CN110607333A - Method for producing lipid-lowering component Monacolin K by using pomelo byproducts through monascus - Google Patents
Method for producing lipid-lowering component Monacolin K by using pomelo byproducts through monascus Download PDFInfo
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Abstract
The invention discloses a method for producing a lipid-lowering component Monacolin K by monascus through grapefruit by-products, belonging to the technical field of microbial fermentation. The Monacolin K is produced by fermenting the pomelo seed sacs and/or the pomelo peels in a solid state by using monascus, and the yield of the Monacolin K is up to 7.413 mg/g. The pomelo fruit sacs and/or the pomelo peel are/is used as main waste in the pomelo processing process, are edible and harmless, are applied to red yeast solid state fermentation to produce a fat-reducing component Monacolin K, can reduce the production cost of red yeast products, meet the requirements of environmental protection and sustainable development, are the embodiment of turning waste into wealth, and have great research significance and economic value.
Description
Technical Field
The invention relates to a method for producing a lipid-lowering component Monacolin K by monascus through grapefruit by-products, belonging to the technical field of microbial fermentation.
Background
Cardiovascular and cerebrovascular diseases become one of the major killers of human health, which has an important relationship with the excessive accumulation of cholesterol in human blood. The Monacolin substance is used as an active substance of fungal metabolism, is a competitive inhibitor of key enzyme HMG-CoA reductase of cholesterol synthesis, and can effectively maintain cholesterol at a lower density level. In the aspect of inhibiting cholesterol synthesis, Monacolin K has excellent effect in Monacolin substances, but according to the existing research, Monacolin K still takes fermentation as a main production mode at present, but the yield is low. In recent years, the morbidity and mortality of cardiovascular diseases jump the top of the world, the demand of lipid-lowering drugs is increasing, the requirements of people on the quality of life are increasing, the demand of functional foods is greatly increased, and the functional food industry is rapidly developed, so that the Monacolin K fermentation production has a wide prospect.
Monascus purpureus is a filamentous fungus used as both medicine and food, has a history of more than one thousand years in China, has been recorded as food by red yeast rice in the early period of eastern Han, and has been recorded with related medicinal value of red yeast rice in the works such as Ben Cao gang mu, Tian Gong Kong Kai, Shen nong Ben Cao Jing and the like. This is closely related to various functional active substances produced by monascus during fermentation, and the most important of them is the physiologically active substance Monacolin K with cholesterol-lowering effect.
The traditional monascus solid-state fermentation mainly uses rice and other grains as fermentation substrates, and at present, many researches on Monacolin K production by monascus solid-state fermentation with agricultural product wastes as substrates exist, for example, Tianjie and other monascus solid-state fermentation with bran as a substrate, and the content of the obtained Monacolin K is only 0.043 mg/g; the monascus is used for brewing yellow wine, such as the jiasheng, and the like, and provides a basis for developing the yellow wine with the lipid-lowering function, but the Monacolin K content of 1.252mg/g is only obtained through optimizing fermentation conditions such as water content, inoculation amount and the like; liu' an Jun et al studied on Monacolin K production by monascus solid fermentation with soybean meal as a matrix, and the highest content of Monacolin K only reaches 0.0325 mg/g. The Monacolin K content was generally lower in these studies.
The pomelo sacs and the pomelo peel are waste generated in the processing process of the pomelo, the pomelo is not only edible pulp, but also a large part of the pomelo sacs and the pomelo peel, the vast majority of the pomelo sacs and the pomelo peel are directly discarded, and a small part of the pomelo sacs are processed into leisure food, but the pomelo sacs are not widely accepted by people. A small amount of pomelo sacs are used for removing peculiar smell or making products such as honey pomelo tea, but the using amount is far less than the production amount. Considerable economic benefit is obtained if the grapefruit seed sacs and the grapefruit peel can be effectively utilized. Meanwhile, the shaddock sacs and shaddock peel are edible and harmless, so that the agricultural product wastes are utilized with high added value, and the effect of making the best use of the things can be achieved. However, the pomelo fruit sac and the pomelo peel mainly comprise dietary fibers, pectin, saponin and the like, and the components of the pomelo fruit sac and the pomelo peel are greatly different from rice, so that the solid fermentation of monascus by using the pomelo fruit sac and the pomelo peel still has some difficulties which need to be solved, such as insufficient nutritional ingredients and the like. The cost of the rice is high, the cost of the grapefruit byproducts is low, the grapefruit byproducts are used for producing active substances through solid state fermentation, the green utilization effect of wastes can be achieved, and the concepts of environmental protection and sustainable development are met. Therefore, the method for treating the grapefruit byproducts is provided, the added value of products is improved, and the method has important application value.
