CN103224958B - Extractive fermentation and regulation and control pH prepare the method for monascorubin - Google Patents
Extractive fermentation and regulation and control pH prepare the method for monascorubin Download PDFInfo
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- 230000004151 fermentation Effects 0.000 title claims abstract description 195
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- Micro-Organisms Or Cultivation Processes Thereof (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention provides a kind of extractive fermentation and regulation and control pH prepares the method for monascorubin, comprise the steps: step 1, monascus ruber is cultivated in seed culture medium, obtains seed liquor; Step 2, is inoculated into described seed liquor in the fermentation culture containing nitrogenous source, carries out fermentation culture, regulates the pH value of fermentation culture, centrifugal, obtains monascus ruber wet cell; Step 3, is inoculated into described monascus ruber wet cell in the fermention medium containing nonionogenic tenside, cultivates, and regulates the pH value of fermention medium, cloud point extraction, is separated.The present invention adopts the pH value regulating fermentation, controls the formation of monascorubin and Citrinin, and application extractive fermentation and cloud point extraction regulate pigment composition to remove by product Citrinin, the monascorubin that obtained citrinin content is lower simultaneously.The inventive method is simple, and easily realize, productive rate is high, and cost is low, and repeatability is strong, and environmental protection, has good application prospect.
Description
Technical field
The present invention relates to a kind of fermentation process of technical field of bioengineering, particularly, relate to a kind of extractive fermentation and regulation and control pH prepares the method for monascorubin.
Background technology
The Red kojic rice that monascus ruber (Monascussp) solid state fermentation is produced is the traditional food color additive of China, is also a kind of traditional Chinese medicine, has had the use history of more than 1,000 year in China.Current employing Modern microbiological liquid submerged fermentation technology produces monascorubin, and this pigment widely uses in East Asian countries as food color additive, as China, Japan, Korea S, Taiwan, Thailand, Vietnam etc.Since having found that monascus ruber fermentative production monascorubin also produces the by product Citrinin with neurotoxicity and renal toxicity, monascorubin has been produced America and Europe and has been forbidden production and selling.Especially, monascorubin product is the mixture of monascorubin, monascorubin, red colouring agent for food, also used as a Chinese medicine citraurin composition.Except the national standard having formulated monascorubin except some countries of East Asia allows its production and selling, monascus yellow pigment and red colouring agent for food, also used as a Chinese medicine citraurin also do not put into effect corresponding national standard.
In order to ensure food safety, control the content of by product in monascorubin product, particularly have the content of the Citrinin of toxic side effect, be the important research content of food microorganisms always.During the fermentation, utilize modern biotechnology means to transform microorganism strains and technically there is feasibility to suppress the formation of Citrinin, but the leavened prod that genetic engineering modified Institute of Micro-biology obtains belongs to Genetic engineering food.Due to the singularity of food safety, it is very loaded down with trivial details and difficult that Genetic engineering food obtains in a lot of country the license produced and sold.Adopt fermentation engineering and reduce the content of Citrinin in conjunction with downstream separation technology and become important means, as regulated the operating parameterss such as dissolved oxygen.
The formation of microbial secondary meta-bolites depends on fermentation condition consumingly.In the fermenting process of monascus ruber, regulate the pH of fermentation media can change point rate of each colour component in monascorubin.Under near-neutral pH condition, point rate of monascorubin is higher, and this and traditional technology adopt rice fermentation Red kojic rice consistent.The pH reducing fermented liquid makes the corresponding increase of point rate of red colouring agent for food, also used as a Chinese medicine citraurin and monascus yellow pigment, causes pigmentary colours to turn yellow.Guangdong “ salt baked chicken in 2012 " food disturbance is exactly because with the addition of monascus yellow pigment.Although the security of monascus yellow pigment obtains the confirmation of research both at home and abroad, pH change but lacks research to the impact that monascus ruber metabolic by-prods is formed.Some scholars infers the security of monascus yellow pigment product from the security of monascus yellow pigment itself, because by product (he is left alone without help etc. as Citrinin, Lip river are cut down) content and toxicity are difficult to obtain conclusive evidence, human consumer can not be allowed to convince, be also difficult to the accreditation obtaining state food administration.
In order to meet the demand of market to monascus yellow pigment, ensure food safety, in the urgent need to the monascus ruber fermentation technique that colour component in low, the monascorubin of exploitation citrinin content is relatively single simultaneously.Find by prior art documents, ZhiqiangHu, XuehongZhang, ZhenqiangWu, HanshiQi, ZhilongWang.ExportofintracellularMonascuspigmentsbytwo-s tagemicrobialfermentationinnonionicsurfactantmicelleaque oussolution.Journalofbiotechnology, 2012, 162:202-209.(Hu Zhiqiang, Zhang Xuehong, Wu Zhenqiang, Qi Hanshi, Wang Zhilong. " in nonionogenic tenside micellar solution, applying monascorubin in two sections of fermentation technique release born of the same parents ". " biotechnology journal ", 2012, introduce 162:202-209), monascorubin in born of the same parents is secreted into extracellular environment by monascus ruber fermentation energy in nonionogenic tenside micellar solution, achieve intracellular product extractive fermentation to born of the same parents' external surfactants micellar solution.But different fermention mediums and fermentation mode, on the impact of monascorubin composition point rate, especially on the impact that by product Citrinin is formed, also not studies have reported that.In further retrieval, also not about extractive fermentation in Free Energy of Surfactant Micelle Solution with regulate and control pH and combine and realize controlling pigment and form and the bibliographical information of citrinin content.
Summary of the invention
For defect of the prior art, the object of this invention is to provide a kind of extractive fermentation and regulation and control pH prepares the method for monascorubin, the method application extractive fermentation with regulate and control pH technological development citrinin content low water-soluble/the serial monascorubin product such as hydrophobicity monascorubin, hydrophobicity red colouring agent for food, also used as a Chinese medicine citraurin, hydrophobicity monascus yellow pigment.
The invention provides a kind of extractive fermentation and regulation and control pH prepares the method for monascorubin, described method by extractive fermentation technology with regulate and control pH technology and combine, prepare the low and monascorubin that monascus pigment component is relatively single of citrinin content; Described method comprises the steps:
Step 1, monascus ruber is cultivated in seed culture medium, obtains seed liquor, and described monascus ruber provides (Monascusanka, CICC5013) for Chinese industrial microorganism strains preservation center;
Step 2, in order to protect the composition regulating pH can control monascorubin, can control the formation of Citrinin simultaneously at low ph conditions, described seed liquor is inoculated in the fermentation culture containing nitrogenous source, carries out fermentation culture, regulate the pH value of fermentation culture, centrifugal, obtain monascus ruber wet cell; Get the pre-treatment of monascus ruber wet cell, release intracellular product, detects fermented liquid pH, the pigment concentration of intraor extracellular, the citrinin content of intraor extracellular;
Wherein, described pre-treatment is specially: get 0.5g wet cell at isopyknic fermented liquid 70%(V/V) ethanol (pH=2) solution in soak 1 hour;
Step 3, in order to the formation protecting extractive fermentation can control monascus yellow pigment further, controls the content of Citrinin simultaneously, described monascus ruber wet cell is inoculated in the fermention medium containing nonionogenic tenside, cultivates, regulate the pH value of fermention medium, cloud point extraction, is separated; Get part cell pretreatment, release intracellular product, detects fermented liquid pH, the pigment concentration of intraor extracellular, the citrinin content of intraor extracellular.
