CN111218487B - Method for promoting monascus to produce monascus yellow pigment by adding alcohol substances - Google Patents
Method for promoting monascus to produce monascus yellow pigment by adding alcohol substances Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
- C12P17/18—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms containing at least two hetero rings condensed among themselves or condensed with a common carbocyclic ring system, e.g. rifamycin
- C12P17/181—Heterocyclic compounds containing oxygen atoms as the only ring heteroatoms in the condensed system, e.g. Salinomycin, Septamycin
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- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L5/00—Preparation or treatment of foods or foodstuffs, in general; Food or foodstuffs obtained thereby; Materials therefor
- A23L5/40—Colouring or decolouring of foods
- A23L5/42—Addition of dyes or pigments, e.g. in combination with optical brighteners
- A23L5/46—Addition of dyes or pigments, e.g. in combination with optical brighteners using dyes or pigments of microbial or algal origin
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/38—Chemical stimulation of growth or activity by addition of chemical compounds which are not essential growth factors; Stimulation of growth by removal of a chemical compound
Abstract
The invention discloses a method for promoting monascus to produce monascus yellow pigment by adding alcohol substances, and belongs to the technical field of microbial fermentation. The invention promotes the increase of the monascus and the synthesis of the monascus pigment by adding the alcohol substances at the initial fermentation stage of the monascus. The method is simple to operate, economical and efficient, does not pollute the environment, does not change the structure of the pigment, does not increase extra labor and equipment, can improve the synthesis of the monascus yellow pigment only by low additional investment, ensures that the color value of the monascus yellow pigment reaches 415U/mL, and has great economic benefit.
Description
Technical Field
The invention relates to a method for promoting monascus to produce monascus yellow pigment by adding alcohol substances, and belongs to the technical field of microbial fermentation.
Background
At present, a microbial fermentation method and a plant extraction method are two main methods for producing the natural yellow pigment. Natural yellow pigments of plant origin such as: the plant-derived pigments face various limitations in the production process, on one hand, organic reagents are adopted in the extraction process, the cost is high, the yield and the application are limited, on the other hand, the seasonality is strong, and the production of the plant-derived pigments causes low utilization rate of equipment under the influence of seasons, so that the resource waste is caused. On the contrary, the method for producing the natural yellow pigment by using the microbial fermentation method is basically safe and nontoxic, and has very wide development prospect.
In order to improve the production level of the monascus yellow pigment, the focus of the papers or patents reported in domestic and foreign literatures is mainly focused on the mutagenesis and screening of high-yield monascus strains, but the effect is not great. For example, the research and development team of Yongsmith in Thailand has bred monascus producing yellow pigment per unit through mutagenesis from the 90 s of 20 th century, and the monascus producing yellow pigment is applied to liquid fermentation and solid fermentation. The wavelength corresponding to the maximum absorption peak of the monascus yellow pigment produced by the strain is 370 nm. However, the production level of the yellow pigment by the liquid fermentation method is very low, and the industrial production is not realized so far. The color value of the monascus yellow (rice) produced by the strain through solid state fermentation for 12 days reaches 2224.63U/gdw, and the fermentation time is too long although the production level of the pigment is high. Monascus yellow pigment producing strain is bred by domestic week et al through mutagenesis, the wavelength corresponding to the maximum absorption peak of the monascus yellow pigment produced by the strain is 410nm, and the color value of the fermentation broth yellow pigment after 7 days of liquid fermentation only reaches 110U/mL. The fermentation pigment concentration is low, the fermentation time is long, and the requirements of industrial production are not met.
In the laboratory of the inventor, in the invention patent of a coupled in-situ extraction fermentation monascus yellow pigment double-liquid-phase fermentation method (patent number 201410116854.6), through designing reasonable carbon-nitrogen source components, controlling the content of free amino acid in a culture medium and a double-liquid-phase fermentation system, the synthesized monascus yellow pigment is rapidly enriched into a fat-soluble solvent (the fat-soluble solvent mainly refers to glyceride substances), thereby greatly improving the production efficiency of the monascus yellow pigment. However, in the actual production process, the lipid substance is found to be high in cost, the pigment is difficult to further separate and extract from the ester substance, the ester substance is difficult to recycle, and the post-treatment step is complex. The invention does not need to add an extracting agent in the fermentation process, can increase the bacterial mass of the monascus and greatly promote the synthesis of monascus yellow pigment by only adding alcohol substances into the fermentation culture medium, and has important research and application values.
