Liquid fermentation culture medium for monascus strain fermentation as well as preparation method and fermentation process of liquid fermentation culture medium

Abstract

The invention discloses a liquid fermentation culture medium for monascus strain fermentation as well as a preparation method and a fermentation process of the liquid fermentation culture medium, and relates to the technical field of microbial fermentation. The liquid fermentation culture medium for monascus strain fermentation comprises a carbon source, corn steep liquor dry powder, peanut protein powder, an inorganic nitrogen source, arginine, ferrous sulfate, inorganic salt and no more than 0.05% of an antifoaming agent according to a specific ratio. The content of protein in the peanut protein powder is 55-65wt%, and the content of grease is 5-6wt%. By optimizing the composition of the liquid fermentation culture medium and under the cooperative action of all the components, the growth of the monascus strain can be promoted, the production capacity of the strain can be improved, and a metabolite with a purple-red appearance and with the maximum absorption peak of 505-510nm of a sample can be obtained.

Description

Technical Field

[0001] The present application relates to the field of microbial fermentation technology, and in particular to a liquid fermentation medium for Monascus species fermentation, a preparation method thereof, and a fermentation process. Background Art

[0002] In the field of Monascus fermentation, the composition of the liquid fermentation medium plays a vital role in the growth, metabolism and color production ability of the strain.

[0003] Monascus red pigment is a metabolite produced during the fermentation process of Monascus species. It is usually a single pigment. The maximum absorption peak of the sample is between 495nm-505nm (excluding 505nm), and the appearance is red. Among them, the test method for the maximum absorption peak of the sample mentioned in this application is as follows: take a Monascus red sample and dissolve it in 100ml of ethanol aqueous solution (1+1) to obtain a sample solution with a color value range of 0.4-0.8u / ml, scan the sample solution under visible light, and record the maximum absorption peak. However, some customers require Monascus red pigment with a purple-red appearance (the maximum absorption peak of the sample is 505-510nm). Therefore, how to stably obtain a Monascus red pigment with a purple-red appearance is one of the goals of this application.

[0004] In addition, bubbles are easily generated during the fermentation process of Monascus species, and the presence of bubbles will affect the metabolic activity of Monascus species, which is not conducive to the improvement of the color production ability of Monascus species. In the related art, defoamers are added to reduce the occurrence of bubbles, but the effect of adding a small amount of defoamer is not obvious, and increasing the amount of defoamer will inhibit the growth of Monascus species, which is not conducive to obtaining metabolites with high color value.

[0005] This application mainly studies how to stably promote the growth of strains from the perspective of liquid fermentation culture medium, obtain metabolites with a purple-red appearance and a maximum absorption peak of 505-510nm while improving the color production ability of the strains, and prevent the generation of a large number of bubbles during the fermentation process. Summary of the invention

[0006] In order to promote the growth of the strain, obtain a metabolite with a purple-red appearance and a maximum absorption peak of the sample at 505-510nm while improving the color production ability of the strain, and prevent the generation of a large number of bubbles during the fermentation process, the present application provides a liquid fermentation medium for the fermentation of Monascus species, a preparation method thereof, and a fermentation process.

[0007] In the first aspect, the present application provides a liquid fermentation medium for Monascus species fermentation using the following technical solution: A liquid fermentation medium for Monascus species fermentation, comprising a carbon source, a nitrogen source, an inorganic salt, trace elements, a defoamer and deionized water; The mass proportion of the carbon source is 9.5-10.5%; The nitrogen source comprises corn steep liquor powder, peanut protein powder and an inorganic nitrogen source, wherein the weight proportion of the corn steep liquor powder is 1.8-2.5%, the weight proportion of the peanut protein powder is 3-3.5%, and the weight proportion of the inorganic nitrogen source is 0.1-0.15%; wherein the protein content of the peanut protein powder is 55-65wt%, and the fat content is 5-6wt%; The trace elements include arginine and ferrous sulfate, wherein the mass proportion of the arginine is 0.4-0.8%, and the mass proportion of the ferrous sulfate is 0.001-0.004%; The mass proportion of the inorganic salt is 0.4-0.8%; The mass proportion of the defoaming agent does not exceed 0.05%.

