CN113278544A - Lactobacillus paracasei, preparation method and application of fermentation liquid of lactobacillus paracasei, and product - Google Patents
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Abstract
The invention discloses lactobacillus paracasei, a preparation method and application of fermentation liquor of the lactobacillus paracasei, and a product. The lactobacillus paracasei is classified and named as Lactobacillus puracasei XS45M3, is preserved in China center for type culture Collection CCTCC at 16 months 4 in 2021, and has a preservation number of CCTCC M2021391. Experiments prove that the lactobacillus paracasei fermentation liquor provided by the invention has no cytotoxicity, and has the functions of promoting collagen proliferation, softening and smoothing skin, removing wrinkles and increasing elasticity. Can be used for preparing topical skin coating agent with effects of improving skin elasticity and scavenging free radicals.
Description
Technical Field
The invention belongs to the field of microorganisms, and particularly relates to lactobacillus paracasei, a preparation method and application of fermentation liquor of the lactobacillus paracasei, and a product.
Background
With the improvement of industrialization degree and living standard of people, a series of skin problems such as aging, sensitivity, pox and the like are paid more and more attention.
Besides having obvious influence on intestinal flora, some probiotic strains can also show strong immune regulation, skin microecology balance and other characteristics on the skin level, and are reflected in the functions of improving neuropeptide-mediated skin inflammation, repairing damaged skin barriers, inhibiting skin harmful flora, regulating skin microecology balance and the like.
The lactobacillus paracasei is a gram-negative bacterium, is an edible probiotic which is approved by the state, is generally applied to dairy products, health products, beverages and infant food, is a market-approved beneficial bacterium, and has the beneficial effect of regulating intestinal health.
In the aspect of improving skin, the effect of evaluating lactobacillus paracasei by adopting an in-vitro skin organ is researched, the lactobacillus paracasei is found to be capable of regulating a reactive inflammation mechanism related to skin and has beneficial effects in key biological processes related to skin barrier function and skin reactivity recovery, the effect of the lactobacillus paracasei in the aspects of improving skin barrier function recovery and reducing local skin inflammation is also reported, in addition, research shows that lactobacillus paracasei fermentation liquor has obvious antioxidant capacity, is a biological surfactant without cytotoxicity and can be used as an excellent skin care product raw material, and the research suggests that the lactobacillus paracasei has huge potential in skin care application.
Disclosure of Invention
In view of the above-mentioned drawbacks or needs for improvement in the prior art, the present invention provides lactobacillus paracasei, a method for preparing a fermentation broth thereof, use of the same, and a product thereof, which are intended to provide an anti-aging or anti-wrinkle component having a low cost and an excellent effect by using an excellent collagen content increase-promoting effect unique to the lactobacillus paracasei fermentation broth as an active ingredient of an external skin pack having an anti-wrinkle or anti-aging effect.
In order to achieve the above object, according to one aspect of the present invention, there is provided Lactobacillus paracasei, which is classified and named Lactobacillus puracaeiss 45M3, and is deposited at the chinese type culture collection CCTCC of CCTCC M2021391 at 16 months 4 and 2021.
According to another aspect of the present invention, there is provided a method for preparing the lactobacillus paracasei fermentation broth, comprising the steps of:
inoculating the activated strain to a fermentation culture medium according to the inoculation amount of 1-3%;
adding rhodiola rosea 1-3 per mill, millennium herb 1-3 per mill and tremella 1-3 per mill by weight percentage into the fermentation culture medium as fermentation nutrient materials.
And filtering and sterilizing after fermentation is finished to obtain filtrate which is the lactobacillus paracasei fermentation liquor.
Preferably, in the preparation method of the lactobacillus paracasei fermentation liquid, rhodiola rosea with the mass fraction of 2 per thousand, millennium with the mass fraction of 3 per thousand and tremella with the mass fraction of 1 per thousand are added into a fermentation culture medium of the lactobacillus paracasei fermentation liquid to serve as fermentation nutrient materials.
