CN104893983A - Liquid state fermentation lower citrinin and high color value monascus red pigment preparing method and product - Google Patents
Liquid state fermentation lower citrinin and high color value monascus red pigment preparing method and product Download PDFInfo
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- CN104893983A CN104893983A CN201410829748.2A CN201410829748A CN104893983A CN 104893983 A CN104893983 A CN 104893983A CN 201410829748 A CN201410829748 A CN 201410829748A CN 104893983 A CN104893983 A CN 104893983A
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Abstract
The invention discloses Monascus anka Yang YZ 301 and a cultivation method thereof, wherein the microbial preservation number of the Monascus anka Yang YZ 301 is CGMCC9707. The invention further discloses a method for preparing lower citrinin and high color value monascus red pigment through liquid state fermentation of the Monascus anka Yang YZ 301. The method includes the first step of strain breeding, the second step of seed cultivation and enlarged cultivation, and the third step of liquid state fermentation, extraction and purification, spray drying and the like. The strains are the Monascus anka Yang YZ 301 obtained by cultivation, repeated UV mutagenesis and repeated isolation and screening of Monascus anka Nakazawa et Sato YZ201 developed by the Tianyi Biological Fermentation Technology Co., Ltd in Dongguan. The invention further discloses a lower citrinin and high color value monascus red pigment product obtained through the preparing method. The provided strains are excellent, and the technology is reasonable and stable. The obtained product is high in yield and stable in quality. The content of citrinin in the product is not more than 0.2 mg/kg (calculated by unit color value of 500 mu/g), and the color value of monascus red pigment ranges from 10,300 mu/g to 10,800 mu/g.
Description
Technical field
The present invention relates to technical field of biological fermentation, particularly the preparation method of a kind of liquid state fermentation low citrinin, High color values monascorubin and goods thereof.
Background technology
Red colouring agent for food, also used as a Chinese medicine belongs to Aspergillaceae fungi monascus parpureus Went, also known as Monascus anka Nakazawa et sato, is form by Monascus anka Nakazawa et sato cultivation and fermentation in rice.Traditionally, red colouring agent for food, also used as a Chinese medicine is widely used in the field of food such as red wine, vinegar.Modern medicine research report is thought, red colouring agent for food, also used as a Chinese medicine has effect that is hypotensive, reducing blood-fat, and contained monacolin K can stop generation cholesterol.The traditional product of monascorubin Ye Shi China, it is by Production by Microorganism Fermentation and the most successfully one of the natural pigment of being used widely.Monascorubin is widely used at food, medicine, daily necessities field as a kind of natural safe food dye from microorganism unique at present.Monascorubin is the alcohol dissolubility pigment of ketone of birdsing of the same feather flock together, wherein there are 6 kinds of compositions: yellow pigment (Monascin, monascus yellow pigment), haematochrome (Pan Hongsu, monascorubin), purpurin (Pan Hong amine, monascorubramine), 6 kinds of constituent structure are close, slightly difference.But, in the process preparing monascorubin, usually can produce a kind ofly have the mycotoxins of carcinogenic, teratogenesis and Citrinin, its genotoxicity produces serious negative impact to monascus product to the popularization in the whole world.
At present, the method for producing monascorubin mainly contains two kinds, and one is produce Red kojic rice by solid fermentation, and then extracts pigment, also can directly apply; Two is produce pigment by liquid fermenting.But the look valency of the monascorubin of solid-fermented technique gained not high and limit its application.Current employing liquid fermentation process produces monascorubin, in product pigment content and yield, finished product look valency etc. all not high, and how to improve monascorubin in fermented liquid look valency, reduce citrinin content, improve product look valency and pigment content, be limited by a great extent the impact of the factors such as bacterial classification, proportioning raw materials, fermenting process, production technique, equipment, Monascus anka Nakazawa et sato produce Citrinin number, outside the Pass having with bacterial classification, also there is close relationship with technological condition for fermentation; But it is actually rare to the optimizing research in the preparation technology of monascorubin in prior art.
Therefore, research and develop a kind of bacterial classification of liquid state fermentation excellent performance, method of cultivation and prepared the method for low citrinin, High color values monascorubin by liquid state fermentation, improve production efficiency and the quality of monascorubin, just become comparatively urgent.
Summary of the invention
For the deficiencies in the prior art, the object of the invention is to, provide a kind of Monascus color aspergillus YZ301 (Monascus anka) obtained after cultivation, ultraviolet mutagenesis, separation screening using the Monascus color aspergillus YZ201 (Monascus anka Nakazawa et Sato) of Dongguan City Tian Yi biofermentation technique company limited exploitation as the bacterial classification producing High color values, low-citrinin monascus pigment.
The present invention also aims to, provide a kind of and be bacterial classification with Monascus color aspergillus YZ301 (Monascus anka), prepared the preparation method of low citrinin, High color values monascorubin by the liquid-state fermentation technology optimized.
The present invention also aims to, a kind of the liquid state fermentation low citrinin, the High color values monascorubin goods that adopt aforesaid method to prepare are provided.
The present invention for the adopted technical scheme that achieves the above object is:
A kind of Monascus color aspergillus YZ301 (Monascus anka), its microbial preservation is numbered CGMCC9707
; Be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center on September 26th, 2014, this depositary institution is positioned at No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City.