[0008] Carbon source is the main energy source for the growth and metabolism of Monascus species. The carbon source mass ratio in this application is controlled to be 9.5-10.5%. Too much carbon source can easily lead to changes in the mycelium morphology, such as mycelium entanglement and agglomeration, which is not conducive to the normal growth and metabolism of Monascus. Nitrogen source is the synthetic raw material of nitrogenous compounds such as protein, nucleic acid, enzyme in Monascus species. In the present application, the nitrogen source of the liquid fermentation medium of Monascus species fermentation includes corn steep liquor powder, peanut protein powder and inorganic nitrogen source. Through the compounding of multiple nitrogen sources, the growth of Monascus species is promoted, and the color production ability of Monascus species is improved. Wherein, corn steep liquor powder is obtained by freeze drying of fresh corn steep liquor, and its water-soluble protein is well preserved, which is conducive to the consumption and absorption of Monascus species. Peanut protein powder is obtained by concentrating and freeze drying the protein solution extracted from peanut cake powder, and the main substance of peanut protein powder is protein, which contains a certain amount of fat, vitamins, etc. synchronously, while promoting the growth and color production ability of Monascus species, it can play a defoaming effect, which is conducive to reducing the addition of defoamer in liquid fermentation medium, to reduce the impact of adding defoamer on the growth of Monascus species. Inorganic nitrogen sources have low costs, and the addition of appropriate concentrations of inorganic nitrogen sources can synergistically promote the growth and development of Monascus species with other nitrogen sources, helping to improve the color production ability of Monascus species.

[0009] Inorganic salts help maintain the osmotic pressure balance inside and outside the Monascus cells and can be added as needed.

[0010] Trace elements include arginine and ferrous sulfate. Through the combined action of arginine and ferrous sulfate, Monascus can obtain purple-red metabolites (the maximum absorption peak of the sample is 505-510nm), which can meet the specific needs of customers.

[0011] In some specific embodiments, the mass proportion of the arginine is 0.55-0.65%, and the mass proportion of the ferrous sulfate is 0.0015-0.0025%.

[0012] In the present application, the mass proportion of arginine is 0.55-0.65%, and the mass proportion of ferrous sulfate is 0.0015-0.0025%, which can further improve the color production ability of Monascus species while obtaining purple-red metabolites.

[0013] In some specific embodiments, the nitrogen source further comprises pancreatic casein, and the mass proportion of the pancreatic casein is 0.1-0.15%.

[0014] In the present application, the nitrogen source of the liquid fermentation medium also includes trypsin casein, which can further promote the growth of Monascus purpurogenum fungi and improve the color production ability of Monascus purpurogenum fungi.

[0015] In some specific embodiments, the carbon source is rice flour.

[0016] In some specific embodiments, the inorganic salt is at least one of magnesium sulfate, sodium chloride, potassium dihydrogen phosphate, and dipotassium hydrogen sulfate.

[0017] In some specific embodiments, the inorganic nitrogen source is at least one of sodium nitrate, ammonium sulfate and ammonium nitrate.

[0018] In a second aspect, the present application provides a method for preparing a liquid fermentation medium for Monascus species fermentation using the following technical scheme: A method for preparing a liquid fermentation medium for Monascus species fermentation comprises the following steps: Add carbon source, corn syrup powder, peanut protein powder, inorganic nitrogen source, inorganic salt, ferrous sulfate and defoamer into part of deionized water, stir until uniformly dispersed, and sterilize at high temperature to obtain material A; After the remaining deionized water is sterilized, arginine is added, stirred until it is evenly dissolved, and filtered through a microfiltration membrane with a pore size of 0.20-0.25 μm. The obtained filtrate is material B; Mix material A and material B evenly, and then adjust the pH to 3.9-4.0 to obtain a liquid fermentation medium.

[0019] Since high temperature will destroy the chemical structure of arginine, causing it to lose its original chemical properties and biological activity, the present application uses a microfiltration membrane to filter impurities from arginine, which is beneficial to fully exert the effect of arginine.

[0020] In some specific embodiments, ferrous sulfate is also added to material A.

[0021] In some specific embodiments, a fermentation process of Monascus species comprises the following steps: S1. Activation of bacterial strains: Activation and cultivation of starting bacterial strains; S2, seed liquid culture: inoculate the activated starting strain into the seed liquid for culture; S3, fermentation culture: after the cultivation in step S2 is completed, the seed liquid of S2 is transferred to any one of the liquid fermentation medium for fermentation of Monascus species described in any one of the above, and cultured in a constant temperature shaker at 30-35°C, with a rotation speed of 150-250r / min, and the pH value of 5.60-5.80 is taken as the fermentation end point.

[0022] In the present application, pH 5.60-5.80 is taken as the fermentation end point of Monascus purpurogenous bacteria. At this time, continued cultivation will no longer increase the pigment, and continued cultivation is not conducive to the extraction of subsequent products.

