CN116762960A - Fermentation product, fermentation product filtrate and preparation method and application thereof - Google Patents

Abstract

The invention discloses a fermentation product, a fermentation product filtrate and a preparation method and application thereof, and belongs to the field of fermentation technology. In the present invention, Monascus pilosa YX-1125 is used as the fermentation strain and sorghum is used as the fermentation substrate. The present invention also filters the fermentation product to obtain fermentation product filtrate (MSFF). Due to its high antioxidant activity, MSFF can reduce cell apoptosis, redox imbalance, production of pro-inflammatory cytokines and expression of MMPs. The extremophile fermentation product provided by the present invention can be used in the health field to resist damage to the body caused by oxygen free radicals and inflammation, and especially has huge application potential in the fields of health food and skin care.

Description

Fermentation product, fermentation product filtrate and preparation method and application thereof

Technical field

The invention belongs to the field of fermentation technology, and specifically relates to a fermentation product, a fermentation product filtrate and a preparation method and application thereof.

Background technique

In order to adapt to survival, microorganisms growing in extreme environments usually have unique bacterial structures, response mechanisms and metabolic systems through natural selection to respond to high levels of oxidative stress (OS) caused by strong stress factors. From a biotechnological perspective, this plasticity of metabolic systems and gene expression makes extremophile applications promising, as they are able to adapt to adverse environments and provide efficient, abundant, and unique metabolites under specific conditions. The huge potential of extremophile fermentation products in combating OS induced by environmental stress has attracted widespread attention from scholars around the world, but current research is mainly concentrated in the environmental field. So far, no literature has been published on the use of extremophile fermentation products in the field of skin health to resist external environmental stress.

Contents of the invention

The object of the present invention is to provide a fermentation product, a fermentation product filtrate and a preparation method and application thereof. The fermentation product and the fermentation product filtrate MSFF are a safe and efficient antioxidant and anti-inflammatory raw material, which can be used in health food and skin. Nursing field.

The invention provides a fermentation product using Monascus pilosus YX-1125 as the fermentation strain and sorghum as the fermentation substrate;

The deposit number of Monascus pilosus YX-1125 is CGMCC 21938.

The invention also provides a method for preparing the above-mentioned fermentation product, which includes the following steps: crushing sorghum and then saccharifying it to obtain a saccharified liquid;

The saccharified liquid and lactic acid-producing bacteria are mixed and cultured, and the culture is stopped when the pH drops to 3.5 to obtain an acidified liquid;

Monascus pilosus YX-1125 is inoculated into the acidified liquid, and after aerobic fermentation, the fermentation liquid contains the fermentation product.

Preferably, the saccharification includes mixing the crushed sorghum with water, and then sequentially digesting the sorghum with α-amylase and complex saccharifying enzyme.

Preferably, the mass-to-volume ratio of sorghum and water is (1g:10mL) to (1g:20mL).

Preferably, when alpha-amylase is used for digestion, the temperature is 80°C and the digestion time is 2 hours;

When using complex glucoamylase for digestion, the temperature is 60°C and the digestion time is 8 hours.

Preferably, the aerobic fermentation temperature is 35°C, the aeration volume is 0.5vvm, and the stirring rate is 160r/min.

The invention also provides a method for preparing fermentation product filtrate, which includes filtering the fermentation product prepared by the above preparation method, and the filtrate is the fermentation product filtrate;

The filtration is to filter out substances exceeding 1000Da.

The present invention also provides the fermentation product filtrate prepared by the above preparation method.

The present invention also provides the use of the above-mentioned fermentation product or the above-mentioned fermentation product filtrate in preparing anti-inflammatory and/or antioxidant materials.

The present invention also provides the use of the above-mentioned fermentation product or the above-mentioned fermentation product filtrate in the preparation of food or skin care products.

Beneficial effects: The present invention provides a fermentation product using Monascus pilosus YX-1125 as the fermentation strain and sorghum as the fermentation substrate. The present invention also filters the fermentation product to obtain the fermentation product filtrate (Monascus and Sorghum Fermentation Filtrate, MSFF). After analysis, the MSFF of the present invention is rich in various short-chain fatty acids, phenolic compounds, amino acids and other biologically active substances, and has strong complex antioxidant potential (80% concentration of MSFF diphenyl bitter hydrazyl free radical scavenging The rate is 67.3%, the copper chelation rate is 59.71%, the iron chelation rate is 42.62%, the hydroxyl radical scavenging rate is 83.31% and the superoxide radical scavenging rate is 55.8%, and the iron ion reducing capacity is equivalent to 1.24mmol/LFeSO ₄ /mL) . In vitro experimental results revealed that 1% (v/v) MSFF can reduce cell apoptosis by 81.4%, remove 35.8% of intracellular reactive oxygen species, and reduce the release of pro-inflammatory factors (interleukin-6 and -8) and up to 83.2%. Inhibits the inflammatory response and the damage it causes by down-regulating the expression of matrix metalloproteinases (metallothionease-2 and -9) by up to 42.5%. Safety results confirmed that MSFF is non-cytotoxic to various types of cells. In summary, MSFF is a safe and efficient antioxidant and anti-inflammatory raw material with huge application potential in the fields of health food and skin care.

