CN104962483A - Monascus purpureus strain and application thereof - Google Patents
Monascus purpureus strain and application thereof Download PDFInfo
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Abstract
The invention discloses a Monascus purpureus strain and application thereof in food production. The Monascus purpureus strain is collected by China General Microbiological Culture Collection Center, and the collection number is CGMCC No.9712. The Monascus purpureus strain almost produces no notalin, has the minimum notalin yield among the known Monascus strains, can generate the natural Monascus pigment, has higher color number, and can be used in the technique for preparing milk products or processing other foods.
Description
Technical field
The present invention relates to microorganism field, be specifically related to a kind of monascus purpureus bacterial strain and uses thereof.
Background technology
Monascus (Monascus sp.) is the very important medicinal fungi of a class, is one of China fungi of being applied in food-processing the earliest.And use range is extensive, is mainly used in culinary art, makes soy cheese, wine brewing and disease therapy etc.The red colouring agent for food, also used as a Chinese medicine Gu adopting Fermentation Condition of Monascus spp rice obtained claims red bent, with a long history, originates from China, and its main pooled applications, in fields such as traditional distiller's yeast, vinegar processed, painted and healthcare products, is the tradition material of dietotherapeutic.Modern medicine study proves, red colouring agent for food, also used as a Chinese medicine has and reduces cholesterol, the effect such as hypoglycemic and hypotensive.In addition, monascus can produce the multiple meta-bolites with physiologically active during the fermentation, such as: monascorubin, citrinin (Monacolin K) class material, γ-aminobutyric acid and multiple enzyme etc.
Although monascus is the edible history of China is longer and pouplarity is higher, but because some monascus species can produce mycotoxins during the fermentation---Citrinin (citrinin), and Citrinin has higher toxicity to human body, therefore limit the range of application of monascus strain to a certain extent.Food safety is the prerequisite of foodstuff production and sale, the content of European and American countries to the Citrinin in imported food especially monascus product has strict restriction, meanwhile, the content also defining Citrinin in Red kojic rice (powder) in People's Republic of China (PRC) light industry standard QB/T2847-2007 must not more than 50 μ g/Kg.The strain variety studying content and the monascus ruber showing Citrinin is closely related, therefore, produce the product of Fermentation Condition of Monascus spp class, need strictly to select bacterial strain and optimized production process, ensures food safety.
Summary of the invention
Technical problem to be solved by this invention is, low for the content lacking at present Citrinin, to meet respective country standard monascus (Monascus sp.) thus be difficult to prepare the content of Citrinin low, safe take monascus as the deficiency of the food of raw material, a kind of monascus purpureus bacterial strain of low-yield citrinin is provided and is preparing the application in food.Described monascus strain is that in existing known monascus bacterial strain, Citrinin output is minimum, and can produce natural monascorubin, can apply to food-processing industry safely.
One of technical scheme of the present invention is: a kind of monascus purpureus (Monascus purpureus), it is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is: CGMCCNo.9712.
Be separated from the red kojic rice powder in Wuhan, China Hubei and obtain described monascus purpureus CGMCCNo.9712.Described monascus purpureus CGMCC No.9712 can produce natural monascorubin, and Citrinin output is lower, can apply to food-processing industry safely.Identify this bacterial strain, result is monascus purpureus Monascus purpureus, called after BD-M-4.This bacterial strain is preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center on October 8th, 2014, and receives preservation center and to register on the books numbering CGMCC No.9712, and it has following Microbiological Characteristics:
1, morphologic characteristic
Very fast at MEA cultured on solid medium, cultivate 10 days under 25 DEG C of dark conditions, colony diameter 45 ~ 50mm.Aerial hyphae is luxuriant, edge white, and middle part is orange; The bacterium colony back side, in secretly orange, dissolves in MEA substratum.
Microscopic examination shows, and produce conidium in a large number, conidiophore specialization is not obvious, and directly or slightly bending, conidium is spherical in shape, colourless, and wall is slightly coarse, and base portion is truncate, concatenates, length 5.5-10.4 μm.Cleistothecium is shallow to brown, most immature, has no thecaspore.
2, the characteristic learned is cultivated
All can grow under 20 ~ 42 DEG C of conditions, proper growth temperature is 25 ~ 32 DEG C, and optimum temperuture is 30 DEG C; Suitable pH is 3.5 ~ 6.0, and optimal pH is 5.5.Suitable growth medium can be PDB liquid nutrient medium, YES liquid nutrient medium or MEA liquid nutrient medium.
3, physiological property
Produce Citrinin hardly, natural monascorubin can be produced, and have higher look valency.
Two of technical scheme of the present invention is: a kind of method preparing described monascus purpureus CGMCC No.9712, it comprises the following steps, cultivates described monascus purpureus CGMCCNo.9712 in the medium.
