CN114874921B - Aureobasidium pullulans from Antarctic source and application thereof - Google Patents
Aureobasidium pullulans from Antarctic source and application thereof Download PDFInfo
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- CN114874921B CN114874921B CN202210625055.6A CN202210625055A CN114874921B CN 114874921 B CN114874921 B CN 114874921B CN 202210625055 A CN202210625055 A CN 202210625055A CN 114874921 B CN114874921 B CN 114874921B
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P17/00—Drugs for dermatological disorders
- A61P17/16—Emollients or protectives, e.g. against radiation
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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Abstract
The invention discloses Aureobasidium pullulans from Antarctic sources and application thereof, relates to the field of microorganisms, and provides Aureobasidium pullulans with a preservation number of CGMCC No.21195, wherein the strain has good cold resistance, permeability resistance, UV resistance and other aspects, and a fermentation product of the strain has good activities of resisting oxidation and UV damage, and can have the effects of resisting oxidation, aging, preventing sunburn and repairing after sun exposure when applied to cosmetics.
Description
Technical Field
The invention relates to the field of microorganisms, in particular to Aureobasidium pullulans from south pole sources and application thereof.
Background
One of the causes of skin aging is free radical aging, and the theory suggests that excessive free radicals are a significant cause of biological aging. According to this theory, excessive active oxygen radicals in the body react with unsaturated fatty acids to produce malondialdehyde, which reacts with proteins on cell membranes to produce brown pigment, which precipitates on the skin to form various color spots. The excessive free radicals can also cause collagen fibers and elastic fibers in the skin to crosslink, denature, become brittle and lose elasticity, and when the moisture of the skin is insufficient, the elastic fibers are easily broken, so that skin aging phenomena such as dark lines, fine lines, wrinkles and the like occur. In addition, the ion radiation in the environment and environmental pollutants such as air pollution and chemicals also cause the continuous generation of free radicals in living organisms. For example, ultraviolet light stimulates fibroblasts and granosomes in the dermis of the skin, releasing superoxide anions, which are converted to other more damaging free radicals. Although the human body has an antioxidant defense system capable of maintaining a balance between oxidation and antioxidation to slow down the generation of active oxygen and free radicals, if the human body is excessively exposed to sunlight for a long period of time, the free radicals generated in a large amount in the human body may cause a decrease in the antioxidant defense ability of the skin, causing skin damage such as photoaging, skin wrinkles, skin immunity disorders, etc. Currently, antioxidants for scavenging free radicals in living bodies such as vitamin E and vitamin C are known, and antioxidants of plant origin such as citrus fruit extracts and the like are reported.
Another important cause of skin aging is photoaging, which causes an increase in the activity of skin elastase due to Ultraviolet (UV) exposure. At present, plant-derived anti-aging actives are known as Polygonum tinctorium, sweet pea, etc.
Although anti-aging substances derived from microorganisms having the effects of scavenging free radicals and resisting aging are gaining attention in the skin care industry, microorganisms having a high ability to resist ultraviolet rays, a high polysaccharide content, or a high antioxidant activity are still lacking.
In view of this, the present invention has been made.
Disclosure of Invention
The invention aims to provide aureobasidium pullulans from south pole sources and application thereof.
The invention is realized in the following way:
in a first aspect, an embodiment of the present invention provides a aureobasidium pullulans, which has a preservation number of CGMCC No.21195.
In a second aspect, embodiments of the present invention provide a method for producing Aureobasidium pullulans, comprising culturing Aureobasidium pullulans as described in the previous embodiments.
In a third aspect, an embodiment of the present invention provides a microbial agent, which includes aureobasidium pullulans as described in the previous embodiment.
In a fourth aspect, embodiments of the present invention provide a ferment obtained from the Aureobasidium pullulans fermentation culture described in the previous embodiments.
In a fifth aspect, embodiments of the present invention also provide a product having a specific function, the product comprising at least one of Aureobasidium pullulans as described in the previous embodiments and ferments as described in the previous embodiments; the specific functions include: at least one of moisturizing, anti-ultraviolet and antioxidant activity.
In a sixth aspect, embodiments of the present invention provide the use of Aureobasidium pullulans as described in the previous embodiments or of a ferment as described in the previous embodiments for the preparation of a product having specific functions, including: at least one of moisturizing, anti-ultraviolet and antioxidant activity.
