Use of rape pollen in promoting high-yield monacolin k production by monascus purpureus and method for promoting high-yield monacolin k production thereby

Abstract

Provided are the use of rape pollen in promoting the high-yield production of monacolin K by Monascus purpureus and a method for promoting the high-yield production of monacolin K thereby. The method comprises: adding 0.6-4.8% of rape pollen to a fermentation medium and performing the fermentative culture of Monascus purpureus. On day 15 of the culture, the content of monacolin K is increased by 3.5-fold, reaching 1524 mg / L.

Description

Application of rapeseed pollen in promoting high monacolin K production by Monascus purpureus and methods for promoting high monacolin K production Technical Field

[0001] This invention relates to the field of Monascus purpureus culture technology, specifically to the application of rapeseed pollen in promoting high Monascus purpureus production of Monacolin K and the method for promoting high Monacolin K production. Background Technology

[0002] Monascus purpureus is a filamentous fungus that produces a variety of secondary metabolites with practical applications, such as red yeast pigment, monacolin K, and γ-aminobutyric acid (GABA). Among them, monacolin K, also known as lovastatin, is a natural statin produced by Monascus purpureus and has good lipid-lowering activity.

[0003] The ability of Monascus purpureus to synthesize monacolin K through fermentation is crucial to the quality of functional red yeast rice. However, the current monacolin K production capacity of Monascus purpureus is relatively low, leading to higher industrial production costs and severely limiting the development of the functional red yeast rice industry. This has also become a major obstacle to the application of Monascus purpureus in food and health products. In recent years, researchers have conducted extensive and in-depth studies on optimizing fermentation conditions, such as improving the composition and ratio of the culture medium to promote monacolin K production by Monascus purpureus. For example, Zhang Chan et al. improved monacolin K synthesis by adding different concentrations of glutamic acid, arginine, honeysuckle, and nicotinamide to the culture medium. However, different exogenous additives are not always positively correlated with the increase of monacolin K, therefore, extensive experiments are needed to screen for promoting substances. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides an application of rapeseed pollen in promoting high monacolin K production by Monascus purpureus and a method for promoting high monacolin K production, which promotes monacolin K production by adding rapeseed pollen to the culture medium.

[0005] To achieve the above objectives, the present invention provides the following technical solution:

[0006] Rapeseed pollen is used to promote high production of Monacolin K by Monascus purpleis.

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

[0008] Preferably, the fermentation medium contains 2.4% rapeseed pollen by mass.

[0009] Preferably, the fermentation medium is a common liquid fermentation medium, and the formula calculated according to the component content 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] This invention provides an application of rapeseed pollen in promoting high monacolin K production by Monascus purpureus and a method for promoting high monacolin K production. Compared with existing technologies, the advantages are:

[0011] Adding rapeseed pollen to the culture medium of Monascus purpureus significantly increased the monacolin K content after 15 days of culture. In particular, when the amount of rapeseed pollen added was 2.4%, the monacolin K content on the 15th day increased by 3.50 times, reaching 1524 mg / L. Attached Figure Description

[0012] Figure 1 is a columnar comparison of the monacolin K yield of purple red Monascus M1 fermented in culture media with different rapeseed pollen contents at different times in Example 1 of the present invention.

[0013] Figure 2 is a columnar comparison of the monacolin K yield of purple red Monascus M1 fermented in culture media with different rapeseed pollen contents at different times in Example 2 of the present invention.

[0014] Figure 3 is a bar chart comparing the monacolin K production of the experimental group and the control group in Example 3 of the present invention.

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

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

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

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

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

[0020] Figure 9 shows electron micrographs of the morphology of Monascus purpureus M1 cultured in the experimental and control groups in Example 3 of the present invention, where a and b are the cell morphology in ordinary culture medium; c and d are the cell morphology in rapeseed pollen culture medium; a and c are magnified at 1000×; b and d are magnified at 10000×; red arrows indicate wrinkles. Detailed Implementation

[0021] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0022] Example 1:

[0023] Detection of monacolin K production using wild-type purple Monascus strain M1 (commercially purchased, preservation number CGMCC 3.0568):

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

[0025] 2. Strain activation: Purple Monascus M1 was cultured on PDA medium at 30℃ for 3 days, and activated for 2 generations;

[0026] 3. Preparation of seed culture: 10 ml of sterile water was added to the PDA culture medium above, and mycelia on the surface of the culture medium were scraped off using a sterile inoculation loop. After filtration with two layers of sterile filter paper, the spore suspension was inoculated into ordinary liquid seed culture at an inoculation rate of 5% and cultured at 30℃ and 200 r / min for 2 days.

[0027] 4. Preparation of fermentation broth: The seed liquid was inoculated into ordinary liquid fermentation medium containing 0%, 1%, 2%, 5%, 10% and 20% rapeseed pollen respectively at an inoculation rate of 10%. The medium was cultured at 30℃ and 150r / min for 2 days, and then cultured at 25℃ and 150r / min for another 2 days.

