CN110964673A

CN110964673A - Pediococcus pentosaceus with high oxidation resistance and whitening effect and application thereof

Info

Publication number: CN110964673A
Application number: CN201911392516.4A
Authority: CN
Inventors: 陈臣; 刘洋; 于海燕; 田怀香; 袁佳杰; 杨茹梦; 郭伟; 陈霜
Original assignee: Shanghai Institute of Technology
Current assignee: Shanghai Institute of Technology
Priority date: 2019-12-30
Filing date: 2019-12-30
Publication date: 2020-04-07
Anticipated expiration: 2039-12-30
Also published as: CN110964673B

Abstract

The invention provides Pediococcus pentosaceus (Pediococcus pentosaceus) with a preservation number of CCTCC M2019323. Tests prove that the DPPP free radical clearance rate of the extracellular supernatant of the pediococcus pentosaceus CCTCC M2019323 is 81.89%, and the tyrosinase inhibition rate is 64.82%. The extracellular supernatant of the pediococcus pentosaceus CCTCC M2019323 is fermented by taking medicinal plants as substrates, and the fermentation liquid has a good effect of inhibiting propionibacterium acnes. Meanwhile, the medicinal plant fermentation liquid can also be applied to the preparation of cosmetics, and the prepared plant fermentation liquid product containing pediococcus pentosaceus CCTCC M2019323 can effectively remove free radicals, promote skin renewal and reduce melanin synthesis, and has a very good application prospect.

Description

Pediococcus pentosaceus with high oxidation resistance and whitening effect and application thereof

Technical Field

The invention relates to screening and identification of lactic acid bacteria with high oxidation resistance and whitening effect and application of the lactic acid bacteria in cosmetics, belonging to the technical field of biology.

Background

Skin care products are always popular for consumption, and the desire of consumers to care skin and seek essence promotes the vigorous development of the market. From the market trend in recent years, the natural plant cosmetics are increased more than the synthetic cosmetics in the global scope, China is the country where the natural plant cosmetics are increased most rapidly, international brands begin to intervene in the biological field besides foreign popular plant components, and the current SK-II magical water has good whitening effect due to the abundant yeast fermentation liquor. Microbial fermentation technology is a process of producing our desired product by using the metabolism of microorganisms under specific circumstances. Modern fermentation technology has wide prospect in skin care, can enrich effective components after fermentation, improve the efficacy of products, reduce toxic and side effects and the like. Which is impossible to do by the traditional solvent extraction method at present. Compared with other extraction technologies, the fermentation technology has the advantages of no pollution, low energy consumption, simple and convenient operation and the like. In addition, partial enzyme generated in the fermentation process of the microorganism plays roles of cleaning the skin, preventing whelk and resisting aging to a certain extent.

Recently, due to the rapid development of probiotic science, the efficacy of probiotics is from the original digestion promoting function to the prevention of allergy, cancer and infection, the improvement of cholesterol metabolism and the like in the medical field. Not only the health care function is achieved by improving the balance of intestinal flora, but also the direct effect of the probiotics on human or animals and plants is very obvious. The probiotics can be used in food, and the fermentation product thereof is widely applied in cosmetics. Lactic acid bacteria are a type of microorganism which is widely applied in industry, and have irreplaceable effect in national economy and better economic benefit. The method relates to various aspects in the industries of light industry, food, medicine, feed and the like. The lactobacillus is widely distributed in nature, the fermentation process of foods such as fermented soybean paste, steamed bread, yoghourt and the like is participated by the lactobacillus, and certain strains of lactobacillus have physiological functions of improving immunity, reducing blood pressure, resisting oxidation, improving intestinal flora and the like. Therefore, as a kind of effective natural antioxidant, lactobacillus having antioxidant function has been researched, and its antioxidant activity has become a hot spot. In recent years, the research on the whitening and skin care of the lactic acid bacteria metabolite is increasing, and the lactic acid bacteria metabolite has a good prospect.

Because the development of probiotic fermentation and skin care in the domestic market is not strong at present, natural skin care products still have the trend of being plant extracts, and the natural skin care products taking the plant extracts as the main components can not meet the consumption requirements of people gradually along with the higher and higher requirements of people on the skin care products. Nowadays, the biotechnology of pure natural cosmetics gradually becomes a new direction of the development of the cosmetic industry, the cosmetic industry formally enters the 4.0 era, and the microorganism, a technology which is not new, begins to enter the visual field of consumers by using 'new face' of the cosmetic industry.