The method of cultivation of described Monascus color aspergillus YZ301 (Monascus anka), it is the Monascus color aspergillus YZ301 (Monascus anka) that the Monascus color aspergillus YZ201 (Monascus anka Nakazawa et Sato) developed by Dongguan City Tian Yi biofermentation technique company limited obtains after cultivation, ultraviolet mutagenesis, separation screening.
Described Monascus color aspergillus YZ301 (Monascus anka) its fungus characteristic is: mycelia: diameter 3 ~ 5um, general 3um, and containing particle, cultivate long afterwards, particle reddens look, therefore mycelium laking; Conidiospore: spherical, pyriform, diameter 6 ~ 9 ~ 12um, colourless, containing particle, usual 2 ~ 3 chainings; Cleistothecium: spherical, diameter 304 ~ 0 (50) um, below 30um person is few; The full son of ascus: oval, colourless 3 x 2um; Pigment: according to Ridgway chromatogram, its color and luster is red garnet (Grenadine Red); Growth temperature: optimum temperuture is 33 ~ 35 DEG C, more than 37 DEG C, less than 30 DEG C poor growths, more than 40 DEG C grow hardly.
Monascus color aspergillus YZ301 (Monascus anka Yang YZ301) AS 3.2636Wa:25 DEG C, 7d, colony diameter 22um, sunset clouds is red; 25d colony diameter 56mm, protuberance is red in scar shape raspberry, and bacterium colony central authorities split.
MEA substratum: 25 DEG C, 7d, colony diameter 8mm, cologne earth look; 25d colony diameter 20mm, Chu Shise; CYA substratum: 25 DEG C, 7d, colony diameter 25rnm, baby pink.
Preferably, described cultivation is that 10 ~ 15 ° of Be, temperature 28 ~ 35 DEG C are cultivated 6 ~ 8 days on wort agar substratum, colony growth is smooth, on koji agar, cultivate 7 days membranaceous bacterium colonies of cutification in 33 ~ 35 DEG C, without aerial hyphae, its shape such as lava flows out, the bright pink look of pomegranate, eugonic on wort agar.
Preferably, described ultraviolet mutagenesis and screening comprise: A, ultraviolet mutagenesis: by the cultivation red colouring agent for food, also used as a Chinese medicine bacterial classification of 7 ~ 9 days, through high temperature 55 ~ 65 DEG C process 2 ~ 10min, high pressure 0.04 ~ 0.07Mpa process 5 ~ 10 hours, put into the triangular flask built with band granulated glass sphere, sterilized water 30 ~ 50ml, be placed in shaking table vibration 1 ~ 2 hour, filter, get 10ml spore suspension, be added in the culture dish of band magnetic bar, carry out uv irradiating 2 ~ 10min under magnetic stirring, be coated with dull and stereotyped, with black cloth wrapping, cultivate 6 ~ 8 days at temperature 32 ~ 35 DEG C;
B, isolation medium: Zulkovsky starch 5%, maltose 3%, peptone 0.1%, adds appropriate lactic acid, adjust ph 5 ~ 6, agar 2%;
C, isolation medium PDA:NaNO
33.0g, KH
2pO
41.0g, MgSO
47H
2o 0.5g, FeSO
47H
2o 0.01g, sucrose 30g, tap water 1000ml, agar 15g;
D, slant medium MEA: wort 20g, glucose 20g, peptone 1.0g, tap water 1000ml, agar 20g.
Adopt aforementioned Monascus anka Nakazawa et sato liquid state fermentation to prepare a method for low citrinin, High color values monascorubin, it comprises the steps:
(1) strain improvement: adopt and be separated the Monascus color aspergillus YZ301 (Monascus anka) that obtains as bacterial classification by ultraviolet mutagenesis, its microbial preservation number is CGMCC9707;
(2) seed culture, enlarged culturing: Monascus color aspergillus YZ301 step (1) seed selection obtained accesses successively in the I and II seeding tank that seed culture medium is housed and cultivates 6 ~ 9 hours respectively, to be inoculated in fermentor tank enlarged culturing afterwards 75 ~ 85 hours, to obtain liquid seed culture fluid;
(3) liquid state fermentation is produced:
(31) employing capacity is 30m
3large Copacity fermentor tank, load liquid fermentation medium;
(32) the liquid seed culture fluid of inoculation step (2) gained, inoculum size is 5 ~ 10%, and access secondary seed tank is cultivated 6 ~ 9 hours at temperature 32 ~ 35 DEG C, obtained fermentation liquid seeds nutrient solution;
(33) after, the fermentation of step (32) gained liquid seeds nutrient solution is accessed in fermentor tank, carry out liquid state fermentation after setting fermentation condition, obtain low citrinin, High color values monascorubin fermented liquid.
Wherein, also comprise in described step (2): Monascus color aspergillus YZ301 is inserted in the triangular flask that sterilized water, granulated glass sphere are housed and smashes 1 ~ 2 hour, by 5 ~ 10% inoculum size access triangular flasks, cultivate 22 ~ 28 hours at temperature 32 ~ 35 DEG C through shaking flask; The raw material composition of the seed culture medium in described step (2) and weight proportion are: starch 4 ~ 6%, NaNO
30.1 ~ 0.3%, MgSO
40.1 ~ 0.3%, KH
2pO
40.1 ~ 0.3%, corn steep liquor 1 ~ 5%.
