CN101942397A - Monascus mutant strain and method for preparing monascus yellow pigment by submerged fermentation thereof and article thereof - Google Patents
Monascus mutant strain and method for preparing monascus yellow pigment by submerged fermentation thereof and article thereof Download PDFInfo
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Abstract
The invention provides a monascus mutant strain, namely Monascus purpureus yang Ty622, and a method for preparing monascus yellow pigment by submerged fermentation of the strain. The method comprises the steps of: selection and breeding of strain, seed culture, large-capacity tank fermentation and extraction of monascus yellow pigment, wherein the seed culture and the fermentation are implemented in a specifically manufactured culture medium, and multistage fermentation is performed in a large-capacity pot fermentation tank, followed by fermentation; the technological conditions of fermentation are as below: the temperature is from 32 to 36 DEG C, the stirring rotational speed is 200 to 400r/min; the ventilation quantity at earlier stage is from 400 to 500L.min and is then regulated to 500 to 600L.min at middle/later stages. The invention further provides an article prepared according to the above method. The preparation method according to the invention has simple technology and is easy for industrialized production, and the resultant fermented liquid has high content of monascus yellow pigment and low content of citrinin.
Description
Technical field
The present invention relates to technical field of bioengineering, be specifically related to method and goods that a kind of monascus mutant strain and submerged fermentation thereof prepare low citrinin monascus yellow pigment.
Background technology
In the prior art, natural yellow pigment generally comprises: the method for Gardenia Yellow, turmeric, sailor yellow, monascus yellow pigment yellow pigment mainly contains extraction process, chemical synthesis and microbe fermentation method.Monascus also claims red colouring agent for food, also used as a Chinese medicine.In natural yellow pigment except that the monascus yellow pigment; other are by plant extract; its production cost height; and the monascus yellow pigment is to utilize microbial fermentation production to be easy to large-scale production; its substratum is a cereal starch with low cost, and light, thermostability are better than the pigment of other plant extraction.Monascus also claims " red bent " " red song ", at Chinese food with pharmaceutically be applied to history in rare thousand, is the raw material of making natural red colouring matter and functional foodstuff.The monascus pigment is the secondary metabolite that monascus ruber produces in process of growth, it is the pure dissolubility pigment of ketone of birdsing of the same feather flock together, composition in 6 is wherein arranged: yellow pigment (monascin, monascus yellow pigment element), haematochrome (Pan Hongsu, monascus red pigment), purpurin (Pan Hong amine, the red amine of monascus), 6 kinds of constituent structure are close, and are slightly variant.
At present, the research and development of monascus yellow pigment pigment are mainly concentrated: (1) filters out the good product yellow pigment monascus strain of character; (2) further study pigment and extract and the purified method, improve pigment stability; (3) further investigate the biochemical metabolism approach of this pigment, understand its metabolic mechanism, thereby consider the generation of pigment such as to be regulated and control at the aspect by the regulation and control mesostate.
Domestic research and development about the monascus pigment also have much.Fujian Agricultural Univeristy discloses the preparation method of mould haematochrome of a kind of water-soluble monascus and monascus yellow pigment pigment in calendar year 2001, the orange red tryptophol dissolubility color red rubroglaucine and the rubropunctatin that produce with monascus ruber are raw material, are converted into water-soluble red colouring matter with specific alkali substance reaction; By containing the oxysulfide reaction, change into water-soluble yellow pigment again.
Patent publication No. discloses the method preparation of adopting solid-state or liquid state fermentation for " preparation method of the mould powder of the plain high color range functional Monascus of a kind of no citrinin " of " CN1448503A ", and its products characteristics is that fermented liquid monascus yellow pigment pigment content is lower.
