Bacillus subtilis as well as fermentation liquor and application thereof

Abstract

The invention provides bacillus subtilis, fermentation liquor prepared from the bacillus subtilis and application of the fermentation liquor. The invention provides bacillus subtilis MPEB0002969, the bacillus subtilis is preserved in Guangdong Microbial Culture Collection Center (GDMCC), and the preservation number is GDMCC No: 63439. The invention further provides fermentation liquor prepared from the bacillus subtilis and application of the fermentation liquor in preparation of cosmetics. The fermentation liquor prepared from the bacillus subtilis provided by the invention has antioxidant, whitening and moisturizing activity, and can be used for preparing cosmetics.

Description

Technical Field

[0001] The present application relates to the field of biotechnology, and more particularly, to Bacillus subtilis, its fermentation broth and applications. Background Art

[0002] Bacillus subtilis is a type of Gram-positive bacteria with slender rod-shaped and endospores, and is an aerobic bacterium widely present in different environments. Bacillus subtilis does not secrete exotoxins and does not contain endotoxins, and is recognized as a biosafe strain (Generally Regarded as Safe, GRAS) by the US Food and Drug Administration (FDA). Bacillus subtilis also has the advantages of relatively simple genetic manipulation, clear physiological and biochemical characteristics, and convenient culture and fermentation. It can be used as a microbial cell factory and is used in the fermentation industry to ferment and produce various fermentation products such as γ-polyglutamic acid, hyaluronic acid, and riboflavin and other target products.

[0003] Poly-γ-glutamic acid (γ-PGA) is a multifunctional peptide polymerized by γ-amide bonds with L-glutamic acid or D-glutamic acid as monomers, and can be obtained by microbial fermentation. It was discovered in 1942 that γ-PGA can be produced by Bacillus subtilis. γ-PGA has the advantages of biocompatibility, water solubility, biodegradability, edibility, non-toxicity to humans and the environment, etc. It can be used as a water retention agent, food additive, bioflocculant, heavy metal absorbent, superabsorbent resin, drug carrier, drug sustained release agent, etc., and has a wide range of applications in the fields of cosmetics, food, medicine, agriculture and wastewater treatment. The international naming of γ-PGA in the cosmetics pharmacopoeia is Natto Gum. Cosmetics containing γ-PGA and γ-PGA hydrogel can improve the moisturizing ability of the skin, have antioxidant properties, and have the effect of skin whitening.

[0004] Synthetic antioxidants, such as butylated hydroxyanisole, are added to cosmetics to reduce oxidative stress, but these compounds are suspected of being toxic. Most of the whitening agents on the market achieve the effect of removing spots and whitening by inhibiting the activity of tyrosinase, blocking the synthesis reaction chain of melanin, and reducing its generation in the skin. Although tyrosinase inhibitors such as L-ascorbic acid, kojic acid, and ellagic acid have been used as skin whitening agents, they have some disadvantages. For example, L-ascorbic acid is heat-sensitive and easily degrades; kojic acid can cause allergic reactions such as contact dermatitis and sensitization, and has carcinogenic potential; ellagic acid is insoluble and has poor bioavailability. Therefore, strengthening the research on natural antioxidants and tyrosinase inhibitors is of great significance for the development and application of natural antioxidants and tyrosinase inhibitors in the cosmetics field. Summary of the Invention

[0005] Bacillus subtilis MPEB0002969 in the present application can produce γ-PGA through fermentation, and the fermentation broth has antioxidant, whitening, and moisturizing activities. This indicates that the fermentation broth has potential uses in the cosmetics industry for developing new products with natural antioxidant, whitening, and moisturizing activities.

[0006] The present application provides a Bacillus subtilis MPEB0002969, which is deposited in the Guangdong Provincial Microbial Culture Collection Center (GDMCC) with the deposit number GDMCC No: 63439, the deposit date of May 8, 2023, and the deposit address at the 5th floor of Building 59, No. 100 compound, Xianlie Middle Road, Guangzhou.

[0007] The present application also provides a fermentation broth prepared from Bacillus subtilis.