Wherein, described substratum is the substratum of this area routine, can grow described monascus purpureus CGMCC No.9712, is preferably PDB liquid nutrient medium, YES liquid nutrient medium or MEA liquid nutrient medium.Described PDB liquid nutrient medium is the PDB liquid nutrient medium of this area routine, and preferably, it comprises 6 ~ 10g/L potato leaching powder and 20 ~ 40g/L glucose.Described YES liquid nutrient medium is the YES liquid nutrient medium of this area routine, and preferably, it comprises the leaching of 4 ~ 6g/L yeast powder, 20 ~ 40g/L glucose, 5 ~ 7g/L potassium primary phosphate, 3 ~ 5g/L SODIUM PHOSPHATE, MONOBASIC and 0.5 ~ 1.5g/L ammonium hydroxide.Described MEA liquid nutrient medium is the MEA liquid nutrient medium of this area routine, and preferably, it comprises 20 ~ 40g/L malt extract and 2 ~ 4g/L soy peptone.The time of described cultivation is the time of this area routine, is preferably 3 ~ 10 days, is more preferably 5 ~ 10 days.The temperature of described cultivation is the temperature of this area routine, is preferably 20 ~ 42 DEG C, is more preferably 25 ~ 30 DEG C.Preferably, described cultivation is concussion cultivation.The rotating speed that described concussion is cultivated is the rotating speed of this area routine, and being preferably 120 ~ 220rpm, is more preferably 180 ~ 220rpm.The inoculum size of described cultivation is the inoculum size of this area routine, is preferably 5 ~ 10%, and be more preferably 5%, described per-cent is volume percent.
Preferably, also comprise before described cultivation and monascus purpureus CGMCC No.9712 is inoculated in the step that seed culture medium carries out seed culture.The time of described seed culture is the time of this area routine, is preferably 5 ~ 10 days, is more preferably 6 ~ 8 days, is 7 days best.The temperature of described seed culture is the temperature of this area routine, is preferably 15 ~ 35 DEG C, is more preferably 25 ~ 32 DEG C, is 30 DEG C best.The substratum of described seed culture is the substratum of this area routine, is preferably PDA solid medium, YES solid medium or MEA solid medium.Described PDA solid medium is the PDA solid medium of this area routine, and preferably, it comprises the leaching of 6 ~ 10g/L potato powder, 20 ~ 40g/L glucose and 20 ~ 30g/L agar.Described YES solid medium is the YES solid medium of this area routine, preferably, it comprises the leaching of 4 ~ 6g/L yeast powder, 20 ~ 40g/L glucose, 5 ~ 7g/L potassium primary phosphate, 3 ~ 5g/L SODIUM PHOSPHATE, MONOBASIC, 0.5 ~ 1.5g/L ammonium hydroxide and 15 ~ 25g/L agar.Described MEA solid medium is the MEA solid medium of this area routine, and preferably, it comprises 20 ~ 40g/L malt extract, 2 ~ 4g/L soy peptone and 12 ~ 20g/L agar.Preferably, described seed culture comprises after the monascus strain just refrigerated slowly heats up, the step of coating or line on seed culture medium.The temperature of described intensification is the temperature of this area routine, is preferably to 15 ~ 25 DEG C.The method of described cultivation is the method for the cultivation of this area routine, is preferably shake-flask culture or fermentor cultivation.
Three of technical scheme provided by the invention is: the described purposes of monascus purpureus CGMCC No.9712 in foodstuff production.
Described food is the food of this area routine, and containing monascus purpureus CGMCC No.9712 or its meta-bolites, being preferably milk-product, is more preferably cheese.
Described monascus purpureus CGMCC No.9712 produces Citrinin hardly, and has higher look valency, can be used for preparing in milk-product or other food processing technology.
Four of technical scheme provided by the invention is: a kind of cheese utilizing Fermentation Condition of Monascus spp obtained, described monascus is monascus purpureus CGMCC No.9712.
Described cheese is monascus cheese, it has this kind of distinctive flavour of cheese and smell, fermenting aroma is strong, there is the distinctive tender texture of such cheese, good, the surperficial corrugationless of shell, shell takes on a red color or purple, and inner in uniform purple or redness, this cheese is applicable to the food habits of Chinese Consumer's.
Five of technical scheme provided by the invention is: a kind of red kojic rice powder utilizing Fermentation Condition of Monascus spp obtained, described monascus is monascus purpureus CGMCC No.9712.
Described red kojic rice powder is red extremely dark violet redness, and quality is crisp, without going mouldy, without obvious macroscopic impurity, in irregular particulate state, has the Qu Xiang that red colouring agent for food, also used as a Chinese medicine is intrinsic.
On the basis meeting this area general knowledge, above-mentioned each optimum condition, can arbitrary combination, obtains the preferred embodiments of the invention.
Agents useful for same of the present invention and raw material are all commercially.
Positive progressive effect of the present invention is: described monascus purpureus CGMCC No.9712 produces Citrinin hardly, is that in existing known monascus bacterial strain, Citrinin output is minimum.And it can produce natural monascorubin, have higher look valency, can be used for preparing in the complete processing of the food such as milk-product, red kojic rice powder.
biomaterial preservation information
Monascus purpureus BD-M-4 of the present invention, be deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on October 8th, 2014, preservation address: No. 3, Yard 1, BeiChen xi Road, Chaoyang District, Beijing City, postcode: 100101, deposit number is: CGMCC No.9712, culture title is BD-M-4, and Classification And Nomenclature is monascus purpureus Monascus purpureus.
Accompanying drawing explanation
Fig. 1 is the situation that monascus purpureus BD-M-4 biomass changes with fermentation time.
The situation of Fig. 2 biomass that is different monascus purpureus bacterial strain when fermentation 10 days.
The situation of Fig. 3 to be different monascus purpureus bacterial strain produce when fermentation 10 days citrinin content.