Preferably, described pigment concentration, its detection method is specially: by cell free fermentation liquid or intracellular product release liquid 70%(V/V) ethanol (pH=2) solution suitably absorb, adopt spectrophotometer measure of spread 410,470, the light absorption value at 510nm place.Above-mentioned light absorption value is multiplied by the concentration absorbing the absorbance unit that obtains of multiple and represent yellow pigment, citraurin and haematochrome respectively.Accordingly, working sample, can absorption spectrum corresponding to check sample in the absorbancy of visible wavelength region (390 ~ 540nm).
Described citrinin content, its detection method is specially: by cell free fermentation liquid or intracellular product release liquid 70%(V/V) ethanol (pH=2) solution suitably absorb, adopt thin-layer chromatography (Merck & Co., Inc. silica gel F254 plate), solvent systems is acetic acid: methyl alcohol, chloroform=9:21:285, determined wavelength is 365nm, is that 0.56 place can detect that the fluorescence of Citrinin is strong and weak at Rf.Meanwhile, under natural light, be respectively 0.8,0.68 and 0 place at Rf and red colouring agent for food, also used as a Chinese medicine citraurin can be detected, monascus yellow pigment and monascorubin.
Preferably, in step 1, described seed culture medium is made up of each component of following content: water 100ml, peptone 2g, yeast powder 2g, glucose 2g.
Preferably, in step 1, described cultivation is 30 DEG C in temperature, carries out in the shaking table of 200rpm, and incubation time is 32 hours.
Preferably, in step 2, described fermentation culture is 30 DEG C in temperature, carries out in the shaking table of 200rpm, and incubation time is 5-7 days.
Preferably, in step 2, the initial pH value of described fermentation culture is 2.5 ~ 6, and final ph is 2.3 ~ 7.
Preferably, in step 3, described cultivation refers to that in temperature be 30 DEG C, and carry out in the shaking table of 200rpm, incubation time is 3 ~ 4 days; Described separation refer in the water-bath of 70 DEG C leave standstill 2 hours.
Preferably, in step 3, the initial pH value of described fermention medium is 3 ~ 5, and final ph is 2.2 ~ 6.2.
Preferably, in step 3, described fermention medium is made up of each component of following content: water 100ml, glucose 5g, KH
2pO
40.4g, MgSO
40.1g, CaCl
20.005g, nonionogenic tenside 1 ~ 4g/100ml, nitrogenous source 5g/100 ~ 15g/100ml.
Preferably, described nitrogenous source includes conventional inorganic nitrogen-sourced and organic natural nitrogenous source.Described nitrogenous source is rice meal, Semen Maydis powder, analysis for soybean powder, Sodium Glutamate, ammonium sulfate, ammonium chloride, SODIUMNITRATE.
Preferably, the micellar solution that present invention uses nonionogenic tenside TritonX-100 achieves extractive fermentation and the cloud point extraction of parachrome.With the nitrogenous source that ammonium sulfate, ammonium chloride etc. are fermention medium, extractive fermentation achieves and intracellular product is extracted into cell free fermentation liquid, and exo-cell pigment is monascus yellow pigment, and citrinin content is lower.Furthermore achieved that the cloud point extraction of cell free fermentation liquid, Citrinin is mainly distributed in tensio-active agent dilute phase and monascus yellow pigment is distributed in tensio-active agent concentrated phase, thus the content of Citrinin in tensio-active agent concentrated phase is reduce further at separation phase, the monascus yellow pigment product of low citrinin content can be obtained by tensio-active agent concentrated phase.
Preferably, the micellar solution that present invention uses nonionogenic tenside TritonX-100 achieves extractive fermentation and the cloud point extraction of parachrome.With the nitrogenous source of the fermention mediums such as SODIUMNITRATE, extractive fermentation achieves and intracellular product is extracted into cell free fermentation liquid, and exo-cell pigment is monascorubin, and citrinin content is higher.Furthermore achieved that the cloud point extraction of cell free fermentation liquid, Citrinin is mainly distributed in tensio-active agent dilute phase and monascorubin is distributed in tensio-active agent concentrated phase, thus the content of Citrinin in tensio-active agent concentrated phase is reduce further at separation phase, the monascorubin product of low citrinin content can be obtained by tensio-active agent concentrated phase.
Preferably, the micellar solution that present invention uses nonionogenic tenside TritonX-100 achieves extractive fermentation and the cloud point extraction of parachrome.With the nitrogenous source that Sodium Glutamate etc. is fermention medium, extractive fermentation achieves and intracellular product is extracted into cell free fermentation liquid, and exo-cell pigment is water-soluble red pigment of red rice derivative, and citrinin content is higher.Further cloud point extraction cell free fermentation liquid, Citrinin is mainly distributed in tensio-active agent dilute phase, water-soluble red pigment of red rice derivative has certain distribution in two-phase, but be mainly distributed in tensio-active agent concentrated phase, thus the content of Citrinin in tensio-active agent concentrated phase is reduce further at separation phase, the water-soluble red pigment of red rice derivative product (wetting ability monascorubin derivative) of low citrinin content can be obtained by tensio-active agent concentrated phase.
The present invention adopts regulation and control pH and the object of extractive fermentation to be to obtain relative single, the serial monascorubin product that citrinin content is low of component, as water-soluble red pigment of red rice, fat-soluble red colouring agent for food, also used as a Chinese medicine citraurin, fat-soluble monascus yellow pigment and fat-soluble monascorubin etc.
The present invention adopts the object of regulation and control pH be to regulate point rate of colour component in monascorubin and control the formation of metabolic by-prods Citrinin.
The present invention adopts the object of extractive fermentation technology to be to change point rate of monascus pigment component, and parachrome is secreted into extracellular.
The present invention adopts extractive fermentation technology, and by intracellular product, the object be secreted into outside born of the same parents is to realize further the cloud point extraction of cell free fermentation liquid.
The present invention adopts the object of cloud point extraction cell free fermentation liquid to be to realize monascorubin and the uneven distribution of Citrinin in biphasic system, reduces the content of Citrinin further.
Compared with prior art, the present invention has following beneficial effect: the present invention adopts fermentation and controls the pH value of fermentation, and then control the formation of monascorubin and Citrinin, the present invention applies extractive fermentation technology simultaneously and cloud point extraction regulates pigment composition and removes by product Citrinin, achieves the hydrophobicity monascorubin of low citrinin content, hydrophobicity red colouring agent for food, also used as a Chinese medicine citraurin, hydrophobicity monascus yellow pigment and water-soluble red pigment of red rice (glutamic acid derivatives of monascorubin) series product development.The inventive method is simple, and easily realize, productive rate is high, and cost is low, and repeatability is strong, and environmental protection, has good application prospect.