Also comprise the liquid fermentation medium first loading in Large Copacity fermentor tank and account for ferment tank 3/4 volume in described step (31), sterilizing 30min under tank temperature 121 ~ 125 DEG C, tank pressure 0.1 ~ 0.15Mpa, cools to 30 ~ 35 DEG C; Raw material composition and the weight proportion of the middle liquid fermentation medium of described step (31) are: ground rice 8 ~ 15%, soybean 0.1 ~ 0.5%, NaNO
30.1% ~ 0.3%, corn steep liquor 1 ~ 5%; Also comprise and regulate fermention medium initial pH value to be 3.0 ~ 4.5 with lactic acid.
Described step (33) fermentation condition is set as follows: temperature for fermentation initial 0 ~ 34h insulation 30 ~ 35 DEG C, to adjust temperature be 33 ~ 36 DEG C to fermentation lasts 35 ~ 85h; Ventilation is that the initial 0 ~ 25h of fermentation is set to 300 ~ 500Lmin, fermentation middle and later periods 26 ~ 85h is adjusted to 550 ~ 700Lmin; Whole fermenting process at the uniform velocity stirs all the time, and rotating speed is 200 ~ 400r/min, tank pressure is 0.04 ~ 0.07Mpa; The incubation time that continuously ferments carries out 75 ~ 85h altogether.
It is 3.0 ~ 4.5 that described step (33) also comprises by lactic acid adjustment fermention medium initial pH value, temperature 32 ~ 36 DEG C; Fermentation lasts carries out feed supplement after within 35 ~ 45 hours, arriving fermentation animated period, and feed supplement amount accounts for 5 ~ 10% of cumulative volume; Described feed supplement is ground rice 10 ~ 20%, soybean 0.1 ~ 0.5%, by lactic acid adjust ph 3.0 ~ 4.5.
The preparation method of aforesaid liquid state fermentation low citrinin, High color values monascorubin, it also comprises the steps:
(4) extraction, purifying: after the dehydration of the low citrinin that described step (33) is obtained, High color values monascorubin fermentation liquor, be 7.0 ~ 9.0 by the mycelium pH stayed, the lixiviate of 60 ~ 80v/v alcohol, secondary press filtration again, by lixiviate filtrate through membrane filtration (i.e. nanofiltration), vacuum concentration (20 ~ 35Be brix), obtain the concentrated solution of low citrinin, High color values monascorubin;
(5) spraying dry: the concentrated solution of step (4) gained is carried out spraying dry at 100 ~ 210 DEG C, obtained low citrinin, High color values monascorubin powder product.
In fermentation culture process, its physical and chemical index of regular sampling analysis also carries out suitable Additional nutrient solution and feed supplement, and fermentation level and output capacity can be made all to be improved, the feed supplement time ferment middle and animated period better.
For in aforesaid fermentation culturing process, adopt the absorbancy of the fermented liquid described in ultraviolet spectral photometer detection, monascorubin finished product, absorption peak wavelength is 495 ~ 505nm.
During the fermentation, detect the content of the Citrinin of described monascorubin finished product, in its finished product, the content of Citrinin is not more than 0.2mg/kg (in unit look valency 500u/g).
The low citrinin adopting aforesaid preparation method to prepare, High color values monascorubin, in its finished product, the content of Citrinin is not more than 0.2mg/kg (in unit look valency 500u/g), monascorubin look valency for 10300 ~ 10800u/g.
Compared with prior art, tool has the following advantages in the present invention:
1, Monascus color aspergillus YZ301 (Monascus anka) provided by the invention, its microbial preservation is numbered CGMCC9707, that contriver is through cultivating, ultraviolet mutagenesis, excellent species is obtained after separating for several times screening, its integrative biology excellent performance, be most suitable for liquid-state fermentation technology, breed stable performance, high yield pigment, this bacterial classification can high temperature resistant (35 ~ 36.5 DEG C) in ultraviolet mutagenesis process, high pressure (0.06 ~ 0.08Mpa), as increased inoculum size and keeping lower pH value in fermented liquid culturing process, fermentation level can be made to stablize, and the content of red colouring agent for food, also used as a Chinese medicine time metabolite Citrinin in fermenting process can be controlled, improve content (i.e. High color values) and the yield of fermented liquid monascorubin, security is high, good stability.
2, the present invention adopts the special bacterial classification cultivated, the conservative control of processing condition and each step, liquid state fermentation is carried out to the Monascus anka Nakazawa et sato mutagenic fungi selected, obtain the monascorubin fermented liquid of high-content, the monascorubin of gained after abstraction process process again, citrinin content is very low, security is high, good stability, monascorubin look valency is high, and the present invention by preferred bacterial classification after seed culture, before liquid state fermentation, additionally use enlarged culturing step by step, thus add the bacterial classification content at fermentation initial stage, improve yeast phase microbic activity, shorten fermentation period, make a large amount of microorganism in yeast phase, be in the growth vigorous stage, keep vigorous metabolism, be beneficial to raising pigment production.
3, the present invention is by extraction, purifying process, effectively high molecular weight protein, small molecular organic acid, sugar is separated with natural pigment, removes part Citrinin, the purity of monascorubin product and stability are significantly improved.
