Preparation method and application of rhizoma polygonati fermentation liquor

Abstract

The preparation method comprises the following steps: mixing saccharomycetes, lactic acid bacteria and bacillus subtilis according to a ratio of 1: 1: 1 to obtain mixed bacteria; 0.25 g of mixed bacteria is taken and dissolved in 5 mL of sterile water for standby application; slicing and crushing the dried rhizoma polygonati, and sieving with a 10-mesh sieve to obtain rhizoma polygonati powder; the preparation method comprises the following steps: mixing water and rhizoma polygonati powder according to a liquid-material ratio of 10mL: 1g, sterilizing at 121 DEG C for 30min, cooling, adding mixed bacteria according to an inoculum size of 3% of the mass of rhizoma polygonati, culturing in a constant-temperature shaking table incubator at 120r. Min <-1 > for 72h at the culture temperature of 035 DEG C, sterilizing at 100 DEG C for 30min after fermentation, cooling and centrifuging to obtain rhizoma polygonati fermentation liquor, and storing at 4 DEG C. The prepared polygonatum sibiricum fermentation liquor is high in active ingredient content, has good cell safety, anti-oxidation, anti-inflammatory and whitening effects, and has the potential of being used for developing functional raw materials of traditional Chinese medicine cosmetics.

Description

Technical Field

[0001] The invention belongs to the technical field of polygonatum deep processing and application, and specifically relates to a preparation method and application of polygonatum fermentation liquid. Background Art

[0002] Polygonatum rhizome has both medicinal and edible functions and is often used as one of the traditional Chinese medicines for anti-aging, health preservation and beauty. Polygonatum rhizome is rich in saponins, polysaccharides, flavonoids, alkaloids, phytosterols and other ingredients, which give Polygonatum rhizome many biological activities, including anti-aging, immunomodulation, antibacterial and antiviral, and hypoglycemic. In 2015, my country approved the use of Polygonatum rhizome extract as a cosmetic raw material. Studies have shown that the topical application of Polygonatum rhizome water extract can promote collagen fiber synthesis, improve skin condition and delay aging. The extract can also reduce skin lesions in diabetic rats induced by streptozotocin and accelerate wound healing. Polygonatum rhizome polysaccharides have an immediate moisturizing effect, and the moisturizing property increases over time. Liquorice and 4,5-dihydroxyflavone in Polygonatum rhizome may act on targets such as CCNA2, CCNE1, and CDK2 to regulate progesterone-mediated oocyte maturation, cell senescence and other related signal pathways to treat chloasma. Studies have shown that the water extract and organic reagent extraction parts of raw Polygonatum sibiricum are non-irritating to rabbit eyes and rabbit skin, but its suspension is moderately irritating to rabbit eyes, skin and mucous membranes, which is presumably due to the irritating calcium oxalate component. In summary, Polygonatum sibiricum extracts are suitable for use as cosmetic raw materials and are expected to be developed into natural Chinese medicine cosmetics. However, there are currently few studies that apply fermentation to Polygonatum sibiricum to enhance its antioxidant, anti-inflammatory and whitening potential, as well as to evaluate whether the extracts are safe.

[0003] Fermentation of Chinese medicinal materials with probiotics can help degrade larger molecules in the medicinal materials into small molecules that are easily absorbed by the skin, and can also produce substances that are beneficial to the skin, such as α-hydroxy acids. Considering that fermentation can also decompose irritating compounds in medicinal materials and reduce their possible irritation to the skin, and that Polygonatum sibiricum is rich in polysaccharides and difficult for the skin to absorb directly, we studied the use of yeast, lactic acid bacteria, Bacillus subtilis and the above mixed bacteria to ferment Polygonatum sibiricum, and determined the changes in components, antioxidant properties, anti-inflammatory effects, anti-tyrosinase activity, and toxicity tests on human immortalized epidermal cells before and after fermentation of Polygonatum sibiricum, and evaluated the efficacy and safety of Polygonatum sibiricum fermentation liquid, providing a scientific basis for the application of Polygonatum sibiricum fermentation liquid as a raw material for functional cosmetics, and proposed a preparation method and application of Polygonatum sibiricum fermentation liquid based on the above research results. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a method for preparing a polygonatum fermented liquid in view of the deficiencies in the prior art. The content of active ingredients in the prepared polygonatum fermented liquid is significantly or extremely significantly increased compared with that in unfermented polygonatum liquid, and the performance in anti-oxidation, anti-inflammatory and inhibition of tyrosinase activity is better. The cell activity of the polygonatum fermented liquid prepared by the present invention is higher than that of the unfermented polygonatum liquid, the toxicity to HaCaT is low, and it shows the potential to promote cell proliferation, has good cell safety, anti-oxidation, anti-inflammatory and whitening effects, and has the potential to be used for the development of functional raw materials for traditional Chinese medicine cosmetics.

