CN115322933A - Acetic acid bacteria and fermentation product and application thereof - Google Patents
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Abstract
The invention discloses acetic acid bacteria and a fermentation product and application thereof. The invention screens and separates a strain acetic acid bacterium Acetobacter sp.XM-1 from saussurea involucrata. The acetic acid bacteria. The survival rates of macrophage and mouse melanoma cells of the fermented extracts at the concentration of 1.25mg/mL at the 3 rd day and the 6 th day are still 100%. The ability to scavenge DPPH free radicals is also better than the ability to resist oxidation without fermentation of the culture medium extract. The fermented extracts at day 3 and day 6 have very strong tyrosinase activity inhibiting ability. When the concentration of the extract on the 3 rd day and the 6 th day is 1.25mg/mL, the inhibition rate of tyrosinase can reach 86 percent and 73 percent respectively. Further, LC-MS analysis of the fermentation extracts on days 3 and 6 revealed that acetic acid bacteria, after fermentation, produced more succinic acid, DL-2-pipecolic acid, citric acid, and gluconic acid than the control group.
Description
Technical Field
The invention relates to a preparation method of an acetic acid bacteria fermentation product with tyrosinase activity inhibition function.
Background
The dark spots and areas of skin pigmentation are a very important topic of research in medical cosmetology. These skin pigments are mainly present in the normally exposed parts of the human body, such as the face or hands, due to the increase of ultraviolet radiation, and the pigmentation also includes chloasma, freckles, post-inflammatory and hormonal pigmentation. Melanin is composed of Eumelanin (Eumelanin) and Pheomelanin (Pheomelanin), and is produced catalytically by Tyrosinase (Tyrosinase) and Tyrosinase-related proteins in mammals and fruits. Tyrosinase is the first rate-limiting step in the initiation of tyrosine oxidation to Dopaquinone (Dopaquinone), which is further converted to DOPA (DOPA) and Dopachrome (Dopachrome). Dopaquinone gradually forms pheomelanin in the presence of glutathione or cysteine. Meanwhile, tyrosinase-related protein-2 (Trp-2) and tyrosinase-related protein-1 (Trp-1) sequentially catalyze the production of Indole-5,6-quinone,5,6-dihydroxyindole-2-carboxylic acid (Indole-5, 6-quinone,5,6-dihydroxyindole-2-carboxylic acid) and Indole-2-carboxylic acid-5,6-quinone (Indole-2-carboxylic acid-5, 6-quinone). These intermediate metabolites are converted to eumelanin by oxidation reactions. Therefore, in order to achieve the purpose of inhibiting melanin production, screening of compounds has been conducted in many studies aiming at inhibiting tyrosinase.
There are many acetic acid bacteria, and in recent years, the research on the acetic acid bacteria mainly aims to obtain high-yield acetic acid and cellulose mycoderm, but the research on the fermentation products of the acetic acid bacteria in the fields of oxidation resistance, whitening and the like is very little. For example, CN201510460020.1 discloses an acetic acid bacterium and application thereof in fermenting apple vinegar; CN201310549668.7 discloses a high-temperature resistant acetobacter pasteurianus and application thereof in acetic acid production by fermentation; CN201711188682.3 Pasteur acetic acid bacteria and application thereof in preparation of fermented fruit and vegetable juice are disclosed; CN202011194208.3 discloses an acetobacter orientalis for cellulose degradation and application thereof.
Disclosure of Invention
The invention aims to provide an acetic acid bacterium strain which is obtained by screening saussurea involucrate. The number is CCTCC M2022983.
Another object of the present invention is to provide an application of the acetic acid bacterium in the preparation of at least one acid selected from the group consisting of succinic acid, pipecolic acid, iminoarginine, 4-acetylbutyric acid, citric acid, gluconic acid, 3-dehydrosphingosine, N-acetyl-leucine, L-Threo-2-pentulose and 6-hydroxycaproic acid.
The invention also aims to provide the application of the acetic acid bacteria in preparing an antioxidant fermentation product.
The invention also aims to provide the application of the acetic acid bacteria in preparing a product for inhibiting tyrosinase fermentation.
The invention also aims to provide the application of the acetic acid bacteria in preparing the whitening fermentation product.
Another objective of the invention is to provide an acetic acid bacteria fermentation product, which is obtained by fermenting the acetic acid bacteria on a GY liquid culture medium, wherein the GY liquid culture medium comprises 4-6% by weight of glucose and 0.4-1.0% by weight of yeast extract powder.
