CN106399121B - A kind of purple red yeast rice bacteria strain - Google Patents
A kind of purple red yeast rice bacteria strain Download PDFInfo
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- CN106399121B CN106399121B CN201610659759.XA CN201610659759A CN106399121B CN 106399121 B CN106399121 B CN 106399121B CN 201610659759 A CN201610659759 A CN 201610659759A CN 106399121 B CN106399121 B CN 106399121B
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- 241000894006 Bacteria Species 0.000 title claims abstract description 21
- 229940026314 red yeast rice Drugs 0.000 title claims abstract description 16
- SWGJCIMEBVHMTA-UHFFFAOYSA-K trisodium;6-oxido-4-sulfo-5-[(4-sulfonatonaphthalen-1-yl)diazenyl]naphthalene-2-sulfonate Chemical compound [Na+].[Na+].[Na+].C1=CC=C2C(N=NC3=C4C(=CC(=CC4=CC=C3O)S([O-])(=O)=O)S([O-])(=O)=O)=CC=C(S([O-])(=O)=O)C2=C1 SWGJCIMEBVHMTA-UHFFFAOYSA-K 0.000 title claims abstract description 7
- 241000228347 Monascus <ascomycete fungus> Species 0.000 claims abstract description 19
- 244000113306 Monascus purpureus Species 0.000 claims abstract description 8
- 235000002322 Monascus purpureus Nutrition 0.000 claims abstract description 8
- 229940057059 monascus purpureus Drugs 0.000 claims abstract description 8
- 238000004321 preservation Methods 0.000 claims abstract description 6
- 240000007594 Oryza sativa Species 0.000 abstract description 29
- 235000007164 Oryza sativa Nutrition 0.000 abstract description 29
- 235000014101 wine Nutrition 0.000 abstract description 23
- 230000001580 bacterial effect Effects 0.000 abstract description 18
- 235000007189 Oryza longistaminata Nutrition 0.000 abstract description 16
- 238000000855 fermentation Methods 0.000 abstract description 16
- 230000004151 fermentation Effects 0.000 abstract description 16
- 238000004821 distillation Methods 0.000 abstract description 10
- 239000002994 raw material Substances 0.000 abstract description 3
- 230000000694 effects Effects 0.000 description 24
- 102000004190 Enzymes Human genes 0.000 description 13
- 108090000790 Enzymes Proteins 0.000 description 13
- 235000009566 rice Nutrition 0.000 description 13
- 239000002609 medium Substances 0.000 description 12
- 238000000034 method Methods 0.000 description 12
- 239000004382 Amylase Substances 0.000 description 11
- 108010065511 Amylases Proteins 0.000 description 11
- 102000013142 Amylases Human genes 0.000 description 11
- 235000019418 amylase Nutrition 0.000 description 11
- 239000001963 growth medium Substances 0.000 description 11
- 239000007788 liquid Substances 0.000 description 10
- 239000000725 suspension Substances 0.000 description 10
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 10
- 229920001817 Agar Polymers 0.000 description 9
- 239000008272 agar Substances 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 8
- 239000000463 material Substances 0.000 description 7
- 238000002360 preparation method Methods 0.000 description 7
- 239000000243 solution Substances 0.000 description 7
- 230000001476 alcoholic effect Effects 0.000 description 6
- 108010089934 carbohydrase Proteins 0.000 description 6
- 238000012216 screening Methods 0.000 description 6
- 230000004913 activation Effects 0.000 description 5
- 241000196324 Embryophyta Species 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- 239000001888 Peptone Substances 0.000 description 4
- 108010080698 Peptones Proteins 0.000 description 4
- 240000004808 Saccharomyces cerevisiae Species 0.000 description 4
- 235000014680 Saccharomyces cerevisiae Nutrition 0.000 description 4
- 229920002472 Starch Polymers 0.000 description 4
- 108090000704 Tubulin Proteins 0.000 description 4
- 235000013339 cereals Nutrition 0.000 description 4
- 230000012010 growth Effects 0.000 description 4
- 238000011534 incubation Methods 0.000 description 4
- 235000019319 peptone Nutrition 0.000 description 4
- 108090000623 proteins and genes Proteins 0.000 description 4
- 235000019991 rice wine Nutrition 0.000 description 4
- 238000011218 seed culture Methods 0.000 description 4
- 239000007787 solid Substances 0.000 description 4
- 235000019698 starch Nutrition 0.000 description 4
- 239000008107 starch Substances 0.000 description 4