[0008] The present application also provides the use of the fermentation broth prepared from Bacillus subtilis in the preparation of cosmetics.

[0009] The fermentation broth prepared from Bacillus subtilis provided by the present application has antioxidant, whitening, and moisturizing activities and can be used in the preparation of cosmetics. Brief Description of the Drawings

[0010] Figure 1A Shows the colony morphology of the strain of the present application.

[0011] Figure 1B Shows the Gram staining result of the strain of the present application.

[0012] Figure 2 Shows the phylogenetic tree based on 16S rDNA of the strain of the present application.

[0013] Figure 3A Shows the wavelength scanning result of γ-PGA.

[0014] Figure 3B Shows the wavelength scanning result of the extract of the fermentation broth of Bacillus subtilis MPEB0002969 of the present application.

[0015] Figure 4 Shows the tyrosinase inhibition curve of the positive control kojic acid.

[0016] Figure 5A Shows the moisturizing rate of the fermentation broth and hyaluronic acid (5 mg / ml) at a relative humidity (RH) of 43%, where, in each group of two columns, the left column corresponds to the fermentation broth and the right column corresponds to hyaluronic acid.

[0017] Figure 5BThe moisture retention rates of the fermentation broth and hyaluronic acid (5 mg / ml) at a relative humidity (RH) of 81% are shown. Among them, in each group of two columns, the left column corresponds to the fermentation broth and the right column corresponds to hyaluronic acid.

[0018] Figure 6 The molecular weight distribution of the proteins contained in the fermentation broth of the strain of the present application is shown. Detailed implementation mode

[0019] The following examples can enable those skilled in the art to understand the present application more comprehensively, but do not limit the present application in any way. The reagents used in the present application are all conventional commercially available reagents unless otherwise specified.

[0020] Preparation of the culture medium:

[0021] MH broth (1 L): Weigh 21.0 g of MH Broth, dissolve it in 1000 mL of ddH2O, and autoclave it at 121 °C for 20 min for standby.

[0022] MH(A) medium (1 L): Weigh 36.5 g of Mueller-Hinton Agar, add 1000 mL of ddH2O, autoclave it at 121 °C for 20 min, cool it to 50 °C, pour it into plates, and after cooling and solidifying, invert it for standby.

[0023] Fermentation medium (1 L): Weigh 15.0 g of glucose, 15.0 g of peptone, 0.6 g of MgSO4·7H2O, 1.0 g of K2HPO4, 0.1 g of MnSO4·H2O, 15.0 g of NHCl4, and 25.0 g of citric acid, add 1000 mL of ddH2O, adjust the pH to 7.0, dispense it according to a liquid loading volume of 20% (V / V), autoclave it at 105 °C for 40 min, and then cool it for standby.

[0024] Identification of the strain

[0025] 10 μL of the MPEB0002969 bacterial solution stored in a glycerol tube was inoculated into MH(A) medium and cultured at 37 °C for 12 h. The colony morphological characteristics and growth status were observed. Single colonies were picked and cultured in MH broth medium at 37 °C with shaking for 12 h to obtain the proliferated bacterial solution of the strain, and Gram staining and 16S rDNA identification were performed (Guangdong Ruibo Technology Co., Ltd., China). The 16S rDNA sequencing results were compared with the NCBI database (https: / / blast.ncbi.nlm.nih.gov / Blast.cgi) using the BLAST search function. The MEGA 10.2.2 software was used for multiple sequence alignment analysis and phylogenetic tree construction by the neighbor-joining method, and iTOL (https: / / itol.embl.de / ) was used for visualizing the phylogenetic tree.

