CN110592155B - Application of arginine in improving monacolin K production by monascus purpureus - Google Patents
Application of arginine in improving monacolin K production by monascus purpureus Download PDFInfo
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Abstract
The invention discloses a fermentation culture medium for producing monacolin K by monascus purpureus, which contains arginine as a component, wherein the arginine is added into the culture medium, and when the addition amount is 0.6%, compared with the original fermentation culture medium, the yield of the monacolin K on day 8 is improved by 3.65 times, and the yield of the monacolin K on day 15 is improved by 2.89 times.
Description
Technical Field
The invention relates to application of arginine in improving monacolin K production by monacolin purple, belonging to the field of preparation of biotechnology.
Background
Monascus is also called monascus, and belongs to the phylum mycomycota, subgenomycota, class of incomplete ascomycete, family of monascus, genus Monascus in the taxonomic classification of fungi. Monascus purpureus can produce various secondary metabolites, such as monascus pigment, monacolin K, gamma-aminobutyric acid, citrinin and the like, wherein a plurality of metabolites are widely applied to the aspects of food pigments, medicines, wine brewing and the like, and the monacolin K is also called lovastatin and is a polyketone substance with physiological activity produced by the monascus. Research finds that the monacolin K has the function of inhibiting the activity of key enzyme HMG-CoA in cholesterol synthesis and can effectively inhibit the cholesterol synthesis, so the monacolin K is regarded as an ideal drug for reducing cholesterol at home and abroad.
Factors such as low monacolin K active ingredient content and high production cost in monascus severely restrict the exertion of the function of functional red yeast rice in the aspects of reducing blood fat and blood pressure and the large-scale industrialized development of functional red yeast rice products. In recent years, in order to improve the quality of functional red yeast rice, researchers have conducted extensive and intensive research works in optimizing fermentation conditions, and the like, and such as rashmi dikshit and LijuanYu, and the like, have conducted response surface methods to optimize the fermentation conditions of a culture medium to improve the yield of monacolin K. Chen Xiaolin, Ma Yi Zhi and Zhao Zhi Xin et al promote the synthesis of monacolin K by adding different inducers to the culture medium. Because of the differences in experimental strains, treatment methods and detection methods, it is not well established which additive is the best inducer.
Disclosure of Invention
The technical problem to be solved by the invention is as follows: provides a fermentation culture medium for producing monacolin K by monascus purpureus, which is mainly added with arginine to stimulate and induce the yield of monacolin K.
Based on the previous metabonomics research result, the content change range of thousands of substances in the synthesis process of monacolin K is found to be large, more than 800 substances are screened out through screening standards FC >2, p <0.05 and VIP >1, more than twenty different metabolites screened out according to superclass classification statistics are screened out, more than ten substances related to citric acid circulation, acetyl-CoA synthesis and the like are screened out, and arginine, malic acid, D-glucose, alpha-ketoglutaric acid, arginine, phenylalanine, L-lactic acid, flavin mononucleotide, thiamine pyrophosphate, lysine and other ten substances are preliminarily planned to be added into a common fermentation culture medium, and the most remarkable additive for improving the yield of monacolin K is screened out through observing the yields of monacolin K at 8 days and 15 days. By adding the above substances at different concentrations, it was found that when arginine was added in an amount of 0.6%, the yield of monacolin K was increased by 3.65 times at day 8 and 2.89 times at day 15, compared to the original fermentation medium. Therefore, the invention mainly researches and researches that arginine with different contents is added into a culture medium, the culture is synchronous with the original culture medium, and related indexes such as color value, biomass, monacolin K and the like are detected.
The invention provides a fermentation medium for producing monacolin K by monascus purpureus, which contains arginine. The content of arginine is 0.4-1.5%.
Further, the content of arginine was 0.6%.
The invention has the beneficial effects that:
according to the invention, arginine is added into the culture medium, and when the addition amount is 0.6%, compared with the original fermentation culture medium, the yield of monacolin K on day 8 is improved by 3.65 times, and the yield of monacolin K on day 15 is improved by 2.89 times.
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The following describes embodiments of the present invention in further detail with reference to the accompanying drawings.
FIG. 1 is a graph comparing the Monacolin K production at different addition levels of arginine.
FIG. 2 is a graph showing the determination of the arginine addition concentration.
FIG. 3 color comparison for different fermentation times.