- CSNNHWWHGAXBCP-UHFFFAOYSA-L Magnesium sulfate Chemical compound [Mg+2].[O-][S+2]([O-])([O-])[O-] CSNNHWWHGAXBCP-UHFFFAOYSA-L 0.000 description 3
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 3
- 238000004458 analytical method Methods 0.000 description 3
- 239000008280 blood Substances 0.000 description 3
- 238000010790 dilution Methods 0.000 description 3
- 239000012895 dilution Substances 0.000 description 3
- 239000000706 filtrate Substances 0.000 description 3
- 238000001914 filtration Methods 0.000 description 3
- 239000008103 glucose Substances 0.000 description 3
- 238000011081 inoculation Methods 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 229910000402 monopotassium phosphate Inorganic materials 0.000 description 3
- 230000000877 morphologic effect Effects 0.000 description 3
- 229930187593 rose bengal Natural products 0.000 description 3
- AZJPTIGZZTZIDR-UHFFFAOYSA-L rose bengal Chemical compound [K+].[K+].[O-]C(=O)C1=C(Cl)C(Cl)=C(Cl)C(Cl)=C1C1=C2C=C(I)C(=O)C(I)=C2OC2=C(I)C([O-])=C(I)C=C21 AZJPTIGZZTZIDR-UHFFFAOYSA-L 0.000 description 3
- 229940081623 rose bengal Drugs 0.000 description 3
- STRXNPAVPKGJQR-UHFFFAOYSA-N rose bengal A Natural products O1C(=O)C(C(=CC=C2Cl)Cl)=C2C21C1=CC(I)=C(O)C(I)=C1OC1=C(I)C(O)=C(I)C=C21 STRXNPAVPKGJQR-UHFFFAOYSA-N 0.000 description 3
- 238000012163 sequencing technique Methods 0.000 description 3
- 230000001954 sterilising effect Effects 0.000 description 3
- 239000002028 Biomass Substances 0.000 description 2
- 239000007836 KH2PO4 Substances 0.000 description 2
- GXCLVBGFBYZDAG-UHFFFAOYSA-N N-[2-(1H-indol-3-yl)ethyl]-N-methylprop-2-en-1-amine Chemical compound CN(CCC1=CNC2=C1C=CC=C2)CC=C GXCLVBGFBYZDAG-UHFFFAOYSA-N 0.000 description 2
- 102000004243 Tubulin Human genes 0.000 description 2
- 208000036815 beta tubulin Diseases 0.000 description 2
- 210000004369 blood Anatomy 0.000 description 2
- 238000009835 boiling Methods 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 238000001035 drying Methods 0.000 description 2
- 230000002255 enzymatic effect Effects 0.000 description 2
- 229960004756 ethanol Drugs 0.000 description 2
- 239000000796 flavoring agent Substances 0.000 description 2
- 235000019634 flavors Nutrition 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 230000006799 invasive growth in response to glucose limitation Effects 0.000 description 2
- JVTAAEKCZFNVCJ-UHFFFAOYSA-N lactic acid Chemical compound CC(O)C(O)=O JVTAAEKCZFNVCJ-UHFFFAOYSA-N 0.000 description 2
- 229910052943 magnesium sulfate Inorganic materials 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 238000000399 optical microscopy Methods 0.000 description 2
- 238000007747 plating Methods 0.000 description 2
- 239000000843 powder Substances 0.000 description 2
- 238000012545 processing Methods 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 238000004445 quantitative analysis Methods 0.000 description 2
- 239000008223 sterile water Substances 0.000 description 2
- 238000012360 testing method Methods 0.000 description 2
- ZCYVEMRRCGMTRW-UHFFFAOYSA-N 7553-56-2 Chemical compound [I] ZCYVEMRRCGMTRW-UHFFFAOYSA-N 0.000 description 1
- 241000228212 Aspergillus Species 0.000 description 1
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 1
- 229910021380 Manganese Chloride Inorganic materials 0.000 description 1
- 241000290967 Monascus aurantiacus Species 0.000 description 1
- 241000589516 Pseudomonas Species 0.000 description 1
- 238000010802 RNA extraction kit Methods 0.000 description 1
- 241000235342 Saccharomycetes Species 0.000 description 1
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 1
- 235000021307 Triticum Nutrition 0.000 description 1
- 241000209140 Triticum Species 0.000 description 1
- 235000009754 Vitis X bourquina Nutrition 0.000 description 1
- 235000012333 Vitis X labruscana Nutrition 0.000 description 1
- 240000006365 Vitis vinifera Species 0.000 description 1
- 235000014787 Vitis vinifera Nutrition 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 235000013334 alcoholic beverage Nutrition 0.000 description 1
- 239000011324 bead Substances 0.000 description 1
- 235000013405 beer Nutrition 0.000 description 1
