1. A method for promoting monascus fermentation to produce monascus yellow pigment is characterized in that fermentation broth or fermentation supernatant of microorganisms is added at the beginning of or during the fermentation of monascus; the microorganism is one or more of bacillus subtilis, lactobacillus, saccharomyces cerevisiae or aspergillus niger.

2. The method of claim 1, wherein the fermentation supernatant is a microbial cell depleted fermentation broth.

3. The method according to claim 1 or 2, wherein the viable count of the fermentation broth is up to 1 x 10⁶～1×10⁸CFU/mL。

4. The method of claim 1, wherein the bacillus subtilis comprises bacillus subtilis ACCC04178, the lactobacillus comprises lactobacillus ACCC 19955, and the saccharomyces cerevisiae comprises saccharomyces cerevisiae ACCC 20039.

5. The method as claimed in claim 1, wherein the Monascus is a Monascus capable of producing Monascus yellow pigment, including but not limited to Monascus purpureus (Monascuspurpureus) ZH106-E, Monascus purpureus (Monascuspurpureus) CICC 40225, Monascus purpureus (Monascuspurpureus) CICC 40937, Monascus ruber (monascu ruber) CGMCC3.465, Monascus ruber (monascu ruber) CGMCC 3.4649.

6. The method according to claim 1, wherein the amount of the microbial fermentation broth added is 1-10% of the total volume of the monascus fermentation system.

7. The method according to claim 1, wherein the microbial fermentation broth is added within 0-96 hours after the monascus inoculation.

8. The method as claimed in claim 1, wherein the fermentation is carried out at a rotation speed of 150-250r/min and a temperature of 25-35 ℃ for at least 4 days.

9. The method of claim 1, wherein the liquid fermentation medium comprises 40-80g/L corn starch, 3-8g/L ammonium sulfate, 2-6g/L sodium nitrate, 0.5-1.5g/L magnesium sulfate, 1.5-3.5g/L potassium dihydrogen phosphate, 1.5-3.5g/L dipotassium hydrogen phosphate, and 0.1-0.5g/L calcium chloride.

10. Use of the method of any one of claims 1 to 9 for the preparation of monascus yellow pigment and food additives containing monascus yellow pigment.

Background

The pigments in the market at present can be mainly classified into natural pigments and non-natural pigments, wherein the non-natural pigments mainly comprise synthetic pigments and semi-synthetic pigments. Synthetic and semi-synthetic pigments on the market account for about 70% of the market share, whereas natural pigments account for only about 30%. The demand for non-natural pigments in the market is decreasing due to their potential toxicity and some other uncertainty. And along with the continuous improvement of living standard of people, people have higher and higher demand for nontoxic and harmless natural pigment. The monascus pigment as a natural colorant has great market prospect in pharmacy, textile and cosmetic industries besides being widely applied to food industries. The monascus red pigment is already industrially produced, while the monascus yellow pigment is not industrially produced due to the low color value.

In recent years, the research of the monascus yellow pigment is receiving more and more attention, and researchers at home and abroad improve the yield of the monascus yellow pigment to different degrees by a fermentation optimization mode. Leejie et al used monascus ruber M-7 as the experimental strain, and controlled the pH of the fermentation medium to 3-4, with 2% sucrose as the optimal carbon source and 0.2% ammonium sulfate as the optimal nitrogen source, so that the water-soluble yellow pigment color number reached 5.56U/mL. Zhou et al rapidly screened important influencing factors by a response surface method and developed a polynomial model to optimize the medium for production of yellow pigment from m.anka mutant MYM as the test strain, at which the monascus yellow pigment yield could reach 88.14U/mL in shake flasks and 92.45U/mL in 5 liter fermentors. After extraction and fermentation in a non-ionic surfactant micelle aqueous solution, Zhong et al prepared monascus yellow pigment from the fermentation broth. First, monascus pigment was transferred from a non-ionic surfactant micellar aqueous solution into an ionic liquid phase by ionic liquid based microemulsion extraction, wherein the recovery of yellow monascus pigment reached 95.5% based on absorbance at 410 nm. Then, the yellow monascus pigment in the ionic liquid phase is recovered by an ionic liquid-butanol two-phase extraction method, and the recovery rate reaches 95%.

It is reported that the co-culture system can provide a more complicated growth environment than pure culture, thereby affecting the growth and metabolism of microorganisms. Multiple studies show that the yield of certain target products can be remarkably improved by constructing a co-culture fermentation system. For example, the yield of Lovastatin can be improved by co-culturing Monascus purpureus MTCC 369 and Monascus ruber MTCC 1880, the yield of bioethanol can be improved by co-culturing Saccharomyces cerevisiae and Pichia stipitis, and the yield of Paracin1.7 can be improved by co-culturing Bacillus subtilis and Lactobacillus paracasei HD 1-7.