Fig. 4 is the morphological specificity of monascus purpureus BD-M-4.Wherein A represents the colonial morphology of monascus purpureus BD-M-4 on wort agar substratum (MEA); B ~ D represents the microscopic features of monascus purpureus BD-M-4.
Embodiment
Mode below by embodiment further illustrates the present invention, but does not therefore limit the present invention among described scope of embodiments.The experimental technique of unreceipted actual conditions in the following example, conventionally and condition, or selects according to catalogue.
Monascus GL-1 in effect example is red colouring agent for food, also used as a Chinese medicine (Monascus sp.), this bacterial strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on May 8th, 2013, bacterial strain deposit number: CGMCC No.7603, announces in CN103444878A.
Monascus purpureus BD-M-1 in effect example is purplish red song (Monascus purpureus), this bacterial strain is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) on August 28th, 2013, bacterial strain deposit number: CGMCC No.8120, announces in CN104585333A.
The acquisition of embodiment 1 monascus purpureus BD-M-4 bacterial strain
Purification procedures is as follows:
(1) PDA solid medium is configured: potato leaching powder 6g/L, glucose 20g/L and agar 20g/L.
(2) coating is cultivated: the red kojic rice powder in the Wuhan, China Hubei that takes a morsel evenly is sprinkled into the PDA solid culture primary surface that step (1) is prepared.Cultivate 2 days in constant incubator for 30 DEG C.
(3) after white fluffy mycelia grows, the line of picking a little mycelia is transferred and is continued to cultivate on another PDA solid medium, cultivates 1 week.Continuous repeating step (2) and step (3) 3 times, obtain the monascus purpureus of homogeneous proterties, called after BD-M-4.
(4) monascus purpureus BD-M-4 bacterial strain to be stored on PDA solid medium and to be placed in 4 DEG C of refrigerators.
By monascus purpureus BD-M-4 on October 8th, 2014 in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC) preservation, acquisition deposit number is: CGMCC No.9712, culture title is BD-M-4, and Classification And Nomenclature is monascus purpureus Monascus purpureus.
The operation of same above-mentioned steps (1) ~ (4), have also obtained the monascus strain of other homogeneous proterties, respectively called after monascus BD-A, monascus BD-B, monascus BD-C and monascus GL-A.
The cultural characters of embodiment 2 monascus purpureus BD-M-4
(1), monascus purpureus BD-M-4 is very fast at MEA cultured on solid medium, and cultivate 10 days under 25 DEG C of dark conditions, colony diameter reaches 45 ~ 50mm.Aerial hyphae is luxuriant, edge white, and middle part is orange; The bacterium colony back side, in secretly orange, dissolves in substratum.Microscopic examination shows, and monascus purpureus BD-M-4 produces a large amount of conidium, and conidiophore specialization is not obvious, and directly or slightly bending, conidium is spherical, colourless, and wall is slightly coarse, and base portion is truncate, concatenates, 5.5-10.4 μm.Cleistothecium is shallow to brown, most immature, has no thecaspore, all meets the feature (see Fig. 4 A ~ D) of monascus.
(2), through cultivating find, BD-M-4 bacterial strain all can grow under 20 ~ 42 DEG C of conditions, and proper growth temperature is 25 ~ 32 DEG C, and optimum temperuture is 30 DEG C; Suitable pH is 3.5 ~ 6.0, and optimal pH is 5.5.Suitable growth medium can be PDB liquid nutrient medium, YES liquid nutrient medium or MEA liquid nutrient medium.Wherein, on MEA liquid nutrient medium and PDB liquid nutrient medium, under 30 DEG C of conditions cultivate 10 days, through 15 generation cultured continuously, its cultural characteristic, morphological specificity etc. have no significant change, and the biological shapes of this bacterium is basicly stable.
Wherein, PDB substratum comprises 30g/L glucose and 10g/L potato leaching powder.YES substratum comprises the leaching of 5g/L yeast powder, 30g/L glucose, 6g/L potassium primary phosphate, 4g/L SODIUM PHOSPHATE, MONOBASIC and 1g/L ammonium hydroxide.MEA substratum comprises 25g/L malt extract and 3g/L soy peptone.
The physiological property of embodiment 3 monascus purpureus BD-M-4
The moiety of PDA solid medium is: the leaching of 6g/L potato powder, 20g/L glucose and 20g/L agar.
The moiety of PDB liquid nutrient medium is: 6g/L potato leaching powder and 20g/L glucose, surplus is water.
Aseptically transfer in PDA solid medium by the monascus purpureus BD-M-4 that 4 DEG C are preserved, 30 DEG C of activation culture to be inoculated in PDA solid medium 30 DEG C of activation culture 5 days after 4 days, obtain first order seed.
Load 50mL PDB liquid nutrient medium at 250mL triangular flask, access diameter is the first order seed bacterium cake of 1cm, and 30 DEG C of shaking tables, 180r/min rotating speed, cultivates 4 days, obtain second order fermentation seed.
Load 100mL PDB liquid nutrient medium at 500mL triangular flask, with the ratio of 5% (v/v) access second order fermentation seed, 30 DEG C of shaking tables, 180r/min rotating speed, cultivates 10 days.
