CN115927104A - Lactococcus lactis, microbial agent, and preparation method and application thereof - Google Patents

Abstract

The invention relates to the field of microorganisms, in particular to lactococcus lactis, a microbial agent, and a preparation method and application thereof. The invention provides Lactococcus lactis JWA32 (Lactococcus lactis JWA 32), which has a deposit number of: CCTCC No. M20221851. The invention provides lactococcus lactis, a microbial agent, and a preparation method and application thereof. The invention provides lactococcus lactis with excellent efficacy for preparing a skin microecological preparation, which can relieve the oxidative damage effect of HaCaT cells and repair epidermal barriers.

Description

Lactococcus lactis, microbial agent and preparation method and application thereof

Technical Field

The invention relates to the field of microorganisms, in particular to lactococcus lactis, a microbial agent, and a preparation method and application thereof.

Background

The skin micro-ecological system is a whole body which is composed of various microorganisms, histiocyte on the surface of the skin, various secretions, microenvironment and the like, maintains the micro-ecological balance of the skin, forms a first biological barrier on the surface of the skin and has important physiological action. At present, the study on the efficacy of probiotics tends to be mature at home and abroad, the probiotics is widely applied to the field of cosmetics, and with the development of a biological fermentation technology, the balance of a skin ecosystem is maintained, so that a skin city wall is firmer, and the skin micro-ecology is repaired on the whole, so that a micro-ecological skin care era for remodeling healthy skin comes. Meanwhile, with the rise of skin care ingredients, the anti-aging effect is younger and more popular in the market, euromonitor data show that the anti-aging skin care product in the Chinese skin care product market reaches 646 billion yuan in 2020 and occupies 28.8 percent of the market share of the skin care product, according to the sori consultation data, in the aspect of concerned effect, the effect of ranking the first overall sample is anti-aging, and consumers are more expected to meet the requirements of slowing down the aging speed of the skin and maintaining the young state through daily skin care.

Skin aging is influenced by many external factors related to environment and life style, ultraviolet radiation is a main factor causing the external skin aging, ultraviolet radiation induces skin photoaging through various signal paths, such as oxidative stress, thereby increasing the generation of free radicals, especially Reactive Oxygen Species (ROS), the toxic attack of the excess free radicals on biological tissues such as chromosomes, mitochondria, cell membranes and connective hoof tissues can cause body damage to cause aging, the excess free radicals in the skin can act on unsaturated fatty acids in the lipid layers of the skin, react to form a lipo-protein complex, namely lipofuscin, and accumulate in cells, and thus, the removal of the excess free radicals is one of effective methods for resisting the skin aging.

The existing probiotics for removing active oxygen free radicals are mainly applied to the fields of foods and medicines, but are less applied to the field of cosmetics.

Disclosure of Invention

In view of the above, the invention provides lactococcus lactis, a microbial agent, and a preparation method and application thereof. The invention provides lactococcus lactis with excellent efficacy for preparing a skin microecological preparation, which can relieve the oxidative damage effect of HaCaT cells and repair epidermal barriers.

In order to achieve the above object, the present invention provides the following technical solutions:

the invention provides Lactococcus lactis JWA32 (Lactococcus lactis JWA 32), which has a deposit number of: CCTCC No. M20221851.

The invention also provides a microbial agent which comprises any one of the following substances and an acceptable auxiliary agent;

the Lactococcus lactis JWA32 (Lactococcus lactis JWA 32) as described above; and/or

(II) an inactivated strain of Lactococcus lactis JWA32 (Lactococcus lactis JWA 32) as described above; and/or

(III) a fermentation broth, culture, exosome, lysate or extract of the above Lactococcus lactis JWA32 (Lactococcus lactis JWA 32).

In some embodiments of the invention, the fermentation broth in the microbial agent comprises a fermentation product filtrate or a fungal fermentation lysate filtrate.

In some embodiments of the present invention, the fermentation product filtrate in the microbial agent is obtained by fermenting, inoculating, culturing and then fermenting seed liquid, centrifuging, filtering, and collecting supernatant.