At present, no research for promoting monascus to produce monascus yellow pigment through co-culture exists, and the purpose of high yield of monascus yellow pigment is difficult to achieve by adopting the components of the common liquid fermentation culture medium. The invention can greatly promote the synthesis of monascus yellow pigment by adding the microbial fermentation liquid into the fermentation culture medium, and has important research and application values.

Disclosure of Invention

The first object of the invention is to provide a method for promoting monascus fermentation to produce monascus yellow pigment, which is characterized in that fermentation liquor or fermentation supernatant of microorganisms is added in the initial fermentation process or the fermentation process of monascus; the microorganism is one or more of bacillus subtilis, lactobacillus, saccharomyces cerevisiae or aspergillus niger.

In one embodiment, the fermentation is a fermentation in a liquid environment.

In one embodiment, the fermentation supernatant is a fermentation broth depleted of microbial cells.

In one embodiment, the viable count of the fermentation broth is up to 1 × 10⁶～1×10⁸CFU/mL。

In one embodiment, the Bacillus subtilis comprises Bacillus subtilis ACCC04178, the Lactobacillus comprises Lactobacillus acidi ACCC 19955, and the Saccharomyces cerevisiae comprises Saccharomyces cerevisiae ACCC 20039.

In one embodiment, the monascus is a monascus capable of producing monascus yellow pigment.

In one embodiment, the Monascus includes, but is not limited to, Monascus purpureus ZH106-E, Monascus purpureus CICC 40225, Monascus purpureus CICC 40937, Monascus ruber CGMCC3.465, or Monascus ruber CGMCC 3.4649.

In one embodiment, the addition amount of the microbial fermentation liquid is 1-10% of the total volume of the monascus fermentation system.

In one embodiment, the microorganism fermentation liquid is added within 0-96 hours after the monascus is inoculated.

In one embodiment, the fermentation is performed at a rotation speed of 150-.

In one embodiment, the fermentation is carried out at a rotation speed of 180 ℃ and a temperature of 30 ℃ for 8 d.

In one embodiment, the fermentation medium contains 40-80g/L corn starch, 3-8g/L ammonium sulfate, 2-6g/L sodium nitrate, 0.5-1.5g/L magnesium sulfate, 1.5-3.5g/L potassium dihydrogen phosphate, 1.5-3.5g/L dipotassium hydrogen phosphate, and 0.1-0.5g/L calcium chloride.

The invention also claims the application of the method in preparing the monascus yellow pigment and the food additive containing the monascus yellow pigment.

The invention has the beneficial effects that:

the invention increases the synthesis of monascus purpureus pigment by adding microbial fermentation liquor. The method is simple to operate, the structure of the pigment is not changed, the synthesis of the monascus yellow pigment can be improved only by low additional investment, the color value of the monascus yellow pigment reaches 535U/mL, and the method has great economic benefit.

Detailed Description

1. The color value is a representation method of the concentration of the pigment, and the value is equal to the product of the dilution factor and the corresponding OD value of the diluted pigment solution at the absorption peak, and the unit is U/mL.

Diluting a certain amount of Monascus ruber fermentation liquid with anhydrous ethanol to a suitable range (to make final absorbance within the range of 0.2-0.8), mixing, standing for a period of time to make pigment completely dissolved in ethanol, and detecting absorbance value of the ethanol dissolved with pigment at 410 nm.

Calculating the formula: yellow pigment color value ═ OD₄₁₀X dilution factor

2. Bacterial strains

B, bacillus subtilis: ACCC 04178.

And (3) lactobacillus: ACCC 19955.

And (3) saccharomyces cerevisiae: ACCC 20039.

Example 1: production of pigment by fermentation of different strains

Seed liquid culture medium (g/L): 60 parts of corn starch, 4 parts of ammonium sulfate, 2 parts of sodium nitrate, 0.5 part of magnesium sulfate, 2 parts of monopotassium phosphate, 2 parts of dipotassium phosphate and 0.1 part of calcium chloride.

Fermentation medium (g/L): 60 parts of corn starch, 4 parts of ammonium sulfate, 2 parts of sodium nitrate, 0.5 part of magnesium sulfate, 2 parts of monopotassium phosphate, 2 parts of dipotassium phosphate, 0.1 part of calcium chloride and 400mL of glyceride.

