Lactobacillus paracasei with anti-inflammatory and soothing effects and application of lactobacillus paracasei in skin protection

Abstract

The invention belongs to the technical field of microorganisms, and discloses lactobacillus paracasei with anti-inflammatory and soothing effects and application of the lactobacillus paracasei in skin protection. The preservation number of the lactobacillus paracasei with the anti-inflammatory and soothing effects is GDMCC No: 63480. The lactobacillus paracasei has the anti-inflammatory and soothing effects. The lactobacillus paracasei LTHINK-0050 strain provided by the invention has high anti-inflammatory activity, has the function of improving and repairing the barrier of skin, and can be used for preparing medicaments, cosmetics and cosmetic raw materials with anti-inflammatory and soothing effects.

Description

Technical Field

[0001] The invention relates to the technical field of microorganisms, and in particular to a Lactobacillus paracasei with anti-inflammatory and soothing effects and application thereof in skin protection. Background Art

[0002] Skin is the largest organ in the human body. It covers the whole body and protects various tissues and organs in the body from physical, mechanical, chemical and pathogenic microbial invasions. Healthy skin condition is a hot topic pursued by most people nowadays. However, under the combined action of internal and external factors, various skin problems will inevitably occur. For example, the appearance of acne is due to the increase in sebum secretion caused by changes in hormone levels. About 90% of people will experience pain caused by acne. Personal living habits, diet, sleep patterns, stress, etc. all lead to the occurrence of acne. In addition, the size of pores is closely related to the vigorous secretion of sebaceous glands, skin aging and increased hair follicle volume. Large pores can make the facial skin look rough, loose and dull. In daily life, sunlight exposure can cause problems such as skin pigmentation. Facial pigmentation is not only related to sunlight exposure, but also to hormone levels and genetics. The appearance of wrinkles is inevitable in the process of skin aging, and is the result of the combined action of endogenous factors (natural aging) and exogenous factors. The skin is home to millions of bacteria, fungi, viruses and mites that make up the skin microbiome, which plays an important role in defending against invading pathogens, cultivating our immune system and breaking down natural products. When the microecological balance is broken, various skin problems will occur.

[0003] Probiotics are living microorganisms that can have beneficial effects on the host when ingested in sufficient amounts. The most studied probiotic microorganisms include Lactobacillus, Bifidobacterium, Bacillus, etc. Lactic acid bacteria are natural residents of the human gastrointestinal tract and play a key role in restoring the homeostasis of the human intestinal microbiota. Lactic acid bacteria can also produce a large amount of lactic acid and other metabolites, such as bacteriocins, short-chain fatty acids, and exopolysaccharides. Therefore, lactic acid bacteria have the ability to withstand human salivary enzymes and low pH values, can colonize intestinal epithelial cells, inhibit pathogens and reactive oxygen species associated with intestinal diseases, and play a physiological role in human health.

[0004] Lactic acid bacteria (LAB) are a type of probiotic that is currently widely studied, including Lactobacillus, Limosilactobacillus and Lactiplantibacillus. Lactic acid bacteria (LAB) are natural symbiotic bacteria in the human gastrointestinal tract, and play a vital role in maintaining the stability of intestinal flora and regulating human nutrition. Lactobacillus intracellular polysaccharides are a type of polysaccharides produced by lactobacilli. These polysaccharides are usually located in the outer wall layer of the cell wall and are composed of multiple monosaccharide units. These polysaccharides play an important role in the physiological functions of lactobacilli. The structure and composition of lactobacillus intracellular polysaccharides have an important influence on their functions. These polysaccharides can be homopolysaccharides or heteropolysaccharides, mainly composed of monosaccharides such as glucose, galactose, rhamnose, mannose, arabinose and xylose. The molecular weight and charge properties of polysaccharides can affect their solubility, viscosity and interaction with host cells.

