CN107723320B - Method for directly preparing crystal monascus orange pigment - Google Patents
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- CN107723320B CN107723320B CN201710808062.9A CN201710808062A CN107723320B CN 107723320 B CN107723320 B CN 107723320B CN 201710808062 A CN201710808062 A CN 201710808062A CN 107723320 B CN107723320 B CN 107723320B
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Abstract
The invention provides a method for directly producing crystal monascus orange pigment, which comprises the following steps: 1, culturing monascus in a seed culture medium to obtain a seed solution; inoculating the seed liquid into a fermentation culture solution for fermentation culture, filtering the fermentation liquid membrane and collecting monascus purpureus mycelia; 3, taking the fermentation culture solution with the same components as the components in the step 2 as a cell suspension culture solution, adjusting the pH value of the cell suspension culture solution, and inoculating monascus mycelium into the cell suspension culture solution to perform cell suspension culture of monascus aurantiol; and 4, filtering the cell suspension culture solution obtained in the step 3 to separate monascus mycelium and fermentation liquor, standing to obtain orange-yellow precipitate, and filtering by using a cellulose membrane to obtain the crystal monascus orange pigment. The method directly produces the extracellular crystal monascus orange pigment, realizes the production of the high-purity monascus pigment, provides important guarantee for improving the quality of the food monascus pigment, and has the advantages of simple method, easy realization, high yield, low cost and strong repeatability.
Description
Technical Field
The invention relates to a fermentation method in the technical field of bioengineering, in particular to a method for directly producing crystal monascus orange pigment.
Background
Red yeast rice produced by Monascus sp solid state fermentation is a traditional food pigment additive in China and is also a traditional Chinese medicine, and the usage history of the red yeast rice in China is more than one thousand years. The monascus red pigment is produced by modern microorganism liquid submerged fermentation technology at present. Typically, monascus pigments produced by monascus fermentation include two monascus yellow pigment components, two monascus orange pigment components, two monascus red pigment components, and derivatives of various amine compounds of monascus red pigment. Commercial monascus color is typically a mixture of the various color components described above.
In order to meet the market demand for single-component natural monascus red pigment, the development of monascus pigment fermentation technology with single component is urgently needed. The monascus pigment with single preparation component is not only an important technical index for regulating and controlling the color of the monascus pigment, but also an important quality index for improving the safety of the monascus pigment food additive. In the fermentation process, the microbial strains are modified by using modern biotechnology means to control the fraction of the monascus pigment, and the method is theoretically feasible, but the fermentation products obtained by modifying the microorganisms through genetic engineering belong to genetically engineered foods. Because of the special property of food safety, it is very tedious and difficult to obtain the production and sale permission rights of the genetically engineered food in many countries. The fermentation engineering technology is adopted to regulate and control the secondary metabolism of the monascus purpureus to become an important means.
Monascus orange pigment and monascus yellow pigment are generally considered cell-bound products, including monascus pigment bound to cell membranes and intracellular monascus pigment. Organic solvents are often used to extract the active components of monascus pigment from the mycelium. It was found through a search of the prior art documents that Kolia SH, Suryawanshi RK, Pailaac CD, Patil SV. Fluconazolete enzyme of red pigments in the fungi Monascus purpureus FEMS Microbiol Lett 2017,364: doi:10.1093/femsle/fnx058(Kolia SH, Suryawansia RK, Patilac CD, Patil SV. Fluconazole treatment enhanced the extracellular release of the red pigments of the mold monascus purpureus, described in European Union of microorganisms Committee microorganisms (Conn.), 2017,364: doi:10.1093/femsle/fnx 058): monascus pigment exists mainly in the form of being bound to cell membranes or inside cells; the antibiotic fluconazole can change the structure of a cell membrane, so that the release of the monascus pigment in cells is accelerated; the release of intracellular monascus pigment enhances the accumulation of monascus pigment. In contrast, no relevant reports have been found on the direct accumulation of extracellular monascus orange pigment during microbial fermentation of monascus pigment.
In further search, no relevant report on the direct acquisition of crystal monascus orange pigment during microbial fermentation of monascus pigment was found.
Disclosure of Invention
Aiming at the difficulty of producing monascus pigment with single component by the existing microbial fermentation, the invention aims to provide a method for directly producing crystal monascus orange pigment.