In some embodiments of the present invention, the microbial agent is obtained by subjecting the filtrate of the cell fermentation lysate of the above microbial agent to fermentation inoculation culture of lactococcus lactis to obtain a seed solution, performing fermentation culture, crushing the cells, centrifuging, and filtering.

In some embodiments of the present invention, the formulation of the microbial agent comprises: one or more of emulsion, aqua, ointment, pellicle or gel.

The invention also provides a preparation method of the microbial agent, which comprises the following steps:

s1: inoculating and culturing the lactococcus lactis to obtain a seed solution;

s2: after the seed liquid is taken for fermentation culture, performing first centrifugation and filtration, and collecting supernatant to obtain the microbial agent; or

And (3) taking the seed liquid for fermentation culture, crushing thalli, performing secondary centrifugation and filtration, and collecting supernatant to obtain the microbial agent.

In some embodiments of the present invention, the culturing time in the above-mentioned production method S1 is 24 hours and the temperature is 37 ℃.

In some embodiments of the present invention, the rotation speed of the first centrifugation in the above preparation method S1 is 6000rpm for 10min.

In some embodiments of the present invention, the amount of inoculation in the fermentation culture in the above-mentioned production method S2 is 10%, and the number of viable bacteria is 1X 10 ⁹ ～10 ¹⁰ CFU/mL。

In some embodiments of the present invention, the fermentation culture time in the above preparation method S2 is 24 hours and 37 ℃.

In some embodiments of the present invention, the rotation speed of the second centrifugation in the above preparation method S1 is 6000rpm for 10min.

The invention also provides application of the lactococcus lactis, the microbial agent or the microbial agent obtained by the preparation method in preparation of products for reducing ROS level, inhibiting hyaluronidase activity, improving ceramide secretion or improving SOD enzyme activity.

The invention also provides application of the lactococcus lactis, the microbial agent or the microbial agent obtained by the preparation method in preparation of products for relieving oxidative damage of HaCaT cells, repairing epidermal barriers or inhibiting harmful bacteria.

In some embodiments of the invention, the harmful bacteria in the above-mentioned applications include staphylococcus aureus or streptococcus albicans.

The invention also provides the application of the lactococcus lactis, the microbial agent or the microbial agent obtained by the preparation method in preparing an antioxidant, anti-aging or anti-wrinkle product.

The invention also provides a product which comprises the lactococcus lactis, the microbial agent or the microbial agent obtained by the preparation method and acceptable auxiliary materials or auxiliary agents.

In some embodiments of the invention, the above product comprises: a cosmetic is provided.

The invention provides Lactococcus lactis JWA32 (Lactococcus lactis JWA 32), which has a deposit number of: CCTCC No. M20221851, the invention also provides a microbial agent, a preparation method and application thereof.

The fermentation filtrate of Lactococcus lactis JWA32 provided by the invention can relieve the oxidative damage of HaCaT cells, has an excellent antioxidant effect, and also has the effect of repairing skin barriers.

Description of biological preservation

Lactococcus lactis JWA32 with preservation date of 11 months and 30 days in 2022, preservation number of CCTCC No. M20221851, and the name of the preservation unit is: china center for type culture Collection, the collection center addresses are: wuhan, wuhan university.

Drawings

In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.

FIG. 1 shows the viability of cells tested by the MTT method;

FIG. 2 shows reactive oxygen species ROS clearance results;

FIG. 3 microscopic fluorescence intensity of the reactive oxygen species-free ROS scavenging results of FIG. 2;

FIG. 4 shows the promotion of ceramide secretion rate;

FIG. 5 shows superoxide dismutase SOD activity;

FIG. 6 shows the results of the inhibition of harmful bacteria; wherein: the upper graph shows the inhibition rate (%) for Staphylococcus aureus, and the lower graph shows the inhibition rate (%) for Streptococcus albus;

FIG. 7 shows hyaluronidase inhibition.

Detailed Description

The invention discloses lactococcus lactis, a microbial agent, and a preparation method and application thereof.

It should be understood that one or more of the expressions "\8230", individually include each of the objects recited after the expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and/or" in connection with three or more of the stated objects shall be understood to have the same meaning unless otherwise understood from the context.

The use of the terms "comprising," "having," or "containing," including grammatical equivalents thereof, is generally to be construed as open and non-limiting, e.g., without excluding other unstated elements or steps unless specifically stated otherwise or otherwise understood from the context.