[0005] The technical solution adopted by the present invention to solve the above technical problems is: a method for preparing polygonatum fermentation liquid, comprising the following steps:

[0006] (1) Yeast, lactic acid bacteria and Bacillus subtilis are mixed in a ratio of 1:1:1 to obtain a mixed bacteria;

[0007] (2) Dissolve 0.25 g of the mixed bacteria in 5 mL of sterile water and set aside;

[0008] (3) Slice and crush the dried polygonatum sibiricum, and pass it through a 10-mesh sieve to obtain polygonatum sibiricum powder; mix water and polygonatum sibiricum powder at a liquid-to-solid ratio of 10 mL:1 g, sterilize at 121°C for 30 min, and cool, then add mixed bacteria at an inoculum amount of 3% based on the mass of the polygonatum sibiricum, and place in a constant temperature shaking incubator at 120 r·min -1 The culture was carried out for 72 hours at a temperature of 35°C. After the fermentation was completed, the mixture was sterilized at 100°C for 30 minutes, cooled and centrifuged to obtain the Polygonatum sibiricum fermentation liquid, which was stored at 4°C.

[0009] The accumulation of free radicals in the body, the sequelae of inflammation, and the activity of tyrosinase are closely related to whitening. ·OH free radicals and DPPH free radicals are highly oxidizing and can attack biological macromolecules in the body, destroy cell structure and function, cause skin spots and wrinkles, and cause skin aging. By measuring the scavenging rate of DPPP, ABTS and hydroxyl free radicals by Polygonatum sibiricum fermented liquid, we found that the scavenging rate of DPPH and ABTS free radicals by Polygonatum sibiricum fermented liquid was significantly higher than that of unfermented Polygonatum sibiricum liquid, but the scavenging rate of ·OH free radicals was significantly lower than that of unfermented Polygonatum sibiricum liquid. It is speculated that the total phenol content is positively correlated with the scavenging ability of DPPH free radicals, the total flavonoid content is also positively correlated with the scavenging ability of DPPH and ABTS free radicals, and the total flavonoids are negatively correlated with the scavenging rate of ·OH free radicals. The change pattern of the scavenging ability of DPPP, ABTS and ·OH free radicals by Polygonatum sibiricum fermented liquid is basically consistent with the change pattern of flavonoids, polyphenols and saponins content after Polygonatum sibiricum fermentation, indicating that the above active ingredients are the material basis for the overall improvement of the antioxidant properties of Polygonatum sibiricum fermented liquid.

[0010] HaCaT cells are one of the components of human epidermal cells and are closely related to epidermal differentiation. By studying the effect of samples on the activity of these cells, the safety of the epidermal barrier can be evaluated. Our study found that the HaCaT cell activity of Polygonatum sibiricum fermented liquid was greater than that of unfermented Polygonatum sibiricum liquid, and the concentration of 1 mg mL -1 The fermented Polygonatum sibiricum liquid was non-toxic to cells, had a certain effect of promoting cell proliferation, and had a high survival rate of treated cells. -1 It is non-toxic to cells. It can be seen that the safety of Polygonatum sibiricum fermented liquid is 10 times higher than that of unfermented Polygonatum sibiricum liquid.

[0011] As a normal physiological reaction of the human body, the inflammatory response plays a physiological barrier and protective role on the skin. Aging is closely related to inflammation, and proinflammatory cytokines such as TNF-a and IL-6 can induce and accelerate cell aging. Anti-inflammatory tests have found that the content of inflammatory factors in the fermented Polygonatum sibiricum liquid prepared by the present invention is significantly lower than that in the unfermented Polygonatum sibiricum liquid, and the content of TNF-a and IL-6 in the fermented Polygonatum sibiricum liquid prepared by the present invention is lower than that in the fermented Polygonatum sibiricum liquid fermented by a single bacteria. This shows that after fermentation, Polygonatum sibiricum can reduce the irritation of Polygonatum sibiricum and relieve inflammation.

[0012] Skin color is closely related to the melanin produced by melanocytes in the basal layer of the epidermis. Tyrosinase is the main rate-limiting enzyme in melanin production, which can inhibit tyrosinase activity and reduce melanin production, thereby exerting a whitening effect. By comparing the inhibitory effect of Polygonatum sibiricum fermented liquid on tyrosinase, we found that the in vitro and intracellular inhibition rate of tyrosinase in Polygonatum sibiricum fermented liquid was significantly higher than that of unfermented Polygonatum sibiricum liquid, and the inhibition rate of tyrosinase was high.

[0013] After testing, it was found that the content of active ingredients such as total polyphenols and total flavonoids in the fermented polygonatum sibiricum broth prepared by the present invention was significantly or extremely significantly increased compared with that in the unfermented polygonatum sibiricum broth, and the performance in anti-oxidation, anti-inflammatory and inhibition of tyrosinase activity was better.

