CN114717130B

CN114717130B - Lactobacillus rhamnosus capable of producing exopolysaccharide at high yield and application of lactobacillus rhamnosus in relieving skin injury

Info

Publication number: CN114717130B
Application number: CN202111043542.3A
Authority: CN
Inventors: 彭书东; 段治; 吴松洁
Original assignee: QINGDAO VLAND BIOTECH Inc; Qingdao Vland Biotech Group Co Ltd
Current assignee: QINGDAO VLAND BIOTECH Inc; Qingdao Vland Biotech Group Co Ltd
Priority date: 2021-09-07
Filing date: 2021-09-07
Publication date: 2023-08-29
Anticipated expiration: 2041-09-07
Also published as: WO2023035634A1; CN114717130A

Abstract

Lactobacillus rhamnosus strain with high extracellular polysaccharide yield and application thereof in relieving skin injury are provided. The invention provides a novel lactobacillus rhamnosus strainLactobacillus rhamnosus) And applications thereof. The lactobacillus rhamnosus can produce extracellular polysaccharide with good antioxidant activity, can effectively inhibit skin from secreting pro-inflammatory cytokines, and relieve damage of ultraviolet UVB to skin, and has a preservation number of CCTCC NO: m2021589. The lactobacillus rhamnosus has no toxic or harmful effect on organisms, is expected to be used for preparing sun-proof cosmetics, and has wide application prospect.

Description

Lactobacillus rhamnosus capable of producing exopolysaccharide at high yield and application of lactobacillus rhamnosus in relieving skin injury

Technical Field

The invention belongs to the technical field of screening and application of probiotics, and particularly relates to lactobacillus rhamnosus with high extracellular polysaccharide yield and application of lactobacillus rhamnosus in relieving skin injury.

Background

Human skin mainly comprises the epidermis layer and the dermis layer 2 tissue. The solar ultraviolet rays irradiated to the skin of the human body contain 90-99% UVA and 1-10% UVB. At the same dose, UVB was more damaging to the skin than UVA by a factor of 1000. And UVB acts mainly on keratinocytes (HaCaT) of the epidermis layer, causing damage to intracellular proteins, DNA and membrane lipids, causing photodamage to the skin due to metabolic disorders, and serious skin cancer. The traditional sun cream can cause side effects such as skin inflammation and allergy. Thus, the use of natural products produced by plants, animals and microorganisms for uv protection has received attention. Studies have now found that the natural flavonoid silymarin protects cells from UVB damage by upregulating the ER alpha and ER beta protein pathways; trehalose resists UVB-induced skin aging by inhibiting the expression of MMPs; extracellular polysaccharide from pantoea agglomerans is able to protect HaCaT cells from UVB damage.

Lactic acid bacteria are ubiquitous probiotics, known as "general safety bacteria", which have been widely used in the fields of foods, medicines, cosmetics, and the like. Lactic acid bacteria can produce natural metabolites such as organic acids, antibacterial peptides, extracellular polysaccharides and the like. Lactic acid bacteria Exopolysaccharide (EPS) refers to macromolecules produced by lactic acid bacteria during their growth and metabolism and secreted outside the cell wall, mainly capsular polysaccharides attached to the cell wall and mucopolysaccharides entering the fermentation broth. In recent years, the research discovers that the lactobacillus and the extracellular polysaccharide thereof have good application effects in the aspects of resisting tumor, resisting inflammation, regulating immunity, resisting bacteria and the like. Functional studies of lactic acid bacteria and their extracellular polysaccharides in terms of resistance to uv damage are also a hotspot in the art.

Disclosure of Invention

The invention aims to provide a novel lactobacillus rhamnosus (Lactobacillus rhamnosus) and application thereof. The lactobacillus rhamnosus high-yield extracellular polysaccharide has good antioxidant activity, can effectively inhibit skin from secreting pro-inflammatory reaction cytokines, and can relieve the damage of ultraviolet UVB to the skin, and the effect is remarkable.

In one aspect, the invention provides lactobacillus rhamnosus, named lactobacillus rhamnosus VHProbi O17 (Lactobacillus rhamnosus VHProbi O), which has been preserved in the China center for type culture collection, university of armed Han and Wuhan in China, for 24 days in 2021, with a preservation number of cctccc NO: m2021589.

In one aspect, the invention provides the use of lactobacillus rhamnosus VHProbi O17 for the manufacture of a product for the prevention and alleviation of skin lesions.

The product is a cosmetic.

In one aspect, the invention provides the use of lactobacillus rhamnosus VHProbi O17 for the production of extracellular polysaccharides.

The invention also provides an extracellular polysaccharide which is produced by taking lactobacillus rhamnosus VHProbi O17 as a fermentation strain.

The extracellular polysaccharide consists of 6 monosaccharides of D-galactosamine hydrochloride, arabinose, glucosamine hydrochloride, galactose, glucose and mannose, and the molar ratio is 0.05:0.03:0.09:0.05:0.19:1.98.

the extracellular polysaccharide contains beta-mannopyranose.