At present, the synthesis approach of producing the monascus yellow pigment by monascus fermentation is still not deeply researched, and the purpose of high yield of the monascus yellow pigment is difficult to achieve by adopting the components of a common liquid fermentation culture medium. Although effective, in situ extraction fermentation is expensive, difficult to recycle and complex in post-treatment steps.
Disclosure of Invention
The first purpose of the invention is to provide a method for promoting monascus fermentation to produce monascus yellow pigment, wherein alcohol substances are added in the fermentation initial stage or the fermentation process of monascus.
In one embodiment, the fermentation is a fermentation in a liquid environment.
In one embodiment, the Monascus is a Monascus capable of producing Monascus yellow pigment, including but not limited to Monascus purpureus (Monascus purpureus) sjs-6, Monascus purpureus (Monascus purpureus) CICC 40225, Monascus purpureus (Monascus purpureus) CICC 40937, Monascus purpureus (Monascus purpureus) CICC 5042, Monascus ruber (Monascus ruber) CGMCC 3.4701, Monascus ruber (Monascus ruber) CGMCC 3.4652, Monascus ruber (Monascus ruber) CGMCC 3.465, and Monascus ruber (monascur) CGMCC 3.4649.
In one embodiment, the alcohol comprises methanol, ethanol, isopropanol, butanol, or mixtures thereof; the alcohol substance can promote the growth of monascus and the synthesis of secondary metabolites, and greatly improve the synthesis capacity and yield of monascus to yellow pigment.
In one embodiment, the alcohol is added in an amount of 1-10% (v/v) of the total volume of the fermentation system.
In one embodiment, the alcohol is added at a timing of 0 to 96 hours after the start of the liquid fermentation culture.
The second purpose of the invention is to provide a fermentation method of monascus yellow pigment, which takes monascus as a fermentation microorganism, adds alcohol substances accounting for 1-10% of the total volume of a fermentation system at the beginning of fermentation or in the fermentation process, and performs fermentation for at least 4 days; the alcohol substance is methanol, ethanol, isopropanol, butanol or their mixture.
In one embodiment, the fermentation is performed at a rotation speed of 150-.
In one embodiment, the fermentation is carried out at a rotation speed of 180 ℃ and a temperature of 30 ℃ for 8 d.
In one embodiment, the fermentation lasts for 8 days.
In one embodiment, the method comprises inoculating monascus seed solution in a liquid fermentation medium for fermentation; adding alcohol substances within 0-96h after the liquid fermentation culture is started.
In one embodiment, the liquid fermentation medium is a minimal medium containing nutrients required by monascus growth, and contains 20-80g/L of corn starch, 3-10g/L of ammonium sulfate, 2-6g/L of sodium nitrate, 0.5-1.5g/L of magnesium sulfate, 0.5-1.5g/L of potassium dihydrogen phosphate, 0.5-1.5g/L of dipotassium hydrogen phosphate and 0.1-0.5g/L of calcium chloride.
The invention also claims the application of the method in the preparation of monascus yellow pigment.
In one embodiment, the method is used to prepare a food colorant.
The invention has the beneficial effects that:
the synthesis of monascus purpureus pigment is increased by adding alcohol substances. The method is simple to operate, economical and efficient, does not pollute the environment, does not change the structure of the pigment, does not increase extra labor and equipment, can improve the synthesis of the monascus yellow pigment only by low additional investment, ensures that the color value of the monascus yellow pigment reaches 415U/mL, and has great economic benefit.
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FIG. 1: alcohol substances are added to promote the liquid fermentation of monascus to produce monascus yellow pigment.
Detailed Description
The color value is a representation method of the concentration of the pigment, and the value is equal to the product of the dilution factor and the corresponding OD value of the diluted pigment solution at the absorption peak, and the unit is U/mL.
The solution to be tested is diluted by proper times by absolute ethyl alcohol (OD value is controlled between 0.2 and 0.8), and full-wavelength scanning is carried out in the range of 200-600nm by taking the absorbance of the absolute ethyl alcohol as a blank control, and the OD value of the peak value between 330-450nm is measured.
Yellow pigment color number (U/mL) ═ OD410nm × total dilution.
Example 1: production of pigment by fermentation of different strains
Seed liquid culture medium (g/L): 60 parts of corn starch, 4 parts of ammonium sulfate, 2 parts of sodium nitrate, 0.5 part of magnesium sulfate, 2 parts of monopotassium phosphate, 2 parts of dipotassium phosphate and 0.1 part of calcium chloride.