Description of drawings

Figure 1 is a schematic flow diagram of the production of MSFF according to the present invention;

Figure 2 shows the infrared spectrum of MSFF;

Figure 3 is a quantitative data chart of different compound types in MSFF;

Figure 4 is a graph showing the results of in vitro evaluation of the effect of MSFF on induced inflammatory cells; (A) and (B) are graphs showing the results of measuring the effects of 0.1%, 0.5% and 1% MSFF on the viability of cells and inflammation-induced cells by the SRB method, respectively; (C) is the result of the effect of 0.1%, 0.5% and 1% MSFF on ROS production in inflammation-induced cells; * and ** are p values <0.05 and <0.01 respectively;

Figure 5 shows the results of the in vitro anti-inflammatory evaluation of MSFF; (A) and (B) respectively represent the results of the effects of 0.1%, 0.5% and 1% MSFF on the production of IL-6 and IL-8 in inflammation-induced cells; (C) and (D) respectively represent the results of the effects of 0.1%, 0.5% and 1% MSFF and 10μM retinoic acid on the expression of MMP-1 and MMP-9 in HaCaT cells induced by ultraviolet irradiation. β-actin is used as an internal control; * and ** are p values <0.05 and <0.01 respectively, indicating statistical significance.

Biological deposit information

Monascus pilosus YX-1125 has been deposited in the General Microbiology Center of China Committee for the Collection of Microbial Cultures (CGMCC) on March 29, 2021. The specific address is No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China Institute of Microbiology, Academy of Sciences, with accession number CGMCC 21938.

Detailed ways

The invention provides a fermentation product using Monascus pilosus YX-1125 as the fermentation strain and sorghum as the fermentation substrate;

The deposit number of Monascus pilosus YX-1125 is CGMCC 21938.

The sorghum of the present invention is preferably sorghum for the production of Maotai-flavor liquor. In the embodiment, the organic waxy sorghum called "Hongyingzi" produced in Maotai Town, Renhuai City (Guizhou, China) is used as an example for illustration. The total starch content of sorghum is 83.40%, of which 96.27% is amylopectin. The sorghum of the present invention is stored in a low temperature environment of 4°C before use. Before fermentation, the sorghum of the present invention preferably further includes crushing, preferably to 100 mesh.

The invention also provides a method for preparing the above-mentioned fermentation product, which includes the following steps: crushing sorghum and then saccharifying it to obtain a saccharified liquid;

The saccharified liquid and lactic acid-producing bacteria are mixed and cultured, and the culture is stopped when the pH drops to 3.5 to obtain an acidified liquid;

Monascus pilosus YX-1125 is inoculated into the acidified liquid, and after aerobic fermentation, the fermentation liquid contains the fermentation product.

In the present invention, sorghum is first saccharified to obtain saccharified liquid. The saccharification of the present invention preferably includes mixing the crushed sorghum with water, and then sequentially digesting the sorghum with α-amylase and complex saccharifying enzyme. In the present invention, it is preferred to mix sorghum crushed to 100 mesh with distilled water, and the solid-liquid mass volume ratio during the mixing is preferably (1g:10mL) to (1g:20mL).

In the present invention, it is preferred to heat the mixed slurry to 80°C, then add α-amylase with a concentration of 140 U/g, and perform pre-saccharification in a rotary shaker (Shanzhi, Shanghai, China) at a speed of 100 r/min. After 2 hours of pre-saccharification, the slurry was cooled to 60°C and saccharified with complex glucoamylase (40 U/g) in a rotary shaker at a speed of 140 r/min for 8 hours to fully release the fermentable sugars in amylopectin. The present invention has no special restrictions on the sources of the α-amylase and complex glucoamylase. Conventional commercially available enzyme preparations in the field can be used. In the examples, they were all purchased from Shandong Agricultural University (Nongda) Biological Products Co., Ltd.

