Preparation method of nitrogen source for improving monascus fermentation efficiency

Abstract

The invention discloses a preparation method of a nitrogen source for improving monascus fermentation efficiency, and belongs to the technical field of fermentation substrate modification. The nitrogen source is prepared by the following method: uniformly spreading soybean meal, and performing microwave treatment to obtain the monascus fermentation nitrogen source. The microwaves are utilized to modify the soybean meal, the nutritional structure of the soybean meal can be well maintained, the soybean meal is used as a nitrogen source, growth and metabolic activity of monascus can be remarkably promoted, accumulation of metabolite such as monascus pigment and monacolin K with important application value is facilitated, and the yield of the monascus is increased. Therefore, the method has good application prospect and value in the technical field of fermentation substrate modification.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of fermentation substrate modification, and particularly relates to a preparation method of a nitrogen source for improving Monascus fermentation efficiency. BACKGROUND

[0002] Monascus has a wide range of applications in many fields such as brewing, vinegar making, and color and flavoring of fermented bean curd. The optimization of its culture and growth conditions has always been the core problem of related research and industrial production. Monascus needs carbon source, nitrogen source, water, inorganic salt and growth factor and other basic nutrients for growth, and the nitrogen source plays a key role in maintaining cell metabolic activity and promoting the growth process. The nitrogen source mainly supports the life activities of microorganisms by synthesizing nitrogen-containing compounds such as amino acids and proteins. At present, the commonly used organic nitrogen sources in industrial production include yeast extract, beef extract, protein peptone, protein hydrolysate and peanut cake powder. Among them, commercial protein peptone is usually made from different raw material proteins such as soybeans, meat, fish and casein. However, traditional animal-based nitrogen sources need to face the risk of disease transmission, cultural taboos and production cost problems in application. In order to cope with these challenges, plant-based nitrogen sources have gradually become the research focus in the field of microbial fermentation in recent years and are widely proposed as an ideal choice to replace traditional animal-based nitrogen sources. Plant-based nitrogen sources have the characteristics of low risk, high safety and wide source. At the same time, the production cost of plant-based nitrogen source is usually low, which helps to reduce the overall cost of Monascus fermentation industry. Therefore, it is of great significance to develop a plant-based nitrogen source. SUMMARY

[0003] The application provides a preparation method of a nitrogen source for improving Monascus fermentation efficiency, and the steps are as follows: uniformly spreading soybean meal, microwave treatment, and obtaining Monascus fermentation nitrogen source.

[0004] In the preparation method, the microwave treatment conditions are as follows: microwave power is 10w-1000w, and microwave time is 10s-300s; preferably, microwave power is 90w, and microwave time is 60s.

[0005] The application provides a Monascus fermentation nitrogen source prepared by the above method.

[0006] The application provides application of the above Monascus fermentation nitrogen source in improving Monascus fermentation efficiency; the fermentation efficiency is growth performance or metabolic performance; wherein the metabolic performance refers to accumulation of secondary metabolites, including accumulation of Monascus pigment and Monacolin K and the like.

[0007] The application provides a Monascus fermentation medium, which comprises the following components: Monascus fermentation nitrogen source 10 g / L, corn flour 60 g / L, glucose 30 g / L, zinc sulfate 0.2 g / L, magnesium sulfate 1 g / L, potassium dihydrogen phosphate 5 g / L.

[0008] The beneficial effects of the present application are: The present application uses microwave to modify the soybean meal, which can better maintain the nutritional structure of the soybean meal, and can significantly promote the growth and metabolic activity of Monascus when the soybean meal is used as a nitrogen source, which is beneficial to the accumulation of Monascus pigment and other metabolites such as monacolin K with important application value, thereby having good application prospect and value in the field of fermentation substrate modification technology. BRIEF DESCRIPTION OF DRAWINGS

[0009] Figure 1 The influence of different nitrogen sources on the pigment yield in the Monascus fermentation process.

[0010] Figure 2 The influence of different nitrogen sources on the monacolin K yield in the Monascus fermentation process.