Disclosure of Invention

The invention aims to: aiming at the market demand of the pure natural cosmetics in biotechnology at present, the lactic acid bacteria with high oxidation resistance and whitening effect and the application thereof are provided.

In order to achieve the above object, the present invention provides Pediococcus pentosaceus having high oxidation resistance and whitening effect, wherein the Pediococcus pentosaceus has a biological name of Pediococcus pentosaceus, is deposited in the chinese type culture collection with a preservation number of CCTCC M2019323.

The invention also provides a preparation method of the plant lactobacillus fermentation liquid, which is characterized in that the pediococcus pentosaceus is inoculated into a fermentation raw material liquid containing plants for fermentation, and the supernatant is obtained by sterilization and centrifugation.

Preferably, the fermentation raw material liquid comprises plants, water and glucose, wherein the mass ratio of the plants to the water is (1-3): 100, and the addition amount of the glucose is 4% -6% of the sum of the masses of the plants and the water.

Preferably, the plant is a plant powder.

More preferably, the plant is a medicinal plant, preferably, platycodon grandiflorum, dandelion, mint, ginseng, angelica sinensis, ginkgo biloba, hemerocallis fulva, lavender, and the like, preferably hemerocallis and lavender, and more preferably hemerocallis and lavender flower.

Preferably, the preparation method of the lactobacillus plantarum fermentation broth specifically comprises the following steps: inoculating 10 times of culture solution of Pediococcus pentosaceus⁶～10⁸And (3) inoculating the CFU/mL into the fermentation raw material liquid, culturing in a constant-temperature incubator at 37 ℃ for 24-48h, sterilizing, centrifuging and taking supernatant, wherein the obtained fermentation supernatant is the plant lactobacillus fermentation liquid with Pediococcus pentosaceus (Pediococcus pentosaceus).

More preferably, the preparation of the seed culture broth comprises: taking a strain of Pediococcus pentosaceus (Pediococcus pentosaceus) which is preserved by a freeze drying tube and dissolved by sterile water by using an inoculating loop, drawing a line on an MRS agar culture medium plate in a loop manner, and culturing for 24-48h in an incubator at 37 ℃ until a single colony grows out, thereby obtaining a plate activated Pediococcus pentosaceus (Pediococcus pentosaceus) strain; inoculating one loop of strain of Pediococcus pentosaceus (Pediococcus pentosaceus) activated by an inoculating loop taking plate into an MRS broth culture medium, and placing the culture medium in an incubator at 37 ℃ for constant-temperature culture for 24-48h to obtain a strain culture solution.

Preferably, the sterilization and centrifugation conditions are sterilization at 115 ℃ for 20min and centrifugation at 4500-5500r/min for 15 min.

The invention also provides the plant lactobacillus fermentation liquor prepared by the method.

The invention also provides application of the pediococcus pentosaceus with high oxidation resistance and whitening effect in preparation of cosmetics.

The invention also provides application of the plant lactobacillus fermentation liquor in preparing cosmetics.

Preferably, said application means adding the lactobacillus plantarum fermentation broth to the cosmetic formulation.

More preferably, the addition amount of the lactobacillus plantarum fermentation broth is 0.2-0.4% (w/w).

Preferably, the cosmetic is an antioxidant cosmetic, a whitening cosmetic, and an anti-acne, anti-acne cosmetic.

Compared with the prior art, the invention has the beneficial effects that:

1) experiments prove that the pediococcus pentosaceus has high DPPH free radical clearance rate and tyrosinase inhibition rate compared with other lactic acid bacteria. Wherein the DPPH free radical clearance rate of the substance in the pediococcus pentosaceus is 56.51%, and the tyrosinase inhibition rate is 28.13%; the DPPH free radical clearance rate of the pediococcus pentosaceus metabolite is 81.89%, and the tyrosinase inhibition rate is 64.82%.

2) The pediococcus pentosaceus can take plants as fermentation substrates, can promote DPPH clearance rate and tyrosinase inhibition rate of plant fermentation liquid, and has better activity compared with DPPH free radical clearance rate of ascorbic acid and tyrosinase inhibition rate of arbutin respectively.

3) The pediococcus pentosaceus and the plant lactic acid bacteria fermentation liquor prepared by using the pediococcus pentosaceus have obvious inhibition effect on acne pathogenic bacteria (propionibacterium acnes).