- 210000000988 bone and bone Anatomy 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 239000001110 calcium chloride Substances 0.000 description 1
- 229910001628 calcium chloride Inorganic materials 0.000 description 1
- 238000004364 calculation method Methods 0.000 description 1
- 229940041514 candida albicans extract Drugs 0.000 description 1
- 238000006243 chemical reaction Methods 0.000 description 1
- WIIZWVCIJKGZOK-RKDXNWHRSA-N chloramphenicol Chemical compound ClC(Cl)C(=O)N[C@H](CO)[C@H](O)C1=CC=C([N+]([O-])=O)C=C1 WIIZWVCIJKGZOK-RKDXNWHRSA-N 0.000 description 1
- 229960005091 chloramphenicol Drugs 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000002425 crystallisation Methods 0.000 description 1
- 230000008025 crystallization Effects 0.000 description 1
- 230000007812 deficiency Effects 0.000 description 1
- 229960000935 dehydrated alcohol Drugs 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000003085 diluting agent Substances 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 229910052564 epsomite Inorganic materials 0.000 description 1
- 210000000416 exudates and transudate Anatomy 0.000 description 1
- 235000013305 food Nutrition 0.000 description 1
- 239000011521 glass Substances 0.000 description 1
- XLYOFNOQVPJJNP-ZSJDYOACSA-N heavy water Substances [2H]O[2H] XLYOFNOQVPJJNP-ZSJDYOACSA-N 0.000 description 1
- 230000002779 inactivation Effects 0.000 description 1
- 229910052740 iodine Inorganic materials 0.000 description 1
- 239000011630 iodine Substances 0.000 description 1
- 239000004310 lactic acid Substances 0.000 description 1
- 235000014655 lactic acid Nutrition 0.000 description 1
- 238000002386 leaching Methods 0.000 description 1
- 230000031700 light absorption Effects 0.000 description 1
- 238000011068 loading method Methods 0.000 description 1
- 235000019341 magnesium sulphate Nutrition 0.000 description 1
- 239000011565 manganese chloride Substances 0.000 description 1
- 238000005374 membrane filtration Methods 0.000 description 1
- 238000009629 microbiological culture Methods 0.000 description 1
- 244000005700 microbiome Species 0.000 description 1
- 235000019796 monopotassium phosphate Nutrition 0.000 description 1
- PJNZPQUBCPKICU-UHFFFAOYSA-N phosphoric acid;potassium Chemical compound [K].OP(O)(O)=O PJNZPQUBCPKICU-UHFFFAOYSA-N 0.000 description 1
- 239000000049 pigment Substances 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 150000003839 salts Chemical class 0.000 description 1
- 238000009738 saturating Methods 0.000 description 1
- 238000007789 sealing Methods 0.000 description 1
- 239000006152 selective media Substances 0.000 description 1
- 238000000926 separation method Methods 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- BHZOKUMUHVTPBX-UHFFFAOYSA-M sodium acetic acid acetate Chemical compound [Na+].CC(O)=O.CC([O-])=O BHZOKUMUHVTPBX-UHFFFAOYSA-M 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- VWDWKYIASSYTQR-UHFFFAOYSA-N sodium nitrate Inorganic materials [Na+].[O-][N+]([O-])=O VWDWKYIASSYTQR-UHFFFAOYSA-N 0.000 description 1
- 238000003892 spreading Methods 0.000 description 1
- 238000004659 sterilization and disinfection Methods 0.000 description 1
- 230000009897 systematic effect Effects 0.000 description 1
- 210000001519 tissue Anatomy 0.000 description 1
- 238000012546 transfer Methods 0.000 description 1
- 238000009423 ventilation Methods 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 239000012138 yeast extract Substances 0.000 description 1
Classifications
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- 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
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Abstract
The present invention provides a kind of purple red yeast rice bacteria strains, which is characterized in that classification naming is purple Monascus FJMR24, and scientific name is Monascus purpureus FJMR24；By China typical culture collection center CCTCC preservation, deposit number NO:M2016192, the deposit date is on April 13rd, 2016, preservation address was the Wuhan Wuhan University, China.The saccharification of the bacterial strain, liquefaction vigor are high, the utilization rate of red rice yellow wine fermentation raw material can be improved, and then improve distillation yield.