In 2005, Fujian Agricultural Univeristy again SEPARATE APPLICATION name be called " the mould haematochrome of acid soluble rice fermented with red yeast and the mould yellow pigment pigment of acid soluble rice fermented with red yeast and preparation method thereof " and " the mould haematochrome of oil solubility rice fermented with red yeast and the mould yellow pigment pigment of oil solubility rice fermented with red yeast and preparation method thereof " patent, the former is a raw material with mould haematochrome of water-soluble monascus and the mould yellow pigment pigment of water-soluble monascus, under catalyst action, can make mould red, the monascus yellow pigment pigment of acid soluble rice fermented with red yeast with organic acid or its anhydride reaction; The latter is a raw material with mould haematochrome of water-soluble monascus and the mould yellow pigment pigment of water-soluble monascus, under catalyst action, with diprotic acid intramolecular anhydride and organic alcohol reaction, transfers mould haematochrome of oil solubility rice fermented with red yeast and monascus yellow pigment pigment to.Know that from above preparation method this method for preparing monascus yellow pigment pigment is mainly based on chemosynthesis, complex process belongs to the downstream technology of biological fermentation.
The patent No. be the Chinese invention patent file of ZL200710032525.3 disclose by physicochemical complex mutation obtained a strain produce yellow pigment at high proportion with the monascus mutant strain Monascus anka mutant MYM2 of high luminance relay valency, it also discloses with this MYM2 is fermentation strain, can produce yellow pigment by liquid state fermentation.This patent has also disclosed the fermented liquid that liquid state fermentation is obtained separate purification after, under 410 and 510 two wavelength, carry out absorbance detection with ultraviolet spectrophotometer, wherein 410nm is a yellow pigment, 510nm is a haematochrome.
People such as south China Polytechnics cycle in 2008, Zhu Mingjun disclose the paper of " seed selection of the mould yellow pigment pigment of high yield monascus bacterial strain and the optimization of fermentation nitrogen source " by name, disclose the strain monascus ruber (Monascus) that its laboratory is preserved particularly and carry out physics and chemically composited mutagenesis, as: utilize the method for ultraviolet, ethyl sulfate, lithium chloride and nitrosoguanidine complex mutation, seed selection is to the monascus mutant strain MYM2 of a strain than the high yield yellow pigment.Prove that through stability experiment the strain stability that mutagenesis obtains is better, liquid state fermentation is tested yellow plain color valency and is up to more than the 100u/ml.This yellow pigment has only a charateristic avsorption band at 410nm between 300～600nm wavelength, stability better between pH 3～8.When the pH value less than 3 monascus yellow pigment pigment instabilities, it is muddy that pigment solution becomes, and has precipitation to produce after the placement.
People such as the Peng Zhen of south China Polytechnics, Wu Zhenqiang disclosed " research of monascus yellow pigment pigment solid state fermentation conditions " achievement in 2009: the optimal culture condition of solid state fermentation production yellow pigment is 55% initial water content, the loading capacity of triangular flask is the bright rice/250ml of 18g, add the ammonium chloride of 0.2mmol/g, initial p H value is 6.0, inoculum size is 10%, 30 ℃ and cultivated 10 days down.The look valency that obtains yellow pigment is about 665.78u/g, and tone is about 4.33, no citrinin.The shortcoming of solid state fermentation be just the solid state fermentation cycle long, wayward, easy microbiological contamination is difficult to reach the suitability for industrialized production requirement.
Japan is in advanced in the world aspect biofermentation technique always, and it also has the product of relevant monascus yellow pigment to sell in market, and as the natural NO0393 of Japan, but the more employing confidentiality of technology is open.And the research and development of domestic monascus ruber also are in the strain improvement stage, yet there are no the report of the suitability for industrialized production of relevant monascus yellow pigment microbial fermentation, its technical bottleneck is not only the selection by mutation of the bacterial classification of high yield yellow pigment, and with the selection of proferment material, technology controlling and process substantial connection is arranged also.
