Method for improving capacity of extreme monascus sorghum fermentation product

Abstract

The invention provides a method for improving the capacity of an extreme monascus sorghum fermentation product. The method comprises the following steps: S1, liquefying; s2, acidification; s3, fermentation: adding an inoculation culture medium containing monascus purpureus YX-1125 spores into the residual solid phase, carrying out immobilized culture for 24 hours by adopting a reactor which is provided with 60 matrix units to form a honeycomb structure, replacing the inoculation culture medium with a collected liquid phase, continuing to culture for 90-100 hours at 35 DEG C, collecting fermentation liquor, and filtering through a 1000Da nanofiltration membrane to obtain a fermentation product. According to the method, the influence of monascus and sorghum fermentation products on resistance to ultraviolet induced damage is greatly improved based on an acid stress means, and the method has a great promotion effect on research on the anti-inflammatory and anti-light aging potential of polar bacteria fermentation products and industrial application of acid stress in the fermentation process; the obtained product has excellent skin anti-inflammatory and anti-photoaging effects, has the potential to become a functional cosmetic raw material, and is used for protecting the skin from environmental stress.

Description

Technical Field

[0001] The invention relates to a method for improving the capacity of extreme Monascus sorghum fermentation products, and belongs to the technical field of synthesizing target compounds using enzyme methods. Background Art

[0002] The skin is an organ that gradually undergoes an irreversible aging process. Unlike other organs, the skin is directly exposed to the external environment and is affected by sunlight. Studies have shown that photoaging is the primary external cause of skin aging, and more than 80% of facial aging is caused by solar ultraviolet (UV) radiation. The most critical histological difference between photoaged skin and naturally aged skin is the excessive accumulation of amorphous elastic fibers in the dermis of photoaged skin, and the obvious abnormal breakage and structural disorder of collagen fibers. After excessive ultraviolet radiation, the integrity of the skin barrier is destroyed and the secretory function is significantly reduced, resulting in a significantly increased risk of skin inflammation and skin malignancies. Therefore, delaying photoaging and protecting the skin barrier are crucial to the prevention and treatment of skin-related diseases, and have important academic value and application potential.

[0003] Extremophiles have developed unique cell structures, reaction mechanisms, and metabolic systems through natural selection to cope with high OS levels caused by strong stressors. From a biotechnological perspective, the plasticity of metabolism and gene expression brings extensive application potential to extremophiles because they can adapt to adverse environments and produce efficient, abundant, and unique metabolites under specific conditions. The potential of extremophilic fermentation products in counteracting OS caused by environmental stress has attracted extensive research interest worldwide. However, current research mainly focuses on environmental issues. The use of extremophilic fermentation products in skin care products to combat external environmental stress has not been documented. Summary of the invention

[0004] In order to solve the above technical problems, the present invention provides a method for improving the capacity of extreme Monascus sorghum fermentation products. The method for improving the capacity of extreme Monascus sorghum fermentation products greatly improves the effect of Monascus and sorghum fermentation products on resisting ultraviolet induced damage based on acid stress, which has a great promoting effect on the research and industrial application of the anti-inflammatory and anti-photoaging potential of extremophilic fermentation products under acidic stress during the fermentation process.

[0005] The present invention is achieved through the following technical solutions.

[0006] The present invention provides a method for improving the capacity of extreme Monascus sorghum fermentation products, comprising the following steps:

[0007] S1, liquefaction: add a mixture of sorghum flour and distilled water into a shaking flask in a water bath at 80°C, add α-amylase to the mixture to a concentration of 140 U / g, pre-saccharify on a rotary shaker for 2 hours, then cool to 60°C, and saccharify with a composite saccharifying enzyme for 8 hours;

[0008] S2, acidification: add 10% lactic acid bacteria inoculum to the saccharified mash, culture under strict static conditions at 42°C until the pH drops to 3.5, and collect the liquid phase; the lactic acid bacteria inoculum is Lactobacillus plantarum KN-819;

[0009] S3. Fermentation: Add the inoculation medium containing the spores of Monascus YX-1125 to the residual solid phase, and carry out immobilized culture for 24 hours in a reactor equipped with a honeycomb structure composed of 60 matrix units. Replace the inoculation medium with the collected liquid phase, and continue to culture at 35°C for 90 to 100 hours. Collect the fermentation broth and filter it through a 1000Da nanofiltration membrane to obtain the fermentation product.

