Application of RHPS4 in Monascus pigment production by Monascus fermentation and application of Monascus fermentation broth

Abstract

The present invention proposes an application of RHPS4 in the production of red pigment by fermentation of Monascus ruber and the application of Monascus fermentation liquid, and relates to the field of microbial fermentation technology, wherein the RHPS4 is 3,11-difluoro-6,8,13-trimethyl-8H-quinoline and [4,3,2-kl] acridinium methyl sulfate. The technical scheme of the present invention co-cultivates RHPS4 with Monascus ruber, and finds that RHPS4 can promote the increase of the unit yield of Monascus ruber through the determination of biomass and Monascus ruber content. When cultured to the 10th day, at a concentration of 2µmol / L, the biomass of Monascus ruber decreased by 49.9%, while the yellow pigment of Monascus ruber increased by 260.5 U / mL in the unit yield, the orange pigment of Monascus ruber increased by 130.3 U / mL in the unit yield, and the red pigment of Monascus ruber increased by 97.2 U / mL in the unit yield.

Description

Technical Field

[0001] The invention relates to the technical field of microbial fermentation, and in particular to an application of RHPS4 in producing monascus pigment by fermentation of monascus and an application of monascus fermentation liquid. Background Art

[0002] Monascus Monascus ) belongs to the phylum Fungi ( Eumycophyta ), a traditional Chinese filamentous fungus used for both medicinal and edible purposes, is widely used in red yeast rice, red yeast wine, etc. During its growth and reproduction, red yeast can produce many beneficial secondary metabolites (SM), including γ-aminobutyric acid, red yeast pigment ( Monascus Among them, Monascus pigments are a class of polyketide compounds (Azaphilones), including red pigments, orange pigments and yellow pigments. So far, about one hundred Monascus pigments have been identified. As a natural pigment resource, Monascus pigments have good color, stability and biological activity, and are widely used in the food processing industry. At the same time, due to the safety of Monascus pigments, it is considered to be a substitute for food additives such as nitrite, and is widely used in the processing of meat products and other foods.

[0003] At present, the red pigment in conventional Monascus fermented products is mainly obtained through liquid fermentation. In the production process, the pigment yield is greatly affected by the fermentation strain and fermentation conditions, resulting in low pigment content and difficulty in extraction and concentration. Therefore, it is of great significance to promote the increase of the content of red pigment in Monascus, find an active compound that can enhance the synthesis of red pigment and study its mechanism of action. Summary of the invention

[0004] The main purpose of the present invention is to propose an application of RHPS4 in producing monascus pigment in Monascus fermentation and an application of Monascus fermentation broth, aiming to increase the unit yield of monascus pigment in the monascus product to obtain a higher performance monascus product.

[0005] To achieve the above object, the present invention proposes an application of RHPS4 in producing red pigment in Monascus fermentation, wherein the RHPS4 is 3,11-difluoro-6,8,13-trimethyl-8H-quinolin[4,3,2-kl]acridinium methyl sulfate, having a chemical formula as described in formula (1),

[0006] ,

[0007] Formula (1).

[0008] In one embodiment, the monascus pigment is a natural pigment.

[0009] In one embodiment, the concentration of RHPS4 is 0-5 µmol / L.

[0010] In one embodiment, the biologic comprises RHPS4.

[0011] In one embodiment, the step of fermenting Monascus to produce Monascus pigment includes: activating and culturing Monascus to enrich it to obtain concentrated Monascus spore liquid; mixing the concentrated Monascus spore liquid with RHPS4 and culturing it to obtain Monascus co-fermentation liquid.

[0012] In one embodiment, the step of activating and culturing Monascus comprises: inoculating Monascus on a PDA culture medium, activating and culturing the Monascus, eluting the Monascus spore liquid, and filtering and centrifuging the Monascus spore liquid to obtain a concentrated Monascus spore liquid.

