Bacillus subtilis BS4-35 and application thereof in preparation of white peony root whitening aqueous extract

Abstract

The invention discloses bacillus subtilis BS4-35 and an application thereof in preparation of a white peony root whitening aqueous extract. The bacillus subtilis BS4-35 is preserved in Guangdong Microbial Culture Collection Center, the preservation number is GDMCC No: 65591, and the preservation date is December 9, 2024. Before the white paeony root whitening aqueous extract is extracted by a hot water ultrasonic method, the bacillus subtilis BS4-35 is added for fermentation treatment, so that the yield of the aqueous extract is remarkably improved, and the activity of inhibiting tyrosinase is also remarkably enhanced. Compared with an unfermented direct hot water ultrasonic extraction method, the yield of the aqueous extract of the radix paeoniae alba can be increased by 31.1%. The contents of polysaccharide and paeoniflorin in the aqueous extract are respectively increased by 18.5% and 11.4%, and the activity of inhibiting tyrosinase is enhanced by 25.3%.

Description

Technical Field

[0001] The invention belongs to the application field of microbial fermentation technology in the extraction of plant effective components, and specifically relates to a strain of Bacillus subtilis BS4-35 and application thereof in the preparation of white peony root whitening water extract. Background Art

[0002] White peony root is the dried root of Paeonia lactiflora, a plant of the Ranunculaceae family. It tastes bitter and sour, is slightly cold in nature, and belongs to the spleen and liver meridians. It has the effects of nourishing blood and regulating menstruation, restraining yin and stopping sweating, softening the liver and relieving pain, and calming liver yang. It is mainly used to treat blood deficiency and chlorosis, irregular menstruation, spontaneous sweating, night sweats, flank pain, abdominal pain, limb cramps, headache and dizziness. The main authentic production areas in my country are Anhui, Zhejiang and Sichuan. The chemical components of white peony root mainly include monoterpenes and their glycoside compounds, triterpenes, polysaccharides, flavonoids and their glycoside compounds, phenylethanol glycosides and volatile oils. Modern pharmacological studies have shown that the active ingredients of white peony root have the effects of protecting damage after cerebral ischemia, protecting myocardial cells, protecting nerves and anti-depression, protecting the liver, protecting the gastrointestinal mucosa, regulating gastrointestinal function, anti-inflammatory, anti-tumor, and regulating autoimmunity.

[0003] Traditional Chinese medicine whitening products are favored by people because of their long-lasting effects, low toxicity and side effects, and high safety. They have become a development trend in the whitening cosmetics industry. White peony extract has been confirmed by many studies to have the effect of removing spots and whitening. White peony contains rich ingredients such as paeoniflorin, polyphenols, polysaccharides and flavonoids. These ingredients can promote skin metabolism, anti-oxidation, inhibit tyrosinase activity, etc., thereby reducing the formation of skin melanin and achieving a whitening effect. Therefore, in recent years, whitening products with white peony extract as the main ingredient have been favored.

[0004] At present, there are many reports on the preparation methods of white peony extracts. According to the solvent used for extraction, the methods can be divided into two categories, namely water extraction and organic solvent extraction. There are great differences in the composition of the substances extracted by the two methods. The water extract mainly contains water-soluble substances such as polysaccharides, oligosaccharides and amino acids; the organic solvent extraction method mainly uses ethanol as the solvent, and the extract mainly contains substances such as paeoniflorin, polyphenols, triterpenes and sterols. In order to improve the yield of the extract, ultrasound, microwave and enzymatic hydrolysis are often used to enhance the extraction. Ultrasonic-assisted extraction has low equipment requirements and will not significantly increase the extraction cost, but the effect of improving the extraction yield is not ideal; microwave-assisted extraction is time-consuming and efficient, but due to the high internal temperature, the structure of the extracted active ingredients is often destroyed; enzymatic hydrolysis-assisted method can not only significantly improve the extraction yield, but also has mild conditions and will not destroy the structure of the active ingredients in the extract; moreover, enzymatic hydrolysis can reduce the molecular weight of polysaccharides and convert some glycosides into aglycones, so the biological activity of the extract will be better. Therefore, in recent years, enzymatic hydrolysis has been increasingly used in the extraction of plant active ingredients.

[0005] Cellulase, pectinase and protease used in enzymatic hydrolysis-assisted extraction are currently produced by microbial fermentation. Therefore, if a suitable microorganism is used to directly ferment white peony root, as long as the growth degree is controlled to produce enzymes to decompose the cell wall of white peony root, but the effective ingredients are not decomposed, the dissolution of the effective ingredients can be promoted and the yield of the extract can be improved. Compared with the enzymatic hydrolysis-assisted extraction method, since microorganisms produce a variety of hydrolases during growth, these enzymes can play a synergistic hydrolysis effect, and the hydrolysis effect on plant cell walls is better. In addition, polysaccharides are properly degraded, the molecular weight is reduced, the activity is improved, and the sugar groups of some glycoside active ingredients are hydrolyzed to release aglycones, and the biological activity of the extract will be better.