Embodiment
Below in conjunction with specific embodiment, the present invention is described in detail.Following examples will contribute to those skilled in the art and understand the present invention further, but not limit the present invention in any form.It should be pointed out that to those skilled in the art, without departing from the inventive concept of the premise, some distortion and improvement can also be made.These all belong to protection scope of the present invention.
embodiment 1
The present embodiment relates to a kind of extractive fermentation and regulation and control pH prepares the method for monascorubin, and described method comprises the steps:
Step 1, monascus ruber is at seed culture medium water (100ml, peptone 2g, yeast powder 2g, glucose 2g) in carry out cultivation 32 hours, obtain seed liquor, described monascus ruber provides (Monascusanka, CICC5013) for Chinese industrial microorganism strains preservation center;
Step 2, it is 4 that described seed liquor is inoculated into initial pH value, is in the fermentation culture of nitrogenous source with SODIUMNITRATE, is 30 DEG C in temperature, carries out the fermentation culture of 5 days in the shaking table of 200rpm, regulates the pH value of fermentation culture, centrifugal, obtains monascus ruber wet cell; Get the pre-treatment of monascus ruber wet cell, release intracellular product, detects fermented liquid pH, the pigment concentration of intraor extracellular, the citrinin content of intraor extracellular; Wherein, described pre-treatment is specially: get 0.5g wet cell at isopyknic fermented liquid 70%(V/V) ethanol (pH=2) solution in soak 1 hour;
The absorbancy analyzing cell free fermentation liquid 410,470 and 510nm place monascorubin is respectively 2,1 and 1.8 absorbance units; Parachrome after ethanolic soln extracts 410,470 and the absorbancy at 510nm place be respectively 35,25 and 32 absorbance units.
Fermented liquid final pH is 6.8, and inside and outside born of the same parents, through thin layer chromatography analysis, citrinin content shows that content is high, has obvious distribution inside and outside born of the same parents, is more prone to be distributed in cell free fermentation liquid.Thin-layer chromatography shows that monascorubin main component is monascorubin.
Step 3, by the cell 0.5g of fermentation culture, adds fermention medium (water 100ml, glucose 5g, KH that initial pH is 4
2pO
40.4g, MgSO
40.1g, CaCl
20.005g, ammonium sulfate 5g/100ml, TritonX-1004g/100ml) middle cultivation is after 5 days, and the absorbancy analyzing cell free fermentation liquid 410,470 and 510nm place monascorubin is respectively 65,45 and 22 absorbance units; Parachrome after ethanolic soln extracts 410,470 and the absorbancy at 510nm place be respectively 25,20 and 15 absorbance units.Fermentation final pH is 2.2, and inside and outside born of the same parents, citrinin content shows substantially not containing Citrinin through thin layer chromatography analysis.Thin-layer chromatography shows that monascorubin main component is monascus yellow pigment.Cloud point extraction cell free fermentation liquid in 70 degree of water-baths further, in tensio-active agent concentrated phase 410,470 and the absorbancy of 510nm place monascorubin be respectively 120,60 and 20 absorbance units; In tensio-active agent dilute phase 410,470 and the absorbancy at 510nm place be respectively 15,8 and 7 absorbance units.
Implementation result: the present embodiment product can obtain through thin layer chromatography analysis result: the existence that Citrinin can be detected in tensio-active agent dilute phase, can't detect in tensio-active agent concentrated phase.
embodiment 2
The present embodiment relates to a kind of extractive fermentation and regulation and control pH prepares the method for monascorubin, and described method comprises the steps:
Step 1, monascus ruber is at seed culture medium (100ml, peptone 2g, yeast powder 2g, glucose 2g) in carry out cultivation 32 hours, obtain seed liquor, described monascus ruber provides (Monascusanka, CICC5013) for Chinese industrial microorganism strains preservation center;
Step 2, it is 4 that described seed liquor is inoculated into initial pH value, is in the fermentation culture of nitrogenous source with Sodium Glutamate, carries out fermentation culture after 7 days, regulates the pH value of fermentation culture, centrifugal, obtains monascus ruber wet cell; Get the pre-treatment of monascus ruber wet cell, release intracellular product, detects fermented liquid pH value, the pigment concentration of intraor extracellular, the citrinin content of intraor extracellular; Wherein, described pre-treatment is specially: get 0.5g wet cell at isopyknic fermented liquid 70%(V/V) ethanol (pH=2) solution in soak 1 hour;
The absorbancy analyzing cell free fermentation liquid 410,470 and 510nm place monascorubin is respectively 10,8 and 12 absorbance units; Parachrome after ethanolic soln extracts 410,470 and the absorbancy at 510nm place be respectively 30,22 and 32 absorbance units.
Fermented liquid final pH is 6.5, and inside and outside born of the same parents, through thin layer chromatography analysis, citrinin content shows that content is high, has obvious distribution inside and outside born of the same parents, is more prone to be distributed in cell free fermentation liquid.Thin-layer chromatography shows that monascorubin main component is monascorubin derivative.
Step 3, by the cell 0.5g of fermentation culture, adds fermention medium (water 100ml, glucose 5g, KH that initial pH is 4
2pO
40.4g, MgSO
40.1g, CaCl
20.005g, ammonium sulfate 15g/100ml, TritonX-1003g/100ml) middle cultivation is after 3 days, and the absorbancy analyzing cell free fermentation liquid 410,470 and 510nm place monascorubin is respectively 45,25 and 20 absorbance units; Parachrome after ethanolic soln extracts 410,470 and the absorbancy at 510nm place be respectively 35,30 and 18 absorbance units.Fermentation final pH is 2.2, and inside and outside born of the same parents, citrinin content shows substantially not containing Citrinin through thin layer chromatography analysis.Thin-layer chromatography shows that monascorubin main component is monascus yellow pigment.Cloud point extraction cell free fermentation liquid in 70 degree of water-baths further, in tensio-active agent concentrated phase 410,470 and the absorbancy of 510nm place monascorubin be respectively 110,60 and 30 absorbance units; In tensio-active agent dilute phase 410,470 and the absorbancy at 510nm place be respectively 15,8 and 7 absorbance units.
Implementation result: the present embodiment product can obtain through thin layer chromatography analysis result: thin layer chromatography analysis can detect the existence of Citrinin in tensio-active agent dilute phase, can't detect in tensio-active agent concentrated phase.
embodiment 3
The present embodiment relates to a kind of extractive fermentation and regulation and control pH prepares the method for monascorubin, and described method comprises the steps:
Step 1, monascus ruber is at seed culture medium (100ml, peptone 2g, yeast powder 2g, glucose 2g) in carry out cultivation 32 hours, obtain seed liquor, described monascus ruber provides (Monascusanka, CICC5013) for Chinese industrial microorganism strains preservation center;
Step 2, it is 6 that described seed liquor is inoculated into initial pH value, is in the fermentation culture of nitrogenous source with Sodium Glutamate, carries out fermentation culture after 7 days, regulates the pH value of fermentation culture, centrifugal, obtains monascus ruber wet cell; Get the pre-treatment of monascus ruber wet cell, release intracellular product, detects fermented liquid pH value, the pigment concentration of intraor extracellular, the citrinin content of intraor extracellular; Wherein, described pre-treatment is specially: get 0.5g wet cell at isopyknic fermented liquid 70%(V/V) ethanol (pH=2) solution in soak 1 hour;
The absorbancy analyzing cell free fermentation liquid 410,470 and 510nm place monascorubin is respectively 5,3 and 5 absorbance units; Parachrome after ethanolic soln extracts 410,470 and the absorbancy at 510nm place be respectively 20,12 and 22 absorbance units.
Fermented liquid final pH is 7, and inside and outside born of the same parents, through thin layer chromatography analysis, citrinin content shows that content is high, has obvious distribution inside and outside born of the same parents, is more prone to be distributed in cell free fermentation liquid.Thin-layer chromatography shows that monascorubin main component is monascorubin derivative.