The molecular weight of the extracellular polysaccharide is 82.4kDa.

The invention also provides application of the extracellular polysaccharide in preparation of products for preventing and relieving skin injury.

The invention also provides a cosmetic which comprises lactobacillus rhamnosus VHProbi O17 and/or exopolysaccharide produced by lactobacillus rhamnosus VHProbi O17 fermentation.

The lactobacillus rhamnosus VHProbi O17 provided by the invention has strong antioxidant activity, and the clearance rate of DPPH and hydroxyl free radical reaches 51.73% and 26.04% respectively.

The lactobacillus rhamnosus VHProbi O17 has no toxic influence on skin cells, has a certain growth promoting effect, and can effectively relieve the damage of UVB irradiation to the cells. After the HaCaT cells are pretreated by the inactivated lactobacillus rhamnosus VHProbi O17 and are irradiated by UVB, the activity reduction amplitude is obviously reduced. Wherein the bacterial count is 8×10 ⁸ At CFU/mL, the activity of HaCaT cells after UVB irradiation is improved by 76.6% compared with that of cells after pretreatment without bacteria. The strain can also remarkably inhibit UVB-induced cells from secreting and producing pro-inflammatory cytokines IL-6 and IL-8, and the secretion amount of IL-6 and IL-8 is respectively reduced by 34.4% and 54.2%, thus unexpected technical effects are achieved.

Lactobacillus rhamnosus VHProbi O17 can produce a novel extracellular polysaccharide OP-2, which consists of 6 monosaccharides of D-galactosamine hydrochloride, arabinose, glucosamine hydrochloride, galactose, glucose and mannose, and can effectively relieve the damage of UVB to the skin. When the irradiation dose is 15mJ/cm ² When the extracellular polysaccharide OP-2 is added to 16mg/mL, the activity of the pretreated HaCaT cells after UVB irradiation is up to 65.7%, and the effect is very remarkable. The extracellular polysaccharide OP-2 can also effectively inhibit UVB from inducing skin cells to secrete IL-1 alpha, IL-6 and IL-8 3 cytokines. After pretreatment of extracellular polysaccharide OP-2, the content of IL-1 alpha in the supernatant is reduced by 20% -91% and the content of IL-6 and IL-8 is reduced by 67% and 23% respectively by UVB induction, thus unexpected technical effects are achieved.

The lactobacillus rhamnosus VHProbi O17 and extracellular polysaccharide produced by the lactobacillus rhamnosus VHProbi O17 can be widely applied to cosmetics and have a wide prospect.

Drawings

FIG. 1 is a colony chart and gram stain chart of strain O17; wherein A is a colony chart and B is a gram staining chart;

FIG. 2 is a protein fingerprint of strain O17;

FIG. 3 is a Riboprinter fingerprint of strain O17;

FIG. 4 is a RAPD fingerprint of strain O17;

FIG. 5 is a rep-PCR fingerprint of strain O17;

FIG. 6 is a graph comparing the effect of Lactobacillus rhamnosus VHProbi O17 on alleviating UVB damage to skin cells;

FIG. 7 shows the growth curve of Lactobacillus rhamnosus VHProbi O17 and the production curve of extracellular polysaccharide;

FIG. 8 is a DEAE- μSphere elution chromatogram of Lactobacillus rhamnosus VHProbi O17 exopolysaccharide;

FIG. 9 is an ion chromatograph of extracellular polysaccharide OP-2;

FIG. 10 is an infrared spectrum analysis of extracellular polysaccharide OP-2;

FIG. 11 is a gel column analysis chart and standard graph of extracellular polysaccharide OP-2; wherein A is a gel column analysis chart, and B is a standard curve chart;

FIG. 12 is a graph comparing the effect of extracellular polysaccharide OP-2 on alleviating UVB damage to skin cells;

FIG. 13 is a graph comparing the inhibition of UVB-induced skin cytokine production by the extracellular polysaccharide OP-2.

Detailed Description

The screening method of the present invention is not limited to the examples, but known screening methods can be used to achieve the screening purpose, and the screening description of the examples is only illustrative of the present invention and is not intended to limit the scope of the present invention. Modifications and substitutions to methods, procedures, or conditions of the present invention without departing from the spirit and nature of the invention are intended to be within the scope of the present invention.

The invention will be further illustrated with reference to specific examples.

EXAMPLE 1 screening of strains for high extracellular polysaccharide production

Preparing MRS agar culture medium: 1000mL of pure water, 10g of peptone, 10g of beef extract, 5.0g of yeast extract, 5g of sodium acetate, 5g of glucose, 2g of monopotassium phosphate, 1.0mL of Tween 80, 2.0g of diamine citrate, 20g of calcium carbonate, 0.58g of magnesium sulfate heptahydrate, 0.25g of manganese sulfate heptahydrate, 15g of agar, pH adjustment of 6.2-6.5 and high-pressure sterilization at 121 ℃ for 15min.