[0026] The surface of the colony of this strain was smooth and showed filamentous when picked up ( Figure 1A ), and the Gram staining was positive ( Figure 1B ). Through the alignment and analysis of the 16S rDNA gene sequence of MPEB0002969, it was found that the identity between strain MPEB0002969 and Bacillus subtilis and Bacillus velezensis was more than 99%. The results of the 16S rDNA phylogenetic tree showed that strain MPEB0002969 and B. subtilis strain R0-2 were in the same evolutionary branch, and the genetic relationship was close at a sequence divergence of 0.0001 ( Figure 2 ). Based on the comprehensive analysis of morphology, 16S rDNA sequence, and phylogenetic tree, strain MPEB0002969 was identified as Bacillus subtilis and deposited in the Guangdong Microbial Culture Collection Center (GDMCC) with the deposit number: GDMCC No: 63439.

[0027] Preparation of fermentation broth

[0028] 10 μL of the proliferated bacterial solution was inoculated into MH broth seed medium and cultured at 37 °C with shaking for 12 h as the seed solution for standby. The seed solution was inoculated into the fermentation medium (200 mL / 1000 mL Erlenmeyer flask) at an inoculation amount of 1% (V / V), and fermented in a constant temperature shaker at 37 °C and 171 r / min for 24 h. The fermentation broth was centrifuged at 8000 r / min at low temperature for 15 min, and the fermentation supernatant was reserved for use.

[0029] Extraction and determination of fermentation crude products

[0030] Take 50 mL of the fermentation supernatant and add three times the volume of absolute ethanol. Place it in the refrigerator and let it stand overnight. Centrifuge at 8000 r / min at low temperature for 15 min. Discard the supernatant. Dissolve the precipitate in distilled water, dialyze, pre-cool, and then freeze-dry to obtain the crude γ-PGA product. The yield of the crude γ-PGA product produced by Bacillus subtilis MPEB0002969 fermentation is 17.79 g / L.

[0031] Prepare a 1 mg / mL crude product solution and a standard γ-PGA solution. Using distilled water as a reference, scan the wavelength range of 190.0 - 400.0 nm in the ultraviolet region with a 10 mm quartz cuvette on an ultraviolet-visible spectrophotometer. The γ-PGA solution ( Figure 3A ) and the crude product solution of Bacillus subtilis MPEB0002969 ( Figure 3B ) have absorption in the wavelength range of 190.0 - 400.0 nm in the ultraviolet region, and the maximum absorption wavelengths of both the γ-PGA solution and the crude fermentation product solution of Bacillus subtilis MPEB0002969 are 205.0 nm.

[0032] Determination of the antioxidant activity of the fermentation broth

[0033] 1. Determination of DPPH radical scavenging activity

[0034] 0.2 mmol / L DPPH solution: Accurately weigh 0.0079 g of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and dissolve it in absolute ethanol and make up the volume to 100 mL. Add 2 mL of the fermentation broth sample and 2 mL of the DPPH solution (0.2 mmol / L) to the test tube. Add 2 mL of the fermentation broth sample and 2 mL of absolute ethanol to the control tube. Add 2 mL of distilled water and 2 mL of the DPPH solution (0.2 mmol / L) to the blank tube. After mixing well, incubate in the dark at room temperature for 30 min, and measure the absorbance of the solution at 517 nm. Measure the DPPH radical scavenging rate of the L-ascorbic acid solution (0.01 mg / mL) according to the same method. Each group of experiments is repeated three times.

[0035] The calculation of the DPPH radical scavenging rate is as follows:

[0036]

[0037] Where A0 is the absorbance of the blank tube; A1 is the absorbance of the test tube; A2 is the absorbance of the control tube.

[0038] 2. Determination of ABTS radical scavenging activity

[0039] 2,2’-Azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS) radical solution: Weigh 0.0384 g of ABTS and dissolve it in 10 mL of distilled water to prepare a 7 mmol / L ABTS solution (Solution A). Then, accurately weigh 0.0066 g of potassium persulfate and dissolve it in 10 mL of distilled water to prepare a 2.45 mmol / L potassium persulfate solution (Solution B). Finally, take 5 mL of Solution A and 5 mL of Solution B, mix them by shaking, and react for 16 h under dark conditions at room temperature to prepare the ABTS radical cation (ABTS·+) stock solution. Before use, dilute the ABTS·+ stock solution with distilled water to make its absorbance at 734 nm be 0.7 ± 0.05 for use. Add 0.2 mL of the fermentation broth sample and 3 mL of the ABTS·+ solution to the test tube, add 0.2 mL of the fermentation broth sample and 3 mL of distilled water to the control tube, and add 0.2 mL of distilled water and 3 mL of the ABTS·+ solution to the blank tube. After mixing well, incubate in the dark at room temperature for 30 min, and measure the absorbance of the solution at 734 nm. Measure the ABTS radical scavenging rate of the L-ascorbic acid solution (0.01 mg / mL) according to the same method. Each experiment is repeated three times.

