention provides the screening technique of above-mentioned meegan enzyme, includes the following steps:
The highland barley Chinese yeast that step 1) is collected in different regions of Tibet, takes and is crushed in right amount, the dilution of sterile working Gradient It is coated in isolation medium, 2~3d of stationary culture in 28 DEG C of incubators, randomly selects with typical mold colony feature Single colonie, scribing line isolate and purify 2-3 times, and inclined-plane saves backup；
Isolated fungal strain is carried out primary dcreening operation, secondary screening and fermentation test by step 2), and it is very high to obtain saccharifying enzymic activity One plant of mould.
In some preferred embodiments of the present invention, prescreening method produces carbohydrase transparent circle using mould in the step 2) It is tested with protease transparent circle, specifically includes the following steps: successively being put and being connected to by fungal strain with sterile toothpick using point connection It on primary dcreening operation culture medium, is cultivated 72 hours at 28 DEG C, measurement colony diameter D, the addition dilute iodine solution of 4mL into culture dish, after 5min, Transparent loop diameter d is measured, selects the biggish bacterial strain point of ratio d/D to be connected to and produces on protease primary dcreening operation culture medium, cultivated at 28 DEG C 72 hours, colony diameter D and transparent loop diameter d is measured, selects the biggish bacterial strain of d/D ratio.It is some preferred in the present invention In embodiment, used primary dcreening operation culture medium and production protease primary dcreening operation culture medium, contained component are respectively as follows:
Primary dcreening operation culture medium: soluble starch 10g/L, yeast extract 1.5g/L, NaNO31.5g/L, K2HPO41g/L, NaCl 0.5g/L, MgSO40.5g/L, FeSO40.01g/L, agar 20g/L；
Produce protease primary dcreening operation culture medium: beef extract 5g/L, peptone 10g/L, NaCl 5g/L, skimmed milk power 3g/L, agar 20g/L。
In some preferred embodiments of the present invention, secondary screening method uses the fermenting property of mould koji-making in the step 2) (saccharifying power, liquefaction power and prolease activity) test, detailed process are as follows: (spore count is 10 by mycotic spore suspension6A/mL Left and right) by 10% inoculum concentration bran mass is accessed, it mixes, 28 DEG C of culture 72h carry out button bottle, continue to cultivate to 6d.In drum In wind drying box, 40 DEG C of drying 12h；Saccharifying power, the liquefaction power that wheat bran is measured according to QB/T 4257-2011, according to GB/T 23527-2009 measurement prolease activities, select the good bacterial strain of fermenting property；In some more preferably embodiments of the present invention In, the component of used bran mass and preparation detailed process are as follows: wheat bran is crossed into 80 meshes, with 40% ratio of water to material by water It is sprayed on wheat bran, mixes, material moistening 30min is sub-packed in 500mL triangular flask, per bottled 30g, 121 DEG C of sterilizing 30min, to temperature When degree is down to 30~35 DEG C, break up spare.
In some preferred embodiments of the present invention, specific step is as follows for fermentation test described in the step 2):
1. simulating the production of highland barley Chinese yeast raw material: being put into basin after highland barley flour is crossed 80 meshes, cover basin lid, 121 DEG C ripe Change 30min；It is uniformly sprayed water with watering can on highland barley flour, is mixed thoroughly according to 40% ratio of water to material after cooling, cross 20 meshes；Wheat bran crosses 80 mesh Water, is sprayed on wheat bran by sieve with 40% ratio of water to material, is mixed, material moistening 30min；Highland barley flour and wheat bran are with the mixing of 1:2 ratio, packing In in 500mL triangular flask, per bottled 30g, 121 DEG C of sterilizing 30min, after culture medium is cooling, break up spare；
2. the simulation small koji fermentation of highland barley: spore suspension is made in the mould that secondary screening in claim 5 is obtained, and presses respectively 10% inoculum concentration accesses highland barley flour bran mass, mixes, 28 DEG C of culture 72h, detains bottle, continues culture to 6d；It is dry in air blast In dry case, 40 DEG C of drying 12h；The saccharifying power of wheat bran is measured according to QB/T 4257-2011, selects relatively high mould of saccharifying power Bacterium Rhizopus oryzae YC666.