According to the first aspect of the invention, the method for directly producing the crystal monascus orange pigment is provided, the method is to directly obtain the crystal monascus orange pigment by applying a cell suspension culture technology, and the method comprises the following steps:
step 1, culturing monascus in a seed culture medium to obtain a seed solution;
step 3, taking the fermentation culture solution with the same components as the components in the step 2 as a cell suspension culture solution, adjusting the pH value of the cell suspension culture solution, inoculating the obtained monascus purpureus mycelia into the cell suspension culture solution, and carrying out cell suspension culture on monascus aurantium;
and 4, filtering the cell suspension culture solution obtained in the step 3 to separate monascus mycelium and fermentation liquor, standing to obtain orange-yellow precipitate, and filtering by using a cellulose membrane to obtain the crystal monascus orange pigment.
Preferably, in step 3: inoculating mycelium into fermentation culture solution with initial pH value of 2-3 and corn flour as carbon source and nitrogen source at stem cell concentration of 8-15 g/l, and performing cell suspension culture in shaker at 30 deg.C and 200rpm for 3-7 days. Preferably, the culture time is 3-4 days, so that the cell density can be increased and the biological reaction time can be shortened.
Preferably, in step 4: filtering the cell suspension culture solution with ceramic membrane with pore diameter of 20-30 μm to separate mycelium and fermentation liquid, standing the cell suspension culture solution directly to obtain orange yellow precipitate, and filtering with cellulose membrane with pore diameter of 5 μm to obtain crystal monascus orange pigment.
Preferably, in step 1: culturing the monascus purpureus in a seed culture medium for 32 hours at 30 ℃ and 300rpm to obtain a seed solution, wherein the seed culture medium is: 100ml of water, 2g of peptone, 2g of yeast powder and 2g of glucose.
Preferably, in the step 2, the separation of monascus mycelium and monascus orange pigment is realized by using a ceramic membrane filtered by a macroporous membrane, wherein the macroporous membrane is used for filtering, and the aperture is 20-30 μm.
According to a second aspect of the present invention there is provided a further method of directly producing crystalline monascus orange pigment by directly obtaining crystalline monascus orange pigment during fermentation comprising the steps of:
step 1, culturing monascus in a seed culture medium to obtain a seed solution;
step 3, filtering the fermented liquid membrane to remove monascus mycelium, and directly obtaining extracellular monascus orange pigment crystals, wherein the crystal monascus orange pigment exists in the filtrate;
and 4, precipitating the filtrate to obtain the crystal monascus orange pigment.
Preferably, in the step 2, the pH needs to be controlled within the range of 2-5, the main pigment component generated by microbial fermentation is monascus orange pigment, and the monascus orange pigment can be secreted to the extracellular environment and directly exists in the extracellular fermentation liquid in a crystal form, so that the operations of separation and the like in the subsequent steps are facilitated.
Preferably, in the step 3, the separation of the thalli and the monascus orange pigment is realized by using a ceramic membrane filtered by a macroporous membrane.
More preferably, the macroporous membrane filters with a pore size of 20-30 μm.
Preferably, the step 4 further comprises the purification of the crystal monascus orange pigment, wherein the purification refers to: washing the crystal monascus orange pigment with water, removing water-soluble impurities on the surface of the crystal monascus orange pigment, dissolving the crystal monascus orange pigment with ethanol, and adding water for recrystallization to obtain the high-purity crystal monascus orange pigment.
Preferably, in step 1: culturing the monascus purpureus in a seed culture medium for 32 hours at 30 ℃ and 300rpm to obtain a seed solution, wherein the seed culture medium is: 100ml of water, 2g of peptone, 2g of yeast powder and 2g of glucose.
Preferably, in step 2: inoculating the seed solution into a fermentation culture solution with an initial pH value of 2-3 and corn flour as a carbon source and a nitrogen source, and performing fermentation culture in a shaking table at the temperature of 30 ℃ and the speed of 200rpm for 4-7 days.