The invention has the following beneficial effects:
aiming at the defects of the existing microorganisms with strong ultraviolet resistance, high polysaccharide content and strong antioxidant activity, the embodiment of the invention provides the aureobasidium pullulans with the preservation number of CGMCC No.21195, the strain has good performance in cold resistance, permeability resistance, UV resistance and other aspects, and the fermentation product has good skin care effect of resisting UV damage.
Drawings
In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings that are needed in the embodiments will be briefly described below, it being understood that the following drawings only illustrate some embodiments of the present invention and therefore should not be considered as limiting the scope, and other related drawings may be obtained according to these drawings without inventive effort for a person skilled in the art.
FIG. 1 shows the strain morphology of Aureobasidium pullulans OSM-NJ01-Gly-1-01 on PDA medium and under microscope;
FIG. 2 shows strain viability in an ultraviolet radiation environment;
FIG. 3 shows strain viability in a low temperature environment;
FIG. 4 is a graph of strain growth in a hypertonic environment;
FIG. 5 shows crude polysaccharide from different strains compared to the quality of the bacteria.
Detailed Description
In order to make the objects, technical solutions and advantages of the embodiments of the present invention more clear, the technical solutions of the embodiments of the present invention will be clearly and completely described below. The specific conditions are not noted in the examples and are carried out according to conventional conditions or conditions recommended by the manufacturer. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
The embodiment of the invention provides Aureobasidium pullulans, which has a preservation number of CGMCC No.21195.
And selecting Aureobasidium pullulans separated from Antarctic soil to obtain Aureobasidium pullulans CGMCC No.21195. The Aureobasidium pullulans CGMCC No.21195 grows well in a PDA liquid culture medium and has the characteristics of strong ultraviolet resistance, high polysaccharide content and strong antioxidant activity.
Biological material preservation information: the Aureobasidium pullulans (Aureobasidium pullulans) of the invention are preserved in China general microbiological culture collection center (China General Microbiological Culture Collection Center, CGMCC) in 11/16/2020, and have the preservation addresses: the microbiological institute of China, national academy of sciences, no. 3, beijing, chaoyang district, north Chen, west way, and post code: 100101, the preservation number is CGMCC No.21195, the culture name is Aureobasidium pullulans OSM-NJ01-Gly-1-01, and the classification name is Aureobasidium pullulans OSM-NJ01-Gly-1-01.
In some embodiments, the morphological features of the aureobasidium pullulans include: culturing in malt juice liquid culture medium at 24-26 deg.c for 2-4 days (preferably 3 days) to form precipitate in the form of at least one of oval, elliptic and oblong with cell size of (3.0-5.7) × (4.7-10.0) microns; culturing wort agar inclined plane at 24-26 ℃, wherein the colony is cheese-shaped, milk white, wet and smooth in surface and glossy, and the colony turns black after two weeks; cultured in corn meal agar Dalmau plates, no pseudohyphae were produced.
In some embodiments, the physiological and biochemical characteristics of the aureobasidium pullulans include: the carbon source which can be utilized by the aureobasidium pullulans comprises at least one of glucose, sucrose, maltose, galactose, raffinose and L-sorbose; non-fermentation of at least one of glucose, galactose, sucrose, lactose, maltose and raffinose; the different carbon sources include at least one of L-rhamnose, glycerol, galactitol, lactose, D-sorbitol, and ribitol.
The invention also provides a production method of the aureobasidium pullulans, which comprises the step of culturing the aureobasidium pullulans in any previous embodiment.
Wherein the culture medium is a culture medium conventional in the art, and can grow the Aureobasidium pullulans CGMCC No.21195, preferably a PDA liquid culture medium, a YPD culture medium or a GMMY culture medium.
The PDA liquid medium is a PDA liquid medium conventional in the art, and preferably comprises 5g/L potato starch, 15g/L glucose and 10g/L peptone. The temperature of the culture is the temperature conventional in the art, and the Aureobasidium pullulans CGMCC No.21195 can be grown, preferably 15-35 ℃, and more preferably 28 ℃. The time for the cultivation is a time conventional in the art, preferably 2 to 7 days, more preferably 2 days. The strain morphology of Aureobasidium pullulans OSM-NJ01-Gly-1-01 on PDA medium and the morphology under microscope are shown in FIG. 1.