[0006] In order to improve the yield and biological activity of white peony root water extract, the present invention performs microbial fermentation on white peony root and then uses hot water ultrasonic extraction, the yield of water extract is significantly improved, the content of polysaccharides and paeoniflorin therein is significantly increased, and the tyrosinase inhibitory activity is also significantly enhanced. Summary of the invention

[0007] The present invention aims to provide a new microbial strain for fermenting white peony root, Bacillus subtilis BS4-35, and its application in preparing white peony root whitening water extract. After white peony root is fermented by the Bacillus subtilis BS4-35 and then extracted by hot water ultrasonic, the yield of water extract is significantly improved, the content of polysaccharide and paeoniflorin therein is significantly increased, and the tyrosinase inhibitory activity is also significantly enhanced.

[0008] The technical solution adopted by the present invention is:

[0009] The present invention provides a new microbial strain, Bacillus subtilis BS4-35, which is deposited in Guangdong Microbial Culture Collection Center with a deposit number of GDMCC No: 65591 and a deposit date of December 9, 2024. The address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province; Postal Code: 510070.

[0010] The Bacillus subtilis BS4-35 is separated from soil, obtained through mutagenesis and screening, and is an excellent strain suitable for fermenting white peony root. The 16S rDNA nucleotide sequence of the strain is shown in SEQ ID NO.1.

[0011] The present invention also provides an application of the Bacillus subtilis BS4-35 in preparing a white peony root whitening water extract, and the application method comprises the following steps: (1) adding a sterile sucrose aqueous solution to white peony root powder, inoculating the bacterial cells of Bacillus subtilis BS4-35 for fermentation, and obtaining a white peony root fermentation product; (2) adding deionized water to the white peony root fermentation product, filtering after hot water ultrasonic extraction, and concentrating the filtrate to obtain a white peony root water extract concentrate; (3) vacuum drying the white peony root water extract concentrate, and crushing it to obtain a white peony root water extract.

[0012] Furthermore, the white peony root in step (1) is a Chinese herbal medicine piece of white peony root, i.e., the dried root of the Ranunculaceae plant Paeonia lactiflora; the white peony root powder is a fine powder of white peony root that is crushed and passed through a 60-mesh sieve, and is sealed and packaged and sterilized in an oven at 105° C. for 1 h.

[0013] Furthermore, the volume of the sterile sucrose aqueous solution added to the white peony powder in the step (1) is 6-8 mL / g based on the mass of the white peony powder; the sterile sucrose aqueous solution has a sucrose concentration of 15-20 g / L and is sterilized by high-pressure steam at 121°C for 15-20 min.

[0014] Furthermore, the fermentation conditions of white peony root in step (1) are as follows: white peony root powder is added to a sterile sucrose aqueous solution, inoculated with Bacillus subtilis BS4-35 bacteria, and then stirred evenly and fermented at 32-36°C and 160-200 r / min for 36-48 h.

[0015] Furthermore, in step (1), the Bacillus subtilis BS4-35 cells are inoculated in the form of seed liquid, and the inoculation amount is 5% to 7% of the volume of the sucrose aqueous solution. The preparation method of the seed liquid is: inoculate the Bacillus subtilis BS4-35 cells into a fresh LB slant culture medium, culture at 35 to 37°C for 24 to 36 hours, and obtain the activated cultured cells of Bacillus subtilis BS4-35; then inoculate the activated cultured cells of Bacillus subtilis BS4-35 into LB liquid culture medium, culture at 30 to 35°C and 160 to 200 r / min for 20 to 24 hours, and obtain OD 600 =3.37~3.62 of Bacillus subtilis BS4-35 seed liquid. The final concentration of the LB slant medium is: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, agar 20 g / L, dissolved in tap water, pH 7.0~7.2; the final concentration of the LB liquid medium is: peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, dissolved in tap water, pH 7.0~7.2.

[0016] Furthermore, in step (2), the preparation method of the white peony root water extract concentrate is as follows: add 12-14 mL / g of deionized water based on the mass of the raw material white peony root powder to the white peony root fermentation product, and perform ultrasonic extraction at 150-200 W for 30-40 min in an ultrasonic cleaning machine at a water temperature of 80-90°C. After the ultrasonic extraction is completed, filter with a hot Buchner funnel, and concentrate the filtrate under reduced pressure at 60°C and -0.1 MPa to 1 / 20-1 / 30 of the original volume to obtain the white peony root water extract concentrate.

[0017] Furthermore, the preparation method of the white peony root aqueous extract in step (3) is as follows: the white peony root aqueous extract concentrate is vacuum dried at 65°C and -0.1 MPa to constant weight, and then ground to obtain the white peony root aqueous extract.

[0018] Compared with the prior art, the beneficial effects of the present invention are mainly reflected in that the present invention isolates a cellulase-producing Bacillus subtilis BS4 strain from soil, and obtains the Bacillus subtilis BS4-35 strain through mutagenesis. After adding sterile sucrose aqueous solution to white peony powder, inoculate Bacillus subtilis BS4-35 for fermentation, and the thalli produces cellulase and other hydrolases, which can hydrolyze cellulose, hemicellulose, pectin and other substances in the cell wall of white peony, and help dissolve the active ingredients bound to cellulose or intracellular in the cell wall. The white peony fermentation product is subjected to hot water ultrasonic extraction, and the yield of the water extract is significantly improved, and the activity of inhibiting tyrosinase is also significantly enhanced. Compared with the direct hot water ultrasonic extraction method without fermentation, the yield of the water extract of white peony can be increased by 31.1%. The contents of polysaccharides and paeoniflorin in the water extract are increased by 18.5% and 11.4% respectively, and the activity of inhibiting tyrosinase is enhanced by 25.3%. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 :Photos of colony morphology of Bacillus subtilis BS4 cultured on LB plate medium at 37℃ for 24 h.