Description
Technical field
The invention belongs to microorganisms technical fields, and in particular to a kind of purple red yeast rice bacteria strain.
Background technique
The yellow rice wine of China is one of alcoholic drink most ancient in the world, in the world three big brewed wine (yellow rice wine, grape wine and beer
Wine) in occupy an important seat.Red rice yellow wine has unusual effect due to being added to red yeast rice and being fermented.But it is existing
There is also many deficiencies for the red rice yellow wine brewing deposited such as: raw material availability is low, and a large amount of vinasse (20%-30%) cannot obtain sufficiently
Effective use is even directly dropped；Traditional handicraft types of spawn is indefinite, and fermentation condition and vinosity are not easy to control；Pure-blood ferment
The characteristic of red rice yellow wine fails to be able to fully demonstrate.
Red yeast rice is one of wine brewing song, it is as the saccharification of red rice yellow wine brewing, fermentation and raw pastil, quality pair
The quality and distillation yield of yellow rice wine all have significant effect, and often have the title of " bone of wine ".Therefore, those skilled in the art there is an urgent need to
Screening obtain it is a kind of it is high liquefaction, saccharification activity bacterial strain, with improve red rice yellow wine brewing materials utilization rate, save be produced into
Originally, increase liquor stability and improve the flavor of red rice yellow wine, while also the initiative for functional purebred compound song provides new bacterium
Source.
Summary of the invention
Technical problem to be solved by the present invention lies in a kind of purple red yeast rice bacteria strain is provided, classification naming is that purple is red
Aspergillus FJMR24, scientific name are Monascus purpureus FJMR24；It is protected by China typical culture collection center (CCTCC)
Hiding, deposit number NO:M2016192, the deposit date is on April 13rd, 2016, preservation address was that China, the Wuhan, the Wuhan is big
It learns.The purple red yeast rice bacteria strain has high liquefaction, high saccharification activity, can improve the utilization rate of red rice yellow wine brewing materials, save
Production cost increases liquor stability and improves the flavor of red rice yellow wine.
One, the screening of purple Monascus FJMR24
1 materials and methods
1.1 material
1.1.1 sample is separated
It collects to make wine from Fujian, Jiangxi and other places by this lab assistant and commonly uses the sort of quyis such as red yeast rice, wheat koji and save.
1.1.2 control strain source
(1) purple Monascus (Monascus purpureus) 41450: it is purchased from Chinese industrial Microbiological Culture Collection management
Center, hereinafter referred to as 41450；
(2) FM23: by Gutian Area, Fujian Province, institute of microbiology is provided, and produces one of common bacterial strain for red rice yellow wine.
1.1.3 culture medium
(1) malt extract medium: 8 ° of brewer's wort, agar 2% (is added) when with solid medium, pH5.6.
(2) rose bengal medium: peptone 0.5%, glucose sugar 1.0%, potassium dihydrogen phosphate 0.1%, magnesium sulfate
0.05%, agar 1.5%, rose-bengal 0.0033%, chloramphenicol 0.01%.
(3) production amylase, carbohydrase fermentation medium: peptone 0.5%, yeast extract 0.5%, soluble starch 3.0%,
CaCl2·2H2O 0.05%, MnCl24H2O 0.05%, MgSO4·7H2O 0.05%, KH2PO40.1%, pH7.0-
7.2。
1.2 method
1.2.1 sample treatment
Sterile working will separate sample and grind, and take 5g in 45mL sterile water, and 150 revs/min of shaking tables vibrate 2h, spare.
1.2.2 single strain isolates and purifies
With log10 dilution rubbing method, by suitable dilution gradient (making the clump count on each plate at 10-50) sample
It is coated on rose-bengal or wort agar medium plate, 30 DEG C are cultivated 1-2 days, choose difference according to colony morphology characteristic
Bacterial strain accesses in another blank plate and continues to cultivate, repeated multiple times up to bacterial strain Economical Purification, and it is oblique in wort agar to transfer
Face, 30 DEG C are cultivated 6-7 days, are covered with 4 DEG C of refrigerators of postposition to mycelia and are saved, spare.
1.2.3 prepared by spore suspension
For picking slant pore into sterile water, being broken up with bead makes spore concentration reach 0.5 × 106A/mL.