In the present invention, the saccharified liquid is acidified, and the acidification is preferably accomplished by using lactic acid-producing bacteria, and the lactic acid-producing bacteria preferably include Lactobacillus plantarum. In the embodiment of the present invention, it is preferable to use Lactobacillus plantarum for inoculation, and culture it under strict static conditions at 42°C until the pH value drops to 3.5. The liquid part is collected for the next fermentation stage. The strain of Lactobacillus plantarum can be Such as Lactobacillus plantarum CICC 6009, Lactobacillus plantarum CICC10345 or Lactobacillus plantarum CICC 6240.

In the present invention, Monascus pilosus YX-1125 is inoculated into the acidified liquid for aerobic fermentation. The strain YX-1125 of the present invention is preferably stored on potato dextrose agar (PDA) at 30°C for 5 days and then stored in a refrigerator at 4°C (short-term) or -20°C (long-term). Spores of strain YX-1125 were transferred to fresh PDA medium every month to maintain viability. Wash the PDA culture medium with sterile physiological saline to obtain fresh inoculated spore suspension. The prepared spore suspension was added to the sterile seed culture medium (glucose 50g/L, yeast extract powder 2.5%), and sterile physiological saline was added to adjust the spore concentration to 10 ⁶ spores/mL. After 8 hours of seed culture at 30°C without ventilation, stirring, or shaking, the seed suspension of strain YX-1125 will be used for inoculation in subsequent fermentations. The aerobic fermentation temperature of the present invention is preferably 35°C, the aeration amount is preferably 0.5vvm, the stirring rate is preferably 160r/min, and the aerobic fermentation time is preferably 96 hours.

The invention also provides a method for preparing fermentation product filtrate, which includes filtering the fermentation product prepared by the above preparation method, and the filtrate is the fermentation product filtrate;

The filtration is to filter out substances exceeding 1000Da.

In the present invention, it is preferred to collect the above-mentioned fermentation broth and filter it through a 1000Da nanofiltration membrane to remove microorganisms and macromolecular impurities in the fermentation broth to obtain MSFF.

The present invention also provides the fermentation product filtrate prepared by the above preparation method.

The MSFF of the present invention contains rich and high-concentration biologically active substances. Fourier transform infrared spectroscopy (FTIR) analysis found that the main bands are distributed in organic acids and phenols related to high antioxidant activity. Regarding similar substances; small molecular substances in MSFF are analyzed through equipment such as GC-MS and multi-parameter bioanalyzers. Among them, organic acids are mainly short-chain fatty acids (SCFAs), accounting for more than 95%. Among the SCFAs in MSFF, the highest proportion is butyric acid (accounting for more than 60% of the total organic acids), followed by lactic acid, propionic acid and acetic acid, and the remaining organic acids are mainly composed of citric acid. Spectroscopic methods were used to measure the composition of phenolic substances in MSFF. The highest content was tannin, with a content of 3.59mgepicacatechin eq/mL MSFF, accounting for more than 50% of the total phenolic substances, followed by flavonoids, with a content of 2.89mgquercetin eq/mL. MSFF, the content of anthocyanins in flavonoids was determined to be 0.05mg cyanidin-3-glucosideeq/g, which is 1.8% of the total flavonoid content. The lowest content is phenolic acids, accounting for 7.2% of the total phenolic content; passed HPLC external standard method analysis showed that MSFF contained a total of 7.53 mg/mL of 17 free amino acids, 37.59 μg/mL of vitamin C, and 53.72 μg/mL of vitamin E.

The present invention also provides the use of the above-mentioned fermentation product or the above-mentioned fermentation product filtrate in preparing anti-inflammatory and/or antioxidant materials.

The fermentation product filtrate of the present invention has strong composite antioxidant potential (80% concentration MSFF has a diphenyl bitter hydrazyl free radical scavenging rate of 67.3%, a copper chelation rate of 59.71%, an iron chelation rate of 42.62%, and a hydroxyl free radical scavenging rate of 67.3%. The radical scavenging rate is 83.31% and the superoxide radical scavenging rate is 55.8%, and the iron ion reducing capacity is equivalent to 1.24mmoL FeSO4/mL). In vitro experimental results revealed that 1% (v/v) MSFF can reduce cell apoptosis by 81.4%, remove 35.8% of intracellular reactive oxygen species, and reduce the release of pro-inflammatory factors (interleukin-6 and -8) and up to 83.2%. Inhibits the inflammatory response and the damage it causes by down-regulating the expression of matrix metalloproteinases (metallothionease-2 and -9) by up to 42.5%.

The present invention also provides the use of the above-mentioned fermentation product or the above-mentioned fermentation product filtrate in the preparation of food or skin care products.

