CN116162552B

CN116162552B - Preparation method and application of extremely red aspergillus sorghum fermentation product

Info

Publication number: CN116162552B
Application number: CN202310036896.8A
Authority: CN
Inventors: 张超龙
Original assignee: Chengdu Baolu Biotechnology Co ltd
Current assignee: Chengdu Baolu Biotechnology Co ltd
Priority date: 2023-01-10
Filing date: 2023-01-10
Publication date: 2023-10-20
Anticipated expiration: 2043-01-10
Also published as: CN116162552A

Abstract

The invention provides a preparation method and application of an extreme monascus sorghum fermentation product, and belongs to the technical field of microbial fermentation. Inoculating Mao Gong Aspergillus YX-1125 seed solution into sorghum fermentation medium, and performing aerobic fermentation under dark condition to obtain extremely red Aspergillus sorghum fermentation product. The extremely monascus sorghum fermentation product is rich in various active ingredients beneficial to skin, has good inhibition effect in various indexes such as skin melanin generation, skin microecological unbalance, skin sensitivity, skin inflammation, skin barrier damage, skin elasticity loss, skin wrinkle generation and the like caused by external stress such as photo-aging and the like, and can be regarded as a safe and excellent skin care product raw material. The invention provides scientific basis for developing the raw materials of the stress-resistant functional cosmetics based on Mao Gong aspergillus YX-1125 which is the Maotai-flavor liquor brewing microorganism and has great economic value and academic significance.

Description

Preparation method and application of extremely red aspergillus sorghum fermentation product

Technical Field

The invention belongs to the technical field of microbial fermentation, and particularly relates to a preparation method and application of an extreme monascus sorghum fermentation product.

Background

The skin may experience irreversible aging over time. And, the skin is directly exposed to the outside environment, and is easily affected by sunlight. Research has shown that photoaging is the primary external cause of skin aging, with more than 80% of facial aging caused by Ultraviolet (UV) light in sunlight. The most important histological difference between photoaged skin and naturally aged skin is that amorphous elastic fibers in the dermis tissue of photoaged skin exhibit an excessively accumulated state, and that collagen fibers exhibit significant abnormal fracture and structural disturbance. In addition to the reduced aesthetics, the aged skin also has a significantly reduced function of defending against damage: the disruption of the integrity of the skin barrier and the significant decrease in secretory function following excessive UV irradiation leads to a significant increase in the risk of developing skin inflammation and even malignant tumors of the skin. Therefore, the composition has the advantages of preventing photoaging, preventing damage to skin barriers, having important significance for preventing and treating skin related diseases, and having obvious academic value and great application potential.

Oxidative stress plays a central role in the development of skin photoaging. When the skin is irradiated with light, the light is absorbed by intracellular dye clusters such as NADH/NADPH, tryptophan or riboflavin, and the dye clusters are activated in the presence of molecular oxygen to form a series of oxidation products and active oxygen including superoxide anions, hydroxyl radicals and O ₂ Derived non-radical compounds such as H ₂ O ₂ Etc. Under UV irradiation of excessive intensity, intracellular ROS production is increasing and exceeds the ability of intracellular redox balance to regulate, leading to two consequences:

(1) High levels of ROS will disrupt the structure and function of the cell and cause the cell to release inflammatory mediators of the interleukin family IL-1, IL-6, IL-8, IL-10 and TNF- α, causing local skin inflammation;

(2) ROS can mediate activation of RTKs signaling cascades, activate multiple apoptosis-related signaling pathways, lead to reduced collagen production and increased synthesis of Matrix Metalloproteinases (MMPs), and finally promote skin aging, leading to skin diseases.