[0040] The ABTS radical scavenging rate is calculated as follows:

[0041]

[0042] Where A0 is the absorbance of the blank tube; A1 is the absorbance of the test tube; A2 is the absorbance of the control tube.

[0043] 3. Determination of hydroxyl radical scavenging activity

[0044] 5 mmol / L H2O2: Accurately weigh 0.0283 g of 30% H2O2, add distilled water to dissolve and make up the volume to 50 mL. 5 mmol / L salicylic acid-ethanol solution: Accurately weigh 0.0345 g of salicylic acid, add anhydrous ethanol to dissolve and make up the volume to 50 mL. 5 mmol / L FeSO4 solution: Accurately weigh 0.0695 g of FeSO4·7H2O, add distilled water to dissolve and make up the volume to 50 mL. The reaction mixture contains 1 mL of FeSO4 (5 mmol / L), 1 mL of salicylic acid-ethanol solution (5 mmol / L), 1 mL of H2O2 (5 mmol / L), and 1 mL of the fermentation broth sample. After incubating at 37 °C for 30 min, measure the absorbance (A1) of the reaction mixture at 562 nm. The blank control measures the absorbance value (A0) by replacing the fermentation broth sample with distilled water; the sample control measures the absorbance value (A2) by replacing the H2O2 solution (5 mmol / L) with distilled water. Measure the hydroxyl radical scavenging rate of the L-ascorbic acid solution (0.01 mg / mL) according to the same method. Each experiment is repeated three times.

[0045] The calculation of the hydroxyl radical scavenging rate is as follows:

[0046]

[0047] Where A0 is the absorbance of the control; A1 is the absorbance of the sample; A2 is the absorbance of the mixed solution without H2O2.

[0048] The antioxidant activity results of the γ-PGA fermentation broth produced by Bacillus subtilis MPEB0002969 (Table 1), and all data are the mean ± standard deviation of three repeated experiments. The DPPH radical scavenging rate of the γ-PGA fermentation broth produced by Bacillus subtilis MPEB0002969 (72.6 ± 0.3%) is close to that of L-ascorbic acid (89.2 ± 0.6%) at 0.01 mg / mL. Compared with L-ascorbic acid (0.01 mg / mL), the γ-PGA fermentation broth produced by Bacillus subtilis MPEB0002969 has stronger ABTS radical and hydroxyl radical scavenging abilities (P < 0.05). The scavenging rate of ABTS radicals (99.8 ± 0.3%) is 5 times higher than that of L-ascorbic acid (19.1 ± 0.2%) at 0.01 mg / mL, and the scavenging rate of hydroxyl radicals (99.8 ± 0.1%) is 6 times higher than that of L-ascorbic acid (14.6 ± 1.7%) at 0.01 mg / mL. Therefore, the γ-PGA fermentation broth produced by Bacillus subtilis MPEB0002969 has strong antioxidant activity.

[0049] Table 1

[0050]

[0051]

[0052] Tyrosinase inhibitory activity of the fermentation broth

[0053] (1) Solution preparation

[0054] 0.5 mg / mL L-tyrosine solution: Weigh 0.005 g of L-tyrosine, dissolve it with PBS buffer solution (pH = 6.8, 0.1 mol / L), assist dissolution with ultrasound, and make up the volume to 10 mL for immediate use. Tyrosinase solution: Weigh 0.005 g of polyphenol oxidase (mushroom) with 1120 u / mg, dissolve it with PBS buffer solution (pH = 6.8, 0.1 mol / L) and make up the volume to 10 mL, store it at -20 °C, and avoid secondary freeze-thaw. Kojic acid solution: The positive control kojic acid is diluted with PBS buffer (pH = 6.8, 0.1 mol / L) into a series of mass concentration gradient solutions of 0.040 g / L, 0.020 g / L, 0.010 g / L, 0.008 g / L, and 0.005 g / L.