4) The cosmetic prepared from the plant lactobacillus fermentation liquor prepared from pediococcus pentosaceus has good whitening and wrinkle-removing effects, and the whitening and skin-lubricating effects are obviously superior to those of similar products sold in the market.

The Pediococcus pentosaceus (Pediococcus pentosaceus) strain 5-4 provided by the invention has a biological name of: pediococcus pentosaceus, deposited in the China center for type culture Collection in 2019, 5 months and 20 days, with the deposition address: wuhan university Collection, Lodoku mountain, Wuchang, Wuhan, Hubei province. The zip code is 430072, and the preservation number is CCTCC M2019323.

Drawings

FIG. 1 is a bar graph showing the measurement of oxidation resistance in Effect example 1 of the present invention;

FIG. 2 bar graph of tyrosinase inhibition assay in effect example 2 of the present invention;

FIG. 3 is a graph showing the measurement of the inhibition ratio of Propionibacterium acnes in Effect example 3 of the present invention; wherein the A part is the bacteriostatic circle of 5-4 pediococcus pentosaceus, and the B, C part is the bacteriostatic circle of 1% methylparaben.

Detailed Description

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Further, it should be understood that various changes or modifications of the present invention may be made by those skilled in the art after reading the teaching of the present invention, and such equivalents may fall within the scope of the present invention as defined in the appended claims.

EXAMPLE 1 Oxidation resistance assay of Lactobacilli extracellular actives

(1) Taking a wine medicine sample to be tested, adding sterile water, diluting in a gradient manner by 10 times, oscillating, taking supernatant, coating the supernatant on a solid culture medium, and culturing for 12-48h to obtain a single bacterial colony serving as a strain to be screened;

(2) under the aseptic condition, taking a strain to be screened, inoculating the strain to a porous cell culture plate containing a liquid culture medium, and activating for 3 generations for later use;

(3) centrifuging the activated bacterial liquid at 4000r/min for 2min, and taking supernatant for later use;

(4) carrying out a preliminary experiment, taking 2mL of DPPH solution, adding a small amount of sample solution into the DPPH solution, gradually adding the sample solution at a small amount and a large amount of sample solution at the same time of adding the sample solution, mixing the sample solution and the DPPH solution at the same time, observing the fading condition of the solution, recording the sample adding amount of the sample when the color of the solution is basically faded, namely the maximum sample adding amount, and taking half of the maximum sample adding amount to measure the antioxidant activity of the sample adding amount;

(5) taking the supernatant of the volume in the A1 and B1 wells of a porous plate, adding absolute ethanol solution into B1 to make the volume reach 200 mu L, then adding 100 mu L of 0.2mmol/L DPPH absolute ethanol solution into A1, supplementing less than 200 mu L of absolute ethanol, simultaneously adding 200 mu L of mixed solution of the absolute ethanol solution and the DPPH absolute ethanol solution into C1, and in C1, the volume ratio of the absolute ethanol solution to the DPPH absolute ethanol solution is 1:1, mixing uniformly, and reacting for 30min in a dark environment;

(6) measuring antioxidant activity of the sample at a wavelength of 517nm by using an enzyme-labeling instrument, and paralleling each sample for at least 3 times, and taking an average value;

(7) the results of the experiment are shown in Table 1. From these, 30 strains with the highest DPPH radical scavenging rate were selected for the next experiment, wherein the DPPH radical scavenging rate of the extracellular supernatant of strains 5-4 was 81.89%.

TABLE 1 DPPH clearance of extract of lactic acid bacteria extracellular supernatant

EXAMPLE 2 determination of tyrosinase inhibitory Activity of lactic acid bacteria extracellular active substances

(1) Taking 30 strains to be tested screened in the embodiment 1, centrifuging the activated bacterial liquid at 4000r/min for 2min, and taking supernatant for later use;

(2) the desired solutions were prepared as follows:

TABLE 2 determination of tyrosinase inhibition

C1, C2, T1 and T2 are added according to the table above, water bath is carried out in a water bath kettle at 37 ℃ for 10min, 1mL of dopa solution is added in the water bath kettle at 37 ℃ for 4min, the solution is immediately placed into a spectrophotometer to measure the light absorption value at 475nm at the time of 5min, and the activity inhibition rate T (%) of the sample on tyrosinase is calculated to be 1- (T1-T2)/(C1-C2). times.100%.