The fermentation products of probiotics and plants, also called metazoans (postbiotics), are widely proven to have the functions of maintaining the health of the epithelial barrier, regulating the immune response and increasing the antioxidant level of the organism, due to the enrichment of metabolites related to human health, such as Short Chain Fatty Acids (SCFAs), alkaloids, active peptides, etc. In recent years, researchers have shown that such fermentation products also have excellent anti-skin photoaging effects. In 2018 Shin and the like, lactobacillus is adopted to ferment the patchouli leaves, which proves that the levels of MMP-2 and MMP-9 can be obviously reduced, and the degradation of collagen after illumination is effectively reduced; the extract of lactobacillus buchneri (found in kimchi) fermented plant is found in Kang et al in 2020 to effectively inhibit the sun-induced elastase activity and the expression of MMPs, and promote the synthesis of skin collagen. According to the analysis of the existing literature, the research on skin photoaging resistance based on probiotics and plant fermentation products is in an initial exploration stage, the strain selection of the probiotics has a large optimization space, key metabolites and regulation modes thereof are not clear, and related action mechanisms are not clear, so that the probiotics and plant fermentation products have great research value.

Microorganisms grown in extreme environments, to accommodate survival, are naturally selected to generally have unique response mechanisms and metabolites to respond to high concentrations of ROS due to strong stress factors. From a biotechnological point of view, the plasticity of this metabolic pathway and gene expression makes extreme microorganisms promising for application, since they are able to adapt to adverse environments and provide efficient enzymes and abundant antioxidant metabolites under specific conditions. The research work of the fermentation of the extremely environmental microorganisms of the authors starts in 2017, and the first discovery of the ability to use nicotine to withstand filamentous fungi to ferment tobacco leaves under high concentration of nicotine stress for the production of highly active saccharifying enzymes is internationally, and in subsequent studies, the extremely microorganism is found to secrete a great deal of antioxidant and anti-inflammatory metabolites such as organic acids and unsaturated fatty acids through metabolic analysis so as to cope with ROS damage caused by environmental stress. Regarding the great potential of the extreme microbial fermentation products in terms of oxidation resistance or anti-inflammatory properties, although a small amount of research is being conducted by the scholars at home and abroad, the method is mainly focused on the environmental field.

However, the use of extremely microbial fermentation products in the field of skin health has not been studied so far.

Disclosure of Invention

In view of the above, the present invention aims to provide a method for preparing and using an extreme monascus sorghum fermentation product, which can be used for skin care.

The invention provides a preparation method of an extreme monascus sorghum fermentation product, which comprises the following steps:

inoculating Mao Gong Aspergillus YX-1125 seed solution into sorghum fermentation medium, and performing aerobic fermentation under dark condition to obtain extremely red Aspergillus sorghum fermentation product;

the accession number of Mao Gong Aspergillus YX-1125 is: CGMCC 21938;

the preparation method of the sorghum fermentation medium comprises the following steps:

mixing sorghum flour and water to obtain a mixture;

and (3) sequentially saccharifying the mixture by saccharifying enzyme and acidifying by lactic acid bacteria, then carrying out solid-liquid separation, collecting liquid components, and sterilizing the liquid components to obtain the sorghum fermentation medium.

Preferably, after the aerobic fermentation, the method further comprises:

carrying out solid-liquid separation on the aerobic fermentation product, and collecting a liquid component;

removing ethanol in the liquid component by rotary evaporation, performing membrane filtration, and collecting filtrate to obtain an extreme monascus sorghum fermentation product;

the pore diameter of the filter membrane adopted by the membrane filtration is 300-5000 Da.

Preferably, the sorghum flour comprises red-tassel sorghum flour.

Preferably, the grain size of the sorghum flour is 100 meshes; the mass ratio of the sorghum flour to the water is 1: (1-20).

Preferably, the saccharification by the saccharification enzyme comprises:

mixing the mixture with alpha-amylase, and performing pre-saccharification for 1-8 hours to obtain slurry;

and cooling the slurry to 20-80 ℃, mixing the slurry with the compound saccharifying enzyme, and saccharifying for 2-24 hours to obtain a saccharified product.

Preferably, the lactic acid bacteria comprise lactobacillus plantarum.

Preferably, after the filtrate is collected, the method further comprises the steps of sterilizing the filtrate, and adding a preservative into the sterilized filtrate; the preservative comprises one or more of p-hydroxyacetophenone, 1, 2-hexanediol and potassium sorbate.

The invention also provides a skin external preparation, which comprises the extreme monascus sorghum fermentation product prepared by the preparation method according to the scheme; the mass percentage of the fermentation product of the extreme monascus sorghum in the skin external preparation is 0.5-99%.