[0005] Therefore, exploring and screening lactic acid bacteria cells with high production of extracellular polysaccharides from the environment is of great research significance and has very broad application value in the fields of food, medicine, medical devices, cosmetics, etc. Summary of the invention

[0006] The purpose of the present invention is to overcome the shortcomings of the prior art and provide a Lactobacillus paracasei LTHINK-0050 with anti-inflammatory and soothing effects and its application in skin protection.

[0007] To achieve the above purpose, the technical solution adopted by the present invention is as follows:

[0008] In the first aspect, the present invention provides a Lactobacillus paracasei with anti-inflammatory and soothing effects, and its preservation number is GDMCC No: 63480. The strain is named Lactobacillus paracasei LTHINK-0050, classified as Lacticaseibacillus paracasei, and was preserved in Guangdong Provincial Microbiological Culture Collection Center on May 19, 2023, and detected as alive; the preservation address is: Guangdong Institute of Microbiology, 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; Contact number: 020-87137633.

[0009] The Lactobacillus paracasei LTHINK-0050 of the present invention has high anti-inflammatory activity, can improve and repair the skin's own barrier function, and can be used to prepare cosmetics and cosmetic raw materials with anti-inflammatory and soothing effects.

[0010] In a second aspect, the present invention provides a fermentation lysate of the Lactobacillus paracasei.

[0011] The yield of extracellular polysaccharide in the fermentation lysate of the Lactobacillus paracasei LTHINK-0050 of the present invention is as high as 172.4±1.2 mg / L.

[0012] In a third aspect, the present invention provides an anti-inflammatory soothing preparation comprising the Lactobacillus paracasei and / or the fermentation lysate.

[0013] In a fourth aspect, the present invention uses the Lactobacillus paracasei and / or the fermentation lysate in the preparation of a medicine or cosmetic for anti-inflammatory and soothing skin.

[0014] In a fifth aspect, the present invention uses the Lactobacillus paracasei and / or the fermentation lysate in the preparation of skin care products.

[0015] In a sixth aspect, the present invention provides a method for preparing a fermentation lysate of Lactobacillus paracasei, comprising the following steps:

[0016] (1) fermenting and culturing the Lactobacillus paracasei to obtain a fermented bacterial liquid;

[0017] (2) taking the fermented bacterial liquid, breaking the bacterial cells, and obtaining a broken liquid;

[0018] (3) taking the crushed liquid and centrifuging it to collect the supernatant, filtering it, and obtaining the fermentation lysate of Lactobacillus paracasei.

[0019] As a preferred embodiment of the preparation method, in step (1), the fermentation culture is: firstly inoculating the Lactobacillus paracasei into a seed culture medium and carrying out seed culture at 37°C for 16h to 24h; then inoculating the Lactobacillus paracasei into a fermentation culture medium and carrying out fermentation culture at 37°C for 24h to 48h;

[0020] As a further preferred embodiment of the preparation method, the seed culture medium is MRS liquid culture medium; the formula of the fermentation culture medium is: peptone 10g / L, beef extract 8g / L, yeast extract powder 4g / L, glucose 20g / L, Tween 80 1.08g / L, dipotassium hydrogen phosphate 2g / L, sodium acetate 5g / L, triammonium citrate 2g / L, magnesium sulfate 0.2g / L, manganese sulfate 0.05g / L, L-cysteine ​​0.5g / L~1.0g / L, ectoine 0.5g / L~2g / L, inulin 0.2g / L~1g / L, oligofructose 1g / L~5g / L, dendrobium polysaccharide 0.5g / L~2g / L, and skimmed milk powder 1g / L~2g / L.

[0021] As a preferred embodiment of the preparation method, in the step (2), the broken bacteria are broken by using a low-temperature and high-pressure homogenizer, the breaking pressure is 800ba to 1000ba, and the homogenization is performed 3 to 5 times.

[0022] As a preferred embodiment of the preparation method, in the step (3), the centrifugation temperature is 0°C to 4°C, the rotation speed is 6000rpm to 8000rpm, and the time is 20min to 30min; and the filtration is performed using a 0.2um filter.