By above-mentioned materials as can be known, in the whole bag of tricks of existing production, preparation monascus yellow pigment, all rest on laboratory stage, and how to control for various harmful byproduct that in preparation process, produces such as Citrinin etc., then there is not relevant report, particularly in the heavy industrialization preparation process, how starting material, processing step, condition are controlled, thereby when realizing efficient suitability for industrialized production, the content of byproducts such as reasonable control Citrinin, make goods meet the food hygienic standard of various strictnesses, do not have the pertinent literature reference especially.
Summary of the invention
One of the object of the invention is, provides through ultraviolet mutagenesis to separate the monascus mutant strain TY622 (Monascus purpureus yang TY622) that obtains, as stable, high yield, secondary metabolites is few and the bacterial classification that is easy to control;
Two of purpose of the present invention is that providing a kind of is that bacterial classification passes through the deep layer liquid-state fermentation technology with monascus mutant strain TY622, realizes the method for the industrialization production of yellow pigment; At monascus yellow pigment complicated process of preparation in the prior art; Yellow pigment content is low, yield poorly; citrinin content height in the fermented liquid; only be not easy the difficult problem of large-scale production, provide a kind of submerged fermentation to prepare the industrialization production method of low citrinin monascus yellow pigment for laboratory stage.
The present invention also aims to, a kind of monascus yellow pigment goods that adopt the low citrinin content of method for preparing are provided.
Monascus mutant strain TY622 provided by the invention (Monascus purpureus yang TY622) is deposited in Chinese common micro-organisms culture presevation administrative center on September 2nd, 2010, and preserving number is CGMCC4110.
The concrete technical scheme that the present invention is adopted for achieving the above object is:
A kind of monascus mutant strain Monascus purpureus yang TY622, its deposit number is CGMCC4110.
A kind of submerged fermentation prepares the method for low citrinin monascus yellow pigment, it is characterized in that, it comprises the steps:
1. strain improvement:
Select for use by ultraviolet mutagenesis and separate the monascus mutant strain TY622 (Monascus purpureus yang TY622) that obtains;
2. seed culture:
Adopt the liquid seeds jar to cultivate the I and II seed earlier, enter again in the large vol fermentor tank after the enlarged culturing and ferment, the steps include:
(21) preparation of seed liquor:, through the I and II seed tank culture 7～8 hours of seed culture medium is housed, obtain the liquid seeds nutrient solution successively with monascus mutant strain TY622 (Monascus purpureus yangTY622);
Wherein the raw material of seed culture medium composition and weight proportion are: starch 3～5%, NaNO
30.1 MgSO～0.3%,
40.1 KH～0.3%,
2PO
40.1～0.3%, corn steep liquor 2～5%;
(22) enlarged culturing: the liquid seeds nutrient solution of step (21) is inoculated into carried out enlarged culturing in the fermentor tank 75～85 hours, the liquid seed culture fluid of the usefulness of must fermenting; Wherein the material component of fermention medium and weight proportion are: rice meal 8～10%, NaNO
30.2%～0.3%, MgSO
40.1%～0.3%, KH
2PO
40.2 NH～0.3%,
4Cl 0.1～0.3%, corn steep liquor 2～5%; Regulating the fermention medium initial pH value with lactic acid is 3.0～4.5.