[0014] Preferably, the method further comprises the step of determining the polysaccharide content in the fermented broth of polygonatum, wherein the determination method is the phenol-sulfuric acid method, and the standard curve using glucose as the standard is as follows:

[0015] A1=0.0087C1+0.0505 (1)

[0016] In formula (1), A1 is the absorbance of the sample to be tested; C1 is the glucose concentration; r 2 =0.9973, linear range 10~80μg·mL -1 .

[0017] Preferably, the method further comprises the step of determining the total saponin content in the fermented broth of polygonatum, wherein the determination method is the vanillin-perchloric acid-glacial acetic acid colorimetric method, and the standard curve using ginsenoside Rb1 as the standard is as follows:

[0018] A2=3.04C2-0.0619 (2)

[0019] In formula (2), A2 is the absorbance of the sample to be tested; C2 is the concentration of ginsenoside Rb1; r 2 =0.9681, linear range 100~400μg·mL -1 .

[0020] Preferably, the method further comprises the step of determining the total polyphenol content in the fermented broth of polygonatum, wherein the determination method is the folin phenol method, and the standard curve using gallic acid as the standard is as follows:

[0021] A3=0.0103C3-0.0111 (3)

[0022] In formula (3), A3 is the absorbance of the sample to be tested; C3 is the concentration of gallic acid; r 2 =0.9991, linear range 10~120μg·mL -1 As a preferred method, the method further comprises the step of determining the total flavonoid content in the fermented broth of Polygonatum cyrtonema.

[0023] The standard curve of NaNO2-Al(NO3)3-NaOH colorimetric method with rutin as the standard is as follows:

[0024] A4=0.006C4+0.0164 (4)

[0025] In formula (4), A4 is the absorbance of the sample to be tested; C4 is the concentration of rutin; r 2 =0.9959, linear range 20~100μg·mL -1 .

[0026] The above-mentioned Polygonatum sibiricum fermented liquid is used as an antioxidant, whitening and anti-inflammatory functional raw material in cosmetics.

[0027] Compared with the prior art, the present invention has the following advantages: the content of active ingredients such as total polyphenols and total flavonoids in the fermented polygonatum sibiricum broth prepared by the present invention is significantly or extremely significantly increased compared with that in the unfermented polygonatum sibiricum broth, and the performance in anti-oxidation, anti-inflammatory and inhibition of tyrosinase activity is better. Cytotoxicity evaluation shows that the cell activity of the fermented polygonatum sibiricum broth prepared by the present invention is higher than that of the unfermented polygonatum sibiricum broth, the toxicity to HaCaT is low, and it shows the potential to promote cell proliferation. Therefore, the fermented polygonatum sibiricum broth fermented by probiotics prepared by the present invention not only has good cell safety, but also has good antioxidant, anti-inflammatory and whitening effects, and has the potential to be used in the development of functional raw materials for traditional Chinese medicine cosmetics. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 to Figure 3 To compare the antioxidant activity of Polygonatum cyrtonema fermented broth from different strains;

[0029] Figure 4 The effect of different concentrations of Polygonatum cyrtonema fermentation broth of different strains on cell survival rate;

[0030] Figure 5 and Figure 6 The effect of Polygonatum sibiricum fermented broth from different strains on the content of TNF-a and IL-6 in HaCaT. DETAILED DESCRIPTION

[0031] 1 Materials and methods

[0032] 1.1 Materials and reagents

[0033] Polygonatum sibiricum, provided by Huangni Zhuguomu Family Farm, Chashan, Ninghai County, Ningbo in 2023. All Polygonatum sibiricum with a growth period of more than 3 years, are washed, sliced, dried, crushed, sieved, sealed and stored for later use, and their moisture content is less than 12.5%. Human immortalized keratinocytes (HaCaT cells, product number HTX2089), Beijing Beina Chuanglian Biotechnology Research Institute.