Step 3, by the cell 0.5g of fermentation culture, adds fermention medium (water 100ml, glucose 5g, KH that initial pH is 5.5
2pO
40.4g, MgSO
40.1g, CaCl
20.005g, SODIUMNITRATE 15g/100ml, TritonX-1003g/100ml) middle cultivation is after 4 days, and the absorbancy analyzing cell free fermentation liquid 410,470 and 510nm place monascorubin is respectively 55,40 and 53 absorbance units; Parachrome after ethanolic soln extracts 410,470 and the absorbancy at 510nm place be respectively 15,8 and 12 absorbance units.Fermentation final pH is 6.2, and inside and outside born of the same parents, citrinin content obviously exists Citrinin through thin layer chromatography analysis at intraor extracellular, and Citrinin is more prone to be distributed in outside born of the same parents.Thin-layer chromatography shows that monascorubin main component is monascorubin.Cloud point extraction cell free fermentation liquid in 70 degree of water-baths further, in tensio-active agent concentrated phase 410,470 and the absorbancy of 510nm place monascorubin be respectively 108,80 and 110 absorbance units; In tensio-active agent dilute phase 410,470 and the absorbancy at 510nm place be respectively 5,2 and 5 absorbance units.
Implementation result: the present embodiment product can obtain through thin layer chromatography analysis result: thin layer chromatography analysis obviously can detect the existence of Citrinin in tensio-active agent dilute phase, almost can't detect in tensio-active agent concentrated phase.
embodiment 4
The present embodiment relates to a kind of extractive fermentation and regulation and control pH prepares the method for monascorubin, and described method comprises the steps:
Step 1, monascus ruber is at seed culture medium (100ml, peptone 2g, yeast powder 2g, glucose 2g) in carry out cultivation 32 hours, obtain seed liquor, described monascus ruber provides (Monascusanka, CICC5013) for Chinese industrial microorganism strains preservation center;
Step 2, it is 2.5 that described seed liquor is inoculated into initial pH value, is in the fermentation culture of nitrogenous source with Sodium Glutamate, carries out fermentation culture after 5 days, regulates the pH value of fermentation culture, centrifugal, obtains monascus ruber wet cell; Get the pre-treatment of monascus ruber wet cell, release intracellular product, detects fermented liquid pH value, the pigment concentration of intraor extracellular, the citrinin content of intraor extracellular; Wherein, described pre-treatment is specially: get 0.5g wet cell at isopyknic fermented liquid 70%(V/V) ethanol (pH=2) solution in soak 1 hour;
The absorbancy analyzing cell free fermentation liquid 410,470 and 510nm place monascorubin is respectively 1,0.3 and 0.8 absorbance unit; Parachrome after ethanolic soln extracts 410,470 and the absorbancy at 510nm place be respectively 30,45 and 12 absorbance units.
Fermented liquid final pH is 2.5, and inside and outside born of the same parents, citrinin content shows substantially not containing Citrinin through thin layer chromatography analysis.Thin-layer chromatography shows that monascorubin main component is red colouring agent for food, also used as a Chinese medicine citraurin.
Step 3, by the cell 0.5g of fermentation culture, adds fermention medium (water 100ml, glucose 5g, KH that initial pH is 3.5
2pO
40.4g, MgSO
40.1g, CaCl
20.005g, SODIUMNITRATE 15g/100ml, TritonX-1003g/100ml) middle cultivation is after 4 days, and the absorbancy analyzing cell free fermentation liquid 410,470 and 510nm place monascorubin is respectively 53,40 and 50 absorbance units; Parachrome after ethanolic soln extracts 410,470 and the absorbancy at 510nm place be respectively 14,9 and 12 absorbance units.Fermentation final pH is 6.2, and inside and outside born of the same parents, citrinin content obviously exists Citrinin through thin layer chromatography analysis at intraor extracellular, and Citrinin is more prone to be distributed in outside born of the same parents.Thin-layer chromatography shows that monascorubin main component is monascorubin.Cloud point extraction cell free fermentation liquid in 70 degree of water-baths further, in tensio-active agent concentrated phase 410,470 and the absorbancy of 510nm place monascorubin be respectively 100,80 and 108 absorbance units; In tensio-active agent dilute phase 410,470 and the absorbancy at 510nm place be respectively 5,2 and 5 absorbance units.
Implementation result: the present embodiment product can obtain through thin layer chromatography analysis result: thin layer chromatography analysis obviously can detect the existence of Citrinin in tensio-active agent dilute phase, almost can't detect in tensio-active agent concentrated phase.
embodiment 5
The present embodiment relates to a kind of extractive fermentation and regulation and control pH prepares the method for monascorubin, and described method comprises the steps:
Step 1, monascus ruber is at seed culture medium (100ml, peptone 2g, yeast powder 2g, glucose 2g) in carry out cultivation 32 hours, obtain seed liquor, described monascus ruber provides (Monascusanka, CICC5013) for Chinese industrial microorganism strains preservation center;
Step 2, it is 6 that described seed liquor is inoculated into initial pH value, is in the fermentation culture of nitrogenous source with Sodium Glutamate, carries out fermentation culture after 5 days, regulates the pH value of fermentation culture, centrifugal, obtains monascus ruber wet cell; Get the pre-treatment of monascus ruber wet cell, release intracellular product, detects fermented liquid pH value, the pigment concentration of intraor extracellular, the citrinin content of intraor extracellular; Wherein, described pre-treatment is specially: get 0.5g wet cell at isopyknic fermented liquid 70%(V/V) ethanol (pH=2) solution in soak 1 hour;
The absorbancy analyzing cell free fermentation liquid 410,470 and 510nm place monascorubin is respectively 5,3 and 5 absorbance units; Parachrome after ethanolic soln extracts 410,470 and the absorbancy at 510nm place be respectively 20,32 and 18 absorbance units.
Fermented liquid final pH is 4, and inside and outside born of the same parents, through thin layer chromatography analysis, citrinin content shows that content is high, has obvious distribution inside and outside born of the same parents, is more prone to be distributed in cell free fermentation liquid.Thin-layer chromatography shows that monascorubin main component is red colouring agent for food, also used as a Chinese medicine citraurin.
Step 3, by the cell 0.5g of fermentation culture, adds fermention medium (water 100ml, glucose 5g, KH that initial pH is 5
2pO
40.4g, MgSO
40.1g, CaCl
20.005g, Sodium Glutamate 15g/100ml, TritonX-1003g/100ml) middle cultivation is after 3 days, and the absorbancy analyzing cell free fermentation liquid 410,470 and 510nm place monascorubin is respectively 50,35 and 50 absorbance units; Parachrome after ethanolic soln extracts 410,470 and the absorbancy at 510nm place be respectively 15,10 and 14 absorbance units.Fermentation final pH is 6.2, and inside and outside born of the same parents, citrinin content obviously exists Citrinin through thin layer chromatography analysis at intraor extracellular, and Citrinin is more prone to be distributed in outside born of the same parents.Thin-layer chromatography shows that monascorubin main component is monascorubin derivative.Cloud point extraction cell free fermentation liquid in 70 degree of water-baths further, in tensio-active agent concentrated phase 410,470 and the absorbancy of 510nm place monascorubin be respectively 90,75 and 98 absorbance units; In tensio-active agent dilute phase 410,470 and the absorbancy at 510nm place be respectively 15,12 and 13 absorbance units.