The invention also provides an application of the extreme monascus sorghum fermentation product prepared by the preparation method in preparing the skin external preparation;

the external preparation for skin is used for inhibiting skin injury caused by exogenous stress;

the skin lesion includes: skin melanin production, skin microecological imbalance, skin sensitivity, skin inflammation, skin barrier damage, skin elasticity loss and skin wrinkle production.

Preferably, the exogenous stress comprises photoaging.

The invention provides a preparation method of an extreme monascus sorghum fermentation product, which comprises the following steps: inoculating Mao Gong Aspergillus YX-1125 seed solution into sorghum fermentation medium, and performing aerobic fermentation under dark condition to obtain extremely red Aspergillus sorghum fermentation product; the accession number of Mao Gong Aspergillus YX-1125 is: CGMCC 21938. The extremely monascus sorghum fermentation product is rich in various active ingredients beneficial to skin, has good inhibition effect in various indexes such as skin melanin generation, skin microecological unbalance, skin sensitivity, skin inflammation, skin barrier damage, skin elasticity loss, skin wrinkle generation and the like caused by external stress such as photo-aging and the like, and can be regarded as a safe and excellent skin care product raw material. The invention provides scientific basis for developing the raw materials of the stress-resistant functional cosmetics based on Mao Gong aspergillus YX-1125 which is the Maotai-flavor liquor brewing microorganism and has great economic value and academic significance.

Description of biological preservation

Mao Gong Aspergillus (Monascus pilosus) YX-1125, deposited in China general microbiological culture Collection center, address Beijing, korea, north Chen Xie Lu 1,3, accession number: CGMCC 21938.

Drawings

FIG. 1 is a graph showing the effect of 2% by mass of fermentation product (left) and 5% by mass of fermentation product (right) on reduction of skin red blood streaks;

FIG. 2 is a graph showing the effect of 2% by mass of fermentation product (left) and 5% by mass of fermentation product (right) on the reduction of skin texture;

FIG. 3 is a graph showing the effect of 2% by mass of the fermentation product (left) and 5% by mass of the fermentation product (right) on the reduction of wrinkles at the corners of eyes.

Detailed Description

the accession number of Mao Gong Aspergillus YX-1125 is: CGMCC 21938.

In the present invention, aspergillus Mao Gong YX-1125 is a strain disclosed in the prior art, see in particular (Monascus pilosus YX-1125:An efficient digester for directly treating ultra-high-strength liquor wastewater and producing short-chain fatty acids under multiple-stress conditions. Bioresource technology.2021,331: 125050.).

In the invention, the preparation method of the seed liquid of the Mao Gong aspergillus YX-1125 preferably comprises the following steps:

inoculating Mao Gong Aspergillus YX-1125 on PDA plate, culturing at 28deg.C in dark until single colony grows on PDA plate; after a single colony grows on a PDA flat plate, scraping spores and hyphae into a liquid seed culture medium, and culturing seeds at 25-45 ℃ to obtain seed liquid;

in the present invention, the temperature of the seed culture is preferably 28 to 40 ℃; the time for the seed culture is preferably 1 to 6 days, more preferably 3 to 4 days.

In the invention, the seed culture medium takes sterile distilled water as a solvent and comprises the following components in concentration: 50g/L glucose and 2.5g/L yeast extract.

In the present invention, the ratio of the volume of the seed liquid of the Mao Gong aspergillus YX-1125 to the mass of the sorghum fermentation medium is preferably 1mL:10g.

In the invention, the preparation method of the sorghum fermentation medium comprises the following steps: mixing sorghum flour and water to obtain a mixture; and (3) sequentially saccharifying the mixture by saccharifying enzyme and acidifying by lactic acid bacteria, then carrying out solid-liquid separation, collecting liquid components, and sterilizing the liquid components to obtain the sorghum fermentation medium.

In the present invention, the sorghum flour preferably comprises red-tassel sorghum flour, more preferably comprises red-cherry organic sorghum; the red-tassel sorghum powder has high content of amylopectin and phenolic substances. In the present invention, the grain size of the sorghum flour is preferably 100 mesh; the mass ratio of the sorghum flour to the water is preferably 1: (1 to 20), more preferably 1: (5-10).

In the present invention, the sorghum medium preferably further comprises sterilization prior to use; the sterilization method is preferably a high temperature sterilization method.