Preferably, the method further comprises the step of seed culture by using a seed culture medium before the culture. The seed culture is conventional in the art. The seed culture medium is a seed culture medium conventional in the art, preferably PDA liquid culture medium. The time for the seed culture is a time conventional in the art, preferably 48 to 60 hours, more preferably 48 hours. The temperature of the seed culture is a temperature conventional in the art, preferably 28 to 30 ℃. The seed culture is inoculated in an amount conventional in the art, preferably 5%, the percentages being by volume.
The embodiment of the invention also provides a microbial agent, which comprises the aureobasidium pullulans according to any of the previous embodiments.
The embodiment of the invention also provides a ferment obtained by fermenting and culturing the aureobasidium pullulans in any embodiment.
In some embodiments, the fermenting comprises: inoculating the seed solution of Aureobasidium pullulans into a liquid culture medium containing pearl powder for culture to obtain a pearl fermentation product.
In some embodiments, the seed fluid is inoculated in an amount of: the volume fraction may be 1% -5%, specifically, may be any one or any range between two of 1%, 2%, 3%, 4% and 5%.
In some embodiments, the mass fraction of the pearl powder in the liquid culture medium is 0.01% -3%, and specifically may be any one or any range between two of 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2% and 3%.
In some embodiments, after inoculation, the culture is performed at 28-30℃for 3-7 days to obtain a culture broth. The culture time can be specifically any one or any two of 3 days, 4 days, 5 days, 6 days and 7 days; the specific temperature of the culture may be in the range of 28℃or 29℃or 30℃or any range between any two.
In some embodiments, the fermenting further comprises: centrifuging the culture solution, collecting supernatant, filtering, and sterilizing to obtain the pearl fermentation product.
In some embodiments, the centrifugation conditions are: 4-10 ℃, 3000-10000 rpm, 1-10 min.
Embodiments of the present invention provide a product having a specific function, the product comprising at least one of Aureobasidium pullulans as described in the previous embodiments and fermentates as described in the previous embodiments; the specific functions include: at least one of moisturizing, anti-ultraviolet and antioxidant activity.
In some embodiments, the type of product is any one of a skin external agent, a cosmetic, a health product, and a pharmaceutical. The external preparation for skin may be a general concept of all components commonly used outside the skin, and may be, for example, cosmetics or medicines. The cosmetic can be basic cosmetic, facial make-up cosmetic, eye cosmetic, head care product, body cosmetic, etc., and the dosage form is not particularly limited, and can be reasonably selected according to different purposes.
In some embodiments, the product is in the form of any one of a gas, a solid, and a liquid. Such product forms include, but are not limited to, aerosol sprays, creams, emulsions, solids, liquids, dispersions, foams, gels, lotions, mousses, ointments, powders, patches, pomades, hand pump sprays, sticks, masks and wet wipes. The skin external preparation of the present invention may be a cosmetic, dermatological or pharmaceutical topical application product, and the preparation method thereof may be a conventional preparation method in the art, as is well known.
In some embodiments, the product may further comprise a carrier, which is a pharmaceutically acceptable carrier conventional in the art, preferably one or more of a preservative, a fragrance, a hydrophilic active agent, and a lipophilic active agent. The pharmaceutically acceptable carrier is present in an amount conventional in the art.
In some embodiments, the skin external preparation may further comprise one or more of the following ingredients: a lipid-forming agent, a dispersant, a viscosity modifier, a solvent, a thickener, a whitening agent, a skin protectant, a chelating agent, a spot-removing agent, a skin feel modifier, an emollient, a cleanser, a moisturizer, a sunscreen agent, a pH modifier, a suspending agent, a solubilizer, a buffer, a reducing agent, an emulsifier, an emulsion stabilizer, a photostabilizer, a cooling agent, a surface modifier, an antioxidant, a film former, or any combination thereof. The amounts of these ingredients may be those conventional in the art.
In some embodiments, the specific function further comprises a high yield polysaccharide.
On the basis of the common general knowledge in the field, the above preferred or optional conditions can be arbitrarily combined to obtain the preferred examples of the invention.
An embodiment of the present invention provides the use of Aureobasidium pullulans as described in any of the preceding embodiments or of a ferment as described in any of the preceding embodiments for the preparation of a product having specific functions, including: at least one of anti-ultraviolet and anti-oxidation activity.