Further, the acetic acid bacteria fermentation product contains 10 components with high content, namely Succinic acid (Succinic acid), pipecolic acid (Pipecolic acid), iminoarginin, ethyl 4-Acetylbutyrate (4-Acetylbutyrate), citric acid (Citric acid), gluconic acid (Gluconic acid), 3-dehydrosphingosine, N-acetyl-leucine (N-Acetylleucine), L-xylulose (L-Threo-2-ketolase) and 6-Hydroxyhexanoic acid (6-Hydroxyhexanoic acid).
Another object of the present invention is to provide an application of the acetic acid bacteria fermentation product in preparing whitening cosmetics, wherein the application comprises:
another objective of the invention is to provide a method for preparing acetic acid bacteria fermentation products, which is obtained by fermenting the acetic acid bacteria on a GY liquid medium, wherein the GY liquid medium comprises 4-6% of glucose and 0.4-1.0% of yeast extract powder by weight.
Further, the inoculation amount of acetic acid bacteria was 10 6 -10 8 CFU/ml. Further, the fermentation time is 3-6 days, and the temperature is 25-37 ℃.
The strain of the invention has the capability of producing various organic acids, and the fermentation product has good oxidation resistance and tyrosinase activity inhibition.
Drawings
The invention is further illustrated by the following figures and examples.
FIG. 1 shows a phylogenetic tree constructed based on 16S rRNA sequences of strains selected from saussurea involucrate.
FIG. 2, effect of different concentrations of acetic acid bacteria fermentation on the activities of RAW264.7 cells (a) and B16-F10 cells (B). control is the extract of the medium without fermentation. 3d is the extract obtained after 3 days of fermentation by acetic acid bacteria. 6d is the extract after 6 days of acetic acid bacteria fermentation.
FIG. 3, analysis of the removal of DPPH free radicals from acetic acid bacteria fermentations at different concentrations. control is the extract of the medium without fermentation. 3d is the extract obtained after 3 days of fermentation by acetic acid bacteria. 6d is the extract after 6 days of acetic acid bacteria fermentation.
FIG. 4, analysis of tyrosinase inhibition by acetic acid bacteria fermentations at different concentrations. control is the extract of the medium without fermentation. 3d is the extract obtained after 3 days of fermentation by acetic acid bacteria. 6d is the extract after 6 days of acetic acid bacteria fermentation.
FIG. 5, MS/MS spectrum analysis of 10 relatively large components in acetic acid bacteria fermentation products at 3 rd and 6 th days. From a to j are Succinic acid (Succinic acid), pipecolic acid (Pipecolic acid), iminoarginin, ethyl 4-Acetylbutyrate (4-Acetylbutyrate), citric acid (Citric acid), gluconic acid (Gluconic acid), 3-dehydrosphingosine, N-acetyl-leucine (N-Acetylleucine), L-xylulose (L-Threo-2-ketonitrate) and 6-Hydroxyhexanoic acid (6-Hydroxyhexanoic acid), respectively.
FIG. 6 analysis of tyrosinase inhibition by different organic acids, including succinic acid (a), citric acid (b) and DL-2-pipecolic acid (c).
Detailed Description
Example 1 isolation and characterization of strains
Commercially available saussurea involucrate is subjected to pure milk fermentation culture, separated by a plate-drawing method, cultured at 37 ℃ for 2 days, and strains on the plate are scraped by an inoculating loop and cultured in a GY liquid culture medium (Glucose Yeast extract, 5% Glucose and 0.5% Yeast extract powder. The preparation method comprises the steps of weighing Glucose and Yeast extract powder with required weight, adding distilled water to 1000mL, and carrying out autoclaving at 121 ℃ for 20 min). The selected strain was subjected to 16S rRNA gene amplification using a 16S rRNA primer (F27-AGAGTTTGATCTMTGGCTCAG; R1492-TACGGYTACCTTGACTT). The PCR stirp is 94 ℃ for 5 minutes; 30 seconds at 94 ℃; 30 seconds at 55 ℃;72 ℃ for 1.5 minutes; 72 ℃ for 7 minutes. The amplified DNA sequence is sent to Jinzhi (Guangzhou) Biotechnology limited to carry out DNA sequencing, and then MEGA software is utilized to construct a phylogenetic tree based on 16S rDNA gene sequence analysis. See fig. 1.
According to the comparison analysis of NCBI database, the obtained strain belongs to acetic acid bacteria and is named Acetobacter sp.XM-1 (figure 1). The strain is preserved in China center for type culture Collection, CCTCC M2022983, of Wuhan university, wuhan City, hubei province, china at 27.06 months in 2022.