[0011] Figure 3 The influence of microwave power on the growth and metabolism of Monascus.

[0012] Figure 4 The influence of microwave time on the growth and metabolism of Monascus. DETAILED DESCRIPTION

[0013] The materials used in the present application are as follows: Soybean meal powder (SMP) from Shandong Wande Fu Company; ultralow-temperature vibration ultrafine grinder (KCW-10S) from Beijing Kujie Yucheng Machinery Equipment Co., Ltd.; high-pressure homogenizer (AH-NANO) from Antao Sina Nano Technology (Suzhou) Co., Ltd.; microwave drying and sterilization equipment (QW-12M3) from Guangzhou Kewei Microwave Co., Ltd.; ultrasonic cell disruptor (SCIENTZ-IID) from Ningbo Xinzhi Biological Technology Co., Ltd.

[0014] Other materials used in the present application, unless otherwise stated, can be obtained through commercial channels. Unless otherwise stated, the other terms used in the present application generally have the meanings commonly understood by those of ordinary skill in the art. The present application will be described in further detail below in conjunction with specific examples and data. The following examples are only intended to illustrate the present application, and do not limit the scope of the present application in any way.

[0015] Example 1 The soybean meal powder was treated by microwave (MW), ultrasonic disruption (UT), ultrafine grinding (UFG), and high-pressure homogenization (HPH), respectively, and the influence of different physical modifications on the growth and metabolic indicators of Monascus was studied. A control group (CK) was set, and the soybean meal powder in the control group was not treated.

[0016] Preparation of plant nitrogen source, the method is as follows: Microwave (MW): 1 kg of soybean meal is evenly and smoothly laid on the conveyor belt, the power of the microwave dryer is adjusted to 900 w, and the time is 300 s. The treated soybean meal powder is placed in a sealed bag and stored in a drying dish.

[0017] Ultrasonic fragmentation (UT): The soybean meal is added to deionized water at a ratio of 1:8 to form a soybean meal solution. The soybean meal solution is placed in an ultrasonic cell disruptor, and the total ultrasonic time is 30 min. After treatment, freeze-drying is performed. The ultrasonic fragmentation conditions are as follows: the power of the ultrasonic cell disruptor is adjusted to 80% of the total power (900 w), the ultrasonic interval is 1 s, the ultrasonic time is 5 s, and the ultrasonic temperature is adjusted to 25 °C.

[0018] Ultrafine grinding (UFG): 1 kg of soybean meal is added to an ultrafine grinder and ground for 40 min. Then it is packed in a sealed bag and stored in a drying dish at room temperature. The ultrafine grinding conditions are as follows: the temperature is adjusted to 0-6 °C.

[0019] High pressure homogenization (HPH): The soybean meal is added to deionized water at a ratio of 1:10 to form a soybean meal solution. The soybean meal solution is subjected to homogenization treatment using a high pressure homogenizer to obtain a soybean meal dispersion, which is freeze-dried. The homogenization conditions are as follows: 40 Mpa, 2 times.

[0020] Example 2 Monascus medium: The Monascus medium includes PDA liquid medium, PDA solid medium and fermentation medium. The formula of the fermentation medium is as follows: nitrogen source 10 g / L, corn flour 60 g / L, glucose 30 g / L, zinc sulfate 0.2 g / L, magnesium sulfate 1 g / L, potassium dihydrogen phosphate 5 g / L.

[0021] Monascus cultivation method: The slant strain of the starting strain Monascus is transferred to PDA solid medium and cultured in a 30 °C constant temperature incubator for 7 days. Two rings are picked from the solid medium and transferred to PDA liquid medium, which is cultured in a 30 °C constant temperature incubator for 20 days to complete activation and be ready for use.

[0022] The soybean meal obtained by the different treatment methods in Example 1 above is used as a microbial nitrogen source. 2 mL of activated bacterial liquid is inoculated into the fermentation medium and placed in a 30 °C constant temperature incubator for culture. After completion of sampling, subsequent detection is performed.