[0013] In one embodiment, during the activated static culture, the culture duration is 7 to 14 days.

[0014] In one embodiment, the step of co-culturing Monascus and RHPS4 comprises: washing spores from the PDA slant culture medium with sterile water to obtain Monascus spore liquid; mixing RHPS4 with the Monascus spore liquid according to the concentration and then culturing at 28° C. to obtain Monascus co-fermentation liquid.

[0015] In one embodiment, the concentration of the Monascus spore solution is 1×10 5 Pieces / mL.

[0016] In one embodiment, in the step of mixing RHPS4 with Monascus spore liquid according to the concentration and then culturing in YES liquid culture medium, the concentration of RHPS4 is 0-5 µmol / L.

[0017] The present invention also provides an application of Monascus fermentation broth in the preparation of food additives, medicines or cosmetics, wherein the Monascus fermentation broth comprises 3,11-difluoro-6,8,13-trimethyl-8H-quinolino[4,3,2-kl]acridinium methyl sulfate.

[0018] The technical solution of the present invention is to co-cultivate 3,11-difluoro-6,8,13-trimethyl-8H-quinolino[4,3,2-kl]acridinium methyl sulfate and Monascus, and through the determination of biomass and Monascus pigment content, it is found that 3,11-difluoro-6,8,13-trimethyl-8H-quinolino[4,3,2-kl]acridinium methyl sulfate can promote the improvement of the unit yield of Monascus pigment. On the 10th day of cultivation, when the concentration of RHPS4 was 2 µmol / L, the biomass of Monascus decreased by 49.9% compared with that at 0 µmol / L, while the unit yield of Monascus yellow pigment increased by 160.4 U / mL, the unit yield of Monascus orange pigment increased by 80.3 U / mL, and the unit yield of Monascus red pigment increased by 59.9 U / mL; at the concentration of 5 µmol / L, compared with that at 0 µmol / L, the biomass of Monascus decreased by 66%, while the unit yield of Monascus yellow pigment increased by 300.5 U / mL, the unit yield of Monascus orange pigment increased by 121.8 U / mL, and the unit yield of Monascus red pigment increased by 75.8 U / mL, indicating that RHPS4 can significantly promote the increase of the unit yield of Monascus pigments during the growth process of Monascus, so as to promote the promotion and application of Monascus products. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other related drawings can be obtained based on these drawings without paying creative work.

[0020] Figure 1 The molecular formula of the RHPS4 compound provided by the present invention;

[0021] Figure 2 The biomass was determined on the 10th day after co-fermentation of 3,11-difluoro-6,8,13-trimethyl-8H-quinolino[4,3,2-kl]acridinium methyl sulfate with Monascus at different concentrations.

[0022] Figure 3 The content of red pigment when different concentrations of 3,11-difluoro-6,8,13-trimethyl-8H-quinolino[4,3,2-kl]acridinium methyl sulfate were co-fermented with Monascus for 10 days;

[0023] Figure 4 The unit yield of Monascus pigment when different concentrations of 3,11-difluoro-6,8,13-trimethyl-8H-quinolino[4,3,2-kl]acridinium methyl sulfate were co-fermented with Monascus on the 10th day.

[0024] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings in conjunction with the embodiments. DETAILED DESCRIPTION

[0025] To make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them.

[0026] It should be noted that, in the embodiments, those without specifying specific conditions are carried out according to normal conditions or conditions recommended by the manufacturer. Those without specifying the manufacturer of reagents or instruments used are conventional products that can be purchased commercially. In addition, the meaning of "and / or" appearing in the full text includes three parallel schemes, and "A and / or B" is taken as an example, including schemes A, B, or A and B that meet the same time. In addition, the technical schemes between the various embodiments can be combined with each other, but must be based on the ability of ordinary technicians in the field to achieve. When the combination of the technical schemes is contradictory or cannot be achieved, it should be considered that the combination of such technical schemes does not exist, and is not within the scope of protection required by the present invention. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in the field without making creative work, all belong to the scope of protection of the present invention.