In some specific embodiments of the present invention, the isolation medium in the step 1) is PDA (containing chloramphenicol) training Base is supported, specifically, the PDA (containing chloramphenicol) nutrient media components are as follows: potato leaching powder 3g/L, glucose 20g/L, agar 20g/L, chloramphenicol 0.1g/L.
In some specific embodiments of the present invention, the inclined-plane Storaged media in the step 1) is PDA culture medium, institute Containing component are as follows: potato leaching powder 3g/L, glucose 20g/L, agar 20g/L.
In some specific embodiments of the present invention, the mycotic spore suspension preparation detailed process is:
The sterile saline of 10mL is poured into the bacterial strain inclined-plane of activation, mycelia on lower inclined plane is scraped with transfer needle, by test tube In spore break up mixing, counted with blood counting chamber, and dilute spore count to 106/mL or so.
In some specific embodiments of the present invention, the detailed process of used bran mass preparation is: by wheat bran 80 meshes are crossed, water is sprayed on wheat bran with 40% ratio of water to material, are mixed, material moistening 30min is sub-packed in 500mL triangular flask, often Bottled 30g, 121 DEG C of sterilizing 30min break up spare when temperature is down to 30 DEG C or so.
The present invention provides a kind of highland barley Chinese yeast yeast-making technology, which uses Rhizopus oryzae Rhizopus provided by the invention Oryzae YC666 carries out highland barley Chinese yeast yeast-making technology；In some specific embodiments of the present invention, by rice provided by the invention After spore suspension is made in head mold Rhizopus oryzae YC666, produced according to highland barley Chinese yeast zymotechnique.
Compared with prior art, the invention has the following advantages:
1. highland barley Chinese yeast after Rhizopus oryzae optimization provided by the invention, saccharifying enzymic activity 1482mg/gh, with commercialization Rhizopus oryzae improves 15% compared to saccharifying enzymic activity, and saccharifying enzymic activity improves 67% compared with traditional highland barley Chinese yeast；Secondly, this The liquefaction enzyme activity for inventing the Rhizopus oryzae provided is 1.3~1.4g/gh, and neutral protease vigor is 800~900 μ g/g Min, acid protease activity are 60~100 μ g/gmin, can be used as saccharolytic in the characteristic highland barley Chinese yeast koji-making of Tibet region It is applied in process optimization, effectively improves the saccharification activity and quality stability of the highland barley Chinese yeast with Tibet region characteristic.
2. Rhizopus oryzae bacterial strain provided by the invention is in the small koji fermentation of industrial production highland barley, mycelia prosperity to be more suitable for using In the fermentation of highland barley Chinese yeast；Secondly bacterial strain color provided by the invention is white, closer to the color of highland barley Chinese yeast.
3. the highland barley Chinese yeast that bent aromatic flavour is made by adding Rhizopus oryzae Rhizopus oryzae YC666 in the present invention.
Specific embodiment
Rhizopus oryzae provided by the invention, the taxology of the mould are named as Rhizopus oryzae, biomaterial are as follows: Rhizopus oryzae YC666 is preserved in Guangdong Province's Culture Collection on April 11st, 2019, and deposit number is GDMCC NO.60628。
The present invention is described in detail below in conjunction with specific embodiment.
Method in following embodiments is unless otherwise instructed conventional method.
The production of simulation highland barley Chinese yeast raw material in following embodiments, detailed process is as follows:
It is put into enamel basin after highland barley flour is crossed 80 meshes, covers basin lid, 121 DEG C of curing 30min.According to 40% after cooling Ratio of water to material is uniformly sprayed water with watering can on highland barley flour, is mixed thoroughly, and 20 meshes are crossed；In addition, wheat bran crosses 80 meshes, it will with 40% ratio of water to material Water is sprayed on wheat bran, is mixed, material moistening 30min.Highland barley flour and wheat bran are sub-packed in 500mL triangular flask with the mixing of 1:2 ratio, Per bottled 30g, 121 DEG C of sterilizing 30min, after culture medium is cooling, break up spare.
The component of bran mass in following embodiments is with preparation detailed process:
Wheat bran is crossed into 80 meshes, water is sprayed on wheat bran with 40% ratio of water to material, is mixed, material moistening 30min is sub-packed in In 500mL triangular flask, per bottled 30g, 121 DEG C of sterilizing 30min break up spare when temperature is down to 30 DEG C or so.