4, the present invention is in fermentation culture process, and its physical and chemical index of regular sampling analysis also in the suitable feed supplement of fermentation animated period, makes fermentation level and output capacity all be improved.
5, the present invention adopts liquid state fermentation technology, raw material environmental protection, pollution-free, safety, formula are reasonable, preparation process easily controls, rational technology stable, efficiency is high, make that constant product quality, effective constituent are high, objectionable constituent are low, meet Safety of Food Quality standard, can large-scale industrial production be realized.
6, in the finished product that obtains of preparation method of the present invention, monascorubin look valency is not more than 0.2mg/kg (in unit look valency 500u/g) up to 10300 ~ 10800u/g, citrinin content.
Above-mentioned is the general introduction of invention technical scheme, and below in conjunction with embodiment, the present invention will be further described.
Embodiment:
The Monascus color aspergillus YZ301 (Monascus anka) that embodiment 1 the present embodiment provides, its microbial preservation is numbered CGMCC9707; The method of cultivation of described Monascus color aspergillus YZ301 (Monascus anka) 1, it is the Monascus color aspergillus that the Monascus color aspergillus YZ201 (Monascus anka Nakazawa et Sato) developed by Dongguan City Tian Yi biofermentation technique company limited obtains after screening through cultivation, ultraviolet mutagenesis, separating for several times, numbering YZ301 (Monascus anka).
Described cultivation is cultivated 6 ~ 8 days in wort agar substratum 10 ~ 15 ° of Be, temperature 28 ~ 35 DEG C, colony growth is smooth, on koji agar, 7 days membranaceous bacterium colonies of cutification are cultivated in 33 ~ 35 DEG C, without aerial hyphae, its shape such as lava flows out, the bright pink look of pomegranate, eugonic on wort agar;
Preferably, described ultraviolet mutagenesis and screening comprise: A, ultraviolet mutagenesis: by the cultivation red colouring agent for food, also used as a Chinese medicine bacterial classification of 7 ~ 9 days, through high temperature 55 ~ 65 DEG C process 2 ~ 10min, high pressure 0.04 ~ 0.07Mpa process 5 ~ 10 hours, put into the 500ml triangular flask built with band granulated glass sphere, sterilized water 80 ~ 100ml, be placed in shaking table vibration 1 ~ 2 hour, filter, get 10ml spore suspension, be added in the culture dish of band magnetic bar, carry out uv irradiating 2 ~ 10min under magnetic stirring, be coated with dull and stereotyped, with black cloth wrapping, cultivate 6 ~ 8 days at temperature 32 ~ 35 DEG C;
B, isolation medium: Zulkovsky starch 5%, maltose 3%, peptone 0.1%, adds appropriate lactic acid, adjust ph 5 ~ 6, agar 2%;
C, isolation medium PDA:NaNO
33.0g, KH
2pO
41.0g, MgSO
47H
2o 0.5g, FeSO
47H
2o 0.01g, sucrose 30g, tap water 1000ml, agar 15g;
D, slant medium MEA: wort 20g, glucose 20g, peptone 1.0g, tap water 1000ml, agar 20g.
Adopt aforementioned Monascus anka Nakazawa et sato liquid state fermentation to prepare a method for low citrinin, High color values monascorubin, it comprises the steps:
(1) strain improvement: adopt and be separated the Monascus color aspergillus YZ301 (Monascus anka) that obtains as bacterial classification by ultraviolet mutagenesis, its preserving number is CGMCC9707;
(2) seed culture, enlarged culturing: Monascus color aspergillus YZ301 step (1) seed selection obtained accesses successively in the I and II seeding tank that seed culture medium is housed and cultivates 6 ~ 9 hours respectively, to be inoculated in fermentor tank enlarged culturing afterwards 75 ~ 85 hours, to obtain liquid seed culture fluid;
(3) liquid state fermentation is produced:
(31) employing capacity is 30m
3large Copacity fermentor tank, load liquid fermentation medium;
(32) the liquid seed culture fluid of inoculation step (2) gained, inoculum size is 5 ~ 10%, and access secondary seed tank is cultivated 6 ~ 9 hours at temperature 32 ~ 35 DEG C, obtained fermentation liquid seeds nutrient solution;
(33) after, the fermentation of step (32) gained liquid seeds nutrient solution is accessed in fermentor tank, carry out liquid state fermentation after setting fermentation condition, obtain low citrinin, High color values monascorubin fermented liquid.
Wherein, also comprise in described step (2): Monascus color aspergillus YZ301 is inserted in the triangular flask that sterilized water, granulated glass sphere are housed and smashes 1 ~ 2 hour, by 5 ~ 10% inoculum size access triangular flasks, cultivate 22 ~ 28 hours at temperature 32 ~ 35 DEG C through shaking flask; The raw material composition of the seed culture medium in described step (2) and weight proportion are: starch 4 ~ 6%, NaNO
30.1 ~ 0.3%, MgSO
40.1 ~ 0.3%, KH
2pO
40.1 ~ 0.3%, corn steep liquor 1 ~ 5%.