(1) Preparation of seed liquid

Selecting purple Monascus ZH106-E (disclosed in a paper with the name of Ethanol addition elements cell reactivity and utilities fermentation of natural yellow pigments in synergistic fermentation of Monascus purpureus), purple Monascus CICC 40225, purple Monascus CICC 40937, red Monascus CGMCC3.465 and red Monascus CGMCC 3.4649 as shake flask strains, respectively inoculating the strains to a seed liquid culture medium after activation, and carrying out shake flask fermentation culture for 2d under the conditions that the rotation speed is 180r/min and the temperature is 30 ℃ to ensure that the bacterial concentration reaches 1 × 10⁷CFU/mL。

(2) Fermentation culture

Inoculating the monascus seed solution in the logarithmic phase prepared in the step (1) into a 500mL triangular flask filled with 50mL fermentation medium in an inoculation amount of 10% by volume, and performing shake-flask fermentation culture for 7d at the rotation speed of 180r/min and the temperature of 30 ℃. After the completion of the test, the color values of the monascus yellow pigments of the eight bacteria ZH106-E, CICC 40225, CICC 40937, CGMCC3.465 and CGMCC 3.4649 are respectively 320U/mL, 20U/mL, 33U/mL, 57U/mL and 30U/mL.

TABLE 1 Effect of different Monascus strains on yellow pigment production by liquid fermentation

Example 2: adding different microbial fermentation liquid (with bacteria) for fermentation to produce pigment

Seed liquid culture medium (g/L): 60 parts of corn starch, 4 parts of ammonium sulfate, 2 parts of sodium nitrate, 0.5 part of magnesium sulfate, 2 parts of monopotassium phosphate, 2 parts of dipotassium phosphate and 0.1 part of calcium chloride.

Fermentation medium (g/L): 60 parts of corn starch, 4 parts of ammonium sulfate, 2 parts of sodium nitrate, 0.5 part of magnesium sulfate, 2 parts of monopotassium phosphate, 2 parts of dipotassium phosphate, 0.1 part of calcium chloride and 400mL of glyceride.

(1) Preparation of seed liquid

Selecting purple monascus ZH106-E as shake flask strain, activating, inoculating to seed liquid culture medium, and shake flask fermentation culturing at 30 deg.C at rotation speed of 180r/min for 2d to obtain monascus liquid seed.

(2) Preparation of microbial fermentation broth

B, bacillus subtilis: and carrying out shake flask fermentation culture for 2d at the rotation speed of 180r/min and the temperature of 30 ℃.

And (3) lactobacillus: and carrying out shake flask fermentation culture for 2d at the rotation speed of 180r/min and the temperature of 30 ℃.

And (3) saccharomyces cerevisiae: and carrying out shake flask fermentation culture for 2d at the rotation speed of 180r/min and the temperature of 30 ℃.

(3) Fermentation culture

Inoculating the monascus liquid seeds in the logarithmic growth phase in the step (1) into a 500mL triangular flask filled with 50mL fermentation medium according to the inoculation amount of 10% by volume, carrying out shake flask fermentation culture for 7d at the rotation speed of 180r/min and the temperature of 30 ℃, and respectively adding different microorganism fermentation liquids (with bacteria) with the volume of 4% in the step (2) into the monascus on the 1 st day of shake flask fermentation. After the completion of the test, the color values of the monascus yellow pigment are 267U/mL, 375U/mL, 300U/mL, 296U/mL and 230U/mL respectively.

TABLE 2 Effect of different microbial additions on the production of yellow pigment by Monascus ZH106-E

Example 3: adding Bacillus subtilis fermentation liquid (with Bacillus subtilis) with different concentrations for fermentation to produce pigment

Seed liquid culture medium (g/L): 60 parts of corn starch, 4 parts of ammonium sulfate, 2 parts of sodium nitrate, 0.5 part of magnesium sulfate, 2 parts of monopotassium phosphate, 2 parts of dipotassium phosphate and 0.1 part of calcium chloride.

Fermentation medium (g/L): 60 parts of corn starch, 4 parts of ammonium sulfate, 2 parts of sodium nitrate, 0.5 part of magnesium sulfate, 2 parts of monopotassium phosphate, 2 parts of dipotassium phosphate, 0.1 part of calcium chloride and 400mL of glyceride.

(1) Preparation of seed liquid

Selecting purple monascus ZH106-E as shake flask strain, activating, inoculating to seed liquid culture medium, and shake flask fermentation culturing at 30 deg.C at rotation speed of 180r/min for 2d to obtain monascus liquid seed.