It will be appreciated that the specific reference to the product in the application may be the same as that described in any of the foregoing embodiments, and will not be repeated.
The features and capabilities of the present invention are described in further detail below in connection with the examples.
Example 1
27 strains were obtained by separation from a soil sample in the intertidal zone (sample number: S02; collection site: south Philippines peninsula region; longitude 058° 57.8225'; latitude 62° 13.0705'; sampling depth: 0 m; weight: 100g; sampling time: 10 th 1 st 2008), one of the strains NJ1P3 (NJ 1P3 is the strain number, the same applies hereinafter) was identified as Aureobasidium pullulans (Aureobasidium pullulans) after separation, and the strain was preserved in China general microbiological culture collection center (China General Microbiological Culture Collection Center, CGMCC) at 11 th month 16 in 2020, the preservation address: the institute of science and microbiology, post code: 100101, the preservation number is CGMCC No.21195, the culture name is Aureobasidium pullulans OSM-NJ01-Gly-1-01, and the classification name is Aureobasidium pullulans OSM-NJ01-Gly-1-01.
The control strain is Aureobasidium pullulans isolated from soil collected from Yunnan, and the strain number is: YN128-1 isolated from Aureobasidium pullulans obtained from Gansu Compositae leaves, strain number: 23-1, which belongs to the extreme climate environment with high altitude, strong ultraviolet radiation and large temperature difference.
(1) Survival test in Ultraviolet (UV) environments
Strain activation: the strain stored at 4℃was transferred to a new slant in an ultra clean bench and cultured at 28℃for two days. Strain culture: colonies on the activated slants were scraped into 50mL YPD medium (10 g/L yeast extract, 20g/L glucose, 20g/L peptone) and cultured on a shaker for 48 hours (28 ℃, 150 rpm).
And (3) obtaining thalli: 10mL of the culture broth was centrifuged (8000 rpm, 10 min) for two days, the supernatant was discarded, 10mL of sterile water was added, and the cells were resuspended.
Ultraviolet irradiation: the obtained thallus suspension is subjected to ultraviolet irradiation for 0s and 20min in a biosafety cabinet (pre-ultraviolet preheating for 10 min), and the thallus suspension is fully mixed in a flat plate after the ultraviolet irradiation and diluted for 10min -3 After that, 20. Mu.L was counted for PDA coating, three replicates each, incubated at 28℃for 48 hours in the absence of light.
Survival rate calculation: and counting the plates after the plate culture is finished, and calculating according to the following formula to obtain the survival rate.
X=1- (a-B)/a×100%; wherein, X: survival rate; a: colony number of 0s plate growth; b: colony count was grown on plates for 20 min.
After 20 minutes of ultraviolet treatment of bacterial suspensions of Aureobasidium pullulans from Antarctica, yunnan and Gansu, the survival rate of NJ1P3 is higher compared with that of control strains YN128-1 and 23-1, indicating that the NJ1P3 has stronger resistance to strong ultraviolet radiation. The results are shown in FIG. 2.
(2) Low temperature survival rate test
Strain activation: strains stored at 4℃were inoculated on a new PDA slope in an ultra clean bench and incubated at 28℃for two days.
Strain culture: colonies on the activated slants were scraped into 50mL YPD medium (10 g/L yeast extract, 20g/L glucose, 20g/L peptone) and cultured on a shaker for 48 hours (28 ℃, 150 rpm).
And (3) obtaining thalli: 10mL of the culture was centrifuged (8000 rpm, 10 min), the supernatant was discarded, 10mL of sterile water was added, and the cells were resuspended.
And (3) low-temperature treatment: diluting 1mL of bacterial liquid to 10 -3 The plates were coated. Placing the bacterial suspension in a refrigerator at-20deg.C for one week, and diluting for 10 times after low temperature treatment -3 mu.L of the cells were PDA-coated, three replicates were made for each strain, and incubated at 28℃for 48 hours.
Survival rate calculation: after the plates had grown to colonies, counts were made and calculated according to the following formula.
X=1- (a-B)/a×100%; wherein, X: survival rate; a: the number of colonies grown on the plate prior to the low temperature treatment; b: number of colonies grown on plates after-20 ℃.