Mycotic spore suspension preparation detailed process in following embodiments is:
The sterile saline of 10mL is poured into the bacterial strain inclined-plane of activation, mycelia on lower inclined plane is scraped with transfer needle, by test tube In spore break up mixing, counted with blood counting chamber, and dilute spore count to 106A/mL or so.
Used primary dcreening operation culture medium and production protease primary dcreening operation culture medium prescription in following embodiments is as follows:
Primary dcreening operation culture medium: soluble starch 10g/L, yeast extract 1.5g/L, NaNO31.5g/L, K2HPO41g/L, NaCl0.5g/L, MgSO40.5g/L, FeSO40.01g/L, agar 20g/L；
Produce protease primary dcreening operation culture medium: beef extract 5g/L, peptone 10g/L, NaCl 5g/L, skimmed milk power 3g/L, agar 20g/L。
PDA culture medium formula in following embodiments is as follows:
Potato leaching powder 3g/L, glucose 20g/L, agar 20g/L.
PDA (containing chloramphenicol) culture medium prescription in following embodiments is as follows:
PDA (containing chloramphenicol) culture medium: potato leaching powder 3g/L, glucose 20g/L, agar 20g/L, chloramphenicol 0.1g/ L。
Separation, purifying and the identification of 1 high-yield glucoamylase mould of embodiment
It separated, identified and is screened to 21 plants of separated fungal strains of different regions of Tibet highland barley Chinese yeast are picked up from.Its Separation method is using dilution plating procedure picking single colonie and is purified twice in succession, and identification method uses traditional form And ITS regional sequence is combined to analyze.
The bacterial strain is through morphology and ITS regional sequence is combined to analyze, and is accredited as the new strain of Rhizopus oryzae, specific qualification result is such as Under:
1. Method of Morphological Observation and result: cultivating by 72h, the bacterium colony grown on PDA (containing chloramphenicol) culture medium For villiform, initially white, after become taupe, dry, big and loose, opaque, mycelia mutually winds, and easily provokes, and spreads Full entire plate.
2. ITS regional sequence analysis method and result:
(1) the extraction of mould genomic DNA uses RNA isolation kit, and specific method is said by plant genome DNA extracts kit Bright book carries out.
The amplification of mould ITS regiospecificity segment using universal primer ITS1/ITS4 by give birth to work bioengineering (on Sea) limited liability company's synthesis；PCR amplification condition is 95 DEG C of initial denaturation 3min, 95 DEG C of denaturation 45s, 53 DEG C of annealing 30s, 72 DEG C Extend 45s (35 circulations), last 72 DEG C of extensions 8min；The composition of PCR amplification system (25uL) are as follows: 10 × PCR Buffer 2.5 μ L, d NTPs 2 μ L, 1 μ L of DNA profiling, 0.3 11 μ L of μ L, ITS4 of μ L, ITS1 of Taq enzyme, with dd H2O is mended to 25 μ L, is mixed It is even.Then pcr amplification product is detected: taking 5uL pcr amplification product and 1uL to mix, point sample is in 1% Ago-Gel On.Electrophoretic buffer is 1 × TAE, and under 120V voltage, electrophoresis 20min takes pictures to electrophorogram by gel imaging system and carried out Analysis, the size of pcr amplification product is judged using 2000bp DNAMarker, and the specific fragment length of mould is about as the result is shown For 600bp.
3. mould ITS sequence is analyzed: pcr amplification product is surveyed by Sangon Biotech (Shanghai) Co., Ltd. Sequence carries out homologous sequence search using BLAST software from Genbank GenBank, compares examination according to sequencing result Test the similarity degree of bacterial strain Yu known fungal strain corresponding sequence.
According to the viewpoint of current mould molecular systematics research field: having same or similar ITS sequence (sequence homology Property >=99%) bacterial strain belong to same species, according to the determining sequence homology with GenBank references bacterial strain of the sequencing results >=99% bacterial strain is Rhizopus oryzae.
The screening of 2 high-yield glucoamylase Rhizopus oryzae of embodiment
Transparent circle primary dcreening operation, secondary screening and fermentation test are passed through to identified bacterial strain, obtaining several plants has excellent brewing characteristic Fungal strain, then simulation highland barley Chinese yeast starter-making materials in 10% inoculum concentration (spore count: 106A/mL) inoculation, It ferments 6 days at 28 DEG C, measures the saccharifying enzymic activity of highland barley Chinese yeast after fermentation, and carry out organoleptic indicator's evaluation.