3. fermentation:
The employing capacity is 20～30m
3Large vol fermentor tank liquid fermenting, learn through the contriver repetition test, adopt 20-30m
3The large vol fermentor tank carry out liquid fermenting, be improve fermentation reaction efficient, improve fermenting speed and quality, one of key factor of reduction Citrinin generation;
(31) at first pack in fermentor tank and account for the liquid fermentation medium of ferment jar 2/3 volume, the 30min that sterilizes under 121～125 ℃ of jar temperature, pressure 0.1Mpa cools to 30～35 ℃;
(32) the described liquid seed culture fluid of inoculation step (22), inoculum size is 5～10%, inserts the secondary seed jar and cultivates 7～8 hours, must ferment and use the liquid seeds nutrient solution;
(33) carry out submerged fermentation jar liquid fermenting: the control fermentation keeps envrionment conditions: temperature is adjusted into 34～36 ℃ then for fermentation initially kept 30～35 ℃ of temperature in 34 hours when fermentation continues 35～85 hours; Remain average rate and stir, mixing speed is 200～400r/min; Remain ventilation, ventilation is initially 400～450Lmin for fermentation, and the fermentation later stage is adjusted into 500～600Lmin; Remaining tank pressure is 0.4～0.7Mpa, carries out fermentation culture continuously 75～85 hours, can obtain low citrinin monascus yellow pigment fermented liquid, contains the monascus yellow pigment in this fermented liquid;
This monascus yellow pigment fermented liquid carries out absorbance detection with the UV-light spectrophotometer, and its charateristic avsorption band wavelength is 465～475nm;
4. extract, purifying monascus yellow pigment:
(41) extract: this low citrinin monascus yellow pigment fermented liquid is carried out processed, and with the edible ethanol lixiviate with 55-70v/v of the mycelium that stays, vacuum concentration (20～35Be hammer degree) is carried out lixiviate filtrate in the secondary press filtration again;
Purifying: utilize concentrated solution heat (30～80 ℃) transform 1～10 hour; Jia Shui, add acid for adjusting pH value, left standstill 5-10 hour, precipitation, filter, add alkali once more and regulate the pH value to 5-10, vacuum concentration (20～35Be hammer degree) to 3-10;
(42) spraying drying: concentrated solution is carried out spraying drying under 100～210 ℃ of conditions, make monascus bloom end pigment finished product.
5. adopt ultraviolet spectral photometer to detect the absorbancy of described monascus yellow pigment finished product.
6. detect the content of the Citrinin of described monascus yellow pigment finished product.
A kind of monascus yellow pigment that adopts aforementioned submerged fermentation to prepare the preparation of low citrinin monascus yellow pigment method is characterized in that the content of Citrinin is not more than 0.2mg/kg in its finished product; Described monascus yellow pigment finished product, detecting its absorbancy with ultraviolet spectral photometer is that the peak value of 468～470nm is higher at wavelength.
Monascus mutant strain TY622 provided by the invention (Monascus purpureus yang TY622), the excellent species that to be the contriver obtain through ultraviolet mutagenesis, long-term breeding, it breeds stable performance, high yield, can control the content of Citrinin during the fermentation, make the content of Citrinin in the yellow pigment finished product that makes be lower than 0.2mg/kg.
Yellow pigment preparation method provided by the invention, be by adopting specific bacterial classification, raw material, processing method and condition control, the monascus ruber mutant strain of selecting for use is carried out multistage cultivation, submerged fermentation, obtain high-load yellow pigment fermented liquid, handle the yellow pigment of back gained again through abstraction process, citrinin content is very low, safe, good stability.
Citrinin belongs to the microbial secondary meta-bolites, the present invention takes multistage cultivation, improve constantly the adaptability of bacterial strain to yeasting, and adopt specific container, material and processing condition, reasonable control by each step is produced Citrinin to reduce yeast phase, finally reaches the purpose that reduces the total body burden of Citrinin.Simultaneously; the present invention is before entering submerged fermentation; the enlarged culturing of carrying out bacterial classification not only can increase the bacterium amount of yeast phase; also help and improve microbic activity in the yeast phase; make a large amount of microorganisms in yeast phase, be in growth phase stationary phase; make its acquisition and keep more vigorous metabolism, be beneficial to raising output, accomplish scale production.
The present invention adopts biofermentation technique to carry out submerged fermentation, and its technology is simple, and condition is easy to control, can realize industrialization, large-scale production.Production process adopts the raw material of environmental protection, realize CR production and safety in production, production unit adopts large vol fermentor tank, production efficiency height, can realize suitability for industrialized production, and make constant product quality, effective constituent height, objectionable constituent low, meet the food quality safety standards.
