CN114686526A - Lactobacillus casei fermentation product and skin care product containing lactobacillus casei fermentation product - Google Patents
Lactobacillus casei fermentation product and skin care product containing lactobacillus casei fermentation product Download PDFInfo
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- CN114686526A CN114686526A CN202111657124.3A CN202111657124A CN114686526A CN 114686526 A CN114686526 A CN 114686526A CN 202111657124 A CN202111657124 A CN 202111657124A CN 114686526 A CN114686526 A CN 114686526A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P1/00—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes
- C12P1/04—Preparation of compounds or compositions, not provided for in groups C12P3/00 - C12P39/00, by using microorganisms or enzymes by using bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K8/00—Cosmetics or similar toiletry preparations
- A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
- A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
- A61K8/99—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from microorganisms other than algae or fungi, e.g. protozoa or bacteria
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q17/00—Barrier preparations; Preparations brought into direct contact with the skin for affording protection against external influences, e.g. sunlight, X-rays or other harmful rays, corrosive materials, bacteria or insect stings
- A61Q17/005—Antimicrobial preparations
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
- A61Q19/00—Preparations for care of the skin
- A61Q19/02—Preparations for care of the skin for chemically bleaching or whitening the skin
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
- A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
- A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
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- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Veterinary Medicine (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Organic Chemistry (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Dermatology (AREA)
- Zoology (AREA)
- Biotechnology (AREA)
- Wood Science & Technology (AREA)
- General Chemical & Material Sciences (AREA)
- Microbiology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Mycology (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Genetics & Genomics (AREA)
- Tropical Medicine & Parasitology (AREA)
- Birds (AREA)
- Epidemiology (AREA)
- Cosmetics (AREA)
- Micro-Organisms Or Cultivation Processes Thereof (AREA)
Abstract
The invention belongs to IPC classification number C12N1/20, and particularly relates to a lactobacillus casei fermentation product and a skin care product containing the lactobacillus casei fermentation product. The lactobacillus casei fermentation product is obtained by fermenting an isolated strain of a food source. The method comprises the following steps of selecting three foods of yoghourt, cheese and pickled vegetable, separating strains on a solid culture medium by using a plate-scribing method, culturing for 24 hours at 37 ℃ to obtain lactobacillus casei, fermenting the lactobacillus casei in a pitera culture medium which takes skim milk as a main component to obtain a lactobacillus casei fermentation product, wherein the lactobacillus casei fermentation product has no influence on the growth of common bacteria such as escherichia coli and the like, has a remarkable inhibition effect on the growth of pathogenic bacteria enterococcus casseliflavus, can promote the growth of resident bacteria staphylococcus epidermidis on the skin, is used in a skin care product, contributes to promoting the activity of resident bacteria on the surface layer of the skin of a human body, reduces the damage of pathogenic bacteria, and provides firm defense for the skin.
Description
Technical Field
The invention belongs to IPC classification number C12N1/20, and particularly relates to a lactobacillus casei fermentation product and a skin care product containing the lactobacillus casei fermentation product.
Background
The traditional fermented food is accompanied with the growth of lactic acid bacteria in the production process. The strain can give flavor to fermented food and inhibit growth of other microorganisms by secreting metabolites such as lactic acid, thereby forming growth advantage of the strain.
Skin is one of the vital organs of the human body, and many microbial populations inhabit the skin, which form a microecological system of the skin with the host skin, associated with the health and physiological functions of the skin. The flora on the skin is divided into resident bacteria and temporary resident bacteria, wherein the resident bacteria comprise staphylococcus, corynebacterium, propionibacterium and the like, are flora living on healthy skin for a long time, and are barriers for protecting the skin. The resident bacteria can secrete antibacterial peptide to prevent pathogenic bacteria infection, decompose debris and lipid of keratinocytes, maintain the acidic environment of the skin, protect the diversity of skin flora and maintain the health of the skin; the transient bacteria are microorganisms caused by the external environment, including staphylococcus aureus, streptococcus hemolyticus, enterococcus and the like, and are main pathogenic bacteria causing skin inflammation and infection.
Patent CN112940968A discloses a Lactobacillus fermentum strain LF8005 cultured in fermented dairy products and pickled vegetables, which has the functions of inhibiting escherichia coli, staphylococcus aureus and candida albicans, scavenging free radicals, resisting oxidation and preventing skin inflammation, but the strains obtained in fermented dairy products and pickled vegetables when cultured in culture bacteria may have certain odor, so that they still have certain odor when used in skin care products, resulting in poor sense of consumers, and although escherichia coli, staphylococcus aureus and candida albicans can keep on, there are some resident bacteria in human skin, which need to promote the growth of these resident bacteria to maintain the skin microstability, but patent CN112940968A does not refer to the same.
Disclosure of Invention
In order to solve the above technical problems, the first aspect of the present invention provides a lactobacillus casei fermentation product obtained by fermenting a strain isolated from a food source.