FIG. 4 Biomass of common strain and arginine-added strain.
FIG. 5 pH values of the ordinary strain and the arginine-added strain.
FIG. 6 shows the form of Monascus purpureus M1 at different magnifications in different media. a and b are the shapes of thalli in a common culture medium; c and d are the thallus form in the arginine culture medium; a. c is 5000 × magnification; b. d is 10000 times magnification; the red arrow is a fold.
FIG. 7a is a graph comparing the red pigment production of the conventional strain with that of the strain supplemented with arginine.
FIG. 7b is a graph comparing the yield of orange pigment in the common strain and the strain added with arginine.
FIG. 7c is a comparison of the yellow pigment production of the common strain and the arginine added strain.
FIG. 8 is a graph comparing the yields of monacolin K from the common strain and the arginine-added strain.
FIG. 9 comparison of the monacolin K production by glutamic acid addition and arginine addition.
FIG. 10 comparison of monascus pigment yields for strains with added glutamic acid versus added arginine.
Detailed Description
Example 1 experiment of the Effect of arginine on the production of Monacolin K
The experimental steps are as follows:
in the early stage of the experiment, through reference of literature, the addition amount of arginine is selected to be 0.5%, 1% and 1.5%, monacolin K is detected by monascus liquid fermentation and HPLC, and the monacolin K yield is found to be reduced on the contrary along with the increase of the addition amount of arginine, so that 0.4%, 0.5% and 0.6% of arginine is respectively selected to be added according to the standard of 0.5%, and the monacolin K yield is found to be the highest when the addition amount of arginine is 0.6%. And then, other physiological index changes after arginine addition, such as monascus pigment, pH value, biomass, scanning electron microscope analysis and the like, are further detected.
And (3) activating the strain, namely culturing the M1 strain on a PDA (PDA) culture medium at 30 ℃ for 3 days, and activating for 2 generations.
The preparation of the seed liquid comprises scraping 1-ring bacterial liquid from the bacteria on the culture medium into a common liquid seed culture medium by using an inoculating ring, and culturing at 30 ℃ and 200 r/min for 2 d.
The fermentation liquid is prepared by inoculating the seed liquid into common liquid fermentation culture medium according to the inoculum size of 10%, culturing at 30 deg.C and 150 r/min for 2d, and culturing at 25 deg.C and 150 r/min for 12 d.
The liquid fermentation medium added with arginine, wherein the addition amount of arginine is 0.6%. See in particular fig. 1-2.
General liquid fermentation Medium (g/L) composition: 90 g of glycerol, ZnSO4 .7H2O2 g, rice flour 20 g, KH2PO42.5 g, peptone 10 g, NaNO3 5 g，MgSO4 .7H2O 1 g。
Arginine was added at 0.6% and the following experiment was performed:
(1) color comparison of fermentation broths
See in particular fig. 3.
(2) Biomass detection
Mycelium biomass was measured by dry weight method. Filtering 5 mL of fermentation liquor by using 3 layers of gauze, washing for 2-3 times by using distilled water, wringing out water, and drying in an oven at 60 ℃ until the weight is constant, namely the dry weight of the mycelium.
Biomass (g/L) = mass of dry matter/volume of fermentation broth. The results are shown in FIG. 4
(3) pH value detection
The pH meter was first calibrated. Washing the electrode with distilled water, washing with fermented liquid once, stirring the solution with glass rod to homogenize the solution, immersing the electrode in the solution to be measured, and reading out its pH value. For each test, the test pieces were rinsed with distilled water and wiped dry. The results are shown in FIG. 5.
(4) Scanning electron microscopy processing
Culturing 8 d monascus thallus, centrifuging at 12000 r/min for 5 min to collect thallus cells, resuspending the cells (blowing with a gun head, taking care not to suck the cells into the gun head to cause cell loss) and fixing in 2.5% glutaraldehyde solution (diluted by PBS buffer solution) for 12 h. The cells were rinsed twice (twice resuspension centrifugation) with 0.1M phosphate buffer (PBS, pH 7.2) and the supernatant discarded. The cells were dehydrated sequentially with ethanol solutions of different concentrations (30%, 50%, 70%, 80%, 90%, 100%), left to stand for 10 min at each concentration, centrifuged at 12000 r/min for 5 min (each concentration was repeated twice), and the supernatant was discarded. Cells were ethanol-displaced by resuspending the cells in isoamyl acetate and ethanol (v: v =1: 1) and then in isoamyl acetate solution. The cells were resuspended in each solvent and allowed to stand for 10 min, centrifuged at 12000 r/min for 5 min, and the supernatant was discarded. Adding solvent HexamethylDisilazane (HMDS) in an amount exceeding that of the sample, placing a centrifugal tube plug on a 60 ℃ oven by using absorbent cotton, and drying until the sample is powdered, and keeping the sample for observation. The results are shown in FIG. 6.