The two strains were placed in a refrigerator at-20℃and after one week, PDA plate coating was performed, colonies growing on the plates were counted, and survival rates were calculated. The result shows that the survival rate of the NJ1P3 under the condition of-20 ℃ is higher than that of the control strains YN128-1 and 23-1, and the NJ1P3 has stronger capability of resisting the low-temperature environment. As shown in fig. 3.
(3) High permeability survival test
Strain activation: strains stored at 4℃were inoculated on a new PDA slope in an ultra clean bench and incubated at 28℃for two days.
Strain culture: colonies on the activated slants were scraped into 50mL YPD medium (10 g/L yeast extract, 20g/L glucose, 20g/L peptone) and cultured on a shaker for 48 hours (28 ℃, 150 rpm).
And (3) obtaining thalli: 10mL of the culture was centrifuged (8000 rpm, 10 min), the supernatant was discarded, 10mL of sterile water was added, and the cells were resuspended.
Culturing in a hypertonic environment: diluting the bacterial suspension 10 -3 Preparing 15% NaCl mother liquor, using YNB medium (67 g/L Bacto Yeast Nitrogen Base, 50g/L glucose) diluted NaCl stock to 0%, 5% and 11% with osmotic pressures near 0mOsm, 2000mOsm and 3142mOsm, respectively (as a comparison, the cell osmotic pressure was about 300mOsm, whereas the normal seawater osmotic pressure was 1000 mOsm). 180 mu L of YNB culture medium and 20 mu L of bacterial suspension are added into a 96-well plate, an enzyme-labeled instrument (Texan Spark) is placed for culture, OD600 is measured every other hour, a growth curve is drawn until reaching a stationary phase, and the growth condition of the strain in different osmotic pressure environments is measured.
Studies have shown that with osmotic pressures approaching 0mOsm, NJ1P3 grew better than YN128-1 and 23-1 and reached equilibrium at around 8 days. With the increase of osmotic pressure, the growth of three strains gradually weakens, but the growth condition of NJ1P3 is always higher than YN128-1 and YN 23-1, and the time for reaching the equilibrium period is also prolonged, which shows that the hypertonic environment can influence the growth of the strains, but the NJ1P3 has stronger resistance to the hypertonic environment. The results are shown in FIG. 4, with the environmental osmotic pressures approaching 0mOsm, 2000mOsm and 3142mOsm from top to bottom.
Example 2
Morphological and cultural characteristics of strains.
The NJ1P3 strain was cultured in malt liquor liquid medium at 25℃for three days, and the cell size (3.0 to 5.7) × (4.7 to 10.0) μm, oval, elliptical, oblong form a precipitate. The wort agar was incubated at 25℃with cheese-like colonies, milky white, moist and smooth surface, glossy and after two weeks the colonies turned black. Corn meal agar Dalmau plates were cultured without producing false hyphae. The determined NJ1P3 strain D1/D2 sequence (shown as SEQ ID No. 1) is subjected to 26S rDNA sequence Blast comparison with NCBI database, has homology of more than 99% with Aureobasidium pullulans strain, has very similar genetic relationship, and is determined to be Aureobasidium pullulans by combining with morphological characteristics of the strain.
The sequence shown in SEQ ID No.1 is as follows:
gcggacctcagtcccggctggccgtattacacactgggctataacacctccaccgaagtggaggccacatt cccagagcctttatccggccgccgaaactgatgctggcctgtccaaactgagtaaaccggtcagaagaccggcc gaacagtttaaacaagtctgattgcaagcgcttccctttcaacaatttcacgtgctttttaactctctttccaaagtgctttt catctttcgatcactctacttgtgcgctatcggtctctcgccaatatttagctttagaagaaatttacctcccatttagagc tgcattcccaaacaactcgactcgtcgaaggagctttacataggttaacatttccagtcacatacgggattctcaccct ctatgacgtcctgttccaaggaacttagactggcgttttacccaaagcatcctctacaaattacaatgcgaacccgaa ggctagctttcaaatttgagctgttgccgcttcactcgccgttactagggcaatccctgttggtttcttttcctccgcttat tgatatgcttaagttcagcgggtatccctacctgatccgaggtcaacctagaaaaataaaggtttcaatcggcagagt tcctctcctttgacagacgttcgaataaattctactacgcctaaagccggagtggcctcgccgaggtctttaaggcgc gcccaactaaggacg。
example 3
Physiological and biochemical characteristics of the strain.
