Application of citrulline in improving monacolin K yield in liquid fermentation of monascus

Abstract

The application provides application of citrulline in improving the yield of monacolin K in liquid-state fermentation of monascus, and the yield and accumulation speed of monacolin K can be greatly improved by applying citrulline in the liquid-state fermentation of monascus for producing monacolin K, so that the application has great popularization and utilization value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of microbial fermentation, and particularly relates to application of citrulline in improving yield of monacolin K produced by liquid fermentation of monascus. BACKGROUND

[0002] Red koji, also known as Danq, originates from Fujian, China, and red koji products are produced in many provinces along the southeast coast of China. Studies have shown that monascus has many secondary metabolites such as red koji pigment, monacolin K, citrinin and gamma-aminobutyric acid. Monacolin K has the functions of regulating blood lipids, blood pressure, enhancing immunity and increasing antibodies.

[0003] Monacolin K, also known as lovastatin, was extracted from monascus culture solution by Japanese scholar Professor Aritomo Endo in 1979. Monacolin K can directly compete with HMG-CoA reductase and effectively inhibit cholesterol synthesis, which is 5 times stronger than compactin. Monacolin K also has the functions of reducing the concentration of cholesterol in stem cells and inhibiting the synthesis and secretion of triglycerides, lipoproteins and the like.

[0004] The ability of red koji to produce monacolin K is still in the weak stage. With the development of biotechnology, liquid fermentation technology for producing monacolin K has become a trend. However, liquid fermentation for producing monacolin K still faces the problems of low yield and not meeting the needs of enterprises and markets. Therefore, it is urgent to study the method for improving the yield of monacolin K. SUMMARY

[0005] In view of the above problems, the application provides application of citrulline in improving yield of monacolin K produced by liquid fermentation of monascus. The application of citrulline in the liquid fermentation of monascus for producing monacolin K can greatly improve the yield and accumulation speed of monacolin K, and has great popularization and utilization value.

[0006] The application is implemented through the following technical scheme.

[0007] Application of citrulline in improving yield of monacolin K produced by liquid fermentation of monascus.

[0008] Specifically, the liquid fermentation medium used in the liquid fermentation of monascus contains citrulline.

[0009] Specifically, citrulline is added to the liquid fermentation medium in the early stage of the liquid fermentation of monascus.

[0010] Specifically, citrulline is added to the liquid fermentation medium before inoculation of the liquid fermentation of monascus.

[0011] Specifically, the formula raw materials of the liquid fermentation medium include rice powder, glycerol, peptone, sodium nitrate, magnesium sulfate, zinc sulfate and potassium dihydrogen phosphate.

[0012] Preferably, the formula raw materials of the liquid fermentation medium include rice powder 15-30 g / L, glycerol 80-100 g / L, peptone 5-15 g / L, sodium nitrate 2-10 g / L, magnesium sulfate 0.5-2 g / L, zinc sulfate 1-4 g / L and potassium dihydrogen phosphate 1-3 g / L.

[0013] Preferably, the content of citrulline is 4-8 g / L in volume of the liquid fermentation medium.

[0014] Specifically, the liquid fermentation conditions of Monascus include culturing for 2-4 days in an air bath at 28-32 ℃ and 170-200 r / min, and then culturing for 10-15 days in an air bath at 24-26 ℃ and 130-160 r / min.

[0015] The present application has the following advantages:

[0016] The present application has the following advantages: BRIEF DESCRIPTION OF DRAWINGS

[0017] The specific embodiments of the present application will be further described in detail below with reference to the accompanying drawings.

[0018] Figure 1 The figure is the high performance liquid chromatogram of the acid type and ester type of Monacolin K.

[0019] Figure 2 The figure is the influence of adding citrulline on the yield of Monacolin K produced by liquid fermentation.

[0020] Figure 3 The figure is the comparison of the yield of Monacolin K after fermentation. DETAILED DESCRIPTION

[0021] The technical solutions in the present application will be described clearly and completely below. Obviously, the described embodiments are only some of the embodiments of the present application, but not all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative work fall within the protection scope of the present application.

[0022] Embodiment 1

[0023] Application of citrulline in improving the yield of monacolin K produced by liquid fermentation of monascus.

[0024] Embodiment 2

[0025] Application of citrulline in improving the yield of monacolin K produced by liquid fermentation of monascus, the specific application steps include: (1) strain activation; (2) preparation of seed liquid; (3) liquid fermentation; wherein, before inoculation of the liquid fermentation of monascus, citrulline is added to the liquid fermentation medium, that is, citrulline is used as a formula component of the liquid fermentation medium, or citrulline is added to the liquid fermentation medium in the early stage of the fermentation of monascus, that is, citrulline is used as a growth factor of the fermentation.

