CN106399121A

CN106399121A - Monascus purpureus strain

Info

Publication number: CN106399121A
Application number: CN201610659759.XA
Authority: CN
Inventors: 任香芸; 何志刚; 林晓婕; 梁璋成; 林晓姿; 李维新; 庄林歆
Original assignee: Institute of Agricultural Engineering Technology of Fujian Academy of Agricultural Sciences
Current assignee: Fujian Academy Of Agricultural Sciences Agricultural Product Processing Research Institute
Priority date: 2016-08-12
Filing date: 2016-08-12
Publication date: 2017-02-15
Anticipated expiration: 2036-08-12
Also published as: CN106399121B

Abstract

The invention provides a monascus purpureus strain. The monascus purpureus strain is characterized in that the strain is classified and named as monascus purpureus FJMR24, and the scientific name of the strain is monascus purpureus FJMR24; and the monascus purpureus strain is preserved in China Center for Type Culture Collection in Wuhan University, Wuhan, China on April 13, 2016 with preservation number of NO: M2016192. The strain is high in saccharifying and liquefying activities; and the utilization rate of red-koji yellow rice wine fermentation raw materials can be enhanced, so that a wind yield is improved.

Description

A kind of Monascus purpureus bacterial strain

Technical field

The invention belongs to microbial technology field is and in particular to a kind of Monascus purpureus bacterial strain.

Background technology

The yellow wine of China is one of alcoholic drink the most ancient in the world, in the world three big brewed wine (yellow wine, wine and beer Wine) in occupy an important seat.Monas cuspurpureus Went yellow wine is had distinguished effect because with the addition of Monas cuspurpureus Went and being fermented.But, existing The Monas cuspurpureus Went yellow wine deposited is brewageed and be there is also many deficiencies such as：Raw material availability is low, and a large amount of distiller grains (20%-30%) can not obtain fully Effectively utilizes are even directly dropped；Traditional handicraft types of spawn is indefinite, and fermentation condition and vinosity are wayward；Pure-blood ferment The characteristic of Monas cuspurpureus Went yellow wine fails to be fully demonstrated.

Monas cuspurpureus Went is one of wine brewing song, saccharifying, fermentation and raw pastil that it is brewageed as Monas cuspurpureus Went yellow wine, its quality pair The quality of yellow wine and distillation yield all have significant effect, and often have the title of " bone of wine ".Therefore, those skilled in the art in the urgent need to Screening obtains a kind of high liquefaction, the bacterial strain of saccharification activity, and to improve the utilization rate of Monas cuspurpureus Went yellow wine brewing materials, saving produces into Originally, increase liquor stability and improve the local flavor of Monas cuspurpureus Went yellow wine, also provide new bacterium for the purebred compound bent initiative of feature simultaneously Source.

Content of the invention

The technical problem to be solved is to provide a kind of Monascus purpureus bacterial strain, and its Classification And Nomenclature is that purple is red Aspergillosiss FJMR24, scientific name is Monascus purpureus FJMR24；Protected by China typical culture collection center (CCTCC) Hide, deposit number is NO：M2016192, preservation date is on April 13rd, 2016, and preservation address is China. Wuhan. Wuhan is big Learn.This Monascus purpureus bacterial strain has high liquefaction, high saccharification activity, can improve utilization rate, the saving of Monas cuspurpureus Went yellow wine brewing materials Production cost, increase liquor stability and improve the local flavor of Monas cuspurpureus Went yellow wine.

First, the screening of Monascus purpureus FJMR24

1 materials and methods

1.1 material

1.1.1 separate sample

Collected the conventional alcoholic fermented product starter such as Monas cuspurpureus Went, malt yeast of wine brewing from Fujian, Jiangxi and other places and preserved by this lab assistant.

1.1.2 control strain source

(1) Monascus purpureus (Monascus purpureus) 41450：It is purchased from the management of Chinese industrial Microbiological Culture Collection Center, hereinafter referred to as 41450；

(2)FM23：By Gutian Area, Fujian Province, institute of microbiology provides, and is that Monas cuspurpureus Went yellow wine produces one of conventional bacterial strain.

1.1.3 culture medium

(1) malt extract medium：8 ° of beerwort, agar 2% (join and add during 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%, chloromycetin 0.01%.

(3) amylase, saccharifying enzyme fermentation medium are produced：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 sterilized water, and 150 revs/min of shaking tables vibrate 2h, standby.