The strain was subjected to physiological and biochemical characterization by reference to Zu Refu, hu Baolong, zhou Deqing, course of microbiology experiment (1993 of the university of double denier press).
As a result, it was found that glucose, sucrose, maltose, galactose, raffinose, L-sorbose and the like as carbon sources can be utilized for the NJ1P3 strain; non-fermentation glucose, galactose, sucrose, lactose, maltose, raffinose; different carbon sources L-rhamnose, glycerol, galactitol, lactose, D-sorbitol and ribitol.
The NJ1P3 strain is preserved in China general microbiological culture collection center (China General Microbiological Culture Collection Center, CGMCC) at 11 and 16 months in 2020, and the preservation address is: the institute of science and microbiology, post code: 100101, the preservation number is CGMCC No.21195, the culture name is Aureobasidium pullulans OSM-NJ01-Gly-1-01, and the classification name is Aureobasidium pullulans OSM-NJ01-Gly-1-01.
Example 4
Culturing the strain.
Inoculating the NJ1P3 strain into a PDA liquid culture medium, and culturing at 28 ℃ for 48 hours to obtain seed liquid;
5% (v/v) seed solution was inoculated into PDA liquid medium, shaking-cultured at 28℃and 200rpm for 2 days.
Example 5
And (3) preparing the pearl fermentation product.
Respectively inoculating the strains YN128-1 and NJ1P3 into a PDA liquid culture medium, and culturing at 28 ℃ for 48 hours to obtain seed liquid;
inoculating 5% (v/v) seed solution into PDA liquid culture medium containing 1% (w/w) pearl powder, shake culturing at 28deg.C at 200rpm for 5 days to obtain culture solution;
centrifuging the culture solution at 8deg.C and 8000rpm for 5 min, collecting supernatant, and filtering for sterilizing to obtain pearl fermentation product.
Example 6
And detecting the polysaccharide content and the molecular weight of the pearl fermentation product.
Fermenting and culturing the two strains to obtain crude polysaccharide, drying thalli and the crude polysaccharide, and weighing. The result shows that the quality of the strain NJ1P3 and the quality of crude polysaccharide are higher than those of YN128-1, and the growth activity of the strain NJ1P3 is higher than that of YN128-1, and the polysaccharide content in the fermentation broth is higher than that of the control strain YN128-1. The results are shown in FIG. 5.
After deproteinizing the crude polysaccharide obtained by fermentation, the molecular weight is measured by a field flow separation technique, and the molecular weight distribution and average molecular weight of the polysaccharide produced by NJ1P3 fermentation are measured. The results are shown in Table 1.
TABLE 1 molecular weight ranges of polysaccharide in fermentation broths
Example 7
And (5) detecting the antioxidant activity of the pearl fermentation product.
Taking Vc as a positive control, taking 1.5mg of Vc, uniformly mixing and dissolving with 1mL of distilled water to obtain a Vc stock solution of 1.5mg/mL, taking 100 mu L of the Vc stock solution, diluting to 30mL to obtain a Vc solution with the concentration of 0.005mg/mL, and obtaining the final concentration of the Vc solution in the reaction of 2.5ppm.
A fermentation sample was prepared as in example 5, and its antioxidant activity for scavenging free radicals was measured by DPPH method.
Diluting each 1mL of test sample with absolute ethyl alcohol for 2.5 times, centrifuging at 3800rpm for 12min, taking supernatant, diluting each 2mL of the supernatant with ultrapure water to 4mL, and obtaining a sample solution with the final concentration of 10%;
2mL of the sample solution was taken and put into a test tube, and 2X 10 was added -4 2mL of mol/L DPPH ethanol solution, uniformly mixing, carrying out light-shielding reaction for 30min at 25 ℃, and measuring absorbance A517 at 517 nm; the clearance was calculated according to the following formula, and the greater the clearance, the stronger the antioxidant capacity, and the results are shown in the following table.
Clearance I (%) = [1- (T-T0)/(C-C0) ] ×100%
Wherein: t: absorbance of 2mL sample solution plus 2mL DPPH solution;
t0: absorbance of 2mL of the sample solution plus 2mL of absolute ethanol solution;
c0: absorbance of 2mL water plus 2mL absolute ethanol;
c: absorbance of 2mL DPPH solution plus 2mL ultrapure water.