[0010] In step S1, sorghum flour is obtained by grinding glutinous sorghum into powder and stored at a temperature below 5°C.

[0011] The spores of Monascus pubescens YX-1125 in step S3 are obtained by culturing Monascus pubescens YX-1125 on potato dextrose agar for 5 days and are stored at low temperature.

[0012] The Lactobacillus plantarum KN-819 is prepared in the following manner: the Lactobacillus plantarum KN-819 is transferred from a 20% glycerol storage medium to an agar medium, maintained on a rotary shaker at 150 rpm for 12 hours, centrifuged for 5 minutes, and then washed.

[0013] The agar medium is de Man, Rogosa and Sharpe medium.

[0014] The low temperature storage requirement of the Monascus pubescens YX-1125 is 4° C. for short-term storage and -20° C. for long-term storage. The spores of the Monascus pubescens YX-1125 are transferred to fresh PDA culture medium every month to maintain vitality.

[0015] The transfer of the Monascus YX-1125 spores to the fresh PDA medium is carried out in the following manner: the PDA medium is washed with sterile saline, the spore suspension of Monascus YX-1125 is added to the sterile inoculation medium, and the spore concentration is adjusted to 106 spores / mL by adding sterile saline.

[0016] The sterile inoculation medium contains 50 g / L glucose, 2.5% yeast extract powder, and the rest is water.

[0017] The Monascus rubrum YX-1125 was maintained at 30° C. for 8 hours before use.

[0018] The beneficial effects of the present invention are as follows: the acid stress-based approach greatly enhances the effect of Monascus and sorghum fermentation products on resistance to ultraviolet-induced damage, and has a great promoting effect on the research and industrial application of the anti-inflammatory and anti-photoaging potential of extremophilic fermentation products under acid stress during the fermentation process; the obtained product has excellent skin anti-inflammatory and anti-photoaging effects, and has the potential to become a functional cosmetic raw material for protecting the skin from environmental stress. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 is a schematic diagram of a process of at least one embodiment of the present invention;

[0020] Figure 2 It is a statistical diagram of the results of the in vitro evaluation of the cytotoxicity of different types and doses of fermented samples on human keratinocytes, UV-induced apoptosis and UV-induced intracellular ROS production;

[0021] Figure 3 It is a statistical diagram of the results of in vitro evaluation of the effects of different types and doses of fermented samples on the levels of IL-6, IL-8, MMP-1 and MMP-9 in human keratinocytes after UVB irradiation;

[0022] Figure 4 Antera3D images acquired at baseline and after 6 weeks to evaluate the anti-inflammatory and anti-photoaging effects of CF and ASF. DETAILED DESCRIPTION

[0023] The technical solution of the present invention is further described below, but the scope of protection claimed is not limited to the description.

[0024] A first embodiment of the present invention relates to Figure 1 A method for improving the capacity of extreme Monascus sorghum fermentation products is shown, comprising the following steps:

[0025] S1, liquefaction: add a mixture of sorghum flour and distilled water into a shaking flask in a water bath at 80°C, add α-amylase to the mixture to a concentration of 140 U / g, pre-saccharify on a rotary shaker for 2 hours, then cool to 60°C, and saccharify with a composite saccharifying enzyme for 8 hours;

[0026] S2, acidification: add 10% lactic acid bacteria inoculum to the saccharified mash, culture under strict static conditions at 42°C until the pH drops to 3.5, and collect the liquid phase; the lactic acid bacteria inoculum is Lactobacillus plantarum KN-819;

[0027] S3. Fermentation: Add the inoculation medium containing the spores of Monascus YX-1125 to the residual solid phase, and carry out immobilized culture for 24 hours in a reactor equipped with a honeycomb structure composed of 60 matrix units. Replace the inoculation medium with the collected liquid phase, and continue to culture at 35°C for 90 to 100 hours. Collect the fermentation broth and filter it through a 1000Da nanofiltration membrane to obtain the fermentation product.

