CN114032190B - Lactobacillus reuteri capable of fermenting dendrobium nobile and effectively repairing solar dermatitis by fermentation liquid thereof - Google Patents
Lactobacillus reuteri capable of fermenting dendrobium nobile and effectively repairing solar dermatitis by fermentation liquid thereof Download PDFInfo
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- CN114032190B CN114032190B CN202110962880.0A CN202110962880A CN114032190B CN 114032190 B CN114032190 B CN 114032190B CN 202110962880 A CN202110962880 A CN 202110962880A CN 114032190 B CN114032190 B CN 114032190B
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- Medicines Containing Material From Animals Or Micro-Organisms (AREA)
Abstract
The invention discloses lactobacillus reuteri capable of fermenting dendrobium nobile and effectively repairing solar dermatitis by fermentation liquor thereof, belonging to the field of fermentation engineering and the technical field of medicines. The lactobacillus reuteri (Lactobacillus reuteri) CCFM1187 can ferment dendrobium, and the dendrobium fermentation liquid has the effect of effectively repairing solar dermatitis, not only improves the survival rate of SDS-induced HaCaT cells, namely improves the repairing effect of skin barriers, but also reduces the NO content of RAW264.7 cells of LPS, namely has the effect of effectively repairing solar dermatitis, so that the lactobacillus reuteri fermentation dendrobium fermentation liquid has a huge application prospect in products aiming at effectively repairing solar dermatitis.
Description
Technical Field
The invention relates to lactobacillus reuteri capable of fermenting dendrobium nobile and effectively repairing solar dermatitis by fermentation liquor thereof, belonging to the technical field of fermentation engineering and medicines.
Background
In recent years, intestinal probiotics have been widely studied and proved to have absolute safety and broad efficacy potential. By utilizing probiotics to ferment traditional Chinese medicine raw materials, strains and fermentation processes capable of fermenting traditional Chinese medicines and improving the activity of fermentation broth after fermentation are excavated, not only can dendrobium resources be saved, but also the effectiveness and application range of the traditional Chinese medicines can be improved.
Dendrobium nobile is a plant of Dendrobium genus of Orchidaceae, and starts to be recorded in Shennong Ben Cao Jing, which is a traditional rare Chinese medicine in China, and has the reputation of gold in medicine since ancient times. Dendrobium nobile is sweet in flavor and slightly cold in nature, and has the effects of benefiting stomach, promoting fluid production, nourishing yin and clearing heat. Researches show that the main active ingredients contained in the dendrobium nobile are dendrobium nobile polysaccharide, dendrobine, amino acid, stilbene and derivatives thereof and various volatile components, and the dendrobium nobile extract has the unique effects of enhancing the immunity of human bodies, relieving physical fatigue, resisting oxidation, inhibiting aging, reducing blood pressure, reducing blood sugar, inhibiting tumor cells and the like.
The research shows that after the dendrobium is fermented, the physiological activity of the dendrobium is enhanced, the molecular weight distribution of total sugar, reducing sugar and polysaccharide in the fermentation liquid is obviously changed, and meanwhile, various enzymes beneficial to human bodies are synthesized in the microorganism growth process. Under certain conditions, the fermented dendrobium extract has the oxidation resistance, the blood sugar reducing capability and the human body fat metabolism regulating capability which are improved to different degrees, the application value of the dendrobium in the aspect of skin care is greatly improved by fermentation, and the improvement of the skin care effect mainly comes from the change of the fermentation on the active ingredients of the dendrobium.
Solar dermatitis is commonly called sunburn, is acute skin inflammation caused by strong sunlight irradiation, is an acute phototoxic reaction on skin due to excessive irradiation of medium-wave ultraviolet rays, and is mostly seen in the late spring and early summer, and women and children are easy to attack. The traditional Chinese medicine is called skin sore, which is considered to be caused by invasion of yang heat toxin into the body surface, accumulation Yu Jifu and skin burn under the condition of sun exposure; if toxic heat is used to block summer-heat and damp or to block internal dampness, swelling, blisters, erosion and liquid seepage occur when the skin is immersed. The causes of solar dermatitis are generally classified into three main types, one type is food factor, one type is cosmetic factor, and the other type is physical factor.
The skin is used as a barrier for protecting the human body from the outside environment, and is a first defense line for resisting various stimuli such as outside machinery, chemistry, physics, biology and the like. Studies have shown that skin proliferation rates of epidermal cells are accelerated after uv irradiation, and that many enzymes or proteins are activated or inhibited to generate a large amount of oxygen radicals, in addition to erythema and/or stains, and that these changes have a significant effect on skin barrier function. While impaired skin barrier function results in a disorder of the skin immune response that aggravates the impaired skin barrier and thus the vicious circle, and the skin immune response reaction occurs simultaneously. Only to maintain the integrity of the skin barrier while promoting the normalization of the skin immune response is an important credential to ensure skin health.
With the increasing demands of people on quality of life, the more annoyance solar dermatitis is brought. According to research, for solar dermatitis, most of the traditional Chinese medicines are selected for repair treatment. However, the effect of the extract directly extracted from the traditional Chinese medicine without fermentation on the aspect of skin care effect is not obviously improved. The active ingredients of the traditional Chinese medicine are improved after glycolysis by utilizing the growth metabolism of microorganisms, so that the original efficacy is improved, or the active ingredients which are not originally possessed are generated, so that new efficacy is generated. In recent years, as the research on active substances and functional components of dendrobium is more and more, the application of dendrobium in cosmetics is increasingly paid attention to. However, at present, a fermentation process for effectively fermenting the dendrobium raw material to ensure that the fermented fermentation liquor has obvious efficacy is not available. Therefore, the limiting factors in the fermentation process are solved by utilizing the modern fermentation engineering technology, the traditional Chinese medicine raw materials are fully developed and utilized, the effective components of the traditional Chinese medicine raw materials are improved, one strain of probiotics is screened out to ferment the dendrobium raw materials, and the fermented extract has important value in the aspect of effectively repairing solar dermatitis.