Also comprise the liquid fermentation medium first loading in Large Copacity fermentor tank and account for ferment tank 3/4 volume in described step (31), sterilizing 30min under tank temperature 121 ~ 125 DEG C, tank pressure 0.1 ~ 0.15Mpa, cools to 30 ~ 35 DEG C; Raw material composition and the weight proportion of the middle liquid fermentation medium of described step (31) are: ground rice 8 ~ 15%, soybean 0.1 ~ 0.5%, NaNO
30.1% ~ 0.3%, corn steep liquor 1 ~ 5%; Also comprise and regulate fermention medium initial pH value to be 3.0 ~ 4.5 with lactic acid.
Described step (33) fermentation condition is set as follows: temperature for fermentation initial 0 ~ 34h insulation 30 ~ 35 DEG C, to adjust temperature be 33 ~ 36 DEG C to fermentation lasts 35 ~ 85h; Ventilation is that the initial 0 ~ 25h of fermentation is set to 300 ~ 500Lmin, fermentation middle and later periods 26 ~ 85h is adjusted to 550 ~ 700Lmin; Whole fermenting process at the uniform velocity stirs all the time, and rotating speed is 200 ~ 400r/min, tank pressure is 0.04 ~ 0.07Mpa; The incubation time that continuously ferments carries out 75 ~ 85h altogether.
It is 3.0 ~ 4.5 that described step (33) also comprises by lactic acid adjustment fermention medium initial pH value, temperature 32 ~ 36 DEG C; Fermentation lasts carries out feed supplement after within 35 ~ 45 hours, arriving fermentation animated period, and feed supplement amount accounts for 5 ~ 10% of cumulative volume; Described feed supplement is ground rice 10 ~ 20%, soybean 0.1 ~ 0.5%, lactic acid adjust ph are 3.0 ~ 4.5.
The preparation method of aforesaid liquid state fermentation low citrinin, High color values monascorubin, it also comprises the steps:
(4) extraction, purifying: after the low citrinin High color values monascorubin fermentation liquor dehydration that described step (33) is obtained, be 7.0 ~ 9.0 by the mycelium pH stayed, the lixiviate of 60 ~ 80v/v alcohol, secondary press filtration, lixiviate filtrate is through membrane filtration (nanofiltration), vacuum concentration (20 ~ 35Be brix) again, obtains the concentrated solution of low citrinin, High color values monascorubin;
(5) spraying dry: the concentrated solution of step (4) gained is carried out spraying dry at 100 ~ 210 DEG C, obtained low citrinin, High color values monascorubin powder product.
In fermentation culture process, its physical and chemical index of regular sampling analysis also carries out suitable Additional nutrient solution and feed supplement, and fermentation level and output capacity can be made all to be improved, the feed supplement time ferment middle and animated period better.
For in aforesaid fermentation culturing process, adopt the absorbancy of the fermented liquid described in ultraviolet spectral photometer detection, monascorubin finished product, absorption peak wavelength is 495 ~ 505nm.
During the fermentation, detect the content of the Citrinin of described monascorubin finished product, in its finished product, the content of Citrinin is not more than 0.2mg/kg (in unit look valency 500u/g).
The low citrinin adopting aforesaid preparation method to prepare, High color values monascorubin, in its finished product, the content of Citrinin is not more than 0.2mg/kg (in unit look valency 500u/g), monascorubin look valency for 10300 ~ 10800u/g.
Below, according to above-mentioned embodiment, four kinds are provided to possess representational specific embodiment.
The Monascus color aspergillus YZ301 (Monascus anka) that embodiment 2 the present embodiment provides, and adopt this Monascus anka Nakazawa et sato liquid state fermentation to prepare method and the goods of low citrinin High color values monascorubin, substantially the same manner as Example 1, its difference is:
A kind of method adopting YZ301 (Monascus anka) liquid state fermentation of Monascus color aspergillus to prepare low citrinin, High color values monascorubin, the difference of its concrete steps is:
(2) seed culture, enlarged culturing: Monascus color aspergillus YZ301 is inserted in the triangular flask that sterilized water, granulated glass sphere are housed and smashes 2 hours, by 10% inoculum size access triangular flask, cultivate 28 hours at temperature 35 DEG C through shaking flask, access is equipped with in the I and II seeding tank of seed culture medium and is cultivated 6 hours respectively successively, to be inoculated in fermentor tank enlarged culturing afterwards 85 hours, to obtain liquid seed culture fluid;
The raw material composition of described seed culture medium and weight proportion are: starch 4%, NaNO
30.2%, MgSO
40.2%, KH
2pO
40.2%, corn steep liquor 1%.
(3) liquid state fermentation is produced:
(31) employing capacity is 30m
3large Copacity fermentor tank, first load in Large Copacity fermentor tank and account for the liquid fermentation medium of ferment tank 3/4 volume, sterilizing 30min under tank temperature 121 DEG C, tank pressure 0.1Mpa, cools to 30 DEG C;
The raw material composition of described liquid fermentation medium and weight proportion are: ground rice 8%, soybean 0.2%, NaNO
30.1%, corn steep liquor 1%; Also comprise and regulate fermention medium initial pH value to be 3.0 ~ 4.5 with lactic acid.