After more than 200 strains of lactic acid bacteria stored in the strain library are respectively activated, the strains are inoculated on an MRS plate and cultured for 48 hours at 37 ℃, the strain 'sticky' phenotype is observed, and the length of a formed wire when a colony is contacted by an inoculating loop is detected. Finally, a lactobacillus strain with high colony viscosity and longest wiredrawing is screened out and named as O17.

Example 2 identification of strains

2.1 colony morphology identification

After the strain O17 is inoculated on an MRS agar medium and cultured for 48 hours at 37 ℃, O17 single colonies are smooth and off-white, the strain O17 single colonies are short-rod-shaped under a microscope, two ends are level, and the photographs of the O17 single colonies and the photographs under an optical microscope are shown in figure 1.

2.2 MALDI-TOF-MS detection of full protein expression of strains

Inoculating fresh bacterial liquid into MRS liquid culture medium according to 0.1% inoculum size, culturing at 37deg.C and 150rpm for 48 hr, collecting bacterial cells, washing with sterile water for 4 times, and air drying surface water. And then a small amount of fresh thalli is uniformly coated on a target plate in a film form, 1 mu L of lysate is added to cover the sample, after the sample is dried, 1 mu L of matrix solution is added to cover the sample, after the sample is dried, the sample target is put into a mass spectrometer for identification. The co-crystallization film formed by the laser irradiation sample and the matrix ionizes the protein in the sample, the ions are accelerated to fly through the flight pipeline under the action of the electric field of 10 KV to 20KV, and the proteins with different molecular weights are detected according to different flight times reaching the detector. Data analysis was performed using the software Autof Acquirer V2.0.59, strain O17 had distinct ion peaks at m/z 2946.082, 3476.573, 3791.097, 4692.724, 5892.635, 6953.051, 7581.626, 9382.474, etc., and the results are shown in FIG. 2. After data comparison analysis, the protein expression similarity of the strain O17 and lactobacillus rhamnosus is highest.

2.3 molecular biological identification

2.4.1 16s rDNA sequence analysis

1. Genomic DNA extraction

Reference was made to the Tiangen bacterial genomic DNA extraction kit (catalog number: DP 302).

2. 16s rDNA Gene amplification

1) Primer sequence:

27F：AGAGTTTGATCCTGGCTCA；

1492R：GGTTACCTTGTTACGACTT。

2) Reaction system (50. Mu.L)

Table 2:16s rDNA PCR amplification System Table

3) Electrophoresis verifies that the PCR product meets the requirement when the nucleic acid electrophoresis result is about 1500 bp.

4) Sequencing of PCR products

The 16s rDNA sequence of the O17 strain, SEQ ID NO. 1, was obtained by sequencing and the sequences were aligned in NCBI database. The results show that SEQ ID NO. 1 has the highest similarity to Lactobacillus rhamnosus (Lactobacillus rhamnosus), and therefore, the O17 strain was initially identified as Lactobacillus rhamnosus (Lactobacillus rhamnosus).

SEQ ID NO:1：

ctcgctccctaaaagggttacgccaccggcttcgggtgttacaaactctcatggtgtgacgggcggtgtgtacaaggcccgggaacgtattcaccgcggcgtgctgatccgcgattactagcgattccgacttcgtgtaggcgagttgcagcctacagtccgaactgagaatggctttaagagattagcttgacctcgcggtctcgcaactcgttgtaccatccattgtagcacgtgtgtagcccaggtcataaggggcatgatgatttgacgtcatccccaccttcctccggtttgtcaccggcagtcttactagagtgcccaactaaatgctggcaactagtcataagggttgcgctcgttgcgggacttaacccaacatctcacgacacgagctgacgacaaccatacaccacctgtcattttgcccccgaaggggaaacctgatctctcaggtgatcaaaagatgtcaagacctggtaaggttcttcgcgttgcttcgaattaaaccacatgctccaccgcttgtgcgggcccccgtcaattcctttgagtttcaaccttgcggtcgtactccccaggcggaatgcttaatgcgttagctgcggcactgaagggcggaaaccctccaacacctagcattcatcgtttacggcatggactaccagggtatctaatcctgttcgctacccatgctttcgagcctcagcgtcagttacagaccagacagccgccttcgccactggtgttcttccatatatctacgcatttcaccgctacacatggagttccactgtcctcttctgcactcaagtttcccagtttccgatgcacttcctcggttaagccgagggctttcacatcagacttaaaaaaccgcctgcgctcgctttacgcccaataaatccggataacgcttgccacctacgtattaccgcggctgctggcacgtagttagccgtggctttctggttggataccgtcacgccgacaacagttactctgccgaccattcttctccaacaacagagttttacgacccgaaagccttcttcactcacgcggcgttgctccatcagacttgcgtccattgtggaagattccctactgctgcctcccgtaggagtttgggccgtgtctcagtcccaatgtggccgatcaacctctcagttcggctacgtatcattgccttggtgagccgttacctcaccaactagctaatacgccgcgggtccatccaaaagcgatagcttacgccatctttcagccaagaaccatgcggttcttggatttatgcggtattagcatctgtttccaaatgttatcccccacttaagggcaggttacccacgtgttactcacccgtccgccactcgttcaaaattaaatcaagatgcaagcacctttcaataatcagaactcgttcgactgc。