Compared with the prior art, the invention has the following beneficial effects:
the invention adopts the steps to directly obtain the extracellular monascus orange pigment, namely the monascus orange pigment can be secreted to the extracellular environment through fermentation or cell suspension culture and directly exists in the fermentation liquor in the form of crystals. In the microbial fermentation process, monascus orange pigment exists in the fermentation medium in the form of crystals, which is beneficial to the separation of thalli and monascus orange pigment and is also beneficial to the separation of monascus orange pigment and other water-soluble impurities in the fermentation medium.
The method for directly producing the extracellular monascus orange pigment realizes the production of the high-purity monascus pigment, provides important guarantee for improving the quality of the food monascus pigment, and has the advantages of simplicity, easiness in implementation, high yield, low cost, strong repeatability and the like.
Drawings
Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:
FIGS. 1a and 1b are photomicrographs of a fermentation flask and a fermentation liquid in a submerged liquid fermentation experiment according to an embodiment of the present invention;
FIGS. 2a and 2b are crystal structures of monascus orange pigment in one embodiment of the present invention;
FIG. 3 and FIG. 4 are the structure identification spectrum and mass spectrogram of monascus orange pigment in one embodiment of the present invention.
Detailed Description
The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.
Referring to the figure, the production of the crystalline monascus orange pigment by microbial fermentation according to the present invention is further illustrated by the following examples.
Example 1
The embodiment relates to a method for producing extracellular monascus orange pigment by direct fermentation, which comprises the following steps:
1. seed culture of microorganisms
Culturing Monascus purpureus provided by Monascus anka, CICC 5013 in seed culture medium (water 100ml, peptone 2g, yeast powder 2g, glucose 2g) at 30 deg.C and 300rpm for 32 hr to obtain seed solution
2. Submerged liquid fermentation
Inoculating the seed solution into a fermentation culture solution with an initial pH value of 2.5 and corn flour as a carbon source and a nitrogen source to serve as a fermentation liquid, and performing fermentation culture in a shaking table at the temperature of 30 ℃ and the speed of 200rpm for 7 days. The broth was observed to be orange yellow and there were many solid particles in the neck of the flask. Further microscopic observation shows that there are many colored lumps on the surface of the mycelium and colored particles in the fermentation broth, as shown in fig. 1a and 1b, which are micrographs (x 400) of the fermentation shake flask and the fermentation broth in the submerged liquid fermentation experiment.
3. Separation of fermentation liquor crystal monascus orange pigment
And (3) filtering the fermentation liquor obtained in the step (2) by using a ceramic membrane with the aperture of 20-30 mu m to separate mycelium and the fermentation liquor. The mycelium residue was washed with water 3 times and the combined washings were allowed to stand with the fermentation broth for 3 hours to give an orange yellow precipitate. Filtering with cellulose membrane with pore diameter of 5 μm to obtain solid pigment, i.e. crystal monascus orange pigment. Further dissolving the precipitate with ethanol, filtering to remove insoluble impurities, evaporating ethanol, and adding water for crystallization to obtain orange yellow crystal monascus orange pigment, as shown in fig. 2a and 2b, wherein fig. 2a is a filter membrane diagram after directly precipitating and filtering the filtrate; FIG. 2b is a micrograph (. times.200) of the precipitate recrystallisation.
In the steps in the embodiment, monascus orange pigment is precipitated in a crystal form in the fermentation process, namely extracellular monascus orange pigment is directly produced, so that the production of high-purity monascus orange pigment is realized, and the method is simple, easy to implement, high in yield and low in cost.
Example 2
The embodiment relates to a method for producing extracellular monascus orange pigment by direct fermentation, which comprises the following steps:
1. seed culture of microorganisms
Culturing Monascus purpureus provided by Monascus anka, CICC 5013 in seed culture medium (water 100ml, peptone 2g, yeast powder 2g, glucose 2g) at 30 deg.C and 300rpm for 32 hr to obtain seed solution
2. Submerged liquid fermentation
Inoculating the seed solution into a fermentation culture solution with an initial pH value of 2.5 and corn flour as a carbon source and a nitrogen source, and performing fermentation culture in a shaking table with the temperature of 30 ℃ and the rpm of 200 for 7 days. The broth was observed to be orange yellow and there were many solid particles in the neck of the flask. Further microscopic observation shows that the mycelium surface has a plurality of colored lumps and colored particles exist in the fermentation broth, and fig. 1a and 1b show micrographs (x 400) of the fermentation shake flask and the fermentation broth in a submerged liquid fermentation experiment.