Disclosure of Invention
In order to solve the technical problems, the shaddock byproduct is applied to the solid-state fermentation of monascus to produce lipid-lowering component Monacolin K, the yield of the Monacolin K is up to 7.4mg/g, the production cost of the monascus product is reduced, the requirements of environmental protection and sustainable development are met, the method is an embodiment of turning waste into wealth, and the method has great research significance and economic value.
The first purpose of the invention is to provide a method for treating pomelo byproducts, which utilizes monascus purpureus 9901 to ferment pomelo byproducts to produce Monacolin K; the grapefruit byproducts comprise grapefruit peel and/or grapefruit sacs; the initial water content of the grapefruit byproducts is 40-55% (m/m); adding carbon source and/or nitrogen source during fermentation.
In one embodiment, the initial moisture content of the grapefruit by-product is preferably 45%.
In one embodiment, the conditions of the fermentation are: solid-state fermentation, activating culture at 25-35 deg.C for 1-3 days, and culturing at 20-30 deg.C for 14-29 days.
In one embodiment, the carbon source is one or more of glycerol, sucrose, lactose, maltose; the nitrogen source is one or more of sodium nitrate, yeast extract and peptone.
In one embodiment, the carbon source is lactose.
In one embodiment, the lactose is added in an amount of 1-5% (m/m).
In one embodiment, the lactose is preferably added in an amount of 3%.
In one embodiment, the nitrogen source is peptone.
In one embodiment, the peptone is added in an amount of 1-3% (m/m).
In one embodiment, the peptone is preferably added in an amount of 1%.
In one embodiment, the grapefruit by-product is: the water content of the shaddock sacs and/or shaddock peel is 40-55%.
In one embodiment, the grapefruit by-product is preferably: pomelo seed bag, water content 45%.
In one embodiment, Monacolin K is produced by inoculating monascus ruber 9901 into a seed culture medium to obtain a seed solution, and then inoculating the seed solution into the pomelo sacs and/or the pomelo peels in an inoculation amount of 5-10% (V/m) for fermentation.
In one embodiment, the amount of inoculation is preferably 10% (V/m).
In one embodiment, the seed medium is (g.L)-1): 40-80 parts of glucose, 10-40 parts of fish meal peptone, 1-5 parts of sodium nitrate, 0.5-2 parts of magnesium sulfate heptahydrate, 0.5-2 parts of dipotassium phosphate and 2-10 parts of corn flour, and the pH value is adjusted to 3-8.
The invention also provides the application of the Monacolin K prepared by the method in the field of pharmacy or food.
The invention has the beneficial effects that:
the Monacolin K is produced by using monascus solid fermentation grapefruit byproducts, and the yield of the Monacolin K is 7.413 mg/g. The pomelo by-product is used as a main waste in the pomelo processing process, is edible and harmless, and the pomelo seed capsules and the pomelo peel are applied to the red yeast solid state fermentation to produce the fat-reducing component Monacolin K, so that the production cost of the red yeast product can be reduced, the requirements of environmental protection and sustainable development are met, the method is an embodiment of turning waste into wealth, and has great research significance and economic value.
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FIG. 1: monacolin K is produced by monascus through the pomelo by-product.
Detailed Description
The method for measuring Monacolin K comprises the following steps:
(1) pretreatment of solid state fermentation samples
Drying monascus by using a shaddock capsule solid state fermentation finished product in an electric heating forced air drying oven, smashing to a powder state by using a grinder, uniformly mixing powder of each sample, putting 0.50g of the sample into a 50mL colorimetric tube, fixing the volume to 50mL by using 70% ethanol, carrying out water bath at 55 ℃ for 1h, shaking once every 20min, cooling to room temperature after the water bath is finished, filtering by using a 0.45 mu m filter membrane, and obtaining filtrate for later use.