[0028] The second embodiment of the present invention is substantially the same as the first embodiment, mainly in that, in step S1, the sorghum flour is obtained by grinding glutinous sorghum into powder and is stored at a temperature below 5°C.

[0029] Furthermore, the spores of Monascus pubescens YX-1125 in step S3 are obtained by culturing Monascus pubescens YX-1125 on potato dextrose agar for 5 days and are preserved at low temperature.

[0030] Furthermore, the low temperature storage requirement of Monascus YX-1125 is 4°C for short-term storage and -20°C for long-term storage. The spores of Monascus YX-1125 are transferred to fresh PDA medium every month to maintain vitality.

[0031] Further, the spores of Monascus YX-1125 were transferred to fresh PDA medium in the following manner: the PDA medium was washed with sterile saline, the spore suspension of Monascus YX-1125 was added to the sterile inoculation medium, and the spore concentration was adjusted to 106 spores / mL by adding sterile saline.

[0032] Furthermore, the sterile inoculation medium is composed of 50 g / L glucose, 2.5% yeast extract powder, and the rest is water.

[0033] The third embodiment of the present invention is substantially the same as the first embodiment, mainly in that Lactobacillus plantarum KN-819 is prepared by transferring Lactobacillus plantarum KN-819 from a 20% glycerol storage medium to an agar medium, maintaining the medium on a rotary shaker at 150 rpm for 12 hours, centrifuging the medium for 5 minutes, and then washing the medium.

[0034] Furthermore, the agar medium is de Man, Rogosa and Sharpe medium.

[0035] Furthermore, Monascus purpurogenum YX-1125 was maintained at 30° C. for 8 hours before use.

[0036] The third embodiment of the present invention is combined with the above embodiment. The raw material is organic glutinous sorghum [Sorghumbicolor (L.) Moench] produced in Maotai Town, Renhuai City, Guizhou Province, named "Hongyingzi", which is the main raw material of Maotai Town MFL, with a total starch content of 83.40%, of which amylopectin is 96.27%. The sorghum was ground into 100 mesh powder and stored at 4°C for standby use. α-Amylase (Bacillus subtilis mesophilic α-amylase, 4000U / g) and amyloglucosidase (Aspergillus niger and Bacillus licheniformis composite saccharifying enzyme, 100000U / mL) were both provided by Shandong Nongda Biological Products Co., Ltd.; these enzymes are used in the liquefaction process of converting starch into glucose. All other chemicals used in the study were of analytical grade and purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (Shanghai, China, www.aladdin-e.com).

[0037] The Monascus rubrum YX-1125 strain was obtained from the MFL Brewery in Maotai Town and is currently stored in the China Microbial Culture Center (No. CGMCC 21938). The Monascus rubrum YX-1125 strain was cultured on potato dextrose agar (PDA) at 30°C for 5 days and then stored in a refrigerator at 4°C (short-term storage) or -20°C (long-term storage). YX-1125 spores were transferred to fresh PDA medium every month to maintain viability. In order to obtain a fresh spore suspension for inoculation, the PDA medium was washed with sterile saline. Next, the spore suspension was added to a sterile inoculation medium (glucose 50g / L, yeast extract powder 2.5%). The spore concentration was adjusted to 106 spores / mL by adding sterile saline. The inoculation suspension of Monascus rubrum YX-1125 was maintained at 30°C for 8 hours without aeration, stirring and shaking, and then used as an inoculum for subsequent fermentation.