According to the research method of the people such as Shao Wei (Shao Wei, Wang Dong, Tang Ming wait " monascus produces proteolytic enzyme characteristic research " [J]. China brewages, 2006,5:34-37.), measure mycelia weight (i.e. biomass), represent its upgrowth situation.As can be seen from Figure 1, monascus purpureus BD-M-4 is in 3 days that cultivate beginning, and thalline generates in a large number, within about the 4th day, thalline reaches maximum value 6.5g/L, and thalli growth is in the steady stage, then the biomass of thalline slowly reduces, and meets the growth characteristics of monascus.
The physiological property of embodiment 4 monascus purpureus BD-M-4
The moiety of PDA solid medium is: the leaching of 10g/L potato powder, 30g/L glucose and 30g/L agar.
The moiety of PDB liquid nutrient medium is: 10g/L potato leaching powder and 30g/L glucose, surplus is water.
Aseptically transfer in PDA solid medium by the monascus purpureus BD-M-4 that 4 DEG C are preserved, 30 DEG C of activation culture to be inoculated in PDA solid medium 30 DEG C of activation culture 5 days after 4 days, obtain first order seed.
Load 50mL PDB liquid nutrient medium at 250mL triangular flask, access diameter is the first order seed bacterium cake of 1cm, and 30 DEG C of shaking tables, 180r/min rotating speed, cultivates 4 days, obtain second order fermentation seed.
Load 100mL PDB liquid nutrient medium at 500mL triangular flask, with the ratio of 10% (v/v) access second order fermentation seed, 30 DEG C of shaking tables, 180r/min rotating speed, cultivates 10 days.
According to permitted Jiangxi honor measuring method (permitted Jiangxi flourish. the detection of red colouring agent for food, also used as a Chinese medicine Citrinin and ferment control technology [D]. Southern Yangtze University's Ph D dissertation, 2004.), adopt Agilent HPLC 1260 to measure, result as shown in Figure 3.As seen from Figure 3, it is almost nil that monascus BD-M-4 produces Citrinin amount, and toxin producing is few, is suitable for preparing cheese.
By the data declaration of embodiment 2 ~ 4, described monascus purpureus CGMCC No.9712 has following Microbiological Characteristics:
1, morphologic characteristic
Very fast at MEA cultured on solid medium, cultivate 10 days under 25 DEG C of dark conditions, colony diameter reaches 45 ~ 50mm.Aerial hyphae is luxuriant, edge white, and middle part is orange; The bacterium colony back side, in secretly orange, dissolves in MEA solid medium.Produce a large amount of conidium, conidiophore specialization is not obvious, and directly or slightly bending, conidium is spherical, colourless, and wall is slightly coarse, and base portion is truncate, concatenates, 5.5-10.4 μm.Cleistothecium is shallow to brown, most immature, has no thecaspore.
2, the characteristic learned is cultivated
All can grow under 20 ~ 42 DEG C of conditions, proper growth temperature is 25 ~ 32 DEG C, and optimum temperuture is 30 DEG C; Suitable pH is 3.5 ~ 6.0, and optimal pH is 5.5.Suitable growth medium can be PDB liquid nutrient medium, YES liquid nutrient medium or MEA liquid nutrient medium.
3, physiological property
Produce Citrinin hardly, natural monascorubin can be produced, and have higher look valency.
The rDNA sequential analysis of embodiment 5 monascus purpureus BD-M-4
Monascus purpureus BD-M-4 is carried out rRNA gene sequencing (being checked order by Institute of Microorganism, Academia Sinica), sequencing result is as shown in SEQ No.1 in sequence table, and this sequence contains the 18S rRNA fragment of monascus purpureus BD-M-4, the complete sequence of ITS1,5.8S rRNA, ITS2 and 28S region sequence fragment.This sequence and the comparison such as document and public database DDBJ, EMBL, GenBank are found, homology is 99%.Therefore, monascus purpureus BD-M-4 belongs to monascus purpureus (Monascus purpureus).
Embodiment 6 monascus purpureus BD-M-4 prepares red kojic rice powder
Batching: long-grained nonglutinous rice 10kg, glucose 40g, potato leaching powder 12g and agar 20g.
The moiety of PDA solid medium is: the leaching of 6g/L potato powder, 20g/L glucose and 20g/L agar.
The moiety of PDB liquid nutrient medium is: 6g/L potato leaching powder and 20g/L glucose, surplus is water.
Preparation process:
(1) activation of bacterial classification and process: aseptically transfer in PDA solid medium by the monascus purpureus BD-M-4 that 4 DEG C are preserved, 30 DEG C of activation culture to be inoculated in PDA solid medium 30 DEG C of activation culture 5 days after 4 days, obtain first order seed.
Load 50mL PDB liquid nutrient medium at 250mL triangular flask, access diameter is the first order seed bacterium cake of 1cm, and 30 DEG C of shaking tables, 180r/min rotating speed, cultivates 4 days, obtain second order fermentation seed.
Load 100mL PDB liquid nutrient medium at 500mL triangular flask, with the ratio of 10% (v/v) access second order fermentation seed, 30 DEG C of shaking tables, 180r/min rotating speed, cultivates 10 days, its pH value is adjusted to 3.0, obtains the bacterial classification of process.
