CN113122583A - Method for improving monascus pigment yield by co-culture of monascus and aspergillus oryzae - Google Patents
Method for improving monascus pigment yield by co-culture of monascus and aspergillus oryzae Download PDFInfo
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Abstract
The invention belongs to the field of microbial fermentation, and particularly relates to a method for improving monascus pigment yield by co-culturing monascus and aspergillus oryzae. The specific technical scheme is as follows: co-culturing Monascus with Aspergillus oryzae. The invention firstly prepares monascus pigment by co-culturing aspergillus oryzae and monascus. In co-culture, when the aspergillus oryzae mycelia and the monascus mycelia are contacted, the stimulation to the monascus mycelia is mild, and the activity of related genes for producing monascus pigment in monascus can be promoted to be more frequent, so that more monascus pigment is produced. The method provided by the invention effectively improves the yield of the monascus pigment, is simple to operate, has low production cost, and is very suitable for industrial production.
Description
Technical Field
The invention belongs to the field of microbial fermentation, and particularly relates to a method for improving monascus pigment yield by co-culturing monascus and aspergillus oryzae.
Background
Monascus pigment is a class of azaphilone compounds produced by monascus, which have bright colors, and can be classified into red, orange and yellow according to color. The monascus pigment is one of natural edible pigments which are allowed to be used in national standards and have higher domestic sales. The monascus pigment not only can be used as an edible pigment, but also has various physiological functions and medical potential.
At present, monascus fermentation is the only method for producing monascus pigment. However, the production efficiency of this method is not high enough. Therefore, the improvement of monascus pigment production by monascus fermentation through various methods becomes a research hotspot. The prior art provides various methods for improving the yield of monascus pigment produced by monascus, such as culture medium optimization, strain transformation, co-culture and the like. Among them, there are research findings: the co-culture of yeast and monascus is expected to improve the yield of monascus pigment. However, this method has two disadvantages: (1) the yeast is taken as unicellular microorganism and is difficult to physically contact with filamentous fungi monascus so as to limit the yield increasing effect; (2) the growth rate of yeast is greatly different from that of monascus, and industrial popularization and application are difficult to carry out.
Therefore, if a new method for improving monascus pigment production by monascus can be developed, the method has important research value and commercial significance.
Disclosure of Invention
The invention aims to provide a method for improving the yield of monascus pigment by co-culturing monascus and aspergillus oryzae.
In order to achieve the purpose of the invention, the technical scheme adopted by the invention is as follows: a method for producing monascus pigment in high yield comprises co-culturing monascus and aspergillus oryzae.
Preferably, the aspergillus oryzae and the monascus are mixed, inoculated and co-cultured according to the spore concentration ratio of 1: 1-1: 10.
Preferably, the aspergillus oryzae and the monascus are mixed, inoculated and co-cultured according to the spore concentration ratio of 1: 2.
Preferably, the co-cultivation conditions are: shaking culture at 26-30 deg.C at 0-250 rpm.
Preferably, the co-cultivation conditions are: shaking at 150rpm at 28 ℃.
Preferably, the spore concentration of the inoculated liquid of the microorganism in the co-culture is 1X 105one/mL.
Preferably, in the co-culture, the volume ratio of the microbial inoculation liquid to the total culture volume is 3: 100-10: 100.
Preferably, in the co-culture, the volume ratio of the microbial inoculation liquid to the total culture volume is 6: 100.
Preferably, the culture medium used for co-culture is PDB medium or GM medium.
Preferably, the method for extracting the monascus pigment comprises the following steps: the mycelium obtained by co-culture was soaked in methanol solution.
The invention has the beneficial effects that: the invention firstly prepares monascus pigment by co-culturing aspergillus oryzae and monascus. Aspergillus oryzae is a safe filamentous fungus that is edible and does not produce toxins or secondary metabolites. In co-culture, when the aspergillus oryzae mycelia and the monascus mycelia are contacted, the stimulation to the monascus mycelia is mild, and the activity of related genes for producing monascus pigment in monascus can be promoted to be more frequent, so that more monascus pigment is produced. The method effectively improves the yield of the monascus pigment, is simple to operate, has low production cost, and is very suitable for industrial production.
Detailed Description
The invention provides a method for improving monascus pigment yield by utilizing co-culture of monascus and aspergillus oryzae, which comprises the step of carrying out mixed inoculation co-culture on aspergillus oryzae and monascus according to a spore concentration ratio of 1: 1-1: 10, preferably 1: 2.