Disclosure of Invention
In order to obtain a method capable of improving the capability of the dendrobium fermentation liquid for obviously repairing solar dermatitis, the invention screens lactobacillus reuteri (Lactobacillus reuteri) CCFM1187. The lactobacillus reuteri CCFM1187 can ferment dendrobium, and the dendrobium fermentation liquid has the effect of effectively repairing solar dermatitis, and is specifically expressed in the following steps: (1) Lactobacillus reuteri CCFM1187 is rapidly proliferated in herba Dendrobii fermentation medium, herba Dendrobii can be fermented, and its viable count is 2.6X10 at 15-16 hr 8 cfu/mL; (2) the dendrobium nobile fermentation liquid has no cytotoxicity to 3T3-L1 cells; (3) Dendrobium nobile fermentation liquorThe survival rate of SDS-induced HaCaT cells is improved, namely the repair effect of skin barriers is improved; (4) The dendrobium nobile fermentation liquid reduces the NO content of RAW264.7 cells of LPS, namely has the effect of effectively repairing solar dermatitis; therefore, lactobacillus reuteri (Lactobacillus reuteri) fermented dendrobium fermented liquid has great application prospect in products aiming at effectively repairing solar dermatitis.
The invention provides lactobacillus reuteri (Lactobacillus reuteri) CCFM1187, wherein the lactobacillus reuteri CCFM1187 is preserved in the Guangdong province microorganism strain collection, and the preservation number is GDMCC No:61729 the preservation date is 2021, 06 and 21, and the preservation address is building 5 of No. 59 of No. 100 university of Mitsui, guangdong province, and the institute of microorganisms.
The lactobacillus reuteri is derived from a sample in Yun Nada and Deng Chuan county, the strain is subjected to sequencing analysis, the 16S rDNA sequence of the strain is shown as SEQ ID NO.1, the sequence obtained by sequencing is subjected to nucleic acid sequence comparison in GeneBank, and the result shows that the similarity between the strain CCFM1187 and the lactobacillus reuteri is as follows: 97.39% and therefore this strain CCFM1187 is Lactobacillus reuteri, designated Lactobacillus reuteri (Lactobacillus reuteri) CCFM1187.
The microbial morphological characteristics of lactobacillus reuteri CCFM1187 are: campylobacter with slightly irregular and rounded ends. The colony is in off-white or milky white, opaque, smooth in surface, convex and neat in edge, and has a diameter of 1-1.5 mm.
The invention also provides application of the lactobacillus reuteri CCFM1187 in fermenting raw materials containing dendrobium.
The invention also provides a dendrobium fermentation liquid, which is prepared by inoculating the lactobacillus reuteri CCFM1187 into a fermentation medium containing dendrobium for fermentation.
In one embodiment of the present invention, the lactobacillus reuteri CCFM1187 is inoculated in the dendrobium fermentation medium in an amount of at least 1.0x10 6 cfu/mL。
In one embodiment of the invention, the lactobacillus reuteri CCFM1187 is inoculated in the dendrobium fermentation medium in an amount of 1.0×10 6 ～1.0×10 7 cfu/mL。
In one embodiment of the invention, the fermentation medium comprises: 40-80 g/L of dendrobium, 10-40 g/L of proliferation factor, 0.1-0.8 g/L of trace element and 0.5-1.5 mL/L of Tween; the proliferation factor is one or more of yeast extract powder, yeast extract, yeast peptone, tryptone, soybean peptone, etc.; the microelements are magnesium sulfate and/or manganese sulfate.
In one embodiment of the invention, the initial pH of the dendrobium fermentation medium is 5.5-7.0.
The invention also provides a method for fermenting the dendrobium raw material and extracting dendrobium raw material fermentation liquid, which comprises the following specific steps:
(1) Inoculating the lactobacillus reuteri CCFM1187 into a culture medium containing dendrobium for fermentation to obtain fermentation liquor;
(2) Centrifuging the fermentation liquor in the step (1) to remove sediment, and obtaining dendrobium nobile fermentation supernatant;
(3) Sterilizing the fermentation supernatant in the step (2) to obtain the sterile dendrobium nobile fermentation supernatant.
In one embodiment of the invention, the dendrobium fermentation medium in the step (1) comprises dendrobium nobile with a dry weight of 40-80 g/L, proliferation factors of 10-40 g/L, microelements of 0.1-0.8 g/L and tween of 0.5-1.5 mL/L.
In one embodiment of the invention, the dendrobium nobile is prepared by crushing fresh or dry dendrobium nobile to particles smaller than 60 mesh.
In one embodiment of the present invention, the proliferation factor is one or more of yeast extract, yeast peptone, tryptone, soybean peptone, and the like.
In one embodiment of the present invention, the trace element is one or both of magnesium sulfate and manganese sulfate.
In one embodiment of the invention, the initial pH of the dendrobium fermentation medium is 5.5-7.0.
In one embodiment of the invention, the sterilization condition is that the temperature is 115-121 ℃ and the time is 15-20 min.