(5) Color value detection
Pretreatment of fermentation liquor: taking 1 mL of fermentation liquor, adding 8 mL of 70% ethanol solution, leaching at 60 ℃ in a constant-temperature water bath for 1 h, centrifuging at 4000 rpm for 15 min, and placing in the dark.
And (3) detection of color value: the absorbance at 410, 448 and 505 nm was measured with an ultraviolet spectrophotometer.
Quantitative formula: monascus pigment color number (U/mL) = absorbance × dilution factor.
The results are shown in FIGS. 7a, 7b, 7 c.
(6) Monacolin K detection
Pretreatment of fermentation liquor: taking 5 mL of fermentation liquid, adding 15 mL of 75% methanol, performing ultrasonic extraction for 30 min, and standing overnight.
Detection of monacolin K: HPLC method, column: InertsilODS-3C 18 (150 mm. times.4.6 mm. times.5 μm), mobile phase: 0.1% phosphoric acid: methanol =1: 3, the flow rate is 1 mL/min, the detector is an ultraviolet detector (PDA), the detection wavelength is 237 nm, the detection temperature is 30 ℃, and the sample injection amount is 10 mu L. The results are shown in FIG. 8.
The specific experimental results are as follows:
1. the yield of monacolin K after arginine addition was increased by 2-3 times compared to the wild type strain.
2. Compared with the original strain, the monascus red pigment, the orange pigment and the yellow pigment on the 15 th day after the arginine is added are respectively improved by 2.50 times, 2.27 times and 2.01 times.
Example 2: comparison of the effects of glutamic acid and arginine:
in the prior art, in the article of analysis of promoting effect of ten additives on monacolin K yield of monacolin, yam powder, orange peel powder, yeast liquid, yeast supernatant, yeast wall-breaking liquid, wall-broken yeast liquid, leucine, glutamic acid, ethanol and other substances are added into original culture medium components in a laboratory, and the yield of secondary metabolites of monacolin in different culture media is measured. The result shows that the addition of glutamic acid can remarkably (p is less than 0.01) improve the yield of monacolin K by 5.60 times.
Glutamate and arginine differ in that: although glutamate is more effective than arginine, glutamate has the disadvantages of: although the monacolin K yield can be obviously improved, the monascus pigment yield is reduced compared with that of the original culture medium, and arginine has an effect of improving the monacolin K yield and the monascus pigment yield.
Glutamic acid fermentation medium (g/L): 90 g of glycerol, 20 g of rice flour, 10 g of peptone and KH2PO4 2.5 g，NaNO3 5 g，MgSO4.7H2O 1 g，ZnSO4.7H2O2 g and glutamic acid 0.15 g.
As shown by the comparison of the yields of monacolin K in the glutamic acid-supplemented and arginine-supplemented strains in FIG. 9, monacolin K is not produced at day 2, and in FIG. 9a, the yield of monacolin K is 203.8 mg/L, which is 5.60 times higher than that of the original culture medium, and in FIG. 9b, the yield is 358.0 mg/L, which is 3.34 times higher than that of the original culture medium.
As shown by the comparison of the monascus pigment yields of the strains added with glutamic acid and the strains added with arginine in the figure 10, the results of the monascus pigment results show that the monascus pigment production is inhibited after the glutamic acid is added, the monascus pigment production is effectively promoted by the addition of the arginine, and the monascus pigment yields are respectively increased by 2.50,2.27 and 2.01 times.
Claims (2)
1. The fermentation culture medium for producing monacolin K by monascus purpureus is characterized by comprising the following components: 90 g of glycerol, ZnSO4 .7H2O2 g, rice flour 20 g, KH2PO42.5 g, peptone 10 g, NaNO3 5 g，MgSO4 .7H2O1 g and arginine with the mass fraction of 0.4-0.6%.
2. The fermentation medium of claim 1, wherein arginine is present in an amount of 0.6%.
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