In some embodiments, a method of screening for an isolated strain in a food source comprises: inoculating sterile water containing food into solid culture medium by plate streaking method, culturing, and screening to obtain the final product (wherein the isolated strains are single colonies).
In some preferred embodiments, the food-containing sterile water is dipped by using an inoculating loop, streaked on a solid MRS medium in steps, and then cultured and screened.
In some embodiments, the food product is selected from one of yogurt, cheese, kimchi.
The pickle, the yoghourt and the cheese can be purchased from supermarket foods.
In some embodiments, when the food can be diluted with sterile water, diluting the food in sterile water using a dilution means to obtain sterile water containing the food; when the food can not be diluted by the sterile water, the food is rinsed in the sterile water by using a rinsing method to obtain the sterile water containing the food.
In some embodiments, the dilution is 0.5 to 2 times (preferably 1 time) the mass of the food; the number of rinsing is more than 5 times (for example, more than 6 times, more than 8 times, more than 9 times, more than 10 times, more than 12 times or 8-12 times).
In some embodiments, the temperature of the culture is 28-37 ℃ (preferably 30 ℃), and the time of the culture is 12-48 h (preferably 20-30 h, and more preferably 24 h).
The method for screening the isolated strains is not limited, and the formed isolated strains can be screened out for nucleic acid amplification, electrophoretic test, sequencing and BLAST comparison analysis, so that the types of the strains to which the isolated strains belong can be accurately determined. However, in subsequent studies, it was found that the screened and isolated bacterial strain was milky, smooth and moist, and the cultured bacterial strain had a smell similar to that of Lactobacillus casei.
The number of the isolated strains obtained by culture can be screened out as required for subsequent detection and verification, and in some embodiments, the number of the isolated strains obtained by screening out and culture is more than 1.
Dipping single colonies by using a gun head, uniformly mixing the single colonies in 10 mu L of sterile water to obtain a colony aqueous solution, taking the colony aqueous solution as a template to perform PCR polymerase chain reaction and nucleic acid gel electrophoresis inspection, sequencing an amplified product, introducing a sequencing result into an NCBI website to perform BLAST sequence comparison, and accurately determining the strain type of the isolated strain, wherein the strain type is the reservation of lactobacillus casei strains.
In some embodiments, the isolated strain has GeneBank ID MH704117.1, MW931847.1, OK 326545.1.
In one embodiment of the present invention, the amplification system in PCR is as follows: template DNA 1. mu.L, Forward primer 1. mu.L, Reverse primer 1. mu. L, dNTP 0.2. mu. L, Buffer 5. mu. L, Phanta 0.2.2. mu. L, ddH2O4. mu.L, a total of 12.4. mu.L.
When the strain type is lactobacillus casei strain, the bacterial colony aqueous solution can be cultured and frozen, in some embodiments, the bacterial colony aqueous solution is subjected to amplification culture and preservation in a manner that the bacterial colony aqueous solution is added into a liquid MRS culture medium for amplification culture (the culture condition is 30 ℃, 200rpm, and culture is carried out for 24 hours) to obtain turbid bacterial liquid; and mixing the turbid bacterial liquid and the frozen stock solution, and storing the mixed solution at-20 ℃ or-80 ℃ to serve as a frozen stock strain.
The ratio of the turbid bacterial liquid to the frozen stock solution and the type of the frozen stock solution can be selected according to the needs, in some embodiments, the frozen stock solution is glycerol with a volume concentration of 50%, and the volume ratio of the turbid bacterial liquid to the glycerol with a volume concentration of 50% is 1: 1.
the fermentation mode of the isolated strain in the food source is as follows: and inoculating the isolated strain with the lactobacillus casei strain in a Pitera liquid culture medium according to the inoculation amount of 3-10% by volume for submerged fermentation shake culture.
In some embodiments, the culture conditions are 30 ℃, 150-200 rpm, and the culture time is 20-30 hours.
In some embodiments, Pierra liquid medium is formulated with skim milk 10.0g/L, yeast extract 1.0g/L, glucose 6.0 g/L.
The Pierra liquid medium is prepared by simple mixing.
The Pierra liquid culture medium can be used after being prepared and sterilized, and in some embodiments, the prepared Pierra liquid culture medium is used after being subjected to continuous high-temperature moist heat sterilization at 115 ℃ for 20 min.
The culture strain uses the pitera culture medium which takes the skim milk as the main component, the culture medium has natural pH value in the culture process, no additional adjustment is needed, the taste of the culture medium product is light, the milk product has faint scent, the culture period is short, no aseptic operation is needed, and the culture medium is rarely polluted by mixed bacteria in the experimental process and meets the sensory requirements of the public on skin care products.
Meanwhile, the specific culture medium is selected for fermentation, so that the problem that the skin care product has poor use sense due to poor odor possibly existing in some bacterial colonies is solved.