Implementation result: the present embodiment product can obtain through thin layer chromatography analysis result: thin layer chromatography analysis obviously can detect the existence of Citrinin in tensio-active agent dilute phase, almost can't detect in tensio-active agent concentrated phase.
embodiment 6
The present embodiment relates to a kind of extractive fermentation and regulation and control pH prepares the method for monascorubin, and described method comprises the steps:
Step 1, monascus ruber is at seed culture medium (100ml, peptone 2g, yeast powder 2g, glucose 2g) in carry out cultivation 32 hours, obtain seed liquor, described monascus ruber provides (Monascusanka, CICC5013) for Chinese industrial microorganism strains preservation center;
Step 2, it is 3.5 that described seed liquor is inoculated into initial pH value, is in the fermentation culture of nitrogenous source with Sodium Glutamate, carries out fermentation culture after 6 days, regulates the pH value of fermentation culture, centrifugal, obtains monascus ruber wet cell; Get the pre-treatment of monascus ruber wet cell, release intracellular product, detects fermented liquid pH value, the pigment concentration of intraor extracellular, the citrinin content of intraor extracellular; Wherein, described pre-treatment is specially: get 0.5g wet cell at isopyknic fermented liquid 70%(V/V) ethanol (pH=2) solution in soak 1 hour;
The absorbancy analyzing cell free fermentation liquid 410,470 and 510nm place monascorubin is respectively 5,2.5 and 5 absorbance units; Parachrome after ethanolic soln extracts 410,470 and the absorbancy at 510nm place be respectively 21,33 and 17 absorbance units.
Fermented liquid final pH is 3.7, and inside and outside born of the same parents, through thin layer chromatography analysis, citrinin content shows that content is high, has obvious distribution inside and outside born of the same parents, is more prone to be distributed in cell free fermentation liquid.Thin-layer chromatography shows that monascorubin main component is red colouring agent for food, also used as a Chinese medicine citraurin.
Step 3, by the cell 0.5g of fermentation culture, adds fermention medium (water 100ml, glucose 5g, KH that initial pH is 3
2pO
40.4g, MgSO
40.1g, CaCl
20.005g, Sodium Glutamate 15g/100ml, TritonX-1003g/100ml) middle cultivation is after 3 days, and the absorbancy analyzing cell free fermentation liquid 410,470 and 510nm place monascorubin is respectively 60,35 and 20 absorbance units; Parachrome after ethanolic soln extracts 410,470 and the absorbancy at 510nm place be respectively 25,10 and 8 absorbance units.Fermentation final pH is 3.5, and inside and outside born of the same parents, citrinin content exists a small amount of Citrinin through thin layer chromatography analysis at intraor extracellular, and Citrinin is more prone to be distributed in outside born of the same parents.Thin-layer chromatography shows that monascorubin main component is red colouring agent for food, also used as a Chinese medicine citraurin.Cloud point extraction cell free fermentation liquid in 70 degree of water-baths further, in tensio-active agent concentrated phase 410,470 and the absorbancy of 510nm place monascorubin be respectively 110,75 and 35 absorbance units; In tensio-active agent dilute phase 410,470 and the absorbancy at 510nm place be respectively 5,2 and 1 absorbance unit.
Implementation result: the present embodiment product can obtain through thin layer chromatography analysis result: thin layer chromatography analysis obviously can detect the existence of Citrinin in tensio-active agent dilute phase, almost can't detect in tensio-active agent concentrated phase.
embodiment 7
The present embodiment relates to a kind of extractive fermentation and regulation and control pH prepares the method for monascorubin, and described method comprises the steps:
Step 1, monascus ruber is at seed culture medium (100ml, peptone 2g, yeast powder 2g, glucose 2g) in carry out cultivation 32 hours, obtain seed liquor, described monascus ruber provides (Monascusanka, CICC5013) for Chinese industrial microorganism strains preservation center;
Step 2, it is 2.5 that described seed liquor is inoculated into initial pH value, is in the fermentation culture of nitrogenous source with Sodium Glutamate, carries out fermentation culture after 6 days, regulates the pH value of fermentation culture, centrifugal, obtains monascus ruber wet cell; Get the pre-treatment of monascus ruber wet cell, release intracellular product, detects fermented liquid pH value, the pigment concentration of intraor extracellular, the citrinin content of intraor extracellular; Wherein, described pre-treatment is specially: get 0.5g wet cell at isopyknic fermented liquid 70%(V/V) ethanol (pH=2) solution in soak 1 hour;
The absorbancy analyzing cell free fermentation liquid 410,470 and 510nm place monascorubin is respectively 3,1 and 1 absorbance unit; Parachrome after ethanolic soln extracts 410,470 and the absorbancy at 510nm place be respectively 24,38 and 18 absorbance units.
Fermented liquid final pH is 2.8, and inside and outside born of the same parents, citrinin content shows content obviously by low through thin layer chromatography analysis, has obvious distribution inside and outside born of the same parents.Thin-layer chromatography shows that monascorubin main component is red colouring agent for food, also used as a Chinese medicine citraurin.
Step 3, by the cell 0.5g of fermentation culture, adds fermention medium (water 100ml, glucose 5g, KH that initial pH is 5
2pO
40.4g, MgSO
40.1g, CaCl
20.005g, rice meal 5g/100ml, TritonX-1003g/100ml) middle cultivation is after 3 days, and the absorbancy analyzing cell free fermentation liquid 410,470 and 510nm place monascorubin is respectively 54,40 and 51 absorbance units; Parachrome after ethanolic soln extracts 410,470 and the absorbancy at 510nm place be respectively 16,9 and 12 absorbance units.Fermentation final pH is 6.2, and inside and outside born of the same parents, citrinin content obviously exists Citrinin through thin layer chromatography analysis at intraor extracellular, and Citrinin is more prone to be distributed in outside born of the same parents.Thin-layer chromatography shows that monascorubin main component is monascorubin.Cloud point extraction cell free fermentation liquid in 70 degree of water-baths further, in tensio-active agent concentrated phase 410,470 and the absorbancy of 510nm place monascorubin be respectively 100,80 and 108 absorbance units; In tensio-active agent dilute phase 410,470 and the absorbancy at 510nm place be respectively 5,2 and 5 absorbance units.
Implementation result: the present embodiment product can obtain through thin layer chromatography analysis result: thin layer chromatography analysis obviously can detect the existence of Citrinin in tensio-active agent dilute phase, almost can't detect in tensio-active agent concentrated phase.
embodiment 8
The present embodiment relates to a kind of extractive fermentation and regulation and control pH prepares the method for monascorubin, and described method comprises the steps:
Step 1, monascus ruber is at seed culture medium (100ml, peptone 2g, yeast powder 2g, glucose 2g) in carry out cultivation 32 hours, obtain seed liquor, described monascus ruber provides (Monascusanka, CICC5013) for Chinese industrial microorganism strains preservation center;
Step 2, it is 6 that described seed liquor is inoculated into initial pH value, is in the fermentation culture of nitrogenous source with Sodium Glutamate, carries out fermentation culture after 6 days, regulates the pH value of fermentation culture, centrifugal, obtains monascus ruber wet cell; Get the pre-treatment of monascus ruber wet cell, release intracellular product, detects fermented liquid pH value, the pigment concentration of intraor extracellular, the citrinin content of intraor extracellular; Wherein, described pre-treatment is specially: get 0.5g wet cell at isopyknic fermented liquid 70%(V/V) ethanol (pH=2) solution in soak 1 hour;
The absorbancy analyzing cell free fermentation liquid 410,470 and 510nm place monascorubin is respectively 5,2 and 6 absorbance units; Parachrome after ethanolic soln extracts 410,470 and the absorbancy at 510nm place be respectively 21,30 and 24 absorbance units.