[0023] Compared with the prior art, the present invention has the following beneficial effects:

[0024] The Lactobacillus paracasei LTHINK-0050 strain of the invention has high anti-inflammatory activity, can control inflammation, improve and repair the skin's own barrier function, and can be used to prepare medicaments, cosmetics and cosmetic raw materials with anti-inflammatory and soothing effects. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is the result of identification of the colony morphology of Lactobacillus paracasei LTHINK-0050;

[0026] Figure 2 is the phylogenetic tree of Lactobacillus paracasei LTHINK-0050;

[0027] Figure 3 is the Gram staining image of Lactobacillus paracasei LTHINK-0050;

[0028] Figure 4 This is an experiment on the soothing and anti-inflammatory effects of Lactobacillus paracasei LTHINK-0050. DETAILED DESCRIPTION

[0030] To better illustrate the purpose, technical solutions and advantages of the present invention, the present invention will be further described below in conjunction with specific embodiments. It should be understood by those skilled in the art that the specific embodiments described herein are only used to explain the present invention and are not intended to limit the present invention.

[0031] Unless otherwise specified, the experimental methods used in the examples are all conventional methods; the materials, reagents, etc. used, unless otherwise specified, can be obtained from commercial channels.

[0032] The culture medium formula used in the embodiment is as follows:

[0033] MRS solid medium: peptone 10g / L, beef extract 10g / L, yeast extract 5g / L, glucose 20g / L, sodium acetate 5g / L, Tween-80 1mL, diammonium hydrogen citrate 2g / L, dipotassium hydrogen phosphate 2g / L, magnesium sulfate heptahydrate 0.58g / L, manganese sulfate monohydrate 0.25g / L, calcium carbonate 4g / L, agar 15g / L;

[0034] MRS liquid culture medium: peptone 10 g / L, beef extract 10 g / L, yeast extract powder 5 g / L, glucose 20 g / L, sodium acetate 5 g / L, Tween-80 1 mL, diammonium hydrogen citrate 2 g / L, dipotassium hydrogen phosphate 2 g / L, magnesium sulfate heptahydrate 0.58 g / L, manganese sulfate monohydrate 0.25 g / L, calcium carbonate 4 g / L.

[0035] Example 1: Isolation and purification of strains

[0036] (1) Separation

[0037] Take about 0.5g of feces from a healthy adult and dilute it with PBS to 10 -1 , 10 -2 , 10 -3 , 10 -4 , 10 -5 , 10 -6 , 10 -7 , 10 -8 The concentration gradient was set, and 100 μL of each concentration was evenly applied to the MRS solid medium with calcium carbonate, and two replicates were made for each concentration. After culturing in a 37°C constant temperature incubator for 48 hours, the colonies and the formation of transparent circles were observed for further separation and purification.

[0038] (2) Purification and culture

[0039] Select a suitable gradient from the above culture concentration gradients, select 8 single colonies with clear boundaries and transparent circles, and mark them as YX-01, YX-02, YX-03, YX-04, YX-05, YX-06, YX-07, and YX-08. The selected colonies are subcultured on a newly prepared MRS solid medium by streaking for 24h to 48h, and then streaked again. This step is repeated three times to obtain a monoclonal strain. After the strain is cultured in MRS liquid medium for 24h, the bacterial solution and 50% glycerol are mixed in a ratio of 1:1 (the final concentration of glycerol is 25%) and stored at -80°C.

[0040] (3) Preparation of intracellular extracts

[0041] Take the above bacteria and centrifuge at 8000r / min for 10min, add PBS, and ultrasonically disrupt in an ice-water bath (power 200W, ultrasonic 3s, interval 10s, repeat 30 times), centrifuge at 4°C and 8000r / min for 10min, take the supernatant, and obtain the intracellular extract. Take the bacteria and add 1mL of the extract, ultrasonically disrupt in an ice bath (power 200W, ultrasonic 3s, interval 10s, repeat 30 times), then centrifuge at 4°C and 10000r / min for 10min, take the supernatant, and determine the intracellular polysaccharide content using the sulfuric acid-phenol method.