In one embodiment of the present invention, the lactobacillus reuteri CCFM1187 of step (1) is inoculated in an amount of 1×10 in the dendrobium fermentation medium 6 ～1.0×10 7 cfu/mL。
In one embodiment of the present invention, the fermentation temperature in step (1) is 32 to 38 ℃.
In one embodiment of the present invention, the fermentation pH in step (1) is 5.5 to 7.0.
In one embodiment of the present invention, the fermentation time in step (1) is 15 to 24 hours.
In one embodiment of the present invention, the centrifugation conditions in step (2) are: 6000-10000 g for 5-20 min.
In one embodiment of the present invention, the conditions of the ultrasonic treatment in step (2) are: the temperature is 25-80 ℃, the ultrasonic power is 200-500 w, and the time is 10-30 min.
The invention also provides a product, which comprises the lactobacillus reuteri CCFM1187 or the dendrobium nobile fermentation broth.
In one embodiment of the invention, the lactobacillus reuteri CCFM1187 is added to the product in an amount of at least 1.0x10 6 cfu/mL。
In one embodiment of the invention, the product is a pharmaceutical or cosmetic product.
In one embodiment of the invention, the pharmaceutical product contains lactobacillus reuteri CCFM1187 or the above dendrobium fermented liquid, pharmaceutical carrier and/or pharmaceutical excipients.
In one embodiment of the invention, the pharmaceutical carrier comprises microcapsules, microspheres, nanoparticles, and liposomes; the pharmaceutical excipients comprise excipients and additives.
In one embodiment of the invention, the cosmetic product is a skin care product or a washing product.
In one embodiment of the invention, the pharmaceutical product comprises a spread-action pharmaceutical product.
In one embodiment of the invention, an additive is also included.
In one embodiment of the invention, the additive is an ointment, a preservative and/or an antioxidant.
In one embodiment of the present invention, the form of the smear medicine is a paste, a film or a gel.
The invention also provides application of the lactobacillus reuteri CCFM1187 or the dendrobium fermented liquid in preparing a product for relieving solar dermatitis symptoms.
In one embodiment of the invention, the product is a pharmaceutical or cosmetic product.
Advantageous effects
The invention provides lactobacillus reuteri (Lactobacillus reuteri) CCFM1187, wherein the lactobacillus reuteri CCFM1187 can ferment dendrobium, and the dendrobium fermentation liquid obtained by fermenting the lactobacillus reuteri CCFM1187 has the effect of effectively repairing solar dermatitis, and is specifically characterized in that:
(1) Lactobacillus reuteri CCFM1187 is rapidly proliferated in herba Dendrobii fermentation medium, herba Dendrobii can be fermented, and its viable count is 2.6X10 at 15-16 hr 8 cfu/mL；
(2) The dendrobium nobile fermentation liquid has no cytotoxicity to 3T3-L1 cells;
(3) The dendrobium fermented liquid improves the survival rate of SDS-induced HaCaT cells, namely improves the repair effect of skin barriers, and is superior to the fermentation liquid of the dendrobium raw material unfermented liquid and the lactobacillus reuteri CCFM1187, and the cell survival rate is 1.11 times and 1.08 times of the fermentation liquid of the dendrobium raw material unfermented liquid and the lactobacillus reuteri CCFM1187 respectively;
(4) The dendrobium fermented liquid reduces the NO content of RAW264.7 cells of LPS, namely has the effect of effectively repairing solar dermatitis, is superior to the fermented liquid of the RAW material unfermented liquid of dendrobium and the fermented liquid of lactobacillus reuteri CCFM1187, and the NO content in the cells is 0.82 times and 0.88 times of the fermented liquid of the RAW material unfermented liquid of dendrobium and the fermented liquid of the lactobacillus reuteri CCFM1187 respectively;
therefore, lactobacillus reuteri (Lactobacillus reuteri) fermented dendrobium fermented liquid has great application prospect in preparing products for preventing or treating solar dermatitis.
Preservation of biological materials
Lactobacillus reuteri (Lactobacillus reuteri) CCFM1187, taxonomic designation Lactobacillus reuteri, was deposited at the Cantonese microorganism strain collection at month 21 of 2021 under accession number GDMCC No:61729 the preservation address is building 5 of Guangdong national institute of microbiology, guangzhou Miao road 100, university 59.
Drawings
Fig. 1: the cytotoxic effects of the different groups on 3T3-L1 were compared.
Fig. 2: comparison of effects of improving SDS-induced HaCaT cell survival in different groups.
Fig. 3: comparison of the inflammatory effects of LPS-induced RAW264.7 cells was reduced in different groups.
Detailed Description
The cells referred to in the following examples (3T 3-L1 cells, haCaT cells, RAW264.7 cells) were purchased from China center for type culture Collection; DMEM medium referred to in the examples below was purchased from sameimer femto instruments limited; fetal Bovine Serum (FBS), penicillin, streptomycin and trypsin referred to in the examples below were purchased from sigma aldrich (Shanghai) trade company, inc.; the MTT working fluids referred to in the examples below were purchased from beijing solibao technologies limited; the NO kit referred to in the examples below was purchased from shanghai bi yun biotechnology limited; the dendrobe referred to in the following examples is purchased from vast sen biotechnology limited, eastern Beijing.