[0020] Figure 2 : Standard curve for determination of polysaccharides by phenol-sulfuric acid method (with glucose as standard).

[0021] Figure 3 : HPLC analysis of paeoniflorin concentration-peak area standard curve DETAILED DESCRIPTION

[0022] The present invention is further described below in conjunction with specific embodiments, but the protection scope of the present invention is not limited thereto.

[0023] In the embodiment of the present invention, the white peony root used is a Chinese herbal medicine piece of white peony root, i.e., the dried root of the Ranunculaceae plant Paeonia lactiflora. White peony root powder is a fine powder of white peony root that is crushed and passed through a 60-mesh sieve, and is sterilized in a 105° C. oven for 1 h after being sealed and packaged.

[0024] In the embodiment of the present invention, the final concentration composition and preparation method of the LB slant medium are: peptone 10 g / L, yeast extract powder 5 g / L, NaCl 10 g / L, agar 20 g / L, dissolved in tap water, pH 7.2. 4 mL of LB slant medium was placed in a test tube, sealed with a silicone stopper, sterilized at 121°C by high-pressure steam for 20 min, and set aside to form a slope before solidification; the final concentration composition and preparation method of the LB liquid medium are: peptone 10 g / L, yeast extract powder 5 g / L, NaCl 10 g / L, dissolved in tap water, pH7.2. 50 mL of LB liquid medium was placed in a 250 mL conical flask, tied with 8 layers of gauze, and sterilized at 121°C by high-pressure steam for 20 min.

[0025] The yield of the white peony root water extract described in the embodiment of the present invention is calculated according to Formula 1:

[0026]

[0027] Example 1: Isolation and screening of microbial strains for fermentation of white peony root

[0028] The original strain of microorganisms for fermenting white peony root is isolated and screened according to the following steps:

[0029] (1) Isolation of strains: Wet soil rich in humus (straw piled and rotten) was collected from farmland, and 5 g of soil was weighed into a triangular flask containing 45 mL of sterile saline and 20 glass beads. The soil microbial suspension was fully shaken for 15 min to prepare a soil microbial suspension. The soil microbial suspension was gradiently diluted to 1×10 -5 , 1×10 -6 and 1×10 -7 , 0.2 mL of the bacterial suspension of the three dilutions was spread on the cellulose plate medium, and cultured at 37°C until the number of colonies no longer increased. Single colonies with different morphology and color were picked and streaked on the cellulose plate medium, and cultured at 37°C for 48 h. 13 pure bacterial strains that can grow on cellulose plate medium were obtained;

[0030] (2) Screening of cellulase-producing strains: Use an inoculation loop to pick up the cells of each pure strain in step (1), and inoculate them on a Congo red cellulose plate medium by the spot-grafting method. After culturing at 37°C for 48 h, measure the diameter of each colony and the diameter of the color-changing circle around it, calculate the ratio of the color-changing circle diameter to the colony diameter, and select colonies with a ratio greater than 1.5 to transfer to LB slant medium to obtain 8 cellulase-producing strains. The strain numbers are shown in Table 1.

[0031] (3) Screening of fermentation strains of white peony: 40 mL of 20 g / L sucrose aqueous solution was added to 9 250-mL flasks, which were sealed with 8 layers of gauze and sterilized at 121°C with high pressure steam for 15 min. 5 g of white peony powder was added to each of the 9 flasks, and 8 flasks were inoculated with 2 loops of slant cells of the 8 strains obtained in step (2), while 1 flask was not inoculated as a blank fermentation control. All flasks were kept at 32°C and 200 r / min for 48 h.

[0032] (4) Extraction: Add 70 mL of deionized water (the solid-liquid ratio of the extraction system is 1:22 (g:mL)) to all the white peony fermentation products in step (3), stir evenly in an ultrasonic cleaner at 80°C, and perform ultrasonic extraction at 100 W for 40 min. After the ultrasonic extraction is completed, filter with a Buchner funnel while it is still hot and collect the filtrate;

[0033] (5) Concentration and drying: The filtrate obtained in step (4) was concentrated under reduced pressure at 60°C and -0.1 MPa to 5.5 mL (1 / 20 of the original volume) to obtain a white peony water extract concentrate. The water extract concentrate was placed in a clean petri dish and vacuum dried at 65°C and -0.1 MPa to constant weight to obtain a white peony water extract. The yields of white peony water extracts fermented by different strains and the control are shown in Table 1.

[0034] Table 1 Yields of water extracts of white peony root fermented by different strains and the control

[0035]

[0036] As can be seen from the data in Table 1, the blank fermentation control without inoculation of bacteria has no significant difference in water extract yield from the direct extraction control due to the basic lack of bacterial growth. The water extract yield of white peony powder after fermentation by different strains is quite different, indicating that there are species differences in the microbial strains used to ferment white peony to improve the water extract yield. After white peony was fermented by strain BS4, the extract yield was 16.7%, which was 17.6% higher than the direct extraction control (14.2%), so the present invention selected strain BS4 as the initial fermentation strain to improve the water extract yield of white peony.