Table 2 free radical clearance of different species fermentation broths (%, n=2)
| Sample of | Vc | YN128-1 pearl ferment | NJ1P3 pearl fermentation product |
| Clearance rate of | 23.75 | 22.89 | 56.07 |
As shown in the table above, the free radical clearance of YN128 pearl ferment is far lower than that of NJ1P3 pearl ferment, and Antarctic bacteria fermentation liquor has stronger antioxidant activity.
Example 8
And testing the activity of the pearl ferment against ultraviolet injury.
A fermentation sample was prepared as in example 5.
The measuring steps are as follows:
inoculating 5000 HaCAT cells in 96-well plate, preparing sample according to the following table when cell fusion degree is about 80%, treating cells, and placing CO 2 The incubator continues to incubate. After 24h, cells were irradiated with UVA or UVB and the non-irradiated group was set as a blank. Immediately sucking the culture medium after UVA irradiation, adding 0.5mg/mL MTT, adding DMSO after 3 hours to extract formazan, and detecting absorbance at 560nm by using an enzyme-labeling instrument; after UVB irradiation, CO is put in 2 The incubator continues to incubate. After 24h, the medium was aspirated, 0.5mg/mL MTT was added, and after 3h, formazan was extracted with DMSO and absorbance at 560nm was measured using a microplate reader.
The results are shown in tables 3 and 4, which demonstrate that the pearl fermentate helps the skin to resist UVA and UVB damage and the effect is similar to the positive control 0.005% Vc effect.
TABLE 3 Activity test of pearl fermentate against UVA injury
TABLE 4 Activity test of pearl fermentate against UVB injury
Example 9
And (5) testing the moisturizing activity of the pearl fermentation product.
A fermentation sample was prepared as in example 5 and tested for moisture retention of a 6% sodium hyaluronate solution and a pearl ferment (5.95% solids) by in vitro weighing.
Accurately weighing 2.5g of sample and sodium hyaluronate solution, placing in a dryer (placing silica gel desiccant), and weighing the mass of the sample after 2, 4, 6, 8 and 24 hours at regular time. The results are shown in Table 5.
TABLE 5 moisture retention of pearl fermentate
| Time (h) | HA moisture retention (%) | Moisture retention of pearl ferment (%) |
| 2 | 76.25 | 78.57 |
| 4 | 73.73 | 76.19 |
| 6 | 71.00 | 73.53 |
| 8 | 68.66 | 70.80 |
| 24 | 49.48 | 52.48 |
As can be seen from Table 5, the sodium hyaluronate solution with a higher moisture retention rate than 6% for the pearl fermentate at each time, demonstrated better moisture retention of the Antarctic bacteria broth.
Example 10
Essence containing pearl ferment is provided.
The essence of example 10 was prepared according to the preparation method of the lotion and the composition of table 6, which are conventional in the art.
TABLE 6 Table of essential components
Example 11
Testing stability of essence containing pearl ferment.
The essence prepared in example 10 was subjected to stability tests of heat resistance, cold resistance, alternation, light irradiation, etc. Wherein, the heat-resistant test conditions are: 45+/-1 ℃, keeping constant temperature and avoiding light for 7 days;
the cold resistance test conditions are as follows: -20+/-1 ℃, keeping the temperature away from light, and 7 days;
the alternating test conditions were: transferring from-20deg.C constant temperature for 3.5 days to 45deg.C constant temperature for 3.5 days, and keeping away from light;
the illumination test conditions are as follows: illumination constant temperature and humidity test box (1.47 x 10) 6 Lux·hr)，7d。
The test results are shown in the following table.
Table 7 stability test
The results show that the cosmetics prepared from the pearl fermentation product have excellent stability.
The above description is only of the preferred embodiments of the present invention and is not intended to limit the present invention, but various modifications and variations can be made to the present invention by those skilled in the art. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Sequence listing
<110> Oshi Diffuse biological Co.Ltd
ZHEJIANG OSM GROUP Co.,Ltd.