Fermented liquid final pH is 4.3, and inside and outside born of the same parents, through thin layer chromatography analysis, citrinin content shows that content is high, has obvious distribution inside and outside born of the same parents, is more prone to be distributed in cell free fermentation liquid.Thin-layer chromatography shows that monascorubin main component is red colouring agent for food, also used as a Chinese medicine citraurin.
Step 3, by the cell 0.5g of fermentation culture, adds fermention medium (water 100ml, glucose 5g, KH that initial pH is 5
2pO
40.4g, MgSO
40.1g, CaCl
20.005g, rice meal 5g/100ml, TritonX-1003g/100ml) middle cultivation is after 3 days, and the absorbancy analyzing cell free fermentation liquid 410,470 and 510nm place monascorubin is respectively 54,40 and 51 absorbance units; Parachrome after ethanolic soln extracts 410,470 and the absorbancy at 510nm place be respectively 16,9 and 12 absorbance units.Fermentation final pH is 6.2, and inside and outside born of the same parents, citrinin content obviously exists Citrinin through thin layer chromatography analysis at intraor extracellular, and Citrinin is more prone to be distributed in outside born of the same parents.Thin-layer chromatography shows that monascorubin main component is monascorubin.Cloud point extraction cell free fermentation liquid in 70 degree of water-baths further, in tensio-active agent concentrated phase 410,470 and the absorbancy of 510nm place monascorubin be respectively 100,80 and 108 absorbance units; In tensio-active agent dilute phase 410,470 and the absorbancy at 510nm place be respectively 5,2 and 5 absorbance units.
Implementation result: the present embodiment product can obtain through thin layer chromatography analysis result: thin layer chromatography analysis obviously can detect the existence of Citrinin in tensio-active agent dilute phase, almost can't detect in tensio-active agent concentrated phase.
embodiment 9
The present embodiment relates to a kind of extractive fermentation and regulation and control pH prepares the method for monascorubin, and described method comprises the steps:
Step 1, monascus ruber is at seed culture medium (100ml, peptone 2g, yeast powder 2g, glucose 2g) in carry out cultivation 32 hours, obtain seed liquor, described monascus ruber provides (Monascusanka, CICC5013) for Chinese industrial microorganism strains preservation center;
Step 2, it is 4 that described seed liquor is inoculated into initial pH value, is in the fermentation culture of nitrogenous source with Sodium Glutamate, carries out fermentation culture after 6 days, regulates the pH value of fermentation culture, centrifugal, obtains monascus ruber wet cell; Get the pre-treatment of monascus ruber wet cell, release intracellular product, detects fermented liquid pH, the pigment concentration of intraor extracellular, the citrinin content of intraor extracellular; Wherein, described pre-treatment is specially: get 0.5g wet cell at isopyknic fermented liquid 70%(V/V) ethanol (pH=2) solution in soak 1 hour;
The absorbancy analyzing cell free fermentation liquid 410,470 and 510nm place monascorubin is respectively 4,1.5 and 0.8 absorbance unit; Parachrome after ethanolic soln extracts 410,470 and the absorbancy at 510nm place be respectively 45,35 and 30 absorbance units.
Fermented liquid final pH is 2.3, and inside and outside born of the same parents, citrinin content shows substantially not containing Citrinin through thin layer chromatography analysis.Thin-layer chromatography shows that monascorubin main component is red colouring agent for food, also used as a Chinese medicine citraurin.
Step 3, by the cell 0.5g of fermentation culture, adds fermention medium (water 100ml, glucose 5g, KH that initial pH is 4
2pO
40.4g, MgSO
40.1g, CaCl
20.005g, Semen Maydis powder 15g/100ml, TritonX-1003g/100ml) middle cultivation is after 3 days, and the absorbancy analyzing cell free fermentation liquid 410,470 and 510nm place monascorubin is respectively 35,15 and 10 absorbance units; Parachrome after ethanolic soln extracts 410,470 and the absorbancy at 510nm place be respectively 15,10 and 7 absorbance units.Fermentation final pH is 2.2, and inside and outside born of the same parents, citrinin content shows substantially not containing Citrinin through thin layer chromatography analysis.Thin-layer chromatography shows that monascorubin main component is monascus yellow pigment.Cloud point extraction cell free fermentation liquid in 70 degree of water-baths further, in tensio-active agent concentrated phase 410,470 and the absorbancy of 510nm place monascorubin be respectively 80,30 and 10 absorbance units; In tensio-active agent dilute phase 410,470 and the absorbancy at 510nm place be respectively 15,8 and 7 absorbance units.
Implementation result: the present embodiment product can obtain through thin layer chromatography analysis result: thin layer chromatography analysis can detect the existence of Citrinin in tensio-active agent dilute phase, can't detect in tensio-active agent concentrated phase.
embodiment 10
The present embodiment relates to a kind of extractive fermentation and regulation and control pH prepares the method for monascorubin, and described method comprises the steps:
Step 1, monascus ruber is at seed culture medium (100ml, peptone 2g, yeast powder 2g, glucose 2g) in carry out cultivation 32 hours, obtain seed liquor, described monascus ruber provides (Monascusanka, CICC5013) for Chinese industrial microorganism strains preservation center;
Step 2, it is 4 that described seed liquor is inoculated into initial pH value, is in the fermentation culture of nitrogenous source with Sodium Glutamate, carries out fermentation culture after 6 days, regulates the pH value of fermentation culture, centrifugal, obtains monascus ruber wet cell; Get the pre-treatment of monascus ruber wet cell, release intracellular product, detects fermented liquid pH value, the pigment concentration of intraor extracellular, the citrinin content of intraor extracellular; Wherein, described pre-treatment is specially: get 0.5g wet cell at isopyknic fermented liquid 70%(V/V) ethanol (pH=2) solution in soak 1 hour;
The absorbancy analyzing cell free fermentation liquid 410,470 and 510nm place monascorubin is respectively 4,1.5 and 0.8 absorbance unit; Parachrome after ethanolic soln extracts 410,470 and the absorbancy at 510nm place be respectively 45,35 and 30 absorbance units.
Fermented liquid final pH is 6.3, and inside and outside born of the same parents, citrinin content shows substantially not containing Citrinin through thin layer chromatography analysis.Thin-layer chromatography shows that monascorubin main component is red colouring agent for food, also used as a Chinese medicine citraurin.
Step 3, by the cell 0.5g of fermentation culture, adds fermention medium (water 100ml, glucose 5g, KH that initial pH is 4
2pO
40.4g, MgSO
40.1g, CaCl
20.005g, analysis for soybean powder 15g/100ml, TritonX-1003g/100ml) middle cultivation is after 3 days, and the absorbancy analyzing cell free fermentation liquid 410,470 and 510nm place monascorubin is respectively 45,25 and 15 absorbance units; Parachrome after ethanolic soln extracts 410,470 and the absorbancy at 510nm place be respectively 18,13 and 6 absorbance units.Fermentation final pH is 2.2, and inside and outside born of the same parents, citrinin content shows substantially not containing Citrinin through thin layer chromatography analysis.Thin-layer chromatography shows that monascorubin main component is monascus yellow pigment.Cloud point extraction cell free fermentation liquid in 70 degree of water-baths further, in tensio-active agent concentrated phase 410,470 and the absorbancy of 510nm place monascorubin be respectively 90,35 and 15 absorbance units; In tensio-active agent dilute phase 410,470 and the absorbancy at 510nm place be respectively 15,8 and 7 absorbance units.
Implementation result: the present embodiment product can obtain through thin layer chromatography analysis result: thin layer chromatography analysis can detect the existence of Citrinin in tensio-active agent dilute phase, can't detect in tensio-active agent concentrated phase.