By repeated screening, verifying and brewing research, isolated one plant of carbohydrase from the highland barley Chinese yeast of Tibet region Vigor is high, and the good Rhizopus oryzae of fermenting property, has very high and stable carbohydrase with the highland barley Chinese yeast that the bacterial strain is made Vigor covers with white hypha with the small curved surface of highland barley that the bacterial strain is made and is evenly distributed, no miscellaneous bacteria, bent thick flavor, no impurity taste.
Specific screening process is as follows:
1. using point connection, fungal strain point the production transparent circle aptitude tests of mould: is connected to production saccharification with sterile toothpick It on enzyme bacterial strain primary dcreening operation culture medium, is cultivated 72 hours at 28 DEG C, measures colony diameter D, the dilute iodine solution of 4mL is added into culture dish, After 5min, transparent loop diameter d is measured, selects the biggish bacterial strain point of ratio d/D to be connected to and produces on protease primary dcreening operation culture medium, at 28 DEG C Lower culture 72 hours, measures colony diameter D and transparent loop diameter d, selects the biggish bacterial strain of ratio d/D.
2. the fermenting property test of mould: mycotic spore suspension is accessed into bran mass by 10% inoculum concentration, is mixed Even, 28 DEG C of culture 72h carry out button bottle, continue culture to 6d.In in air dry oven, 40 DEG C of drying 12h；According to QB/T The saccharifying power of 4257-2011 measurement wheat brans, liquefaction power measure prolease activity, selection fermentation according to GB/T 23527-2009 Bacterial strain of good performance.
3. fermentation test: being made spore suspension for mould, accesses the culture of highland barley flour wheat bran by 10% inoculum concentration respectively Base mixes, and 28 DEG C of culture 72h carry out button bottle, continues culture to 6d.In in air dry oven, 40 DEG C of drying 12h；According to QB/ T4257-2011 measures the saccharifying power of wheat bran, selects the highest Rhizopus oryzae of saccharifying power.
The separation of 1 business Rhizopus oryzae of comparative example
Koji gradient dilution purchased from Angel Yeast Co., Ltd is coated in isolation medium, is trained in 28 DEG C Stationary culture 3d in case is supported, the single colonie with Rhizopus oryzae colony characteristics is chosen, scribing line isolates and purifies 2-3 times, and inclined-plane saves standby With.
The small-sized koji-making comparative test of embodiment 3
Embodiment 2 obtains high-yield glucoamylase Rhizopus oryzae and comparative example acquisition business Rhizopus oryzae is small in highland barley Chinese yeast raw material Type koji-making comparative test.
It fills 500g highland barley flour bran mass respectively in 3 5L wide-mouth bottles, is separately added into the high yield rice root that screening obtains Mould, commercialization Rhizopus oryzae and Tibet region producing region highland barley Chinese yeast, make its spore suspension final concentration reach 106A/mL or so, It is produced according to highland barley Chinese yeast fermentation test technique.Saccharifying enzymic activity and organoleptic indicator's evaluation are measured after fermentation.
1 two kinds of Rhizopus oryzae bacterial strains of table and Tibet region highland barley Chinese yeast diastasimetry result
Organoleptic indicator's evaluation result of 2 two kinds of Rhizopus oryzae bacterial strains of table and Tibet region highland barley Chinese yeast production highland barley Chinese yeast
Highland barley Chinese yeast made of high-yield glucoamylase Rhizopus oryzae is added, saccharifying power highest is 1482 ± 77.38mg/gh, Curved surface mycelia is uniform, and surface is white, no miscellaneous bacteria.Therefore the bacterial strain of screening meets the requirement of industrial fermentation, while screening bacterium The saccharifying enzymic activity of strain is above business Rhizopus oryzae and Tibet producing region highland barley Chinese yeast；The bent thick flavor for highland barley Chinese yeast of fermenting, nothing Peculiar smell is relatively beneficial to preparation Tibet characteristic highland barley wine product.