It should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Further, two or more steps or actions may be performed simultaneously.

The use of any and all examples, or exemplary language such as "for example" or "including" herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed

And (4) limiting. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Moreover, the numerical ranges and parameters setting forth the invention are approximations that may have numerical values that are within the numerical ranges specified in the specific examples. Any numerical value, however, inherently contains certain standard deviations found in their respective testing measurements. Accordingly, unless expressly stated otherwise, it is understood that all ranges, amounts, values and percentages used in this disclosure are by weight modified by "about". As used herein, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a particular value or range.

The invention provides Lactococcus lactis JWA32 (Lactococcus lactis JWA 32) with a preservation number (CCTCC No. M20221851), which has the functions of relieving oxidative damage of HaCaT cells and secreting ceramide, namely the functions of resisting oxidation and repairing skin barriers, and inhibiting harmful bacteria such as staphylococcus aureus and candida albicans, can be applied to skin microecologics, particularly anti-aging preparations, is preferably applied to anti-aging cosmetics, and can relieve skin photoaging.

The skin probiotics provided by the invention comprise fermentation filtrate of lactococcus lactis, and the fermentation filtrate can comprise fermentation product filtrate or thallus fermentation lysate filtrate.

The preparation method of the fermentation filtrate comprises the following steps: inoculating lactococcus lactis into a seed culture medium for culture to obtain a seed solution; inoculating the seed liquid into a fermentation culture medium for fermentation to obtain a fermentation liquid; and centrifuging the fermentation liquor, filtering and sterilizing to obtain fermentation product filtrate.

The preparation method of the thallus fermentation lysate filtrate comprises the following steps: inoculating lactococcus lactis into a seed culture medium for culture to obtain a seed solution; inoculating the seed liquid into a fermentation culture medium for fermentation, crushing thalli after fermentation, and performing centrifugal filtration to obtain thalli fermentation lysate filtrate.

The skin microecological preparation provided by the invention can be in the dosage forms of emulsion, aqua, paste, film or gel and the like.

In examples 1 and 2 and verification examples 1 to 6 of the present invention, the raw materials and reagents used were all commercially available.

The invention is further illustrated by the following examples:

EXAMPLE 1 isolation, purification and identification of Strain (providing 16S)

Collecting feces samples of college students in Wuhan region, diluting with sterile oxygen-free water, selecting suitable gradient diluent, coating on MRS solid culture medium, and anaerobic culturing at 37 deg.C

Culturing for 48 hours, selecting typical colonies, streaking on MRS solid medium for 2-3 times, and culturing to obtain pure colonies. 16S rDNA molecular identification is carried out, a universal primer is adopted for strain identification,

the upstream primer was 27F: AGAGTTTGATCCTGGCTCAG (shown in SEQ ID NO: 1);

the downstream primer is 1492R: TACGGCTACCTTGTTACGACTT (shown as SEQ ID NO: 2), after nucleotide sequence alignment in NCBI, showed a strain of Lactococcus lactis, named Lactococcus lactis JWA32 (Lactococcus lactis JWA 32), which was deposited in China Center for Type Culture Collection (CCTCC) at 11/30.2022.