2.4.2 Riboprinter fingerprint

The purified single colony is dipped from an agar culture medium flat plate by a fungus taking rod, the single colony is placed into a sample tube with buffer solution, the single colony is stirred by a hand-held stirrer to be suspended in the buffer solution, then the sample holder is placed into a heater for inactivation and then placed into a Riboprinter system, and the Riboprinter fingerprint of the strain O17 is obtained after DNA preparation, film transfer, imaging detection and data processing of the sample (figure 3).

2.4.3 RAPD and rep-PCR fingerprint identification

1. RAPD fingerprint identification

1) Primer sequence: m13 (5'-GAGGGTGGCGGTTCT-3');

2) RAPD reaction system

TABLE 3 RAPD reaction System

3) Electrophoresis

1.5% agarose gel plates were prepared, DL2000 DNA markers were used as a result control, 100V electrophoresis was performed for 80min at a constant pressure, and finally the electropherograms were detected using a gel imaging system. RAPD finger-print of strain O17 is shown in FIG. 4.

2. rep-PCR fingerprint

1) rep-PCR primer

CTACGGCAAGGCGACGCTGACG。

2) reaction system of rep-PCR

TABLE 4 rep-PCR reaction System

3) Electrophoresis

DL2000 DNA Marker served as a result control. Detecting the amplification result by 100V voltage and 80min electrophoresis time. The rep-PCR fingerprint of strain O17 is shown in FIG. 5.

In summary, by combining colony morphological characteristics, protein fingerprint patterns and molecular biological identification results of the O17 strain, it can be concluded that the O17 strain provided by the invention is a novel lactobacillus rhamnosus strain, and is named as lactobacillus rhamnosus VHProbi O17 (Lactobacillus rhamnosus VHProbi O17).

Example 3 antioxidant Activity of Lactobacillus rhamnosus VHProbi O17

1. Determination of DPPH free radical scavenging ability of Strain

1) Preparation of PBS bacterial suspension

Single colony with excellent growth state is inoculated into 3mL of MRS liquid culture medium, and is cultured for 24h at 37 ℃, the culture solution is taken as an inoculating solution, and is inoculated into 50mL of MRS liquid culture medium according to the inoculating amount of 2 percent, and the culture solution of the strain is obtained by standing and culturing for 24h. After 1mL of bacterial liquid is sucked up and bacterial cells are collected, the bacterial cells are washed by 1mL of buffer solution for 2 times, and then 2mL of buffer solution is added to resuspend the bacterial cells for standby.

2) Determination of DPPH free radical scavenging ability of Strain

1mL of PBS bacterial suspension of the strain to be detected is taken, 1mL of 0.4mM DPPH free radical solution is added, after uniform mixing, the mixture is placed at room temperature for shading reaction for 30min, then the absorbance A sample of the sample at the wavelength of 517nm is measured, and the sample is measured for 3 times of parallelism. The control samples were zeroed with equal volumes of PBS and DPPH ethanol mixed solution and with equal volumes of PBS and ethanol mixed solution. The clearance is calculated according to the following formula: clearance% = [1- (a) _{Sample of} -A _{Blank space} )/A _Control ]×100％。

2. Determination of the ability of the Strain to scavenge hydroxyl radicals

100. Mu.L of 5mM sodium salicylate-ethanol solution, 100. Mu.L of 5mM ferrous sulfate, 500. Mu.L of deionized water and 200. Mu.L of lactic acid bacteria PSP solution were mixed uniformly, 100. Mu.L of hydrogen peroxide solution (3 mM) was added thereto, and after 15min in a 37℃water bath, the supernatant was collected by centrifugation at 4000rpm for 10min and the absorbance of the sample was measured at a wavelength of 510 nm. The hydroxyl radical scavenging rate was calculated according to the following formula.

Clearance% = (a _{Sample of} -A _{Control of} )/(A _{Blank space} -A _{Control of} )×100％。

Wherein: a is that _{Control of} Is the absorbance of the mixed solution of ferrous sulfate, hydrogen peroxide and sodium salicylate, A _{Blank space} Is the absorbance of the mixed solution of ferrous sulfate and sodium salicylate.

TABLE 5 antioxidant Activity

From the data in Table 5, it can be seen that the Lactobacillus rhamnosus VHProbi O17 provided by the invention can effectively remove DPPH and hydroxyl free radicals, and the removal rates respectively reach 51.73% and 26.04%. Thus, the lactobacillus rhamnosus VHProbi O17 has strong antioxidant activity and achieves unexpected technical effects.