(2) long-grained nonglutinous rice is soaked 12 hours in clear water, make feed moisture content be 50%, obtain soaked long-grained nonglutinous rice;
(3) soaked long-grained nonglutinous rice is put into high-pressure steam sterilizing pan, 121 DEG C of steamed rice 20min obtain steamed long-grained nonglutinous rice meal, and make long-grained nonglutinous rice ripe without the white heart, uniformity is without caking;
(4) poured out by steamed long-grained nonglutinous rice meal, spreading for cooling is also sieved, in 38 DEG C of inoculations.The bacterial classification of the process of every 10kg long-grained nonglutinous rice inoculation 0.04g step (1) gained, mix thoroughly, put into fixed temperature and humidity incubator, constant temperature 30 DEG C, constant relative humidity 80% cultivate 6 days, period turned over song and moisturizing every 4 hours, rate of water make-up is 10%, and described per-cent is mass percent, stops moisturizing when being cultured to the 5th day.Taking-up oven dry grinds, and obtains red kojic rice powder.
Obtained red kojic rice powder is red extremely dark violet redness, and quality is crisp, without going mouldy, without obvious macroscopic impurity, in irregular particulate state, has the Qu Xiang that red colouring agent for food, also used as a Chinese medicine is intrinsic.
Embodiment 7 monascus purpureus BD-M-4 prepares cheese
Batching: fresh cow milk 100L (purchased from bright milk industry), MA14 (purchased from Danisco (China) company limited product) starter 3g, calf stomach rennin (purchased from Chr. Hansen A/S) 1g, salt 0.75kg, glucose 40g and potato leaching powder 6g.
(1), after getting 100L fresh cow milk filtering and impurity removing matter, 72 DEG C of sterilizing 30s, are then cooled to 30 DEG C, must process breast.To described process Ruzhong inoculating starter MA14, inoculum size is 3g/100L, and add 1g calf stomach rennin when being cultured to pH6.5 under 30 DEG C of constant temperature, curdled milk 30min obtains curdled milk;
(2) curd cutting of step (1) gained is become block, stir 10min discharging whey; In curdled milk, add the salt of 1.5% after whey to be discharged, described per-cent is the mass percent that described salt accounts for described curdled milk, enters square dies after stirring;
(3) curdled milk enters mold forming and obtains curd pieces and regularly upset, and frequency be every upset in 2 hours 1 time, lasting 24 hours, whey is discharged further;
(4) the curd pieces surface uniform obtained toward step (3) sprays the Monascus fermentation broth that thickness is about 1mm, loads ripe container, and constant incubator is ripe, and the temperature of described maturation is: the initial stage 25 DEG C, relative humidity 90%, the middle and later periods 10 DEG C; The time of described maturation is: 2 weeks initial stages, 2 weeks middle and later periods.
Described Monascus fermentation broth is obtained by following method: monascus purpureus BD-M-4 to be inoculated in PDB liquid nutrient medium 30 DEG C and to cultivate 2 days, the formula of described PDB liquid nutrient medium is: glucose 40g/L, potato leaching powder 6g/L, surplus is water.
Obtained monascus cheese has the distinctive flavour of monascus cheese and smell, and fermenting aroma is strong, has the distinctive tender texture of monascus cheese, good, the surperficial corrugationless of shell, shell takes on a red color or purple, inner in uniform purple or redness, is applicable to the food habits of Chinese Consumer's.
Embodiment 8 monascus purpureus BD-M-4 prepares cheese
Batching: fresh sheep breast 100L (purchased from bright milk industry), MA14 (purchased from Danisco (China) company limited product) starter 3g, microbial rennet (purchased from Chr. Hansen A/S) 1g, salt 0.5kg, glucose 40g and potato leaching powder 6g.
(1), after getting 100L fresh sheep breast filtering and impurity removing matter, 72 DEG C of sterilizing 15s, are then cooled to 28 DEG C, must process breast.To described process Ruzhong inoculating starter MA14, inoculum size is 3g/100L, and add 1g microbial rennet when being cultured to pH6.5 under 28 DEG C of constant temperature, curdled milk 30min obtains curdled milk;
(2) curd cutting of step (1) gained is become block, stir 10min discharging whey; In curdled milk, add the salt of 1% after whey to be discharged, described per-cent is the mass percent that described salt accounts for described curdled milk, enters square dies after stirring;
(3) curdled milk enters mold forming and obtains curd pieces and regularly upset, and frequency be every upset in 2 hours 1 time, lasting 24 hours, whey is discharged further;
(4) the curd pieces surface uniform obtained toward step (3) sprays the Monascus fermentation broth that thickness is about 1.5mm, loads ripe container, and constant incubator is ripe, and the temperature of described maturation is: the initial stage 20 DEG C, relative humidity 95%, the middle and later periods 8 DEG C; The time of described maturation is: 2 weeks initial stages, 2 weeks middle and later periods.
Described Monascus fermentation broth is obtained by following method: monascus purpureus BD-M-4 to be inoculated in PDB liquid nutrient medium 30 DEG C and to cultivate 2 days, the formula of described PDB liquid nutrient medium is: the formula of described monascus substratum is: glucose 40g/L, potato leaching powder 6g/L, surplus is water.
Embodiment 9 monascus purpureus BD-M-4 prepares cheese
Batching: fresh horse breast 100L (purchased from bright milk industry), MA14 (purchased from Danisco (China) company limited product) starter 3g, calf stomach rennin (purchased from Chr. Hansen A/S) 1g, salt 1.5kg, glucose 40g and potato leaching powder 6g.