In the present invention, the saccharification by the saccharifying enzyme preferably comprises: mixing the mixture with alpha-amylase, and performing pre-saccharification for 1-8 hours to obtain slurry; and cooling the slurry to 20-80 ℃, mixing the slurry with the compound saccharifying enzyme, and saccharifying for 2-24 hours to obtain a saccharified product.

In the present invention, the complex saccharification enzyme is purchased from Shandong Bioengineering Co.

In the present invention, the pre-saccharification is carried out in a preferably water bath; the temperature of the pre-saccharification is preferably 50-100 ℃, more preferably 80 ℃; the working concentration of the alpha-amylase is preferably 50-200U/g, more preferably 100-150U/g; the pre-saccharification is preferably carried out in a rotary shaker, preferably with a rotational speed of 50 to 200rpm, more preferably 100 to 150rpm. In the present invention, the time for the pre-saccharification is preferably 2 to 5 hours.

In the present invention, the temperature of the slurry after cooling is preferably 40 to 60 ℃. In the present invention, the working concentration of the complex saccharification enzyme is preferably 40U/g. In the present invention, the saccharification is preferably carried out in a rotary shaking table, and the rotation speed of the rotary shaking table is preferably 50 to 200rpm, more preferably 100 to 150rpm. In the present invention, the saccharification time is preferably 8 to 20 hours.

In the present invention, the lactic acid bacteria preferably include lactobacillus plantarum.

In the present invention, the lactic acid bacteria acidification preferably includes: mixing the saccharification product with lactobacillus, and acidifying to obtain an acidified product; the acidification time is preferably 2 to 24 hours, more preferably 8 to 20 hours.

In the present invention, the temperature of the aerobic fermentation is preferably 32℃and the time is preferably 3d.

After the aerobic fermentation, the invention preferably further comprises: carrying out solid-liquid separation on the aerobic fermentation product, and collecting a liquid component; removing ethanol in the liquid component by rotary evaporation, performing membrane filtration, and collecting filtrate to obtain an extreme monascus sorghum fermentation product; the pore diameter of the filter membrane adopted by the membrane filtration is 300-5000 Da. In the present invention, the solid-liquid separation is performed to remove the bacterial cells; the solid-liquid separation is preferably performed by centrifugation. In the present invention, the pore size of the filter membrane used for the membrane filtration is preferably 1000Da.

After collecting the filtrate, the invention preferably further comprises sterilizing the filtrate, and adding a preservative to the sterilized filtrate. In the present invention, the sterilization method is preferably a high-temperature secondary sterilization or a filtration sterilization method. In the present invention, the preservative preferably includes one or more of p-hydroxyacetophenone, 1, 2-hexanediol and potassium sorbate.

In the invention, the mass percentage of the fermentation product of the extreme monascus sorghum in the skin external preparation is preferably 1-5%.

The invention also provides an application of the extreme monascus sorghum fermentation product prepared by the preparation method in preparing the skin external preparation.

The extremely monascus sorghum fermentation product of the present invention can be used for skin care. In the present invention, the extremely red aspergillus kawachii fermentation product is used as an active ingredient in the skin external agent; the active ingredient has inhibition effect on metallothionein, inflammatory factor, intracellular oxygen free radical, melanin and epidermic harmful microorganism, and has no toxicity proved by cytotoxics, human body patch, chick embryo allantoic membrane test and animal test.

In the present invention, the skin external preparation is used for inhibiting skin damage caused by exogenous stress; the skin lesions preferably comprise: skin melanin production, skin microecological imbalance, skin sensitivity, skin inflammation, skin barrier damage, skin elasticity loss and skin wrinkle production.

In the present invention, the exogenous stress preferably includes photoaging.

In the present invention, the skin external preparation preferably includes a mask, essence, toner, cream, shampoo, body lotion, or private part care solution.

The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention.