Claims (9)

1. the Rhizopus oryzae of one plant of high-yield glucoamylase, the taxology of the Rhizopus oryzae is named as Rhizopus oryzae, and deposit number is GDMCC NO.60628。
2. the screening technique of Rhizopus oryzae as described in claim 1, which is characterized in that screening technique includes the following steps:
The highland barley Chinese yeast that step 1) is collected in different regions of Tibet, takes and is crushed in right amount, sterile working Gradient dilution spread In isolation medium, 2~3d of stationary culture in 28 DEG C of incubators randomly selects the single bacterium with typical mold colony feature It falls, scribing line isolates and purifies 2-3 times, and inclined-plane saves backup；
Isolated fungal strain is carried out primary dcreening operation, secondary screening and fermentation test by step 2), obtains one plant of high mould of saccharifying enzymic activity.
3. screening technique as claimed in claim 2, which is characterized in that the prescreening method in the step 2) produces sugar using mould Change enzyme transparent circle and the test of protease transparent circle, specifically includes the following steps: using a point connection, with sterile toothpick by fungal strain Successively point is connected on primary dcreening operation culture medium, is cultivated 72 hours at 28 DEG C, and colony diameter D is measured, and the dilute iodine of 4mL is added into culture dish Liquid after 5min, measures transparent loop diameter d, selects the biggish bacterial strain point of ratio d/D to be connected to and produces on protease primary dcreening operation culture medium, It is cultivated 72 hours at 28 DEG C, measures colony diameter D and transparent loop diameter d, select the biggish bacterial strain of d/D ratio.
4. screening technique as claimed in claim 3, which is characterized in that used primary dcreening operation culture medium and production protease primary dcreening operation training Base is supported, contained component is respectively as follows:
Primary dcreening operation culture medium: soluble starch 10g/L, yeast extract 1.5g/L, NaNO31.5g/L, K2HPO41g/L, NaCl 0.5g/L, MgSO40.5g/L, FeSO40.01g/L, agar 20g/L；
Produce protease primary dcreening operation culture medium: beef extract 5g/L, peptone 10g/L, NaCl 5g/L, skimmed milk power 3g/L, agar 20g/ L。
5. screening technique as claimed in claim 2, which is characterized in that the detailed process of secondary screening in the step 2) are as follows: will be mould Bacterium spore suspension accesses bran mass by 10% inoculum concentration, mixes, and 28 DEG C of culture 72h carry out button bottle, continues culture extremely 6d；In in air dry oven, 40 DEG C of drying 12h；Saccharifying power, the liquefaction power that wheat bran is measured according to QB/T 4257-2011, according to GB/T 23527-2009 measures prolease activity, selects the good bacterial strain of fermenting property.
6. screening technique as claimed in claim 5, which is characterized in that the detailed process of used bran mass preparation Are as follows: wheat bran is crossed into 80 meshes, water is sprayed on wheat bran with 40% ratio of water to material, is mixed, material moistening 30min is sub-packed in 500mL tri- In the bottle of angle, per bottled 30g, 121 DEG C of sterilizing 30min break up spare when temperature is down to 30~35 DEG C.
7. screening technique as claimed in claim 5, which is characterized in that the specific steps of fermentation test are such as in the step 2) Under:
1. simulating the production of highland barley Chinese yeast raw material: being put into basin after highland barley flour is crossed 80 meshes, cover basin lid, 121 DEG C of curings 30min；It is uniformly sprayed water with watering can on highland barley flour, is mixed thoroughly according to 40% ratio of water to material after cooling, cross 20 meshes；Wheat bran crosses 80 mesh Water, is sprayed on wheat bran by sieve with 40% ratio of water to material, is mixed, material moistening 30min；Highland barley flour and wheat bran are with the mixing of 1:2 ratio, packing In in 500mL triangular flask, per bottled 30g, 121 DEG C of sterilizing 30min, after culture medium is cooling, break up spare；
2. the simulation small koji fermentation of highland barley: spore suspension is made in the mould that secondary screening in claim 5 is obtained, respectively by 10% Inoculum concentration accesses highland barley flour bran mass, mixes, 28 DEG C of culture 72h, detains bottle, continues culture to 6d；In in air dry oven, 40 DEG C of drying 12h；The saccharifying power of wheat bran is measured according to QB/T 4257-2011, the mould for selecting saccharifying power relatively high.
8. application of the Rhizopus oryzae as described in claim 1 in highland barley Chinese yeast yeast-making technology.
9. application as claimed in claim 8 after spore suspension is made in Rhizopus oryzae, is carried out according to highland barley Chinese yeast zymotechnique Production.
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