[0034] Yeast (20211010), lactic acid bacteria (20211018), Angel Yeast Co., Ltd. (Yichang); Bacillus subtilis (20210927), Shandong Yingrun Biotechnology Co., Ltd.; rutin standard (L0204-5G), gallic acid (LA20P33), Beijing Bailingwei Technology Co., Ltd.; 2,2-azinobis-(3-ethylbenzthiazoline-6-sulfonic acid) diammonium salt [2,2'-azinobis-(3-ethylbenzthiazoline-6-sulphonate, ABTS] (AK112912), ginsenoside Rb1 standard, high performance liquid chromatography (HPLC) Purity>98% (GR0180815) determined by chromatography, HPLC, Hefei Bomei Biotechnology Co., Ltd.; 1,1-diphenyl-2-picrylhydrazyl (DPPH) (GTBXB-QP), Shanghai Chemical Industry Development Co., Ltd.; Folin (210910), Hefei BASF Biotechnology Co., Ltd.; L-DOPA (201424), Shanghai MacLean Biochemical Technology Co., Ltd.; Dulbecco's modified eagle medium (DMEM) culture medium (PM150210), fetal bovine serum (164210), phosphate buffer solution (PBS) (PB180327), Wuhan Pronosai Life Science Co., Ltd.; cell rapid lysis solution (20211215), Beijing Solebow Technology Co., Ltd.; tumor necrosis factor (TNF) alpha, TNF-α) kit (E-EL-H0109c), interleukin 6 (IL-6) kit (E-SOEL-H0001), Wuhan Elaruite Biotechnology Co., Ltd.; glucose, vanillin, glacial acetic acid, perchloric acid, sodium nitrite, aluminum chloride hexahydrate, ferrous sulfate, sodium nitrate, phenol, concentrated sulfuric acid, potassium persulfate, salicylic acid, hydrogen peroxide, etc., Sinopharm Group (Beijing).

[0035] 1.2 Instruments and Equipment

[0036] TU-1810 UV spectrophotometer, Beijing Puxi General Instrument Co., Ltd.; TDZ4-WS low-speed desktop centrifuge, Hunan Xiangyi Laboratory Instrument Co., Ltd.; BSA124S electronic balance, Sartorius Scientific Instrument Co., Ltd., Germany; LKTC-B1-T gas bath constant temperature oscillator, Changzhou Guohua Instrument Manufacturing Co., Ltd.; YXQ-LS-18SI steam pressure sterilizer, Shanghai Boxun Medical Biological Instrument Co., Ltd.; XL-925 multi-function wall-breaking food processor, Dongguan Xiangling Machinery Equipment Co., Ltd.; GT221156 carbon dioxide incubator, Thermo Fisher Scientific Co., Ltd., USA; SPECTRAMAX-190 full-wavelength microplate reader, Shanghai Meigu Molecular Instrument Co., Ltd.; SW-CJ-2F clean workbench, Suzhou Antai Air Technology Co., Ltd.

[0037] 1.3 Test methods

[0038] 1.3.1 Preparation of Polygonatum sibiricum fermentation broth

[0039] Yeast, lactic acid bacteria and Bacillus subtilis were mixed at a ratio of 1:1:1 to obtain mixed bacteria; 0.25g of yeast, lactic acid bacteria, Bacillus subtilis and mixed bacteria were taken respectively, dissolved in 5mL of sterile water respectively, and set aside; dried polygonatum was sliced ​​and crushed, and passed through a 10-mesh sieve to obtain polygonatum powder; water and polygonatum powder were mixed at a liquid-to-solid ratio of 10mL:1g (V:m), sterilized at 121℃ for 30min and cooled, and then the yeast, lactic acid bacteria, Bacillus subtilis and mixed bacteria were added in an inoculation amount of 3% according to the mass of polygonatum, and placed in a constant temperature shaking incubator at 120r·min -1 Cultivate for 72 hours (culture temperature: 28°C for yeast, 35°C for lactic acid bacteria, Bacillus subtilis and mixed bacteria); after fermentation, sterilize at 100°C for 30 minutes, cool and centrifuge to obtain the Polygonatum cyrtonema fermentation liquid, which is stored at 4°C. The unfermented group was used as the control group.

[0040] 1.3.2 Determination of components in Polygonatum cyrtonema fermentation broth

[0041] When determining the components in the Polygonatum sibiricum fermentation broth below, the sample is appropriately diluted according to the specific situation.

[0042] Determination of polysaccharide content: The determination method is the phenol-sulfuric acid method, and the standard curve using glucose as the standard is as follows:

[0043] A1=0.0087C1+0.0505 (1)

[0044] In formula (1), A1 is the absorbance of the sample to be tested; C1 is the glucose concentration; r 2 =0.9973, linear range 10~80μg·mL -1 .

[0045] Determination of total saponin content: The determination method is vanillin-perchloric acid-acetic acid colorimetry, and the standard curve using ginsenoside Rb1 as the standard is as follows:

[0046] A2=3.04C2-0.0619 (2)

[0047] In formula (2), A2 is the absorbance of the sample to be tested; C2 is the concentration of ginsenoside Rb1; r 2 =0.9681, linear range 100~400μg·mL -1 .

[0048] Determination of total polyphenol content: The determination method is the Folin phenol method, and the standard curve using gallic acid as the standard is as follows:

[0049] A3=0.0103C3-0.0111 (3)

[0050] In formula (3), A3 is the absorbance of the sample to be tested; C3 is the concentration of gallic acid; r 2 =0.9991, linear range 10~120μg·mL -1 .