Method provided by the invention can realize the large-scale industrial production of monascus yellow pigment, its composition of raw materials is reasonable, technical process succinct, condition control is easy, fermentation reaction is stable, reaction efficiency is high, in preparation process and goods, prepared Yellow pigment content height, citrinin content are low.
The present invention is further described below in conjunction with specific embodiment.
Embodiment
Embodiment 1:
The monascus mutant strain Monascus purpureus yang TY622 that present embodiment provides, its deposit number is CGMCC4110.
Adopt aforementioned monascus mutant strain TY622 submerged fermentation to prepare the method for the monascus yellow pigment of low citrinin, it comprises the steps:
1. strain improvement:
Select for use by ultraviolet mutagenesis and separate the monascus mutant strain TY622 (Monascus purpureus yang TY622) that obtains;
2. seed culture:
Adopt the liquid seeds jar to cultivate the I and II seed earlier, enter again in the large vol fermentor tank after the enlarged culturing and ferment, the steps include:
(21) preparation of seed liquor: adopt high yield monascus trichoderma strain TY622, through I and II seed tank culture that seed culture medium is housed 7～8 hours, obtain the liquid seeds nutrient solution successively; Wherein the raw material of seed culture medium composition and weight proportion are: starch 3～5%, NaNO
30.1 MgSO～0.3%,
40.1 KH～0.3%,
2PO
40.1～0.3%, corn steep liquor 2～5%;
(22) enlarged culturing: the liquid seeds nutrient solution of step 2.1 is inoculated into carried out enlarged culturing in the fermentor tank 75～85 hours, make the liquid seed culture fluid of fermentation usefulness;
3. fermentation:
The employing capacity is 20～30m
3Large vol fermentor tank liquid fermenting, learn through the contriver repetition test, adopt 20-30m
3The large vol fermentor tank carry out liquid fermenting, be improve fermentation reaction efficient, improve fermenting speed and quality, one of key factor of reduction Citrinin generation;
(31) at first pack in fermentor tank and account for the liquid fermentation medium of ferment jar 2/3 volume, the 30min that sterilizes under 121～125 ℃ of jar temperature, pressure 0.1Mpa cools to 30～35 ℃; The material component of described fermention medium and weight proportion are: rice meal 8～10%, NaNO
30.2%～0.3%, MgSO
40.1%～0.3%, KH
2PO
40.2 NH～0.3%,
4Cl 0.1～0.3%, corn steep liquor 2～5%; Regulating the fermention medium initial pH value with lactic acid is 3.0～4.5.
(32) inoculation step 2.2 described liquid seed culture fluids, inoculum size is 5～10%, inserts the secondary seed jar and cultivates 7～8 hours, makes fermentation liquid seeds nutrient solution;
(33) carry out submerged fermentation jar liquid fermenting: the control fermentation keeps envrionment conditions: temperature is adjusted into 33～36 ℃ then for fermentation initially kept 30～35 ℃ of temperature in 34 hours when fermentation continues 35～85 hours; Remain average rate and stir, mixing speed is 200～400r/min; Remain ventilation, ventilation is initially 400～450Lmin for fermentation, and the fermentation later stage is adjusted into 500～600Lmin; Remaining tank pressure is 0.4～0.7Mpa, carries out fermentation culture continuously 75～85 hours, can obtain low citrinin monascus yellow pigment fermented liquid, contains the monascus yellow pigment in this fermented liquid; This monascus yellow pigment fermented liquid carries out absorbance detection with the UV-light spectrophotometer, and its charateristic avsorption band wavelength is 465～475nm.
4. extract, purifying monascus yellow pigment:
(41) extract: this low citrinin monascus yellow pigment fermented liquid is carried out processed, and with the edible ethanol lixiviate with 55-70v/v of the mycelium that stays, vacuum concentration (20～35Be hammer degree) is carried out lixiviate filtrate in the secondary press filtration again;
Purifying: utilize concentrated solution heat (30～80 ℃) transform 1～10 hour; Jia Shui, add acid for adjusting pH value, left standstill 5-10 hour, precipitation, filter, add alkali once more and regulate the pH value to 5-10, vacuum concentration (20～35Be hammer degree) to 3-10;
(42) spraying drying: concentrated solution is carried out spraying drying under 100～210 ℃ of conditions, make monascus bloom end pigment finished product.