(2) Preparation of Bacillus subtilis fermented liquid

The same as in example 2.

(3) Fermentation culture

Inoculating the monascus liquid seeds in the logarithmic growth phase in the step (1) into a 500mL triangular flask filled with 50mL fermentation medium according to the inoculation amount of 10% by volume, carrying out shake flask fermentation culture for 7d at the rotation speed of 180r/min and the temperature of 30 ℃, and respectively adding the hay bacillus fermentation liquid (with bacteria) in the step (2) into the fermentation medium according to the addition amounts of 0% (namely no addition), 2%, 4%, 6%, 8% and 10% (v/v) of the culture system volume on the 1 st day of the monascus shake flask fermentation. The color values of the monascus yellow pigment measured after 7d of shake flask fermentation culture are 260U/mL, 371U/mL, 381U/mL, 186U/mL, 214U/mL and 204U/mL respectively.

TABLE 3 influence of different addition amounts of Bacillus subtilis fermentation broth (with bacteria) on the production of yellow pigment by Monascus ZH106-E

Example 4: adding Bacillus subtilis fermentation liquid (without Bacillus subtilis) with different concentrations for fermentation to produce pigment

Seed liquid culture medium (g/L): 60 parts of corn starch, 4 parts of ammonium sulfate, 2 parts of sodium nitrate, 0.5 part of magnesium sulfate, 2 parts of monopotassium phosphate, 2 parts of dipotassium phosphate and 0.1 part of calcium chloride.

Fermentation medium (g/L): 60 parts of corn starch, 4 parts of ammonium sulfate, 2 parts of sodium nitrate, 0.5 part of magnesium sulfate, 2 parts of monopotassium phosphate, 2 parts of dipotassium phosphate, 0.1 part of calcium chloride and 400mL of glyceride.

(1) Preparation of seed liquid

Selecting purple monascus ZH106-E as shake flask strain, activating, inoculating to seed liquid culture medium, and shake flask fermentation culturing at 30 deg.C at rotation speed of 180r/min for 2d to obtain monascus liquid seed.

(2) Preparation of Bacillus subtilis fermented liquid (aseptic)

Fermentation of Bacillus subtilis in the same manner as in example 2, a Bacillus subtilis fermentation broth (with bacteria) was obtained and centrifuged to remove the bacteria and obtain a Bacillus subtilis fermentation broth (sterile).

(3) Fermentation culture

Inoculating the monascus liquid seeds in the logarithmic growth phase in the step (1) into a 500mL triangular flask filled with 50mL fermentation medium according to the inoculation amount of 10% by volume, carrying out shake flask fermentation culture for 7d at the rotation speed of 180r/min and the temperature of 30 ℃, and respectively adding the hay fermentation liquid (sterile) in the step (2) into the fermentation medium according to the addition amounts of 0% (namely no addition), 2%, 4%, 6%, 8% and 10% of the volume of the culture system on the 1 st day of the monascus shake flask fermentation. The color values of the monascus yellow pigment measured after 7d of shake flask fermentation culture are 282U/mL, 374U/mL, 485U/mL, 515U/mL, 503U/mL and 494U/mL respectively.

TABLE 4 influence of different addition amounts of Bacillus subtilis fermentation broth (sterile) on the production of yellow pigment by Monascus ZH106-E

Example 5: adding 6 percent of hay fermentation liquor (sterile) at different time for fermentation to produce pigment

Seed liquid culture medium (g/L): 60 parts of corn starch, 4 parts of ammonium sulfate, 2 parts of sodium nitrate, 0.5 part of magnesium sulfate, 2 parts of monopotassium phosphate, 2 parts of dipotassium phosphate and 0.1 part of calcium chloride.

Fermentation medium (g/L): 60 parts of corn starch, 4 parts of ammonium sulfate, 2 parts of sodium nitrate, 0.5 part of magnesium sulfate, 2 parts of monopotassium phosphate, 2 parts of dipotassium phosphate, 0.1 part of calcium chloride and 400mL of glyceride.

(1) Preparation of seed liquid

Selecting purple monascus ZH106-E as shake flask strain, activating, inoculating to seed liquid culture medium, and shake flask fermentation culturing at 30 deg.C at rotation speed of 180r/min for 2d to obtain monascus liquid seed.

(2) Preparation of Bacillus subtilis fermented liquid (aseptic)

The same as in example 4.