[0042] The experimental results are shown in Table 1, which show that the total polysaccharide content of the fermentation lysate of YX-05 is the highest.

[0043] Table 1 Total intracellular polysaccharide content of different strains

[0044] Strain number Total intracellular polysaccharide content (mg / L) YX-01 71.2 YX-02 54.2 YX-03 66.3 YX-04 102.4 YX-05 146.1 YX-06 112.4 YX-07 78.6 YX-08 141.6

[0045] Example 2: Identification of bacterial strains

[0046] (1) Observation of colony and bacterial morphology

[0047] The strains screened in Example 1 were tested for morphological characteristics, Gram staining and other indicators, referring to the methods of Tsai Miao-ying and Dong Xiu-zhu. The details are as follows:

[0048] The selected strain YX-05 was subcultured on a newly prepared MRS solid medium by streaking for 24 to 48 hours, and then streaked again. This step was repeated three times to obtain a monoclonal strain. After the strain was cultured in MRS liquid medium for 24 hours, the bacterial solution was mixed with 50% glycerol in a ratio of 1:1 (the final concentration of glycerol was 25%) and stored at -80°C. The morphology of the colony of the strain in this example is shown in the figure below. Figure 1 shown.

[0049] (2) Molecular Biological Identification

[0050] DNA of strain YX-05 was extracted and PCR amplified using universal primers for bacterial 16S rRNA. The PCR products were sequenced at Suzhou Hongxun Biotechnology Co., Ltd. The nucleotide sequences of the universal primer pair for 16S rRNA were as follows: the upstream primer was 27F: AGAGTTTGATCMTGGCTCAG; the downstream primer was 1492R: GGTTACCTTGTTACGACTT. The amplification system is shown in Table 2. Reaction conditions: ABI 2720PCR instrument was used for amplification and sequencing. Amplification program: 96℃5min; 96℃30s, 58℃30s, 72℃90s, 5 cycles; 72℃7min; 4℃ storage.

[0051] Table 2 Amplification system

[0052] 2*Phanta Max Master Mix (Novozymes amplification kit) 12.5μL 27F (Suzhou Hongxun Biosynthesis) 0.5μL 1492R (Suzhou Hongxun Biosynthesis) 0.5μL Genomic DNA 1.5μL <![CDATA[ddH2O]]> Add to 25μL

[0053] The PCR amplification products were identified by agarose gel electrophoresis. The PCR products were sequenced by Suzhou Hongxun Biotechnology Co., Ltd., and the nucleotide sequence of 16S rDNA is shown in SEQ ID No: 1. The sequencing results were entered into the NCBI database for homology comparison. The 16S rRNA gene sequence of strain YX-05 was submitted to the GenBank database, and the sequences with high homology were searched using BLAST. The sequences were compared and analyzed using CLUSTAL X 1.81 and MEGA6.0 software, and a phylogenetic tree was established, as shown in Figure 1. Figure 2 The 16S rDNA sequence homology of the strain and Lactobacillus paracasei reaches 99%, and the strain isolated in this example is identified as Lactobacillus paracasei.

[0054] (3) Physiological and biochemical identification:

[0055] Gram staining and physiological and biochemical experiments of strain YX-05. The specific experimental methods are as follows:

[0056] a. Pick out different colonies with transparent calcium melting rings, round, white or yellow, and translucent, and separate them by streaking on the MRS plate. After Gram staining, examine under a microscope. If the bacteria are blue-purple under the microscope, they are Gram-positive bacteria. Figure 3 shown.

[0057] b. Indole test: Inoculate the bacteria to be tested into Dunhen's peptone solution, place in a 37°C constant temperature incubator for 24-48 hours, add 2 mL of xylene to the culture solution, mix well and let stand for 5 minutes, add 2 mL of indole reagent along the tube wall, and the appearance of red precipitation is positive. No red is a negative strain.