1.2.4 bacterial strain primary dcreening operation
Using plate transparent circle method.Spore liquid is subjected to 10 times of gradient dilutions, acceptable diluent sample is selected to be coated on equivalent
On amylase, carbohydrase culture medium flat plate, 30 DEG C constant temperature incubation 1-2 days, when newly formed bacterium colony and conidium not yet generate,
Select clump count in the plate of 5-6, iodine solution be added dropwise, the transparent loop diameter (H) presented with vernier caliper measurement periphery of bacterial colonies with
Colony diameter (C), and ratio calculated (H/C value), analysis determine more excellent bacterial strain as secondary screening bacterium, are connected to brewer's wort inclined-plane, 30 DEG C,
It 6-7 days, after mycelia is covered with, is saved in refrigerator, it is spare.
1.2.5 the activation of secondary screening bacterium
The spore liquid of secondary screening bacterium is inoculated in seed culture medium with 10%, 150 revs/min, 30 DEG C are cultivated 2 to 3 days, standby
With.
1.2.6 sample preparation and enzyme activity determination
Enter 150mL amylase, carbohydrase selective medium, the seed of 5% activation of access respectively into 500mL triangular flask
Liquid, 30 DEG C, 150 revs/min shaking table culture 6 days, with 0.45um membrane filtration fermentation liquid, filtrate is the sample of enzyme activity detection
Product, remaining mycelia measure its biomass after drying to constant weight.
The measuring method of biomass:
Wash with distilled water by filtered mycelium, it is weighed after 80 DEG C of drying to constant weights, calculates mycelium dry weight, g/
L。
Enzyme activity determination method:
A, amylase activity measures
Using RNA isolation kit, concrete operations are carried out according to the description of product.Amylase caused by 1g mycelium 40 DEG C,
Under conditions of pH6.0,1h liquefaction 1mg soluble starch is defined as 1 enzyme activity unit.
B, saccharifying enzymic activity measures
Preparation of reagents is carried out referring to GB 8276-2006.Two 50mL colorimetric cylinders (A, B pipe) is taken, soluble shallow lake is separately added into
Powder solution 10mL and acetic acid-sodium acetate buffer solution 8mL, shakes up.5min-10min is preheated in 40 DEG C of waters bath with thermostatic control.In B pipe
Enzyme solution 10.0mL to be measured is added, timing immediately shakes up.At this temperature after accurate response 60min, respectively add into A, B pipe immediately
Sodium hydroxide solution 0.2mL, shakes up, while two pipes being taken out, and is water-cooled rapidly, and enzyme solution to be measured is added in A pipe
10.0mL as blank control.The reaction solution drawn in above-mentioned two pipe of A, B respectively dilutes certain multiple, takes 0.5m to be added respectively
1.5mL days NS reagents, taking-up is rapidly cooled to room temperature after 15min is heated in boiling water bath, 10mL distilled water is added, in wavelength
Light absorption value is measured at 480nm.
Under conditions of 40 DEG C, pH4.6,1h hydrolyzes soluble starch and generates the Portugal 1mg carbohydrase caused by 1g mycelium
Grape sugar, is defined as 1 enzyme activity unit.
2 results and analysis
The separation of 2.1 mould single plants and primary dcreening operation
It is isolated from the sample of collection to 67 plants of moulds, number is respectively MR1-MR32, MB1-MB19, MW1-MW16.
The characteristic for decomposing transparent circle can be generated in periphery of bacterial colonies using starch according to amylase, carbohydrase bacterial strain is produced, it herein will be saturating
Bright loop diameter and colony diameter ratio (H/C) are as the initial indication for judging strain enzyme-producing ability.The result shows that different strains exist
Transparent circle and bacterium colony on plate is (such as Fig. 1) not of uniform size, and there is also large change (such as tables 1) for H/C value.Isolated strains
FJMR10, FJMR24, FJMW8, FJMR1, FJMB12, FJMR20, FJMR18, MR32, FJMW15, FJMW13 and control bacterium FM23
Enzymatic productivity be above another pair according to bacterium purple Monascus 41450, and significant difference；Other strain enzyme-producing abilities are weaker even
It can't detect transparent circle.
Producing enzyme situation of 1 isolated strains of table on plate
Note: 1) "-" indicates to can't detect transparent circle on plate；2) only the bacterial strain bigger than 41450 to H/C value carries out this table
Difference analysis；3) lowercase " a-j " indicates significant difference (P≤0.05), similarly hereinafter.