[0038] Lactobacillus plantarum (No. KN-819) comes from the Resources and Environment Department of Maotai Liquor Research Institute. The strain was screened from the MFL Brewery in Maotai Town. Due to its strong lactic acid production ability, the strain was used for pre-fermentation of Monascus rubrum YX-1125 to simulate an acidic stress environment similar to MFL brewing. Before use, the KN-819 strain was transferred from a 20% glycerol storage medium to de Man, Rogosa and Sharpe medium. To obtain the inoculum, the inoculum culture was maintained on a rotary shaker at 30°C and 150rpm for 12 hours, then centrifuged at 14,000×g for 5 minutes and subsequently washed with sterile saline.

[0039] The preparation included three stages: liquefaction, acidification, and fermentation. The first stage—liquefaction—included pre-saccharification and saccharification. Briefly, a mixture of distilled water and sorghum flour [solid-to-liquid ratio of 1:10 (w / v)] was added to a shake flask in an 80°C water bath. Then, α-amylase was added to the mixture to a final concentration of 140 U / g, and pre-saccharification was performed on a rotary shaker (Shanzhi, Shanghai, China) at 100 rpm for 2 h. The mixture was then cooled to 60°C and saccharified using a composite saccharifying enzyme (40 U / g) at 140 rpm for 8 h to completely release fermentable sugars from amylopectin.

[0040] Acidification is then carried out to create a highly acidic environment for subsequent stress fermentation using lactic acid bacteria: after the saccharification mash is cooled, 10% lactic acid bacteria inoculum is added and cultured under strict static conditions at 42°C until the pH drops to 3.5, and then the liquid phase is collected for the subsequent fermentation stage.

[0041] Finally, the acid fermentation (ASF) samples were produced by fermentation according to the method of the prior art, which was carried out in a modified 5L spherical column bioreactor (Shanghai Baoxing). In order to immobilize the filamentous fungus Monascus pubescens YX-1125, the reactor was equipped with a honeycomb structure consisting of 60 matrix units. After 24 hours of immobilization culture, the inoculation medium was replaced with the collected acidified liquid. The culture was continued at 35°C with an aeration rate of 0.5vvm and a stirring rate of 160rpm. After 96 hours, the fermentation broth was collected and filtered through a 1000Da nanofiltration membrane to remove microorganisms and macromolecular impurities and obtain the ASF sample.

[0042] In addition, through the above steps, the acidification step can be skipped to obtain a conventional fermentation (CF) sample, and the fermentation step can be skipped to obtain an acidic environment control (AEC) sample.

[0043] Experimental Example 1

[0044] The metabolites produced were analyzed using gas chromatography-mass spectrometry (GC-MS). For liquid-liquid extraction, dichloromethane was used as the solvent to obtain the organic phase, followed by the identification of active substances in extreme Monascus sorghum fermentation product (MSFF) using an Agilent 7890N gas chromatograph combined with a 5977B quadrupole mass spectrometer (Agilent Technologies, Palo Alto, California, USA) in electron impact ionization mode (70 eV). Then, a DB-WAX capillary column (30 m × 0.25 mm × 0.25 μm) was used. Data analysis was performed using Agilent MSD ChemStation software. The presence of bioactive substances in the samples was determined by comparing the data with the mass spectrometry database maintained by the National Institute of Standards and Technology.

[0045] As shown in Table 1, in ASF and CF samples, butyrate became the main metabolite (accounting for more than 60% of short-chain fatty acids (SCFAs)), followed by propionate. It is noteworthy that ASF significantly improved the short-chain fatty acid (SCFA) production efficiency of Monascus rubrum YX-1125 during fermentation. In contrast, CF shifted the metabolic flux to the production of by-products, especially alcohols (ethanol and propylene glycol). This change was confirmed by a 5-fold increase in ethanol production, from 2.77 g / L to 13.82 g / L. In addition, the production and proportion of SCFAs in CF decreased significantly compared with ASF. In addition, the metabolites in the AEC sample were basically lactic acid (data not shown). This result indicates that the ability of Monascus rubrum YX-1125 to produce SCFA using sorghum can be improved by acid stress.