[0058] c. Nitrate reduction test: Inoculate the bacteria to be tested into nitrate culture medium and culture at 37℃ for 24h~48h. First add 0.1mL of reagent A to each tube, then add a few drops of reagent B. If red appears, it is a positive bacteria, and if no red appears, it is a negative bacteria.

[0059] d. Gelatin liquefaction test: select the bacteria to be tested that have been cultured for 18h-24h, and inoculate a large amount of them into the gelatin layer at about two-thirds of its depth, and culture them in a 37℃ constant temperature incubator for several days. Take another medium that has not been inoculated with the strain and culture it at the same time as a control. Take it out every day and place it in the refrigerator for 20-30min to observe the results. If the gelatin liquefies, it is positive, and if the gelatin does not liquefy, it is negative.

[0060] Table 3 Physiological and biochemical experimental results

[0061]

[0062]

[0063] Based on the colony and bacterial morphology, physiological and biochemical characteristics and molecular biological identification results of strain YX-05 in this embodiment, strain YX-05 can be determined to be Lactobacillus paracasei, and it is named Lactobacillus paracasei LTHINK-0050. It was deposited in Guangdong Microbiological Culture Collection Center on May 19, 2023, with a deposit number of GDMCC No: 63480, and was classified as Lacticaseibacillus paracasei, and was detected as alive on May 19, 2023; the deposit address is: 5th Floor, Building 59, No. 100 Xianlie Middle Road, Guangzhou; Contact number: 020-87137633.

[0064] Example 3: Preparation of Lactobacillus paracasei LTHINK-0050 fermentation lysate

[0065] The present embodiment provides a method for preparing a fermentation lysate of Lactobacillus paracasei LTHINK-0050, comprising the following steps:

[0066] (1) Fermentation: first, Lactobacillus paracasei LTHINK-0050 is inoculated into a seed culture medium and cultured at 37°C for 16 h-24 h; then, it is inoculated into a fermentation culture medium and cultured at 37°C for 24 h-48 h to obtain bacteria; the seed culture medium is an MRS liquid culture medium, and the fermentation culture medium is peptone 10 g / L, beef extract 8 g / L, yeast extract powder 4 g / L, glucose 20 g / L, Tween 80 1.08 g / L, dipotassium hydrogen phosphate 2 g / L, sodium acetate 5 g / L, ammonium citrate tribasic 2 g / L, magnesium sulfate 0.2 g / L, manganese sulfate 0.05 g / L, L-cysteine ​​1.0 g / L, ectoine 2 g / L, inulin 1 g / L, oligofructose 5 g / L, dendrobium polysaccharide 2 g / L, and skimmed milk powder 2 g / L.

[0067] (2) Bacterial cell disruption: The bacterial cells obtained from the fermentation in step (1) are resuspended in a bacterial disruption buffer, and the bacterial cells are disrupted using a high-pressure homogenizer at a bacterial disruption pressure of 1000 bar. The cells are homogenized 3 to 5 times to obtain a disruption solution; the bacterial disruption buffer comprises 20 mM sodium dihydrogen phosphate and 20 mM disodium hydrogen phosphate.

[0068] (3) Clarification and filtration: The crushed liquid obtained in step (2) is centrifuged at 4° C. and 8000 rpm for 30 min, the supernatant is collected, and filtered through a 0.2 μm filter to obtain the Lactobacillus paracasei LTHINK-0050 fermentation lysate.

[0069] Example 4: Optimization of conditions for preparing lactobacillus fermentation lysate by Lactobacillus paracasei LTHINK-0050

[0070] The method for preparing lactobacillus fermentation lysate from Lactobacillus paracasei LTHINK-0050 was optimized. The fermentation medium was optimized. Example 3 was used as test group 1, and the following test groups and comparison groups were set:

[0071] In order to further confirm the optimal culture medium, a single-factor experiment was used to analyze the concentrations of key components in the culture medium that affect polysaccharide production, such as the osmotic pressure regulator ectoine; growth-promoting polysaccharides inulin, oligofructose, and dendrobium polysaccharides; and skimmed milk powder.

