Application of rapeseed pollen in promoting high yield of monacolin K in purple Monascus and method for promoting high yield of monacolin K

Abstract

The present invention provides an application of rapeseed pollen in promoting high yield of monacolin K in purple Monascus and a method for promoting high yield of monacolin K, and relates to the technical field of purple Monascus culture. The method is to add 0.6%-4.8% rapeseed pollen to a fermentation medium to ferment and culture purple Monascus. The present invention overcomes the deficiencies of the prior art, and by adding rapeseed pollen to a culture medium of purple Monascus, the content of monacolin K can be significantly increased after 15 days of culture, especially when the amount of rapeseed pollen added is 2.4%, the content of monacolin K on the 15th day is increased by 3.50 times to 1524 mg / L.

Description

Technical Field

[0001] The invention relates to the technical field of purple Monascus cultivation, and in particular to application of rapeseed pollen in promoting high yield of monacolin K in purple Monascus and a method for promoting high yield of monacolin K. Background Art

[0002] Monascus is a filamentous fungus that can produce a variety of secondary metabolites with application value, such as monascus pigment, monacolin K, γ-aminobutyric acid, etc. Among them, monacolin K, also known as "lovastatin", is a natural statin produced by Monascus and has good lipid-lowering activity.

[0003] The ability of Monascus to ferment and synthesize monacolin K is the key to determining the quality of functional Monascus. However, the existing Monascus production capacity of monacolin K is relatively low, resulting in a corresponding increase in the cost of industrial production, which seriously limits the development of the functional Monascus industry and has become a major obstacle to the application of Monascus in the fields of food and health products. In recent years, researchers have done a lot of in-depth research on optimizing fermentation conditions, such as promoting the production of monacolin K by Monascus by improving the composition and proportion of the culture medium. For example, Zhang Chan and others increased the synthesis of monacolin K by adding different concentrations of glutamic acid, arginine, honeysuckle, nicotinamide and other substances to the culture medium. However, different exogenous additives are not always positively correlated with the growth of monacolin K, so a large number of experiments are needed to screen promoting substances. Summary of the invention

[0004] In view of the shortcomings of the prior art, the present invention provides an application of rapeseed pollen in promoting high yield of monacolin K in purple Monascus and a method for promoting high yield of monacolin K, and the yield of monacolin K is promoted by adding rapeseed pollen to a culture medium.

[0005] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0006] Rapeseed pollen is used to promote high production of monacolin K in Monascus purpurogenum.

[0007] Preferably, the method comprises adding 0.6%-4.8% rapeseed pollen by mass to the fermentation medium, and then inoculating purple Monascus into the fermentation medium for culturing.

[0008] Preferably, 2.4% rapeseed pollen by mass is added to the fermentation medium.

[0009] Preferably, the fermentation medium is a common liquid fermentation medium, and the formula calculated according to the content of ingredients per liter is: 90g glycerol, 0.2g ZnSO4·7H2O, 20g rice flour, 2.5g KH2PO4, 10g peptone, 5g NaNO3, 1g MgSO4·7H2O, and the balance is water.

[0010] The present invention provides an application of rapeseed pollen in promoting high yield of monacolin K in purple Monascus and a method for promoting high yield of monacolin K. Compared with the prior art, the advantages are:

[0011] Adding rapeseed pollen to the culture medium of purple Monascus can significantly increase the monacolin K content after 15 days of culture. Especially when the rapeseed pollen addition amount is 2.4%, the monacolin K content on the 15th day increased by 3.50 times to 1524 mg / L. BRIEF DESCRIPTION OF THE DRAWINGS

[0012] Figure 1 It is a bar chart comparing the monacolin K yields of purple Monascus M1 fermented in culture medium with different rape pollen contents at different times in Example 1 of the present invention;

[0013] Figure 2 It is a bar chart comparing the monacolin K yields of purple Monascus M1 fermented in culture medium with different rapeseed pollen contents at different times in Example 2 of the present invention;

[0014] Figure 3 The figure is a bar chart comparing the monacolin K yields of the experimental group and the control group in Example 3 of the present invention;

[0015] Figure 4 This is a comparison chart of the yellow pigment color levels of the experimental group and the control group in Example 3 of the present invention;

[0016] Figure 5 This is a comparison chart of the orange color scales of the experimental group and the control group in Example 3 of the present invention;