1.2.2 single strain isolates and purifies

Use log10 dilution rubbing method, by suitable dilution gradient (making the clump count on each flat board at 10-50) sample Coat on rose-bengal or wort agar culture medium flat plate, cultivate 1-2 days for 30 DEG C, difference is chosen according to colony morphology characteristic Bacterial strain, accesses and continues culture in another blank plate, repeated multiple times until bacterial strain Economical Purification, and it is oblique in wort agar to transfer Face, cultivates 6-7 days for 30 DEG C, treats that mycelia covers with rearmounted 4 DEG C of Refrigerator stores, standby.

1.2.3 spore suspension preparation

To sterilized water, broken up with bead makes spore concentration reach 0.5 × 10 to picking slant pore⁶Individual/mL.

1.2.4 bacterial strain primary dcreening operation

Using flat board transparent circle method.Spore liquid is carried out 10 times of gradient dilutions, selects acceptable diluent sample to coat equivalent On amylase, saccharifying enzyme culture medium flat plate, 30 DEG C of constant temperature culture 1-2 days, when newly formed bacterium colony conidium not yet produces, Select clump count in the flat board of 5-6, Deca iodine solution, the transparent loop diameter (H) being presented with vernier caliper measurement periphery of bacterial colonies with Colony diameter (C), and ratio calculated (H/C value), analysis determines that more excellent bacterial strain, as secondary screening bacterium, is connected to beerwort inclined-plane, 30 DEG C, 6-7 days, after mycelia is covered with, in Refrigerator store, standby.

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, cultivates 2 to 3 days for 30 DEG C, standby With.

1.2.6 sample preparation and enzyme activity determination

Enter 150mL amylase, saccharifying enzyme selective medium respectively in 500mL triangular flask, access the seed of 5% activation Liquid, 30 DEG C, 150 revs/min of shaking table cultures 6 days, with 0.45um membrane filtration fermentation liquid, filtrate is the sample of enzyme activity detection Product, remaining mycelia is dried to constant weight and measures its Biomass.

The assay method of Biomass：

Mycelium after filtering is cleaned with distilled water, 80 DEG C of dryings weigh to constant weight, calculate mycelium dry weight, g/ L.

Enzyme activity determination method：

A, amylase activity measure

Using RNA isolation kit, concrete operations are carried out according to the description of product.Amylase produced by 1g mycelium 40 DEG C, Under conditions of pH6.0,1h liquefaction 1mg soluble starch, it is defined as 1 enzyme activity unit.

B, saccharifying enzymic activity measure

Preparation of reagents is carried out with reference to GB 8276-2006.Take two 50mL color comparison tubes (A, B manage), be separately added into solubility and form sediment Powder solution 10mL and acetic acid-sodium acetate buffer solution 8mL, shake up.5min-10min is preheated in 40 DEG C of waters bath with thermostatic control.In B pipe Add enzyme liquid 10.0mL to be measured, immediately timing, shake up.At this temperature after accurate response 60min, respectively add in A, B pipe immediately Sodium hydroxide solution 0.2mL, shakes up, and takes out two pipes simultaneously, is water-cooled rapidly, and adds enzyme liquid to be measured in A pipe 10.0mL as blank.The reactant liquor drawn in above-mentioned A, B two pipe respectively dilutes certain multiple, takes 0.5m to add respectively 1.5mL days NS reagent, takes out after heating 15min in boiling water bath and is rapidly cooled to room temperature, add 10mL distilled water, in wavelength Light absorption value is measured at 480nm.

Under conditions of 40 DEG C, pH4.6,1h hydrolysis soluble starch produces 1mg Portugal to saccharifying enzyme produced by 1g mycelium Grape sugar, is defined as 1 enzyme activity unit.

2 results and analysis

The separation of 2.1 mycete individual plants and primary dcreening operation

Isolate 67 plants of mycetes from the sample collected, numbering is respectively MR1-MR32, MB1-MB19, MW1-MW16. According to producing amylase, saccharifying enzyme bacterial strain can utilize starch and producing the characteristic decomposing transparent circle in periphery of bacterial colonies, herein will be thoroughly Bright loop diameter and colony diameter ratio (H/C) are as the initial indication judging strain enzyme-producing ability.Result shows, different strains exist Transparent circle on flat board and bacterium colony (as Fig. 1) not of uniform size, its H/C value there is also large change (as table 1).Isolated strains FJMR10, FJMR24, FJMW8, FJMR1, FJMB12, FJMR20, FJMR18, MR32, FJMW15, FJMW13 and comparison bacterium FM23 Enzymatic productivity be above another comparison bacterium Monascus purpureus 41450, and significant difference；Other strain enzyme-producing abilities are compared with overly soft pulse extremely Can't detect transparent circle.