[0046] Table 1. Contents of metabolites in ASF and CF samples

[0047]

[0048] N / D: Not detected.

[0049] *Indicates significant differences among the same metabolites (p<0.05).

[0050] Experimental Example 2

[0051] Four major phenolic compounds in the fermentation samples were quantified: (i) total soluble phenolic acid content was determined using a gallic acid standard curve and expressed as mg gallic acid eq / mL; (ii) total flavonoid content was determined using a quercetin standard curve and expressed as mg quercetin eq / mL; (iii) total tannin content was determined by an epicatechin standard curve and expressed as mg epicatechin eq / mL; (iv) total anthocyanin content was determined by spectrophotometry and expressed as μg glucoside cyanidin-3-glucoside (C3G) per mL of sample (μg cyanidin-3-glucoside eq / mL). All measurements were repeated three times independently.

[0052] Spectroscopic analysis was used to quantify the phenolic content in the three different samples. The results are detailed in Table 2. It can be seen that the concentration of phenols in different fermentation samples varies. In the ASF sample, total tannins had the highest concentration among the total phenols (3.59 mg epicatechin equivalent / mL), and phenolic acids were the second most abundant phenolic compound with a content of 1.52 mg gallic acid equivalent / mL. In contrast, the concentration of all phenolic compounds except total tannins in the AEC sample was higher than that of other samples. The study also pointed out that the phenolic acid content in both ASF and CF samples decreased after fermentation with Monascus pilosula YX-1125, indicating that Monascus pilosula YX-1125 has the potential to degrade phenols. In conclusion, due to the negative impact of fermentation on phenolic content, the content of ASF samples was lower than that of CF and AEC.

[0053] Table 2. Phenolic contents in AEC, CF and ASF samples determined by spectrometry

[0054]

[0055] TDs: total 3-deoxyanthocyanidins; TAs: total anthocyanidins; TFs: total flavonoids; PAs: phenolic acids; TTs: total tannins.

[0056] Different lowercase letters in the same item indicate significant differences (p<0.05).

[0057] Experimental Example 3

[0058] Five criteria were used to evaluate the antioxidant activity: (i) ferrous reducing antioxidant power (FRAP) test, expressed as mmoL eqFeSO4 / mL; (ii) 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging activity; (iii) copper and iron chelating ability; (vi) superoxide radical (O2·-) scavenging activity; and (v) hydroxyl radical (HO·) scavenging activity.

[0059] The in vitro antioxidant potential of the fermented samples was evaluated using a variety of assays; the results are listed in Table 3. The formation of hydroxyl radicals, one of the most common ROS triggered by UV irradiation, is associated with transition metals, particularly copper and iron. In the absence of these ions, hydrogen peroxide is relatively stable. When the homeostasis of these metals is disturbed, hydroxyl radicals are formed via the Fenton reaction. Hydroxyl radicals are considered to be the most active ROS and can interact with almost any biomolecule, such as proteins and DNA, to form toxic compounds, leading to cell damage. The experiments showed that all fermented samples had high metal chelating capacity (especially for copper) and hydroxyl radical scavenging capacity. Since the values ​​of hydroxyl radical scavenging activity were significantly greater than those of metal chelating activity, it is speculated that the fermented samples may prevent the formation and cascade of ROS at the initial stage of oxidation through multiple mechanisms, such as the removal of metal ions. In addition, the fermented samples showed strong free radical scavenging activity, with DPPH and superoxide scavenging rates of 60.24% and 40.55% for ASF, 63.20% and 48.51% for CF, and 67.38% and 55.68% for AEC, respectively, demonstrating their extensive ROS scavenging abilities. In addition, AEC had the strongest antioxidant capacity with a FRAP value of 1.24 mmol eq.FeSO4 / mL, followed by CF. The strongest antioxidant capacity of AEC may be attributed to its highest content of phenolic substances, such as tannins and anthocyanins. These findings indicate that all samples are excellent multi-target antioxidants with great potential to prevent UV-induced OS by inhibiting free radicals and acting on the initial stages of oxidation of cellular compounds.