[0023] In some specific embodiments, in S1, the starting strain is any one of the Monascus purpurogenum strains numbered CGMCC No.3.15548, CGMCC No.3.15547, CGMCC No.3.15546, CGMCC No.3.898, and CGMCC No.3.896 purchased from the Institute of Microbiology, Chinese Academy of Sciences.

[0024] Different Monascus species have different color production abilities and the colors of their metabolites. The liquid fermentation medium in the present application is developed based on the above-mentioned numbered Monascus species.

[0025] In some specific embodiments, in S3, the inoculation amount of the seed liquid is 4.5%-5.5% (v / v) of the liquid fermentation medium for fermentation of Monascus species.

[0026] In summary, this application at least includes the following beneficial technical effects: (1) The present application optimizes the composition of the liquid fermentation medium and, under the coordinated action of various components, can promote the growth of Monascus species, improve the production capacity of the species, and obtain a metabolite with a purple-red appearance and a maximum absorption peak of the sample at 505-510nm.

[0027] (2) In the present application, the mass proportion of arginine is 0.55-0.65%, and the mass proportion of ferrous sulfate is 0.0015-0.0025%, which can further improve the color production ability of Monascus species while obtaining purple-red metabolites. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1It is a schematic diagram of the fermentation process of a Monascus species of the present application.

[0029] Description of reference numerals: S1, bacterial strain activation; S2, seed liquid culture; S3, fermentation culture. DETAILED DESCRIPTION

[0030] The present application is further described below in conjunction with specific experiments. Example

[0031] [Example 1] A liquid fermentation medium for fermentation of Monascus species, prepared from the following raw materials in percentage by weight: Rice flour: 9.5%; Corn syrup powder: 2.5%; Peanut protein powder: 3.5%; in peanut protein powder, the protein content is 55wt%, and the fat content is 5wt%; Sodium nitrate: 0.10%; Magnesium sulfate: 0.4%; Arginine: 0.4%; Ferrous sulfate: 0.004%; Defoamer: 0.02%; the defoamer uses silicone defoamer, model BYK-024; Deionized water: balance.

[0032] In this embodiment, the method for preparing a liquid fermentation medium for Monascus species fermentation comprises the following steps: Add rice flour, corn syrup powder, peanut protein powder, sodium nitrate, magnesium sulfate, ferrous sulfate and defoaming agent into 85% deionized water, stir until uniformly dispersed, and sterilize at 121° C. to obtain material A; After the remaining 15% of deionized water is sterilized at 121°C, arginine is added, stirred until evenly dissolved, and filtered through a microfiltration membrane with a pore size of 0.20-0.25 μm. The obtained filtrate is material B; Mix material A and material B evenly, and then adjust the pH to 3.9-4.0 to obtain a liquid fermentation medium.

[0033] [Example 2] A liquid fermentation medium for fermentation of Monascus species, prepared from the following raw materials in percentage by weight: Rice flour: 10.5%; Corn syrup powder: 1.8%; Peanut protein powder: 3%; in peanut protein powder, the protein content is 55wt%, and the fat content is 5wt%; Ammonium sulfate: 0.15%; Magnesium sulfate: 0.4%; Arginine: 0.8%; Ferrous sulfate: 0.001%; Defoamer: 0.03%; the defoamer uses silicone defoamer, model BYK-024; Deionized water: balance.

[0034] In this embodiment, the method for preparing a liquid fermentation medium for Monascus species fermentation comprises the following steps: Add rice flour, corn syrup powder, peanut protein powder, ammonium nitrate, magnesium sulfate, ferrous sulfate and defoaming agent into 85% deionized water, stir until uniformly dispersed, and sterilize at 121° C. to obtain material A; After the remaining 15% of deionized water is sterilized at 121°C, arginine is added, stirred until evenly dissolved, and filtered through a microfiltration membrane with a pore size of 0.20-0.25 μm. The obtained filtrate is material B; Mix material A and material B evenly, and then adjust the pH to 3.9-4.0 to obtain a liquid fermentation medium.

[0035] [Example 3] A liquid fermentation medium for fermentation of Monascus species, which differs from [Example 1] in that: The weight percentage of arginine is 0.55%, and the weight percentage of ferrous sulfate is 0.0015%.

[0036] [Example 4] A liquid fermentation medium for fermentation of Monascus species, which differs from [Example 1] in that: The weight percentage of arginine is 0.65%, and the weight percentage of ferrous sulfate is 0.0025%.