[0023] I. Study on the metabolic status of Monascus 1. Determination of Monascus pigment Monascus pigment is a kind of natural multi-component pigment produced by Monascus during fermentation process, mainly showing yellow, orange, red and other color tones, with good coloring and color-enhancing application value, and thus widely used in food natural coloring agents.

[0024] The fermentation broth was centrifuged at 5000 r / min for 20 min, and the supernatant was reserved; the precipitate was mixed with 95% ethanol, and ultrasonic extraction (ultrasonic frequency 10 kHz) was performed for 60 min. The extraction liquid was centrifuged at 5000 r / min for 20 min, and the precipitate at the bottom was collected again, and extracted with 95% ethanol and centrifuged, repeated three times. The fermentation supernatant and the alcohol extraction supernatant were combined to obtain the pigment extraction liquid. The pigment extraction liquid was appropriately diluted, 95% ethanol solution was used as a blank control, and the OD values at the maximum absorption wavelengths of red pigment, orange pigment and yellow pigment (505 nm, 448 nm and 420 nm) were determined by ultraviolet spectrophotometer, and the color values of red pigment, orange pigment and yellow pigment were calculated. The formula for calculating the color value of pigment is: color value of pigment = OD* wavelength, and the unit of color value is AU / mL.

[0025] The test results are shown in Table 1. Figure 1 During the cultivation of Monascus, different nitrogen sources showed significant differences in the synthesis of the three pigments (red pigment, orange pigment and yellow pigment), and the content change trend was closely related to the metabolic characteristics of the nitrogen source. Among them, when MW and UT were used as the nitrogen source, the content of the three pigments was higher.

[0026] 2. Determination of Monacolin K Monacolin K is a common natural active ingredient in Monascus, and its chemical structure is similar to that of Lovastatin, a lipid-lowering drug. It mainly inhibits HMG-CoA reductase, a key pathway, reduces the synthesis of cholesterol in the body, and thus helps to reduce cholesterol in the blood, which has certain value in improving blood lipid levels, supporting cardiovascular health and reducing the risk of atherosclerosis.

[0027] The detection of acid Monacolin K content adopts HPLC method.

[0028] (1) Sample processing The fermentation broth was placed in a petri dish and freeze-dried into powder. 0.04 g of powder was added to a colorimetric tube with a capacity of 10 mL. In order to further increase the contact area of the sample and the reagent, and to ensure accurate control, 70% ethanol solution was used to make up to 6 mL. Under the condition of constant temperature 55 ℃, the sample was placed in water bath for 1 h. During this period, the sample was shaken every 20 min to ensure uniform mixing. After the completion of water bath, the extraction liquid was taken out and filtered using a 0.22 μm filter membrane to obtain a sample solution (4 mL) for standby. ​

[0029] (2) Draw the standard curve of acid Monacolin K Take 20 mg of Monacolin K standard, and place it in a 100 mL volumetric flask. Add 90 mL of 70% ethanol solution at 55°C, adjust the pH of the solution to 7.7 using 20% sodium hydroxide solution, and dilute to volume with 70% ethanol to obtain a standard solution with a mass concentration of 200 mg / L. Dilute the 200 mg / L standard solution with 70% ethanol to obtain standard working solutions with concentrations of 0, 50 mg / L, 100 mg / L, 150 mg / L, and 200 mg / L, and pass them through a 0.22 μm filter membrane for high performance liquid chromatography determination.

[0030] High performance liquid chromatography conditions: detection wavelength 238 nm, C18 chromatographic column (150 mm x 4.6 mm x 5 μm), column temperature 28°C, mobile phase water and acetonitrile (45:55, V / V), H3PO4 solution as buffer, pH 2.5, injection volume 20 μL. Flow rate 1.0 mL / min.

[0031] Draw the standard curve with the mass concentration (X) of acid Monacolin K as the abscissa and the peak area (Y) as the ordinate, and obtain the linear regression equation Y=60.892X+49.56, with a correlation coefficient R 2 0.999.