Fermentation medium (g/L): 60 parts of corn starch, 4 parts of ammonium sulfate, 2 parts of sodium nitrate, 0.5 part of magnesium sulfate, 2 parts of monopotassium phosphate, 2 parts of dipotassium phosphate and 0.1 part of calcium chloride.
(1) Preparation of seed liquid
Selecting purple Monascus sjs-6 (disclosed in the paper of effective biosynthesizes of Natural Yellow Pigments by Monascus purpureus in a Novel Integrated Fermentation System), purple Monascus CICC 40225, purple Monascus CICC 40937, purple Monascus CICC 5042, and red Monascus CGMCC 3.4701, red Monascus CGMCC 3.4652, red Monascus CGMCC 3.465, and red Monascus CGMCC 3.4649 as shake flask strains, respectively inoculating to seed liquid culture medium after activation, and shake flask Fermentation culturing at the rotation speed of 180r/min and the temperature of 30 ℃ for 2d to make the concentration of the strains reach 1 × 107CFU/mL。
(2) Fermentation culture
Inoculating the monascus seed solution in the logarithmic phase prepared in the step (1) into a 500mL triangular flask filled with 50mL fermentation medium in an inoculation amount of 10% by volume, and performing shake-flask fermentation culture for 7d at the rotation speed of 180r/min and the temperature of 30 ℃. After the completion of the test, the color values of the monascus yellow pigments of the eight bacteria sjs-6, CICC 40225, CICC 40937, CICC 5042, CGMCC 3.4701, CGMCC 3.4652, CGMCC 3.465 and CGMCC 3.4649 are respectively 238U/mL, 15U/mL, 23U/mL, 24U/mL, 42U/mL, 45U/mL, 66U/mL and 26U/mL.
TABLE 1 Effect of different Monascus strains on yellow pigment production by liquid fermentation
Example 2: adding different alcohols to ferment and produce pigment
Seed liquid culture medium (g/L): 60 parts of corn starch, 4 parts of ammonium sulfate, 2 parts of sodium nitrate, 0.5 part of magnesium sulfate, 2 parts of monopotassium phosphate, 2 parts of dipotassium phosphate and 0.1 part of calcium chloride.
Fermentation medium (g/L): 60 parts of corn starch, 4 parts of ammonium sulfate, 2 parts of sodium nitrate, 0.5 part of magnesium sulfate, 2 parts of monopotassium phosphate, 2 parts of dipotassium phosphate and 0.1 part of calcium chloride.
(1) Preparation of seed liquid
The monascus purpureus sjs-6 is selected as a shake flask strain, activated and inoculated to a seed liquid culture medium, and shake flask fermentation culture is carried out for 2d under the conditions that the rotation speed is 180r/min and the temperature is 30 ℃.
(2) Fermentation culture
Inoculating the monascus liquid seeds in logarithmic phase into a 500mL triangular flask filled with 50mL fermentation medium according to the inoculation amount of 10% by volume, carrying out shake flask fermentation culture for 7d at the rotation speed of 180r/min and the temperature of 30 ℃, and respectively adding sterile water, methanol, ethanol, n-propanol, isopropanol, n-butanol, isoamylol and 1-octanol with the culture system volume of 2% (v/v) on the 3 rd day. After the completion of the test, the color numbers of the monascus yellow pigment are respectively 220U/mL, 201U/mL, 304U/mL, 108U/mL, 240U/mL, 94U/mL, 111U/mL and 82U/mL.
TABLE 2 influence of different alcohols on the production of yellow pigment by Monascus purpureus sjs-6
Example 3: production of pigment by different addition of ethanol through fermentation
Seed liquid culture medium (g/L): 60 parts of corn starch, 4 parts of ammonium sulfate, 2 parts of sodium nitrate, 0.5 part of magnesium sulfate, 2 parts of monopotassium phosphate, 2 parts of dipotassium phosphate and 0.1 part of calcium chloride.
Fermentation medium (g/L): 60 parts of corn starch, 4 parts of ammonium sulfate, 2 parts of sodium nitrate, 0.5 part of magnesium sulfate, 2 parts of monopotassium phosphate, 2 parts of dipotassium phosphate and 0.1 part of calcium chloride.
(1) Preparation of seed liquid
The monascus purpureus sjs-6 is selected as a shake flask strain, activated and inoculated to a seed liquid culture medium, and shake flask fermentation culture is carried out for 2d under the conditions that the rotation speed is 180r/min and the temperature is 30 ℃.