[0037] [Example 5] A liquid fermentation medium for fermentation of Monascus species, which differs from [Example 1] in that: The weight percentage of arginine is 0.8%, and the weight percentage of ferrous sulfate is 0.002%.

[0038] Comparative Example [Comparative Example 1] A liquid fermentation medium for fermentation of Monascus species, which differs from [Example 1] in that arginine is replaced by an equal amount of glycine.

[0039] [Comparative Example 2] A liquid fermentation medium for fermentation of Monascus species, which differs from [Example 1] in that arginine is replaced by an equal amount of glutamic acid.

[0040] [Comparative Example 3] A liquid fermentation medium for fermentation of Monascus species, which differs from [Example 1] in that arginine is replaced by an equal amount of deionized water.

[0041] [Comparative Example 4] A liquid fermentation medium for fermenting Monascus species, which differs from [Example 1] in that ferrous sulfate is replaced by an equal amount of sodium sulfate.

[0042] [Comparative Example 5] A liquid fermentation medium for fermenting Monascus species, which differs from [Example 1] in that ferrous sulfate is replaced by deionized water of equal mass.

[0043] [Comparative Example 6] A liquid fermentation medium for fermenting Monascus species, which differs from [Example 1] in that peanut protein powder is replaced by an equal amount of soybean protein powder.

[0044] [Comparative Example 7] A liquid fermentation medium for fermentation of Monascus species, which differs from [Example 6] in that the mass proportion of the defoaming agent is 0.8%.

[0045] Application Examples A fermentation process of Monascus purpurogenum, using any one of the Monascus purpurogenum species numbered CGMCC No.3.15548, CGMCC No.3.15547, CGMCC No.3.15546, CGMCC No.3.898, and CGMCC No.3.896 purchased from the Institute of Microbiology, Chinese Academy of Sciences as a starting strain, wherein, Figure 1 The fermentation process of Monascus species specifically includes the following steps: S1. Activation of bacterial strains: Activation and cultivation of starting bacterial strains; S2, seed liquid culture: inoculate the activated starting strain into the seed liquid for culture; S3, fermentation culture: after the completion of the culture in step S2, the seed solution of S2 is transferred to the liquid fermentation medium, the inoculation amount of the seed solution is 4.5%-5.5% (v / v) of the liquid fermentation medium, and the culture is carried out in a constant temperature shaker at 30-35°C, the rotation speed is 150-250r / min, and the pH value of 5.60-5.80 is taken as the fermentation end point.

[0046] The following is a specific application example, in which the starting strain is specifically the Monascus purpurogenum strain numbered CGMCC No.3.15548.

[0047]

Application Example 1

[0048]

Application Example 2

[0049]

Application Example 3

[0050]

Application example 4

[0051]

Application Example 5

[0052] Comparative Application Examples

Comparative Application Example 1

[0053]

Comparative Application Example 2

[0054]

Comparative Application Example 3

[0055]

Comparative Application Example 4

[0056]

Comparative Application Example 5

[0057]

Comparative Application Example 6

[0058]

Comparative Application Example 7

[0059] Performance testing Average color value: The color value of the fermentation liquid was determined according to the method specified in the national standard GB / T 1886.181-2016, and the test wavelength was 505-510nm. Each sample was tested three times in parallel and the average value was recorded.

[0060] Maximum absorption peak: dissolve the fermentation broth in 100 ml of ethanol-water solution (1+1) to obtain a sample solution with a color value ranging from 0.4-0.8 u / ml. Scan the sample solution under visible light and record the maximum absorption peak.

[0061] Fermentation time: record the time it takes to reach the end point of fermentation.

[0062] Fermentation foam height: record the highest height of the foam during the fermentation process.

[0063] Table 1

[0064] Combining the above-mentioned application examples 1-5 and the detection data in Table 1, it can be seen that the use of the liquid fermentation medium in Examples 1-5 to ferment a specific Monascus species is conducive to obtaining a product with a high color value and a maximum absorption peak at 505-510nm (purple-red appearance), and at the same time, less foam is produced during the fermentation process.

[0065] Combining the above Application Example 1 with Comparative Application Examples 1-5 and the test data in Table 1, it can be seen that when arginine and ferrous sulfate work together with other raw materials, the specific Monascus species of the present application can metabolize a product with a maximum absorption peak at 505-510nm (purple-red appearance).

[0066] Combining the above Application Example 1 and Comparative Application Example 6 and the test data in Table 1, it can be seen that when soybean protein powder is used instead of peanut protein powder, the color value of the metabolic product is reduced, and the foam production in the fermentation process is increased.