The method specifically comprises the following steps:
1. aspergillus oryzae was inoculated on PDA slant medium and expanded at 28 ℃ for 5 days. Washing Aspergillus oryzae mycelia with sterile water, collecting spore solution, adjusting spore concentration to 1 × 105one/mL.
2. Inoculating Monascus purpureus into PDA slant culture medium, and performing amplification culture at 30 deg.C for 10 days. Washing Monascus mycelium with sterile water, collecting spore solution, and adjusting spore concentration to 1 × 105one/mL.
3. Adding the aspergillus oryzae spore liquid and the monascus spore liquid into a fermentation liquid culture medium, and performing shake culture at the temperature of 26-30 ℃ and the rpm of 0-250 for 10 days. After the fermentation is finished, filtering to obtain mycelium, and discarding the filtrate. The fermentation temperature is preferably 28 ℃ and the shaker speed is preferably 150 rpm. The volume ratio of the spore liquid to the total culture volume (spore liquid + culture medium) is 3: 100-10: 100, and preferably 6: 100.
4. Soaking the mycelium with 80% (v/v) methanol solution at 40 deg.C for 1h, and extracting monascus pigment into the methanol solution. Filtering to remove mycelium to obtain methanol solution as monascus pigment solution.
Step 3 the fermentation medium includes but is not limited to: PDB culture medium (potato glucose culture medium) or GM culture medium (glucose 30g/L, ammonium sulfate 10g/L, ferrous sulfate 0.1g/L, calcium chloride 0.1 g/L).
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Examples
1. And (4) preparing a seed solution. Inoculating Monascus purpureus or Aspergillus oryzae mycelia on the slant to a fresh PDA slant culture, culturing in a test tube with slant at 28 deg.C for 10 days, and culturing Aspergillus oryzae for 5 days. Then, respectively washing and diluting monascus and aspergillus oryzae by using sterile water, wherein the specific dilution method comprises the following steps: 5mL of distilled water previously sterilized and cooled was added to a test tube containing Monascus purpureus or Aspergillus oryzae, and the mycelia were repeatedly washed with sterile water with gentle shaking to dissolve the spores in the water. The spore solution was collected under sterile conditions and the spore concentration was calculated under a microscope using a hemocytometer. Diluting Aspergillus oryzae spore liquid and Monascus ruber spore liquid to 1 × 10 with fresh sterile water5one/mL.
2. And (3) co-culturing monascus and aspergillus oryzae. PDB medium was grown and sterilized, and 47mL of fresh PDB medium was added to a pre-sterilized 250mL triangular shake flask. Measuring Aspergillus oryzae spore liquid and Monascus purpureus spore liquid, adding into the prepared PDB culture medium, and mixing. Setting 4 treatments, wherein the volume ratio of the aspergillus oryzae spore liquid to the monascus spore liquid is respectively as follows: 1:1 (treatment group 1), 1:2 (treatment group 2), 1:5 (treatment group 3), and 1:10 (treatment group 4), and rice was added to each treatmentThe sum of the volume of the aspergillus spore liquid and the monascus spore liquid is 3 mL. 3mL of Monascus purpureus spore liquid (without Aspergillus oryzae) was cultured as a blank control. Simultaneously taking 1mL of penicillium spore liquid (the spore concentration is 1 multiplied by 10)5one/mL) and 2mL of Monascus purpureus spore solution were mixed and cultured to obtain a positive control. When each group was cultured at 28 ℃ for 10 days with shaking at 150rpm, it was observed that Aspergillus oryzae and Aspergillus kawachii form a complex mycelium pellet and the color of the medium changed from light yellow to deep red.
3. And (4) extracting the monascus pigment. After the fermentation is finished, filtering with nylon filter cloth to obtain each group of mycelium, and discarding the filtrate to obtain wet mycelium. Preparing a methanol solution with the volume fraction of 80%, mixing the methanol solution with the volume 5 times of that of each group of wet mycelia with each group of wet mycelia respectively, and then slightly stirring the mycelium pellets by using a glass rod to loosen the mycelia, so as to be beneficial to the extraction of monascus pigment. The mixture was kept at 40 ℃ for 1 hour, and the intracellular monascus pigment was substantially extracted into the extracellular methanol solution, to obtain monascus pigment solutions prepared for each group.
4. And (5) measuring the color value of the monascus pigment. The absorbance of each group of methanol solutions containing monascus pigment was measured at 370nm, 410nm and 510nm, respectively, and is called color number. In the measurement results, 1U was calculated as an absorbance equal to 1.0. The total color value of each group of monascus pigment is equal to the sum of the color values of the 3 parts.