Producing enzyme situation on flat board for table 1 isolated strains

Note：1) "-" represents and can't detect transparent circle on flat board；2) this table is only carried out to the H/C value bacterial strain bigger than 41450 Difference analysis；3) lower case " a-j " represents significant difference (P≤0.05), similarly hereinafter.

The quantitative analyses of 2.2 primary dcreening operation strain enzyme-producing abilities

2.2.1 amylase, saccharifying enzyme ability are produced

With FM23 for compareing bacterium, 10 plants of bacterium that primary dcreening operation is obtained carry out enzyme activity quantitative analyses, result such as table 2.Except FJMR1 Outside FJMW13, the amylase activity of other 8 plants of bacterium is all remarkably higher than FM23；Saccharifying enzymic activity aspect, FJMR24, FJMR10, The enzymatic productivity of FJMW8, FJMR32, FJMR20 and FJMB12 is 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 both have concordance；The shallow lake of FJMR24 The equal highest of powder enzyme, saccharifying enzymic activity, reaches 2200 ± 18.5U/g and 2330.4 ± 31.9U/g respectively.

The amylase of table 2 primary dcreening operation bacterial strain, saccharifying enzymic activity

From upper table 2, Monascus purpureus FJMR24 amylase activity, saccharifying enzymic activity highest, show that this bacterial strain has High liquefaction, high saccharification activity, can improve the utilization rate of Monas cuspurpureus Went yellow wine brewing materials, increase the distillation yield of Monas cuspurpureus Went yellow wine.

During the Monascus purpureus FJMR24 (scientific name is Monascus purpureus FJMR24) that screening is obtained is preserved in State Type Tissue Collection CCTCC, deposit number is NO：M2016192, preservation date is on April 13rd, 2016, preservation Address is China. Wuhan. Wuhan University.

2nd, the morphological feature of Monascus purpureus FJMR24, gene sequencing

1 culture medium and the preparation of spore suspension

1.1 culture medium

1.1.1MEA culture medium：Powdery Fructus Hordei Germinatus extract 2%, peptone 1%, glucose 2%, agar 1.5%.

1.1.2 malt extract medium：8 ° of beerwort, agar 2% (join and add during solid medium), pH5.6.

The preparation of 1.2 spore suspensions

By FJMR24 streak inoculation in wort agar inclined-plane, 30 DEG C of constant temperature culture 7 days, will with 10mL physiological saline solution Spore on inclined-plane is washed down, and vortex oscillator mixes, and 4 layers of lens paper filter, and are counted by blood counting chamber method, and filtrate is adjusted It is made into 1 × 10⁶The spore suspension of individual/mL.

The morphological feature of 2FJMR24

2.1 bacterium colonies and microscopic morphology observational method

2.1.1 colony morphological observation：Using dibbling method, the FJMR24 of activation is inoculated in MEA flat board, in 30 DEG C of constant temperature Culture, observes colonial morphology, growing state in 10 days, and is recorded in time.

2.1.2 microscopic morphology is observed：Using slide glass culture method, the spore liquid of FJMR24 is seeded in MEA agar thin slice week Enclose, after 30 DEG C of constant temperature culture 6-7 days, with shapes such as observation by light microscope hypha form, conidium, ascospore, cleistotheciums State feature, and carry out taking pictures, analyze by image processing software.

2.2 colony morphology characteristic

FJMR24 is cultivated on MEA culture medium, temperature is 30 DEG C, cultivates 10 days.The colonial morphology of FJMR24 such as Fig. 2 institute Show, bacterium colony is circular, diameter 28-30mm, edge is complete, orange；Surface is flat, center projections；Quality felt is velvet-like；Reverse side central authorities are shallow Redness, orange around；No liquid body exudate produces, and no soluble pigment produces.

2.3 individual morphology features

Form under optical microscope and scanning electron microscope for the bacterial strain FJMR24, as shown in Figure 3.Mycelia is irregularly divided , transparent, there is obvious oil droplet, have tabula, tool excipuliform crystallization on wall, width is 3-5 μm；Conidium is singly raw or chain is given birth to, and Give birth on mycelia top or side shoot top, pyriform or spherical, 7-11 μm；Ascospore ellipse or oval, (4-6) μ m (3-4.5) μm, wall smooths；Cleistothecium is subsphaeroidal, 20-50 μm, is coated brown.