[0067] Combining the above-mentioned comparative application examples 6 and 7 and the test data in Table 1, it can be seen that when soy protein powder is used instead of peanut protein powder, increasing the amount of defoaming agent can reduce the amount of foam generated during the fermentation process. However, the color value of the metabolites of the specific Monascus species of the present application is further reduced. At the same time, the appearance of the metabolites is not purple-red, which is not conducive to obtaining metabolites with a purple-red appearance and a high color value.

[0068] This specific implementation manner is merely an explanation of the present application and is not a limitation of the present application. After reading this specification, those skilled in the art may make non-creative modifications to the specific implementation manner as needed, but such modifications are protected by the patent law as long as they are within the scope of the claims of the present application.

Claims

1. A liquid fermentation medium for Monascus species fermentation, characterized in that: Including carbon source, nitrogen source, inorganic salt, trace elements, defoaming agent and deionized water; The mass proportion of the carbon source is 9.5-10.5%; The nitrogen source comprises corn steep liquor powder, peanut protein powder and an inorganic nitrogen source, wherein the weight proportion of the corn steep liquor powder is 1.8-2.5%, the weight proportion of the peanut protein powder is 3-3.5%, and the weight proportion of the inorganic nitrogen source is 1-1.5%; wherein the protein content of the peanut protein powder is 55-65wt%, and the fat content is 5-6wt%; The trace elements include arginine and ferrous sulfate, wherein the mass proportion of the arginine is 0.4-0.8%, and the mass proportion of the ferrous sulfate is 0.001-0.004%; The mass proportion of the inorganic salt is 0.4-0.8%; The mass proportion of the defoaming agent does not exceed 0.05%.

2. The liquid fermentation medium for Monascus species fermentation according to claim 1, characterized in that: The mass proportion of the arginine is 0.55-0.65%, and the mass proportion of the ferrous sulfate is 0.0015-0.0025%.

3. The liquid fermentation medium for Monascus species fermentation according to claim 1, characterized in that: The nitrogen source also includes pancreatic casein, and the mass proportion of the pancreatic casein is 0.1-0.15%.

4. The liquid fermentation medium for Monascus species fermentation according to claim 1, characterized in that: The carbon source is rice flour.

5. The liquid fermentation medium for Monascus species fermentation according to claim 1, characterized in that: The inorganic salt is at least one of magnesium sulfate, sodium chloride, potassium dihydrogen phosphate and dipotassium hydrogen sulfate.

6. The liquid fermentation medium for Monascus species fermentation according to claim 1, characterized in that: The inorganic nitrogen source is at least one of sodium nitrate, ammonium sulfate and ammonium nitrate.

7. A method for preparing a liquid fermentation medium for Monascus species fermentation according to any one of claims 1 to 6, characterized in that: The following steps are involved: Add carbon source, corn syrup powder, peanut protein powder, inorganic nitrogen source, inorganic salt, ferrous sulfate and defoamer into part of deionized water, stir until uniformly dispersed, and sterilize at high temperature to obtain material A; After the remaining deionized water is sterilized, arginine is added, stirred until evenly dissolved, and filtered through a microfiltration membrane with a pore size of 0.20-0.25 μm. The obtained filtrate is material B; Mix material A and material B evenly, and then adjust the pH to 3.9-4.0 to obtain a liquid fermentation medium.

8. A fermentation process of Monascus species, characterized in that: The following steps are involved: S1. Activation of bacterial strains: Activation and cultivation of starting bacterial strains; S2, seed liquid culture: inoculate the activated starting strain into the seed liquid for culture; S3, fermentation culture: after the cultivation in step S2 is completed, the seed liquid of S2 is transferred to a liquid fermentation medium for fermentation of Monascus species according to any one of claims 1 to 6, and cultured in a constant temperature shaker at 30-35°C, with a rotation speed of 150-250 r / min, and the pH value of 5.60-5.80 is taken as the fermentation end point.

9. The fermentation process of a Monascus species according to claim 8, characterized in that: In S1, the starting strain is any one of the Monascus purpurogenum strains numbered CGMCC No.3.15548, CGMCC No.3.15547, CGMCC No.3.15546, CGMCC No.3.898, and CGMCC No.3.

896.

10. The fermentation process of a Monascus species according to claim 8, characterized in that: In S3, the inoculation amount of the seed liquid is 4.5%-5.5% of the liquid fermentation medium for fermentation of Monascus species.

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