[0037] The final concentration of the cellulose plate medium is: sodium carboxymethyl cellulose (CMC-Na) 10 g / L, peptone 10 g / L, yeast extract 5 g / L, NaCl 10 g / L, KH2PO4 1 g / L, dissolved in tap water, pH 7.2; the final concentration of the Congo red cellulose plate medium is: CMC-Na 10 g / L, (NH4)2SO4 2 g / L, KH2PO4 1 g / L, NaCl 1g / L, MgSO4·7H2O 0.5 g / L, Congo red 0.2 g / L, agar 20 g / L, dissolved in distilled water, pH natural. The above culture medium is in a triangular flask, tied with 8 layers of gauze, sterilized at 121°C by high-pressure steam for 20 min, and poured into a sterile culture dish with a diameter of 9 cm before solidification, 15-20 mL per dish.

[0038] Example 2: Classification and Identification of Strain BS4

[0039] After the strain BS4 was streaked on LB plate medium and cultured at 37℃ for 24 hours, the colonies were milky white, opaque, rough on the surface, and irregular on the edges. Gram staining was positive. Under an optical microscope, the bacteria were observed to be short rod-shaped, 0.8~1.0×2~3μm in size, and could form spores, which were located in the center or slightly off the center of the bacteria and 0.6~0.8×1.0~1.5 μm in size. The colony morphology of the strain BS4 cultured on LB plate medium at 37℃ for 24 hours is shown in Figure 1 .

[0040] The 16S rDNA nucleotide sequence of strain BS4 was measured as shown in SEQ ID NO.1. The sequence was compared by BLAST in NCBI (National Center for Biotechnology Information, https: / / www.ncbi.nlm.nih.gov) (Database: rRNA / ITS databsese-16s rRNA, Limit to: Sequences fromtype materail). Among the four strains with the highest homology (99.92%), three were Bacillus subtilis. Therefore, based on the morphological characteristics of strain BS4 and the comparison results of 16S rDNA sequence, it can be determined that strain BS4-35 is a strain of Bacillus subtilis. Therefore, the classification of BS4 belongs to (refer to NCBI, http: / / www.ncbi.nlm.nih.gov): Bacteria, Bacillota, Bacilli, Bacillales, Bacillaceae, Bacillus, Bacillus subtilis.

[0041] The 16S rDNA nucleotide sequence of the strain BS4 is:

[0042]

[0043] Example 3: Mutation breeding of microbial strains for fermentation of white peony root

[0044] (1) UV mutagenesis: After the strain BS4 was activated and cultured in LB slant medium for 24 h, a loop of bacteria was picked up with an inoculation loop and placed in 25 mL of sterile saline (50 mL centrifuge tube). The centrifuge tube was shaken on a mixer for 1 min to fully suspend the bacteria. Based on the results of the preliminary experiment on the count of bacteria in the bacterial solution (dilution coating method), the bacterial solution was diluted with sterile saline to a concentration of 1×10 8 About 1000 cells / mL. Under red light illumination, take 2 mL of bacterial solution and put it in 5 sterile culture dishes with a diameter of 6 cm. Put a sterile paper clip in each culture dish. Place the culture dishes on a magnetic stirrer and irradiate them for 30, 45, 60, 75, and 90 s at a distance of 30 cm from a 15 W ultraviolet lamp preheated for 30 min. After the bacterial solution after the above irradiation treatment is diluted appropriately, 0.1 mL is transferred to the cellulose plate culture medium. The inoculated cellulose plate culture medium is wrapped with black cloth, inverted and cultured at 37°C for 48 h, and the colonies on the plate are counted. The bacterial solution that has not been irradiated with ultraviolet light is used as a control to calculate the mortality rate;

[0045] (2) Primary screening: Select colonies on plates with a lethality rate of more than 90% and inoculate them into Congo red cellulose agar medium. After culturing at 37°C for 48 h, measure the colony diameter of each strain and the diameter of the color change circle around it. Select colonies with a ratio of greater than 2.0 and transfer them to LB slant medium to obtain 15 mutant strains.

[0046] (3) Rescreening: Add 40 mL of 20 g / L sucrose aqueous solution to 15 250-mL flasks, tie the flasks with 8 layers of gauze, and sterilize them at 121°C with high pressure steam for 15 min. Add 5 g of white peony powder to each flask, inoculate 2 loops of the slant cells of the 15 strains obtained in step (2), and place them for constant temperature fermentation at 32°C and 200 r / min for 48 h;

[0047] (4) Extraction: Add 70 mL of deionized water (the solid-liquid ratio of the extraction system is 1:22 (g:mL)) to all the white peony fermentation products in step (3), stir evenly in an ultrasonic cleaner at 80°C, and perform ultrasonic extraction at 100 W for 40 min. After the ultrasonic extraction is completed, filter with a Buchner funnel while it is still hot and collect the filtrate;

[0048] (5) Concentration and drying: The filtrate obtained in step (4) was concentrated under reduced pressure at 60°C and -0.1 MPa to 5.5 mL (1 / 20 of the original volume) to obtain a white peony water extract concentrate. The water extract concentrate was placed in a clean petri dish and vacuum dried at 65°C and -0.1 MPa to constant weight to obtain a white peony water extract. The yields of white peony water extracts fermented by different mutant strains and the control are shown in Table 2.