EXAMPLE 2 preparation of acetic acid bacteria fermentation product
After activating acetic acid bacteria, the activated strain was mixed in a volume ratio of 1:100 inoculated on GY liquid medium (the inoculum size of acetic acid bacteria is about 10) 6 -10 7 CFU/ml); shake-culturing at 37 deg.C for 3 days and 6 days, extracting the obtained fermentation broth with ethyl acetate, removing ethyl acetate from the extractive solution with rotary evaporator, and freeze drying to obtain extract.
Example 3 cytotoxicity assay
Security evaluation was performed using MTT detection. CO at 37 ℃ and 5% 2 Culturing macrophage (RAW 264.7) and mouse melanoma cells (B16-F10) in incubator for 24 hr, adding different concentrations of fermented extract, culturing for 24 hr, adding 6 μ L MTT (3%) solution, reacting, and determining CO content at 37 deg.C and 5% 2 After 4 hours of reaction, the culture medium was removed, 200. Mu.L of DMSO was added to dissolve the precipitate, and the 490nm absorbance was analyzed by a microplate reader.
Example 4 Oxidation resistance analysis
A solution of 0.25mM DPPH was prepared. And adding 180 mu L of DPPH solution into 20 mu L of acetic acid bacteria fermentation extracts with different concentrations, shaking and mixing, and standing at room temperature for reaction. The absorbance at 517nm was analyzed using a microplate reader. Calculated clearance (%) = [ control at 517nm absorbance-sample reaction after 517nm absorbance/control at 517nm absorbance ] + 100.
Example 5 inhibition tyrosinase assay
Acetic acid bacteria fermentation extracts of different concentrations were added to 96-well plates, 100. Mu.L of mushroom tyrosinase (400U/mL) and L-DOPA (10 mM) were added to the mixture, and the mixture was allowed to stand at room temperature for reaction. The absorbance was measured at OD475 with a microplate reader. The inhibition of tyrosinase was calculated as follows: relative inhibition of mushroom tyrosinase activity (%) = [ control at 475nm absorbance-sample reacted at 475nm absorbance/control at 475nm absorbance ] + 100.
Example 6 analysis of Compounds in acetic acid bacteria fermentation
The analysis of the fermentation extract was carried out by entrusted Suzhou Jinwei Zhi Biotech Co., ltd for qualitative and quantitative detection, and the experimental procedure was as follows. Adding 600 μ L methanol (containing 2-chloro-L-phenylalanine (4 ppm)) into the fermented extract, grinding with tissue grinder, performing ultrasonic treatment at room temperature for 15 min, centrifuging, filtering, and detecting by LC/MS with chromatographic column using ACQUITY
HSS T3 column (2.1X 150mm,1.8 μm, waters Corp.), positive ion mode, mobile phase 0.1% formic acid acetonitrile (C) and 0.1% formic acid water (D)The gradient elution procedure was: 0 to 1min,2% by weight of C; 1-9min, 2% -50% of C; 9-12min, 50% -98% C;12 to 13.5min,98% by weight; 13.5-14min, 98% -2% C;14 to 20min,2% of C. Anion mode, mobile phase acetonitrile (a) and 5mM ammonium formate water (B), gradient elution procedure: 0 to 1min,2% by weight A; 1-9min, 2% -50% A; 9-12min, 50% -98% A;12 to 13.5min,98% by weight of A; 13.5-14min, 98% -2% A; 14-17min, 2% by weight of A. The flow rate was 0.25mL/min. The mass spectrum adopts an electrospray ionization (ESI) source, the positive ion spray voltage is 3.50kV, the negative ion spray voltage is-2.50 kV, the sheath gas is 30arb, and the auxiliary gas is 10arb. The temperature of the capillary is 325 ℃, primary full scanning is carried out with the resolution of 60000, the scanning range of primary ions is 100-1000, secondary cracking is carried out by adopting HCD, the collision voltage is 30 percent, the secondary resolution is 15000, the first 4 ions of the signal are collected for fragmentation, and unnecessary MS/MS information is removed by adopting dynamic elimination.
Table 1, day 3 and day 6 acetic acid bacteria fermentations contain relatively high amounts of the first 10 components.
a ratio of acetic acid bacteria fermentation products to non-fermented culture medium extracts at day 3 and day 6.