The present invention is combined with regulation and control pH technology by extractive fermentation technology, achieve fermentable produce the low and monascus pigment component of citrinin content relatively single water-soluble/the serial monascorubin product such as hydrophobicity monascorubin, hydrophobicity red colouring agent for food, also used as a Chinese medicine citraurin, hydrophobicity monascus yellow pigment.Adopt extractive fermentation technology, monascorubin in born of the same parents can be discharged into born of the same parents' external surfactants micellar solution by microorganism fermentation culture in nonionogenic tenside micellar solution, and produces the outer monascus yellow pigment of the low born of the same parents of citrinin content under control fermented liquid low pH condition.Raised temperature can realize cloud point extraction, and cloud point extraction can make that Citrinin is distributed in tensio-active agent dilute phase and monascorubin is distributed in tensio-active agent concentrated phase.When low pH extractive fermentation, form the outer yellow pigment fermented liquid of the low born of the same parents of citrinin content, cloud point extraction can be removed Citrinin wherein further and obtain the low hydrophobicity monascus yellow pigment of citrinin content.When high pH extractive fermentation, formed citrinin content high water-soluble/hydrophobicity monascorubin fermented liquid, cloud point extraction also can remove Citrinin wherein and obtain citrinin content low water-soluble/hydrophobicity monascorubin.
It is more than the description of the preferred embodiment of the present invention, should be understood that, enforcement of the present invention is not limited to above-mentioned situation, the present invention is particularly suitable for the fermenting process of thin intracellular product, as the production etc. of oil compounds in the production of small molecule organic compound in the production of intracellular microbe enzyme, cell and cell, effectively can remove intracellular product to suppress, improve the concentration of microbial fermentation product and regulate the composition of fermentable secondary metabolite.Thus improve the volume productivity of fermentable and improve the efficiency of the processes such as intracellular product fermentation, product release.
Above specific embodiments of the invention are described.It is to be appreciated that the present invention is not limited to above-mentioned particular implementation, those skilled in the art can make various distortion or amendment within the scope of the claims, and this does not affect flesh and blood of the present invention.
Claims (4)
1. extractive fermentation and regulation and control pH prepare a method for monascorubin, and it is characterized in that, described method comprises the steps:
Step 1, monascus ruber carries out cultivation 32 hours in seed culture medium, obtains seed liquor, and described monascus ruber provides for Chinese industrial microorganism strains preservation center, and Classification system is Monascusanka, and deposit number is CICC5013; Described seed culture medium is made up of each component of following content: water 100ml, peptone 2g, yeast powder 2g, glucose 2g;
Step 2, it is 4 that described seed liquor is inoculated into initial pH value, is in the fermentation culture of nitrogenous source with SODIUMNITRATE, is 30 DEG C in temperature, carries out the fermentation culture of 5 days in the shaking table of 200rpm, regulates the pH value of fermentation culture, centrifugal, obtains monascus ruber wet cell; Get the pre-treatment of monascus ruber wet cell, release intracellular product, detects fermented liquid pH, the pigment concentration of intraor extracellular, the citrinin content of intraor extracellular; Wherein, described pre-treatment is specially: get 0.5g wet cell and soak 1 hour in the ethanolic soln of isopyknic fermented liquid 70% (V/V), the pH=2 of ethanol;
The absorbancy analyzing cell free fermentation liquid 410,470 and 510nm place monascorubin is respectively 2,1 and 1.8 absorbance units; Parachrome after ethanolic soln extracts 410,470 and the absorbancy at 510nm place be respectively 35,25 and 32 absorbance units;
Fermented liquid final pH is 6.8, and inside and outside born of the same parents, through thin layer chromatography analysis, citrinin content shows that content is high, has obvious distribution inside and outside born of the same parents, is more prone to be distributed in cell free fermentation liquid, and thin-layer chromatography shows that monascorubin main component is monascorubin;
Step 3, by the cell 0.5g of fermentation culture, add initial pH and cultivate after 5 days in the fermention medium of 4, described fermention medium is made up of each component of following content: water 100ml, glucose 5g, KH
2pO
40.4g, MgSO
40.1g, CaCl
20.005g, ammonium sulfate 5g/100ml, TritonX-1004g/100ml; The absorbancy analyzing cell free fermentation liquid 410,470 and 510nm place monascorubin is respectively 65,45 and 22 absorbance units; Parachrome after ethanolic soln extracts 410,470 and the absorbancy at 510nm place be respectively 25,20 and 15 absorbance units; Fermentation final pH is 2.2, and inside and outside born of the same parents, through thin layer chromatography analysis, citrinin content shows that thin-layer chromatography shows that monascorubin main component is monascus yellow pigment substantially not containing Citrinin; Cloud point extraction cell free fermentation liquid in 70 degree of water-baths further, in tensio-active agent concentrated phase 410,470 and the absorbancy of 510nm place monascorubin be respectively 120,60 and 20 absorbance units; In tensio-active agent dilute phase 410,470 and the absorbancy at 510nm place be respectively 15,8 and 7 absorbance units.
2. extractive fermentation and regulation and control pH prepare a method for monascorubin, and it is characterized in that, described method comprises the steps:
Step 1, monascus ruber carries out cultivation 32 hours in seed culture medium, obtains seed liquor, and described monascus ruber provides for Chinese industrial microorganism strains preservation center, and Classification system is Monascusanka, and deposit number is CICC5013; Described seed culture medium is made up of each component of following content: water 100ml, peptone 2g, yeast powder 2g, glucose 2g;
Step 2, it is 4 that described seed liquor is inoculated into initial pH value, is in the fermentation culture of nitrogenous source with Sodium Glutamate, carries out fermentation culture after 7 days, regulates the pH value of fermentation culture, centrifugal, obtains monascus ruber wet cell; Get the pre-treatment of monascus ruber wet cell, release intracellular product, detects fermented liquid pH value, the pigment concentration of intraor extracellular, the citrinin content of intraor extracellular; Wherein, described pre-treatment is specially: get 0.5g wet cell and soak 1 hour in the ethanolic soln of isopyknic fermented liquid 70% (V/V), the pH=2 of ethanol;
The absorbancy analyzing cell free fermentation liquid 410,470 and 510nm place monascorubin is respectively 10,8 and 12 absorbance units; Parachrome after ethanolic soln extracts 410,470 and the absorbancy at 510nm place be respectively 30,22 and 32 absorbance units;
Fermented liquid final pH is 6.5, and inside and outside born of the same parents, through thin layer chromatography analysis, citrinin content shows that content is high, has obvious distribution inside and outside born of the same parents, is more prone to be distributed in cell free fermentation liquid.Thin-layer chromatography shows that monascorubin main component is monascorubin derivative;
Step 3, by the cell 0.5g of fermentation culture, add initial pH and cultivate after 3 days in the fermention medium of 4, described fermention medium is made up of each component of following content: water 100ml, glucose 5g, KH
2pO
40.4g, MgSO
40.1g, CaCl
20.005g, ammonium sulfate 15g/100ml, TritonX-1003g/100ml; The absorbancy analyzing cell free fermentation liquid 410,470 and 510nm place monascorubin is respectively 45,25 and 20 absorbance units; Parachrome after ethanolic soln extracts 410,470 and the absorbancy at 510nm place be respectively 35,30 and 18 absorbance units;
Fermentation final pH is 2.2, and inside and outside born of the same parents, through thin layer chromatography analysis, citrinin content shows that thin-layer chromatography shows that monascorubin main component is monascus yellow pigment substantially not containing Citrinin; Cloud point extraction cell free fermentation liquid in 70 degree of water-baths further, in tensio-active agent concentrated phase 410,470 and the absorbancy of 510nm place monascorubin be respectively 110,60 and 30 absorbance units; In tensio-active agent dilute phase 410,470 and the absorbancy at 510nm place be respectively 15,8 and 7 absorbance units.