5. adopt ultraviolet spectral photometer to detect the absorbancy of described monascus yellow pigment finished product.
6. detect the content of the Citrinin of described monascus yellow pigment finished product.
Adopt the monascus yellow pigment of preceding method preparation, the content of Citrinin is not more than 0.2mg/k in its finished product; Detecting its absorbancy with ultraviolet spectral photometer is that the peak value of 468～470nm is higher at wavelength.
Below, provide according to such scheme again, and four of carrying out possess representational specific embodiment.
Embodiment 2:
Bacterial strain that present embodiment provides and preparation method, goods are substantially the same manner as Example 1, and its difference is:
Two, be equipped with substratum
2. substratum is equipped with
2.1 the raw material of liquid seed culture medium is formed and proportioning: starch 4%, NaNO
30.2%, MgSO
40.2%, KH
2PO
40.2%, corn steep liquor 3%;
2.2 the material component of fermention medium and proportioning are: rice meal 9%, NaNO
30.15%, MgSO
40.2%, KH
2PO
40.25%, NH
4Cl 0.2%, corn steep liquor 4%%; Regulating the fermented liquid initial pH value with lactic acid is 3.0～4.0.
Three, zymotechnique
3.1 the preparation of seed liquor: adopt the mould mutant strain of monascus of high yield yellow pigment, process I and II seed tank culture 7～8 hours obtains liquid seed culture fluid;
3.2 enlarged culturing: the liquid seed culture fluid of step 2.1 is inoculated into carries out enlarged culturing in the fermentor tank, the liquid seed culture fluid that must be used to ferment;
3.3 fermentation: the employing volume is 30m
3The fermentor tank liquid fermenting, at first at 30m
3Fermentor tank in the 20m that packs into
3The aforesaid liquid fermention medium, 121 ℃ of jar temperature, pressure 0.1Mpa is sterilization 30min down, cools after 30～35 ℃;
Inoculate the above-mentioned liquid seed culture fluid that is used for fermenting in the secondary culture tank, inoculum size was cultivated 7～8 hours according to 7%, must ferment and use the liquid seeds nutrient solution;
Inoculate above-mentioned fermentation and use the liquid seeds nutrient solution in fermentor tank, inoculum size enters fermentation culture, processing condition again by 7%: 1. temperature control: 30～35 ℃ of initial temperatures were adjusted into 33～36 ℃ after 35 hours; 2. mixing speed: 200～400r/min; 3. ventilation: be initially 400～450 (Lmin), after 35 hours, be adjusted into 500～600/ (Lmin) then; 4. tank pressure: 0.4～0.7MPa.Obtain the monascus yellow pigment fermented liquid of low citrinin after 75～85 hours.
At fermenting process, regularly get fermented liquid and survey its pH and detect absorbancy and at the look valency of wavelength 465～475nm with ultraviolet spectral photometer.
Four, extract monascus yellow pigment finished product:
4.1 extract: this low citrinin monascus yellow pigment fermented liquid is carried out processed, and with the edible ethanol lixiviate with 55-70v/v of the mycelium that stays, vacuum concentration (20～35Be hammer degree) is carried out lixiviate filtrate in the secondary press filtration again; Purifying: utilize concentrated solution heat (30～80 ℃) transform 1～10 hour; Jia Shui, add acid for adjusting pH value, left standstill 5-10 hour, precipitation, filter, add alkali once more and regulate the pH value to 5-10, vacuum concentration (20～35Be hammer degree) to 3-10;
4.2 spraying drying: concentrated solution is carried out spraying drying under 100～210 ℃ of conditions, make monascus bloom end pigment finished product.