[0027] Monascus Monascus ) belongs to the phylum Fungi ( Eumycophyta ), a traditional Chinese filamentous fungus used for both medicinal and edible purposes, is widely used in red yeast rice, red yeast wine, etc. During its growth and reproduction, red yeast can produce many beneficial secondary metabolites (SM), including γ-aminobutyric acid, red yeast pigment ( Monascus Among them, Monascus pigments are a class of polyketide compounds (Azaphilones), including red pigments, orange pigments and yellow pigments. So far, about one hundred Monascus pigments have been identified. As a natural pigment resource, Monascus pigments have good color, stability and biological activity, and are widely used in the food processing industry. At the same time, due to the safety of Monascus pigments, it is considered to be a substitute for food additives such as nitrite, and is widely used in the processing of meat products and other foods.

[0028] At present, the red pigment in conventional Monascus fermented products is mainly obtained through liquid fermentation. In the production process, the pigment yield is greatly affected by the fermentation strain and fermentation conditions, resulting in low pigment content and difficulty in extraction and concentration. Therefore, it is of great significance to promote the increase of the content of red pigment in Monascus, find an active compound that can enhance the synthesis of red pigment and study its mechanism of action.

[0029] For this reason, please see Figures 1 to 4 The present invention provides an application of RHPS4 in the production of red pigment by fermentation of Monascus spp., wherein the RHPS4 is 3,11-difluoro-6,8,13-trimethyl-8H-quinolin[4,3,2-kl]acridinium methyl sulfate, and the chemical formula is shown in formula (1).

[0030] ,

[0031] Formula (1).

[0032] The technical solution of the present invention is to co-cultivate 3,11-difluoro-6,8,13-trimethyl-8H-quinolino[4,3,2-kl]acridinium methyl sulfate and Monascus, and through the determination of biomass and Monascus pigment content, it is found that 3,11-difluoro-6,8,13-trimethyl-8H-quinolino[4,3,2-kl]acridinium methyl sulfate can promote the improvement of the unit yield of Monascus pigment. On the 10th day of cultivation, when the concentration of RHPS4 was 2 µmol / L, the biomass of Monascus decreased by 49.9% compared with that at 0 µmol / L, while the unit yield of Monascus yellow pigment increased by 160.4 U / mL, the unit yield of Monascus orange pigment increased by 80.3 U / mL, and the unit yield of Monascus red pigment increased by 59.9 U / mL; at the concentration of 5 µmol / L, compared with that at 0 µmol / L, the biomass of Monascus decreased by 66%, while the unit yield of Monascus yellow pigment increased by 300.5 U / mL, the unit yield of Monascus orange pigment increased by 121.8 U / mL, and the unit yield of Monascus red pigment increased by 75.8 U / mL, indicating that RHPS4 can significantly promote the increase of the unit yield of Monascus pigments during the growth process of Monascus, so as to promote the promotion and application of Monascus products.

[0033] In any embodiment of the present invention, the monascus pigment is a natural pigment, and RHPS4 has the effect of promoting the increase of unit yield.

[0034] In any embodiment of the present invention, the concentration of RHPS4 is 0-5 µmol / L, which may be 2 µmol / L or 5 µmol / L. Within this concentration range, as the concentration of RHPS4 increases, the unit yield of monascus pigment increases.

[0035] In one embodiment, the step of producing Monascus pigment by Monascus includes: activating and culturing Monascus to enrich it to obtain concentrated Monascus spore liquid; mixing the concentrated Monascus spore liquid with RHPS4 and culturing it to obtain Monascus co-fermentation liquid.

[0036] The technical solution of the present invention enhances the growth vitality of Monascus by activating and culturing Monascus, and ensures a good growth state of the strain in the subsequent fermentation process; and the unit yield of Monascus pigment can be increased by adding RHPS4 and mixing with Monascus spores for culturing.