3. extractive fermentation and regulation and control pH prepare a method for monascorubin, and it is characterized in that, described method comprises the steps:
Step 1, monascus ruber carries out cultivation 32 hours in seed culture medium, obtains seed liquor, and described monascus ruber provides for Chinese industrial microorganism strains preservation center, and Classification system is Monascusanka, and deposit number is CICC5013; Described seed culture medium is made up of each component of following content: water 100ml, peptone 2g, yeast powder 2g, glucose 2g;
Step 2, it is 4 that described seed liquor is inoculated into initial pH value, is in the fermentation culture of nitrogenous source with Sodium Glutamate, carries out fermentation culture after 6 days, regulates the pH value of fermentation culture, centrifugal, obtains monascus ruber wet cell; Get the pre-treatment of monascus ruber wet cell, release intracellular product, detects fermented liquid pH, the pigment concentration of intraor extracellular, the citrinin content of intraor extracellular; Wherein, described pre-treatment is specially: get 0.5g wet cell and soak 1 hour in the ethanolic soln of isopyknic fermented liquid 70% (V/V), the pH=2 of ethanol;
The absorbancy analyzing cell free fermentation liquid 410,470 and 510nm place monascorubin is respectively 4,1.5 and 0.8 absorbance unit; Parachrome after ethanolic soln extracts 410,470 and the absorbancy at 510nm place be respectively 45,35 and 30 absorbance units;
Fermented liquid final pH is 2.3, and inside and outside born of the same parents, through thin layer chromatography analysis, citrinin content shows that thin-layer chromatography shows that monascorubin main component is red colouring agent for food, also used as a Chinese medicine citraurin substantially not containing Citrinin;
Step 3, by the cell 0.5g of fermentation culture, add initial pH and cultivate after 3 days in the fermention medium of 4, described fermention medium is made up of each component of following content: water 100ml, glucose 5g, KH
2pO
40.4g, MgSO
40.1g, CaCl
20.005g, Semen Maydis powder 15g/100ml, TritonX-1003g/100ml; The absorbancy analyzing cell free fermentation liquid 410,470 and 510nm place monascorubin is respectively 35,15 and 10 absorbance units; Parachrome after ethanolic soln extracts 410,470 and the absorbancy at 510nm place be respectively 15,10 and 7 absorbance units;
Fermentation final pH is 2.2, and inside and outside born of the same parents, through thin layer chromatography analysis, citrinin content shows that thin-layer chromatography shows that monascorubin main component is monascus yellow pigment substantially not containing Citrinin; Cloud point extraction cell free fermentation liquid in 70 degree of water-baths further, in tensio-active agent concentrated phase 410,470 and the absorbancy of 510nm place monascorubin be respectively 80,30 and 10 absorbance units; In tensio-active agent dilute phase 410,470 and the absorbancy at 510nm place be respectively 15,8 and 7 absorbance units.
4. extractive fermentation and regulation and control pH prepare a method for monascorubin, and it is characterized in that, described method comprises the steps:
Step 1, monascus ruber carries out cultivation 32 hours in seed culture medium, obtains seed liquor, and described monascus ruber provides for Chinese industrial microorganism strains preservation center, and Classification system is Monascusanka, and deposit number is CICC5013; Described seed culture medium is made up of each component of following content: water 100ml, peptone 2g, yeast powder 2g, glucose 2g;
Step 2, it is 4 that described seed liquor is inoculated into initial pH value, is in the fermentation culture of nitrogenous source with Sodium Glutamate, carries out fermentation culture after 6 days, regulates the pH value of fermentation culture, centrifugal, obtains monascus ruber wet cell; Get the pre-treatment of monascus ruber wet cell, release intracellular product, detects fermented liquid pH value, the pigment concentration of intraor extracellular, the citrinin content of intraor extracellular; Wherein, described pre-treatment is specially: get 0.5g wet cell and soak 1 hour in the ethanolic soln of isopyknic fermented liquid 70% (V/V), the pH=2 of ethanol;
The absorbancy analyzing cell free fermentation liquid 410,470 and 510nm place monascorubin is respectively 4,1.5 and 0.8 absorbance unit; Parachrome after ethanolic soln extracts 410,470 and the absorbancy at 510nm place be respectively 45,35 and 30 absorbance units;
Fermented liquid final pH is 6.3, and inside and outside born of the same parents, through thin layer chromatography analysis, citrinin content shows that thin-layer chromatography shows that monascorubin main component is red colouring agent for food, also used as a Chinese medicine citraurin substantially not containing Citrinin;
Step 3, by the cell 0.5g of fermentation culture, add initial pH and cultivate after 3 days in the fermention medium of 4, described fermention medium is made up of each component of following content: water 100ml, glucose 5g, KH
2pO
40.4g, MgSO
40.1g, CaCl
20.005g, analysis for soybean powder 15g/100ml, TritonX-1003g/100ml;
The absorbancy analyzing cell free fermentation liquid 410,470 and 510nm place monascorubin is respectively 45,25 and 15 absorbance units; Parachrome after ethanolic soln extracts 410,470 and the absorbancy at 510nm place be respectively 18,13 and 6 absorbance units;
Fermentation final pH is 2.2, and inside and outside born of the same parents, through thin layer chromatography analysis, citrinin content shows that thin-layer chromatography shows that monascorubin main component is monascus yellow pigment substantially not containing Citrinin; Cloud point extraction cell free fermentation liquid in 70 degree of water-baths further, in tensio-active agent concentrated phase 410,470 and the absorbancy of 510nm place monascorubin be respectively 90,35 and 15 absorbance units; In tensio-active agent dilute phase 410,470 and the absorbancy at 510nm place be respectively 15,8 and 7 absorbance units.
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| CN107723320B (en) * | 2017-09-08 | 2020-09-18 | 广东科隆生物科技有限公司 | Method for directly preparing crystal monascus orange pigment |
| CN109371053B (en) * | 2018-12-24 | 2021-08-06 | 江西科技师范大学 | Construction method of monascus pigment producing strain |
| CN110592158A (en) * | 2019-09-19 | 2019-12-20 | 长江大学 | Liquid fermentation method for improving purity of monascus yellow pigment Monascin and Ankaflavin |
| CN112042858B (en) * | 2020-09-15 | 2022-11-29 | 江西理工大学 | Method for extracting biosurfactant of monascus pigment |
| CN114507698A (en) * | 2020-11-17 | 2022-05-17 | 天津科技大学 | Liquid state fermentation method for increasing monascus orange pigment yield |
| CN112481327B (en) * | 2020-12-01 | 2023-04-14 | 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 |
| 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 |
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| Effect of pH and nonionic surfactant on profile of intracellular and extracellular monascus pigments;Biyu K et al;《Process Biochemistry》;20130406;第48卷(第5-6期);第759-767页 * |
| Effect of pH on citrinin and red pigments production by Monascus purpureus CCT3802;Sandra Fernanda Bilbao Orozco等;《World J Microbiol Biotechnol》;20080229;第24卷(第2期);第263-268页 * |
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