Lactobacillus plantarum QS6-12, which is described in the examples below, is disclosed in Mao Bingyong, yin Ruimin, li Xiaoshu, cui Shomao, zhang Hao, zhao Jianxin, chen Wei. Comparative Genomic Analysis of Lactiplantibacillus plantarum Isolated from Different Niches [ J ]. Genes,2021,12 (2): paper; the related lactobacillus casei 2-L2 is preserved in the food biotechnology center of the university of south of the Yangtze river and is obtained by screening from an inner Mongolian Hulen bell yoghourt sample; the related lactobacillus reuteri DL5-6 is preserved in the food biotechnology center of the university of Jiangnan and is screened from the milk cow samples of Yunnan university Deng Chuanxian.
The calculation method of the following examples is as follows:
1) The method for detecting the number of living bacteria comprises the following steps: the national standard GB 4789.35-2016 food safety national standard food microbiology detection of lactobacillus detection is adopted.
2) Cell viability = (dosing OD-blank OD)/(negative OD-blank OD) ×100%.
3) Cell inhibition = 1-cell viability.
The following examples relate to the following media:
MRS liquid medium: 10g/L peptone, 10g/L beef extract, 20g/L glucose, 2g/L anhydrous sodium acetate, 5g/L yeast powder, 2g/L, K diammonium hydrogen citrate 2 PO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 0 0.1g/L、MnSO 4 0.05g/L, tween-80 1ml/L, and pH 6.2-6.4.
MRS solid medium: agar (20 g/L) was added on the basis of MRS broth.
Dendrobium nobile fermentation medium: 40g/L of dendrobium raw material, 10g/L of yeast extract powder, 0.58g/L of magnesium sulfate heptahydrate, 0.25g/L of manganese sulfate and 1ml/L of Tween 80 are uniformly mixed, purified water is added for constant volume, and the pH value is regulated to 7.0.
Complete medium: 10% Fetal Bovine Serum (FBS), 100U/mL penicillin, 100mg/mL streptomycin, 90% DMEM medium.
Example 1: screening and identification of strains
The method comprises the following specific steps:
1. screening
The sample is obtained from Yun Nada and Deng Chuan county, the sample is pretreated, stored in a refrigerator at the temperature of minus 80 ℃ in 20% glycerol, taken out for thawing, uniformly mixed and absorbed with 0.5mL of sample, added into 4.5mL of physiological saline, subjected to gradient dilution by 9g/L of physiological saline, selected to be coated on an MRS solid culture medium, cultured for 48 hours at the temperature of 37 ℃, picked up to be streaked and purified on the MRS solid culture medium, picked up to be transferred to the MRS liquid culture medium for enrichment, and 30% of glycerol is preserved to obtain the strain named CCFM1187; wherein, the typical colony of the lactobacillus reuteri is in off-white or milky white, opaque, smooth in surface, convex and neat in edge, and has a diameter of 1-1.5 mm.
2. Authentication
Extracting genome of the strain CCFM1187, amplifying and sequencing 16S rDNA of the strain CCFM1187 (the nucleotide sequence of the 16S rDNA of the strain CCFM1187 is shown as SEQ ID NO.1 by Jin Weizhi biotechnology Co., ltd.) and comparing the sequence with nucleic acid sequence in NCBI, and the result shows that the similarity of the strain CCFM1187 and lactobacillus reuteri is: 97.39% and therefore this strain CCFM1187 is Lactobacillus reuteri, designated Lactobacillus reuteri (Lactobacillus reuteri) CCFM1187.
Example 2: dendrobium nobile fermentation liquid prepared by fermenting dendrobium nobile with lactobacillus reuteri
The method comprises the following specific steps:
1. activation and culture of strains
(1) All containers and tools were sterilized.
(2) And (3) after the strain CCFM1187 preservation tube is uniformly vibrated, a small amount of bacterial liquid is dipped in a sterile inoculating loop, and the bacterial liquid is streaked and purified in an MRS solid culture medium. After standing for a period of time, the plates were placed in an incubator at 37℃for 48 hours. After the culture is finished, single colony is selected and inoculated into 5mL of MRS liquid culture medium, and the culture medium is placed in a constant temperature incubator at 37 ℃ for culture for 12-18 hours; the activating solution of the strain CCFM1187 is obtained, the activating solution prepared is activated for two generations continuously according to the method, and the inoculation amount of each time is 2% (v/v); preparing the activated seed liquid.
2. Preparation of lactobacillus reuteri CCFM1187 fermented dendrobium fermentation liquid
(1) Preparing a dendrobium fermentation medium: mixing herba Dendrobii 40g/L, yeast extract 10g/L, magnesium sulfate heptahydrate 0.58g/L, manganese sulfate 0.25g/L and Tween 80 1mL/L, adding purified water to constant volume, adjusting pH to 7.0, heating at 115deg.C for 20min, and sterilizing;
(2) Cooling the sterilized herba Dendrobii fermentation medium in step (1) to below 40deg.C, and sterilizing in sterile environment at a final concentration of 1×10 6 Addition amount of cfu/mL adding step1, fermenting the lactobacillus reuteri CCFM1187 seed liquid prepared in step 1 for 15-16 hours at a constant temperature and a constant pH value of 7.0 at a temperature of 37 ℃, wherein the viable count reaches 2.6X10 8 cfu/mL; continuing fermentation for 24 hours until the fermentation end point is reached, and obtaining a fermentation liquor containing dendrobium, wherein the viable count of lactobacillus reuteri CCFM1187 is as follows: 7.2X10 8 cfu/mL。
(3) And (3) centrifuging 8000g of the fermentation broth containing dendrobium nobile in the step (2) for 5min after ultrasonic treatment for 10min at the power of 500w and the temperature of 80 ℃ in an ultrasonic cleaning machine, and centrifuging to remove thalli and residues of the dendrobium nobile fermentation broth, thereby obtaining a fermentation supernatant containing dendrobium nobile. Heating the fermentation supernatant containing herba Dendrobii at 115deg.C for 20min for sterilization to obtain sterile herba Dendrobii fermentation broth, wherein the herba Dendrobii fermentation broth contains no live lactobacillus reuteri.