[0072] (1) Effects of different concentrations of osmotic pressure regulator ectoine on the total polysaccharide content of lysate.

[0073] Using Example 3 as the basic medium, the fermentation medium used 0 g / L, 0.5 g / L, 1.0 g / L, 2.0 g / L, and 4.0 g / L ectoine concentrations, respectively, and the remaining parameters were prepared according to Example 3. The total polysaccharide in the lysate of each experimental group and the control group was detected by the sulfuric acid-phenol method. The test results are as follows:

[0074] The fermentation medium of experimental group 1 was 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 1.08 g / L Tween 80, 2 g / L potassium dihydrogen phosphate, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1.0 g / L L-cysteine, 2 g / L ectoine, 1 g / L inulin, 5 g / L fructooligosaccharide, 2 g / L dendrobium polysaccharide, and 2 g / L skimmed milk powder.

[0075] The fermentation medium of experimental group 2 was 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 1.08 g / L Tween 80, 2 g / L potassium dihydrogen phosphate, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1.0 g / L L-cysteine, 0.5 g / L ectoine, 1 g / L inulin, 5 g / L fructooligosaccharide, 2 g / L dendrobium polysaccharide, and 2 g / L skimmed milk powder.

[0076] The fermentation medium of experimental group 3 was 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 1.08 g / L Tween 80, 2 g / L potassium dihydrogen phosphate, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1.0 g / L L-cysteine, 1 g / L ectoine, 1 g / L inulin, 5 g / L fructooligosaccharide, 2 g / L dendrobium polysaccharide, and 2 g / L skimmed milk powder.

[0077] The fermentation medium of control group 1 was 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 1.08 g / L Tween 80, 2 g / L potassium dihydrogen phosphate, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1.0 g / L L-cysteine, 1 g / L inulin, 5 g / L oligofructose, 2 g / L dendrobium polysaccharide, and 2 g / L skimmed milk powder.

[0078] The fermentation medium of control group 2 was 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 1.08 g / L Tween 80, 2 g / L potassium dihydrogen phosphate, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1.0 g / L L-cysteine, 4 g / L ectoine, 1 g / L inulin, 5 g / L fructooligosaccharide, 2 g / L dendrobium polysaccharide, and 2 g / L skimmed milk powder.

[0079] Table 4 Total polysaccharide content of lysates prepared from medium with different concentrations of ectoine

[0080] Experimental groups Total intracellular polysaccharide content (mg / L) Experimental Group 1 184.2 Experimental Group 2 168.3 Experimental Group 3 172.4 Comparison group 1 143.4 Comparison group 2 142.5

[0081] (2) Effect of growth-promoting polysaccharides on the total polysaccharide content of lysates

[0082] Using Example 3 as the basic culture medium, different concentrations of inulin, oligofructose, and dendrobium polysaccharide were analyzed, and the remaining parameters were prepared according to Example 3. The total polysaccharide in the lysate of each experimental group and the control group was detected by sulfuric acid-phenol method. The test results are as follows:

[0083] The fermentation medium of experimental group 1 was 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 1.08 g / L Tween 80, 2 g / L potassium dihydrogen phosphate, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1.0 g / L L-cysteine, 2 g / L ectoine, 1 g / L inulin, 5 g / L fructooligosaccharide, 2 g / L dendrobium polysaccharide, and 2 g / L skimmed milk powder.

[0084] The fermentation medium of experimental group 4 was 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 1.08 g / L Tween 80, 2 g / L potassium dihydrogen phosphate, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1.0 g / L L-cysteine, 2 g / L ectoine, 0.2 g / L inulin, 1 g / L fructooligosaccharide, 0.5 g / L dendrobium polysaccharide, and 2 g / L skimmed milk powder.

[0085] The fermentation medium of experimental group 5 was 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 1.08 g / L Tween 80, 2 g / L potassium dihydrogen phosphate, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1.0 g / L L-cysteine, 2 g / L ectoine, 0.5 g / L inulin, 2.5 g / L oligofructose, 1 g / L dendrobium polysaccharide, and 2 g / L skimmed milk powder.