[0060] Table 3. Evaluation results of in vitro antioxidant activity test of fermentation samples.

[0061]

[0062] FRAP: ferrous iron reducing antioxidant; DPPH: 1,1-diphenyl-2-picrylhydrazyl.

[0063] Different lowercase letters in the same item indicate significant differences (p<0.05).

[0064] Experimental Example 4

[0065] Human keratinocytes (HaCaT cells, provided by the Stem Cell Bank of the Chinese Academy of Sciences) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) containing 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin at 37°C, 5% CO2, and controlled humidity. HaCaT cells were seeded at a density of 7.5 × 104 cells per well overnight and subsequently pretreated in DMEM containing 0.5% FBS for 6 h after the addition of different concentrations of MSFF or sterile distilled water (as a control). After pretreatment, cells were washed twice with phosphate-buffered saline (PBS) to remove any residual DMEM and covered with a thin layer of PBS before being exposed to 15 mJ / cm2 of UVB using a UVB generator (UVPCL-1000 UV crosslinker, Upland, CA, USA). 2 of UVB radiation.

[0066] The viability of human keratinocytes was determined using the SRB method. The fermentation samples were applied to HaCaT cells at three different concentrations (0.1%, 0.5%, and 1%). Figure 2 As shown in panel A, the viability of HaCaT cells treated with these doses was comparable to that of the control group, confirming that the fermentation samples at these doses were non-cytotoxic to HaCaT cells (>80% cell viability). Therefore, these nine fermentation sample doses were non-cytotoxic and could be used for subsequent experiments.

[0067] HaCaT cells were pretreated with various types and doses of fermentation samples and then treated with 15 mJ / cm 2 to evaluate their protective ability against UV-induced cell apoptosis. Figure 2 (B in the middle). UVB exposure significantly reduced the viability of HaCaT cells not treated with fermentation samples (control group) to 55.4% compared to non-exposure. It was observed that the survival rate of cells pretreated with ASF samples was significantly higher than that of cells treated with equal doses of CF and AEC samples. In addition, the increase in HaCaT cell survival was concentration-dependent. Cells pretreated with 1.0% ASF samples achieved the highest survival rate of UVB-irradiated HaCaT cells, reaching 90.4%. In contrast, the AEC sample group had the lowest cell survival rate, indicating that fermentation products induced by Monascus acid stress (such as SCFA) have a stronger anti-apoptotic effect. Therefore, the results indicate that acid stress enhances the ability of fermented samples to inhibit UVB-induced apoptosis in human keratinocytes.

[0068] Experimental Example 5

[0069] Intracellular reactive oxygen species (ROS) were measured using 2′,7′-dichlorodihydrofluorescein diacetate (DCF-DA). Human keratinocytes (HaCaT cells) were pretreated for 6 hours before UVB irradiation and then incubated for 2 hours. These cells were then stained with 5 μM DCF-DA for 30 minutes at 37°C, rinsed twice with PBS, and lysed with 90% dimethyl sulfoxide in PBS. Fluorescence was measured at excitation / emission wavelengths of 485 / 525 nm using a fluorescence microplate reader.

[0070] Considering that UVB induces excessive ROS production, which can cause inflammation and photoaging, the effects of different fermentation samples on UVB-induced intracellular ROS production in HaCaT cells were studied. The DCF-DA fluorescent probe was used to detect the intracellular ROS level. Figure 2 As shown in the middle C panel, the fluorescence intensity of UVB-irradiated cells (control) was significantly higher than that of unirradiated cells (174.5%). In contrast, the levels of UVB-induced intracellular ROS in cells pretreated with all fermentation samples were significantly reduced. This effect depended on the concentration of the sample. The ROS level in cells treated with 1% AEC reached the minimum value (110.1%) compared with the other pretreated cells. The results showed that all fermentation samples could effectively inhibit the generation of ROS in UVB-induced human skin cells; this finding is consistent with the previous conclusion that all fermentation samples have a strong ROS scavenging ability.