The MSFF of the present invention has no cytotoxicity to various types of cells, that is, MSFF is a safe and efficient antioxidant and anti-inflammatory raw material that can be used in the fields of health food and skin care.

In order to further illustrate the present invention, a fermentation product, fermentation product filtrate and preparation methods and applications provided by the present invention are described in detail below in conjunction with the accompanying drawings and examples, but they should not be understood as limiting the scope of the present invention.

For data processing and statistical data processing in the embodiment of the present invention, GraphPad Prism software v6.0 (GraphPad Software, San Diego, CA, USA) is used. Sample analysis methods include parametric analysis of variance (ANOVA) test and Tukey test.

Example 1

The production of MSFF requires three stages: liquefaction, acidification and fermentation as shown in Figure 1.

The first stage of liquefaction is divided into pre-saccharification step and saccharification step. First, a mixture of distilled water and sorghum powder (solid-liquid ratio 1:10 (w/v)) was added to the shake flask, and the temperature was controlled at 80°C (water bath). Subsequently, α-amylase with a concentration of 140 U/g was added to the prepared slurry, and presaccharification was performed in a rotary shaker (Shanzhi, Shanghai, China) at a speed of 100 r/min. After 2 hours of pre-saccharification, the slurry was cooled to 60°C and saccharified with complex glucoamylase (40 U/g) in a rotary shaker at a speed of 140 r/min for 8 hours to fully release the fermentable sugars in amylopectin.

The second stage is acidification, where Lactobacillus plantarum is used to create an extremely acidic environment for subsequent stress fermentation. After the saccharified slurry is cooled, 10% Lactobacillus plantarum inoculation suspension is added, and the culture is terminated under strict static conditions at 42°C until the pH value drops to 3.5. The liquid part is collected for the next fermentation stage.

Before use, the glycerol-preserved strains of Lactobacillus plantarum (Lactobacillus plantarum) were transferred to MRS medium. After culturing the seeds for 12 hours on a rotary shaker at 30°C and 150 rpm, centrifuge at 14,000 × g for 5 minutes, and then wash once with sterile physiological saline to obtain an inoculation suspension.

The third stage was fermentation, which was carried out in a modified 5-L bubbling (BC) bioreactor (Baoxing, Shanghai, China). The reactor is equipped with 60 matrix units to form a honeycomb structure for the immobilization of filamentous fungus YX-1125. After 24 h of immobilized culture, the seed culture medium was replaced with the collected acidified liquid and maintained at 35°C, with an aeration volume of 0.5 vvm and a stirring rate of 160 r/min. After 96 hours, the fermentation broth was collected and filtered through a 1000Da nanofiltration membrane to remove microorganisms and macromolecular impurities in the fermentation broth to obtain MSFF.

Example 2

Composition analysis of MSFF

2.1 Fourier transform infrared spectroscopy (FTIR)

MSFF was analyzed by FTIR on a Nicolet 6700 spectrometer. The samples were analyzed in transmission mode and the spectra were recorded in the range 4500-400cm-1. Scans were collected at a resolution of 4 cm and a scan speed of 1 cm/s, with background spectra collected before each measurement.

The FTIR spectrum of MSFF is shown in Figure 2. The large absorption band appearing near 3359.55 cm ^-1 corresponds to strong water absorption, accompanied by HOH stretching vibration. The smaller signal at 2938.32cm ^-1 is attributed to the CH stretching vibration of the methyl and methylene groups of carbohydrates or organic acids. Organic acids are identified from C=O stretching vibrations at 1735.40 cm ^-1 and 1675.01 cm ^-1 . The value of the CC stretching vibration band of the phenyl group in aromatic compounds is 1412.68cm ^-1 , and the signal at 1215.49cm ^-1 is related to the CO stretching vibration. That is, the main bands of MSFF samples are distributed on organic acids and phenolic substances, and these main bioactive components are related to high antioxidant activity.

2.2 Gas chromatographymass spectrometry (GC-MS)

GC-MS was used to analyze MSFF, especially the organic acid composition in it. According to the research of Mellinas, I SOLABERRIETA, C J PELEGRíN, et al. Valorization of agro-industrialwastes by ultrasound-assisted extraction as a source of proteins, antioxidants and cutin :A cascade approach[J].Antioxidants(Basel)11(2022).), and improved: use dichloromethane as the solvent to perform liquid-liquid extraction to obtain the organic phase. An Agilent 7890N gas chromatograph coupled with a 5977B quadrupole mass spectrometer (MS) (Agilent Technologies, Palo Alto, CA, USA) was used to identify active substances in MSFF under the electron impact (EI) ionization mode (70eV). A DB-WAX capillary column (30m×0.25mm×0.25μm) was used. Data analysis was performed using Agilent MSDChemStation software. The biologically active substances present in the samples were determined by matching with the National Institute of Standards and Technology (NIST) mass spectrometry database.