Preferably, the lactobacillus paracasei fermentation broth is prepared by a method in which the fermentation medium is prepared according to one of the following methods:
fermentation medium 1: taking 5-10g of soybean protein powder, 3-5g of soybean meal, 1-5g of starch, 1-5g of NaCl and 0.5-1g of MgSO4Fixing the volume to 1L, and adjusting the pH value to be between 4 and 7; or
Fermentation medium 2: every 30mL-50mL of oat enzymolysis liquid, 1-5g of collagen powder, 1-5g of soybean meal, 0.5-2g of glucose, 1-5g of NaCl and 0.1-0.5g of FeSO4Fixing the volume to 1L, and adjusting the pH value to be between 5 and 6; the preparation method of the oat enzymolysis liquid comprises the following steps: pulverizing herba Avenae Fatuae 5-10g, adding 100mL water, mixing, heating to 40 deg.C, adding 2-5g starch, and performing enzymolysis for 3-5 hr.
Preferably, the preparation method of the lactobacillus paracasei fermentation liquor comprises the following steps of:
fermentation medium 1: every 5g of soybean protein powder, 3g of soybean meal, 5g of starch, 5g of NaCl and 0.5MgSO 24Fixing the volume to 1L, and adjusting the pH value to 6; or
Fermentation medium 2: each 30mL of oat enzymolysis liquid, 5g of collagen powder, 5g of soybean meal, 2g of glucose, 5g of NaCl and 0.1g of FeSO4Fixing the volume to 1L, and adjusting the pH value to 6; the preparation method of the oat enzymolysis liquid comprises the following steps: every 5g of oat is crushed, then 100mL of water is added, the mixture is uniformly mixed, heated to 40 ℃, and 2g of starch is added for enzymolysis for 5 hours.
Preferably, the fermentation conditions of the preparation method of the lactobacillus paracasei fermentation liquor are as follows: the cells were incubated at 37 ℃ for 48 hours with 120rpm shaking.
According to another aspect of the present invention, there is provided the use of the lactobacillus paracasei fermentation broth, characterized by being used for preparing a coating agent for external application to the skin.
Preferably, the lactobacillus paracasei fermentation liquor is applied to preparing an anti-aging external skin coating agent for improving skin elasticity and scavenging free radicals of the skin.
According to another aspect of the present invention, there is provided an external skin coating agent comprising the lactobacillus paracasei fermented liquid.
Preferably, the external skin coating agent contains 5% to 20% of the lactobacillus paracasei fermented liquid.
In general, compared with the prior art, the above technical solution contemplated by the present invention can achieve the following beneficial effects:
experiments prove that the lactobacillus paracasei fermentation liquor provided by the invention has no cytotoxicity, and has the functions of promoting collagen proliferation, softening and smoothing skin, removing wrinkles and increasing elasticity. Can be used for preparing topical skin coating agent with effects of improving skin elasticity and scavenging free radicals.
Preferably, the prebiotics with obvious growth promoting effect on the lactobacillus paracasei XS45m3 are selected: the millennium herb, the rhodiola rosea and the tremella are used as a nutrition bag in proper proportion to promote the fermentation effect of the lactobacillus paracasei, and the result shows that the content of the collagen is promoted to be improved by 15 percent, and particularly the improvement effect is obvious and even exceeds 50 percent when the fermentation culture medium 2 containing the oat enzymolysis liquid is matched. The components of the fermentation medium containing prebiotics in proper proportion have great influence on the growth of lactobacillus paracasei XS45m3 and the capability of the fermentation liquid thereof in promoting the increase of the collagen content.
Detailed Description
In order to make the objects, technical solutions and advantages of the present invention more apparent, the present invention is further described in detail with reference to the following embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention. In addition, the technical features involved in the embodiments of the present invention described below may be combined with each other as long as they do not conflict with each other.
The Lactobacillus paracasei provided by the invention is classified and named as Lactobacillus puracaei XS45M3, is preserved in China center for type culture Collection CCTCC at 16 months 4 and 2021, and has a preservation number of CCTCC M2021391.