Four, the citrinin content of detection monascus yellow pigment finished product reaches at the look valency of wavelength at 465～475nm.
Five, detect the content of the Citrinin of described monascus yellow pigment finished product.
Embodiment 3:
Bacterial strain that present embodiment provides and preparation method, goods and embodiment 1 or 2 are basic identical, and its difference is:
Two, zymotechnique " step (3) be: at 20m
3Fermentor tank in the 13.3m that packs into
3The aforesaid liquid fermention medium; Inoculate in fermentor tank, inoculum size is 5%.Other steps regularly detect the pH and the look valency of fermentation technology process fermented liquid with embodiment 1, and extract monascus yellow pigment its citrinin content of finished product detection and look valency.
Embodiment 4:
The method of present embodiment and goods are basic identical with embodiment 1 or 2 or 3, and its difference is:
One, is equipped with substratum
1. substratum is equipped with
1.1 the raw material of liquid seed culture medium is formed and proportioning: starch 3%, NaNO
30.1%, MgSO
40.1%, KH
2PO
40.1%, corn steep liquor 2%;
1.2 the material component of fermention medium and proportioning are: rice meal 9%, NaNO
30.15%, MgSO
40.2%, KH
2PO
40.25%, NH
4Cl 0.2%, corn steep liquor 4%%; Regulating the fermented liquid initial pH value with lactic acid is 3.8;
Detect the pH and the toner of fermentation technology process fermented liquid; Detect monascus yellow pigment pigment finished product citrinin content and look valency.
Embodiment 5:
The method of present embodiment and goods are basic identical with embodiment 1 or 2 or 3 or 4, and its difference is:
One, is equipped with substratum
1. substratum is equipped with
1.1 the raw material of liquid seed culture medium is formed and proportioning: starch 4%, NaNO
30.2%, MgSO
40.2%, KH
2PO
40.2%, corn steep liquor 3%;
1.2 the material component of fermention medium and proportioning are: rice meal 8%, NaNO
30.2%, MgSO
40.1%, KH
2PO
40.2%, NH
4Cl0.1%, corn steep liquor 2%; Regulating the fermented liquid initial pH value with lactic acid is 3.0～4.0.
The The performance test results of the monascus yellow pigment goods that the present invention obtains sees Table 1.
The The performance test results of table 1 monascus yellow pigment
As described in above-mentioned embodiment, the present invention is not limited to the foregoing description, and the technical scheme that all employings are same as the previously described embodiments or similar reaches other schemes of the object of the invention, all within protection domain of the present invention.
Claims (10)
1. monascus mutant strain Monascus purpureus yang TY622, its deposit number is CGMCC4110.
2. a method that adopts the submerged fermentation of the described monascus mutant strain of claim 1 to prepare the monascus yellow pigment is characterized in that it comprises the steps:
(1) strain improvement:
Select for use the monascus mutant strain TY622 that separates acquisition by ultraviolet mutagenesis as bacterial classification, its preserving number is CGMCC4110;
(2) seed culture:
Adopt the liquid seeds jar to cultivate I and II bacterial classification earlier, enter again in the large vol fermentor tank after the enlarged culturing and ferment, make the liquid seed culture fluid of fermentation usefulness;
(3) fermentation:
The employing capacity is 20～30m
3The large vol fermentor tank carry out deep fermentation, obtain low citrinin monascus yellow pigment fermented liquid, and the monascus yellow pigment that contains in the fermented liquid.