The 16S rDNA sequence of lactococcus lactis JWA32 (shown in SEQ ID NO: 3): <xnotran> TTA GGAGCGCCCTCCTTGCGGTTAGGCAACCTACTTCGGGTACTCCCAACTCCCGTGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGGCGTGCTGATCCGCGATTACTAGCGATTCCGACTTCATGTAGGCGAGTTGCAGCCTACAATCCGAACTGAGAATGGTTTTAAGAGATTAGCTAAACATCACTGTCTCGCGACTCGTTGTACCATCCATTGTAGCACGTGTGTAGCCCAGGTCATAAGGGGCATGATGATTTGACGTCATCCCCACCTTCCTCCGGTTTATCACCGGCAGTCTCGTTAGAGTGCCCAACTTAATGATGGCAACTAACAATAGGGGTTGCGCTCGTTGCGGGACTTAACCCAACATCTCACGACACGAGCTGACGACAACCATGCACCACCTGTATCCCGTGTCCCGAAGGAACTTCCTATCTCTAGGAATAGCACGAGTATGTCAAGACCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGCTCCACCGCTTGTGCGGGCCCCCGTCAATTCCTTTGAGTTTCAACCTTGCGGTCGTACTCCCCAGGCGGAGTGCTTATTGCGTTAGCTGCGATACAGAGAACTTATAGCTCCCTACATCTAGCACTCATCGTTTACGGCGTGGACTACCAGGGTATCTAATCCTGTTTGCTCCCCACGCTTTCGAGCCTCAGTGTCAGTTACAGGCCAGAGAGCCGCTTTCGCCACCGGTGTTCCTCCATATATCTACGCATTTCACCGCTACACATGGAATTCCACTCTCCTCTCCTGCACTCAAGTCTACCAGTTTCCAATGCATACAATGGTTGAGCCACTGCCTTTTACACCAGACTTAATAAACCACCTGCGCTCGCTTTACGCCCAATAAATCCGGACAACGCTCGGGACCTACGTATTACCGCGGCTGCTGGCACGTAGTTAGCCGTCCCTTTCTGGGTAGTTACCGTCACTTGATGAGCTTTCCACTCTCACCAACGTTCTTCTCTACCAA </xnotran>

CAGAGTTTTACGATCCGAAAACCTTCTTCACTCACGCGGCGTTGCTCGG

TCAGACTTTCGTCCATTGCCGAAGATTCCCTACTGCTGCCTCCCGTAGG

AGTTTGGGCCGTGTCTCAGTCCCAATGTGGCCGATCACCCTCTCAGGTC

GGCTATGTATCATCGCCTTGGTGAGCCTTTACCTCACCAACTAGCTAAT

ACAACGCGGGATCATCTTTGAGTGATGCAATTGCATCTTTCAAACTTAA

AACTTGTGTTTAAAGTTTTTATGCGGTATTAGCATTCGTTTCCAAATGTT

GTCCCCCGCTCAAAGGCAGATTCCCCACGCGTTACTCACCCGTTCGCTGCTCATCCAGTTGGTACAAGTACCAACCTTCAGCGCTCAACTG。

Example 2 preparation of fermentation filtrate

Examples of the invention1 taking out the glycerol cryopreservation tube of the obtained strain from a refrigerator at minus 80 ℃, putting the tube in a room temperature (25 +/-5 ℃) for melting, picking the strain by using an inoculating loop in a sterile environment, streaking and inoculating the strain on an MRS solid plate, culturing for 48 hours at 37 ℃, observing the colony morphology on the plate to ensure that lactococcus lactis is recognized and is free of pollution, picking single bacteria on the plate according to the inoculation amount of 10 percent and inoculating the single bacteria to a corresponding MRS liquid culture medium, putting the plate in a 37 ℃ culture box for standing and culturing for 24 to 48 hours until the bacteria grows turbid to obtain an activated bacteria liquid (1 multiplied by 10) which is a culture liquid ⁹ ～10 ¹⁰ CFU/mL), transferring the bacterial liquid into a corresponding MRS fermentation culture medium for two times according to the inoculation amount of 10%, placing the MRS fermentation culture medium in an incubator at 37 ℃ for standing culture for 24 hours, obtaining a lactococcus lactis culture after fermentation is completed, obtaining a supernatant through centrifugation (the rotating speed is 6000rpm, the time is 10 min), and obtaining a fermentation filtrate through filtering the supernatant. Verification example 1MTT method for detecting cell viability of fermentation broth

After the HaCaT cells are inoculated on a 96-well plate and cultured for 24h, the supernatant is sucked, fermentation filtrate of the lactococcus lactis JWA32 obtained in example 2 with different concentrations is added to continue culturing for 24h, and the cell survival rate is detected by adopting an MTT method.