(2) Fermentation culture
Inoculating the liquid seed of the monascus in the logarithmic growth phase into a 500mL triangular flask containing 50mL of fermentation medium by the inoculation amount of 10% by volume, carrying out shake flask fermentation culture for 7d at the rotation speed of 180r/min and the temperature of 30 ℃, and respectively adding ethanol with the volume of 0% (not added), 1%, 2%, 3%, 4%, 5% and 6% of the culture system on the 3 rd day. After the completion of the test, the color numbers of the monascus yellow pigment are respectively 212U/mL, 207U/mL, 315U/mL, 356U/mL, 325U/mL, 290U/mL and 211U/mL.
TABLE 3 influence of different addition amounts of ethanol on the production of yellow pigment by Monascus purpureus sjs-6
Example 4: adding 3% ethanol at different time for fermentation to produce pigment
Seed liquid culture medium (g/L): 60 parts of corn starch, 4 parts of ammonium sulfate, 2 parts of sodium nitrate, 0.5 part of magnesium sulfate, 2 parts of monopotassium phosphate, 2 parts of dipotassium phosphate and 0.1 part of calcium chloride.
Fermentation medium (g/L): 60 parts of corn starch, 4 parts of ammonium sulfate, 2 parts of sodium nitrate, 0.5 part of magnesium sulfate, 2 parts of monopotassium phosphate, 2 parts of dipotassium phosphate and 0.1 part of calcium chloride.
(1) Preparation of seed liquid
The monascus purpureus sjs-6 is selected as a shake flask strain, activated and inoculated to a seed liquid culture medium, and shake flask fermentation culture is carried out for 2d under the conditions that the rotation speed is 180r/min and the temperature is 30 ℃.
(2) Fermentation culture
Inoculating the liquid seed of the monascus in the logarithmic growth phase into a 500mL triangular flask filled with 50mL of fermentation medium in an inoculation amount of 10% by volume, carrying out shake flask fermentation culture for 7d at the rotation speed of 180r/min and the temperature of 30 ℃, and adding 3% (v/v) ethanol in the volume of the culture system at the 0 th day, the 1 st day, the 2 nd day, the 3 rd day, the 4 th day, the 5 th day and the 6 th day respectively. After the completion of the test, the color values of the monascus yellow pigment are respectively 410U/mL, 350U/mL, 348U/mL, 333U/mL, 372U/mL, 343U/mL and 336U/mL.
TABLE 4 Effect of adding 3% (v/v) ethanol at different times on the production of yellow pigment sjs-6
Example 5: the yield of monascus yellow pigment in different fermentation time without adding alcohol substances
Seed liquid culture medium (g/L): 60 parts of corn starch, 4 parts of ammonium sulfate, 2 parts of sodium nitrate, 0.5 part of magnesium sulfate, 2 parts of monopotassium phosphate, 2 parts of dipotassium phosphate and 0.1 part of calcium chloride.
Fermentation medium (g/L): 60 parts of corn starch, 4 parts of ammonium sulfate, 2 parts of sodium nitrate, 0.5 part of magnesium sulfate, 2 parts of monopotassium phosphate, 2 parts of dipotassium phosphate and 0.1 part of calcium chloride.
(1) Preparation of seed liquid
The monascus purpureus sjs-6 is selected as a shake flask strain, activated and inoculated to a seed liquid culture medium, and shake flask fermentation culture is carried out for 2d under the conditions that the rotation speed is 180r/min and the temperature is 30 ℃.
(2) Fermentation culture
Inoculating the liquid seed of the monascus in the logarithmic growth phase into a 500mL triangular flask filled with 50mL fermentation medium according to the inoculation amount of 10% by volume, and respectively measuring the time color values of 0 day, 1 day, 2 day, 3 day, 4 day, 5 day, 6 day, 7 day and 8 day at the rotating speed of 180r/min and the temperature of 30 ℃. The measured color numbers of the monascus yellow pigment are 2U/mL, 6U/mL, 50U/mL, 81U/mL, 121U/mL, 210U/mL, 238U/mL, 235U/mL and 230U/mL respectively.
TABLE 5 results of fermentation without addition of alcohol for various periods of time
Example 6: the yield of monascus yellow pigment in different fermentation time is increased when ethanol is added
Seed liquid culture medium (g/L): 60 parts of corn starch, 4 parts of ammonium sulfate, 2 parts of sodium nitrate, 0.5 part of magnesium sulfate, 2 parts of monopotassium phosphate, 2 parts of dipotassium phosphate and 0.1 part of calcium chloride.