<120> Aureobasidium pullulans of Antarctic origin and use thereof
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 720
<212> DNA
<213> Aureobasidium pullulans strain
<400> 1
gcggacctca gtcccggctg gccgtattac acactgggct ataacacctc caccgaagtg 60
gaggccacat tcccagagcc tttatccggc cgccgaaact gatgctggcc tgtccaaact 120
gagtaaaccg gtcagaagac cggccgaaca gtttaaacaa gtctgattgc aagcgcttcc 180
ctttcaacaa tttcacgtgc tttttaactc tctttccaaa gtgcttttca tctttcgatc 240
actctacttg tgcgctatcg gtctctcgcc aatatttagc tttagaagaa atttacctcc 300
catttagagc tgcattccca aacaactcga ctcgtcgaag gagctttaca taggttaaca 360
tttccagtca catacgggat tctcaccctc tatgacgtcc tgttccaagg aacttagact 420
ggcgttttac ccaaagcatc ctctacaaat tacaatgcga acccgaaggc tagctttcaa 480
atttgagctg ttgccgcttc actcgccgtt actagggcaa tccctgttgg tttcttttcc 540
tccgcttatt gatatgctta agttcagcgg gtatccctac ctgatccgag gtcaacctag 600
aaaaataaag gtttcaatcg gcagagttcc tctcctttga cagacgttcg aataaattct 660
actacgccta aagccggagt ggcctcgccg aggtctttaa ggcgcgccca actaaggacg 720
Claims (10)
1. Aureobasidium pullulans is characterized in that the preservation number is CGMCC No.21195.
2. A production method of aureobasidium pullulans, characterized in that it comprises culturing aureobasidium pullulans as defined in claim 1.
3. The method according to claim 2, wherein the culture medium used for the culture is at least one selected from the group consisting of PDA liquid medium, YPD medium and GMMY medium.
4. A production method according to claim 2 or 3, wherein the conditions of the cultivation are; 15-35 ℃ for 2-7 days.
5. A microbial agent comprising the aureobasidium pullulans as defined in claim 1.
6. A ferment obtained by fermentation culture of aureobasidium pullulans as defined in claim 1;
the fermentation comprises: inoculating the seed solution of Aureobasidium pullulans into a liquid culture medium containing pearl powder for culture to obtain a pearl fermentation product;
the inoculation amount of the seed liquid is as follows: 1% -5% of volume fraction;
the pearl powder comprises at least one of submicron pearl powder, water-soluble pearl powder and hydrolyzed pearl lyophilized powder;
the mass fraction of the pearl powder in the liquid culture medium is 1% -3%;
after inoculation, culturing for 3-7 days at the temperature of 28-30 ℃ to obtain a culture solution;
the fermentation further comprises: centrifuging the culture solution, taking supernatant, filtering and sterilizing to obtain a pearl fermentation product;
the centrifugation conditions are as follows: 4-10deg.C, 3000-10000 rpm, 1-10 min.
7. A product with specific functions, characterized in that it comprises a ferment according to claim 6; the specific functions are as follows: at least one of anti-ultraviolet, antioxidant activity and moisture retention;
the product is any one of external preparation for skin and cosmetic.
8. The product of claim 7, wherein the product is in any one of a gaseous state, a solid state, and a liquid state.
9. Use of a ferment according to claim 6 for the preparation of a product with a specific function, wherein the specific function is: at least one of moisturizing, anti-uv and anti-oxidant activity;
the product is any one of external preparation for skin and cosmetic.
10. The use according to claim 9, wherein the product is in the form of any one of a gas, a solid and a liquid.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102994395A (en) * | 2012-10-15 | 2013-03-27 | 苏州大学 | Aureobasidium pullulans and application thereof |
| CN107485598A (en) * | 2017-08-30 | 2017-12-19 | 广州市科能化妆品科研有限公司 | A kind of pearl toner of thickening certainly and preparation method thereof |
| KR20190104956A (en) * | 2019-08-27 | 2019-09-11 | 주식회사 아리바이오 | Aureobasidium pullulans for highly producing melanin free beta-glucan and method for culturing |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN102994395A (en) * | 2012-10-15 | 2013-03-27 | 苏州大学 | Aureobasidium pullulans and application thereof |
| CN107485598A (en) * | 2017-08-30 | 2017-12-19 | 广州市科能化妆品科研有限公司 | A kind of pearl toner of thickening certainly and preparation method thereof |
| KR20190104956A (en) * | 2019-08-27 | 2019-09-11 | 주식회사 아리바이오 | Aureobasidium pullulans for highly producing melanin free beta-glucan and method for culturing |
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