Example 4 Effect of Lactobacillus rhamnosus VHProbi O17 against UVB radiation

1. HaCaT cell culture

HaCaT keratinocytes were anaerobically cultured in high-sugar DMEM medium containing 10% (v/v) FBS and 1% (v/v) penicillin-streptomycin. Culture conditions: the carbon dioxide incubator was charged with 5% (v/v) carbon dioxide, and the medium was changed every 3 days at 37 ℃.

2. Toxicity analysis of Lactobacillus rhamnosus VHProbi O17 on cells

Lactobacillus rhamnosus VHProbi O17 was inoculated in 100 mmlmrs medium and aerobically fermented at 37 ℃ for 40h; centrifuging at 6000 Xg for 10min, and discarding supernatant; washing the thalli for 2 times by using PBS buffer solution; pure water was added to prepare 1.6X10 concentrations respectively ⁷ 、8×10 ⁷ 、4×10 ⁸ 、2×10 ⁹ And 1X 10 ¹⁰ CFU/mL bacterial liquid; heating the bacterial liquid at 100deg.C for 20min to inactivate the bacterial; and freeze-drying bacterial solutions with different concentrations for later use, and respectively dissolving the bacterial solutions with equal volumes of DEME culture medium.

HaCaT cells were cultured in 24 well plates for 24h, and the medium was removed; adding the above inactivated Lactobacillus rhamnosus VHProbi O17 (bacterial amount of 0,1×10) ⁸ ，2×10 ⁸ ，4×10 ⁸ ，8×10 ⁸ And 16X 10 ⁸ CFU/mL) of DMEM medium, incubating for 2h; then, each well was replaced with 500. Mu.L of fresh DMEM medium and incubated for 24 hours. The toxicity of lactobacillus rhamnosus VHProbi O17 to cells was evaluated by MTT method, specifically: 50 mu L of MTT (thiazole blue) solution with concentration of 3mg/mL is added to each well of a 24-well plate, and incubated for 3 hours in a dark place; the solution was aspirated from the wells and 500 μl of dimethyl sulfoxide was added to each well to dissolve and absorbance was detected at 450nm using a microplate reader.

3. UVB-induced HaCat cells secrete pro-inflammatory cytokines

HaCaT cells were cultured in 24 well plates for 24h, and the medium was removed; adding the above inactivated Lactobacillus rhamnosus VHProbi O17 (bacterial amounts are 0,1×10 respectively) ⁸ ，2×10 ⁸ ，4×10 ⁸ ，8×10 ⁸ CFU/mL) of DMEM medium, incubating for 2h; the medium was aspirated and 150 μl PBS buffer was added to each well; irradiating with ultraviolet lamp with ultraviolet intensity of 250 μw/cm ² The irradiation dose was 15mJ/cm. After culturing for 24 hours in DMEM medium, the cell activity was measured by MTT method. HaCaT cells that were not pre-treated with UV irradiation and Lactobacillus rhamnosus VHProbi O17 were used as controls.

4. Cytokine analysis

Taking the cell culture supernatant after ultraviolet irradiation, respectively detecting the concentrations of pro-inflammatory cytokines interleukin-6 (IL-6) and interleukin-8 (IL-8) by using an ELISA kit, and referring to the operation instruction. The results are shown in FIG. 6.

As is clear from the results of FIG. 6, the bacterial count was 8X 10 or less ⁸ At CFU/mL, the inactivated lactobacillus rhamnosus VHProbi O17 has no inhibition effect on the growth of HaCaT cells; at 2X 10 ⁸ ～8×10 ⁸ In the CFU/mL range, the inactivated lactobacillus rhamnosus VHProbi O17 has a certain promotion effect on cell growth with the increase of the bacterial amount (FIG. 6A).

After UVB irradiation, the activity of HaCaT cells was reduced to different extents. However, after the HaCaT cells are pretreated by the inactivated lactobacillus rhamnosus VHProbi O17 and are irradiated by UVB, the activity reduction amplitude is obviously reduced. Wherein the bacterial count is 8×10 ⁸ At CFU/mL, the activity of HaCaT cells after UVB irradiation was 76.6% higher than that without pretreatment (FIG. 6B).

Furthermore, pretreatment with inactivated lactobacillus rhamnosus VHProbi O17 can significantly inhibit UVB-induced HaCaT cells from secreting pro-inflammatory cytokines IL-6 and IL-8. After UVB irradiation, the content of IL-6 and IL-8 in the culture supernatant of the HaCaT cells increases sharply, reaching 478.41pg/mL and 522.583pg/mL at the highest; however, the bacterial load was 8X 10 ⁸ After CFU/mL of the inactivated Lactobacillus rhamnosus VHProbi O17 pretreatment, IL-6 and IL-8 production was significantly reduced by 34.4% and 54.2%, respectively (FIGS. 6C, 6D).

In conclusion, the lactobacillus rhamnosus VHProbi O17 provided by the invention has no toxicity to cells, can effectively relieve the damage of UVB irradiation to the cells, and achieves unexpected technical effects.