Fermentation medium (g/L): 60 parts of corn starch, 4 parts of ammonium sulfate, 2 parts of sodium nitrate, 0.5 part of magnesium sulfate, 2 parts of monopotassium phosphate, 2 parts of dipotassium phosphate and 0.1 part of calcium chloride.
(1) Preparation of seed liquid
The monascus purpureus sjs-6 is selected as a shake flask strain, activated and inoculated to a seed liquid culture medium, and shake flask fermentation culture is carried out for 2d under the conditions that the rotation speed is 180r/min and the temperature is 30 ℃.
(2) Fermentation culture
Inoculating the monascus liquid seeds in logarithmic growth phase into a 500mL triangular flask filled with 50mL fermentation medium by the inoculation amount of 10% by volume, adding 3% ethanol (v/v) of the volume of the culture system on the 0 th day under the conditions that the rotation speed is 180r/min and the temperature is 30 ℃, and measuring the color value every 24h after the color value. The results show that the color values of the monascus yellow pigment measured on days 0, 1, 2, 3, 4, 5, 6, 7 and 8 are respectively 2U/mL, 6U/mL, 13U/mL, 32U/mL, 143U/mL, 228U/mL, 319U/mL, 390U/mL and 415U/mL, and the bacterial autolysis phenomenon appears when the fermentation time is continuously prolonged.
TABLE 6 results of fermentation with ethanol addition for various times
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
Claims (8)
1. A method for promoting monascus fermentation to produce monascus yellow pigment is characterized in that alcohol substances are added at the beginning of fermentation or in the fermentation process of monascus; the alcohol substance is ethanol, isopropanol or a mixture thereof;
the fermentation is carried out in a liquid environment;
the addition amount of the alcohol substances is 3% -6% of the total volume of the fermentation system.
2. The method according to claim 1, wherein the alcohol is added at a timing of 0 to 96 hours after the start of the liquid fermentation culture.
3. A fermentation method of monascus yellow pigment is characterized in that monascus is used as a fermentation microorganism, alcohol substances accounting for 3% -6% of the total volume of a fermentation system are added at the beginning of fermentation or in the fermentation process, and fermentation is carried out for 4-8 days; the alcohol substance is ethanol, isopropanol or a mixture thereof.
4. The method of claim 3, wherein the Monascus bacteria is Monascus bacteria capable of producing Monascus yellow pigment, including but not limited to Monascus purpureus (Monascus purpureus) sjs-6, Monascus purpureus (Monascus purpureus) CICC 40225, Monascus purpureus (Monascus purpureus) CICC 40937, Monascus purpureus (Monascus purpureus) CICC 5042, Monascus ruber (Monascus ruber) CGMCC 3.4701, Monascus ruber (Monascus ruber) CGMCC 3.4652, Monascus ruber (Monascus ruber) CGMCC 3.465, and Monascus ruber (Monascus ruber) CGMCC 3.4649.
5. The method as claimed in claim 4, wherein the fermentation is carried out at a rotation speed of 150-250r/min and a temperature of 25-35 ℃.
6. The method according to any one of claims 3 to 5, wherein the method comprises inoculating monascus seed solution in a liquid fermentation medium for fermentation; adding alcohol substances within 0-96h after the liquid fermentation culture is started.
7. The method of claim 6, wherein the liquid fermentation medium comprises 20-80g/L of corn starch, 3-10g/L of ammonium sulfate, 2-6g/L of sodium nitrate, 0.5-1.5g/L of magnesium sulfate, 0.5-1.5g/L of potassium dihydrogen phosphate, 0.5-1.5g/L of dipotassium hydrogen phosphate, and 0.1-0.5g/L of calcium chloride.
8. Use of the method of any one of claims 1 to 7 for the preparation of monascus yellow pigment and food additives containing monascus yellow pigment.
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| CN113186236A (en) * | 2021-05-28 | 2021-07-30 | 汕头大学 | Method for increasing yield of monascus yellow pigment by adding particles into monascus fermentation broth |
| CN113403354B (en) * | 2021-06-07 | 2022-11-22 | 汕头大学 | Method for promoting monascus to produce yellow pigment through co-culture |
| CN114058648B (en) * | 2021-12-14 | 2023-11-21 | 广东天益生物科技有限公司 | Method for fermenting high-yield extracellular monascus red pigment by using monascus purpureus |
| CN114891841A (en) * | 2022-05-20 | 2022-08-12 | 南昌大学 | Method for increasing yield of yellow pigment in liquid fermentation of monascus and yam powder |
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