Control:
the fermentation liquor containing dendrobium without lactobacillus reuteri fermentation is named as a dendrobium raw material unfermented liquor, and the preparation method is as follows:
(1) Preparing a dendrobium raw material unfermented liquid: mixing herba Dendrobii 40g/L, yeast extract 10g/L, magnesium sulfate heptahydrate 0.58g/L, manganese sulfate 0.25g/L and Tween 80 1mL/L, adding purified water to constant volume, adjusting pH to 7.0, heating at 115deg.C for 20min, and sterilizing;
(2) And (3) centrifuging 8000g of the fermentation liquor containing the dendrobium nobile in the step (1) for 5min after ultrasonic treatment for 10min at the power of 500w at the temperature of 80 ℃ in an ultrasonic cleaning machine, and centrifuging to remove residues of the unfermented liquor of the dendrobium nobile raw material, thereby obtaining a supernatant containing the dendrobium nobile. Heating the supernatant containing herba Dendrobii at 115deg.C for 20min for sterilization to obtain sterile herba Dendrobii supernatant, wherein the unfermented herba Dendrobii liquid contains no viable bacteria.
3. Preparation of lactobacillus reuteri CCFM1187 fermentation broth
(1) Preparing a fermentation medium of lactobacillus reuteri CCFM 1187: 10g/L peptone, 10g/L beef extract, 20g/L glucose, 2g/L anhydrous sodium acetate, 5g/L yeast powder, 2g/L, K diammonium hydrogen citrate 2 PO 4 ·3H 2 O 2.6g/L、MgSO 4 ·7H 2 0 0.1g/L、MnSO 4 0.05g/L, tween 80 1mL/L, pH 6.2-6.4,heating at 115deg.C for 20min for sterilization;
(2) Cooling the sterilized fermentation medium of step (1) to a temperature below 40deg.C, and sterilizing in a sterile environment at a final concentration of 1×10Xin the fermentation medium of Lactobacillus reuteri CCFM1187 6 Adding cfu/mL of the seed solution of the lactobacillus reuteri CCFM1187 prepared in the step 1, fermenting for 12-18 hours in a constant temperature incubator at 37 ℃, wherein the viable count reaches 2.6X10 8 cfu/mL to obtain bacterial liquid;
(3) Lactobacillus reuteri CCFM1187 fermentation broth: centrifuging the bacterial liquid prepared in the step (2) for 15min at 8000rpm to obtain a supernatant; adjusting the pH to 7.2-7.4; filtering with 0.22 μm disposable filter, sterilizing, packaging, and storing in refrigerator at-20deg.C.
Example 3: effect of Lactobacillus reuteri CCFM1187 fermented Dendrobium broth on 3T3-L1 cell survival
The method comprises the following specific steps:
(1) Culturing 3T3-L1 cells
3T3-L1 cells were seeded at 25cm 2 In a cell culture flask of (2), the whole culture was performed at 37℃with 5% CO 2 Conventional culture is carried out under the condition of 80% humidity, when the cells reach 80% fusion, 3T3-L1 cells with good growth state are selected, and 2.5g/L trypsin is used for digesting the cells for resuspension, so as to prepare cell suspension. The cell suspension was mixed at 4X 10 3 Each well was inoculated in 96-well cell culture plates at 100. Mu.L per well and cultured for 24 hours.
(2) Discarding the stock culture solution in a 96-well cell culture plate, and adding a dendrobium nobile fermentation broth supernatant (hereinafter referred to as dendrobium nobile fermentation broth) fermented by lactobacillus reuteri CCFM1187 into each well, wherein the culture conditions are as follows: 37 ℃ and 5% CO 2 80% humidity; the culture time is as follows: 24h; the addition amount of the dendrobium nobile fermentation liquid is as follows: 100. Mu.L; the dendrobium fermentation liquid and the complete culture medium are respectively added into a 96-well cell culture plate containing 3T3-L1 cells according to the volume ratio of (1%: 99%), the volume ratio of (0.5%: 99.5%) and the volume ratio of (0.25%: 99.75%).
And taking the cells without the dendrobium fermented liquid as a normal control group.
(3) The culture plates were removed, and the cell viability was measured by MTT method, and the results are shown in FIG. 1 and Table 1.
Table 1: effect of Lactobacillus reuteri fermentation broth on 3T3-L1 cell viability
| Group/concentration (v/v)% | Cell viability (%) |
| Normal control group | 100.00±1.32 |
| Lactobacillus reuteri CCFM1187 fermented dendrobium nobile fermentation liquor group/1% | 114.13±7.54 |
| Lactobacillus reuteri CCFM1187 fermented dendrobium fermented liquid group/0.50% | 105.74±1.56 |
| Lactobacillus reuteri CCFM1187 fermented dendrobium nobile fermentation liquor group/0.25% | 97.51±3.89 |
As can be seen from FIG. 1 and Table 1, the ratio of the volume ratio of the dendrobe fermentation broth to the total culture medium is (1%: 99%), the ratio of the volume ratio of the dendrobe fermentation broth to the total culture medium is (0.5%: 99.5%) and the ratio of the volume ratio of the dendrobe fermentation broth to the total culture medium is (0.25%: 99.75%) to the proliferation survival ratio of the 3T3-L1 cells is 114.13%, 105.74% and 97.51% (P > 0.005), respectively.