[0032] (3) Monacolin K content calculation Determine the sample solution by high performance liquid chromatography, calculate the acid Monacolin K content according to the standard curve regression equation, and the calculation formula is as follows: ; In the formula, A0 is the peak area of acid MK; 4 is the sample volume, mL; C0 is the mass concentration of acid MK standard, μg / mL; A is the peak area of acid standard; and 0.04 is the powder mass, g.

[0033] The test results are shown in Table 1: Figure 2 During the cultivation of Monascus, different nitrogen sources have a significant impact on the synthesis of Monacolin K, and the content change is closely related to the metabolic characteristics of the nitrogen source and the cultivation stage. Among them, when MW is used as the nitrogen source, the content of Monacolin K is the highest.

[0034] ​The above experiment takes the yield of the characteristic metabolite of Monascus as the main evaluation index to screen the optimal nitrogen source type for Monascus fermentation. With the goal of maximizing the yield of metabolites, microwave soybean meal (MW) is ultimately determined as the optimal nitrogen source for Monascus to produce red pigment / Monacolin K, thereby providing a theoretical basis and process reference for subsequent optimization of soybean meal nitrogen source composition and directional high yield of model strains.

[0035] II. Optimization of nitrogen source preparation method For the microwave treatment method, further explore the influence of soybean meal on the growth and metabolism of Monascus under different microwave power and microwave time treatment conditions. The research indicators include fermentation broth pH, Monascus biomass, protein content, polypeptide content, red pigment content, and Monacolin K content.

[0036] The determination methods of the above indicators are as follows: Fermentation broth pH: use a pH meter to determine the pH value of the fermentation broth.

[0037] Monascus biomass: take 10 mL of Monascus fermentation broth in a centrifuge tube, centrifuge at 10000 r / min for 10 min, and measure the volume V of the supernatant. The mycelium concentration calculation formula is as follows: .

[0038] Protein content: refer to GB 5009.5-2016 "National Food Safety Standard Determination of Protein in Food" to determine the total nitrogen content by Kjeldahl nitrogen determination method, and calculate the protein content.

[0039] Polypeptide content: refer to the method of Shi Xiaodan (Shi Xiaodan, 2022), mix the fermentation broth with 20% trichloroacetic acid in equal volume, centrifuge at 10000 r / min for 10 min, take 200 µL of supernatant, add 600 µL of biuret reagent A, shake uniformly, add 200 µL of biuret reagent B, measure the absorbance at wavelength 540 nm, and substitute into the bovine serum protein standard curve to obtain the concentration of polypeptides in the sample.

[0040] Standard curve: take 200 µL of standard bovine serum protein solution with different concentration gradients, add 600 µL of biuret reagent A, shake uniformly, add 200 µL of biuret reagent B, mix well, and measure the absorbance at wavelength 540 nm to draw the bovine serum protein standard curve.

[0041] Red pigment content: refer to the above red pigment determination method of Monascus.

[0042] Monacolin K content: refer to the above Monacolin K determination method.

[0043] (I) Microwave power selection of soybean meal The power of the microwave dryer was adjusted to 10%, 30%, 50%, 80%, and 100% of the total power (900w), and the soybean meal was evenly spread in the microwave dryer. The total microwave time was 1 min.

[0044] According to the fermentation method of Example 2, Monascus was cultured, and the fermentation time was 168 h. The pH of the fermentation broth, the biomass of Monascus, the protein content, the polypeptide content, the red pigment content, and the monacolin K content were determined.

[0045] The test results are shown in Table 1. Figure 3 The pH values were not significantly different among the treatment groups, all of which were between 6.0 and 6.35 (Figure A). The biomass reached the highest level (21.67 g / L) under the 10% power treatment, which was significantly higher than that of the other groups (p < 0.05), indicating that the soybean meal treated with low power microwave was more conducive to the growth of the bacteria (Figure B). The protein concentration in the soybean meal also reached the highest level (2.78 mg / mL) under the 10% power treatment, and the protein concentration in the soybean meal showed a downward trend as the power increased, suggesting that high power led to protein thermal denaturation (Figure C). The polypeptide content in the soybean meal reached the highest level (more than 18 mg / mL) under the 10% and 50% power treatments, which was higher than that of the other groups (Figure D). The red pigment yield reached the peak value (69.45 AU / mL) under the 10% power treatment, and the other groups were slightly lower (Figure E). Monacolin K reached the highest level (76.17 μg / L) under the 10% power treatment, which was significantly better than that of the other treatments (p < 0.05) (Figure F).