(32) the liquid seed culture fluid of inoculation step (2) gained, inoculum size is 5%, and access secondary seed tank is cultivated 9 hours at temperature 32 DEG C, obtained fermentation liquid seeds nutrient solution;
(33) after, the fermentation of step (32) gained liquid seeds nutrient solution is accessed in fermentor tank, obtain low citrinin, High color values monascorubin fermented liquid; The fermentation condition wherein set is: 1. temperature controls: initial temperature 30 ~ 35 DEG C, is adjusted to 33 ~ 36 DEG C after 35 hours; 2. mixing speed: 220 ~ 400r/min; 3. ventilation: be initially 350 ~ 500 (Lmin), after 35 hours, is then adjusted to 550 ~ 700/ (Lmin); 4. tank pressure: 0.04 ~ 0.07MPa.
It is 3.0 ~ 4.5 that described step (33) also comprises by lactic acid adjustment fermention medium initial pH value, temperature 32 ~ 36 DEG C; Fermentation lasts carried out feed supplement after 35 ~ 45 hours, and feed supplement amount accounts for 5 ~ 10% of cumulative volume, complete through 75 ~ 85 hours fermentation, can obtain low citrinin, the high fermented liquid containing monascorubin; Described feed supplement is ground rice 10 ~ 20%, soybean 0.1 ~ 0.5%, lactic acid adjust ph are 3.0 ~ 4.5.
(4) extraction, purifying: after the low citrinin obtained, High color values monascorubin fermentation liquor are dewatered, be 7.0 ~ 9.0 by the mycelium pH stayed, the lixiviate of 60 ~ 80v/v alcohol, through secondary press filtration, lixiviate filtrate is through membrane filtration (nanofiltration), vacuum concentration (20 ~ 35Be brix) again, obtains the concentrated solution of low citrinin, High color values monascorubin;
(5) spraying dry: the concentrated solution of gained is carried out spraying dry at 100 ~ 210 DEG C, obtained low citrinin High color values monascorubin powder product.
At fermenting process, in periodic detection product, the content of Citrinin, detects absorbancy with ultraviolet spectral photometer, evaluates the look valency of monascorubin in finished product.
The Monascus color aspergillus YZ301 (Monascus anka) that embodiment 3 the present embodiment provides, and adopt this Monascus anka Nakazawa et sato liquid state fermentation to prepare low citrinin, the method for High color values monascorubin and goods, substantially identical with embodiment 1,2, its difference is:
Prepare in the method for low citrinin, High color values monascorubin, during concrete steps (3) liquid state fermentation is produced: raw material composition and the weight proportion of the middle liquid fermentation medium of described step (31) are adjusted to: ground rice 10%, soybean 0.2%, NaNO
30.1%, corn steep liquor 3%; Also comprise and regulate fermention medium initial pH value to be 3.0 ~ 4.5 with lactic acid.The liquid seed culture fluid of inoculation step (2) gained in described step (32), inoculum size is 6%, and access secondary seed tank is cultivated 6 hours at temperature 32 DEG C.
Other steps are substantially identical with embodiment 1 or 2.At fermenting process, in periodic detection product, the content of Citrinin, detects absorbancy with ultraviolet spectral photometer, evaluates the look valency of monascorubin in finished product.
The Monascus color aspergillus YZ301 (Monascus anka) that embodiment 4 the present embodiment provides, and adopt this Monascus anka Nakazawa et sato liquid state fermentation to prepare low citrinin, the method for High color values monascorubin and goods, substantially identical with embodiment 1,2,3, its difference is:
Prepare in the method for low citrinin, High color values monascorubin, concrete steps (2) seed culture, enlarged culturing: the raw material composition of described liquid seed culture medium and proportioning: starch 4%, NaNO
30.1%, MgSO
40.1%, KH
2pO
40.1%, corn steep liquor 1%; During concrete steps (3) liquid state fermentation is produced, raw material composition and the weight proportion of the middle liquid fermentation medium of described step (31) are adjusted to: ground rice 12%, soybean 0.2%, NaNO
30.1%, corn steep liquor 5%; Also comprise and regulate fermention medium initial pH value to be 3.0 ~ 4.5 with lactic acid.The liquid seed culture fluid of inoculation step (2) gained in described step (32), inoculum size is 8%, and access secondary seed tank is cultivated 6 hours at temperature 34 DEG C.Described step (33) also comprises fermentation lasts and carries out feed supplement after 35 ~ 45 hours, and feed supplement amount accounts for 10% of cumulative volume; Described feed supplement is ground rice 10%, soybean 0.1%, lactic acid adjust ph are 3.0 ~ 4.5.
Other steps are substantially identical with embodiment 1 or 2 or 3.At fermenting process, in periodic detection product, the content of Citrinin, detects absorbancy with ultraviolet spectral photometer, evaluates the look valency of monascorubin in finished product.
The Monascus color aspergillus YZ301 (Monascus anka) that embodiment 5 the present embodiment provides, and adopt this Monascus anka Nakazawa et sato liquid state fermentation to prepare low citrinin, the method for High color values monascorubin and goods, substantially identical with embodiment 1,2,3,4, its difference is:
Prepare in the method for low citrinin, High color values monascorubin, concrete steps (2) seed culture, enlarged culturing: the raw material composition of described liquid seed culture medium and proportioning: starch 4%, NaNO
30.1%, MgSO
40.1%, KH
2pO
40.1%, corn steep liquor 1%; During concrete steps (3) liquid state fermentation is produced, raw material composition and the weight proportion of the middle liquid fermentation medium of described step (31) are adjusted to: ground rice 15%, soybean 0.3%, NaNO
30.1%, corn steep liquor 5%; Also comprise and regulate fermention medium initial pH value to be 3.0 ~ 4.5 with lactic acid.The liquid seed culture fluid of inoculation step (2) gained in described step (32), inoculum size is 10%, and access secondary seed tank is cultivated 8 hours at temperature 35 DEG C.Described step (33) also comprises fermentation lasts and carries out feed supplement after 35 ~ 45 hours, and feed supplement amount accounts for 5% of cumulative volume; Described feed supplement is ground rice 10%, soybean 0.5%, be 3.0 ~ 4.5 by lactic acid adjust ph.