The preparation method of the yeast liquid comprises the following steps:
inoculating the activated strain to a fermentation culture medium according to the inoculation amount of 1-3%, and culturing for 48 hours in a shaking table at 37 ℃ and at 120 rpm;
the activated strain is prepared according to the following method:
taking out a lactobacillus paracasei glycerol cryopreservation tube stored in a refrigerator at the temperature of-80 ℃, placing the tube in a room temperature to melt, picking strains by using an inoculating loop in a sterile environment, streaking and inoculating the strains on an MRS solid plate, culturing for 24 hours at the temperature of 37 ℃, observing the colony morphology on the plate to confirm that the lactobacillus paracasei is free of pollution, picking single strains on the plate to inoculate the single strains into an MRS liquid culture medium, and culturing for 24 hours at the temperature of 37 ℃ to obtain the activated strains.
The fermentation medium is prepared according to the following method:
fermentation medium 1: 5g of soybean protein powder, 3g of soybean meal, 5g of starch, 5g of NaCl, 0.5g of MgSO4The volume is fixed to 1L, and the ph is adjusted to 4-7.
Fermentation medium 2: 30mL of oat enzymolysis liquid (5g of crushed oat, 100mL of water is added, the mixture is uniformly mixed and heated to 40 ℃, 2g of starch is added for enzymolysis for 5 hours), 5g of collagen powder, 5g of bean pulp, 2g of glucose,5g NaCl、0.1g FeSO4The volume is fixed to 1L, and the ph is adjusted to 5-6.
During fermentation, 1-3 per mill rhodiola rosea, 1-3 per mill millennium herb and 1-3 per mill tremella are added in dry weight percentage to serve as fermentation nutrient materials; preferably, fermentation nutrient material containing 2 per mill of rhodiola rosea, 3 per mill of millennium herb and 1 per mill of tremella is added.
And filtering and sterilizing after fermentation is finished to obtain filtrate which is the lactobacillus paracasei fermentation liquor.
The lactobacillus paracasei coating agent provided by the invention can be used for preparing skin external coating agents, such as face cream, facial mask and the like, and has the effects of improving skin elasticity, removing skin free radicals and resisting skin aging; wherein the addition proportion of the lactobacillus paracasei fermentation liquor is between 5 and 20 percent.
The following are examples:
example 1 isolation, purification and conservation of Probiotics
Facial skin microorganism samples of healthy people of 18-28 years old are collected in a laboratory, diluted by sterile oxygen-free water in a gradient manner, and then diluted solution with a proper gradient is selected and coated on an MRS solid culture medium for culturing for 48 hours at 37 ℃. And selecting typical colonies, streaking on MRS solid culture medium for 2-3 times, and culturing to obtain pure colonies. The strain is identified as a strain of Lactobacillus paracasei by 16S rDNA molecules, and is named as Lactobacillus paracasei XS45m3(Lactobacillus puracaeisi XS45m3), and the strain is preserved in China Center for Type Culture Collection (CCTCC) at 16 months 4 and 2021.
Example 2
Selecting 7 materials of tremella, lucid ganoderma, chickpea, rhodiola rosea, poria cocos, bitter gourd and millennium herb, crushing the 7 materials, adding the crushed materials into an MRS culture medium according to the mass ratio of 5 per thousand respectively, taking the blank MRS culture medium as a reference, inoculating 2% of activated lactobacillus paracasei XS45m3 respectively, performing gradient dilution after culturing for 24 hours, and coating and counting a plate with proper dilution concentration.
The results are shown in the table above, different plant components added in MRS culture have different effects on the growth of lactobacillus paracasei, wherein tremella, rhodiola rosea, poria cocos and millennium herb have promoting effects on the growth of the lactobacillus paracasei, and lucid ganoderma, chickpea and bitter gourd have inhibiting effects on the growth of the ganoderma, chickpea and bitter gourd. The invention selects three plant components with most obvious growth promotion effect on lactobacillus paracasei as nutrition bags during fermentation, namely tremella, rhodiola rosea and millennia root.
Example 3
(1) Strain activation
Taking out a lactobacillus paracasei XS45m3 glycerol freezing tube stored in a refrigerator at the temperature of-80 ℃, placing the tube in a sterile environment after melting at room temperature, picking strains by using an inoculating loop, streaking and inoculating the strains on an MRS solid plate, culturing for 48 hours at the temperature of 37 ℃, observing the colony morphology on the plate to confirm that the lactobacillus paracasei is bacteria and has no pollution, picking single bacteria on the plate and inoculating the single bacteria into an MRS liquid culture medium, and culturing for 24 hours at the temperature of 37 ℃ for later use.