[0017] Figure 6 This is a comparison chart of red pigment levels between the experimental group and the control group in Example 3 of the present invention;

[0018] Figure 7 This is a comparison chart of biomass between the experimental group and the control group in Example 3 of the present invention;

[0019] Figure 8 This is a comparison chart of pH changes among the experimental group, the control group and the blank group in Example 3 of the present invention;

[0020] Fig. 9The electron microscope images of the morphology of Monascus M1 cultured in the experimental group and the control group in Example 3 of the present invention, wherein a and b are the bacterial morphology in ordinary culture medium; c and d are the bacterial morphology in rape pollen culture medium; a and c are magnified 1000×; b and d are magnified 10000×; and the red arrows are wrinkles. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention is clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Embodiment 1:

[0023] Detection of monacolin K produced by culturing wild-type Monascus purpurogenum strain M1 (commercially available, with a deposit number of CGMCC 3.0568):

[0024] 1. Ingredients of ordinary liquid fermentation medium (g / L): glycerol 90g, ZnSO4·7H2O 0.2g, rice flour 20g, KH2PO4 2.5g, peptone 10g, NaNO3 5g, MgSO4·7H2O 1g;

[0025] 2. Strain activation: Culture the purple Monascus M1 on PDA medium at 30°C for 3 days and activate for 2 generations;

[0026] 3. Preparation of seed solution: inject 10 ml of sterile water into the above PDA medium, and use a sterile inoculation loop to scrape the mycelium on the surface of the medium, then filter it with two layers of sterile filter paper, inoculate the spore suspension into ordinary liquid seed culture at an inoculum rate of 5%, and culture at 30°C and 200 r / min for 2 days;

[0027] 4. Preparation of fermentation liquid: inoculate the seed liquid into common liquid fermentation medium with 0%, 1%, 2%, 5%, 10% and 20% rapeseed pollen according to the inoculation amount of 10%, culture at 30℃, 150r / min for 2d, and then continue to culture at 25℃, 150r / min;

[0028] Detection of monacolin K content in each group of culture medium:

[0029] The specific detection process is:

[0030] Pretreatment of fermentation broth: Take 2 mL of fermentation broth from each group, add 6 mL of 75% methanol, perform ultrasonic extraction for 30 min, let stand overnight, then filter into a liquid phase vial using a 0.22 μm filter membrane and store in a 4°C refrigerator.

[0031] Detection of monacolin K: HPLC method was used, chromatographic column: Inertsi10DS-3 C18 (150mm×4.6mm×5μm), mobile phase: 0.1% phosphoric acid: methanol = 1:3, flow rate 1mL / min, detector was ultraviolet detector (PDA), detection wavelength was 237nm, detection temperature was 30℃, injection volume was 10μL.

[0032] Test results are shown in Figure 1 When the addition amount of rapeseed pollen was 5%, the content of monacolin K decreased after 15 days of cultivation compared with that without adding rapeseed pollen.

[0033] Embodiment 2:

[0034] Referring to the above Example 1, the seed liquid was inoculated into a common liquid fermentation medium supplemented with 0%, 0.6%, 1.2%, 1.8%, 2%, 2.4%, 3%, 3.6%, 4.2% and 4.8% rapeseed pollen according to an inoculum amount of 10%, and cultured at 30°C and 150 r / min for 2 days, and then continued to be cultured at 25°C and 150 r / min;

[0035] The content of monacolin K in each group of culture medium was detected. The specific results are as follows Figure 2 As shown, the ordinary liquid fermentation medium with 2.4% rapeseed pollen added had the highest monacolin K content at 15d, and the monacolin K content increased by 3.50 times compared with the medium with 0% rapeseed pollen added, reaching 1524 mg / L.

[0036] Embodiment 3:

[0037] Referring to the above Example 2, the seed liquid was inoculated into ordinary liquid fermentation medium with 0% and 2.4% rape pollen added according to an inoculum amount of 10%, and the medium with 0% rape pollen added was set as the control group, the medium with 2.4% rape pollen added was set as the experimental group, and the pure ordinary liquid fermentation medium without inoculation was set as the blank group; the culture was carried out at 30°C and 150r / min for 2d, and then continued to be cultured at 25°C and 150r / min, and the following tests were performed:

[0038] 1. Detection of monacolin K. Referring to the detection method of Example 1 above, the monacolin K content of the control group and the experimental group at different culture times was detected. The specific results are as follows: Figure 3As shown, the monacolinK content in the experimental group was significantly higher than that in the control group during the 5-18 days of fermentation.