Cell survival rate detection by MTT method: after cell treatment, the medium was discarded, 100. Mu.L of 5mg/mL MTT working solution prepared in serum-free medium was added to each well, the mixture was incubated at 37 ℃ for 4 hours, the MTT working solution was discarded, 100. Mu.L of DMSO was added to each well, and the mixture was shaken in a dark place at low speed for 10 minutes, and the absorbance was measured at 490nm using a microplate reader. Calculating the formula: relative cell survival = An/A0 (note: an is absorbance of experimental group, A0 is absorbance of negative control group)

TABLE 1 cell viability by MTT assay

Group of	Cell survival rate
		Normal control (100. Mu.L serum-free medium)	100％
2.5% fermentation filtrate	89.65％
		5% fermentation filtrate	92.27％
7.5% fermentation filtrate	109.58％
		10% fermentation filtrate	112.02％

Table 1 corresponds to the data of fig. 1;

as shown in fig. 1 and table 1, according to the toxicity evaluation criteria described in ISO 10993-5, the cell viability was defined as no cytotoxicity when the cell viability was higher than 70%, after HaCaT cells were cultured with different concentrations of the fermentation filtrate of lactococcus lactis JWA32, the cell viability was not cytotoxic, and when the concentrations were 7.5% and 10%, the cell viability was improved compared with the control group, which indicates that the fermentation filtrate at the concentrations had a certain proliferation promoting effect on HaCaT cells.

Validation example 2HaCaT keratinocyte ROS scavenging assay

Cell: primary human keratinocytes HaCaT;

materials: DCFH-DA, 3% hydrogen peroxide (H) ₂ O ₂ ) A solution;

stimulation conditions: hydrogen peroxide (H) ₂ O ₂ ) The ROS content in the keratinocyte is induced to rise, the using concentration of the hydrogen peroxide is 120 mu M, the treatment time is 1h, and the test can meet the following quality standards: the ROS content of the negative control under the action of the stimulus is increased by more than or equal to 30 percent compared with the ROS content of the blank/solvent control;

positive control: 0.05% vitamin C (Vc).

The test method comprises the following steps: activated cells at 1X 10 ⁴ Inoculating 200 μ L of the mixture into a 96-well plate (fusion degree reaches 45% -60% in 24h after inoculation), and placing in CO ₂ Culturing in an incubator for 24h +/-2 h. The medium in each well of the well plate was discarded, and the experimental group was added with the medium containing the fermentation filtrate obtained in example 2 prepared as described above at different concentrations, the medium containing Vc at a concentration of 0.05% was added to the positive control wells, and the normal medium was added to the blank control wells and the model wells. Placing the pore plate in CO after sample adding ₂ The incubator is 24h +/-2 h. After incubation, the solution was not discarded, except for the blank control, all the wells were subjected to H ₂ O ₂ Induction, H ₂ O ₂ The induction time of (1) was 1h and the induction dose was 120. Mu.M. Discarding the culture medium in the wells, washing with PBS for 3 times, adding 100 μ L of DCFH-DA working solution (sampling in Biyuntian active oxygen detection kit), and CO ₂ Incubating in incubator for 30min, and incubating in serum-free DMEM

And (3) cleaning cells in each hole for 3 times, placing the 96-hole plate after the operation is finished on a detection table of a fluorescence microplate reader, setting the wavelength of incident light to be 525nm, setting the wavelength of exciting light to be 488nm, and reading.

Table 2 reactive oxygen species ROS scavenging results (x ± s, n = 3)

Group of	ROS level (%)
		Blank control (DCFH-DA)	100％
Model group (H) 2 O 2 Induction + DCFH-DA)	163.67±6.85％ ***
		Positive control Vc0.05%	131.33±3.30％ **
2.5% of the experimental groups	112.33±2.05％ ***
		5% of the test groups	103.33±1.70％ ***
10% of the experimental groups	100.33±3.09％ ***

Table 2 corresponds to the data of fig. 2;

as can be seen from FIG. 2 and Table 2 and the results of fluorescence microscopy photographs in FIG. 3, the ROS levels in the model group were significantly increased compared to the blank cell group, indicating H ₂ O ₂ The oxidative stress model for inducing the HaCaT cells is successfully established, compared with a model group, the ROS level of a positive control group (Vc) is obviously weakened, the positive control group can reduce the excessive ROS free radicals in the HaCaT cells, and meanwhile, the ROS level of the HaCaT cells of a fermentation liquor group of the lactococcus lactis JWA32 is also obviously weakened, so that the ROS level of the HaCaT cells can be reduced in the oxidative stress model of the HaCaT cells, and the purpose of relieving the oxidative damage of the HaCaT cells is achieved.