(3) Fermentation culture

Inoculating the monascus liquid seeds in the logarithmic growth phase in the step (1) into a 500mL triangular flask filled with 50mL fermentation medium according to the inoculation amount of 10% by volume, carrying out shake flask fermentation culture for 7d at the rotation speed of 180r/min and the temperature of 30 ℃, and adding the hay fermentation broth (sterile) in the step (2) with the culture system volume of 6% (v/v) at the time of 0 day, 1 day, 2 days, 3 days, 4 days, 5 days and 6 days of the monascus shake flask fermentation respectively. The color values of the monascus yellow pigment measured after 7d of shake flask fermentation culture are 525U/mL, 495U/mL, 467U/mL, 381U/mL, 282U/mL, 267U/mL and 243U/mL respectively.

TABLE 5 Effect of 6% (v/v) addition of Bacillus subtilis fermentation broth (sterile) at different times on the production of yellow pigment from ZH106-E

Example 6: the yield of monascus yellow pigment in different fermentation time without adding the hay fermentation liquid

Seed liquid culture medium (g/L): 60 parts of corn starch, 4 parts of ammonium sulfate, 2 parts of sodium nitrate, 0.5 part of magnesium sulfate, 2 parts of monopotassium phosphate, 2 parts of dipotassium phosphate and 0.1 part of calcium chloride.

Fermentation medium (g/L): 60 parts of corn starch, 4 parts of ammonium sulfate, 2 parts of sodium nitrate, 0.5 part of magnesium sulfate, 2 parts of monopotassium phosphate, 2 parts of dipotassium phosphate, 0.1 part of calcium chloride and 400mL of glyceride.

(1) Preparation of seed liquid

Selecting purple monascus ZH106-E as shake flask strain, activating, inoculating to seed liquid culture medium, and shake flask fermentation culturing at 30 deg.C at rotation speed of 180r/min for 2d to obtain monascus liquid seed.

(2) Fermentation culture

Inoculating the monascus liquid seeds in the logarithmic growth phase in the step (1) into a 500mL triangular flask filled with 50mL fermentation medium according to the inoculation amount of 10% by volume, and respectively measuring the color values of the fermentation medium at 0 th day, 1 st day, 2 nd day, 3 rd day, 4 th day, 5 th day, 6 th day and 7 th day in a shaking flask at the rotation speed of 180r/min and the temperature of 30 ℃. The color number of the monascus yellow pigment is measured to be 2U/mL, 6U/mL, 52U/mL, 101U/mL, 153U/mL, 210U/mL, 225U/mL and 235U/mL respectively.

TABLE 6 results of fermentation without addition of Bacillus subtilis fermentation broth for various times

Example 7: when the hay fermentation liquor (sterile) is added, the yield of the monascus yellow pigment is increased at different times of fermentation

Seed liquid culture medium (g/L): 60 parts of corn starch, 4 parts of ammonium sulfate, 2 parts of sodium nitrate, 0.5 part of magnesium sulfate, 2 parts of monopotassium phosphate, 2 parts of dipotassium phosphate and 0.1 part of calcium chloride.

Fermentation medium (g/L): 60 parts of corn starch, 4 parts of ammonium sulfate, 2 parts of sodium nitrate, 0.5 part of magnesium sulfate, 2 parts of monopotassium phosphate, 2 parts of dipotassium phosphate, 0.1 part of calcium chloride and 400mL of glyceride.

(1) Preparation of seed liquid

Selecting purple monascus ZH106-E as shake flask strain, activating, inoculating to seed liquid culture medium, and shake flask fermentation culturing at 30 deg.C at rotation speed of 180r/min for 2d to obtain monascus liquid seed.

(2) Preparation of Bacillus subtilis fermented liquid (aseptic)

The same as in example 4.

(3) Fermentation culture

Inoculating the monascus liquid seeds in the logarithmic growth phase in the step (1) into a 500mL triangular flask filled with 50mL fermentation medium according to the inoculation amount of 10% by volume, adding 6% of the volume of the culture system in the step (2) of the hay fermentation broth (sterile) in the shake flask fermentation on the 0 th day at the rotation speed of 180r/min and the temperature of 30 ℃, and then measuring the color value every 24 hours. The results show that the measured color values of the monascus yellow pigment at 0 th day, 1 st day, 2 nd day, 3 rd day, 4 th day, 5 th day, 6 th day and 7 th day of the shake flask fermentation are 2U/mL, 6U/mL, 100U/mL, 210U/mL, 301U/mL, 352U/mL, 431U/mL and 535U/mL respectively.

TABLE 7 results of fermentation with addition of Bacillus subtilis fermentation broth for various times

Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.