[0051] Determination of total flavonoid content: The determination method is NaNO2-Al(NO3)3-NaOH colorimetric method, and the standard curve with rutin as the standard is as follows:

[0052] A4=0.006C4+0.0164 (4)

[0053] In formula (4), A4 is the absorbance of the sample to be tested; C4 is the concentration of rutin; r 2 =0.9959, linear range 20~100μg·mL -1 1.3.3 Determination of Antioxidant Capacity of Polygonatum cyrtonema Fermentation Broth

[0054] DPPH free radical scavenging: 1mL Polygonatum sibiricum liquid, 1mL 0.8mmol·L -1 DPPH solution was used as the basis, and deionized water was added to a total volume of 5 mL as the test group, and placed in the dark for 30 minutes, and the absorbance at 517 nm was measured. Deionized water was used instead of Polygonatum sibiricum solution as the blank group, and 95% ethanol was used instead of DPPH solution as the background group.

[0055] ABTS free radical scavenging: 0.5 mL of Polygonatum sibiricum liquid and 4 mL of ABTS solution (A 734nm The absorbance at 734 nm was measured by substituting deionized water for the Huangjing solution as the blank group, and substituting deionized water for the ABTS solution as the background group.

[0056] Hydroxyl radicals (·OH) scavenging: 1mL Polygonatum sibiricum liquid, 1mL 9mmol·L -1 FeSO4·7H2O, 1mL 9mmol·L -1 Salicylic acid, 1mL 9mmol·L -1 H2O2 was used as the basis, and deionized water was added to a total volume of 10 mL as the test group. After being kept at 37°C for 10 minutes, the absorbance at 510 nm was measured. Deionized water was used instead of Polygonatum odoratum liquid as the blank group, and deionized water was used instead of H2O2 as the background group.

[0057] The free radical scavenging rate was calculated as follows:

[0058] Free radical scavenging rate = [1-(A 待测 -A 本底 ) / A 空白 ]×100%

[0059] In the formula, A 待测 A is the absorbance of the sample and other reagents; 本底 A is the absorbance of the sample and other reagents without DPPH, ABTS or H2O2; 空白 The absorbance of the sample was replaced by deionized water.

[0060] 1.3.4 Activity detection of Polygonatum sibiricum fermentation broth on HaCaT cells

[0061] HaCaT cells in good logarithmic growth phase were used as test subjects, and 5×10 3 Cells were inoculated into 96-well culture plates and cultured overnight in a 37°C, 5% CO2 incubator. A cell control group (no treatment), a blank group (culture medium without cells), and a sample group containing different concentrations of Polygonatum sibiricum extract were set up and cultured for 48 hours. During the test, the supernatant was discarded, the prepared CCK-8 working solution was added, and the cells were cultured at 37°C in the dark. After 4 hours, the A value was read at 450nm on a microplate reader. The formula for calculating cell viability is as follows:

[0062] Cell survival rate = (A 样品组 -A 空白组 ) / (A 对照组 -A 空白组 )×100%

[0063] In the formula, A 样品组 A is the absorbance of the sample, cells, and CCK-8 working solution; 空白组 A is the absorbance of the sample and CCK-8 working solution; 对照组 is the absorbance of the CCK-8 working solution containing cells.

[0064] 1.3.5 Determination of anti-inflammatory efficacy of Polygonatum sibiricum fermented liquid

[0065] 2×10 per well 5 HaCaT cells were added to a 6-well culture plate and cultured overnight in a cell culture incubator. Three groups were set up: a blank control group, which only added 2 mL of culture medium; a model group, which added 2 mL of 20 μg mL -1 Lipopolysaccharide (LPS) culture medium; sample group, mass fraction 1 mg mL -1 Polygonatum cyrtonema fermented by different strains and the final concentration of LPS was 20 μg·mL -1 After culturing for 24 hours, the residual cells were removed by centrifugation, and the supernatant was collected. The contents of inflammatory factors were subsequently determined according to the instructions of the TNF-α and IL-6 enzyme-linked immunosorbent assay (ELISA) kits.

[0066] 1.3.6 Determination of the whitening effect of Polygonatum sibiricum fermented liquid

[0067] In vitro tyrosinase activity inhibition assay: References

[20] And improve. Add each reagent from top to bottom in the order of Table 1, mix well, place at 37℃ away from light for 30 minutes, and measure the absorbance of each group at a wavelength of 470nm. The calculation formula is as follows:

[0068] Tyrosinase inhibition rate = [(A A -A B )-(A C -A D )] / (A A -A B )×100%

[0069] In the formula, A A , A B , A C , A D They are the absorbances of groups A, B, C and D in Table 1, respectively.