Other steps are substantially identical with embodiment 1 or 2 or 3 or 4.At fermenting process, in periodic detection product, the content of Citrinin, detects absorbancy with ultraviolet spectral photometer, evaluates the look valency of monascorubin in finished product.
The low citrinin that the present invention obtains, the performance test results of High color values monascorubin goods are in table 1.
The performance test results of table 1 low citrinin, High color values monascorubin
The present invention is not limited to above-mentioned embodiment, adopts raw material, the technique identical or approximate with the above embodiment of the present invention, and the preparation method of the low citrinin obtained, High color values monascorubin and goods, all within scope.
Claims (10)
1. a Monascus color aspergillus YZ301 (Monascus anka), its microbial preservation is numbered CGMCC9707.
2. the method for cultivation of Monascus color aspergillus YZ301 (Monascus anka) according to claim 1, it is characterized in that, obtained after screening through cultivation, ultraviolet mutagenesis, separating for several times by Monascus color aspergillus YZ201 (Monascus anka Nakazawa et Sato).
3. the method for cultivation of Monascus color aspergillus YZ301 (Monascus anka) according to claim 2, it is characterized in that, described cultivation is that 10 ~ 15 ° of Be, temperature 28 ~ 35 DEG C are cultivated 6 ~ 8 days on wort agar substratum, colony growth is smooth, on koji agar, cultivates 7 days membranaceous bacterium colonies of cutification in 33 ~ 35 DEG C, without aerial hyphae, its shape such as lava flows out, the bright pink look of pomegranate, eugonic on wort agar;
Described ultraviolet mutagenesis and screening comprise:
A, ultraviolet mutagenesis: by the cultivation red colouring agent for food, also used as a Chinese medicine bacterial classification of 7 ~ 9 days, through high temperature 55 ~ 65 DEG C process 2 ~ 10min, high pressure 0.05 ~ 0.10Mpa process 5 ~ 10 hours, put into the 500ml triangular flask built with band granulated glass sphere, sterilized water 80 ~ 100ml, be placed in shaking table vibration 1 ~ 2 hour, filter, get 10ml spore suspension, be added in the culture dish of band magnetic bar, carry out uv irradiating 2 ~ 10min under magnetic stirring, be coated with dull and stereotyped, with black cloth wrapping, cultivate 6 ~ 8 days at temperature 32 ~ 35 DEG C;
B, isolation medium: Zulkovsky starch 5%, maltose 3%, peptone 0.1%, adds appropriate lactic acid, adjust ph 5 ~ 6, agar 2%;
C, isolation medium PDA:NaNO
33.0g, KH
2pO
41.0g, MgSO
47H
2o 0.5g, FeSO
47H
2o 0.01g, sucrose 30g, tap water 1000ml, agar 15g;
D, slant medium MEA: wort 20g, glucose 20g, peptone 1.0g, tap water 1000ml, agar 20g.
4. adopt the described Monascus anka Nakazawa et sato liquid state fermentation of one of claim 1-3 to prepare a method for low citrinin, High color values monascorubin, it is characterized in that, comprise the steps:
(1) strain improvement: adopt and be separated the Monascus color aspergillus YZ301 (Monascus anka) that obtains as bacterial classification by ultraviolet mutagenesis, its microbial preservation number is CGMCC9707;
(2) seed culture, enlarged culturing: Monascus color aspergillus YZ301 step (1) seed selection obtained accesses successively in the I and II seeding tank that seed culture medium is housed and cultivates 6 ~ 9 hours respectively, to be inoculated in fermentor tank enlarged culturing afterwards 75 ~ 85 hours, to obtain liquid seed culture fluid;
(3) liquid state fermentation is produced:
(31) employing capacity is 30m
3large Copacity fermentor tank, load liquid fermentation medium;
(32) the liquid seed culture fluid of inoculation step (2) gained, inoculum size is 5 ~ 10%, and access secondary seed tank is cultivated 6 ~ 9 hours at temperature 32 ~ 35 DEG C, obtained fermentation liquid seeds nutrient solution;
(33) after, the fermentation of step (32) gained liquid seeds nutrient solution is accessed in fermentor tank, carry out liquid state fermentation after setting fermentation condition, obtain low citrinin, High color values monascorubin fermented liquid.
5. the preparation method of liquid state fermentation low citrinin according to claim 4, High color values monascorubin, it is characterized in that, also comprise in described step (2): Monascus color aspergillus YZ301 is inserted in the triangular flask that sterilized water, granulated glass sphere are housed and smashes 1 ~ 2 hour, by 5 ~ 10% inoculum size access triangular flasks, cultivate 22 ~ 28 hours at temperature 32 ~ 35 DEG C through shaking flask; The raw material composition of the seed culture medium in described step (2) and weight proportion are: starch 4 ~ 6 %, NaNO
30.1 ~ 0.3%, MgSO
40.1 ~ 0.3%, KH
2pO
40.1 ~ 0.3%, corn steep liquor 1 ~ 5%.