Verification example 3 detection of ceramide

(1) Culturing Hacat cells: 3X 10 addition per well in 96-well plates ⁴ Cells, culture volume 200. Mu.L/well, 5% CO at 37 ℃% ₂ Culturing for 16-24 hours (cell fusion is 70%);

(2) Grouping and inducing cell models: negative control: discarding the supernatant, and adding fresh DMEM medium 180 μ L/well; model group: discard the supernatant and add 180. Mu.L/well of fresh medium containing 3mg/mL SDS; experimental groups: the supernatant was discarded and 180. Mu.L/well of fresh medium containing 3mg/mL SDS was added.

(3) Addition of sample, negative control: add 20. Mu.L of PBS; model group: add 20. Mu.L of PBS; experimental groups: 20 μ L of the fermentation filtrate obtained in example 2 was added, and the content of CO was 5% at 37 ℃% ₂ Cultured under the conditions of (1) for 24 hours.

(4) Cell culture supernatants were collected and centrifuged at 7500rpm for 10min to collect the supernatants.

(5) ELISA kit for detecting ceramide content

Sample adding of the standard: setting standard product holes and sample holes, wherein 50 mu L of standard products with different concentrations are added into the standard product holes respectively, and 50 mu L of sample diluent is added into the standard 0 holes;

sample adding: blank holes (the blank reference holes are not added with the sample and the enzyme labeling reagent, and the rest steps are operated in the same way) and sample holes to be detected are respectively arranged. Adding 50 mu L of sample into a sample hole to be detected on the enzyme-labeled coated plate, adding the sample to the bottom of the hole of the enzyme-labeled plate, keeping the hole wall as far as possible, and slightly shaking and uniformly mixing;

adding an enzyme: adding 100 mu L of enzyme-labeled reagent into each hole except for blank holes;

incubation: sealing the plate with a sealing plate film, and then incubating for 60 minutes at 37 ℃;

preparing liquid: diluting 20 times of the concentrated washing liquid with 20 times of distilled water for later use;

washing: carefully uncovering the sealing plate film, discarding liquid, spin-drying, filling washing liquid into each hole, standing for 60 seconds, discarding, repeating the steps for 5 times, and patting dry;

color development: adding 50 mu L of color developing agent A into each hole, then adding 50 mu L of color developing agent B, lightly shaking and uniformly mixing, and developing for 15 minutes at 37 ℃ in a dark place;

and (4) terminating: adding 50 mu L of stop solution into each well to stop the reaction (at the moment, the blue color immediately turns to yellow);

and (3) determination: zeroing with blank well, sequentially measuring absorbance (OD value) of each well at 450nm wavelength, measuring within 15min after adding stop solution, and measuring OD ₄₅₀ The values are input into ELISA data processing software to obtain data.

TABLE 3 promoting ceramide secretion Rate

Table 3 corresponds to the data of fig. 4;

as shown in fig. 4 and table 3, compared with the model group, the addition of the fermentation filtrate of lactococcus lactis JWA32 of example 2 significantly promoted the secretion of Ceramide in HaCaT cells under the same culture conditions, and Ceramide (Ceramide) is an important component of human stratum corneum lipid, and has the effects of moisturizing, maintaining stratum corneum structure stability (adhesion), repairing damage, and the like, and the content of Ceramide affects the quality of skin barrier function, which indicates that the fermentation filtrate prepared by the present invention can supplement skin stratum corneum lipid and enhance skin barrier epidermal barrier function.