(1), after getting 100L fresh horse breast filtering and impurity removing matter, 72 DEG C of sterilizing 30s, are then cooled to 32 DEG C, must process breast.To described process Ruzhong inoculating starter MA14, inoculum size is 3g/100L, and add 1g calf stomach rennin when being cultured to pH6.5 under 32 DEG C of constant temperature, curdled milk 30min obtains curdled milk;
(2) curd cutting of step (1) gained is become block, stir 10min discharging whey; In curdled milk, add the salt of 1.25% after whey to be discharged, described per-cent is the mass percent that described salt accounts for described curdled milk, enters square dies after stirring;
(3) curdled milk enters mold forming and obtains curd pieces and regularly upset, and frequency be every upset in 2 hours 1 time, lasting 24 hours, whey is discharged further;
(4) the curd pieces surface uniform obtained toward step (3) sprays the Monascus fermentation broth that thickness is about 1mm, loads ripe container, and constant incubator is ripe, and the temperature of described maturation is: the initial stage 22 DEG C, relative humidity 85%, the middle and later periods 12 DEG C; The time of described maturation is: 2 weeks initial stages, 2 weeks middle and later periods.
Described Monascus fermentation broth is obtained by following method: monascus purpureus BD-M-4 to be inoculated in PDB liquid nutrient medium 30 DEG C and to cultivate 2 days, the formula of described PDB liquid nutrient medium is: glucose 40g/L, potato leaching powder 6g/L, surplus is water.
Embodiment 10 monascus purpureus BD-M-4 prepares cheese
Batching: the newborn 100L of fresh camel (purchased from bright milk industry), MA14 (purchased from Danisco (China) company limited product) starter 3g, calf stomach rennin (purchased from Chr. Hansen A/S) 1g, salt 0.5kg, glucose 40g and potato leaching powder 6g.
(1), after getting the newborn filtering and impurity removing matter of the fresh camel of 100L, 72 DEG C of sterilizing 30s, are then cooled to 30 DEG C, must process breast.To described process Ruzhong inoculating starter MA14, inoculum size is 3g/100L, and add 1g calf stomach rennin when being cultured to pH6.5 under 30 DEG C of constant temperature, curdled milk 30min obtains curdled milk;
(2) curd cutting of step (1) gained is become block, stir 10min discharging whey; In curdled milk, add the salt of 1% after whey to be discharged, described per-cent is the mass percent that described salt accounts for described curdled milk, enters square dies after stirring;
(3) curdled milk enters mold forming and obtains curd pieces and regularly upset, and frequency be every upset in 2 hours 1 time, lasting 24 hours, whey is discharged further;
(4) the curd pieces surface uniform obtained toward step (3) sprays the Monascus fermentation broth that thickness is about 1mm, loads ripe container, and constant incubator is ripe, and the temperature of described maturation is: the initial stage 25 DEG C, relative humidity 90%, the middle and later periods 10 DEG C; The time of described maturation is: 2 weeks initial stages, 2 weeks middle and later periods.
Described Monascus fermentation broth is obtained by following method: monascus purpureus BD-M-4 to be inoculated in PDB liquid nutrient medium 30 DEG C and to cultivate 2 days, the formula of described PDB liquid nutrient medium is: glucose 40g/L, potato leaching powder 6g/L, surplus is water.
Embodiment 11 monascus purpureus BD-M-4 ferments
Seed culture: the monascus purpureus BD-M-4 that 4 DEG C are preserved slowly is warming up to 15 DEG C, aseptically by intensifications afterwards bacterial strain rule on PDA solid medium, 30 DEG C of activation culture 4 days, obtain seed liquor.
PDA solid medium is: the leaching of 6g/L potato powder, 20g/L glucose and 20g/L agar.
Fermentation culture: seed liquor is inoculated in PDB liquid nutrient medium with 5%, 42 DEG C shake cultivation 3 days, and rotating speed is 220rpm, and described per-cent is volume percent.
PDB liquid nutrient medium is: 6g/L potato leaching powder and 20g/L glucose, surplus is water.
Embodiment 12 monascus purpureus BD-M-4 ferments
Seed culture: the monascus purpureus BD-M-4 that 4 DEG C are preserved slowly is warming up to 25 DEG C, aseptically by intensifications afterwards bacterial strain rule on YES solid medium, 30 DEG C of activation culture 7 days, obtain seed liquor.
YES solid medium is: the leaching of 5g/L yeast powder, 30g/L glucose, 6g/L potassium primary phosphate, 4g/L SODIUM PHOSPHATE, MONOBASIC, 1g/L ammonium hydroxide and 20g/L agar.
Fermentation culture: seed liquor is inoculated in YES liquid nutrient medium with 5%, 20 DEG C shake cultivation 10 days, and rotating speed is 120rpm, and described per-cent is volume percent.
YES liquid nutrient medium is: the leaching of 5g/L yeast powder, 30g/L glucose, 6g/L potassium primary phosphate, 4g/L SODIUM PHOSPHATE, MONOBASIC and 1g/L ammonium hydroxide, surplus is water.
Embodiment 13 monascus purpureus BD-M-4 ferments
Seed culture: the monascus purpureus BD-M-4 that 4 DEG C are preserved slowly is warming up to 15 DEG C, aseptically by intensifications afterwards bacterial strain rule on YES solid medium, 25 DEG C of activation culture 8 days, obtain seed liquor.