[0055] (2) Experimental grouping

[0056] In a 96-well microplate, set up solvent background wells (Ta), solvent reaction wells (Tb), sample background wells (Tc), and sample reaction wells (Td). Among them, Ta is the solvent background group, without adding the substrate L-tyrosine solution and without adding the sample solution; the Tb group is the solvent reaction group, adding the substrate L-tyrosinase solution but without adding the sample solution; the Tc group is the sample background group, without adding the substrate L-tyrosine solution and adding the sample solution; the Td group is the sample reaction group, adding both the substrate L-tyrosine solution and the sample solution. Each group is replicated three times.

[0057] (3) Experimental procedures

[0058] Referring to the reagent addition amounts in Table 2, sequentially add the L-tyrosine solution, sample solution / reagent, and PBS buffer to each well, mix thoroughly, incubate in a constant temperature environment at 37 °C for 10 min, then sequentially add 20 μL of tyrosinase solution to each well, mix at 37 °C for 5 min, and then place it in a microplate reader for measurement at a wavelength of 475 nm.

[0059]

[0060] Where A d is the absorbance of the sample reaction well; Ac is the absorbance of the sample background well; A b is the average absorbance of the solvent reaction wells; Aa is the average absorbance of the solvent background wells.

[0061] Table 2

[0062]

[0063] Measure the kojic acid solution of positive control samples at different concentrations. Using the kojic acid solution concentration as the abscissa and the tyrosinase inhibition rate as the ordinate, plot a curve ( Figure 4 ), and its regression equation is y = 1597.1x + 29.087, with a correlation coefficient R 2 = 0.961. Calculate the sample concentration corresponding to a tyrosinase inhibition rate of 50% according to the regression equation. The IC 50 value of the positive control kojic acid is 0.0131. The γ-PGA fermentation broth produced by Bacillus subtilis MPEB0002969 provided in this application has a certain inhibitory effect on tyrosinase, with an inhibition rate of 70.7 ± 0.9%, indicating that the γ-PGA fermentation broth produced by Bacillus subtilis MPEB0002969 has a good whitening effect.

[0064] Determination of the moisturizing activity of the fermentation broth

[0065] Prepare 50 mL of saturated (NH4)2SO4 and K2CO3 solutions, place them in a desiccator, and let stand overnight. Thus, a saturated (NH4)2SO4 solution with a relative humidity (RH) of 81% and a saturated K2CO3 solution with a relative humidity (RH) of 43% are prepared. Dry the weighing bottle to a constant weight and saturate it in the saturated solutions at the above relative humidities. Add 1.000 g of the sample solution to the weighing bottle, place it in a desiccator humidified with dry silica gel, set the ambient temperature to 25 °C, and weigh the mass change of the sample at the 2nd h, 4th h, 6th h, 8th h, 10th h, and 12th h. Use a 5.0 mg / mL hyaluronic acid (HA) solution as a control.

[0066] The calculation formula for the moisture retention rate is:

[0067] Where Wn is the sample amount (g) at a certain moment; W0 is the initial mass (g) of the sample.

[0068] The moisture retention effect of the γ-PGA fermentation broth produced by Bacillus subtilis MPEB0002969 in an environment with a relative humidity (RH) of 43% ( Figure 5A ) and 81% ( Figure 5B ) is similar to that of the hyaluronic acid (HA) solution (5 mg / mL) (P > 0.05), indicating that the γ-PGA fermentation broth produced by Bacillus subtilis MPEB0002969 has a good moisture retention effect.