[0046] In summary, 10% microwave power is the most conducive to maintaining the nutritional structure of soybean meal, promoting the growth of Monascus, and accumulating active metabolites, providing an effective reference for nitrogen source modification.

[0047] (II) Microwave time screening of soybean meal The microwave time was set to 30s, 60s, 90s, 120s, and 180s, and the soybean meal was evenly spread in the microwave dryer. The microwave power was 50% (total power 900w).

[0048] According to the fermentation method of Example 2, Monascus was cultured, and the fermentation time was 168 h. The pH of the fermentation broth, the biomass of Monascus, the protein content, the polypeptide content, the red pigment content, and the monacolin K content were determined.

[0049] The test results are shown in Table 1. Figure 4 ​​The microwave time had little effect on the pH of the fermentation broth, and the pH of each treatment group was maintained at about 6 (Figure A). The biomass reached a peak (about 19.22 g / L) at a microwave treatment time of 60 s, which was significantly higher than that of other groups (p < 0.05) (Figure B), and the monacolin K yield was also the highest (83.83 mg / L) (Figure F), indicating that 60 s treatment was beneficial to activate the secondary metabolic pathway and improve physiological activity. The red pigment yield was better at a microwave treatment time of 30 s, suggesting that short-time treatment helps to preserve the pigment precursor (Figure E). The protein concentration in soybean meal showed a decreasing trend with the extension of microwave time, decreasing from 2.23 mg / mL in the 30 s treatment group to 1.02 mg / mL in the 180 s group (p < 0.05), which may be related to protein thermal denaturation or degradation (Figure C). The polypeptide content in soybean meal showed no significant difference among all treatment groups, indicating that its release was relatively stable (Figure D).

[0050] In summary, 60 s microwave treatment has the advantages of promoting cell growth and functional metabolite accumulation, providing an effective strategy for optimizing soybean meal pretreatment process and improving the fermentation efficiency of Monascus.

[0051] The above description is only a preferred embodiment of the present application, and is not intended to limit other forms of the present application. Any skilled person in the art can use the disclosed technical content to make changes or modifications to equivalent embodiments. However, any simple modification, equivalent change and modification made according to the technical essence of the present application to the above embodiments, without departing from the technical solution of the present application, still falls within the protection scope of the present application.

Claims

1. A method for preparing a nitrogen source for improving Monascus fermentation efficiency, characterized in that, The steps are as follows: evenly spread the soybean meal, microwave treatment, obtain monascus fermentation nitrogen source.

2. The production method according to claim 1, characterized by, The microwave treatment conditions are: microwave power 10w~1000w, microwave time 10s~300s.

3. The production method according to claim 2, characterized by, The microwave treatment conditions are: microwave power 90w, microwave time 60s.

4. The monascus fermentation nitrogen source prepared by the method of any one of claims 1-3.

5. The monascus fermentation nitrogen source of claim 4 is used to improve the fermentation efficiency of monascus.

6. Use according to claim 5, characterized in that, The fermentation efficiency is growth performance or metabolic performance.

7. Use according to claim 6, characterized in that, The metabolic performance refers to the accumulation of secondary metabolites.

8. Use according to claim 7, characterized in that, The secondary metabolites include monascus pigments, monacolin K.

9. A Monascus fermentation medium, characterized in that, Comprise the following components: The monascus fermentation nitrogen source of claim 4 is 10 g / L, corn meal is 60 g / L, glucose is 30 g / L, zinc sulfate is 0.2 g / L, magnesium sulfate is 1 g / L, potassium dihydrogen phosphate is 5 g / L.

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