3. Cell suspension culture
And (3) filtering the fermentation liquor obtained in the step (2) by using a ceramic membrane with the aperture of 20-30 mu m to separate mycelium and the fermentation liquor. Taking the same fermentation culture solution as the above components as cell suspension culture solution, adjusting pH to 2.5, adding mycelium into the fermentation culture solution at dry thallus concentration of 10g/l, and performing cell suspension culture in shaker at 30 deg.C and 200rpm for 4 days. It was also observed that the broth was orange yellow and there were many solid particles in the neck of the flask.
4. Separation of fermentation liquor crystal monascus orange pigment
And (3) filtering the cell suspension culture solution in the step (3) by using a ceramic membrane with the pore diameter of 20-30 mu m to separate mycelium, culture medium residues and fermentation liquor. The mycelium and the culture medium residue were washed with water for 3 times, and the washing solution and the fermentation broth were combined and directly left to stand for 3 hours to obtain an orange-yellow precipitate. Filtering with cellulose membrane with pore diameter of 5 μm to obtain solid pigment, i.e. crystal monascus orange pigment. Further dissolving the precipitate with ethanol, filtering to remove insoluble impurities, evaporating ethanol, and adding water for crystallization to obtain orange yellow crystals, as shown in FIGS. 2a and 2b, wherein FIG. 2a is a filter membrane diagram of the filtrate directly precipitated and filtered; FIG. 2b is a micrograph (. times.200) of the precipitate recrystallisation.
Example 3
The embodiment relates to a method for producing extracellular monascus orange pigment through direct fermentation, which comprises the following steps:
step 1, culturing Monascus purpureus in a seed culture medium (100 ml of water, 2g of peptone, 2g of yeast powder and 2g of glucose) at 30 ℃ and 300rpm for 32 hours to obtain a seed solution, wherein the Monascus purpureus is provided by the China center for industrial microorganism strain preservation (Monascus anka, CICC 5013);
and 3, filtering and separating the mycelium and the fermentation liquor by using a ceramic membrane with the aperture of 20-30 mu m. The fermentation broth was directly left to stand for 3 hours to obtain an orange-yellow precipitate. Filtering with cellulose membrane with pore diameter of 5 μm to obtain solid pigment, i.e. crystal monascus orange pigment. Further using ethanol to dissolve and analyze to obtain the monascus orange pigment with the concentration of 1.2g/l in the fermentation liquor.
Example 4
The embodiment relates to a method for producing extracellular monascus orange pigment through direct fermentation, which comprises the following steps:
step 1, culturing Monascus purpureus in a seed culture medium (100 ml of water, 2g of peptone, 2g of yeast powder and 2g of glucose) at 30 ℃ and 300rpm for 32 hours to obtain a seed solution, wherein the Monascus purpureus is provided by the China center for industrial microorganism strain preservation (Monascus anka, CICC 5013);
and 3, filtering and separating the mycelium and the fermentation liquor by using a ceramic membrane with the aperture of 20-30 mu m. The fermentation broth was directly left to stand for 3 hours to obtain an orange-yellow precipitate. Filtering with cellulose membrane with pore diameter of 5 μm to obtain solid pigment, i.e. crystal monascus orange pigment. Further using ethanol to dissolve and analyze to obtain the monascus orange pigment with the concentration of 0.9g/l in the fermentation liquor.
Example 5
The embodiment relates to a method for producing extracellular monascus orange pigment through direct fermentation, which comprises the following steps:
step 1, culturing Monascus purpureus in a seed culture medium (100 ml of water, 2g of peptone, 2g of yeast powder and 2g of glucose) at 30 ℃ and 300rpm for 32 hours to obtain a seed solution, wherein the Monascus purpureus is provided by the China center for industrial microorganism strain preservation (Monascus anka, CICC 5013);
and 3, filtering and separating the mycelium and the fermentation liquor by using a ceramic membrane with the aperture of 20-30 mu m. The fermentation broth was directly left to stand for 3 hours to obtain an orange-yellow precipitate. Filtering with cellulose membrane with pore diameter of 5 μm to obtain solid pigment, i.e. crystal monascus orange pigment. Further using ethanol to dissolve and analyze to obtain the monascus orange pigment with the concentration of 1.4g/l in the fermentation liquor.