[0026] Embodiment 3

[0027] Application of citrulline in improving the yield of monacolin K produced by liquid fermentation of monascus, the specific application steps include:

[0028] (1) strain activation: the monascus strain is cultured on PDA medium for activation;

[0029] (2) preparation of seed liquid: the inoculation ring is used to scrape the bacterial liquid from the culture medium of step (1) to the liquid seed culture medium, so as to prepare the seed liquid;

[0030] (3) liquid fermentation: the seed liquid is inoculated into the liquid fermentation medium at a volume ratio of 8-15% (v / v), and is first cultured in an air bath at 28-32℃ and 170-200 r / min for 2-4 days, and then is transferred to an air bath at 24-26℃ and 130-160 r / min for 10-15 days; the formula raw materials of the liquid fermentation medium include rice powder 15-30 g / L, glycerol 80-100 g / L, peptone 5-15 g / L, sodium nitrate 2-10 g / L, magnesium sulfate 0.5-2 g / L, zinc sulfate 1-4 g / L, and potassium dihydrogen phosphate 1-3 g / L;

[0031] Wherein, the citrulline can be added into the liquid fermentation medium before inoculation of the Monascus liquid fermentation, i.e. the citrulline is added as a formula component of the liquid fermentation medium, i.e. the formula raw materials of the liquid fermentation medium are as follows: rice powder 15-30 g / L, glycerol 80-100 g / L, peptone 5-15 g / L, sodium nitrate 2-10 g / L, magnesium sulfate 0.5-2 g / L, zinc sulfate 1-4 g / L, potassium dihydrogen phosphate 1-3 g / L, and citrulline 4-8 g / L; or the citrulline is added into the liquid fermentation medium in the early stage of the Monascus liquid fermentation, i.e. the citrulline is used as a growth factor of the fermentation, and the content of the citrulline is controlled to be 4-8 g / L based on the volume of the liquid fermentation medium.

[0032] Example 4

[0033] Effect of citrulline on the production of monacolin K of Monascus.

[0034] 1. Experimental procedure

[0035] Strain activation: the Monascus strain is cultured on PDA medium at 30°C for 4 days, and activated for 2 generations.

[0036] Preparation of seed liquid: 1 ring of bacteria liquid is scraped from the culture medium with an inoculation ring into the liquid seed culture medium, and the culture conditions are as follows: temperature 30°C, rotation speed 180 r / min, and air bath culture for 3 days.

[0037] Liquid fermentation: the seed liquid is inoculated into the liquid fermentation medium at a volume ratio of 10% (v / v), and cultured at 30°C and 180 r / min for 3 days, and then transferred to an air bath at 25°C and 150 r / min for 15 days, and the content of monacolin K is detected periodically during the culture process; wherein, the content of citrulline (analytical pure, produced by Shanghai Yuan Ye Biological Technology Co., Ltd.) in the liquid fermentation medium is controlled to be 4 g / L, 6 g / L and 8 g / L respectively during preparation of the liquid fermentation medium, so as to investigate the effect of different contents of citrulline in the liquid fermentation medium on the production of monacolin K, and the detection results are shown in Figure 2 and Figure 3 ;

[0038] 2. Detection of monacolin K

[0039] Pre-treatment: 5 mL of the fermentation liquid is taken in a 50 mL centrifuge tube, 70% ethanol is added at a volume ratio of 1:4 with respect to the fermentation liquid, ultrasonic extraction is performed for one hour, and then the sample is left overnight, and then filtered to a liquid phase vial by using a 0.45 μm organic microporous filter membrane, and the filtrate is used for detection of monacolin K.

[0040] Detection: HPLC was used with a Shim-pack GIST column (2.1×100mm, 2μm), column temperature 25℃, UV detector at 238nm, mobile phase: acetonitrile:water:phosphoric acid = 650:350:0.6 (v / v); flow rate: 1.0mL / min, injection volume 10μL. Detection was performed at 238nm using a UV detector. Qualitative analysis was based on the retention time of the analyte and standard, and quantification was based on the peak area ratio of the analyte and standard. The ultra-high performance liquid chromatograph used was an LC-30A, manufactured by Shimadzu Corporation, Japan; Monacoline K standard was chromatographically pure and manufactured by Shanghai Maclean Biotechnology Co., Ltd.

[0041] 3. Results Analysis

[0042] Depend on Figure 2 and Figure 3 It was found that when citrulline was added at concentrations of 4 g / L, 6 g / L, and 8 g / L, the yield and accumulation rate of monacolin K in the fermentation broth significantly increased, indicating that citrulline has a promoting effect on the production of monacolin K through liquid fermentation. At the end of fermentation, i.e., on day 15 of cultivation, the yield of monacolin K in the fermentation broth without citrulline was only 85.40 mg / L, while the yields of monacolin K in the fermentation broth with citrulline additions of 4 g / L, 6 g / L, and 8 g / L were 270.74 mg / L, 223.87 mg / L, and 265.29 mg / L, respectively, approximately three times that of the group without citrulline. Therefore, applying citrulline to the liquid fermentation of Monascus purpureus to produce monacolin K can significantly increase the yield and accumulation rate of monacolin K, and has significant potential for promotion and utilization.

Claims

1. The application of citrulline in increasing the yield of Monakolin K produced by liquid fermentation of Monascus purpureus, characterized in that, The liquid fermentation culture medium formula includes rice flour 15-30 g / L, glycerol 80-100 g / L, peptone 5-15 g / L, sodium nitrate 2-10 g / L, magnesium sulfate 0.5-2 g / L, zinc sulfate 1-4 g / L, potassium dihydrogen phosphate 1-3 g / L, and citrulline 4-8 g / L.

2. The application as described in claim 1, characterized in that, Citrulline was added to the liquid fermentation medium during the initial stage of the Monascus purpureus liquid fermentation.

3. The application as described in claim 1, characterized in that, Citrulline was added to the liquid fermentation medium before the Monascus purpureus liquid fermentation was carried out.

4. The application as described in claim 1, characterized in that, The conditions for liquid fermentation of Monascus purpureus are as follows: first, culture in an air bath at 28–32°C and 170–200 r / min for 2–4 days, and then transfer to an air bath at 24–26°C and 130–160 r / min for 10–15 days.

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