[0037] The Monascus fermentation broth prepared by the method of the present invention can be used in more fields, such as food processing, medicine and cosmetics, due to its unique ingredients and higher activity, and its scope of use can be expanded.

[0038] In one embodiment, the step of activating and culturing Monascus comprises: inoculating Monascus on a PDA culture medium, activating and culturing the Monascus, eluting the Monascus spore liquid, and filtering and centrifuging the Monascus spore liquid to obtain a concentrated Monascus spore liquid.

[0039] The technical solution of the present invention comprises the following steps: washing spores with sterile water, breaking the spores with glass beads, determining the number of spores with a microscope and a blood cell counting plate, and diluting the spore liquid; and further obtaining a concentrated Monascus spore liquid by filtering and centrifugation, thereby improving the purity and concentration of the Monascus spores, thereby ensuring uniform distribution and efficient reproduction of the Monascus in subsequent culturing processes.

[0040] In one embodiment, during the activated static culture, the culture duration is 7 to 14 days.

[0041] The technical solution of the present invention can ensure that Monascus is fully activated on the PDA culture medium and reaches an optimal growth state by setting the culture time to 7 to 14 days, thereby improving the activity and reproduction capacity of Monascus, and increasing the yield and maturity of spores. At the same time, it also avoids the problem of spore aging caused by too long a culture period, which helps to improve production efficiency.

[0042] In one embodiment, the step of co-culturing Monascus and RHPS4 comprises: washing spores from the PDA slant culture medium with sterile water to obtain Monascus spore liquid; mixing RHPS4 with the Monascus spore liquid according to the concentration and then culturing at 28° C. to obtain Monascus co-fermentation liquid.

[0043] The technical solution of the present invention co-cultivates the Monascus spore liquid with RHPS4, and RHPS4 can regulate the metabolic pathway of Monascus, promote the synthesis of Monascus pigment, and increase the unit yield of Monascus pigment.

[0044] In one embodiment, the concentration of Monascus spores in the Monascus spore liquid is 1×105 Pieces / mL.

[0045] In one embodiment, in the step of mixing RHPS4 with Monascus spore liquid according to the concentration and then culturing in YES liquid culture medium, the concentration of RHPS4 is 0-5 µmol / L.

[0046] The present invention also provides an application of Monascus fermentation broth in the preparation of food additives, medicines or cosmetics, wherein the Monascus fermentation broth comprises 3,11-difluoro-6,8,13-trimethyl-8H-quinolino[4,3,2-kl]acridinium methyl sulfate.

[0047] Since the present application adopts all the technical solutions of all the above embodiments, it has at least all the beneficial effects brought by the technical solutions of the above embodiments, which will not be described one by one here.

[0048] The technical solution of the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the following embodiments are only used to explain the present invention, and are not used to limit the present invention.

[0049] Experimental Materials

[0050] Monascus is red Monascus M7 ( Monascusruber M7), the deposit number is CCAM 070120;

[0051] RHPS4 was purchased from Aladdin Biochemical Technology Co., Ltd., with the catalog number R287983-25g;

[0052] Potato dextrose agar (PDA) Medium preparation: 200 g peeled potatoes, cut into 1 cm 3 size, add distilled water and boil for 30 min, collect the filtrate through gauze, add 20 g glucose and 15-20 g agar powder, add 1 L of distilled water, and use it for Monascus culture;

[0053] Preparation of YES medium: 160 g sucrose, 40 g yeast extract, add 1 L of distilled water and mix, sterilize at 121℃, and use for liquid fermentation of Monascus.