Compared with a normal control group, the proliferation survival rate of the lactobacillus reuteri CCFM1187 fermented dendrobium fermentation liquid group with different concentrations is not obviously different, and the lactobacillus reuteri CCFM1187 fermented dendrobium fermentation liquid has no toxicity to cells and can be used for repairing solar dermatitis.
Example 4: effect of Lactobacillus reuteri fermentation broth on SDS-induced HaCaT cell survival
The method comprises the following specific steps:
1. culturing HaCaT cells
Re-warming the frozen HaCaT cells at 37 ℃, centrifuging at 1000rpm for 5min, and taking a precipitate; washing the sediment once with a complete culture medium, and re-suspending the cells with the complete culture medium to obtain a cell re-suspension; inoculating the cell heavy suspension into 10cm culture dish, and gas phase containing 5% (v/v) CO at 37deg.C 2 The culture in the cell incubator of (2) was continued by replacing the complete medium the next day at 37℃with a gas phase containing 5% (v/v) CO 2 Is cultured in a cell culture incubator. The cells are subcultured when growing to 70-80% density of the culture dish, and the culture method comprises the following steps: the gas phase contains 5% (v/v) CO at 37deg.C 2 Is cultured in a cell culture incubator.
Selecting HaCaT cells with good growth state, digesting the HaCaT cells by trypsin with the concentration of 2.5g/L, centrifuging, and re-suspending by using a complete culture medium, and performing cell counting to obtain a cell re-suspension; the cell resuspension was followed by 5X 10 3 Inoculating each well into a 96-well culture plate, wherein each well is 100 mu L; the 96-well culture plate was placed at 37℃and the gas phase contained 5% (v/v) CO 2 Culturing for 24 hours in a cell culture box; cells were treated with a complete medium containing 50. Mu.M SDS for 24h.
2. Preparation of different dendrobium nobile fermentation broths:
lactobacillus reuteri CCFM1187 fermented dendrobium fermented liquid group: lactobacillus reuteri CCFM1187 fermented dendrobium fermented liquid is prepared according to the method of example 2;
lactobacillus plantarum QS6-12 fermented dendrobium nobile fermentation broth group: the method of example 2 was followed, except that lactobacillus reuteri CCFM1187 was adjusted to lactobacillus plantarum QS6-12 to prepare lactobacillus plantarum QS6-12 fermented dendrobium broth;
lactobacillus casei 2-L2 fermented dendrobium nobile fermentation broth group according to the method of example 2, except that lactobacillus reuteri CCFM1187 is adjusted to lactobacillus casei 2-L2, and lactobacillus casei 2-L2 fermented dendrobium nobile fermentation broth is prepared;
lactobacillus reuteri DL5-6 fermented dendrobium fermented liquid group, according to the method of example 2, except that lactobacillus reuteri CCFM1187 is adjusted to lactobacillus reuteri DL5-6, and lactobacillus reuteri DL5-6 fermented dendrobium fermented liquid is prepared;
dendrobium raw material unfermented liquor group: a dendrobium raw material unfermented liquor group prepared by the control method in example 2;
lactobacillus reuteri CCFM1187 broth group: a Lactobacillus reuteri CCFM1187 broth set was prepared as described in example 2.
3. Influence of different dendrobe fermentation liquids on HaCaT cell survival rate
(1) The groups are as follows:
blank control group: only MTT working solution is contained, and no cell is used as a blank control group;
negative control group: the HaCaT cells which are not treated by SDS and are prepared in the step 1 are used as a negative control group;
model group: the HaCaT cells treated by SDS for 24 hours prepared in the step 1 are used as a model group;
(2) Preparation of different lactobacillus fermented dendrobium fermented liquid groups:
respectively mixing the unfermented dendrobium raw material prepared in the step 2, lactobacillus reuteri CCFM1187 fermentation liquor, lactobacillus plantarum QS6-12 fermentation dendrobium fermentation liquor, lactobacillus casei 2-L2 fermentation dendrobium fermentation liquor, lactobacillus reuteri DL5-6 fermentation dendrobium fermentation liquor supernatant and a complete culture medium according to the volume ratio of 10%: mixing at a ratio of 90% to obtain mixed solutions;
(3) After mixing, 100 μl of the mixed solution was added to the HaCaT cell 96-well plate treated with SDS in step 1, and cultured for 24 hours;
adding 100 mu L of complete culture medium into the negative control group and the model group, and culturing for 24 hours;
the culture conditions of each group are as follows: the gas phase contains 5% (v/v) CO at 37deg.C 2 Is cultured in a cell culture incubator.
Each group was provided with 5 duplicate wells and the MTT assay was used to measure cell viability. The results are shown in Table 2 and FIG. 2.