Verification example 4 measurement of superoxide dismutase SOD

HaCaT cells in logarithmic growth phase at 1X 10 ⁵ Density of individual/mL in 24-well plates, 1mL per well, then placing them in 5% CO ₂ Adherent culture is carried out for 24h at 37 ℃ in an incubator. The culture medium was discarded and the experiment was divided into a normal control group, a positive control group, a model group and a sample group. The fermentation filtrate obtained in example 2 was added to the cell culture solution of each well of the sample group at a ratio of 10%, vc of 1% was added to the positive control group, and equal volume of the blank medium was added to the normal control group and the model group, and after 24 hours of culture, the cells were washed 2 times with PBS. Sample group, positive control group and H ₂ O ₂ H with the final concentration of 60 mu mol/L is added into the cell culture solution of the model group ₂ O ₂ And (3) solution, inducing cells to generate oxidative stress injury, adding an equal volume of DMEM medium without FBS into cell culture solution of each hole of the blank group and the normal control group, continuously culturing for 1h, washing the cells by PBS, and repeating for 3 times. The cells were lysed at 4 ℃ for 10min, and the lysed cells were collected and the supernatant obtained according to the protocol of the kit. According to the kit operation method, SOD activity is measured, and protein concentration is measured by a BCA protein concentration measuring method.

TABLE 4

Table 4 corresponds to the data of fig. 5;

as can be seen from table 4 and fig. 5, the 10% fermentation filtrate prepared in example 2 has a significant difference from the normal control group, which indicates that it can significantly improve the SOD enzyme activity in HaCaT cells compared to the normal control group.

Verification example 5 test for inhibiting harmful bacteria

Skin pathogenic bacteria (10) cultured to logarithmic growth phase ⁷ -10 ⁸ CFU/mL) is inoculated into a blank culture medium containing 10% of a substance to be detected according to the inoculation amount of 1%, and the number or OD of pathogenic bacteria in the culture medium is detected after 24h ₆₀₀ The absorbance value of (c).

The substance to be tested: example 2 fermentation filtrate obtained

Bacterial suspension 1: taking staphylococcus aureus bacterial liquid cultured to logarithmic phase, diluting with blank LB culture medium to obtain staphylococcus aureus 10 ⁶ CFU/mL of bacterial suspension.

Bacterial suspension 2: diluting the white streptococci bacterial liquid cultured to logarithmic phase with blank YPD culture medium to obtain white streptococci-containing 10 ⁶ CFU/mL of bacterial suspension.

Respectively adding 10% of the substance to be detected into the bacterial suspensions 1 and 2, adding a blank culture medium with the same volume into the control group, culturing at 37 ℃, and measuring the light absorption values at different time points of 0h, 15h, 19h and 23 h.

Inhibition (%) =1- (OD) _{Control group} -OD _{Experimental group} )/OD _{Control group} ×100％

As shown in FIG. 6, the fermentation filtrate prepared in example 2 has effective inhibitory effect on Staphylococcus aureus and Candida albicans, and the bacteriostatic rate after 23h can be maintained at above 70%.

Verification example 6 inhibition of Hyaluronidase Activity

Hyaluronidase is capable of indiscriminately hydrolyzing the 1, 4-linkage between β -N-acetylglucosamine and D-glucuronide in hyaluronic acid to yield β -N-acetylglucosamine which can be condensed with acetylacetone under alkaline conditions to form a chromogen 2-methyl-3-diacetylpyrrole derivative, which is chromogen and p-dimethylaminobenzaldehyde in concentrated hydrochloric acid ethanol.

Reagent: MRS, calcium chloride, citric acid, sodium citrate, hyaluronidase, sodium hyaluronate, acetylacetone, dopa, p-dimethylaminobenzaldehyde, calcium chloride, sodium hydroxide, concentrated hydrochloric acid, acetic acid and absolute ethyl alcohol, wherein the preparation of the reagent is as follows:

0.2mol/L acetic acid and 0.2mol/L sodium acetate were prepared, and 4.8mL of acetic acid and 45.2mL of sodium acetate were mixed to prepare an acetic acid buffer (pH = 5.6). 0.5mL of the fermentation filtrate (10%) prepared in example 2 and 0.5mL of hyaluronidase solution (prepared by dissolving hyaluronidase in acetate buffer solution to a concentration of 500U/mL) were incubated in 37 ℃ water bath for 20min; 0.1mL of CaCl was added ₂ Keeping the temperature of the solution (2.5 mol/L) for 20min at 37 ℃; adding 0.5mL sodium hyaluronate (sodium hyaluronate is dissolved in acetic acid buffer solution to prepare a concentration of 0.5 mg/mL), keeping the temperature for 40min at 37 ℃, taking out and placing for 10min at room temperature (25 +/-5 ℃); 0.1mL of NaOH solution (5 mol/L) and 0.5mL of acetylacetone were added