The quantitative analysis of 2.2 primary dcreening operation strain enzyme-producing abilities
2.2.1 amylase, carbohydrase ability are produced
It is control bacterium with FM23, enzyme activity quantitative analysis is carried out to 10 plants of bacterium that primary dcreening operation obtains, as a result such as table 2.Except FJMR1
Outside FJMW13, the amylase activity of other 8 plants of bacterium is all remarkably higher than FM23；In terms of saccharifying enzymic activity, FJMR24, FJMR10,
The enzymatic productivity of FJMW8, FJMR32, FJMR20 and FJMB12 are better than FM23, wherein FJMR24, FJMR10, FJMW8 difference therewith
Significantly；On the whole, its saccharifying enzymic activity of the high bacterial strain of amylase activity is also higher, and the two is with uniformity；The shallow lake of FJMR24
The equal highest of powder enzyme, saccharifying enzymic activity, respectively up to 2200 ± 18.5U/g and 2330.4 ± 31.9U/g.
Amylase, the saccharifying enzymic activity of 2 primary dcreening operation bacterial strain of table
By upper table 2 it is found that purple Monascus FJMR24 amylase activity, saccharifying enzymic activity highest, show that the bacterial strain has
Height liquefaction, high saccharification activity, can improve the utilization rate of red rice yellow wine brewing materials, increase the distillation yield of red rice yellow wine.
It will be during purple Monascus FJMR24 (scientific name be Monascus purpureus FJMR24) that screening obtains be preserved in
State Type Tissue Collection CCTCC, deposit number NO:M2016192, the deposit date is on April 13rd, 2016, preservations
Address is the Wuhan Wuhan University, China.
Two, the morphological feature of purple Monascus FJMR24, gene sequencing
The preparation of 1 culture medium and spore suspension
1.1 culture medium
1.1.1MEA culture medium: powdery malt extract 2%, peptone 1%, glucose 2%, agar 1.5%.
1.1.2 malt extract medium: 8 ° of brewer's wort, agar 2% (is added) when with solid medium, pH5.6.
The preparation of 1.2 spore suspensions
By FJMR24 streak inoculation in wort agar inclined-plane, 30 DEG C constant temperature incubation 7 days, will with 10mL sterile saline
Spore on inclined-plane is washed down, and vortex oscillator mixes, 4 layers of lens wiping paper filtering, and is counted by blood counting chamber method, by filtrate tune
It is made into 1 × 106The spore suspension of a/mL.
The morphological feature of 2FJMR24
2.1 bacterium colonies and microscopic morphology observation
2.1.1 colony morphological observation: dibbling method is used, the FJMR24 of activation is inoculated in MEA plate, in 30 DEG C of constant temperature
Culture is observed colonial morphology, growing state in 10 days, and is recorded in time.
2.1.2 microscopic morphology is observed: utilizing slide glass cultivation, the spore liquid of FJMR24 is seeded in MEA agar thin slice week
It encloses, 30 DEG C after constant temperature incubation 6-7 days, with shapes such as optical microscopy observation hypha form, conidium, ascospore, cleistotheciums
State feature, and taken pictures by image processing software, analyzed.
2.2 colony morphology characteristic
FJMR24 is cultivated on MEA culture medium, temperature is 30 DEG C, is cultivated 10 days.The colonial morphology of FJMR24 such as Fig. 2 institute
Show, bacterium colony is round, and diameter 28-30mm, edge is complete, orange；Surface is flat, center projections；Quality felt is velvet-like；Reverse side center is shallow
Red, surrounding are orange；No liquid body exudate generates, and no soluble pigment generates.
2.3 individual morphology features
Form of the bacterial strain FJMR24 under optical microscopy and scanning electron microscope, as shown in Figure 3.Mycelia is irregularly divided
Branch, it is transparent, there is obvious oil droplet, there is tabula, tool excipuliform crystallization on wall, width is 3-5 μm；The life of conidium list or chain are raw,
It is raw on mycelia top or side shoot top, pyriform or spherical shape, 7-11 μm；Ascospore ellipse or oval, (4-6) μ m
(3-4.5) μm, wall is smooth；Cleistothecium is subsphaeroidal, 20-50 μm, is coated with brown.
The gene sequencing of 3FJMR24
3.1 phylogenetic trees based on the building of ITS rDNA gene order
The ITS rDNA sequencing length of FJMR24 is 501bp, compares on NCBI and constructs systematic evolution tree such as Fig. 4.