(2) Preparation of fermentation Medium
Fermentation medium 1: 5g of soybean protein powder, 3g of soybean meal, 5g of starch, 5g of NaCl, 0.5g of MgSO4The volume of the ionized water is adjusted to 1L, and the ph is adjusted to 6.
Fermentation medium 2: 30mL of oat enzymolysis liquid (5g of crushed oat, 100mL of ionized water is added, the mixture is uniformly mixed and heated to 40 ℃, 2g of starch is added for enzymolysis for 5 hours), 5g of collagen powder, 5g of bean pulp, 2g of glucose, 5g of NaCl and 0.1g of FeSO4The volume of the ionized water is fixed to 1L, and the ph is adjusted to 6.
(3) Preparation of fermented nutritional material
Weighing 1-3g of rhodiola rosea, 1-3g of millennium herb and 1-3g of tremella, respectively adding the rhodiola rosea, the millennium herb and the tremella into the fermentation culture medium 1 and the fermentation culture medium 2, and uniformly mixing to obtain the fermentation nutrient material.
(4) Fermentation of probiotics
Inoculating the bacterial liquid obtained by activating the strain into a fermentation culture medium according to the inoculation amount of 1-3%, adding different fermentation nutrient materials, and culturing for 48 hours in a shaking table at 37 ℃ and at 120 rpm.
(5) Filtering the fermentation liquor
And filtering after the probiotic fermentation is finished, wherein the filtrate is the probiotic fermentation liquor.
Example 4 cytotoxicity evaluation test
At 3X 104Cell/well Density HaCaT cells were seeded in 96-well plates at 37 ℃ with 5% CO2Culturing for 24 hours under the condition, then sucking and removing the supernatant, grouping according to an experimental scheme, and respectively adding 100 mu L of serum-free DMEM culture solution containing 5% of the fermentation liquid S1-S6 prepared in example 3 into each hole of the experimental group; the negative control group used serum-free DMEM medium. 37 ℃ and 5% CO2After 24 hours of incubation under these conditions, the supernatant was aspirated, 100. mu.L of a medium containing 5mg/mL of MTT solution was added, incubated at 37 ℃ for 4 hours, the MTT solution was removed, 100. mu.L of DMSO was added, and the mixture was shaken in the dark for 10 minutes at a low speed, and the absorbance was measured at 490 nm. Each set of experiments was set up in 3 replicates and the results are expressed as mean ± standard deviation.
Relative cell survival rate ═ an/A0
Note: a. thenFor the absorbance of the test group, A0The absorbance of the negative control group is shown.
Cell viability above 70% may be defined as no cytotoxicity according to the toxicity assessment criteria described in ISO 10993-5: 2009; the above results confirmed that the fermentation broth prepared in example 3 was not cytotoxic.
Example 5 fermentation broth-stimulated collagen proliferation assay
At 5X 103Cell/well Density HSF cells were seeded into 96-well plates at 37 ℃ with 5% CO2Culturing for 24 hr, sucking supernatant, and grouping according to experiment
Experimental groups: each well was filled with 100. mu.L of DMEM (serum-free) medium containing 5% of the fermentation broth A1-A6 prepared in example 3;
control group: adding 100 mu L of DMEM (serum-free) culture medium into each well;
37℃,5%CO2after culturing for 48 hours under the conditions, the culture medium supernatant was collected and type I collagen was detected using an ELISA kit. Each set of experiments was set up in 3 replicates and the results are expressed as mean ± standard deviation.
Note: "+" indicates P <0.05 compared to control.
The increased collagen content can make skin tender, smooth, wrinkle-removing, and elasticity-increasing. As can be seen from the table, the collagen content of the HSF cells treated with the fermentation broth S1-S6 prepared in example 3 was increased compared to the collagen produced by the untreated HSF cells, and the difference was significant compared to the control group, wherein the collagen content of S2, S4, and S6 in the experimental group was higher than that of S1, S3, and S5. The fermentation broth S1-S6 can stimulate the HSF cells to produce collagen, and compared with the fermentation broth prepared by fermenting the fermentation medium 2, the fermentation broth prepared by fermenting the fermentation medium 2 stimulates the HSF cells to produce collagen with higher content. The most preferred combination is S4.