Table 2: influence of dendrobium fermentation liquid obtained by fermenting different strains on cell survival rate
| Group of | Cell viability (%) |
| |
100 |
| Model group | 59.80±0.61 |
| Dendrobium raw material unfermented liquor group | 66.48±0.47 |
| Lactobacillus reuteri CCFM1187 fermentation broth group | 68.29±0.24 |
| Lactobacillus reuteri CCFM1187 fermented dendrobium nobile fermentation liquid group | 73.68±0.35 |
| Lactobacillus plantarum QS6-12 fermented dendrobium nobile fermentation liquor group | 66.75±0.35 |
| Lactobacillus casei 2-L2 fermented dendrobium nobile fermentation liquor group | 56.53±0.48 |
| Lactobacillus reuteri DL5-6 fermented dendrobium nobile fermentation liquor group | 54.10±0.32 |
As can be seen from fig. 2 and table 2, the proliferation survival rate of the negative control group is 100.00%, the proliferation survival rate of the model group is 59.80%, the survival rate of the dendrobium raw material unfermented liquid group is 66.48%, the survival rate of the lactobacillus reuteri CCFM1187 fermented liquid group is 68.29%, the survival rate of the lactobacillus reuteri CCFM1187 fermented dendrobium fermented liquid group is 73.68%, which is 1.23 times of the proliferation survival rate of the model group, which is 1.11 times of the survival rate of the dendrobium raw material unfermented liquid group, which is 1.08 times of the survival rate of the lactobacillus reuteri CCFM1187 fermented liquid group, and which significantly promotes the proliferation of cells (P < 0.001). The survival rate of the lactobacillus plantarum QS6-12 fermented dendrobium fermentation liquid group is 66.75%, the survival rate of the lactobacillus casei 2-L2 fermented dendrobium fermentation liquid group is 56.53%, and the survival rate of the lactobacillus reuteri DL5-6 fermented dendrobium fermentation liquid group is 54.10%.
Compared with a dendrobium raw material unfermented liquor group, a lactobacillus reuteri CCFM1187 fermentation liquor group, a lactobacillus plantarum QS6-12 fermentation dendrobium fermentation liquor group, a lactobacillus casei 2-L2 fermentation dendrobium fermentation liquor group, a lactobacillus reuteri DL5-6 fermentation dendrobium fermentation liquor group and a lactobacillus reuteri CCFM1187 fermentation dendrobium raw material fermentation liquor can obviously promote HaCaT cell growth and alleviate cell barrier damage.
Example 5: the effect of lactobacillus reuteri fermentation dendrobium on LPS-induced NO content of RAW264.7 cells is specifically as follows:
1. culturing RAW264.7 cells
Culturing RAW264.7 cells, inoculating to 25cm 2 In a cell culture flask of (2), the whole culture was performed at 37℃with 5% CO 2 Conventional culture was performed under 80% humidity. When the cells reach 80% fusion, RAW264.7 cells with good growth state are selected, RAW264.7 cells are collected by a cell scraper, and the cells are resuspended by a complete culture medium to prepare a cell suspension. The cell suspension was mixed at 5X 10 4 Each well was inoculated into 24-well cell culture plates at 500. Mu.L per well and cultured for 24 hours.
2. Influence of different dendrobe fermentation solutions on NO content of RAW264.7 cells
(1) Mixing a dendrobium raw material unfermented liquor group, a lactobacillus reuteri CCFM1187 fermentation liquor group, a lactobacillus plantarum QS6-12 fermentation dendrobium fermentation liquor group, a lactobacillus casei 2-L2 fermentation dendrobium fermentation liquor group, a lactobacillus reuteri DL5-6 fermentation dendrobium fermentation liquor supernatant and a complete culture medium which are prepared according to the method of the step 2 of the example 2 according to the volume ratio of (1%: 99%) respectively to prepare mixed liquor;
(2) After mixing, 500. Mu.L of the mixture was added to each well of the 24-well plate containing RAW264.7 cells obtained in step 1, and the culture plate was placed at 37℃with 5% CO 2 Culturing in an incubator for 24 hours.
Setting each group:
negative control group: sucking the culture medium of each hole on the 24-hole plate after the culture in the step (2), and adding 500 mu L of complete culture medium per hole;
model group: adding 5 mug/mL of LPS solution prepared in the step 1 to a 24-well plate containing RAW264.7 cells, 500 mug/well;
sample group (each lactobacillus fermented dendrobium fermented liquid group): sucking the culture medium of each well on the 24-well plate after the culture in the step (2), and adding 5 mug/mL of LPS solution prepared completely, wherein each well is 500 mug;
the 24-well plates of each group were placed in an incubator, and after further culturing at 37℃for 24 hours, 50. Mu.L of each supernatant was aspirated into 96 plates, 5 wells each, and the NO content of the cells was measured by the Gris reagent method, and the results are shown in Table 3 and FIG. 3.
TABLE 3 influence of Dendrobium nobile fermentation broths obtained by fermentation of different strains on NO content of cells
As can be seen from fig. 3 and table 3, lactobacillus reuteri CCFM1187 fermented dendrobium fermented liquid group can significantly reduce the NO content of cells. NO is an important mediator in the immunomodulation process, and its production in excess may trigger immunopathogenic processes causing tissue damage, leading to skin inflammation. The normal control group has the NO content of 12.23 mu M, the model group has the NO content of 55.80 mu M, the non-fermented liquid group of the dendrobium is 49.03 mu M, the NO content of the lactobacillus reuteri CCFM1187 fermented liquid group is 45.42 mu M, the NO content of the lactobacillus reuteri CCFM1187 fermented dendrobium is 40.19 mu M, the NO content of the model group is 0.72 times that of the model group, the NO content of the non-fermented liquid group of the dendrobium is 0.82 times that of the non-fermented liquid group of the dendrobium, the NO content of the non-fermented liquid group of the lactobacillus reuteri CCFM1187 is 0.88 times that of the non-fermented liquid group of the lactobacillus reuteri CCFM1187, and the NO content of cells is obviously reduced (P is less than 0.001). The NO content of the lactobacillus plantarum QS6-12 fermented dendrobium fermentation liquid group is 48.23 mu M, the NO content of the lactobacillus casei 2-L2 fermented dendrobium fermentation liquid group is 51.54 mu M, and the NO content of the lactobacillus reuteri DL5-6 fermented dendrobium fermentation liquid group is 54.26 mu M.