FJMR24 and Monascus purpureus ATCC 16379 (AY498573), Monascus aurantiacus CICC
5014 (AY629435) gather in same branch, and homology is up to 100%, and the Pseudomonas is in monascus van tieghem (Monascussp.).
3.2 phylogenetic trees based on the building of β-tubulin gene order
For the types of spawn for further confirming that FJMR24, the β-tubulin of the bacterium is sequenced to obtain the sequence of 901bp
Column, to phylogenetic tree such as Fig. 5 of related kind of β-tubulin sequence.FJMR24 and Monascus purpureus ATCC
16379 (AY498573) gather in same branch, and homology analyzes result in conjunction with ITS rDNA and morphology is special up to 100%
Sign can determine that FJMR24 is purple Monascus (Monascus purpureus).
Detailed description of the invention
The transparent circle that the part Fig. 1 bacterial strain generates on plate；
Fig. 2 FJMR24 colonial morphology；
Fig. 3 FJMR24 individual morphology；
The phylogenetic tree that Fig. 4 is constructed based on ITS rDNA gene order；
The phylogenetic tree that Fig. 5 is constructed based on β-tubulin gene order.
Specific embodiment
In order to describe the technical content, the structural feature, the achieved object and the effect of this invention in detail, below in conjunction with specific implementation
Mode is explained in detail.
Embodiment 1
The fermentation character of FJMR24
1 thermal adaptability
FJMR24 spore suspension is inoculated into malt extract liquid and plating medium, 15 DEG C, 20 DEG C, 25 are respectively placed in
DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C of incubator stationary cultures.
The influence that 3 fermentation temperature of table grows FJMR24
Note: "-" is not grow；" ± " is slightly to grow；"+" is that growth is general；" ++ " is well-grown；" +++ " makes a living
Length is vigorous；The volume that increment measures sample is 50mL.
From table 3 it can be seen that purple Monascus FJMR24 thermal adaptability is strong；Being 15 DEG C to 45 DEG C in temperature can give birth to
It is long, yeasting can be well adapted to.
2pH adaptability
It is 2.0,3.0,4.0,5.0,6.0,7.0,8.0 that FJMR24 spore suspension is inoculated into pH value (lactic acid adjusting) respectively
In malt juice liquid medium, it is placed in 30 DEG C of constant incubator stationary cultures.
The influence that the fermentation of table 4 pH grows FJMR24
Note: "-" is not grow；" ± " is slightly to grow；"+" is that growth is general；" ++ " is well-grown；" +++ " makes a living
Length is vigorous；The volume that increment measures sample is 50mL.
From table 4, it can be seen that purple Monascus FJMR24 is adaptable to pH；It can be grown in pH for 2 to 8, that is,
It says that purple Monascus FJMR24 can be grown under highly acid and weak basic condition, yeasting can be well adapted to.
3 ethanol tolerances
The dehydrated alcohol of filtration sterilization is added in 45 DEG C or so of sterile malt juice body and plating medium, makes to train
The concentration of alcohol for supporting base is respectively 8%, 10%, 12%, 15%, 18%, 20%, is inoculated with FJMR24 spore suspension, is placed in 30 DEG C
Constant incubator stationary culture.
The influence that 5 alcoholic strength of table grows FJMR24
Note: "-" is not grow；" ± " is slightly to grow；"+" is that growth is general；" ++ " is well-grown；" +++ " makes a living
Length is vigorous；The volume that increment measures sample is 50mL.
As can be seen from Table 5, purple Monascus FJMR24 is good to ethanol tolerance, alcoholic strength at 8% to 18%,
FJMR24 can be grown, and can well adapt to yeasting.
Embodiment 2
It is as follows that FJMR24 forced fermentation tests concrete technology method:
1 material
1.1 culture medium
1.1.1 seed culture medium: NaNO30.2%, KH2PO40.15%, MgSO40.1%, glucose 5%, peptone
1.5 ﹪, pH are natural.
1.1.2 malt extract medium: 10 ° of wave woods brewer's wort 1L, pH 5.4.
1.2 long-grained nonglutinous rices: commercially available
1.3 bacterial strain
1.3.1FM23: being provided by Institute of Micro-biology, Gutian Area, Fujian Province county, produce one of common bacterial strain for red rice yellow wine；
1.3.2 it saccharomyces cerevisiae JH301: by this laboratory breeding and saves.