[0054] Example 1

[0055] 1) Monascus was inoculated on PDA medium for activation and cultured in a 28°C incubator for 14 days;

[0056] 2) Preparation of Monascus spore suspension: After 14 days of culture, add 5 mL of sterile water to wash the spores from the slant, collect the spore liquid after filtering through a sterile double-layer lens paper, centrifuge at 8000 rpm for 10 min, concentrate the spore liquid, and use a hemocytometer to determine the spore count. Dilute the spore liquid with sterile water to a concentration of 1×10 5 spores / mL of Monascus spore suspension;

[0057] 3) Co-fermentation culture: 500 μL of Monascus spore suspension and RHPS4 were inoculated into YES liquid medium with a final concentration of 2 μmol / L and cultured at 28°C;

[0058] 4) Sampling: 3,11-difluoro-6,8,13-trimethyl-8H-quinolin[4,3,2-kl]acridinium methyl sulfate was co-fermented and cultured with Monascus for 10 days. Mycelium and fermentation liquid were separated by gauze filtration.

[0059] Example 2

[0060] 1) Monascus was inoculated on PDA medium for activation and cultured in a 28°C incubator for 14 days;

[0061] 2) Preparation of Monascus spore suspension: After 14 days of culture, add 5 mL of sterile water to wash the spores from the slant, collect the spore liquid after filtering through a sterile double-layer lens paper, centrifuge at 8000 rpm for 10 min, concentrate the spore liquid, and use a hemocytometer to determine the spore count. Dilute the spore liquid with sterile water to a concentration of 1×10 5 spores / mL of Monascus spore suspension;

[0062] 3) Co-fermentation culture: 500 μL of Monascus spore suspension and RHPS4 were inoculated into YES liquid medium with a final concentration of 5 μmol / L and cultured at 28°C;

[0063] 4) Sampling: 3,11-difluoro-6,8,13-trimethyl-8H-quinolin[4,3,2-kl]acridinium methyl sulfate was co-fermented and cultured with Monascus for 10 days. Mycelium and fermentation liquid were separated by gauze filtration.

[0064] Comparative Example 1

[0065] 1) Monascus was inoculated on PDA medium for activation and cultured in a 28°C incubator for 14 days;

[0066] 2) Preparation of Monascus spore suspension: After 14 days of culture, add 5 mL of sterile water to wash the spores from the slant, collect the spore liquid after filtering through a sterile double-layer lens paper, centrifuge at 8000 rpm for 10 min, concentrate the spore liquid, and use a hemocytometer to determine the spore count. Dilute the spore liquid with sterile water to a concentration of 1×10 5 spores / mL of Monascus spore suspension;

[0067] 3) Co-fermentation culture: 500 μL of Monascus spore suspension was inoculated into YES liquid medium without adding RHPS4 (i.e., the final concentration was 0 μmol / L), and cultured at 28°C;

[0068] 4) Sampling: 3,11-difluoro-6,8,13-trimethyl-8H-quinolin[4,3,2-kl]acridinium methyl sulfate was co-fermented and cultured with Monascus for 10 days. Mycelium and fermentation liquid were separated by gauze filtration.

[0069] Performance Testing

[0070] The mycelium and fermentation liquid of Monascus obtained by co-fermentation and culture for 10 days in Examples 1 to 2 and Comparative Example 1 were taken to detect the biomass and pigment content thereof. The detection method is as follows:

[0071] Determination of biomass: Filter the Monascus fermentation liquid through gauze, collect the liquid and mycelium respectively. Wash the mycelium with distilled water three times, put it in an oven to dry for 4 hours, take it out and weigh it with a balance, and record the mycelium biomass (g);

[0072] Determination of pigment content: The filtered fermentation broth was diluted at a ratio of 100 µL fermentation broth: 900 µL sterile water. For comparative example 1, the liquid culture medium diluted by the same multiple was used as the control group. The yield (U / mL) of extracellular yellow, orange, and red pigments produced by the Monascus strain was calculated by multiplying the absorbance values ​​measured at 390 nm, 470 nm, and 520 nm by the dilution multiple.