The gene sequencing of 3FJMR24

3.1 phylogenetic trees being built based on ITS rDNA gene order

The ITS rDNA sequencing length of FJMR24 is 501bp, compares and constructing system cladogram such as Fig. 4 on NCBI. FJMR24 and Monascus purpureus ATCC 16379 (AY498573), Monascus aurantiacus CICC 5014 (AY629435) gather in same branch, and homology is up to 100%, and this Pseudomonas is in monascus van tieghem (Monascussp.).

3.2 phylogenetic trees being built based on β-tubulin gene order

For further confirming that the types of spawn of FJMR24, the β-tubulin of this bacterium is carried out being sequenced obtains the sequence of 901bp Row, the phylogenetic tree such as Fig. 5 to related kind β-tubulin sequence.FJMR24 and Monascus purpureus ATCC 16379 (AY498573) gather in same branch, and homology reaches 100%, special in conjunction with ITS rDNA analysis result and morphology Levy it may be determined that FJMR24 is Monascus purpureus (Monascus purpureus).

Brief description

The transparent circle that Fig. 1 part bacterial strain produces on flat board；

Fig. 2 FJMR24 colonial morphology；

Fig. 3 FJMR24 individual morphology；

The phylogenetic tree that Fig. 4 is built based on ITS rDNA gene order；

The phylogenetic tree that Fig. 5 is built based on β-tubulin gene order.

Specific embodiment

By describing the technology contents of the present invention, structural features in detail, being realized purpose and effect, below in conjunction with being embodied as Mode is explained in detail.

Embodiment 1

The fermentation character of FJMR24

1 thermal adaptability

FJMR24 spore suspension is inoculated in malt extract liquid and plating medium, be respectively placed in 15 DEG C, 20 DEG C, 25 DEG C, 30 DEG C, 35 DEG C, 40 DEG C, 45 DEG C, 50 DEG C of incubator quiescent culture.

The impact that table 3 fermentation temperature grows to 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 of increment determination sample is 50mL.

From table 3 it can be seen that Monascus purpureus FJMR24 thermal adaptability is strong；All can give birth to for 15 DEG C to 45 DEG C in temperature 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 regulation) respectively In malt juice liquid medium, it is placed in 30 DEG C of constant incubator quiescent culture.

The impact that table 4 fermentation pH grows to FJMR24

From table 4, it can be seen that Monascus purpureus FJMR24 is to pH strong adaptability；It is 2 to 8 equal energy growths in pH, that is, Say that Monascus purpureus FJMR24 all can grow 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 about of sterile malt juice body and plating medium, makes training The concentration of alcohol of foster base is respectively 8%, 10%, 12%, 15%, 18%, 20%, inoculates FJMR24 spore suspension, is placed in 30 DEG C Constant incubator quiescent culture.

The impact that table 5 alcoholic strength grows to FJMR24

As can be seen from Table 5, Monascus purpureus FJMR24 is good to ethanol tolerance, alcoholic strength when 8% to 18%, FJMR24 can grow, and can well adapt to yeasting.

Embodiment 2

FJMR24 forced fermentation test concrete technology method is as follows：

1 material

1.1 culture medium

1.1.1 seed culture medium：NaNO₃0.2%, KH₂PO₄0.15%, MgSO₄0.1%, glucose 5%, peptone 1.5, pH is natural.

1.1.2 malt extract medium：10 ° of ripple woods beerwort 1L, pH 5.4.

1.2 Semen Oryzae：Commercially available

1.3 bacterial strain

1.3.1FM23：There is provided by Institute of Micro-biology of Gutian Area, Fujian Province county, be that Monas cuspurpureus Went yellow wine produces one of conventional bacterial strain；

1.3.2 saccharomyces cerevisiae JH301：By the selection-breeding of this laboratory and preserve.

2 methods

2.1 the preparation of spore suspension

By FJMR24 or FM23 streak inoculation in wort agar inclined-plane, 30 DEG C of constant temperature culture 7 days, use 10mL sterile physiological Spore on inclined-plane is washed down by saline, and vortex oscillator mixes, and 4 layers of lens paper filter, and are counted by blood counting chamber method, will Filtrate is adjusted to 1 × 10⁶The spore suspension of individual/mL.