(2) Liquid chromatography detection (HPLC detection)
The instrument comprises the following steps: waters 2487; the type of the chromatographic column: 20RBA × SB-C18; mobile phase: acetonitrile, water 55:45(V/V), phosphoric acid to adjust pH to 2.5; flow rate: 1.0 mL/min-1(ii) a Column temperature: 30 ℃; sample introduction amount: 20-30 μ L; a detector: an ultraviolet detector; detection wavelength: 238 nm;
(3) calculation of the Monacolin K content: the Monacolin K content (mg/g) ═ c (mg/L) × 0.05(L)/0.5(g) in the formula:
c: the concentration of Monacolin K in the extract (mg/L);
0.05: 50mL was converted to 0.05L;
0.5: 0.5g sample mass.
Example 1: monacolin K production by fermentation of different strains
Seed liquid culture medium: glucose 60 g.L-125 g.L fish meal peptone-1Sodium nitrate 2 g.L-1Magnesium sulfate heptahydrate 1 g.L-1Dipotassium hydrogen phosphate 1 g.L-1Corn flour 6 g.L-1The pH was adjusted to 6.0.
Fermentation medium: pomelo seed bag (cut into small pieces, such as cubes with a side length of 0.5-1 cm), and has 50% water content.
(1) Preparation of seed liquid
Red koji 9901 (refer to study on solid fermentation high yield Monacolin K of Monascus ruber by Sophora Subcorgensis university thesis 2015-12-31, published on,), red koji with CICC number 41649 and CICC number 41232, purple koji with CICC number 41601 and CICC number 5046, and Monascus pilosus with CGMCC number 3.4633 and CGMCC number 3.4647 are respectively activated and inoculated to a seed liquid culture medium, and shake flask fermentation culture is carried out for 2d at the rotation speed of 180r/min and the temperature of 30 ℃.
(2) Fermentation culture
Respectively inoculating the red monascus 9901, the CICC 41649, the CICC 41232, the CICC 41601, the CICC 5046, the CGMCC 3.4633 and the CGMCC 3.4647 liquid seeds in the logarithmic growth phase obtained in the step (1) into a 250mL triangular flask filled with 10g of pomelo seed capsules in an inoculation amount of 10% (V/m), wherein the initial water content is 50%; fermenting and culturing for 2d at the temperature of 30 ℃; then fermenting and culturing for 18d at the temperature of 25 ℃. After completion, the Monacolin K content was found to be 5.322mg/g, 1.253mg/g, 1.853mg/g, 3.559mg/g, 4.235mg/g, 0.253mg/g, 0.552mg/g, respectively (see Table 1).
TABLE 1 results of different Monascus fermentations
Example 2: fermentation production of Monacolin K with different initial water contents
Fermentation medium: pomelo seed bags, different initial water content.
Inoculating the liquid red monascus 9901 seeds in logarithmic phase obtained in example 1 into a 250mL triangular flask filled with 10g of pomelo seed capsules in an inoculation amount of 10% by volume, wherein the initial water content of a fermentation medium is 40%, 45%, 50%, 55%, 60% and 65%, and performing fermentation culture for 2d at the temperature of 30 ℃; fermenting and culturing for 18d at the temperature of 25 ℃. After the fermentation was completed, the Monacolin K contents were 5.514mg/g, 6.372mg/g, 5.762mg/g, 5.625mg/g, 5.310mg/g, 4.760mg/g, respectively (see Table 2).
TABLE 2 results of fermentations with different initial water contents
Example 3: monacolin K fermentation production with different inoculation amounts
Fermentation medium: pomelo seed bag, 45% water content.
Liquid red koji 9901 seeds obtained in example 1 in the logarithmic growth phase were inoculated in an amount of 5%, 10%, 15%, 20%, 25%, 30% by volume into a 250mL Erlenmeyer flask containing 10g of grapefruit seed capsules, respectively, at an initial water content of 45%. Fermenting and culturing for 2d at the temperature of 30 ℃; fermenting and culturing for 18d at the temperature of 25 ℃. After the fermentation was completed, the Monacolin K content was found to be 6.383mg/g, 6.682mg/g, 5.942mg/g, 5.821mg/g, 6.114mg/g, 4.745mg/g, respectively (see Table 3).
TABLE 3 results of different inoculum size fermentations
Example 4: fermentation production of Monacolin K by adding different carbon sources
Fermentation medium: pomelo seed sacs, 45% moisture content, 5% content of different carbon sources were added.