EXAMPLE 5 preparation of Lactobacillus rhamnosus VHProbi O17 exopolysaccharide

1. Preparation of extracellular polysaccharide

Inoculating activated lactobacillus rhamnosus VHProbi O17 into 100mL of MRS culture medium, culturing at 37 ℃ for 56h, taking 5mL of bacterial liquid every 8h, and centrifuging at 6000 Xg for 10min to obtain supernatant. The supernatants were dialyzed against 1000Da dialysis bags for 48 hours, with pure water changed every 12 hours. And detecting the content of extracellular polysaccharide by using a phenol-sulfuric acid method after the dialysis is finished. As a result, as shown in FIG. 7, lactobacillus rhamnosus VHProbi O17 reached the stationary phase at 32h and the extracellular polysaccharide yield reached the maximum value at 40h, which was 0.603mg/mL.

2. Separation and extraction of extracellular polysaccharide

Lactobacillus rhamnosus VHProbi O17 was inoculated into 1L of MRS medium, aerobically fermented at 37 ℃ for 40h, centrifuged at 6000×g for 10min, and the supernatant was obtained. Concentrating the supernatant under reduced pressure for 5 times, adding 2 times of cold absolute ethyl alcohol, and standing at 4 ℃ overnight; centrifuging at 12000 Xg for 15min to obtain crude polysaccharide precipitate; the alcohol precipitation step was repeated 1 time. Dissolving the crude polysaccharide precipitate with pure water, adding 4% (w/v) trichloroacetic acid, standing at 4deg.C for 2 hr, centrifuging at 8000 Xg for 15min, and removing protein. After deproteinization, the pH of the solution was adjusted to 4 with 6mol/L NaOH to avoid degradation of the polysaccharide under low pH conditions, and then 2 volumes of cold absolute ethanol were added and allowed to stand overnight with 12000 Xg centrifugation for 15min to give crude polysaccharide. The crude polysaccharide obtained was dissolved and dialyzed with 1000Da dialysis bags for 48 hours, with pure water replaced every 12 hours, and after the dialysis was completed, the pH of the solution was adjusted to 6.5 with 1mol/L NaOH.

The dialyzed solution was put into a column packed with DEAE-. Mu.Sphere (1.2X11.4 cm), and polysaccharide was eluted stepwise with pure water and NaCl of different concentrations (0.05,0.1,0.2,0.4,0.6M) at a flow rate of 1mL/min, after which the eluate was collected at 5mL per tube, and the absorbance at 490nm per tube was measured by phenol-sulfuric acid method; the polysaccharide-containing collection tubes were pooled, and samples were dialyzed and lyophilized for concentration for further study.

The results are shown in FIG. 8: the most active extracellular polysaccharide eluted from 0.1M NaCl was identified as OP-2.

Example 6 structural characterization of Lactobacillus rhamnosus VHProbi O17 exopolysaccharide production

1) Monosaccharide composition

In a closed tube, about 5mg of the extracellular polysaccharide OP-2 sample was hydrolyzed with 2M trifluoroacetic acid at 105℃for 6h and the sample was dried with nitrogen. Then methanol is added for washing and drying is carried out, and the washing is repeated for 3 times. The residue was redissolved in deionized water and filtered through a 0.22 μm microporous filter membrane, and analyzed using ICS5000 ion chromatography detection. Chromatographic parameters: using Dionex ^TM CarboPac ^TM PA10 (250×4.0mm,10 um) liquid chromatography column; the sample loading was 5uL. Mobile phase a (0.1M NaOH), mobile phase B (0.1M NaOH,0.2M NaAc), flow rate 0.5mL/min; the column temperature is 30 ℃; elution gradient: 0min A/B (95:5, v/v), 30min A/B (80:20 v/v), 30.1min A/B (60:40 v/v), 45min A/B (60:40 v/v), 45.1min A/B (95:5 v), 60min A/B (95:5 v). Finally, chromatographic data analysis was performed using software Chromeleon 7.2, and the results are shown in fig. 9.

2) Infrared spectroscopic analysis

The dried 2mg of extracellular polysaccharide OP-2 was mixed with 200mg of KBr powder, ground to homogeneity and tableted. FT-IR spectrometry is used for infrared scanning of samples, with a wavelength in the range 4000-400cm ^-1 . An infrared spectrum was recorded as shown in fig. 10.

3) Molecular weight

Extracellular polysaccharide OP-2 was formulated as a 5mg/mL solution, centrifuged at 12000 Xg for 10min, the supernatant was filtered through a 0.22 μm microporous filter membrane, and the sample was transferred to a 1.8mL sample-in vial. A20 uL sample was injected into a high performance liquid chromatograph with a BRT105-104-102 serial gel column and differential detector for analysis. The molecular weight of each sample was calculated from the standard (molecular weight: 1152, 5000, 11600, 23800, 48600, 80900, 148000, 273000, 409800, 667800 Da) curve by deriving a calculation formula, as shown in FIG. 11.