YES solid medium is: the leaching of 4g/L yeast powder, 20g/L glucose, 5g/L potassium primary phosphate, 3g/L SODIUM PHOSPHATE, MONOBASIC, 0.5g/L ammonium hydroxide and 15g/L agar.
Fermentation culture: seed liquor is inoculated in YES liquid nutrient medium with 10%, 25 DEG C shake cultivation 5 days, and rotating speed is 220rpm, and described per-cent is volume percent.
YES liquid nutrient medium is: the leaching of 4g/L yeast powder, 20g/L glucose, 5g/L potassium primary phosphate, 3g/L SODIUM PHOSPHATE, MONOBASIC and 0.5g/L ammonium hydroxide, surplus is water.
Embodiment 14 monascus purpureus BD-M-4 ferments
Seed culture: the monascus purpureus BD-M-4 that 4 DEG C are preserved slowly is warming up to 15 DEG C, aseptically by intensifications afterwards bacterial strain rule on YES solid medium, 25 DEG C of activation culture 8 days, obtain seed liquor.
YES solid medium is: the leaching of 6g/L yeast powder, 40g/L glucose, 7g/L potassium primary phosphate, 5g/L SODIUM PHOSPHATE, MONOBASIC, 1.5g/L ammonium hydroxide and 25g/L agar.
Fermentation culture: seed liquor is inoculated in YES liquid nutrient medium with 10%, 30 DEG C shake cultivation 5 days, and rotating speed is 180rpm, and described per-cent is volume percent.
YES liquid nutrient medium is: the leaching of 6g/L yeast powder, 40g/L glucose, 7g/L potassium primary phosphate, 5g/L SODIUM PHOSPHATE, MONOBASIC and 1.5g/L ammonium hydroxide, surplus is water.
Embodiment 15 monascus purpureus BD-M-4 ferments
Seed culture: the monascus purpureus BD-M-4 that 4 DEG C are preserved slowly is warming up to 20 DEG C, aseptically by intensifications afterwards bacterial strain rule on MEA solid medium, 15 DEG C of activation culture 10 days, obtain seed liquor.
MEA solid medium is: 25g/L malt extract, 3g/L soy peptone and 15g/L agar.
Fermentation culture: seed liquor is inoculated in MEA liquid nutrient medium with 5%, 30 DEG C shake cultivation 10 days, and rotating speed is 180rpm, and described per-cent is volume percent.
MEA liquid nutrient medium is: 25g/L malt extract and 3g/L soy peptone, and surplus is water.
Embodiment 16 monascus purpureus BD-M-4 ferments
Seed culture: the monascus purpureus BD-M-4 that 4 DEG C are preserved slowly is warming up to 20 DEG C, aseptically by intensifications afterwards bacterial strain rule on MEA solid medium, 15 DEG C of activation culture 10 days, obtain seed liquor.
MEA solid medium is: 20g/L malt extract, 2g/L soy peptone and 12g/L agar.
Fermentation culture: seed liquor is inoculated in MEA liquid nutrient medium with 8%, 42 DEG C shake cultivation 3 days, and rotating speed is 120rpm, and described per-cent is volume percent.
MEA liquid nutrient medium is: 20g/L malt extract and 2g/L soy peptone, and surplus is water.
Embodiment 17 monascus purpureus BD-M-4 ferments
Seed culture: the monascus purpureus BD-M-4 that 4 DEG C are preserved slowly is warming up to 20 DEG C, aseptically by intensifications afterwards bacterial strain rule on MEA solid medium, 15 DEG C of activation culture 10 days, obtain seed liquor.
MEA solid medium is: 40g/L malt extract, 4g/L soy peptone and 20g/L agar.
Fermentation culture: seed liquor is inoculated in MEA liquid nutrient medium with 10%, 42 DEG C shake cultivation 3 days, and rotating speed is 150rpm, and described per-cent is volume percent.
MEA liquid nutrient medium is: 40g/L malt extract and 4g/L soy peptone, and surplus is water.
Effect example 1
Purplish red bent BD-M-4, monascus BD-A, monascus BD-B, monascus BD-C, monascus GL-A, monascus GL-1 and monascus purpureus BD-M-1 are carried out the step activating, cultivate according to the step of embodiment 3, measure mycelia weight, result as shown in Figure 2 and Table 1.
The result of Fig. 2 illustrates, with compared with the biomass of other bacterial strains compare, the maximum biomass of monascus strain BD-M-4 is comparatively large, has significant difference, under 30 DEG C of environment, has obvious growth vigor, be particularly suitable for preparing cheese with other bacterial strains.
The situation of the different monascus purpureus bacterial strain of table 1 biomass when fermentation 10 days
| Strain name | Biomass (g/L) |
| Purplish red bent BD-M-4 | 6.5 |
| Monascus BD-A | 1.5 |
| Monascus BD-B | 1.8 |
| Monascus BD-C | 3 |
| Monascus GL-A | 1.3 |
| Monascus GL-1 | 1.6 |
| Monascus purpureus BD-M-1 | 3.2 |
Effect example 2
Purplish red bent BD-M-4, monascus BD-A, monascus BD-B, monascus BD-C, monascus GL-A, monascus GL-1 and monascus purpureus BD-M-1 are carried out the step activating, cultivate according to the step of embodiment 4, measure citrinin content, result as shown in figure 3 and table 2.