The liquid red koji 9901 seeds obtained in example 1 in the logarithmic growth phase were inoculated into a 250mL Erlenmeyer flask containing 10g of pomelo seed capsules at an inoculum size of 10% by volume, and the initial water content was 45%, and a carbon source was additionally added in an amount of 5% (m/m) in each of glycerol, glucose, sucrose, lactose and maltose. Fermenting and culturing for 2d at the temperature of 30 ℃; fermenting and culturing for 18d at the temperature of 25 ℃. After the fermentation was completed, the Monacolin K contents were determined to be 7.588mg/g, 6.226mg/g, 7.558mg/g, 7.763mg/g, 7.441mg/g (see Table 4).
TABLE 4 results of fermentation with different carbon sources added
Example 5: monacolin K fermentation production with different lactose adding amounts
Fermentation medium: pomelo seed capsules, 45% moisture content, different amounts of lactose were added.
The liquid red koji 9901 seeds obtained in example 1 in the logarithmic growth phase were inoculated into a 250mL Erlenmeyer flask containing 10g of pomelo seed capsules at an inoculum size of 10% by volume, and lactose was additionally added at an initial water content of 45% in an amount of 1%, 3%, 5%, 7%, 9%, respectively. Fermenting and culturing for 2d at the temperature of 30 ℃; fermenting and culturing for 18d at the temperature of 25 ℃. After completion, the Monacolin K contents were determined to be 7.093mg/g, 7.413mg/g, 6.692mg/g, 6.305mg/g, 5.686mg/g (see Table 5).
TABLE 5 results of fermentation with different lactose addition levels
Example 6: fermentation production of Monacolin K by adding different nitrogen sources
Fermentation medium: pomelo seed sacs, 45% moisture content, 2% content of different nitrogen sources were added.
The liquid red koji 9901 seeds obtained in example 1 in the logarithmic growth phase were inoculated into a 250mL Erlenmeyer flask containing 10g of spent grains at an inoculation amount of 10% by volume, the initial water content was 45%, and the nitrogen sources added were ammonium sulfate, sodium nitrate, corn flour, yeast extract, peptone, and 2% by volume, respectively. Fermenting and culturing for 2d at the temperature of 30 ℃; the Monacolin K content was measured at 25 ℃ at 18d after the end of the fermentation culture at 3.815mg/g, 5.981mg/g, 4.184mg/g, 6.240mg/g, 6.506mg/g, respectively (see Table 6).
TABLE 6 results of fermentation with different nitrogen sources added
Example 7: monacolin K fermentation production by different peptone adding amounts
Fermentation medium: pomelo seed capsules, 45% moisture content, different contents of sodium nitrate were added.
The liquid red koji fungus 9901 seeds obtained in example 1 in the logarithmic growth phase were inoculated into a 250mL Erlenmeyer flask containing 10g of spent grains at an inoculation amount of 10% by volume, the initial water content was 45%, and a nitrogen source of peptone type was additionally added at an addition amount of 1%, 1.5%, 2%, 2.5%, 3%, respectively. Fermenting and culturing for 2d at the temperature of 30 ℃; fermenting and culturing for 18d at the temperature of 25 ℃. After completion, the Monacolin K contents were determined to be 7.088mg/g, 5.561mg/g, 5.954mg/g, 6.095mg/g, 5.904mg/g (see Table 7).
TABLE 7 results of fermentation with different amounts of peptone added
Example 8: monacolin K produced by fermenting other wastes
The fermentation parameters for the fermentative production of Monacolin K from different wastes were optimized with reference to the methods of examples 1 to 7. The highest yield of Monacolin K was determined.
(1) Monacolin K produced by monascus through shaddock peel fermentation
Fermentation medium: shaddock peel, 45% moisture content.
Liquid seeds of monascus ruber 9901 obtained in example 1 in the logarithmic growth phase were inoculated into 250mL triangular flasks containing 10g of shaddock peel at an inoculum size of 10% by volume, respectively, and the initial water content was 45%. Lactose was additionally added as a carbon source at 3%. Fermenting and culturing for 2d at the temperature of 30 ℃; fermenting and culturing for 18d at the temperature of 25 ℃. After completion, the Monacolin K content was found to be 4.423 mg/g.
(2) Monacolin K produced by fermenting monascus with orange peel
Fermentation medium: orange peel, 45% moisture content.