[0049] Table 2 The yields of water extracts of white peony root fermented by different mutant strains and the control are shown in Table 2.

[0050]

[0051] It can be seen from the data in Table 2 that among the 15 screened strains, the water extract yield of the strain numbered BS4-35 was 18.8% after fermenting white peony root, which was 12.6% higher than the 16.7% of the wild strain BS4 and 32.4% higher than the yield of the direct extraction control of 14.2%. Therefore, the present invention selected the BS4-35 strain as the microbial strain for fermenting white peony root.

[0052] In summary, the cellulase-producing strain BS4 was isolated from the humus-rich soil. After identification, it was determined that the strain was a Bacillus subtilis. After ultraviolet mutagenesis and screening, the Bacillus subtilis BS4-35 strain was obtained. After the white peony was fermented by this strain, the yield of the water extract was increased by 32.4% compared with the unfermented control. The Bacillus subtilis BS4-35 strain is deposited in the Guangdong Provincial Microbiological Culture Collection Center, with the deposit number GDMCC No: 65591, the deposit date December 9, 2024, and the address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou City, Guangdong Province; Postal Code 510070.

[0053] Example 4: Application of Bacillus subtilis BS4-35 in the preparation of white peony root water extract 1

[0054] Bacillus subtilis BS4-35 is used in the preparation of white peony root water extract, which can be carried out according to the following steps:

[0055] (1) Freeze-dried Bacillus subtilis BS4-35 cells were inoculated on fresh LB slant medium and cultured at 35°C for 36 h. Two loops of cells from the slant were inoculated into 50 mL of LB liquid medium and cultured at 30°C and 200 r / min for 24 h. The OD 600 =3.37 of seed solution;

[0056] (2) Add 80 mL of 20 g / L sucrose aqueous solution to a 250-mL flask, tie the flask with 8 layers of gauze, and sterilize with high-pressure steam at 121°C for 15 min. Add 10 g of white peony powder to the flask, then add 4 mL of the seed solution in step (1) (inoculation amount 4%), tie the flask with 8 layers of gauze, and ferment at 32°C and 200 r / min for 48 h to obtain white peony fermentation product;

[0057] (3) Add 140 mL of deionized water [the solid-liquid ratio of the extraction system is 1:22 (g:mL)] to all the white peony fermentation products in step (2), stir evenly, place in an ultrasonic cleaner at a water temperature of 80°C, perform ultrasonic extraction at 100 W for 40 min, filter with a Buchner funnel while hot, and collect all the filtrate;

[0058] (4) The filtrate prepared in step (3) was concentrated under reduced pressure at 60°C and -0.1 MPa to 11 mL (1 / 20 of the original filtrate volume) to obtain a white peony water extract concentrate. The entire water extract concentrate was placed in a clean petri dish, vacuum dried at 65°C and -0.1 MPa to constant weight, and crushed to obtain a white peony water extract.

[0059] According to the above steps, 1.86 g of water extract was obtained from 10 g of white peony root, with a yield of 18.6%.

[0060] Comparative Example 1: Preparation of white peony root water extract by conventional hot water ultrasonic method (compared with Example 4)

[0061] (1) 10 g of white peony powder was placed in a 250-mL Erlenmeyer flask, and 220 mL of deionized water was added [the solid-liquid ratio of the extraction system was 1:22 (g:mL)]. After stirring evenly, the mixture was placed in an ultrasonic cleaner at 80 °C and subjected to 100 W ultrasonic extraction for 40 min. The filtrate was filtered with a Buchner funnel while still hot and all the filtrate was collected.

[0062] (2) The filtrate prepared in step (1) was concentrated under reduced pressure at 60°C and -0.1 MPa to 11 mL (1 / 20 of the original filtrate volume) to obtain a white peony water extract concentrate. The entire water extract concentrate was placed in a clean petri dish, vacuum dried at 65°C and -0.1 MPa to constant weight, and crushed to obtain a white peony water extract.

[0063] According to the above steps, 1.45% water extract was obtained from 10 g of white peony root, with a yield of 14.5%.

[0064] Comparison of the results of Example 4 and Comparative Example 1 shows that, before hot water ultrasonic extraction of white peony root, fermentation with Bacillus subtilis BS4-35 was added, and the preparation rate of the water extract was increased from 14.5% of the unfermented extract to 18.6%, an increase of 28.3%.