[0028] The content of monacolin K in the culture medium of each group was detected:

[0029] The specific testing process is as follows:

[0030] Pretreatment of fermentation broth: Take 2 mL of each group of fermentation broth, add 6 mL of 75% methanol, extract with ultrasound for 30 min, let stand overnight, then filter through a 0.22 μm filter membrane into a liquid chromatography vial and store at 4℃.

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

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

[0033] Example 2:

[0034] Referring to Example 1 above, the seed liquid was inoculated into ordinary liquid fermentation medium containing 0%, 0.6%, 1.2%, 1.8%, 2%, 2.4%, 3%, 3.6%, 4.2%, and 4.8% rapeseed pollen at an inoculation rate of 10%, respectively, and cultured at 30°C and 150 r / min for 2 days, and then cultured at 25°C and 150 r / min for the rest of the time.

[0035] The content of monacolin K in each culture medium was detected, and the specific results are shown in Figure 2. The ordinary liquid fermentation medium with 2.4% rapeseed pollen added had the highest monacolin K content at 15 days, and the monacolin K content was 3.50 times higher than that of the medium with 0% rapeseed pollen added, reaching 1524 mg / L.

[0036] Example 3:

[0037] Referring to Example 2 above, the seed culture was inoculated at a rate of 10% into ordinary liquid fermentation medium containing 0% and 2.4% rapeseed pollen, respectively. The medium containing 0% rapeseed pollen served as the control group, the medium containing 2.4% rapeseed pollen served as the experimental group, and the pure ordinary liquid fermentation medium without inoculation served as the blank group. The culture was carried out at 30℃ and 150 rpm for 2 days, followed by continued culture at 25℃ and 150 rpm. The following tests were performed:

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

[0039] 2. Color value detection

[0040] Fermentation broth pretreatment: Take 1 ml of each group of fermentation broth, add 6 ml of 70% ethanol solution, then incubate in a constant temperature water bath at 60℃ for 1 h, centrifuge at 4000 rpm for 15 min, and place in the dark.

[0041] Color value detection: The absorbance values ​​at 410, 448, and 505 nm were measured using a UV spectrophotometer.

[0042] Quantitative formula: Red yeast rice pigment color value (U / mL) = absorbance value × dilution factor.

[0043] The results are shown in Figures 4, 5, and 6. The yellow, orange, and red pigment levels in the experimental group were significantly higher than those in the control group after 5 days of fermentation.

[0044] 3. Biomass detection

[0045] Mycelial biomass was determined using the dry weight method. 3 mL of fermentation broth from each group was placed in a centrifuge tube, transferred to filter paper for filtration, and then washed three times with ultrapure water. After filtering out as much water as possible, the tubes were dried in a 60°C oven until constant weight was achieved; this constant weight of the mycelium was the final determination.

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

[0047] The results are shown in Figure 7, where the biomass of the experimental group was significantly higher than that of the control group.

[0048] 4. pH value detection

[0049] pH meters were used for testing, and 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 microscopy processing

[0051] Red Monascus cells cultured for 12 days were collected by centrifugation at 12000 rpm for 5 min. The cells were resuspended (using a pipette tip, being careful not to aspirate cells into the tip and cause cell loss) and fixed for 12 h in 2.5% glutaraldehyde solution (diluted with PBS buffer). The cells were washed twice with 0.1 M phosphate-buffered saline (PBS, pH 7.2) (resuspended and centrifuged twice), and the supernatant was discarded. The cells were then dehydrated sequentially with different concentrations of ethanol (30%, 50%, 70%, 80%, 90%, 100%), incubated for 10 min at each concentration, and centrifuged at 12000 rpm for 5 min (each concentration was repeated twice), 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 ethanol. Cells were resuspended in each solvent and allowed to stand for 10 min, then centrifuged at 12000 rpm for 5 min, and the supernatant was discarded. Hexamethyl disilazane (HMDS) was added until the sample was completely submerged. The top of the centrifuge tube was plugged with absorbent cotton to improve absorbency, and the tubes were dried in a 60˚C oven until powdery. The samples were then observed using a scanning electron microscope. The specific results are shown in Figure 9, where a and b represent the cell morphology in the control group; c and d represent the cell morphology in the experimental group; a and c are magnified at 1000×; b and d are magnified at 10000×; red arrows indicate wrinkles; indicating that the Monascus purpureus cultured in the experimental group had better morphology.

[0052] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit it. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. Application of rapeseed pollen in promoting high monacolin K production by Monascus purpureus.

2. A method for promoting high production of monacolin K by Monascus purpureus, characterized in that: The method involves adding 0.6%-4.8% rapeseed pollen by mass to a fermentation medium, and then inoculating the fermentation medium with purple red Monascus for cultivation.

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

4. The method for promoting high production of monacolin K by Monascus purpleis according to claim 2, characterized in that: The fermentation medium is a common liquid fermentation medium, and the formula calculated according to the component content 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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