[0071] Additionally, to determine whether the fermented samples inhibited UV-induced pro-inflammatory mediators, the expression levels of pro-inflammatory cytokines such as IL-6 and IL-8 were investigated. Overall, UV irradiation upregulated the expression of pro-inflammatory cytokines that induce inflammation and photoaging ( Figure 3 (A and B). Addition of all fermentation samples to HaCaT cells significantly inhibited UV-induced IL-6 and -8 expression in a dose-dependent manner compared with the negative control. Notably, ASF-pretreated cells exhibited lower levels of proinflammatory cytokines at the same dose levels compared with cells pretreated with CF and AEC, especially at higher dose levels. It is noteworthy that 1.0% ASF treatment achieved an 85.2% UV-mediated IL-6 inhibition rate. These results indicate that acidic stress enhances the ability of fermentation samples to inhibit UVB-induced inflammation in human keratinocytes by reducing the levels of proinflammatory cytokines.

[0072] The anti-photoaging effects of various types and doses of fermented samples were compared with 10 μM retinoic acid (RA) to evaluate whether it attenuated UV-induced MMP-1 and MMP-9 expression. All groups except the control group were treated with 5 J / cm 2 The expression levels of MMPs were Figure 3As shown in panels C and D, UV irradiation significantly increased MMP-1 expression to 267.2%, while fermented samples and RA treatment significantly reduced its expression, and the MMP-1 expression levels in 1% ASF-treated and RA-treated cells were reduced to 147.8% and 107.0%, respectively, compared with UV-induced cells. Overall, MMP-1 expression decreased with increasing sample concentrations, and the MMP-1 expression level in the ASF-treated group was significantly higher than that in the CF and AEC groups. MMP-9 expression levels increased significantly to 197.5% after UV irradiation, but decreased significantly after treatment with ASF, CF, AEC, and RA. Notably, after UV irradiation, the MMP-9 expression levels in RA-treated cells were not significantly different from those observed in cells treated with ASF at concentrations higher than 0.5%. These findings suggest that acid stress can effectively enhance the anti-photoaging ability of fermented samples by inhibiting the expression of MMPs related to UV-induced photoaging.

[0073] Experimental Example 6

[0074] Four clinical factors were evaluated to test the in vivo effects of the fermented samples: facial wrinkles, periorbital wrinkles, melanin, and facial redness. The study was conducted on 36 healthy volunteers aged 35 to 65 years whose jobs required them to spend more than 6 hours a day outdoors. Volunteers were excluded if: 1) they were pregnant, breastfeeding, or smokers; experienced skin infections, inflammatory acne, or photosensitive skin diseases; or showed a tendency to scar; 2) if they participated in a study and received topical vitamin A treatment within 6 months before entering the study; 3) if they studied or used systemic retinoids in the past 12 months; or 4) if they received ablative / non-ablative laser skin resurfacing treatment within 3 months before the study period and were allergic or sensitive to weak acid solutions. This study was a randomized, double-blind, placebo-controlled trial.

[0075] First, the Antera3D system was used to compare the effects of CF and ASF samples with placebo on PW. Figure 4 As shown in Figure A. Compared with the initial PW values, a significant decrease was observed after 6 weeks of treatment with CF and ASF, and the decrease rate in the ASF sample was higher than that in the CF sample (10.14 vs. 6.24%). In contrast, there was no significant difference between the initial and final periorbital wrinkle values ​​in the placebo group. Next, the Antera3D system was used to compare the effects of the two different samples with the placebo on pigmentation. Figure 4As shown in panel B. After 6 weeks of treatment, the skin melanin values ​​of the CF group and the ASF group decreased slightly compared with the initial skin melanin values, but the difference was not significant. It is worth noting that the melanin in the placebo group increased compared with the initial value, indicating that both ASF and CF can moderately alleviate the overexpression of melanin caused by ultraviolet rays.