[0086] The fermentation medium of control group 3 was 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract powder, 20 g / L glucose, 1.08 g / L Tween 80, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 2.0 g / L L-cysteine, and 2 g / L skimmed milk powder.

[0087] The fermentation medium of control group 4 was 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 1.08 g / L Tween 80, 2 g / L potassium dihydrogen phosphate, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1.0 g / L L-cysteine, 2 g / L ectoine, 2 g / L inulin, 8 g / L fructooligosaccharide, 4 g / L dendrobium polysaccharide, and 2 g / L skimmed milk powder.

[0088] Table 5 Total polysaccharide content of lysates prepared from polysaccharide culture media with different concentrations

[0089] Experimental groups Total intracellular polysaccharide content (mg / L) Experimental Group 1 184.2 Experimental Group 4 169.1 Experimental Group 5 175.3 Comparison group 3 151.7 Comparison group 4 159.5

[0090] (3) Effect of skimmed milk powder on the total polysaccharide content of lysate

[0091] Using Example 3 as the basic culture medium, different concentrations of skimmed milk powder were analyzed, and the remaining parameters were prepared according to Example 3. The total polysaccharide of the lysate of each experimental group and the control group was detected by sulfuric acid-phenol method. The test results are as follows:

[0092] The fermentation medium of experimental group 1 was 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 1.08 g / L Tween 80, 2 g / L potassium dihydrogen phosphate, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1.0 g / L L-cysteine, 2 g / L ectoine, 1 g / L inulin, 5 g / L fructooligosaccharide, 2 g / L dendrobium polysaccharide, and 2 g / L skimmed milk powder.

[0093] The fermentation medium of experimental group 6 was 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 1.08 g / L Tween 80, 2 g / L potassium dihydrogen phosphate, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1.0 g / L L-cysteine, 2 g / L ectoine, 1 g / L inulin, 5 g / L fructooligosaccharide, 2 g / L dendrobium polysaccharide, and 1 g / L skimmed milk powder.

[0094] The fermentation medium of control group 5 was 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 1.08 g / L Tween 80, 2 g / L potassium dihydrogen phosphate, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1.0 g / L L-cysteine, 2 g / L ectoine, 1 g / L inulin, 5 g / L fructooligosaccharide, and 2 g / L dendrobium polysaccharide.

[0095] The fermentation medium of control group 6 was 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 1.08 g / L Tween 80, 2 g / L potassium dihydrogen phosphate, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 1.0 g / L L-cysteine, 2 g / L ectoine, 2 g / L inulin, 8 g / L fructooligosaccharide, 4 g / L dendrobium polysaccharide, and 3 g / L skimmed milk powder.

[0096] Table 6 Total polysaccharide content of lysates from different groups

[0097] Experimental groups Total intracellular polysaccharide content (mg / L) Experimental Group 1 184.2 Experimental Group 6 181.6 Comparison group 5 158.4 Comparison group 6 153.7

[0098] The fermentation medium of the experimental group was used to ferment Lactobacillus paracasei LTHINK-0050 to prepare lactobacillus fermentation lysate, and the fermentation lysate had a high content of intracellular polysaccharides. Growth-promoting factors such as ectoine, dendrobium polysaccharide, inulin, and oligofructose in the fermentation medium components cooperated with other basic components in the culture medium to better promote the production of intracellular polysaccharides.

[0099] Example 5: Fermentation lysate of Lactobacillus paracasei LTHINK-0050 inhibits inflammatory factors

[0100] The fermentation lysate of Lactobacillus paracasei LTHINK-0050 prepared in Example 3 was used for anti-inflammatory testing based on the LPS-induced RAW264.7 cell inflammation model.