Compared with a dendrobium RAW material unfermented liquid group, the lactobacillus reuteri CCFM1187 fermentation liquid group, the lactobacillus plantarum QS6-12 fermentation dendrobium fermentation liquid group, the lactobacillus casei 2-L2 fermentation dendrobium fermentation liquid group, the lactobacillus reuteri DL5-6 fermentation dendrobium fermentation liquid group and the lactobacillus reuteri CCFM1187 fermentation dendrobium RAW material fermentation liquid can obviously inhibit the NO content of RAW264.7 cells, and improve cell inflammation injury.
While the invention has been described with reference to the preferred embodiments, it is not limited thereto, and various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of Jiangnan
<120> Lactobacillus reuteri strain capable of fermenting herba Dendrobii and its fermentation broth for effectively repairing solar dermatitis
<130> BAA210990A
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 1418
<212> DNA
<213> artificial sequence
<400> 1
ccgactttgg gcgttacaaa ctcccatggt gtgacgggcg gtgtgtacaa ggcccgggaa 60
cgtattcacc gcggcatgct gatccgcgat tactagcgat tccgacttcg tgtaggcgag 120
ttgcagccta cagtccgaac tgagaacggc tttaagagat tagcttactc tcgcgagttt 180
gcgactcgtt gtaccgtcca ttgtagcacg tgtgtagccc aggtcataag gggcatgatg 240
atctgacgtc gtccccacct tcctccggtt tgtcaccggc agtctcacta gagtgcccaa 300
ctcaatgctg gcaactagta acaagggttg cgctcgttgc gggacttaac ccaacatctc 360
acgacacgag ctgacgacga ccatgcacca cctgtcattg cgtccccgaa gggaacgcct 420
tatctctaag gttagcgcaa gatgtcaaga cctggtaagg ttcttcgcgt agcttcgaat 480
taaaccacat gctccaccgc ttgtgcgggc ccccgtcaat tcctttgagt ttcaaccttg 540
cggtcgtact ccccaggcgg agtgcttaat gcgttagctc cggcactgaa gggcggaaac 600
cctccaacac ctagcactca tcgtttacgg catggactac cagggtatct aatcctgttc 660
gctacccatg ctttcgagcc tcagcgtcag ttgcagacca gacagccgcc ttcgccactg 720
gtgttcttcc atatatctac gcattccacc gctacacatg gagttccact gtcctcttct 780
gcactcaagt cgcccggttt ccgatgcact tcttcggtta agccgaaggc tttcacatca 840
gacctaagca accgcctgcg ctcgctttac gcccaataaa tccggataac gcttgccacc 900
tacgtattac cgcggctgct ggcacgtagt tagccgtgac tttctggttg gataccgtca 960
ctgcgtgaac agttactctc acgcacgttc ttctccaaca acagagcttt acgagccgaa 1020
acccttcttc actcacgcgg tgttgctcca tcaggcttgc gcccattgtg gaagattccc 1080
tactgctgcc tcccgtagga gtatggaccg tgtctcagtt ccattgtggc cgatcagtct 1140
ctcaactcgg ctatgcatca tcgccttggt aagccgttac cttaccaact agctaatgca 1200
ccgcaggtcc atcccagagt gatagccaaa gccatctttc aaacaaaagc catgcggctt 1260
ttgttgttat gcggtattag catctgtttc caaatgttat cccccgctcc ggggcaggtt 1320
acctacgtgt tactcacccg tccgccactc actggtgatc catcgtcaat caggtgcaag 1380
caccatcaat cagtcgggct cagtgcgtac gacttgca 1418
Claims (8)
1. Lactobacillus reuteri (Lactobacillus reuteri) CCFM1187, wherein lactobacillus reuteri (Lactobacillus reuteri) CCFM1187 is deposited with the collection of microorganism strains in the cantonese province under the accession number GDMCCNo:61729, the preservation date is 2021, 06 and 21.
2. Use of lactobacillus reuteri CCFM1187 according to claim 1 for fermenting a raw material comprising dendrobium.
3. A dendrobium fermentation broth, which is characterized in that lactobacillus reuteri CCFM1187 of claim 1 is inoculated into a fermentation medium containing dendrobium for fermentation.
4. A dendrobium fermentation broth according to claim 3, wherein lactobacillus reuteri CCFM1187 is inoculated in a fermentation medium containing dendrobium in an amount of at least 1.0 x 10 6 cfu/mL。
5. The dendrobium fermentation broth of claim 3 or 4, wherein the fermentation medium comprises: 40-80 g/L of dendrobium, 10-40 g/L of proliferation factor, 0.1-0.8 g/L of trace element and 0.5-1.5 mL/L of Tween; the proliferation factor is one or more of yeast extract powder, yeast extract, yeast peptone, tryptone and soybean peptone; the microelements are magnesium sulfate and/or manganese sulfate.
6. A product comprising lactobacillus reuteri CCFM1187 of claim 1 or comprising a dendrobium fermentation broth of any of claims 3-5, wherein the product is a pharmaceutical or cosmetic product.
7. The product of claim 6, wherein lactobacillus reuteri CCFM1187 is added in an amount of at least 1.0 x 10 6 cfu/mL。
8. Use of lactobacillus reuteri CCFM1187 according to claim 1 or a dendrobium fermentation broth according to any of claims 3-5 for the preparation of a product for alleviating the symptoms of solar dermatitis, said product being a pharmaceutical or cosmetic product.
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