As shown by the chromatographic analysis result, the extracellular polysaccharide OP-2 produced by the lactobacillus rhamnosus VHProbi O17 provided by the invention consists of 6 monosaccharides of D-galactosamine hydrochloride, arabinose, glucosamine hydrochloride, galactose, glucose and mannose, and the molar ratio is 0.05:0.03:0.09:0.05:0.19:1.98. the infrared spectrum scan shows that the extracellular polysaccharide OP-2 exists in beta-mannopyranose. In addition, the molecular weight of the extracellular polysaccharide OP-2 was found to be 82.4kDa by gel column analysis.

The above results show that the extracellular polysaccharide OP-2 produced by Lactobacillus rhamnosus VHProbi O17 has a different structural composition compared with the known extracellular polysaccharide, and therefore the polysaccharide is a novel lactobacillus extracellular polysaccharide.

Example 7 Effect of extracellular polysaccharide OP-2 on HaCaT cells

1. HaCaT cell culture

Reference example 4.

2. Toxicity of extracellular polysaccharide OP-2 to cells

HaCatT cells were cultured in 24-well plates for 24h, the medium was removed, DMEM medium containing 0,0.5,1,2,4,8, 16mg/mL purified extracellular polysaccharide OP-2 was added, respectively, and incubated for 2h; then each well was replaced with 500. Mu.L of fresh DMEM medium for 24h. After the cell culture is finished, observing the cell morphology by an inverted microscope, and evaluating the toxicity of the extracellular polysaccharide OP-2 to the HaCaT cells by adopting an MTT method.

From the results of FIG. 12A, it can be seen that the activity of HaCat cells treated with the exopolysaccharide OP-2 was not significantly reduced, thus indicating that the exopolysaccharide OP-2 had no toxic effect on the HaCat cells.

3. UVB-induced HaCat cells

HaCat cells cultured in 24-well plates for 24h were first incubated for 2h in DMEM medium containing 0,1, 2,4,8 and 16mg/mL of extracellular polysaccharide OP-2, respectively; then the medium was aspirated and 150 μl of PBS buffer was added to each well; irradiating with ultraviolet lamp with ultraviolet intensity of 250 μw/cm ² Irradiation doses were 7.5, 15 and 30mJ/cm, respectively ² . After irradiation, haCat cells were continuously cultured in DMEM medium for 24 hours, and then cell activity was detected by MTT method. HaCat cells not pre-treated with UV irradiation and extracellular polysaccharide OP-2 were used as controls.

From the results of FIGS. 12B,12C,12D, it can be seen that UVB irradiation significantly reduced the activity of HaCaT cells, but that after pretreatment with the extracellular polysaccharide OP-2, UVB damage to the cells was alleviated. When the irradiation dose is 15mJ/cm ² When the extracellular polysaccharide OP-2 is added to 16mg/mL, the activity of the pretreated HaCaT cells after UVB irradiation is up to 65.7%, and unexpected technical effects are achieved.

4. Cytokine analysis

The irradiation dose is 15mJ/cm ² The concentrations of the four pro-inflammatory cytokines IL-1α, IL-1β, IL-6, IL-8 were measured using ELISA kit, and the results are shown in FIG. 13.

From the results of FIG. 13, it can be seen that the pro-inflammatory factors IL-1α, IL-6 and IL-8 secreted by HaCaT cells were dramatically increased after UVB induction, while the secretion amount of the inflammatory factor IL-1β was not significantly changed. The HaCaT cells pretreated by the exopolysaccharide OP-2 are induced by UVB to secrete IL-1 alpha, IL-6 and IL-8, which are inhibited to a certain extent. Taking IL-1α as an example, after UVB induction, the concentration of IL-1α in HaCat cell supernatants increased from 1.41pg/mL to 74.97pg/mL; the content of IL-1α in the supernatant was reduced by 20% -91% by pretreatment of HaCat cells with exopolysaccharide OP-2 (1-16 mg/mL) and then UVB induction, and the content was reduced to 6.73pg/mL (FIG. 13A). Similarly, IL-6 and IL-8 have similar results, with 67% and 23% reduction (FIGS. 13C, 13D), respectively, with unexpected technical results.

In conclusion, the lactobacillus rhamnosus VHProbi O17 provided by the invention has strong antioxidant activity, and the clearance rate of DPPH and hydroxyl free radical reaches 51.73% and 26.04% respectively. The strain has no toxic effect on skin cells and has a certain growth promoting effect. Lactobacillus rhamnosus VHProbi O17 can effectively reduce the damage of UVB irradiation to cells, remarkably inhibit secretion of cells induced by UVB to produce pro-inflammatory cytokines IL-6 and IL-8, and reduce the secretion amounts of IL-6 and IL-8 by 34.4% and 54.2%, respectively. Lactobacillus rhamnosus VHProbi O17 is able to produce a new extracellular polysaccharide OP-2 consisting of 6 monosaccharides D-galactosamine hydrochloride, arabinose, glucosamine hydrochloride, galactose, glucose and mannose. The extracellular polysaccharide can obviously inhibit UVB-induced skin cells from secreting IL-1 alpha, IL-6 and IL-8 3 cytokines, and relieve the damage of UVB to skin. The lactobacillus rhamnosus VHProbi O17 and extracellular polysaccharide produced by the lactobacillus rhamnosus VHProbi O17 can be widely applied to cosmetics and have a wide prospect.