[0076] Compared with the initial skin redness values, the redness of the CF group, the ASF group and the placebo group decreased. Figure 4 As shown in Figure C. The reduction in redness was significantly greater in the CF and ASF treatment groups compared to the placebo treatment group. In addition, the reduction rate in the ASF group was significantly higher than that in the CF group (9.78% vs. 5.06%). Finally, ASF and CF were evaluated in improving photoaging-induced skin roughness. After 6 weeks of treatment, the TI in the CF and ASF treatment groups decreased by 10.99% and 3.52%, respectively, compared to the initial values. Figure 4 In addition, the TI of some volunteers treated with ASF decreased by more than 20%. Overall, consistent with the in vitro results, acid stress significantly enhanced the ability of fermented samples to inhibit various manifestations of skin inflammation and photoaging.

Claims

1. A method for improving the capacity of extreme Monascus sorghum fermentation products, characterized in that: The following steps are involved: S1, liquefaction: add a mixture of sorghum flour and distilled water into a shaking flask in a water bath at 80°C, add α-amylase to the mixture to a concentration of 140 U / g, pre-saccharify on a rotary shaker for 2 hours, then cool to 60°C, and saccharify with a composite saccharifying enzyme for 8 hours; S2, acidification: add 10% lactic acid bacteria inoculum to the saccharified mash, culture under strict static conditions at 42°C until the pH drops to 3.5, and collect the liquid phase; the lactic acid bacteria inoculum is Lactobacillus plantarum KN-819; S3. Fermentation: Add the inoculation medium containing the spores of Monascus YX-1125 to the residual solid phase, and carry out immobilized culture for 24 hours in a reactor equipped with a honeycomb structure composed of 60 matrix units. Replace the inoculation medium with the collected liquid phase, and continue to culture at 35°C for 90 to 100 hours. Collect the fermentation broth and filter it through a 1000Da nanofiltration membrane to obtain the fermentation product.

2. The method for improving the capacity of extreme Monascus sorghum fermentation products according to claim 1, characterized in that: In step S1, sorghum flour is obtained by grinding glutinous sorghum into powder and stored at a temperature below 5°C.

3. The method for improving the capacity of extreme Monascus sorghum fermentation products according to claim 1, characterized in that: The spores of Monascus pubescens YX-1125 in step S3 are obtained by culturing Monascus pubescens YX-1125 on potato dextrose agar for 5 days and are stored at low temperature.

4. The method for improving the capacity of extreme Monascus sorghum fermentation products according to claim 1, characterized in that: The Lactobacillus plantarum KN-819 is prepared in the following manner: the Lactobacillus plantarum KN-819 is transferred from a 20% glycerol storage medium to an agar medium, maintained on a rotary shaker at 150 rpm for 12 hours, centrifuged for 5 minutes, and then washed.

5. The method for improving the capacity of extreme Monascus sorghum fermentation products according to claim 4, characterized in that: The agar medium is de Man, Rogosa and Sharpe medium.

6. The method for improving the capacity of extreme Monascus sorghum fermentation products according to claim 3, characterized in that: The low temperature storage requirement of the Monascus pubescens YX-1125 is 4° C. for short-term storage and -20° C. for long-term storage. The spores of the Monascus pubescens YX-1125 are transferred to fresh PDA culture medium every month to maintain vitality.

7. The method for improving the capacity of extreme Monascus sorghum fermentation products according to claim 6, characterized in that: The transfer of the Monascus YX-1125 spores to the fresh PDA medium is carried out in the following manner: the PDA medium is washed with sterile saline, the spore suspension of Monascus YX-1125 is added to the sterile inoculation medium, and the spore concentration is adjusted to 106 spores / mL by adding sterile saline.

8. The method for improving the capacity of extreme Monascus sorghum fermentation products according to claim 7, characterized in that: The sterile inoculation medium contains 50 g / L glucose, 2.5% yeast extract powder, and the rest is water.

9. The method for improving the capacity of extreme Monascus sorghum fermentation products according to claim 1 or 2, characterized in that: The Monascus rubrum YX-1125 was maintained at 30° C. for 8 hours before use.

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