The composition of organic acids is shown in Figure 3. Organic acids are mainly short-chain fatty acids (SCFAs), accounting for more than 95%. Among the SCFAs in MSFF, the highest proportion is butyric acid (accounting for more than 60% of the total organic acids), followed by lactic acid, propionic acid and acetic acid, and the remaining organic acids are mainly composed of citric acid.

2.3 Determination of phenolic substances

Reference for quantitative methods of the four main phenolic compounds in MSFF (V P DIA, Z WANG, MWEST, et al. Processing method and corn cultivar affected anthocyanin concentration from dried distillers grains with solubles[J].J.Agric.Food Chem.63 (2015)3205-3218.;VP DIA,P PANGLOLI,L JONES,et al.Phytochemical concentrations andbiological activities of Sorghum bicolor alcoholic extracts[J].Food Funct.7(2016)3410-3420.;T J HERALD,P GADGIL ,R PERUMAL,et al.High-throughput micro-plate HCl-vanillin assay for screening tannin content in sorghum grain[J].J.Sci.FoodAgric.94(2014)2133-2136.)：

(i) Total Solublephenolic acids, calculated according to the gallic acid standard curve, expressed as mg gallic acid equivalents per mLMSFF, unit mg gallic acid eq/mL;

(ii) Total flavonoids, quantified using the quercetin standard curve, expressed as mg quercetin equivalents per mL MSFF, in mg quercetin eq/mL;

(iii) Total Tannins (TotalTannins), measured using the epicatechin standard curve, expressed as mg epicatechin per mLMSFF, unit: mg epicatechin eq/mL;

(iv) Total anthocyanins, measured by spectrophotometry, expressed as μg glucoside per mL MSFF, and the unit is μgcyanidin-3-glucoside eq/g. All measurements were repeated three times independently.

Spectroscopic methods were used to measure the composition of phenolic substances in MSFF, and the results are shown in Figure 3. The highest content of phenolic substances is tannin, with a content of 3.59 mg epicatechin eq/mL MSFF, accounting for more than 50% of the total phenolic substances, followed by flavonoids, with a content of 2.89 mg quercetin eq/mL MSFF. Among flavonoids, anthocyanins The measured content is 0.05mgcyanidin-3-glucoside eq/g, which is 1.8% of the total flavonoid content. The lowest content is phenolic acids, accounting for 7.2% of the total phenolic content.

2.4HPLC and biochemical analyzers

Vitamin and amino acid content and composition in MSFF were determined by HPLC. A Shim-pack GIST C(18) chromatographic column (4.6mm×250mm, 5μm) is used; the mobile phase is acetonitrile and 0.1mol/L potassium dihydrogen phosphate aqueous solution, the ratio is 80:20; the flow rate: 1mL/min; the detection wavelength is 246 nm, injection volume 10 μL, column temperature 40°C. Identification of amino acids and vitamins was confirmed by retention time comparison with purchased commercial standards. A multi-parameter biochemical analyzer (Biostream, Netherlands) was used to measure various hydroxy acids such as lactic acid, citric acid, tartaric acid, and malic acid in MSFF.

The HPLC external standard method analysis results are shown in Figure 3. MSFF contains a total of 7.53 mg/mL of 17 free amino acids: tyrosine 0.25 mg/mL, arginine 0.18 mg/mL, and phenylalanine 0.40 mg/mL. , histidine 0.11mg/mL, methionine 0.09mg/mL, glutamic acid 1.06mg/mL, lysine 0.31mg/mL, aspartic acid 0.67mg/mL, asparagine 0.10mg/mL, isoleucine Acid 0.50mg/mL, leucine 0.65mg/mL, threonine 0.33mg/mL, valine 0.61mg/mL, proline 0.90mg/mL, serine 0.49mg/mL, alanine 0.46mg /mL and glycine 0.42mg/mL. Cysteine, glutamine, and tryptophan were not detected. In addition, MSFF also detected vitamin C and vitamin E at 37.59 μg/mL and 53.72 μg/mL respectively. In summary, MSFF contains abundant and high concentrations of biologically active substances.