(3) The results of the experiment are shown in Table 3. As can be seen from the table, the strain 5-4 was the strain with the highest tyrosinase inhibition rate, which was 64.82%.

TABLE 3 tyrosinase inhibition of extract of extracellular supernatant of lactic acid bacteria

Bacterial strains	Whiteness degree	Bacterial strains	Whiteness degree	Bacterial strains	Whiteness degree	Bacterial strains	Whiteness degree
								2-28	37.00％	3-27	25.67％	4-3	26.18％	4-4	32.26％
5-16	29.58％	4-2	33.48％	5-15	25.64％	5-4	64.82％
								4-1	28.43％	5-25	21.06％	4-5	38.43％	4-7	36.11％
1-22	24.08％	3-4	22.31％	3-18	28.43％	3-23	61.37％
								2-13	25.70％	3-30	35.12％	5-30	24.53％	2-28	50.25％
3-2	29.25％	4-17	28.31％	5-27	47.19％	5-29	49.81％
								3-6	29.89％	4-6	46.99％	2-12	31.95％	3-5	12.70％
5-9	24.91％

EXAMPLE 3 determination of antioxidative Activity and tyrosinase inhibition efficiency of intracellular active substance of lactic acid bacterium

(1) Taking a strain to be tested 5-4, centrifuging the activated bacterial liquid at 4000r/min for 2min, and completely emptying the centrifuged supernatant (namely the mixed liquid of the MRS culture medium and extracellular secretion) in a sterile operating platform;

(2) adding 0.9ml of sterile water, completely mixing the sterile water with a micro vortex mixer, covering an EP tube, centrifuging at low temperature and low speed for a short time again, wherein the centrifugation aims to clean the residual mixed liquid of the MRS culture medium and extracellular secretion in the EP tube, and repeating the experimental steps for 2-3 times until the culture medium in the EP tube is completely cleaned;

(3) a small glass beaker is found, a proper amount of ice-water mixture and a small foam pore plate are added into the small glass beaker, an EP tube is opened and inserted into the small foam pore plate, after an ultrasonic rotor of an ultrasonic crusher is cleaned, the EP tube is placed into the ultrasonic crusher, the corresponding position between the ultrasonic rotor and the EP tube is adjusted (the ultrasonic rotor can not be inserted too deep into the liquid surface and can not be positioned outside the liquid surface), and the ultrasonic crusher is utilized to carry out cell wall breaking work;

(4) the wall breaking power is 30% power, each treatment is 2S, the process is stopped for 2S, and the total wall breaking time is 30 min. After the wall breaking is completed, taking out the EP pipe, and washing the ultrasonic rotor by using clear water;

(5) dripping the crushed liquid drops on a glass slide by using a liquid transfer gun, dripping a drop of crystal violet solution, baking on an alcohol lamp until no liquid drops exist, observing the crushing state of the liquid drops by using an optical microscope, checking whether the liquid drops are completely crushed, repeating the work of the second step again if the liquid drops are not completely crushed, performing wall breaking work again on the liquid drops, and centrifuging the liquid drops through a centrifuge if the liquid drops are completely broken to obtain supernatant;

(6) the DPPH radical clearance was again determined by the same method as described in example 1, and the DPPH radical clearance of the intracellular active substances of the strains 5-4 was found to be 56.51%;

(7) the tyrosinase inhibition was again determined by the same method as described in example 2, and the intracellular active substance of strain 5-4 was determined to have a tyrosinase inhibition of 28.13%.

Example 4 identification of Strain 5-4

(1) Colony characteristics: the strain 5-4 is streaked and separated on an MRS plate, and is cultured for 48 hours at 37 ℃, so that the strain 5-4 grows well, the colony color is milky white, the circular bulge is formed, and the edge is neat;

(2) the characteristics of the thallus are as follows: the strain 5-4 is gram-positive coccus, has no catalase or oxidase, has no motility, does not produce spores, and can grow in aerobic and anaerobic environments. The temperature suitable for growth is 35-40 ℃, the pH value range of the growth is 4.5-8.0, and the acid tolerance is realized;

(3) 16S rDNA sequence analysis of Strain 5-4

The extraction method of genome DNA of strain 5-4 comprises selecting purified strain 5-4, inoculating single colony into 10mM MRS liquid culture medium, culturing at 37 deg.C for 8 hr, centrifuging (4000r/min, 15min), and collecting thallus