[0070] Table 1 Composition of reaction solution for anti-tyrosinase activity assay

[0071]

[0072] Tyrosinase activity inhibition test in HaCaT cells: 3×10 HaCaT cells in logarithmic growth phase were taken per well. 4Inoculate in a 24-well plate, add DMEM culture medium, place in a 37°C, 5% carbon dioxide incubator for overnight culture, add 250μL Polygonatum fermentation broth as the sample group, and use the untreated group as the control group, continue to culture for 48h, remove the cells, wash once with PBS buffer with a pH value of 7.2, pour it out, add 100μL lysis solution to each well, continue to culture for 5min, make the cells fully lyse, remove and collect the cells, centrifuge to take the supernatant, take 50μL supernatant to a 96-well plate, add 50μL 1% L-dopa solution to each well, react at 37°C for 1h, and measure its absorbance at a wavelength of 475nm with an enzyme marker. The calculation formula of tyrosinase inhibition rate is as follows:

[0073] Tyrosinase inhibition rate = (A 样品组 -A 空白组 ) / (A 对照组 -A 空白组 )×100%

[0074] In the formula, A 样品组 A is the absorbance of cells after adding Polygonatum cyrtonema fermentation broth; 对照组 A is the absorbance of cells not treated with Polygonatum cyrtonema fermentation broth; 空白组 It is the absorbance of the fermentation broth containing Polygonatum sibiricum but not containing cells.

[0075] 1.4 Data Processing

[0076] Data are expressed as mean ± standard deviation, and each experiment was repeated 3 times. SPSS Statistics 27 software was used for data analysis by one-way ANOVA, and GraphPad Prism 5.0 software was used to process data and plot graphs. P < 0.05 indicated a significant difference.

[0077] 2 Results and analysis

[0078] 2.1 Content of each component in Polygonatum cyrtonema fermentation broth

[0079] Table 2 shows the content of active ingredients in the fermented liquor of Polygonatum cyrtonema of different strains. As shown in Table 2, the polysaccharide content of the fermented group was significantly lower than that of the unfermented group (P<0.0001), and the polysaccharide content in the yeast fermented liquor decreased the most; while the total saponin, total polyphenol and total flavonoid content of the fermented group increased to varying degrees compared with the unfermented group, and the total polyphenol and total flavonoid content were mixed bacteria> Bacillus subtilis> lactic acid bacteria> yeast> unfermented. Among them, the total flavonoid content increased significantly relative to other active ingredients, and the mixed bacteria fermented liquor increased the most, increasing by 57.94% compared with the unfermented group.

[0080] Table 2 The content of active ingredients in the fermented broth of Polygonatum cyrtonema from different strains

[0081]

[0082] Note: ***, **** indicate significant and extremely significant differences compared with the non-fermented group at P<0.001 and P<0.000 1 levels, respectively. Figure 1 , Figure 5 , Figure 6 , Same as Table 3.

[0083] 2.2 Antioxidant activity of Polygonatum sibiricum fermentation broth

[0084] Figure 1 to Figure 3 The antioxidant activity of Polygonatum sibiricum fermented broth from different strains was compared. Figure 1 to Figure 3 It can be seen that the fermentation group had significantly higher scavenging ability for DPPH and ABTS free radicals than the non-fermentation group (P<0.0001), while the scavenging ability for hydroxyl free radicals was reduced. Among them, compared with the non-fermentation group, the DPPH free radical scavenging rate of the mixed bacteria fermentation group was the highest at 87.65%; the ABTS free radical scavenging rate of the Bacillus subtilis fermentation group was the highest at 72.5%; the hydroxyl free radical scavenging rate of the mixed bacteria fermentation group was the lowest, which was 14.43% lower than that of the non-fermentation group.

[0085] 2.3 Toxicity of Polygonatum sibiricum fermentation broth on HaCaT cells

[0086] Figure 4 The effect of different concentrations of Polygonatum sibiricum fermentation broth from different strains on cell survival rate. Figure 4 It can be seen that the mass fraction is 0.01~1mg·mL -1 The cell activity of the Polygonatum cyrtonema fermentation liquid group was higher than that of the non-fermentation group. The Polygonatum cyrtonema fermentation liquid had no toxicity to HaCaT cells and had a certain proliferation effect on HaCaT cells. -1 The fermented broth of Polygonatum cyrtonema and the mass fraction of 0.1 mg·mL -1 The unfermented Polygonatum sibiricum was used for subsequent anti-inflammatory efficacy evaluation and whitening efficacy evaluation tests.

[0087] 2.4 Effects of Polygonatum sibiricum fermented liquid on inflammatory factors in inflammatory cells

[0088] Figure 5 and Figure 6 The effect of different strains of Polygonatum sibiricum fermented broth on the content of TNF-a and IL-6 in HaCaT. Figure 5 and Figure 6It can be seen that after LPS induction, the levels of TNF-α and IL-6 in the model group were significantly higher than those in the blank group (P<0.000 1), indicating that the HaCaT cell inflammation model was successfully established. The levels of the two inflammatory factors in the fermentation group were significantly or extremely significantly lower than those in the model group (P<0.001 or P<0.000 1), and the fermentation group had better anti-inflammatory effects than the non-fermentation group. Among them, the levels of TNF-α and IL-6 inflammatory factors in the mixed bacteria fermentation group were 50.15 and 3.12 pg·mL, respectively. -1 , the decrease was the largest compared with the non-fermented group, indicating that the fermented liquid has better anti-inflammatory effect.