2 methods
The preparation of 2.1 spore suspensions
By FJMR24 or FM23 streak inoculation in wort agar inclined-plane, 30 DEG C constant temperature incubation 7 days, with 10mL sterile physiological
Salt water washes down the spore on inclined-plane, and vortex oscillator mixes, 4 layers of lens wiping paper filtering, and is counted by blood counting chamber method, will
Filtrate is adjusted to 1 × 106The spore suspension of a/mL.
2.2 seed culture
The loading 50mL seed culture medium into 250mL triangular flask, 10% inoculation FJMR24 or FM23 spore suspension, 30 DEG C,
150 revs/min shaking table culture 2-3 days, 4 layers of gauze filter off spare after mycelia.
The preparation of 2.3 solid fermentation culture mediums
Commercially available long-grained nonglutinous rice is impregnated into 3h at room temperature, leaching is clear, drain away the water after be put in boiling 30min in food steamer, dispensed after spreading for cooling
Enter in 250mL triangular flask, per bottled 30g, wraps up 8 layers of gauze sealing, 121 DEG C of sterilizing 20min.Taking-up is cooled to 40 DEG C or so,
Shaking triangular flask keeps the grain of rice loose, while adsorbing the condensed water of bottle wall.
2.4FJMR24 the preparation with FM23 pure red koji rice
The FJMR24 of activation or FM23 is inoculated in solid fermentation culture medium with 5%, is piled up one foot of bottom of bottle, 30 DEG C
Occur Monascus mycelium on culture to the grain of rice, shaking flask 1 time, adsorbs the condensed water in bottle wall, and meter Qu is shakeout；To grain of rice table
Face is paved with red mycelium, and shaking flask 1 time, and appropriate amounts of sterilized water is added according to grain of rice dry tack free degree；Daily shaking flask 2-3 later
It is secondary, to adjust red yeast rice ventilation character and keep growth uniform.The red yeast rice cultivated 10 days is put in 40 DEG C of baking ovens and is dried to moisture content <
15%.
The activation of 2.5 saccharomycete
A ring is taken to be inoculated in 100mL malt extract medium saccharomyces cerevisiae JH301 hook, 30 DEG C of culture 10-12h are spare.
The forced fermentation of 2.6FJMR24 is tested
It is the basic sort of quyi with FM23 red yeast rice, is to strengthen song with FJMR24 pure red koji rice, using the glutinous rice of sterilizing as base-material,
Following processing is done respectively:
1:10g FM23 red yeast rice+10g FJMR24 pure red koji rice (inactivation)+2mL yeast activated liquid is handled, is inoculated in
In 100g glutinous rice, 30 DEG C ferment 15 days.
2:10g FM23 red yeast rice+10g FJMR24 pure red koji rice+2mL yeast activated liquid is handled, it is glutinous to be inoculated in 100g
Meter Zhong, 30 DEG C ferment 15 days.
To the alcoholic strength and the indexs such as distillation yield in fermentation ends test sample.
The measurement of 2.7 alcoholic strengths
It is carried out according to GB/T13662-2008 " yellow rice wine ".
The measurement of 2.8 distillation yields
Standard atmospheric pressure, 20 DEG C, the alcohol output that the content of a unit institute output is 20%.Distillation yield calculation formula is
Distillation yield %=(alcoholic strength % × go out capacity for liquor kg/ rice dosage kg/20% alcohol).
3 results
It is as shown in table 6 that purple Monascus FJMR24 in FM23 red yeast rice fermentation prepares the application effect in red rice yellow wine, adds
After entering purple Monascus FJMR24, the alcoholic strength and distillation yield of red rice yellow wine are improved, and compared with control (processing 1), wine
Precision reaches significant difference (α < 0.05), and distillation yield reaches extremely significant sex differernce (α < 0.01)；Illustrate purple Monascus FJMR24
High liquefaction, saccharification characteristic improves the utilization rate of fermentation raw material, and then increases the distillation yield of red rice yellow wine.
6 purple Monascus FJMR24 forced fermentation effect of table
The above description is only an embodiment of the present invention, is not intended to limit the scope of the invention, all to utilize this hair
Equivalent structure or equivalent flow shift made by bright description or Figure of description, is applied directly or indirectly in other
Relevant technical field, is included within the scope of the present invention.
Claims (1)
1. a kind of purple red yeast rice bacteria strain, which is characterized in that classification naming is purple Monascus FJMR24, and scientific name is
Monascuspurpureus FJMR24；By China typical culture collection center (CCTCC) preservation, deposit number NO:
M2016192, the deposit date is on April 13rd, 2016, preservation address was the Wuhan Wuhan University, China.
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