[0073] The mycelium biomass and pigment yield of the Monascus fermentation broth in Examples 1 to 2 and Comparative Example 1 were obtained as shown in Table 1. Figure 2 , Figure 3 and Figure 4 shown.

[0074] Table 1 Biomass and pigment yield of Monascus fermentation broth in Comparative Example 1 and Examples 1 to 2

[0075]

[0076] In Table 1, unit yield of yellow pigment = yellow pigment yield / biomass, unit yield of orange pigment = orange pigment yield / biomass, unit yield of red pigment = red pigment yield / biomass.

[0077] Figure 2 The biomass was measured on the 10th day of co-fermentation of RHPS4 and Monascus in Examples 1 to 2 of the present invention and Comparative Example 1.

[0078] according to Figure 2 It can be seen that at a concentration of 2 µmol / L, the biomass of RHPS4 was reduced by about 49.9% compared with the control group; at a concentration of 5 µmol / L, the biomass of RHPS4 was reduced by about 66.0% compared with the control group, indicating that RHPS4 inhibits the growth of Monascus mycelium, and the higher the concentration, the lower the biomass.

[0079] Figure 3 It is the content of Monascus pigment when RHPS4 and Monascus are co-fermented on the 10th day in Examples 1 to 2 of the present invention and Comparative Example 1.

[0080] according to Figure 3 It can be seen that as the concentration of RHPS4 increases, the contents of red, orange, and yellow pigments show a downward trend.

[0081] Figure 4 It is the unit yield of Monascus pigment when RHPS4 and Monascus are co-fermented on the 10th day in Examples 1 to 2 and Comparative Example 1 of the present invention.

[0082] according to Figure 4 It can be seen that compared with the control group, the unit yields of yellow, orange and red pigments increased with the increase of RHPS4 concentration. Among them, at a concentration of 2 µmol / L of RHPS4, the unit yield of yellow pigment increased by 69.8%; the unit yield of orange pigment increased by 78.4%; and the unit yield of red pigment increased by 84.6%, indicating that RHPS4 can effectively increase the unit yield of red pigment.

[0083] The above description is only a preferred embodiment of the present invention, and does not limit the patent scope of the present invention. All equivalent structural changes made by using the contents of the present invention specification under the inventive concept of the present invention, or directly / indirectly applied in other related technical fields are included in the patent protection scope of the present invention.

Claims

1. An application of RHPS4 in the production of monascus pigment by fermentation of Monascus spp., characterized in that: The RHPS4 is 3,11-difluoro-6,8,13-trimethyl-8H-quinolino[4,3,2-kl]acridinium methyl sulfate; The steps of producing monascus pigment by fermentation of Monascus koji include: Activating and culturing Monascus to enrich the Monascus spores to obtain a concentrated Monascus spore liquid; The concentrated Monascus spore liquid is mixed with RHPS4 and then cultured to obtain Monascus co-fermentation liquid; Wherein, in the step of mixing the concentrated Monascus spore liquid with RHPS4 and then culturing, the concentration of RHPS4 is 0-5 µmol / L; The Monascus is red Monascus M7; The monascus pigment is selected from red pigment, orange pigment and yellow pigment.

2. The use according to claim 1, characterized in that The steps of activating and culturing Monascus and enriching it include: Monascus is inoculated on PDA medium, activated and statically cultured, and eluted to obtain Monascus spore liquid; The Monascus spore liquid is filtered and centrifuged to obtain concentrated Monascus spore liquid.

3. The use according to claim 2, characterized in that In activated static culture, the culture time is 7 to 14 days.

4. The use according to claim 2, characterized in that RHPS4 was mixed with Monascus spore liquid according to the concentration and then cultured in YES liquid medium to obtain Monascus co-fermentation liquid.

5. The use according to claim 4, characterized in that The concentration of Monascus spores in the Monascus spore solution is 1×10 5 Pieces / mL.

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