Through calculation, the monascus pigment yield of the treatment group 1 is 15.4U/mL, the monascus pigment yield of the treatment group 2 is 17.6U/mL, the monascus pigment yield of the treatment group 3 is 14.2U/mL, the monascus pigment yield of the treatment group 4 is 12.3U/mL, the monascus pigment yield of the blank control group is 11.2U/mL, and the monascus pigment yield of the positive control group is 5.2U/mL. Compared with the blank control group, the yield of the monascus pigment is improved by 57.1 percent.
The above-described embodiments are merely illustrative of the preferred embodiments of the present invention, and do not limit the scope of the present invention, and various changes, modifications, alterations, and substitutions which may be made by those skilled in the art without departing from the spirit of the present invention shall fall within the protection scope defined by the claims of the present invention.
Claims (10)
1. A method for producing monascus pigment with high yield is characterized by comprising the following steps: and (3) co-culturing monascus and aspergillus oryzae.
2. The method for producing monascus pigment in high yield according to claim 1, wherein the monascus pigment is selected from the group consisting of: the Aspergillus oryzae and the Monascus purpureus are mixed according to the spore concentration ratio of 1: 1-1: 10, inoculated and co-cultured.
3. The method for producing monascus pigment in high yield according to claim 1, wherein the monascus pigment is selected from the group consisting of: the aspergillus oryzae and the monascus are mixed, inoculated and co-cultured according to the spore concentration ratio of 1: 2.
4. The method for producing monascus pigment in high yield according to claim 1, wherein the monascus pigment is selected from the group consisting of: the co-culture conditions are as follows: shaking culture at 26-30 deg.C at 0-250 rpm.
5. The method for producing monascus pigment in high yield according to claim 4, wherein the monascus pigment is selected from the group consisting of: the co-culture conditions are as follows: shaking at 150rpm at 28 ℃.
6. The method for producing monascus pigment in high yield according to claim 1, wherein the monascus pigment is selected from the group consisting of: in the co-culture, the spore concentration of the microbial seed solution is 1 × 105one/mL.
7. The method for producing monascus pigment in high yield according to claim 6, wherein the monascus pigment is selected from the group consisting of: in the co-culture, the volume ratio of the microbial inoculation liquid to the total culture volume is 3: 100-10: 100.
8. The method for producing monascus pigment in high yield according to claim 7, wherein: in the co-culture, the volume ratio of the microbial inoculation liquid to the total culture volume is 6: 100.
9. The method for producing monascus pigment in high yield according to claim 1, wherein the monascus pigment is selected from the group consisting of: the culture medium used for co-culture is PDB culture medium or GM culture medium.
10. The method for producing monascus pigment in high yield according to claim 1, wherein the monascus pigment is selected from the group consisting of: the method for extracting the monascus pigment comprises the following steps: the mycelium obtained by co-culture was soaked in methanol solution.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116063420A (en) * | 2022-08-25 | 2023-05-05 | 华中农业大学 | Transcription factor MrMrl3 mutant and application thereof |
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| CN107574173A (en) * | 2017-11-02 | 2018-01-12 | 江西科技师范大学 | A kind of recombinant plasmid and its method for building High-productive Monascus Pigment Strain |
| CN108034667A (en) * | 2018-01-15 | 2018-05-15 | 江西科技师范大学 | A kind of red monascus alpha-amylase gene, its preparation method and application |
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| CN107574173A (en) * | 2017-11-02 | 2018-01-12 | 江西科技师范大学 | A kind of recombinant plasmid and its method for building High-productive Monascus Pigment Strain |
| CN108034667A (en) * | 2018-01-15 | 2018-05-15 | 江西科技师范大学 | A kind of red monascus alpha-amylase gene, its preparation method and application |
Non-Patent Citations (4)
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| CHUL S. SHIN等: "Morphological Change and Enhanced Pigment Production of Monascus When Cocultured with Saccharomyces cerevisiae or Aspergillus oryzae", 《BIOTECHNOLOGY AND BIOENGINEERING》 * |
| 张建玲: "混菌发酵对红曲霉产色素及桔霉素的影响", 《中国优秀硕士学位论文全文数据库（电子期刊网）》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116063420A (en) * | 2022-08-25 | 2023-05-05 | 华中农业大学 | Transcription factor MrMrl3 mutant and application thereof |
| CN116063420B (en) * | 2022-08-25 | 2024-01-26 | 华中农业大学 | Transcription factor MrMrl3 mutant and application thereof |
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