Example 1

The preparation method of the fermentation product of the aspergillus terreus and sorghum comprises the following steps:

1. preparing spores: inoculating Mao Gong Aspergillus YX-1125 onto PDA plate, and culturing at 28deg.C in dark for 3 days;

2. seed liquid preparation: after single colony grows on the PDA plate, the spores and hyphae are scraped into a liquid seed culture medium, and the liquid seed culture medium is cultured for 3 days at the temperature of 28 ℃ to obtain seed liquid, wherein the seed liquid consists of 50g/L glucose and 2.5 yeast extract added with sterile distilled water;

3. and (3) strain fermentation: inoculating the seed solution into the prepared sorghum fermentation medium according to the proportion of 1mL of the seed solution to 10g of the medium, and performing aerobic fermentation for 3 days at 32 ℃ under dark conditions;

the sorghum fermentation medium is prepared by the following steps: crushing red-leaf sorghum to a particle size of 100 meshes, and adding sterile water, wherein the mass ratio of the red-leaf sorghum to the sterile water is 1:5 adding alpha-amylase with concentration of 100U/g into a mixture of red-tassel sorghum and sterile water, pre-saccharifying at 150rpm in a rotary table under the water bath condition of 50 ℃, cooling the slurry to 60 ℃ after 1h of pre-saccharification, saccharifying for 8h with a compound saccharifying enzyme with the speed of 100rpm in the rotary table, and then adding the effective viable count of 10 ⁶ cfu/ml; acidifying for 2h, removing solid matters in the acidified product, and sterilizing at high temperature.

4. And (3) extracting and purifying fermentation products: removing thalli by solid-liquid separation, removing ethanol in the solution by rotary evaporation, filtering by using a 1000Da membrane, performing high-temperature secondary sterilization, and adding hydroxyacetophenone with the volume percentage concentration of 0.1-0.5%, thereby obtaining the extremely red aspergillus sorghum fermentation product.

The fermentation product of the aspergillus terreus can be used for skin care, and is used as an active ingredient in the external skin preparation; the active ingredients are metallothionein, inflammatory factors, intracellular oxygen free radicals, melanin and epidermis harmful microorganisms, and have no toxicity through cytotoxics, human body patches, chicken embryo allantois tests and animal tests.

Wherein (1) MTT assay: after 24h of cell transfection, the cells were seeded in 96-well plates and the plates were incubated in an incubator. At the time points of 0h, 24h, 48h, 72h, 20. Mu.l MTT solution (5 mg/ml, sigma) was added to each well and after further incubation for 4h, a concentration of fermentation product was added to each well. The absorbance was measured at 490nm using an enzyme-linked immunosorbent assay, and a graph of cell growth was drawn.

(2) Acute skin irritation test in animals: cosmetic safety Specification 2015; number of animals/sex: test methods for 4 male and female halves (female not pregnant and not born): the test animals were fed in a single cage and were acclimatized to at least 3d in the laboratory environment prior to testing. The back vertebrae of the test animals were dehaired 24 hours before the test, and the dehairing range was about 3cm by 3cm. A0.5 mL sample was applied to one side of the skin 2.5cm by 2.5cm dehaired skin, the other side of the skin was used as a control, and after 4 hours the skin irritation was observed. The results showed no irritation.

(3) HET-CAM test: 6 perfect 9-day-old chick embryos are selected, a certain amount of the test substance is directly contacted with chick embryo allantois, the change condition of chorioallantoic membrane blood vessels is observed after a period of action, and positive and negative controls are simultaneously made.