The situation of the different monascus purpureus bacterial strain of table 2 citrinin content when fermentation 10 days
| Strain name | Citrinin content (μ g/L) |
| Purplish red bent BD-M-4 | 10 |
| Monascus BD-A | 100 |
| Monascus BD-B | 500 |
| Monascus BD-C | 1200 |
| Monascus GL-A | 20 |
| Monascus GL-1 | 15 |
| Monascus purpureus BD-M-1 | 15 |
Table 2 illustrates, the citrinin content that purplish red bent BD-M-4 fermentation produces is minimum in the existing monascus prepared for food, relative to other monascuses, the Citrinin that purplish red bent BD-M-4 fermentation produces is very little, can be widely used in the complete processing preparing the food such as milk-product, red kojic rice powder.
Effect example 3
By purplish red bent BD-M-4, monascus BD-A, monascus BD-B, monascus BD-C, monascus GL-A, monascus GL-1 and monascus purpureus BD-M-1 is inoculated in YES substratum with the ratio of 5% (v/v) and (comprises 5g/L yeast leaching powder, 30g/L glucose, 6g/L potassium primary phosphate, 4g/L SODIUM PHOSPHATE, MONOBASIC and 1g/L ammonium hydroxide) in, fermented liquid is cultivated 5 days to obtain at 28 DEG C, according to permitted Jiangxi honor measuring method (permitted Jiangxi flourish. the detection of red colouring agent for food, also used as a Chinese medicine Citrinin and ferment control technology [D]. Southern Yangtze University's Ph D dissertation, 2004.), get the isopyknic 70% alcohol extraction 30min of 10mL fermented liquid, filter, after filtrate is suitably diluted, in the interscan of 190-700nm wavelength region.
The calculation formula of look valency: look valency (CV)=OD
505× extension rate (U/ml).Result is as shown in table 3:
The look valency situation of the different monascus purpureus bacterial strain of table 3 when fermentation 5 days
| Strain name | Look valency (CV) |
| Purplish red bent BD-M-4 | 6 |
| Monascus BD-A | 4 |
| Monascus BD-B | 4 |
| Monascus BD-C | 5 |
| Monascus GL-A | 5 |
| Monascus GL-1 | 4 |
| Monascus purpureus BD-M-1 | 5 |
Table 3 illustrates, the look valency that purplish red bent BD-M-4 produces monascorubin is high, illustrates that the ability of producing Monascouruarin is strong.
Should be understood that those skilled in the art can make various changes or modifications the present invention after having read foregoing of the present invention, these equivalent form of values fall within the application's appended claims limited range equally.
Claims (10)
1. a monascus purpureus (Monascus purpureus), it is characterized in that, it is deposited in China Committee for Culture Collection of Microorganisms's common micro-organisms center, and preserving number is: CGMCC No.9712.
2. prepare a method of monascus purpureus CGMCC No.9712, it is characterized in that, it comprises the following steps, cultivates monascus purpureus CGMCC No.9712 in the medium.
3. method as claimed in claim 2, it is characterized in that, described substratum is PDB liquid nutrient medium, YES liquid nutrient medium or MEA liquid nutrient medium; Described PDB liquid nutrient medium comprises 6 ~ 10g/L potato leaching powder and 20 ~ 40g/L glucose; Described YES liquid nutrient medium comprises the leaching of 4 ~ 6g/L yeast powder, 20 ~ 40g/L glucose, 5 ~ 7g/L potassium primary phosphate, 3 ~ 5g/L SODIUM PHOSPHATE, MONOBASIC and 0.5 ~ 1.5g/L ammonium hydroxide; Described MEA liquid nutrient medium comprises 20 ~ 40g/L malt extract and 2 ~ 4g/L soy peptone; The time of described cultivation is 3 ~ 10 days, is preferably 5 ~ 10 days; The temperature of described cultivation is 20 ~ 42 DEG C, is preferably 25 ~ 30 DEG C; Described cultivation is concussion cultivation, and the rotating speed that described concussion is cultivated is 120 ~ 220rpm, is preferably 180 ~ 220rpm; And/or the inoculum size of described cultivation is 5 ~ 10%, and described per-cent is volume percent.
4. method as claimed in claim 2, is characterized in that, also comprise monascus purpureus CGMCC No.9712 is inoculated in the step that seed culture medium carries out seed culture before described cultivation.
5. method as claimed in claim 4, it is characterized in that, the time of described seed culture is 5 ~ 10 days, is preferably 6 ~ 8 days; The temperature of described seed culture is 15 ~ 35 DEG C, is preferably 25 ~ 32 DEG C; And/or the substratum of described seed culture is PDA solid medium, YES solid medium or MEA solid medium.
6. method as claimed in claim 2, it is characterized in that, described seed culture comprises the step slowly heated up by the monascus strain of refrigeration.
7. method as claimed in claim 6, it is characterized in that, the temperature of described intensification is 15 ~ 25 DEG C.
8. the purposes of monascus purpureus CGMCC No.9712 in foodstuff production as claimed in claim 1.
9. utilize the cheese that Fermentation Condition of Monascus spp is obtained, it is characterized in that, described monascus is monascus purpureus CGMCC No.9712.
10. utilize the red kojic rice powder that Fermentation Condition of Monascus spp is obtained, it is characterized in that, described monascus is monascus purpureus CGMCC No.9712.
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