Example 6
At 5X 103Cell/well Density HSF cells were seeded into 96-well plates at 37 ℃ with 5% CO2Incubate under conditions for 24 hours, aspirate the supernatant and group according to the experiment.
Experimental groups: each hole is added with a catalyst containing H2O2100 μ L of (400 μmol/L) DMEM (serum-free) medium, after 1 hour of treatment, the supernatant was aspirated and washed with PBS to remove H2O2Thereafter, 100. mu.L of DMEM (serum-free) medium containing 5% of the fermentation broth S1-S6 prepared in example 3 was added to each well;
model group: each hole is added with a catalyst containing H2O2100 μ L of (400 μmol/L) DMEM (serum-free) medium, after 1 hour of treatment, the supernatant was aspirated and washed with PBS to remove H2O2Then, 100 μ L of DMEM (serum-free) medium was added;
control group: adding 100 mu L of DMEM (serum-free) culture medium into each well;
after 24h incubation, cells were washed with PBS and incubated with 40. mu. M H2DCFDA for 30 min at 37 ℃. The cells were then placed on a multifunctional microplate reader and the fluorescence intensity of each well was measured at an excitation wavelength of 485nm and an emission wavelength of 528 nm. Each set of experiments was set up in 3 replicates and the results are expressed as mean ± standard deviation.
Note: "+" indicates P <0.05 compared to control.
As can be seen from the table, when the ROS content in the H2O 2-treated HSF cells (model group and experimental group) is compared with that in the untreated HSF cells (control group), the ROS content in the model group and the experimental group is significantly higher than that in the control group, wherein the ROS content in the model group is the highest, and the ROS content in the experimental group is significantly reduced and is lower than that in the model group after the fermentation broth S1-S6 prepared in example 3 is dried; and in the same fermentation process, the intracellular ROS content of the fermentation broth prepared by fermenting the fermentation medium 2 is lower than that of the fermentation broth prepared by fermenting the fermentation medium 1. Indicates H2O2Can stimulate HSF cells to generate a large amount of ROS, intervene by lactobacillus paracasei fermentation liquor, can effectively remove the ROS content in the cells, and the fermentation liquor prepared by fermenting the fermentation medium 2 is superior to the fermentation liquor prepared by fermenting the fermentation medium 1 in removing the ROS in the cells.
Example 7 population experiments
The fermentation broth S4 prepared in example 3 was added to the cream at 5% and applied to the skin surface of human body. Subjects of 28-45 years of age were selected as 10 people, divided into 2 groups of 5 subjects per group of samples. After the face of the subject is cleaned in the morning and evening every day, the face cream is uniformly smeared on the face for continuous use for 2 weeks, and the subject does not use other skin care products and medicines during the test period and keeps normal work and rest and diet. The skin of the subject was evaluated before the start and after the end of the test, respectively. Cream bases were provided by Chang E pharmaceutical Co., Ltd, Hubei.
The fermentation broth S4 prepared in example 3 was added to the mask solution at 10%, and the mask paper was soaked in 25mL of the mask solution containing the fermentation broth. 10 subjects of 28-45 years of age were selected and divided into 2 groups of 5 subjects per group of samples, 2 times weekly for 4 weeks, and during the test period subjects were not on other skin care products and drugs and kept on normal work and diet. The skin of the subject was evaluated before (0d), during (14d) and after (28d) the test was started. The base material of the facial mask liquid is provided by Chang E pharmaceutical industry Co.
The results of the experiment are as follows:
experimental results on face cream
Experimental results of the mask
Note: the larger the number of pores "+" is, the larger the number of pores is; the more the number of the elasticity "+" is, the better the skin elasticity is; more gloss "+" indicates better skin gloss; the more wrinkles "+" the more skin wrinkles; more melanin "+" indicates more melanin pigmentation of the skin.
According to experimental results, the cream and the mask containing the lactobacillus paracasei fermentation liquor S4 prepared in example 3 have the effects of increasing skin elasticity, improving skin luster, improving skin pores and reducing wrinkles, and the fermentation liquor S4 is an effective component for resisting skin aging.