Sequence listing

<110> Qingdao blue organism Co., ltd

<120> Lactobacillus rhamnosus with high extracellular polysaccharide yield and application thereof in relieving skin injury

<160> 1

<170> SIPOSequenceListing 1.0

<210> 1

<211> 1432

<212> DNA

<213> Lactobacillus rhamnosus (Lactobacillus rhamnosus)

<400> 1

ctcgctccct aaaagggtta cgccaccggc ttcgggtgtt acaaactctc atggtgtgac 60

gggcggtgtg tacaaggccc gggaacgtat tcaccgcggc gtgctgatcc gcgattacta 120

gcgattccga cttcgtgtag gcgagttgca gcctacagtc cgaactgaga atggctttaa 180

gagattagct tgacctcgcg gtctcgcaac tcgttgtacc atccattgta gcacgtgtgt 240

agcccaggtc ataaggggca tgatgatttg acgtcatccc caccttcctc cggtttgtca 300

ccggcagtct tactagagtg cccaactaaa tgctggcaac tagtcataag ggttgcgctc 360

gttgcgggac ttaacccaac atctcacgac acgagctgac gacaaccata caccacctgt 420

cattttgccc ccgaagggga aacctgatct ctcaggtgat caaaagatgt caagacctgg 480

taaggttctt cgcgttgctt cgaattaaac cacatgctcc accgcttgtg cgggcccccg 540

tcaattcctt tgagtttcaa ccttgcggtc gtactcccca ggcggaatgc ttaatgcgtt 600

agctgcggca ctgaagggcg gaaaccctcc aacacctagc attcatcgtt tacggcatgg 660

actaccaggg tatctaatcc tgttcgctac ccatgctttc gagcctcagc gtcagttaca 720

gaccagacag ccgccttcgc cactggtgtt cttccatata tctacgcatt tcaccgctac 780

acatggagtt ccactgtcct cttctgcact caagtttccc agtttccgat gcacttcctc 840

ggttaagccg agggctttca catcagactt aaaaaaccgc ctgcgctcgc tttacgccca 900

ataaatccgg ataacgcttg ccacctacgt attaccgcgg ctgctggcac gtagttagcc 960

gtggctttct ggttggatac cgtcacgccg acaacagtta ctctgccgac cattcttctc 1020

caacaacaga gttttacgac ccgaaagcct tcttcactca cgcggcgttg ctccatcaga 1080

cttgcgtcca ttgtggaaga ttccctactg ctgcctcccg taggagtttg ggccgtgtct 1140

cagtcccaat gtggccgatc aacctctcag ttcggctacg tatcattgcc ttggtgagcc 1200

gttacctcac caactagcta atacgccgcg ggtccatcca aaagcgatag cttacgccat 1260

ctttcagcca agaaccatgc ggttcttgga tttatgcggt attagcatct gtttccaaat 1320

gttatccccc acttaagggc aggttaccca cgtgttactc acccgtccgc cactcgttca 1380

aaattaaatc aagatgcaag cacctttcaa taatcagaac tcgttcgact gc 1432

Claims

1. Lactobacillus rhamnosus strainLactobacillus rhamnosus) VHProbi O17, characterized in that, the preservation number of lactobacillus rhamnosus is CCTCC NO: m2021589.

2. Use of lactobacillus rhamnosus as claimed in claim 1 for the manufacture of a product for preventing or alleviating skin damage, characterised in that the lactobacillus rhamnosus is an inactivated thallus.

3. The use according to claim 2, wherein the article is a cosmetic product.

4. An extracellular polysaccharide, which is produced by using the lactobacillus rhamnosus as defined in claim 1 as a fermentation strain, and consists of 6 monosaccharides of D-galactosamine hydrochloride, arabinose, glucosamine hydrochloride, galactose, glucose and mannose, wherein the molar ratio is 0.05:0.03:0.09:0.05:0.19:1.98.

5. the extracellular polysaccharide according to claim 4, wherein said extracellular polysaccharide comprises β -mannopyranose.

6. The extracellular polysaccharide according to claim 4 or 5, wherein the extracellular polysaccharide has a molecular weight of 82.4kDa.

7. Use of an extracellular polysaccharide according to any one of claims 4 to 6 for the preparation of a product for preventing or alleviating skin lesions.

8. A cosmetic product comprising the inactivated bacterial cell of lactobacillus rhamnosus of claim 1 and/or the extracellular polysaccharide of any of claims 4-6.