[0101] Set up the following treatment groups:

[0102] Model: 200 μL PBS and LPS (final concentration 1 μg / mL) were added to a 6-well plate; Control: 200 μL PBS was added to a 6-well plate; Test sample: 200 μL filtered test sample (Lactobacillus paracasei LTHINK-0050 lysate with mass percentages of 3.13%, 6.25%, 12.5%, and 25%, respectively) and LPS (final concentration 1 μg / mL) were added to a 6-well plate; 3 replicate wells were set up for each treatment group, and the cell supernatant was collected after incubation for 24 hours, and the operation was performed according to the instructions of the ELISA kit.

[0103] The results are shown in Table 5:

[0104] Table 5 Inflammatory factor detection results

[0105]

[0106] Effects of different concentrations of Synthetic biology LAC+ on the expression level of IL-6 in RAW264.7 cells induced by LPS Figure 4 shown.

[0107] In summary, the Lactobacillus paracasei LTHINK-0050 strain of the present invention has high anti-inflammatory activity and can be used to prepare cosmetics and cosmetic raw materials with anti-inflammatory and soothing effects.

[0108] Finally, it should be noted that the above embodiments are only used to illustrate the technical solution of the present invention rather than to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solution of the present invention can be modified or replaced by equivalents without departing from the essence and scope of the technical solution of the present invention.

Claims

1. A Lactobacillus paracasei with anti-inflammatory and soothing effects, characterized in that: Its deposit number is GDMCC No:63480.

2. A fermentation lysate of Lactobacillus paracasei according to claim 1.

3. An anti-inflammatory soothing preparation, characterized in that: The method comprises the Lactobacillus paracasei according to claim 1 and / or the fermentation lysate according to claim 2.

4. Use of the Lactobacillus paracasei described in claim 1 and / or the fermentation lysate described in claim 2 in preparing medicines or cosmetics for anti-inflammatory and soothing skin.

5. Use of the Lactobacillus paracasei described in claim 1 and / or the fermentation lysate described in claim 2 in preparing skin care.

6. A method for preparing a fermentation lysate of Lactobacillus paracasei according to claim 2, characterized in that: The following steps are involved: (1) fermenting and culturing the Lactobacillus paracasei to obtain a fermented bacterial liquid; (2) taking the fermented bacterial liquid, breaking the bacterial cells, and obtaining a broken liquid; (3) taking the crushed liquid and centrifuging it to collect the supernatant, filtering it, and obtaining the fermentation lysate of Lactobacillus paracasei.

7. The preparation method according to claim 6, characterized in that: In the step (1), the fermentation culture is as follows: firstly the Lactobacillus paracasei is inoculated into a seed culture medium and subjected to seed culture at 37° C. for 16 h to 24 h; and then inoculated into a fermentation culture medium and subjected to fermentation culture at 37° C. for 24 h to 48 h.

8. The preparation method according to claim 7, wherein the seed culture medium is an MRS liquid culture medium; the formula of the fermentation culture medium is: 10 g / L peptone, 8 g / L beef extract, 4 g / L yeast extract, 20 g / L glucose, 1.08 g / L Tween 80, 2 g / L dipotassium hydrogen phosphate, 5 g / L sodium acetate, 2 g / L triammonium citrate, 0.2 g / L magnesium sulfate, 0.05 g / L manganese sulfate, 0.5 g / L-1.0 g / L L-cysteine, 0.5 g / L-2 g / L ectoine, 0.2 g / L-1 g / L inulin, 1 g / L-5 g / L fructooligosaccharide, 0.5 g / L-2 g / L dendrobium polysaccharide, and 1 g / L-2 g / L skimmed milk powder.

9. The preparation method according to claim 6, characterized in that: In the step (2), the broken bacteria are broken by a low-temperature and high-pressure homogenizer, the breaking pressure is 800ba to 1000ba, and the homogenization is performed 3 to 5 times.

10. The preparation method according to claim 6, characterized in that: In the step (3), the centrifugation temperature is 0°C to 4°C, the rotation speed is 6000rpm to 8000rpm, and the time is 20min to 30min; the filtration is performed using a 0.2um filter.

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