Example 6
The embodiment relates to a method for producing extracellular monascus orange pigment by applying a cell suspension culture technology, which comprises the following steps:
step 1, culturing Monascus purpureus in a seed culture medium (100 ml of water, 2g of peptone, 2g of yeast powder and 2g of glucose) at 30 ℃ and 300rpm for 32 hours to obtain a seed solution, wherein the Monascus purpureus is provided by the China center for industrial microorganism strain preservation (Monascus anka, CICC 5013);
and 2, inoculating the seed solution into a fermentation culture solution with an initial pH value of 3 and corn flour as a carbon source and a nitrogen source, and performing fermentation culture in a shaking table at the temperature of 30 ℃ and the speed of 200rpm for 5 days. Filtering the fermentation liquid with ceramic membrane with pore diameter of 20-30 μm to separate mycelium and fermentation liquid.
And 3, taking the fermentation culture solution with the same components as the components as a cell suspension culture solution, adjusting the pH value to 2.7, inoculating the separated mycelium into the cell suspension culture solution at the stem cell concentration of 8g/l, and carrying out cell suspension culture in a shaking table at the temperature of 30 ℃ and the speed of 200rpm for 4 days.
And 4, filtering the cell suspension culture solution by using a ceramic membrane with the pore diameter of 20-30 mu m to separate mycelium and fermentation liquor. The culture was directly left to stand for 3 hours to obtain an orange-yellow precipitate. Filtering with cellulose membrane with pore diameter of 5 μm to obtain solid pigment, i.e. crystal monascus orange pigment. Further using ethanol to dissolve and analyze to obtain the monascus orange pigment with the concentration of 1.5g/l in the fermentation liquor.
Example 7
The embodiment relates to a method for producing extracellular monascus orange pigment by applying a cell suspension culture technology, which comprises the following steps:
step 1, culturing Monascus purpureus in a seed culture medium (100 ml of water, 2g of peptone, 2g of yeast powder and 2g of glucose) at 30 ℃ and 300rpm for 32 hours to obtain a seed solution, wherein the Monascus purpureus is provided by the China center for industrial microorganism strain preservation (Monascus anka, CICC 5013);
and 2, inoculating the seed solution into a fermentation culture solution with an initial pH value of 4.5 and using corn flour as a carbon source and a nitrogen source, and performing fermentation culture for 4 days in a shaking table with the temperature of 30 ℃ and the rpm of 200. Filtering the fermentation liquid with ceramic membrane with pore diameter of 20-30 μm to separate mycelium and fermentation liquid.
And 3, taking the fermentation culture solution with the same components as the components as a cell suspension culture solution, adjusting the pH value to 2, inoculating the separated mycelium into the cell suspension culture solution at the stem cell concentration of 10g/l, and carrying out cell suspension culture in a shaking table at the temperature of 30 ℃ and the speed of 200rpm for 4 days.
And 4, filtering the cell suspension culture solution by using a ceramic membrane with the pore diameter of 20-30 mu m to separate mycelium and fermentation liquor. The culture was directly left to stand for 3 hours to obtain an orange-yellow precipitate. Filtering with cellulose membrane with pore diameter of 5 μm to obtain solid pigment, i.e. crystal monascus orange pigment. Further using ethanol to dissolve and analyze to obtain the monascus orange pigment with the concentration of 1.3g/l in the fermentation liquor.
Example 8
The embodiment relates to a method for producing extracellular monascus orange pigment by applying a cell suspension culture technology, which comprises the following steps:
step 1, culturing Monascus purpureus in a seed culture medium (100 ml of water, 2g of peptone, 2g of yeast powder and 2g of glucose) at 30 ℃ and 300rpm for 32 hours to obtain a seed solution, wherein the Monascus purpureus is provided by the China center for industrial microorganism strain preservation (Monascus anka, CICC 5013);
and 2, inoculating the seed solution into a fermentation culture solution with an initial pH value of 5 and corn flour as a carbon source and a nitrogen source, and performing fermentation culture in a shaking table with the temperature of 30 ℃ and the rpm of 200 for 4.5 days. Filtering the fermentation liquid with ceramic membrane with pore diameter of 20-30 μm to separate mycelium and fermentation liquid.