Analysis of antioxidant properties of 2.5MSFF

ReferenceB GULLON,P GULLON,TA et al.Optimization of solventextraction of antioxidants from Eucalyptus globulus leaves by responsesurface methodology:Characterization and assessment of their bioactiveproperties[J].Ind.Crops Prod.108(2017)649-659. Five different methods were used to evaluate antioxidant activity:

(i) FRAP (ferric reducing antioxidant power) antioxidant power test, unit is mmoL eqFeSO ₄ /mL;

(ii) DPPH (1,1-diphenyl-2-picroylhydrazyl) free radical scavenging activity;

(iii) Ability to chelate copper and iron;

(vi) Superoxide radical (O ^2·- ) scavenging activity;

(v) Hydroxyl radical (HO·) removal activity.

The results of evaluating the in vitro antioxidant potential of MSFF through multiple detection methods are shown in Table 1. MSFF has both high metal chelating ability (especially copper) and hydroxyl radical scavenging ability. Since the scavenging activity of hydroxyl radicals is numerically much higher than the metal chelating activity, it is speculated that MSFF can hinder the formation of oxygen free radicals and cascade reactions at the initial stage of oxidation through various mechanisms such as removing metal ions.

MSFF also has a good effect in other free radical scavenging. The DPPH and O ^2- scavenging rates of 80% of MSFF reached 67.3% and 55.8%, proving the comprehensive reactive oxygen species scavenging ability of MSFF. In addition, in terms of antioxidant capacity, MSFF also has a high value. The FRAP value of 80% MSFF is 1.24mmoleq.FeSO ₄ /mL MSFF, which is equivalent to 102.8g of vitamin C per liter of MSFF (per 100mg The antioxidant activity of vitamin C is 1.51mmol eq.FeSO ₄ ). The high antioxidant capacity of MSFF may be related to its high level of phenolic substances (such as tannins and anthocyanins). The above test results all prove that MSFF can inhibit free radicals and act on the initial stage of oxidation of cellular compounds. It is an excellent multi-targeted antioxidant with huge antioxidant potential.

Table 1 MSFF in vitro antioxidant activity test evaluation results

2.6 In vitro cell experiments

2.6.1 Keratinocytes (HaCaT cells) and mouse mononuclear macrophages (RAW264.7, catalog number BNCC342033), purchased from the Chinese Academy of Sciences, cultured in Dulbecco's culture medium with 10% fetal calf serum and 1% penicillin/streptomycin in Dulbecco's modified Eagle's medium (DMEM) under humidified conditions of 37°C and 5% _CO2 .

Cells were seeded in a 96-well plate at 1.5 × 10 ⁴ cells/well, 100 μL per well, and cultured in a 37°C, 5% CO ₂ incubator. After the cells were incubated for 24 hours, the culture medium was aspirated and washed with fetal calf blood to remove the residual culture medium. 100 μL DMEM was added to the cells in the control group and blank group, and 100 μL MSFF of different concentrations (DMEM dilution) was added to the cells in the sample group. Each group had 6 parallel cells. Incubate in the box for 18 hours. They were then stimulated with 1 μg/mL lipopolysaccharides (LPS) or irradiated with a UV generator (UVP CL-1000 Ultraviolet Crosslinker, Upland, CA, USA) to a dose of 5 mJ/cm ² to induce an inflammatory response.

2.6.2 Cell viability test

According to the method of reference (EA ORELLANA, A LKASINSKI. Sulforhodamine B (SRB) assay in cell culture to investigate cell proliferation [J]. Bio Protoc. 6 (2016) e1984.), sulforhodamine B was used to determine cell viability. After the incubation period, cells were washed once with sterile fetal calf serum and then fixed into each well with 250 μL of 10% (w/v) trichloroacetic acid (TCA) and incubated at 4°C for 1 hour. Then, rinse the plate with slow tap water, remove excess water with paper towels, and allow to dry at room temperature (20-25°C). Add 250 μL of 0.057% (w/v) sulforhodamine B solution to each well and stain for 30 min. Unbound dye was washed with 1% (v/v) acetic acid. The protein-binding dye was dissolved in 250 μL of 10 mM triethanolamine buffered saline solution, and 100 μL was transferred to a 96-well plate, and the OD value at 510 nm was measured in a microplate reader.

RAW264.7 cells were treated with three different concentrations of MSFF (0.1%, 0.5% and 1%). The results are shown in A in Figure 4. The viability of the treated HaCaT cells was comparable to that of the control group, confirming that MSFF has a strong No cytotoxicity to HaCaT cells (>80% cell survival).

HaCaT cells were pretreated with the above three concentrations of MSFF respectively, and then the inflammatory response of HaCaT cells was induced. The results are shown in Figure 4, B. Compared with cells that did not produce inflammation, the inflammatory response significantly reduced the viability of HaCaT cells to 55.4%. Pretreating cells with 0.5% MSFF increased the viability of UV-exposed HaCaT cells to 81.4%. In addition, the improvement of HaCaT cell viability was concentration-dependent. Therefore, the resulting data indicate that MSFF is non-cytotoxic at 1% concentration and can effectively reduce human keratinocyte apoptosis under UV irradiation.