[0065] Example 5: Application of Bacillus subtilis BS4-35 in the preparation of white peony root water extract 2

[0066] Bacillus subtilis BS4-35 is used in the preparation of white peony root water extract, which can be carried out according to the following steps:

[0067] (1) Inoculate the slant of Bacillus subtilis BS4-35 stored in a refrigerator at 4°C on a fresh LB slant medium and culture at 36°C for 30 h. Pick two loops of cells from the slant and inoculate 50 mL of LB liquid medium. Culture at 32°C and 180 r / min for 22 h to obtain the OD 600 =3.51 of seed solution;

[0068] (2) Add 140 mL of 17.5 g / L sucrose aqueous solution to a 500-mL flask, tie the flask with 8 layers of gauze, and sterilize with high-pressure steam at 121°C for 15 min. Add 20 g of white peony powder to the flask, then add 8.4 mL of the seed solution in step (1) (inoculation amount 6%), tie the flask with 8 layers of gauze, and ferment at 34°C and 180 r / min for 42 h to obtain white peony fermentation product;

[0069] (3) Add 260 mL of deionized water [the solid-liquid ratio of the extraction system is 1:20 (g:mL)] to all the white peony fermentation products in step (2), stir evenly, place in an ultrasonic cleaner at a water temperature of 85°C, perform ultrasonic extraction at 150 W for 35 min, filter with a Buchner funnel while hot, and collect all the filtrate;

[0070] (4) The filtrate prepared in step (3) was concentrated under reduced pressure at 60°C and -0.1 MPa to 16 mL (1 / 25 of the original filtrate volume) to obtain a white peony root aqueous extract concentrate. The entire aqueous extract concentrate was placed in a clean petri dish, vacuum dried at 65°C and -0.1 MPa to constant weight, and crushed to obtain a white peony root aqueous extract.

[0071] According to the above steps, 3.96 g of water extract was obtained from 20 g of white peony root, with a yield of 19.8%.

[0072] Comparative Example 2: Preparation of white peony root water extract by conventional hot water ultrasonic method (compared with Example 5)

[0073] (1) 20 g of white peony powder was placed in a 500-mL conical flask, and 400 mL of deionized water was added [the solid-liquid ratio of the extraction system was 1:20 (g:mL)]. After stirring evenly, the mixture was placed in an ultrasonic cleaner at 85 °C and ultrasonically extracted at 150 W for 35 min. The filtrate was filtered with a Buchner funnel while still hot and all the filtrate was collected.

[0074] (2) The filtrate prepared in step (1) was concentrated under reduced pressure at 60°C and -0.1 MPa to 16 mL (1 / 25 of the original filtrate volume) to obtain a white peony water extract concentrate. The entire water extract concentrate was placed in a clean petri dish, vacuum dried at 65°C and -0.1 MPa to constant weight, and crushed to obtain a white peony water extract.

[0075] According to the above steps, 3.02 g of water extract was obtained from 20 g of white peony root, with a yield of 15.1%.

[0076] By comparing the results of Example 5 and Comparative Example 2, it can be seen that before hot water ultrasonic extraction of white peony root, fermentation treatment with Bacillus subtilis BS4-35 was added, and the preparation rate of the water extract was increased from 15.1% of the unfermented one to 19.8%, an increase of 31.1%.

[0077] Example 6: Application of Bacillus subtilis BS4-35 in the preparation of white peony root water extract

[0078] Bacillus subtilis BS4-35 is used in the preparation of white peony root water extract, which can be carried out according to the following steps:

[0079] (1) Inoculate the slant of Bacillus subtilis BS4-35 stored in a refrigerator at 4°C on a fresh LB slant medium and culture at 37°C for 24 h. Pick two loops of the slant and inoculate 50 mL of LB liquid medium. Culture at 34°C and 160 r / min for 20 h. Obtain the OD 600 =3.62 of seed solution;

[0080] (2) Add 300 mL of 15 g / L sucrose aqueous solution to a 1-L flask, seal the flask with 8 layers of gauze, and sterilize with high-pressure steam at 121°C for 20 min. Add 50 g of white peony powder to the flask, then add 21 mL of the seed solution in step (1) (inoculation amount 7%), seal the flask with 8 layers of gauze, and ferment at 36°C and 160 r / min for 36 h to obtain white peony fermentation product;

[0081] (3) Add 600 mL of deionized water [the solid-liquid ratio of the extraction system is 1:18 (g:mL)] to all the white peony fermentation products in step (2), stir evenly, place in an ultrasonic cleaner at a water temperature of 90°C, and perform ultrasonic extraction at 200 W for 30 min. Filter with a Buchner funnel while hot to collect all the filtrate;

[0082] (4) The filtrate prepared in step (3) was concentrated under reduced pressure at 60°C and -0.1 MPa to 30 mL (1 / 30 of the original filtrate volume) to obtain a white peony water extract concentrate. The entire water extract concentrate was placed in a clean petri dish, vacuum dried at 65°C and -0.1 MPa to constant weight, and crushed to obtain a white peony water extract.

[0083] According to the above steps, 9.73 g of water extract was obtained from 50 g of white peony root, with a yield of 19.5%.

[0084] Comparative Example 3: Preparation of white peony root water extract by conventional hot water ultrasonic method (compared with Example 6)

[0085] (1) 50 g of white peony powder was placed in a 1-L beaker, and 750 mL of deionized water was added [the solid-liquid ratio of the extraction system was 1:18 (g:mL)]. After stirring evenly, the mixture was placed in an ultrasonic cleaner at 90 °C and ultrasonically extracted at 200 W for 30 min. The filtrate was filtered with a Buchner funnel while still hot, and all the filtrate was collected.