And 3, taking the fermentation culture solution with the same components as the components as a cell suspension culture solution, adjusting the pH value to 3, inoculating the separated mycelium into the cell suspension culture solution at the stem cell concentration of 9g/l, and carrying out cell suspension culture in a shaking table at the temperature of 30 ℃ and the speed of 200rpm for 4 days.
And 4, filtering the cell suspension culture solution by using a ceramic membrane with the pore diameter of 20-30 mu m to separate mycelium and fermentation liquor. The culture was directly left to stand for 3 hours to obtain an orange-yellow precipitate. Filtering with cellulose membrane with pore diameter of 5 μm to obtain solid pigment, i.e. crystal monascus orange pigment. Further using ethanol to dissolve and analyze to obtain the monascus orange pigment with the concentration of 1.3g/l in the fermentation liquor.
Example 9
The embodiment relates to a method for producing extracellular monascus orange pigment by applying a cell suspension culture technology, which comprises the following steps:
step 1, culturing Monascus purpureus in a seed culture medium (100 ml of water, 2g of peptone, 2g of yeast powder and 2g of glucose) at 30 ℃ and 300rpm for 32 hours to obtain a seed solution, wherein the Monascus purpureus is provided by the China center for industrial microorganism strain preservation (Monascus anka, CICC 5013);
and 2, inoculating the seed solution into a fermentation culture solution with an initial pH value of 4.5 and using corn flour as a carbon source and a nitrogen source, and performing fermentation culture for 4 days in a shaking table with the temperature of 30 ℃ and the rpm of 200. Filtering the fermentation liquid with ceramic membrane with pore diameter of 20-30 μm to separate mycelium and fermentation liquid.
And 3, taking the fermentation culture solution with the same components as the components as a cell suspension culture solution, adjusting the pH value to 3, inoculating the separated mycelium into the cell suspension culture solution at the stem cell concentration of 15g/l, and carrying out cell suspension culture in a shaking table at the temperature of 30 ℃ and the speed of 200rpm for 3 days.
And 4, filtering the cell suspension culture solution by using a ceramic membrane with the pore diameter of 20-30 mu m to separate mycelium and fermentation liquor. The culture was directly left to stand for 3 hours to obtain an orange-yellow precipitate. Filtering with cellulose membrane with pore diameter of 5 μm to obtain solid pigment, i.e. crystal monascus orange pigment. Further using ethanol to dissolve and analyze to obtain the monascus orange pigment with the concentration of 1.4g/l in the fermentation liquor.
Practice ofExample 10
The embodiment relates to a method for producing extracellular monascus orange pigment through direct fermentation, which comprises the following steps:
step 1, culturing Monascus purpureus in a seed culture medium (100 ml of water, 2g of peptone, 2g of yeast powder and 2g of glucose) at 30 ℃ and 300rpm for 32 hours to obtain a seed solution, wherein the Monascus purpureus is provided by the China center for industrial microorganism strain preservation (Monascus anka, CICC 5013);
and 3, filtering and separating the mycelium and the fermentation liquor by using a ceramic membrane with the aperture of 20-30 mu m. The fermentation broth was directly left to stand for 3 hours to obtain an orange-yellow precipitate. Filtering with cellulose membrane with pore diameter of 5 μm to obtain solid pigment, i.e. crystal monascus orange pigment. The precipitate was further dissolved in ethanol and centrifuged to remove insoluble material. Evaporating the solvent from the supernatant until the volume of the solvent is one fourth of the original volume, and adding water to the original volume. Placed in a 4 degree refrigerator overnight. Orange yellow needle crystals are finally obtained.
In the above examples, the structural identification of high purity crystal monascus orange pigment: dissolving the crystal pigment in ethanol solution, and further performing mass spectrometry to obtain a spectrogram and a mass spectrum of the pigment, as shown in FIGS. 3 and 4. In the figure, the monascus orange pigment has the maximum absorption peak at 468nm, and meanwhile, the mass spectrogram of a positive ion mode shows that the molecular weight is 354 daltons and is consistent with the molecular weight of the monascus orange pigment.
While the present invention has been described in detail with reference to the preferred embodiments, it should be understood that the above description should not be taken as limiting the invention. Various modifications and alterations to this invention will become apparent to those skilled in the art upon reading the foregoing description. Accordingly, the scope of the invention should be determined from the following claims.