The DNA is extracted by a genome DNA extraction kit (biological engineering (Shanghai) Co., Ltd.). Two synthetic universal primers (27F: GAGAGTTTGATCCTGGCTCAG, specifically shown as SEQ ID NO: 1; 1492R: TACGGCTACCTTGTTACGAC, specifically shown as SEQ ID NO:2) are used for PCR amplification, and PCR products are recovered by using a column type PCR product purification kit (Shanghai Biotechnology engineering, Inc.), and are sent to the Shanghai Biotechnology engineering, Inc. for sequencing after purification. The 16S rDNA nucleotide sequence of the obtained strain 5-4 is shown as SEQ ID NO. 3 in the sequence table and is 1073 bp. Sent to GenBank (GenBank accession number: GQ359860) for Blast analysis. Strain 5-4 the most homologous strain was Pediococcus pentosaceus 90.8(Sequence ID: MK611241.1), with a homology of 100%. According to the DNA described by Goodfellow and O' Donnell, species having a G + C (mol%) of 10% to 12% and a sequence homology of 16S of 95% or more were classified as a genus, and Embley and Stackelbraggdt considered that species having a sequence homology of 16S of 97% or more were considered as a species. It can be concluded from this that strain 5-4 belongs to the same species as Pediococcus pentosaceus 90.8. Strain 5-4 was identified as Pediococcus pentosaceus. The lactobacillus CCTCC M2019323 is identified as Pediococcus pentosaceus (Pediococcus pentosaceus) according to microbiological characteristics such as morphological characteristics, physiological and biochemical characteristics and the like and genetic characteristics 16S rDNA thereof, the strain is preserved in China center for type culture Collection (CCTCC for short) in 2019, 5 months and 20 days, and the preservation number is CCTCC M2019323.

Example 5 preparation of Hemerocallis fulva lactic acid bacteria fermentation supernatant I

Adding glucose 4% of the mixed solution mass into the mixed solution of the daylily flower powder and the deionized water with the material taking liquid ratio (mass) of 1:100, and adding edible Na₂CO₃Adjusting the initial pH to 4.5 and adding the solution to a concentration of 10⁶-10⁸5-4 parts of CFU/mL pediococcus pentosaceus, wherein the volume percentage concentration of the zymocyte liquid is 4 percent, and the zymocyte liquid is cultured in a constant-temperature incubator at 37 ℃ for 24 hours. Sterilizing in a high-pressure steam sterilizing pot at 121 deg.C for 15min after fermentation. Centrifuging at 5000r/min for 15min, and collecting supernatant, namely day lily lactobacillus fermentation supernatant I.

Example 6 preparation of Hemerocallis fulva lactic acid bacteria fermentation supernatant II

The difference between the embodiment and the embodiment 5 is only that the mass ratio of the feed liquid is 2:100, and the other parts are the same as the embodiment 5.

Example 7 preparation of Hemerocallis fulva lactic acid bacteria fermentation supernatant III

The difference between the embodiment and the embodiment 5 is only that the mass ratio of the material liquid is 3:100, and the other parts are the same as the embodiment 5.

Example 8 preparation of Lavender lactic acid bacteria fermentation broth I

This example differs from example 5 in that the plant selected was lavender pollen, and the rest was the same as example 5.

Example 9 preparation of Lavender lactic acid bacteria fermentation broth II

The difference between the embodiment and the embodiment 8 is only that the mass ratio of the feed liquid is 2:100, and the other parts are the same as the embodiment 8.

Example 10 preparation of Lavender lactic acid bacteria fermentation broth III

The difference between the embodiment and the embodiment 8 is only that the mass ratio of the material liquid is 3:100, and the other parts are the same as the embodiment 8.

Example 11 preparation of lactic acid bacteria fermented Hemerocallis Fulvii cleansing cosmetic

The extracellular active substance of lactic acid bacteria was formulated into an emulsion according to the following formulation, which is shown in the following table.