3. described submerged fermentation prepares the method for low citrinin monascus yellow pigment according to claim 2, it is characterized in that the concrete steps of described step (2) are:
(21) preparation of seed liquor:, through the I and II seed tank culture 7～8 hours of seed culture medium is housed, obtain the liquid seeds nutrient solution successively with monascus mutant strain TY622;
(22) enlarged culturing: the liquid seeds nutrient solution of step (21) is inoculated into carried out enlarged culturing in the fermentor tank 75～85 hours, the liquid seed culture fluid of the usefulness of must fermenting;
4. described submerged fermentation prepares the method for the monascus yellow pigment of low citrinin according to claim 3, it is characterized in that, the raw material of the seed culture medium in the described step (21) is formed and weight proportion is: starch 3～5%, NaNO
30.1 MgSO～0.3%,
40.1 KH～0.3%,
2PO
40.1～0.3%, corn steep liquor 2～5%;
The material component and the weight proportion of the described fermention medium in the described step (22) are: rice meal 8～10%, NaNO
30.2%～0.3%, MgSO
40.1%～0.3%, KH
2PO
40.2 NH～0.3%,
4C10.1～0.3%, corn steep liquor 2～5%; Regulating the fermention medium initial pH value with lactic acid is 3.0～4.5.
5. described submerged fermentation prepares the method for the monascus yellow pigment of low citrinin according to claim 2, it is characterized in that the concrete steps of described step (3) are:
(31) at first pack in fermentor tank and account for the liquid fermentation medium of ferment jar 2/3 volume, the 30min that sterilizes under 121～125 ℃ of jar temperature, pressure 0.1Mpa cools to 30～35 ℃;
(32) the described liquid seed culture fluid of inoculation step (22), inoculum size is 5～10%, inserts the secondary seed jar and cultivates 7～8 hours, must ferment and use the liquid seeds nutrient solution;
(33) carry out submerged fermentation jar liquid fermenting:
Control fermentation keeps envrionment conditions: temperature keeps 30～35 ℃ of temperature for fermentation in initial 0 to 34 hour, continues to be adjusted into 33～36 ℃ in 35～85 hours in fermentation then;
Remain average rate and stir, mixing speed is 200～400r/min;
Remain ventilation, ventilation is initially 400～450Lmin for fermentation, and the fermentation later stage is adjusted into 500～600Lmin;
Remaining tank pressure is 0.4～0.7Mpa, carries out fermentation culture continuously 75～85 hours, can obtain low citrinin monascus yellow pigment fermented liquid, and the monascus yellow pigment that contains in the fermented liquid.
6. described submerged fermentation prepares the method for the monascus yellow pigment of low citrinin according to claim 5, it is characterized in that, the monascus yellow pigment fermented liquid of described step (33) carries out absorbance detection with the UV-light spectrophotometer, and its charateristic avsorption band wavelength is 465～475nm.
7. described submerged fermentation prepares the method for low citrinin monascus yellow pigment according to claim 2, it is characterized in that it also comprises the steps:
(4) extraction, purifying monascus yellow pigment:
(41) extract: this low citrinin monascus yellow pigment fermented liquid is carried out processed, and with the edible ethanol lixiviate with 55-70v/v of the mycelium that stays, 20～35Be hammer degree vacuum concentration is carried out lixiviate filtrate in the secondary press filtration again;
(42) purifying: utilize to concentrate to be warmed to 30～80 ℃, transform 1～8 hour, add acid for adjusting pH value, through leaving standstill, precipitate, filtering, carry out 20～35Be hammer degree vacuum concentration more successively, get concentrated solution to 3-10;
(43) spraying drying: the described concentrated solution of step (42) is carried out spraying drying under 100～210 ℃ of conditions, make monascus yellow pigment finished product.
8. described submerged fermentation prepares the method for low citrinin monascus yellow pigment according to claim 2, it is characterized in that it also comprises the steps:
(5) adopt ultraviolet spectral photometer to detect the absorbancy of described monascus yellow pigment finished product.
(6) content of the Citrinin of the described monascus yellow pigment finished product of detection.
9. according to the monascus yellow pigment of the described method preparation of one of claim 2-8, it is characterized in that the content of Citrinin is not more than 0.2mg/kg in its finished product.
10. monascus yellow pigment according to claim 9 is characterized in that, described monascus yellow pigment, and detecting its absorbancy with ultraviolet spectral photometer is that the peak value of 468～470nm is higher at wavelength.
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