[0089] 2.5 Inhibition rate of Polygonatum sibiricum fermentation broth on tyrosinase

[0090] Table 3 shows the effects of different strains of Polygonatum sibiricum fermented liquid on tyrosinase activity in vivo and in vitro. As shown in Table 3, different strains of Polygonatum sibiricum fermented liquid have inhibitory effects on tyrosinase, and the inhibition rate of the fermented group is significantly higher than that of the unfermented group (P<0.0001). Among them, the in vitro and intracellular inhibition rates of the mixed bacteria fermentation group were 92.39% and 65.50%, respectively, which were the largest increase compared with the unfermented group. In summary, Polygonatum sibiricum fermented liquid, especially the mixed bacteria group, has the potential to be used as a whitening raw material.

[0091] Table 3 Effects of Polygonatum sibiricum fermentation broth from different strains on tyrosinase activity in vivo and in vitro (n=3)

[0092]

[0093] 3 Discussions

[0094] Our study found that the polysaccharide content in the fermentation group was significantly lower than that in the non-fermentation group, with the yeast fermentation group showing the largest decrease. This is presumably because polysaccharides can serve as a carbon source for microbial growth during the fermentation process.

[22] . However, compared with the unfermented group, the content of total saponins, total polyphenols and total flavonoids in the fermented liquor of Polygonatum sibiricum increased, and the content of total polyphenols and total flavonoids was significantly or extremely significantly higher than that of the unfermented group. The content of total polyphenols and flavonoids in the mixed bacteria fermentation group increased by 33.21% and 57.94%, respectively. It is speculated that the cellulase produced by microorganisms during the fermentation process degraded the cell wall, which helped the dissolution of intracellular substances, thereby increasing the content of active ingredients in the sample.

[0095] The accumulation of free radicals in the body, the sequelae of inflammation, and the activity of tyrosinase are closely related to whitening. ·OH free radicals and DPPH free radicals are highly oxidizing and can attack biological macromolecules in the body, destroy cell structure and function, cause skin spots and wrinkles, and cause skin aging. By measuring the scavenging rate of DPPP, ABTS and hydroxyl free radicals by Polygonatum sibiricum fermentation liquid, we found that the scavenging rate of DPPH and ABTS free radicals by Polygonatum sibiricum fermentation liquid was significantly higher than that of unfermented Polygonatum sibiricum liquid, but the scavenging rate of ·OH free radicals was significantly lower than that of the unfermented group. It is speculated that the total phenol content is positively correlated with the scavenging ability of DPPH free radicals, the total flavonoid content is also positively correlated with the scavenging ability of DPPH and ABTS free radicals, and the total flavonoids are negatively correlated with the scavenging rate of ·OH free radicals. In the results of this experiment, the change pattern of the scavenging ability of DPPP, ABTS and ·OH free radicals in the Polygonatum sibiricum fermentation group is basically consistent with the change pattern of flavonoids, polyphenols and saponins content after Polygonatum sibiricum fermentation, indicating that the above active ingredients are the material basis for the overall improvement of the antioxidant performance of the Polygonatum sibiricum fermentation group.

[0096] HaCaT cells are one of the components of human epidermal cells and are closely related to epidermal differentiation. By studying the effect of samples on the activity of these cells, the safety of the epidermal barrier can be evaluated. This study showed that the activity of HaCaT cells in the fermentation group was greater than that in the non-fermentation group, and the concentration was 1 mg mL -1 The fermented Polygonatum sibiricum liquid was non-toxic to cells and had a certain effect of promoting cell proliferation. The survival rate of cells treated with the mixed bacteria group was the highest. -1 It is non-toxic to cells, which shows that the safety of Polygonatum sibiricum fermented liquid is 10 times higher than that of unfermented liquid.

[0097] As a normal physiological reaction of the human body, inflammatory response plays a physiological barrier and protective role on the skin. Aging is closely related to inflammation, and proinflammatory cytokines such as TNF-a and IL-6 can induce and accelerate cell aging. Anti-inflammatory tests found that the content of inflammatory factors in the Polygonatum sibiricum fermentation liquid group was significantly lower than that in the non-fermented group, and the content of TNF-a and IL-6 in the mixed bacteria fermentation group was lower than that in the Polygonatum sibiricum fermentation liquid group with three single bacteria. This shows that after fermentation, Polygonatum sibiricum can reduce the irritation of Polygonatum sibiricum and relieve inflammation.