TABLE 4 Hemerocallis fulva lactic acid bacteria fermented upper cleansing cosmetic formula

The preparation method of the emulsion comprises the following steps:

(1) accurately weighing each component in the phase A into a beaker A;

(2) accurately weighing deionized water in a beaker B, and dispersing the rest components in the phase B in a water phase one by one;

(3) placing the beaker A phase beaker in a water bath at 90 ℃, and simultaneously heating the aqueous solution in the beaker B to 90 ℃ while stirring;

(4) when the temperature of the two phases reaches about 90 ℃, slowly pouring the sample in the beaker A into the beaker B under the condition of stirring the aqueous solution of the phase B, and continuously stirring for 5 min;

(5) then homogenizing for 2min under the condition that the homogenizing speed is 10000 rpm;

(6) then slowing down the stirring speed, and adding the C phase component when the temperature is reduced to 55 ℃;

(7) and (5) continuously stirring for 20-30 min, and obtaining the uniform system.

Example 12 preparation of Hemerocallis fulva lactic acid bacteria fermentation broth cosmetic

The difference between this example and example 11 is that the amount of the daylily lactobacillus fermentation liquid added is 0.3% (w/w), and the other portions are the same as example 11.

Example 13 preparation of Hemerocallis fulva lactic acid bacteria fermentation broth cosmetic

The difference between this example and example 11 is that the amount of the daylily lactobacillus fermentation liquid added is 0.4% (w/w), and the other portions are the same as example 11.

Comparative example 1

This comparative example differs from example 5 only in that fermentation was not conducted with Pediococcus pentosaceus 5-4 in the production process, and the other portions are the same as example 5.

Comparative example 2

This comparative example is different from example 8 only in that fermentation was not conducted with Pediococcus sacchari 5-4 in the preparation process, and the other part is the same as example 8.

Comparative example 3

The difference between the comparative example and the example 11 is that the hemerocallis fulva lactobacillus fermentation liquid is not added in the formula of the emulsion, and the rest parts are the same as the example 11.

Effect example 1 measurement of Oxidation resistance

The enzyme assay was used to measure the antioxidant activity of 0.01% ascorbic acid (VC) samples, 10% Hemerocallis broth from examples 5-7 and the plant stock solution from comparative example 1, 10% plant stock solutions from examples 8-10 and comparative example 2, as described in example 1. The results are shown in table 5:

TABLE 5 DPPH clearance assay results for various samples

As shown in fig. 1, the anti-oxidation effects of the daylily flower lactobacillus fermentation liquids of 10% in examples 5 to 7 provided by the present invention are respectively equivalent to the effects of Vc of 0.0037%, 0.0043% and 0.0049%, and the anti-oxidation effects of the lavender flower lactobacillus fermentation liquids of 10% in examples 8 to 10 are respectively equivalent to the effects of Vc of 0.0032%, 0.0040% and 0.0046%, and the prepared plant fermentation liquids have certain wrinkle-removing effects. More importantly, in comparative examples 1 and 2, which did not add 10% of the hemerocallis fulva fermentation liquids prepared in examples 5-7 of the present invention, the DPPH clearance was only about 20%, while the DPPH clearance increased with the addition of different ratios of the plant fermentation liquids of Pediococcus pentosaceus, which was up to 39.95%, the DPPH clearance of 10% of examples 5-7 was significantly higher than that of comparative example 1, and the DPPH clearance of examples 8-10 was also significantly higher than that of comparative example 2. Therefore, the pediococcus pentosaceus provided by the invention has high oxidation resistance, and can also obviously improve the oxidation resistance of the medicinal plant fermentation liquor.

Effect example 2 measurement of tyrosinase inhibition Rate

The tyrosinase inhibition was measured by the enzyme assay on 0.01% ascorbic acid (VC), 10% day lily fermentation broth of examples 5-7 and the plant stock of comparative example 1, 10% plant stock of examples 8-10 and comparative example 2, as described in example 2. The results are shown in Table 6:

TABLE 6 determination of tyrosinase inhibition efficiency for various samples

As shown in FIG. 2, the results of the significance analysis show that the whitening effect of the daylily flower lactic acid bacteria fermentation liquid of 10% provided in examples 5-7 of the present invention is equivalent to the effect of arbutin of 0.38%, 0.45% and 0.51% respectively,

the whitening effect of the 10% lavender flower lactic acid bacteria fermentation broth of examples 8-10 is equivalent to that of 0.32%, 0.38%, and 0.47% arbutin, respectively, and has certain whitening effect. More importantly, in comparative examples 1 and 2 in which 10% of the day lily fermentation liquid prepared in examples 5 to 7 of the present invention was not added, the tyrosinase inhibition rate was only about 20%, whereas the plant fermentation liquid added with pediococcus pentosaceus in different ratios had the tyrosinase inhibition rate increased with the increase of the addition ratio, wherein the tyrosinase inhibition rate was as high as 49.64%, and the inhibition rates of examples 5 to 7 were significantly higher than those of comparative example 1, and the inhibition rates of examples 8 to 10 were also significantly higher than those of comparative example 2. Therefore, the pediococcus pentosaceus provided by the invention has high tyrosinase inhibition rate, and can also obviously improve the tyrosinase inhibition capability of the medicinal plant fermentation liquid.