It will be understood by those skilled in the art that the foregoing is only a preferred embodiment of the present invention, and is not intended to limit the invention, and that any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the scope of the present invention.
Claims (10)
1. The Lactobacillus paracasei is characterized by being classified and named as Lactobacillus puracaei XS45M3 and being preserved in China center for type culture Collection CCTCC at 16 months 4 and 2021 with the preservation number of CCTCC M2021391.
2. The method of preparing a lactobacillus paracasei fermentation broth of claim 1 comprising the steps of:
inoculating the activated strain to a fermentation culture medium according to the inoculation amount of 1-3%;
adding rhodiola rosea 1-3 per mill, millennium herb 1-3 per mill and tremella 1-3 per mill by weight percentage into the fermentation culture medium as fermentation nutrient materials.
And filtering and sterilizing after fermentation is finished to obtain filtrate which is the lactobacillus paracasei fermentation liquor.
3. The method for preparing lactobacillus paracasei fermentation broth according to claim 2, wherein rhodiola rosea 2 per mill, millennium 3 per mill and tremella 1 per mill are added into the fermentation medium by weight percentage as fermentation nutrient materials.
4. The method of preparing a lactobacillus paracasei fermentation broth of claim 2 wherein the fermentation medium is prepared according to one of the following methods:
fermentation medium 1: taking 5-10g of soybean protein powder, 3-5g of soybean meal, 1-5g of starch, 1-5g of NaCl and 0.5-1g of MgSO4Fixing the volume to 1L, and adjusting the pH value to be between 4 and 7; or
Fermentation medium 2: every 30mL-50mL of oat enzymolysis liquid, 1-5g of collagen powder, 1-5g of soybean meal, 0.5-2g of glucose, 1-5g of NaCl and 0.1-0.5g of FeSO4Fixing the volume to 1L, and adjusting the pH value to be between 5 and 6; the preparation method of the oat enzymolysis liquid comprises the following steps: pulverizing herba Avenae Fatuae 5-10g, adding 100mL water, mixing, heating to 40 deg.C, adding 2-5g starch, and performing enzymolysis for 3-5 hr.
5. The method of preparing a lactobacillus paracasei fermentation broth according to claim 4, wherein the fermentation medium is prepared according to the following method:
fermentation medium 1: every 5g of soybean protein powder, 3g of soybean meal, 5g of starch, 5g of NaCl and 0.5MgSO 24Fixing the volume to 1L, and adjusting the pH value to 6; or
Fermentation medium 2: each 30mL of oat enzymolysis liquid, 5g of collagen powder, 5g of soybean meal, 2g of glucose, 5g of NaCl and 0.1g of FeSO4Fixing the volume to 1L, and adjusting the pH value to 6; the preparation method of the oat enzymolysis liquid comprises the following steps: every 5g of oat is crushed, then 100mL of water is added, the mixture is uniformly mixed, heated to 40 ℃, and 2g of starch is added for enzymolysis for 5 hours.
6. The method for preparing a lactobacillus paracasei fermentation broth according to claim 2, wherein the fermentation conditions are: the cells were incubated at 37 ℃ for 48 hours with 120rpm shaking.
7. Use of a lactobacillus paracasei fermentation broth according to any of claims 2 to 6 for the preparation of a coating agent for external application to the skin.
8. The use of a lactobacillus paracasei fermented liquid according to claim 7 for preparing an external skin coating agent for anti-aging including improvement of skin elasticity and scavenging of skin free radicals.
9. An external skin coating agent comprising the lactobacillus paracasei fermented liquid according to any one of claims 2 to 6.
10. The external dermal coating agent according to claim 9, characterized by containing 5% to 20% of the lactobacillus paracasei fermentation broth according to any one of claims 2 to 6.
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CN114561331A (en) * | 2022-04-29 | 2022-05-31 | 山东锦鲤生物工程有限公司 | Lactobacillus paracasei and application thereof |
CN114686526A (en) * | 2021-12-30 | 2022-07-01 | 广州君研生物科技有限公司 | Lactobacillus casei fermentation product and skin care product containing lactobacillus casei fermentation product |
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