[0086] (2) The filtrate prepared in step (1) was concentrated under reduced pressure at 60°C and -0.1 MPa to 30 mL (1 / 30 of the original filtrate volume) to obtain a white peony water extract concentrate. The entire water extract concentrate was placed in a clean petri dish, vacuum dried at 65°C and -0.1 MPa to constant weight, and crushed to obtain a white peony water extract.

[0087] According to the above steps, 7.64 g of water extract was obtained from 50 g of white peony root, with a yield of 15.3%.

[0088] By comparing the results of Example 6 and Comparative Example 3, it can be seen that before hot water ultrasonic extraction of white peony root, fermentation treatment with Bacillus subtilis BS4-35 was added, and the preparation yield of the water extract was increased from 15.3% of the unfermented extract to 19.5%, an increase of 27.5%.

[0089] Example 7: Analysis of polysaccharide and paeoniflorin contents in white peony root water extract

[0090] The polysaccharide and paeoniflorin contents of the white peony root aqueous extracts prepared in Examples 4 to 6 and Comparative Examples 1 to 3 were determined by the following methods.

[0091] (1) Analysis of polysaccharide content: The polysaccharide content in the white peony water extract was determined by the phenol-sulfuric acid method. The specific method is as follows: the white peony water extract is prepared into an aqueous solution with a concentration of 0.5 g / L with deionized water, and then 1 mL is taken into a 10 mL centrifuge tube, and 4 mL of anhydrous ethanol is added. After sufficient shaking, it is placed in a refrigerator at 4°C for 12 h, and then centrifuged at 8000 r / min for 10 min. The supernatant is poured off, and 5 mL of deionized water is added to dissolve the precipitate and used as the sample to be tested. 1 mL of the sample solution to be tested is taken into a 10 mL stoppered tube, and 1 mL of 5% phenol aqueous solution is added. After shaking, 5 mL of concentrated sulfuric acid (mass concentration 98%) is quickly added. After shaking, it is heated in a boiling water bath for 15 min and cooled to room temperature. The absorbance (A) at a wavelength of 490 nm is measured using 1 mL of deionized water instead of the sample solution as a reference. 490). The same method was used to determine the A of glucose aqueous solutions with different concentrations. 490 , plot glucose concentration—A 490 Standard curve ( Figure 2 ), and the regression equation is obtained, and the polysaccharide content in the sample is calculated by the regression equation.

[0092] (2) Analysis of paeoniflorin content: The paeoniflorin content in the white peony water extract was determined by HPLC. The specific method is as follows: the white peony water extract was prepared into an aqueous solution with a concentration of 0.2 mg / mL with deionized water, and then filtered through a 0.45 µm microporous filter membrane for testing. The high performance liquid chromatograph was LC-20AD (Shimadzu Instrument Co., Ltd., Japan), the chromatographic column was Aglilent Extend-C18 column (5 µm, 4.6 mm × 250 mm), and the column temperature was room temperature; the mobile phase was acetonitrile and 0.1% phosphoric acid aqueous solution in a volume ratio of 14:86, the flow rate was 1.0 mL / min, the detection wavelength was 230 nm, and the injection volume was 10 μL. The peak areas of the standard paeoniflorin methanol solutions of different concentrations were determined under the above HPLC conditions, and the paeoniflorin concentration-peak area standard curve was drawn ( Figure 3 ), and the regression equation was obtained, and the concentration of paeoniflorin in the sample was calculated by the regression equation.

[0093] The polysaccharide and paeoniflorin contents in the white peony water extracts prepared in Examples 4 to 6 and Comparative Examples 1 to 3 are shown in Table 3

[0094] Table 3 Polysaccharide and paeoniflorin contents in the aqueous extracts of white peony prepared in the examples and comparative examples

[0095]

[0096] From the results in Table 3, it can be seen that in the white peony water extracts prepared in Examples 4 to 6, the average polysaccharide content is 58.4%, and the paeoniflorin content is 14.7%, which are increased by 18.5% and 11.4% respectively compared with the polysaccharide (49.3%) and paeoniflorin (13.2%) contents in the white peony water extracts prepared in Comparative Examples 1 to 3.

[0097] Example 8: Inhibitory activity of white peony root aqueous extract on tyrosinase

[0098] White peony root water extracts were prepared in Examples 4-6 and Comparative Examples 1-3, and prepared into aqueous solutions with mass concentrations of 0.5, 1.0, and 2.0 mg / mL using deionized water. Their inhibitory activities against tyrosinase were determined by the following method.

[0099] Prepare 0.2 mol / L, pH 6.8 sodium dihydrogen phosphate-potassium dihydrogen phosphate buffer (PB), 1 mg / mL L-tyrosine aqueous solution, and 35 U / mL tyrosinase aqueous solution. Set up the sample loading as follows:

[0100] Test sample ① (A1): 2.0 mL PB + 0.5 mL tyrosinase aqueous solution + 0.5 mL L-tyrosine aqueous solution;

[0101] Test sample ② (A2): 2.5 mL PB + 0.5 mL tyrosinase aqueous solution;

[0102] Test sample ③ (A3): 0.5 mL sample solution + 1.5 mL PB + 0.5 mL tyrosinase aqueous solution + 0.5 mL L-tyrosine aqueous solution;

[0103] Test sample ④ (A4): 0.5 mL sample solution + 2.0 mL PB + 0.5 mL tyrosinase aqueous solution.