The solution (3.5 mL of acetylacetone dissolved in 50mL of 1mol/L sodium carbonate) is transferred to an ice water bath for 10min after being put in a boiling water bath for 15min, and then is placed for 10min at room temperature (25 +/-5 ℃); 1.0mL of P-DAB color-developing agent (0.8 g dimethylaminobenzaldehyde dissolved in 15mL of concentrated hydrochloric acid, and then 15mL of absolute ethyl alcohol are added and mixed uniformly) is added dropwise, and diluted with 4.3mL of absolute ethyl alcohol, and the mixture is placed at room temperature (25 ℃ +/-5 ℃) for 30min for color development, and the absorbance value at the wavelength of 530nm is measured by a spectrophotometer.

Hyaluronidase inhibition (%) = [ (C-D) - (A-B) ]/(C-D) × 100%

Wherein, a is the absorbance value of (hyaluronidase + sample + sodium hyaluronate) sample solution, B is the absorbance value of (acetate buffer + sample + acetate buffer) blank sample, C is the absorbance value of (hyaluronidase + distilled water + sodium hyaluronate) control solution, D is the absorbance value of (acetate buffer + distilled water + acetate buffer) control blank, and the test results table 5 and fig. 7 show:

TABLE 5

Table 5 corresponds to the data of fig. 7;

compared with a control MRS, the fermentation filtrate has obvious hyaluronidase inhibition activity, the hyaluronidase inhibition rate is as high as about 94.71 percent, and the fermentation filtrate can be used for anti-wrinkle functional daily chemicals.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims (10)

1. Lactococcus lactis JWA32 (Lactococcus lactis JWA 32), characterized in that it has the deposit number: CCTCC No. M20221851.

2. The microbial agent is characterized by comprising any one of the following components and acceptable auxiliary agents;

lactococcus lactis JWA32 (Lactococcus lactis JWA 32) according to claim 1; and/or

(II) an inactivated Lactococcus lactis JWA32 (Lactococcus lactis JWA 32) as set forth in claim 1; and/or

(III) a fermentation broth, culture, exosome, lysate or extract of Lactococcus lactis JWA32 (Lactococcus lactis JWA 32) as claimed in claim 1.

3. The microbial inoculant of claim 2, wherein said fermentation broth comprises a fermentation product filtrate or a fungal fermentation lysate filtrate.

4. The microbial inoculant according to claim 2 or 3, wherein the microbial inoculant is in a dosage form comprising: one or more of emulsion, aqua, ointment, pellicle or gel.

5. The process for the preparation of a microbial inoculant according to any one of claims 2 to 4, comprising the steps of:

s1: inoculating and culturing the lactococcus lactis to obtain a seed solution;

s2: taking the seed liquid for fermentation culture, performing first centrifugation and filtration, and collecting supernatant to obtain the microbial agent; or

And (3) taking the seed liquid for fermentation culture, crushing thalli, performing secondary centrifugation and filtration, and collecting supernatant to obtain the microbial agent.

6. Use of lactococcus lactis according to claim 1, the microbial agent according to any one of claims 2 to 4 or the microbial agent obtained by the production method according to claim 5 for producing a product which reduces ROS levels, inhibits hyaluronidase activity, increases ceramide secretion or increases sodase activity.

7. Use of lactococcus lactis according to claim 1, the microbial agent according to any one of claims 2 to 4 or the microbial agent obtained by the production method according to claim 5 for producing a product for alleviating oxidative damage of HaCaT cells, repairing epidermal barriers or inhibiting harmful bacteria.

8. Use of lactococcus lactis according to claim 1, a microbial agent according to any one of claims 2 to 4 or a microbial agent obtained by the production method according to claim 5 for producing an antioxidant, anti-aging or anti-wrinkle product.

9. Product comprising lactococcus lactis according to claim 1, a microbial agent according to any one of claims 2 to 4 or obtained by the preparation process according to claim 5, and acceptable adjuvants or adjuvants.

10. The product of claim 9, comprising: a cosmetic is provided.

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