2.6.3 Determination of intracellular reactive oxygen species

References (HE Y, HU Y, JIANG X, et al. Cyanidin-3-O-glucoside inhibits theUVB-induced ROS/COX-2pathway in HaCaT cells[J].J.Photochem.Photobiol.B.177(2017) 24-31.;PM D J FERNANDO,M J PIAO,K A KANG,et al.Rosmarinic acidattenuates cell damage against UVB radiation-induced oxidative stress viaenhancing antioxidant effects in human HaCaT cells[J].Biomol.Ther.24(2016)75- 84.) method, using reactive oxygen species fluorescent probe (2',7'-dichlorodihydrofluorescein-diacetate, DCF-DA) to measure intracellular reactive oxygen species. Briefly, HaCaT cells were pretreated for 6 h before UV irradiation and then incubated for 2 h. After incubation, HaCaT cells were stained with 5 μM DCF-DA at 37°C for 30 min, then washed twice with fetal calf serum, followed by lysing the cells with 90% dimethyl sulfoxide (DMSO) in fetal calf serum. Fluorescence microplate readers measure fluorescence at excitation/emission wavelengths of 485/525nm.

Inflammation can induce excessive production of reactive oxygen species, which in turn triggers the photoaging process. Therefore, monitoring the intracellular reactive oxygen species generation can be used to study the effect of MSFF on inflammation-induced oxidative stress in RAW264.7 cells. The DCF-DA fluorescent probe was used to detect the generation of intracellular reactive oxygen species. The results are shown in C in Figure 4. The fluorescence intensity of reactive oxygen species in inflammatory cells (171.5%) was significantly higher than that in non-UV-irradiated cells (100%). However, after MSFF treated cells, the reactive oxygen species in inflammatory cells were reduced to less than 130% in a concentration-dependent manner. Compared to inflammatory cells, 1% MSFF treated cells decreased by 35.8% (171.5% vs. 110.1%). These results indicate that MSFF has the ability to reduce the generation of reactive oxygen species in cells.

2.6.4 Enzyme linked immunosorbent assay (ELISA)

The production of IL-6 and IL-8 in the culture medium was quantified using ELISA kits (Biolegend, San Diego, CA, USA) according to the manufacturer's instructions. A microplate reader reads absorbance at 450 nm.

The results are shown in Figure 5, A and B. LPS stimulation upregulates the expression of pro-inflammatory cytokines that induce inflammation and photoaging. Compared with the negative control, adding MSFF to RAW264.7 cells can significantly inhibit the IL-6 induced by LPS stimulation. expression in a dose-dependent manner, in which the inhibitory effect of 1.0% MSFF on IL-6 reached 83.2%. In addition, MSFF also gradually strengthened its inhibitory effect on LPS-induced IL-8 at concentrations of 0.1%, 0.5% and 1.0%, showing a dose-dependent phenomenon. These results indicate that MSFF can effectively inhibit the generation of inflammation by reducing the content of pro-inflammatory cytokines.

2.6.5 Western blotting

Western blotting was used to detect the expression of metallothioneases (Matrix metalloproteinases, MMPs). The treated cells were collected, washed with fetal calf serum, and then lysed using radioimmunoprecipitation to obtain whole cell lysate. The supernatant was collected and the protein was quantified using the Bradford protein method to determine the standard curve. Cell lysates were separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis, and proteins were subsequently transferred to NC membranes. Incubate overnight at 4°C with primary antibodies against β-actin (Santa Cruz, USA), MMP-1 (Abcam, USA), and MMP-9 (Abcam, USA). Finally, the membrane was washed three times with triethanolamine buffered saline solution plus Tween-20 (each wash was 5 min) to remove the primary antibody, and then incubated with the corresponding coupled secondary antibody (Proteintech, China) for 1 h at room temperature. Immunoreactive protein bands were visualized by chemiluminescence detection (BeyoECL Plus, Millipore).

Evaluate whether 0.1%, 0.5% and 1.0% MSFF can attenuate the expression of MMP-1 and -9 caused by UV to test its alleviating effect on inflammation-induced wrinkles and skin structure damage, and compare it with retinoic acid (Retinoic acid, RA) were compared at the 10 μM dose. Except for the control group, all other groups were treated with UV irradiation at a dose of 5J/cm2.