Effect example 3 measurement of inhibition ratio of Propionibacterium acnes

Comparison of the effect of 1% methylparaben on Propionibacterium acnes with Hemerocallis fulva lactic acid bacteria fermentation broth of example 8 was performed using the Oxford cup bacteriostasis test.

The specific method comprises the following steps: the sterilized culture medium of the acne propionibacterium acnes is heated to be completely melted, poured into a culture dish and solidified. After complete cooling and solidification, 100. mu.L of Propionibacterium acnes suspension was pipetted with a pipette tip sterilized and placed on a petri dish and spread evenly with a spreading bar until almost completely dry. Directly and vertically placing 3-4 Oxford cups (circular small tubes with the inner diameter of 6mm, the outer diameter of 8mm and the height of 10mm, wherein two ends of the tubes are smooth, and glass tubes and porcelain tubes can also be used) on the surface of the culture medium in a sterile operation, slightly pressurizing to ensure that the tubes are in contact with the culture medium without gaps, and adding 200 mu L of a sample to be detected (1% of methylparaben) into the cups. After the mixture is filled, the mixture is cultured for 16 to 18 hours at 37 ℃, and the observation result shows that the diameter of the zone of inhibition is measured by a ruler, and the outer diameter of the Oxford cup is subtracted from the diameter of the zone of inhibition.

The results are shown in fig. 3, where the part a is the inhibition zone of pediococcus pentosaceus 5-4, and the part B, C is the inhibition zone of 1% methylparaben, it can be clearly seen that the daylily flower lactobacillus fermentation liquid has indeed the inhibition effect on propionibacterium acnes, and the inhibition capability is obvious, and the diameter of the inhibition zone is about 15.0 mm.

Effect example 4

To further verify the efficacy of the Hemerocallis fulva lactic acid bacteria fermentation broth in cosmetics, the emulsions prepared in examples 11-13 were subjected to corresponding human experiments. The specific process is as follows:

1. sample (I)

Emulsions from examples 11 to 13

2. Experimental methods

Selecting 100 volunteers (50 male and 50 female, age 18-25 years), with dark yellow and dry skin on face, and no cosmetic allergy history. Volunteers were divided into 2 groups of 50 persons each, with half the ratio between men and women.

A first group, trial implementation 11 product; second, trial example 12; third, trial example 13; and fourthly, trying a certain commercially available whitening emulsion.

The using method comprises the following steps: after cleaning the face in the morning and evening, a proper amount of suitable product is uniformly smeared on the face, and the face is gently tapped and massaged for 8 weeks for continuous trial. The subjective evaluation of the product used by the test subjects was investigated in the form of a questionnaire. The results are shown in Table 7.

Table 7 evaluation of test subjects on products

Table 7 shows the results of the evaluation of the trial products by the test subjects, and the results in the table show that after the daylily lactobacillus fermentation liquid is added, the examples 11 to 13 of the present invention have good whitening and wrinkle removing effects, and the whitening and skin lubricating effects are significantly better than those of the similar products on the market.

Unless specifically stated otherwise, the numerical values set forth in these examples do not limit the scope of the invention. In all examples shown and described herein, unless otherwise specified, any particular value should be construed as merely illustrative, and not restrictive, and thus other examples of example embodiments may have different values.

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art that various changes in form and details may be made therein without departing from the spirit and scope of the invention; such modifications and substitutions do not depart from the spirit and scope of the present invention, and they should be construed as being included in the following claims and description.