[0104] Each test sample was shaken and kept in a 37°C water bath for 30 min, and then the absorbance was immediately measured at a wavelength of 475 nm. The absorbances of the above four test samples were measured to be A1, A2, A3 and A4, respectively. The tyrosinase inhibition rate (I) was calculated according to Formula 2.

[0105]

[0106] The inhibition rates of the white peony root aqueous extracts prepared in Examples 4 to 6 and Comparative Examples 1 to 3 on tyrosinase activity at concentrations of 0.5, 1.0 and 2.0 mg / mL are shown in Table 4.

[0107] Table 4 Inhibition rate of tyrosinase activity of white peony root aqueous extracts prepared in Examples and Comparative Examples at different concentrations

[0108]

[0109] It can be seen from the data in Table 4 that the white peony water extracts prepared in Examples 4 to 6 have higher inhibition rates on tyrosinase activity at different concentrations than those in Comparative Examples 1 to 3. Compared with the white peony water extracts prepared in Comparative Examples 1 to 3, the white peony water extracts prepared in Examples 4 to 6 have an average inhibition rate on tyrosinase activity of 25.3% at three concentrations.

[0110] Tyrosinase is a polyphenol oxidase. In the process of melanin formation, it participates in catalyzing the hydroxylation of monophenol to form o-dihydroxy compounds and oxidizing catechol to form o-quinone. It is a key enzyme in the production of melanin. Its activity determines the amount of melanin. Therefore, the strength of the inhibitory effect on tyrosinase is the main indicator for evaluating the whitening effect of a compound. Compared with the unfermented white peony water extract, the white peony water extract prepared with the assistance of Bacillus subtilis BS4-35 fermentation has significantly improved the inhibitory activity of tyrosinase, indicating that the white peony water extract prepared with the assistance of Bacillus subtilis BS4-35 fermentation has better freckle removal and whitening effects.

[0111] The above description is only a preferred embodiment of the present invention and does not limit the present invention in any way. Any simple modification, change, enlargement and equivalent transformation made to the above embodiment based on the technical essence of the present invention still falls within the protection scope of the technical solution of the present invention.

Claims

1. A strain of Bacillus subtilis BS4-35, characterized in that: It is deposited in Guangdong Provincial Microbiological Culture Collection Center, the culture collection number is GDMCC No: 65591, and the collection date is December 9, 2024.

2. The Bacillus subtilis BS4-35 of claim 1 is isolated from soil and obtained through mutagenesis and screening, and is characterized in that: The 16S rDNA nucleotide sequence of the Bacillus subtilis BS4-35 is as shown in SEQ ID NO.

1.

3. Use of the Bacillus subtilis BS4-35 described in claim 1 in preparing white peony root whitening water extract.

4. A method for preparing whitening water extract of white peony root by fermentation with Bacillus subtilis BS4-35, comprising the following steps: (1) adding a sterile sucrose aqueous solution to white peony root powder, inoculating the bacterial cells of Bacillus subtilis BS4-35 described in claim 1 for fermentation, and obtaining a white peony root fermentation product; (2) adding deionized water to the fermented white peony root, subjecting the mixture to hot water ultrasonic extraction and filtering, and concentrating the filtrate to obtain a white peony root water extract concentrate; (3) The white peony root aqueous extract concentrate is vacuum dried and crushed to obtain the white peony root aqueous extract.

5. The method according to claim 4, characterized in that The volume of the sterile sucrose aqueous solution added to the white peony powder in the step (1) is 6-8 mL / g based on the mass of the white peony powder; the sterile sucrose aqueous solution has a sucrose concentration of 15-20 g / L and is sterilized by high-pressure steam at 121° C. for 15-20 min.

6. The method according to claim 4, characterized in that In the step (1), the inoculation amount of Bacillus subtilis BS4-35 bacteria is 5% to 7% of the volume of the sterile sucrose aqueous solution.

7. The method according to claim 4, characterized in that The fermentation conditions of white peony root in step (1) are as follows: adding sterile sucrose aqueous solution to white peony root powder, inoculating Bacillus subtilis BS4-35 bacteria, stirring evenly, and fermenting at a constant temperature of 32-36° C. and 160-200 r / min for 36-48 h.

8. The method according to claim 4, characterized in that The white peony root powder in step (1) is the fine powder of white peony root after being crushed and passed through a 60-mesh sieve, and is sealed and packaged and sterilized in an oven at 105° C. for 1 h.

9. The method according to claim 4, characterized in that In the step (2), the preparation method of the white peony root water extract concentrate is as follows: add 12-14 mL / g of deionized water based on the mass of the raw material white peony root powder to the white peony root fermentation product, perform ultrasonic extraction at 150-200 W for 30-40 min in an ultrasonic cleaning machine at a water temperature of 80-90°C; after the ultrasonic extraction is completed, filter while hot, and reduce the pressure to concentrate the filtrate to 1 / 20-1 / 30 of the original volume to obtain the white peony root water extract concentrate.

10. The method according to claim 4, characterized in that The preparation method of the white peony root aqueous extract in step (3) is as follows: the white peony root aqueous extract concentrate is dried at 65° C. and −0.1 MPa in a vacuum oven until constant weight is reached, and then ground to obtain the white peony root aqueous extract.

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