Liquid seeds of monascus ruber 9901 obtained in example 1 in the logarithmic growth phase were inoculated into 250mL triangular flasks containing 10g of orange peel at an inoculation amount of 10% by volume, respectively, and the initial water content was 45%. Lactose was additionally added as a carbon source at 3%. Fermenting and culturing for 2d at the temperature of 30 ℃; fermenting and culturing for 18d at the temperature of 25 ℃. After that, the Monacolin K content is measured to be 2.232 mg/g.
(3) Monacolin K produced by fermenting monascus with peanut meal
Fermentation medium: peanut meal, 45% moisture content.
The liquid red koji fungus 9901 seeds in the logarithmic growth phase obtained in example 1 were inoculated into 250mL Erlenmeyer flasks containing 10g of peanut meal at an inoculation amount of 10% by volume, respectively, and the initial water content was 45%. Lactose was additionally added as a carbon source at 3%. Fermenting and culturing for 2d at the temperature of 30 ℃; fermenting and culturing for 18d at the temperature of 25 ℃. After completion, the Monacolin K content was found to be 3.128mg/g (see Table 8).
TABLE 8 fermentation production of Monacolin K from different wastes
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 (10)
1. A method for treating pomelo byproducts is characterized in that Monacolin K is produced by fermenting pomelo byproducts by red monascus 9901; the pomelo by-product comprises pomelo seed sacs and/or pomelo peel; the initial water content of the grapefruit byproducts is 40-55% (m/m); adding carbon source and/or nitrogen source during fermentation.
2. The method of claim 1, wherein the fermentation conditions are: solid-state fermentation, activating culture at 25-35 deg.C for 1-3 days, and culturing at 20-30 deg.C for 14-29 days.
3. The method of claim 1, wherein the carbon source is one or more of glycerol, sucrose, lactose, maltose.
4. The method of claim 1, wherein the nitrogen source is one or more of sodium nitrate, yeast extract, peptone.
5. The method of claim 1, wherein the carbon source is added in an amount of 1-5% (m/m).
6. The method of claim 1, wherein the nitrogen source is added in an amount of 1-3% (m/m).
7. The method of claim 1, wherein the grapefruit byproduct is: the water content of the shaddock sacs and/or shaddock peel is 40-55% (m/m).
8. The method of claim 1, wherein Monacolin K is produced by inoculating monascus ruber 9901 into a seed culture medium to obtain a seed solution, and then inoculating the seed solution into the grapefruit seed sacs and/or grapefruit peel in an inoculation amount of 5-10% (V/m) for fermentation.
9. The method of claim 8, wherein in one embodiment the seed medium is (g-L)-1): 40-80 parts of glucose, 10-40 parts of fish meal peptone, 1-5 parts of sodium nitrate, 0.5-2 parts of magnesium sulfate heptahydrate, 0.5-2 parts of dipotassium phosphate and 2-10 parts of corn flour, and the pH value is adjusted to 3-8.
10. Use of the method of any one of claims 1 to 9 in the pharmaceutical or food field.
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116870101A (en) * | 2023-06-25 | 2023-10-13 | 汕头大学 | Application of monascus fermentation product in regulating lipid metabolism |
| CN116942768A (en) * | 2023-06-20 | 2023-10-27 | 汕头大学 | Application of fermented pomelo ascus in regulating intestinal flora |
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| CN109497265A (en) * | 2018-12-17 | 2019-03-22 | 江苏春之雨生物科技发展有限公司 | It is a kind of using pomace, vinasse as the biological feedstuff mixed fungus fermentation method of raw material |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN109497265A (en) * | 2018-12-17 | 2019-03-22 | 江苏春之雨生物科技发展有限公司 | It is a kind of using pomace, vinasse as the biological feedstuff mixed fungus fermentation method of raw material |
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| CHEN ZHI等: "Enhancement of monacolin K production via intergeneric protoplast fusion between Aspergillus terreus and Monascus anka", 《PROGRESS IN NATURAL SCIENCE》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN116942768A (en) * | 2023-06-20 | 2023-10-27 | 汕头大学 | Application of fermented pomelo ascus in regulating intestinal flora |
| CN116870101A (en) * | 2023-06-25 | 2023-10-13 | 汕头大学 | Application of monascus fermentation product in regulating lipid metabolism |
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