Claims (6)
1. A method for directly producing crystal monascus orange pigment is characterized in that: the method directly obtains the crystal monascus orange pigment by applying a cell suspension culture technology, and comprises the following steps:
step 1, culturing monascus in a seed culture medium to obtain a seed solution;
culturing the monascus purpureus in a seed culture medium for 32 hours at 30 ℃ and 300rpm to obtain a seed solution, wherein the seed culture medium is as follows: 100ml of water, 2g of peptone, 2g of yeast powder and 2g of glucose;
step 2, inoculating the seed liquid into a fermentation culture solution for fermentation culture, and filtering a fermentation liquid membrane to collect monascus purpureus mycelia; the initial pH value is 3-5;
step 3, taking the fermentation culture solution with the same components as the components in the step 2 as a cell suspension culture solution, adjusting the pH value of the cell suspension culture solution, inoculating the obtained monascus purpureus mycelia into the cell suspension culture solution, and carrying out cell suspension culture on monascus aurantium;
in the step 3: inoculating mycelium into fermentation culture solution with initial pH value of 2-3 and corn flour as carbon source and nitrogen source at stem cell concentration of 8-15 g/l, and performing cell suspension culture in shaker at 30 deg.C and 200rpm for 3-7 days;
and 4, filtering the cell suspension culture solution obtained in the step 3 to separate monascus mycelium and fermentation liquor, standing to obtain orange-yellow precipitate, and filtering by using a cellulose membrane to obtain the crystal monascus orange pigment.
2. The method for directly producing crystalline monascus orange pigment according to claim 1, wherein in step 4: filtering the cell suspension culture solution with ceramic membrane with pore diameter of 20-30 μm to separate mycelium and fermentation liquid, standing the cell suspension culture solution directly to obtain orange yellow precipitate, and filtering with cellulose membrane with pore diameter of 5 μm to obtain crystal monascus orange pigment.
3. The method for directly producing crystalline monascus orange pigment according to any one of claims 1-2, wherein in step 2, the separation of monascus mycelium and monascus orange pigment is achieved by using a ceramic membrane for macroporous membrane filtration, wherein the macroporous membrane filtration has a pore size of 20-30 μm.
4. A method for directly producing crystal monascus orange pigment is characterized in that: the method directly obtains the crystal monascus orange pigment in the fermentation process, and comprises the following steps:
step 1, culturing monascus in a seed culture medium to obtain a seed solution;
culturing the monascus purpureus in a seed culture medium for 32 hours at 30 ℃ and 300rpm to obtain a seed solution, wherein the seed culture medium is as follows: 100ml of water, 2g of peptone, 2g of yeast powder and 2g of glucose;
step 2, inoculating the seed liquid into a fermentation culture solution containing a carbon source and a nitrogen source, adjusting the pH value of the fermentation culture solution, and performing fermentation culture;
in the step 2: inoculating the seed solution into a fermentation culture solution with an initial pH value of 2-3 and corn flour as a carbon source and a nitrogen source, and performing fermentation culture in a shaking table at the temperature of 30 ℃ and the speed of 200rpm for 7 days; the pH value is controlled within the range of 2-5 in the whole process of fermentation culture;
step 3, filtering the fermentation culture liquid membrane in the step 2 to remove monascus mycelium, and directly obtaining extracellular monascus orange pigment crystals, wherein the crystal monascus orange pigment exists in the filtrate;
and 4, precipitating the filtrate obtained in the step 3 to obtain crystal monascus orange pigment.
5. The method for directly producing crystalline monascus orange pigment according to claim 4, wherein in step 3, the separation of monascus mycelium and monascus orange pigment is achieved by using a ceramic membrane for macroporous membrane filtration, wherein the macroporous membrane filtration is performed, and the pore size is 20-30 μm.
6. The method for directly producing crystalline monascus orange pigment according to claim 4, wherein said step 4 further comprises the purification of said crystalline monascus orange pigment, said purification being: washing the crystal monascus orange pigment with water, removing water-soluble impurities on the surface of the crystal monascus orange pigment, dissolving the crystal monascus orange pigment with ethanol, and adding water for recrystallization to obtain the high-purity crystal monascus orange pigment.
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