2.2 seed culture

The loading 50mL seed culture medium in 250mL triangular flask, 10% inoculation FJMR24 or FM23 spore suspension, 30 DEG C, 150 revs/min of shaking table cultures 2-3 days, standby after 4 layers of gauzes elimination mycelia.

The preparation of 2.3 solid fermentation culture medium

3h will be soaked under commercially available Semen Oryzae room temperature, drench clear, drain away the water after be put in steaming and decocting 30min in food steamer, subpackage after spreading for cooling Enter in 250mL triangular flask, often bottled 30g, 8 layers of gauze sealing of wrapping, 121 DEG C of sterilizing 20min.Taking-up is cooled to 40 DEG C about, Shake triangular flask makes the grain of rice loose, adsorbs the condensed water of bottle wall simultaneously.

2.4FJMR24 and the preparation of FM23 pure red koji rice

FJMR24 or FM23 of activation is inoculated in solid fermentation culture medium with 5%, is piled up bottom of bottle one foot, 30 DEG C Cultivate and Monascus mycelium is occurred to the grain of rice, shaking flask 1 time, the condensed water in absorption bottle wall, and rice-koji is shakeout；Treat grain of rice table Face is paved with red mycelium, shaking flask 1 time, and adds appropriate amounts of sterilized water according to grain of rice dry tack free degree；Daily shaking flask 2-3 afterwards Secondary, to adjust Monas cuspurpureus Went ventilation character and to make growth uniform.The culture Monas cuspurpureus Went of 10 days is put in 40 DEG C of baking ovens and is dried to moisture ＜ 15%.

2.5 saccharomycetic activation

Saccharomyces cerevisiae JH301 is hooked takes a ring to be inoculated in 100mL malt extract medium, 30 DEG C of culture 10-12h, standby.

The forced fermentation test of 2.6FJMR24

The alcoholic fermented product starter based on FM23 Monas cuspurpureus Went, bent for strengthening with FJMR24 pure red koji rice, with the Oryza glutinosa of sterilizing as base material, Do following process respectively：

Process 1：10g FM23 Monas cuspurpureus Went+10g FJMR24 pure red koji rice (inactivation)+2mL yeast activated liquid, is inoculated in In 100g Oryza glutinosa, 30 DEG C ferment 15 days.

Process 2：10g FM23 Monas cuspurpureus Went+10g FJMR24 pure red koji rice+2mL yeast activated liquid, is inoculated in 100g glutinous Meter Zhong, 30 DEG C ferment 15 days.

The index such as the alcoholic strength in detection of end sample to be fermented and distillation yield.

The mensure of 2.7 alcoholic strengths

According to GB/T13662-2008《Yellow wine》Carry out.

The mensure of 2.8 distillation yields

Normal atmosphere, 20 DEG C, the content of a unit institute output is 20% alcohol output.Distillation yield computing formula is Distillation yield %=(alcoholic strength % × go out capacity for liquor kg/ rice consumption kg/20% ethanol).

3 results

The application effect that Monascus purpureus FJMR24 is prepared in Monas cuspurpureus Went yellow wine in FM23 Monas cuspurpureus Went fermentation is as shown in table 6, plus After entering Monascus purpureus FJMR24, the alcoholic strength of Monas cuspurpureus Went yellow wine and distillation yield are improved, and compared with comparison (processing 1), wine Precision reaches significant difference (α ＜ 0.05), and distillation yield reaches pole significant difference (α ＜ 0.01)；Monascus purpureus FJMR24 is described High liquefaction, saccharifying characteristic improves the utilization rate of fermentation raw material, and then increased the distillation yield of Monas cuspurpureus Went yellow wine.

Table 6 Monascus purpureus FJMR24 forced fermentation effect

The foregoing is only embodiments of the invention, not thereby limit the present invention the scope of the claims, every using this Equivalent structure or equivalent flow conversion that bright description or Figure of description are made, or directly or indirectly it is used in other Related technical field, is included within the scope of the present invention.

Claims

1. it is characterised in that Classification And Nomenclature is Monascus purpureus FJMR24, scientific name is a kind of Monascus purpureus bacterial strain Monascuspurpureus FJMR24；By China typical culture collection center (CCTCC) preservation, deposit number is NO： M2016192, preservation date is on April 13rd, 2016, and preservation address is China. Wuhan. Wuhan University.