[0039] 2. Color price detection

[0040] Fermentation broth pretreatment: Take 1 ml of fermentation broth from each group, add 6 ml of 70% ethanol solution, then place in a constant temperature water bath at 60°C for 1 hour, centrifuge at 4000 rpm for 15 minutes, and place away from light.

[0041] Color value detection: Use a UV spectrophotometer to measure the absorbance at 410, 448, and 505 nm.

[0042] Quantitative formula: Monascus pigment color value (U / mL) = absorbance value × dilution multiple.

[0043] Results Figure 4 , Figure 5 , Figure 6 Among them, the yellow pigment level, orange pigment level and red pigment level of the experimental group were significantly higher than those of the control group after 5 days of fermentation.

[0044] 3. Biomass detection

[0045] The mycelial biomass was determined by dry weight method. 3 mL of fermentation liquid was taken from each group into a centrifuge tube, then transferred to filter paper for filtration, and then washed with ultrapure water for 3 times. After the water was filtered out as much as possible, it was placed in a 60°C oven and dried to constant weight, which was the mycelial constant weight.

[0046] Biomass (g / L) = (dry matter weight / fermentation broth volume) * 1000.

[0047] Results Figure 7 , among which the biomass of the experimental group was significantly higher than that of the control group.

[0048] 4. pH value detection

[0049] The pH meter was used for testing. The specific test results are shown in Figure 8 When the fermentation time was less than 8 days, the pH of the experimental group was higher than that of the control group, and when the fermentation time was more than 8 days, the pH of the experimental group was lower than that of the control group.

[0050] 5. Scanning electron microscope processing

[0051] The Monascus cells of each group that had been cultured for 12 days were centrifuged at 12000r / min for 5min to collect the bacterial cells, and the cells were resuspended (blown with a pipette tip, and be careful not to suck the cells into the pipette tip to cause cell loss) in a 2.5% glutaraldehyde solution (diluted with PBS buffer solution) and fixed for 12h. The cells were rinsed twice with 0.1M phosphate buffer (PBS, pH7.2) (resuspended and centrifuged twice) and the supernatant was discarded. The cells were dehydrated with ethanol solutions of different concentrations (30%, 50%, 70%, 80%, 90%, 100%) in sequence, and each concentration was allowed to stand for 10min, and centrifuged at 12000r / min for 5min) (repeated twice for each concentration), and the supernatant was discarded. The cells were first resuspended in isoamyl acetate and ethanol (v:v=1:1), and then resuspended in isoamyl acetate to replace the cells with ethanol. The cells were resuspended in each solvent and allowed to stand for 10 minutes. The samples were centrifuged at 12000r / min for 5 minutes and the supernatant was discarded. The solvent hexamethyl disilazane (HMDS) was added until the sample was submerged. The top of the centrifuge tube was plugged with absorbent cotton to increase water absorption. The sample was dried in a 60˚C oven until it became powder. The sample was sent for scanning electron microscopy observation. The specific results are as follows: Fig. 9 As shown, a and b are the bacterial morphology in the control group; c and d are the bacterial morphology in the experimental group; a and c are magnified 1000×; b and d are magnified 10000×; the red arrows are wrinkles; this indicates that the Monascus bacteria cultured in the experimental group have a better morphology.

[0052] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Rapeseed pollen promotes purple Monascus ( Monascus purpureus ) Application on high-yield monacolin K.

2. A method for promoting high production of monacolin K by purple Monascus, characterized in that: The method comprises the following steps: adding rapeseed pollen in a mass percentage of 0.6% to 4.8% into a fermentation medium, and then inoculating purple Monascus into the fermentation medium for culturing.

3. A method for promoting high production of monacolin K by purple Monascus according to claim 2, characterized in that: The fermentation medium is added with 2.4% rapeseed pollen by mass.

4. A method for promoting high production of monacolin K by purple Monascus according to claim 2, characterized in that: The fermentation medium is a common liquid fermentation medium, and the formula calculated according to the content of components per liter is: 90g glycerol, 0.2g ZnSO4·7H2O, 20g rice flour, 2.5g KH2PO4, 10g peptone, 5g NaNO3, 1g MgSO4·7H2O, and the balance is water.

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