SEQUENCE LISTING

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ccattgtagc acgtgtgtag cccaggtcat aaggggcatg atgatttgac gtcgtcccca 300

ccttcctccg gtttgtcacc ggcagtctca ctagagtgcc caacttaatg ctggcaacta 360

gtaataaggg ttgcgctcgt tgcgggactt aacccaacat ctcacgacac gagctgacga 420

caaccatgca ccacctgtca ttctgtcccc gaagggaacc tctaatctct tagactgtca 480

gaagatgtca agacctggta aggttcttcg cgtagcttcg aattaaacca catgctccac 540

cgcttgtgcg ggcccccgtc aattcttttg agtttcaacc ttgcggtcgt actccccagg 600

cggattactt aatgcgttag ctgcagcact gaagggcgga aaccctccaa cacttagtaa 660

tcatcgttta cggcatggac taccagggta tctaatcctg ttcgctaccc atgctttcga 720

gcctcagcgt cagttgcaga ccagacagcc gccttcgcca ctggtgttct tccatatatc 780

tacgcatttc accgctacac atggagttcc actgtcctct tctgcactca agtctcccag 840

tttccaatgc acttcttcgg ttgagccgaa agctttcaca ttagacttaa aagaccgcct 900

gcgctcgctt tacgcccaat aaatccggat aacgcttgcc acctacgtat taccgcggct 960

gctggcacgt agttagccgt ggctttctgg ttaaataccg tcactggtaa acagttactc 1020

ttacccacgt tcttctttaa caacagagct ttacgagccg aaacccttct tca 1073

Claims

1. The Pediococcus pentosaceus with high oxidation resistance and whitening effect is characterized in that the Pediococcus pentosaceus has a biological name of Pediococcus pentosaceus and is preserved in China center for type culture Collection with the preservation number of CCTCC M2019323.

2. A method for producing a lactic acid bacteria fermentation broth for plants, comprising inoculating the Pediococcus pentosaceus strain of claim 1 to a fermentation raw material solution containing plants, fermenting, sterilizing, centrifuging, and collecting the supernatant.

3. The method for preparing a plant lactic acid bacteria fermentation broth according to claim 2, wherein the fermentation raw material broth comprises a plant, water and glucose, wherein the mass ratio of the plant to the water is (1-3): 100, and the addition amount of the glucose is 4% -6% of the total mass of the plant and the water.

4. The method for preparing a lactobacillus plantarum fermentation broth according to claim 2, wherein the plant is a plant powder; the plant is medicinal plant.

5. The method of preparing a fermented liquid of lactic acid bacteria according to claim 4, wherein the medicinal plant is one or more of platycodon grandiflorum, dandelion, mint, ginseng, angelica sinensis, ginkgo biloba, hemerocallis or lavender.

6. The method for preparing a lactobacillus plantarum fermentation broth according to claim 2, wherein the lactobacillus plantarum fermentation brothThe preparation of the bacterial fermentation liquid specifically comprises the following steps: inoculating 10 times of culture solution of Pediococcus pentosaceus⁶～10⁸C FU/mL is inoculated into the fermentation raw material liquid, the fermentation raw material liquid is cultured in a constant-temperature incubator at 37 ℃ for 24h-48h, then the sterilization and centrifugation are carried out, the supernatant liquid is taken, and the obtained fermentation supernatant liquid is the plant lactobacillus fermentation liquid with pediococcus pentosaceus; the sterilization and centrifugation conditions are that the sterilization is carried out for 20min at the temperature of 115 ℃ and the centrifugation is carried out for 15min at the speed of 4500-5500 r/min.

7. The method according to claim 6, wherein the inoculation is a culture solution of a strain inoculated with Pediococcus pentosaceus, and the preparation of the culture solution of the strain comprises: taking a loop of pediococcus pentosaceus strain stored in a freeze-drying tube dissolved by sterile water by using an inoculating loop, streaking the loop on an MRS agar culture medium plate, and culturing the streaked pediococcus pentosaceus strain in an incubator at 37 ℃ for 24-48h until a single colony grows out to obtain a plate-activated pediococcus pentosaceus strain; inoculating the strain of Pediococcus pentosaceus activated by the inoculating loop taking flat plate into an MRS broth culture medium in a loop manner, and placing the culture medium in an incubator at 37 ℃ for constant-temperature culture for 24-48h to obtain a strain culture solution.

8. A lactobacillus plantarum fermentation broth prepared according to the method of any one of claims 2-7.

9. Use of pediococcus pentosaceus having high antioxidative and whitening effects according to claim 1 for the preparation of cosmetics.

10. Use of the lactobacillus plantarum fermentation broth according to claim 8 in the preparation of cosmetics.