The immobilized continuous fermentation system is utilized to simulate the stress brewing process of the Maotai wine by taking the sorghum hydrolysate as a raw material, so that the production of an extreme monascus sorghum fermentation product is realized; in clinical experiments, the protection and repair effects of the fermentation product on the skin barrier can be comprehensively evaluated through 4 indexes, namely short-term moisturizing evaluation, long-term moisturizing evaluation, short-term oil control evaluation and long-term oil control evaluation. Both 2% and 5% concentrations were used to test their effect on skin barrier. Experimental results show that in the short-term moisture preservation evaluation, the moisture content of the skin is rapidly reduced to about 2 after 6 hours from the average difference value of more than 20 just coated along with the change of time, and the 2% concentration fermentation liquor has stronger water supplementing capability than the 5% fermentation liquor; whereas the TEWL value of 2% strength broth is highest in terms of trans-epidermal water loss (TEWL), the results indicate that 2% broth possesses a stronger short-term moisturizing effect. In the long-term moisturizing effect, although 5% fermentation broth showed better skin moisturizing ability and higher TEWL value on day 2, 2% concentration fermentation broth was more excellent in terms of data after 7 days. In short term oil control, 2% fermentation broth showed some oil control effect and the trend was not significant, however, it is noted that 5% fermentation broth had some oil content enhancing effect, which may be related to a lower pH of higher concentration fermentation broth, which is more capable of promoting skin oil secretion in a short period of time. In the aspect of long-term oil control, 2% and 5% of fermentation liquor show remarkable oil control effect, and the oil control effect is in a direct proportion to the concentration of the fermentation liquor, which shows that the fermentation liquor effectively inhibits the grease secretion of the skin, and presumably, the reason is that the skin barrier injury is repaired and the balance of epidermis flora is maintained. In summary, the fermentation liquor has better skin barrier protection effect, and can effectively maintain the integrity and elasticity of skin barrier, wherein the 2% concentration fermentation liquor has stronger long-term skin barrier repairing capability, and the 5% fermentation liquor has stronger short-term skin barrier repairing capability and skin oil control capability, and the result suggests that the fermentation liquor with different concentrations can be used in skin barrier maintenance related skin care products with different purposes, as shown in figure 1.

By the Antera3D technique, we tested the effect of 2% and 5% fermentation broth on skin red blood streaks, melanin, skin texture, canthus wrinkles, and canthus fine lines. The results showed that after 42 days of fermentation broth use, the subject had various degrees of reduction in red blood streaks (FIG. 1), skin texture (FIG. 2), and corner wrinkles (FIG. 3), with overall better results for both 2% and 5% fermentation broth, with 5% being more pronounced but less improved and not directly proportional to broth concentration. The possible reason why the melanin-related effect is not obvious is that the trace of the subject is limited to 16/21 after the use, and most of the subjects have long-time sunlight irradiation experience in daily life, and meanwhile, the improvement of red blood streaks, skin textures and wrinkles at the corners of eyes under the condition can be seen, so that the fermentation liquor has stronger anti-photoaging effect. In conclusion, the fermentation liquor has good inhibition effect in various indexes such as skin sensitivity, skin inflammation, damaged skin barrier, loss of skin elasticity, skin wrinkle generation and the like caused by photo-aging, and can be considered as an excellent raw material of potential skin care products.

Although the foregoing embodiments have been described in some, but not all, embodiments of the invention, according to which one can obtain other embodiments without inventiveness, these embodiments are all within the scope of the invention.

Claims

1. A method for preparing an extreme monascus sorghum fermentation product, which is characterized by comprising the following steps:

the accession number of Mao Gong Aspergillus YX-1125 is: CGMCC 21938;

mixing sorghum flour and water to obtain a mixture;

2. The method according to claim 1, further comprising, after the aerobic fermentation:

the pore diameter of a filter membrane used for membrane filtration is 300-5000 Da.

3. The method of claim 1, wherein the sorghum flour is red-tassel sorghum flour.

4. A method of preparation according to claim 1 or 3, wherein the sorghum flour has a particle size of 100 mesh; the mass ratio of the sorghum flour to the water is 1: (1-20).

5. The method of claim 1, wherein saccharifying the saccharifying enzyme comprises:

6. The method according to claim 1, wherein the lactic acid bacteria are lactobacillus plantarum.

7. The preparation method according to claim 2, further comprising sterilizing the filtrate after collecting the filtrate, and adding a preservative to the sterilized filtrate; the preservative is one or more of p-hydroxyacetophenone, 1, 2-hexanediol and potassium sorbate.

8. A skin external preparation comprising the fermentation product of monascus purpureus prepared by the preparation method of any one of claims 1 to 7; the mass percentage of the fermentation product of the extreme monascus sorghum in the skin external preparation is 0.5% -99%.

9. The use of the fermentation product of extremely red aspergillus kawachii prepared by the preparation method of any one of claims 1 to 7 for preparing an external preparation for skin;

the skin lesions are: one or more of skin microecological imbalance, skin sensitivity, skin inflammation, damaged skin barrier, loss of skin elasticity and skin wrinkle.

10. The use of claim 9, wherein the exogenous stress is photoaging.