[0098] Skin color is closely related to the melanin produced by melanocytes in the basal layer of the epidermis. Tyrosinase is the main rate-limiting enzyme in melanin production, which can inhibit tyrosinase activity and reduce melanin production, thereby exerting a whitening effect. This study compared the inhibitory effect of Polygonatum sibiricum fermented liquid on tyrosinase and found that the in vitro and intracellular inhibition rate of Polygonatum sibiricum fermented liquid on tyrosinase was significantly higher than that of the unfermented group, and the mixed fermented liquid had the highest inhibition rate on tyrosinase.

[0099] 4 Conclusion

[0100] The amount of active ingredients in the fermented Polygonatum sibiricum liquid, such as total polyphenols and total flavonoids, increased significantly or extremely significantly compared with the unfermented group, and the performance in antioxidant, anti-inflammatory and tyrosinase inhibition was better. Especially after fermentation with a mixed bacteria composed of yeast, lactic acid bacteria and Bacillus subtilis, the above effects of the Polygonatum sibiricum fermented liquid were more prominent. Cytotoxicity evaluation showed that the cell activity of the Polygonatum sibiricum fermented liquid was higher than that of the unfermented group, and the mixed bacteria group had the lowest toxicity to HaCaT, and showed the potential to promote cell proliferation. Therefore, the Polygonatum sibiricum fermented liquid fermented with probiotics not only has good cell safety, but also has good antioxidant, anti-inflammatory and whitening effects, and has the potential to be used in the development of functional raw materials for Chinese medicine cosmetics.

Claims

1. A method for preparing a fermented polygonatum sibiricum broth, characterized in that: The following steps are involved: (1) Yeast, lactic acid bacteria and Bacillus subtilis are mixed in a ratio of 1:1:1 to obtain a mixed bacteria; (2) Dissolve 0.25 g of the mixed bacteria in 5 mL of sterile water and set aside; (3) Slice and crush the dried polygonatum sibiricum, and pass it through a 10-mesh sieve to obtain polygonatum sibiricum powder; mix water and polygonatum sibiricum powder at a liquid-to-solid ratio of 10 mL:1 g, sterilize at 121°C for 30 min, and cool, then add mixed bacteria at an inoculum amount of 3% based on the mass of the polygonatum sibiricum, and place in a constant temperature shaking incubator at 120 r·min -1 The culture was carried out for 72 hours at a temperature of 35°C. After the fermentation was completed, the mixture was sterilized at 100°C for 30 minutes, cooled and centrifuged to obtain the Polygonatum sibiricum fermentation liquid, which was stored at 4°C.

2. The method for preparing a Polygonatum sibiricum fermentation broth according to claim 1, characterized in that: The method also includes the step of determining the polysaccharide content in the fermented broth of polygonatum, wherein the determination method is the phenol-sulfuric acid method, and the standard curve using glucose as the standard is as follows: A1=0.0087C1+0.0505 (1) In formula (1), A1 is the absorbance of the sample to be tested; C1 is the glucose concentration; r 2 =0.9973, linear range 10~80μg·mL -1 .

3. The method for preparing a Polygonatum sibiricum fermentation broth according to claim 1, characterized in that: The method also includes the step of determining the total saponin content in the fermented broth of polygonatum, and the determination method is the vanillin-perchloric acid-glacial acetic acid colorimetric method, and the standard curve using ginsenoside Rb1 as the standard is as follows: A2=3.04C2-0.0619 (2) In formula (2), A2 is the absorbance of the sample to be tested; C2 is the concentration of ginsenoside Rb1; r 2 =0.9681, linear range 100~400μg·mL -1 .

4. The method for preparing a Polygonatum cyrtonema fermentation broth according to claim 1, characterized in that: The method also includes the step of determining the total polyphenol content in the fermented liquor of Polygonatum cyrtonema. The determination method is the Folin phenol method. The standard curve using gallic acid as the standard is as follows: A3=0.0103C3-0.0111 (3) In formula (3), A3 is the absorbance of the sample to be tested; C3 is the concentration of gallic acid; r 2 =0.9991, linear range 10~120μg·mL -1 .

5. The method for preparing a Polygonatum cyrtonema fermentation liquid according to claim 1, characterized in that: The method also includes the step of determining the total flavonoid content in the fermented liquor of polygonatum, wherein the determination method is the NaNO2-Al(NO3)3-NaOH colorimetric method, and the standard curve with rutin as the standard is as follows: A4=0.006C4+0.0164 (4) In formula (4), A4 is the absorbance of the sample to be tested; C4 is the concentration of rutin; r 2 =0.9959, linear range 20~100μg·mL -1 .

6. Use of the polygonatum fermented liquid according to claim 1 as a functional raw material for anti-oxidation, whitening and anti-inflammatory effects in cosmetics.

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