[0069] Identification and analysis of the fermentation broth proteome

[0070] Add an appropriate amount of protein to a final concentration of 5 mmol / L dithiothreitol (DTT), incubate at 37 °C for 1 h, and return to room temperature. Add a final concentration of 10 mmol / L iodoacetamide and incubate at room temperature in the dark for 45 min. Dilute the sample 4-fold with 25 mmol / L ammonium bicarbonate, add trypsin according to the protein-to-trypsin ratio of 50:1, incubate overnight at 37 °C; add formic acid to adjust the pH to less than 3 to terminate the enzymatic digestion. Use a C18 desalting column to desalt the sample, activate the desalting column with 100% acetonitrile, equilibrate the column with 0.1% formic acid, load the sample onto the column, then wash the column with 0.1% formic acid to wash away impurities, and finally elute with 70% acetonitrile, collect the flow-through, and lyophilize. Dissolve the lyophilized powder with 10 μL of solution (100% water, 0.1% formic acid), centrifuge at 14000 g for 20 min at 4 °C, take 1 μg of the supernatant for injection, and perform liquid chromatography-mass spectrometry detection (Jiaan Jianda Medical Technology (Shanghai) Co., Ltd.).

[0071] Types of proteins

[0072] The total number of identified peptide segments in the γ-PGA fermentation broth produced by Bacillus subtilis MPEB0002969 in this application is 721, and the total number of identified proteins is 397. Among them, the total number of enzymes is 267, accounting for 57.25%. These enzymes include enzymes such as superoxide dismutase (SOD), alkyl hydroperoxide reductase C (Ahpc), non-ribosomal peptide synthetase, and polyketide synthase (PKS).

[0073] Superoxide dismutase (SOD) has the function of catalyzing the dismutation reaction of superoxide anion radicals and balancing oxygen free radicals in the body. It is a very good antioxidant enzyme for eliminating skin lesions and scavenging superoxide ion radicals. It is one of the important enzymes for organisms to effectively scavenge reactive oxygen species and is called the first line of defense of the organism's antioxidant system, playing important roles such as anti-aging, antioxidant, and sunscreen. Peroxiredoxin can degrade reactive oxygen species (ROS) such as superoxide anion, hydrogen peroxide, and hydroxyl radicals. Alkyl hydroperoxide reductase C (Ahpc) is a kind of peroxiredoxin and has the activity of decomposing organic peroxides. Bacillus subtilis can produce different kinds of natural antibacterial substances during its growth process, and can form lipopeptide antibiotics through the hybridization of non-ribosomal peptides (NRPSs) and polyketides (PKSs) and other pathways. Polyketide synthase (PKS) is a multifunctional complex enzyme with a modular structure and is responsible for catalyzing the biosynthesis of polyketide compounds (PKs). Polyketide compounds have rich physiological activities, including antibacterial activity, antitumor activity, and antioxidant activity, etc.

[0074] Relative molecular weight distribution of proteins

[0075] Statistical analysis was carried out on the relative molecular weights of the 397 identified proteins, and the results ( Figure 6 ) showed that the minimum relative molecular weight of the 397 proteins was 6.1 kDa (M4KR26: 50S ribosomal protein), and the maximum was 606.8 kDa (Q6YK40: Bacillomycin D synthetase B). A total of 370 proteins had relative molecular weights distributed between 0 - 100 kDa (accounting for 93.20%), 14 between 100 - 200 kDa (accounting for 3.53%), and 13 between 200 - 610 kDa (accounting for 3.27%).

[0076] In summary, the fermentation broth prepared from Bacillus subtilis provided by this application has antioxidant, whitening, and moisturizing activities and can be used to prepare cosmetics.

[0077] Those skilled in the art should understand that the above embodiments are merely exemplary embodiments, and various changes, substitutions, and alterations can be made without departing from the spirit and scope of this application.

Claims

1. A Bacillus subtilis, which is deposited in the Guangdong Microbial Culture Collection Center (GDMCC) with the deposit number of GDMCC No: 63439.

2. A fermentation broth prepared from the Bacillus subtilis according to claim 1.

3. Use of the fermentation broth according to claim 2 in the preparation of cosmetics.

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