The results are shown in Figure 5 C-D. The expression level of MMP-1 significantly increased to 267.2% after UV irradiation, but the expression of MMP-1 caused by UV was significantly down-regulated after MSFF and RA treatment. Overall, as the concentration of MSFF increased, the expression of MMP-1 gradually decreased. The MMP-1 corresponding to 1% MSFF and RA were slightly higher than the levels in the control group without any UV irradiation. MMP-9 levels significantly increased to 192.5% after UV irradiation but decreased to 110.7%, 96.2%, 89.6% and 92.3% after treatment with 0.1%, 0.5%, 1.0% MSFF and RA. Under normal conditions without UV irradiation, the expression of MMP-9 is comparable to that of 0.5% MSFF plus UV irradiation. These results indicate that MSFF can effectively attenuate the expression of MMPs related to UV-induced wrinkle formation.

2.6.6 Security Assessment

References (S HONG, P PANGLOLI, R PERUMAL, et al. A comparative study onphenolic content, antioxidant activity and anti-inflammatory capacity of aqueous and ethanolic extracts of sorghum in lipopolysaccharide-induced RAW264.7macrophages[J].Antioxidants.9(2020 )1297.;WK CHO,H KIM,S KIM,H H SEO,etal.Anti-aging effects ofLeontopodium alpinum(Edelweiss)callus culture ex-tract through transcriptome profiling[J].Genes.11(2020)230.) , using RAW264.7 and HaCaT cells to evaluate the cellular safety of MSFF.

The cells were seeded in a 96-well plate with 200 μL culture medium (2.5 × 10 ⁴ cells/well). After overnight attachment, the cells were treated with different concentrations (0.1 to 50.0 mg/mL) of the prepared extract and 1 μg/mL lipopolysaccharide for 24 hours. The medium without MSFF and lipopolysaccharide was used as a negative control, and the medium without lipopolysaccharide was used as a positive control. Cell viability was analyzed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) method and measured based on the absorbance at a wavelength of 490 nm. MTT was purchased from Sigma-Aldrich (St. Louis, MO, USA).

The viability of two cell lines, HaCaT and RAW264.7, was evaluated using the MTT assay in the concentration range of 0.1 to 50.0 mg/mL after MSFF treatment. No significant effects were observed for either extract at all doses evaluated (no significant change in absorbance data, p>0.05), indicating that MSFF has higher cytocompatibility in both cells.

Although the above embodiments describe the present invention in detail, they are only part of the embodiments of the present invention, not all embodiments. People can also obtain other embodiments based on this embodiment without any inventive step. These embodiments All belong to the protection scope of the present invention.

Claims (10)

1. A fermentation product is characterized in that Monascus pilosus YX-1125 is used as a fermentation strain, and sorghum is used as a fermentation substrate;

the preservation number of the monascus comingensis YX-1125 is CGMCC21938.

2. The process for producing a fermentation product according to claim 1, comprising the steps of: crushing sorghum, and saccharifying to obtain saccharified liquid;

mixing and culturing the saccharification liquid and lactic acid-producing bacteria, and stopping culturing when the pH value is reduced to 3.5 to obtain an acidizing fluid;

inoculating monascus comingii YX-1125 into the acidized liquid, wherein the fermentation liquid contains the fermentation product after aerobic fermentation.

3. The method according to claim 2, wherein saccharification comprises mixing the crushed sorghum with water and then digesting with an alpha-amylase and a complex saccharification enzyme in sequence.

4. The method according to claim 3, wherein the mass-to-volume ratio of sorghum to water is (1 g:10 ml) to (1 g:20 ml).

5. The process according to claim 3, wherein the digestion with alpha-amylase is carried out at a temperature of 80℃for a period of 2 hours;

when the composite saccharifying enzyme is used for digestion, the temperature is 60 ℃ and the digestion time is 8 hours.

6. The preparation method according to claim 2, wherein the aerobic fermentation temperature is 35 ℃, the aeration rate is 0.5vvm, and the stirring rate is 160r/min.

7. A method for preparing a fermentation product filtrate, which is characterized by comprising the steps of filtering a fermentation product prepared by the preparation method according to any one of claims 2 to 6, wherein the filtrate is the fermentation product filtrate;

the filtration is to filter out substances exceeding 1000 Da.

8. A fermentation product filtrate obtained by the preparation method of claim 7.

9. Use of the fermentation product of claim 1 or the fermentation product filtrate of claim 8 for the preparation of an anti-inflammatory and/or antioxidant material.

10. Use of the fermentation product of claim 1 or the fermentation product filtrate of claim 8 for the preparation of a food or skin care product.