6. the preparation method of liquid state fermentation low citrinin according to claim 4, High color values monascorubin, it is characterized in that, the liquid fermentation medium first loading in Large Copacity fermentor tank and account for ferment tank 3/4 volume is also comprised in described step (31), sterilizing 30min under tank temperature 121 ~ 125 DEG C, tank pressure 0.1 ~ 0.15Mpa, cools to 30 ~ 35 DEG C; Raw material composition and the weight proportion of the middle liquid fermentation medium of described step (31) are: ground rice 8 ~ 15%, soybean 0.1 ~ 0.5%, NaNO
30.1% ~ 0.3%, corn steep liquor 1 ~ 5%; Also comprise and regulate fermention medium initial pH value to be 3.0 ~ 4.5 with lactic acid.
7. the preparation method of liquid state fermentation low citrinin according to claim 4, High color values monascorubin, it is characterized in that, described step (33) fermentation condition is set as follows: temperature for fermentation initial 0 ~ 34h insulation 30 ~ 35 DEG C, to adjust temperature be 33 ~ 36 DEG C to fermentation lasts 35 ~ 85h; Ventilation is that the initial 0 ~ 25h of fermentation is set to 300 ~ 500Lmin, fermentation middle and later periods 26 ~ 85h is adjusted to 550 ~ 700Lmin; Whole fermenting process at the uniform velocity stirs all the time, and rotating speed is 200 ~ 400r/min, tank pressure is 0.04 ~ 0.07Mpa; The incubation time that continuously ferments carries out 75 ~ 85h altogether.
8. the preparation method of liquid state fermentation low citrinin according to claim 4, High color values monascorubin, is characterized in that, it is 3.0 ~ 4.5 that described step (33) also comprises by lactic acid adjustment fermention medium initial pH value, temperature 32 ~ 36 DEG C; Fermentation lasts carries out feed supplement after within 35 ~ 45 hours, arriving fermentation animated period, and feed supplement amount accounts for 5 ~ 10% of cumulative volume; Described feed supplement is ground rice 10 ~ 20%, soybean 0.1 ~ 0.5%, by lactic acid adjust ph 3.0 ~ 4.5.
9. the preparation method of liquid state fermentation low citrinin according to claim 4, High color values monascorubin, is characterized in that, also comprise the steps:
(4) extraction, purifying: after the dehydration of the low citrinin that described step (33) is obtained, High color values monascorubin fermentation liquor, be 7.0 ~ 9.0 by the mycelium pH stayed, the lixiviate of 60 ~ 80v/v alcohol, secondary press filtration again, lixiviate filtrate is carried out film process (nanofiltration), vacuum concentration, obtain the concentrated solution (20 ~ 35Be brix) of low citrinin, High color values monascorubin;
(5) spraying dry: the concentrated solution of step (4) gained is carried out spraying dry at 100 ~ 210 DEG C, obtained low citrinin High color values monascorubin powder product.
10. adopt low citrinin, High color values monascorubin prepared by the described method of one of claim 4-9, it is characterized in that, it adopts Monascus color aspergillus YZ301 (Monascus anka) to obtain as bacterial classification, and its microbial preservation is numbered CGMCC9707; In its finished product, the content of Citrinin is not more than 0.2mg/kg (in unit look valency 500u/g), monascorubin look valency for 10300 ~ 10800u/g.
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| CN106929428A (en) * | 2017-01-18 | 2017-07-07 | 东莞市双红生物技术有限公司 | The method and product of Monascus polysaccharides are produced in liquid state fermentation |
| CN108486164A (en) * | 2018-03-30 | 2018-09-04 | 福建省农业科学院农业工程技术研究所 | The production technology of low citrinin Monascus color |
| CN111394404A (en) * | 2019-05-14 | 2020-07-10 | 江苏臻大天园健康科技有限公司 | Method for producing coenzyme Q10 by using red yeast rice |
| CN110195022A (en) * | 2019-06-28 | 2019-09-03 | 福州大学 | A method of it reducing monascus ruber bulb diameter and improves biomass and amount of pigment simultaneously |
| CN112430635A (en) * | 2020-12-01 | 2021-03-02 | 广东肇庆星湖生物科技股份有限公司 | Fermentation method for low-yield citrinin and high-yield water-soluble monascus yellow pigment |
| CN112481327A (en) * | 2020-12-01 | 2021-03-12 | 广东肇庆星湖生物科技股份有限公司 | Fermentation regulation and control method of water-soluble monascus yellow pigment |
| CN114058648A (en) * | 2021-12-14 | 2022-02-18 | 广东天益生物科技有限公司 | Method for high-yield fermentation of extracellular monascus red pigment by using monascus purpureus |
| CN114058648B (en) * | 2021-12-14 | 2023-11-21 | 广东天益生物科技有限公司 | Method for fermenting high-yield extracellular monascus red pigment by using monascus purpureus |
| CN114540340A (en) * | 2022-04-18 | 2022-05-27 | 安徽农业大学 | Method for improving ultraviolet mutagenesis effect of monascus |
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