2. The fermentation products of day 3 and day 6 of the present invention still showed 100% survival rate on macrophages (RAW 264.7) and mouse melanoma cells (B16-F10) at a concentration of 1.25mg/mL, indicating that the fermentation products did not have toxic effect on macrophages and mouse melanoma cells (FIG. 2). When the concentration is increased to 2.5mg/mL, the effect on mouse melanoma cells is still unaffected, but the effect on macrophages is meant. The extract without acetic acid bacteria fermentation has larger influence on cells.
3. The fermentation products of day 3 and day 6 of the present invention had 40% and 36% of DPPH radical removal capacity at a concentration of 2.5mg/mL, respectively, and 28% and 21% of DPPH radical removal capacity at a concentration of 1.25 mg/mL. It showed better antioxidant capacity relative to the media extract without fermentation (fig. 3).
4. The fermentation product of day 3 of the invention has 90% of tyrosinase inhibiting ability under the condition of 5mg/mL concentration. Under the condition that the concentration is 1.25mg/mL, the tyrosinase inhibition ability is still more than 86%. The fermentation product of the day 6 of the invention has more than 90 percent of tyrosinase inhibiting capacity under the condition of 5mg/mL concentration. At a concentration of 1.25mg/mL, there was 73% inhibition of tyrosinase (FIG. 4). The medium extract without fermentation, at a concentration of 5mg/mL, had only 21% inhibition of tyrosinase.
5. According to the present invention, the metabolites of the fermented product of acetic acid bacteria were analyzed by LC-MS (Table 1), and it was found that 10 components contained in the fermented product, which are relatively large in content, were Succinic acid (Succinic acid), pipecolic acid (Pipecolic acid), immunoarginin, ethyl 4-Acetylbutyrate (4-Acetylbutyrate), citric acid (Citric acid), gluconic acid (Gluconic acid), 3-dehydrosphingosine, N-acetyl-leucine (N-Acetylleucine), L-xylulose (L-Threo-2-ketolase) and 6-Hydroxyhexanoic acid (6-Hydroxyhexanoic acid), respectively, relative to the culture medium extract which had not been fermented, as shown in FIG. 5.
6. Further analyzing the tyrosinase inhibiting ability of the organic acid with a high content ratio, the tyrosinase inhibiting rates of 0.5mg/mL Succinic acid (Succinic acid), DL-2-Pipecolic acid (Pipecolic acid) and Citric acid (Citricic acid) are respectively 43%, 18% and 57%, and the tyrosinase inhibiting ability of 1mg/mL calcium gluconate (Gluconic acid calcium salt) is weak. Succinate and citrate performed best in inhibiting tyrosinase (figure 6).
Claims (10)
1. An acetic acid bacterium, which is characterized in that: XM-1 in China center for type culture Collection has a serial number of CCTCC M2022983.
2. Use of the acetic acid bacterium according to claim 1 for the preparation of at least one acid selected from the group consisting of succinic acid, pipecolic acid, iminoarginin, 4-acetylbutyric acid, citric acid, gluconic acid, 3-dehydrosphingosine, N-acetyl-leucine, L-Threo-2-pentulose and 6-hydroxycaproic acid.
3. Use of the acetic acid bacterium according to claim 1 for preparing an antioxidant fermentation product.
4. The use of acetic acid bacteria according to claim 1 for preparing a product inhibiting tyrosinase fermentation.
5. The use of the acetic acid bacteria of claim 1 in the preparation of a whitening fermented product.
6. A fermentation product of acetic acid bacteria, which is obtained by fermenting the acetic acid bacteria of claim 1 on a GY liquid medium comprising 4-6% by weight of glucose and 0.4-1.0% by weight of yeast extract powder.
7. The acetic acid bacteria fermentation product according to claim 6, wherein: the 10 components with high content in the fermentation product are succinic acid, pipecolic acid, iminoarginin, 4-acetyl ethyl butyrate, citric acid, gluconic acid, 3-dehydrosphingosine, N-acetyl-leucine, L-xylulose and 6-hydroxycaproic acid respectively.
8. The use of the acetic acid bacteria fermentation product according to claim 6 or 7 in the preparation of whitening cosmetics.
9. A method for preparing acetic acid bacteria fermentation products is characterized by comprising the following steps: the product is obtained by fermenting the acetic acid bacteria of claim 1 on GY liquid medium, which comprises glucose 4-6 wt% and yeast extract powder 0.4-1.0 wt%.
10. The method of claim 9, wherein the fermentation product of acetic acid bacteria is prepared by: the inoculation amount of acetic acid bacteria is 10 6 -10 8 CFU/ml, fermentation time is 3-6 days,the temperature is 25-37 ℃.
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