According to a second aspect of the invention, the skin care product comprises a lactobacillus casei fermentation product filtrate obtained by filtering and sterilizing the lactobacillus casei fermentation product.
In some embodiments, the lactobacillus casei fermentation product filtrate is used after homogenization; in some embodiments, the homogenization condition of the lactobacillus casei fermentation product filtrate is that the homogenization is carried out at the rotating speed of 6000-9000rpm for 2-10 min.
In some embodiments, the lactobacillus casei fermentation product filtrate comprises greater than 0.5% (e.g., greater than 0.5%, greater than 2%, greater than 4%, greater than 6%, greater than 8%, greater than 10%, greater than 15%, greater than 20%, or 5-20%) by mass of the skin care product.
The skin care product comprises but is not limited to one of moisturizing water, lotion, essence water, cream, eye cream, facial cream and facial mask.
The third aspect of the invention provides moisturizing water, which comprises, by mass, 0.01-0.05% of a chelating agent, 0.05-0.3% of a humectant, 0.1-0.5% of an emollient, 5-7% of a small molecular polyol, 0-0.5% of a preservative, 0.5-1.5% of nicotinamide, 5-20% of a lactobacillus casei fermentation product filtrate, and water to 100%.
Chelating agents include, but are not limited to, disodium EDTA.
The moisturizer is a moisturizer commonly used in the field of skin care products, such as active molecules with moisturizing function, plant extracts with moisturizing function and the like. In some embodiments, the humectant is a combination of TREMELLA FUCIFORMIS (TREMELLA FUCIFORMIS) fruiting body extract, sodium hyaluronate, oat (AVENA SATIVA) beta-glucan in a weight ratio of 1: 1: 1.
emollients are commonly used in the skin care product art and include, but are not limited to, bis-PEG-18 methyl ether dimethylsilane.
The small molecule polyol serves primarily as one of moisturizing and solubilizing, and in some embodiments is a combination of butanediol and 1.2-hexanediol in a weight ratio of 10: 1.
the preservative is commonly used in the field of skin care products, such as potassium sorbate, p-hydroxyacetophenone, chlorhexidine and the like.
In some embodiments, the moisturizing water is prepared by:
(1) weighing a chelating agent, a humectant, an emollient, micromolecular polyhydric alcohol, a preservative, nicotinamide, lactobacillus casei fermentation product filtrate and water according to mass fraction for later use, and taking butanediol in the micromolecular polyhydric alcohol according to a weight ratio of 5: 1, dividing the mixture into butanediol A and butanediol B, wherein the butanediol A is marked as micromolecular polyalcohol A, the butanediol B and the rest micromolecular polyalcohol are marked as micromolecular polyalcohol B;
(2) mixing and stirring water, a chelating agent, micromolecular polyol A and a preservative at 70-80 ℃ until no solid matter exists to obtain a material A;
(3) and (3) after the material A is cooled to 35-45 ℃, adding the rest raw materials in sequence, uniformly stirring, and discharging to obtain the moisturizing lotion.
Compared with the prior art, the invention has at least the following beneficial effects:
1. the production process of the fermented food is accompanied with the growth of lactic acid bacteria, and the strains give the fermented food flavor and inhibit the growth of other microorganisms by secreting metabolic products such as lactic acid and the like to form the growth advantages of the strains; the lactobacillus casei is obtained by separating food and is used in skin care products;
2. the lactobacillus casei fermentation product has no influence on the growth of common bacteria such as escherichia coli, has a remarkable inhibition effect on the growth of pathogenic bacteria enterococcus casseliflavus, and can promote the growth of skin resident bacteria staphylococcus epidermidis;
3. the lactobacillus casei fermentation product filtrate is subjected to homogenizing wall breaking treatment and is combined with other components of the skin care product, so that the skin care product has the function of improving skin microecology;
4. most skin care products on the market at present are oil-water formulas, living probiotics cannot be directly added, the quantity of the probiotics is difficult to maintain, and the quality of products is difficult to control, so the probiotics are rarely added into the skin care products;
5. the product disclosed by the invention has the functions of purifying, relieving, conditioning and repairing skin by natural active ingredients, is beneficial to promoting the activity of resident flora on the surface layer of human skin, reducing the damage of pathogenic flora and providing firm defense for the skin, has the effects of moisturizing, whitening, diminishing inflammation and inhibiting bacteria, conforms to market demands and has a considerable development prospect.
Drawings
FIG. 1 shows the sequencing results of the 16SrDNA amplification products in example 1;
FIG. 2 shows the alignment of gene sequences in example 1;
FIG. 3 is a photograph of the Pierra broth obtained in examples 1 to 3;
FIG. 4 is a photograph of a fermentation product of Lactobacillus casei obtained by fermentation in example 1;
FIG. 5 shows the sequencing result of the 16SrDNA amplification product in example 2;
FIG. 6 shows the alignment of gene sequences in example 2;
FIG. 7 shows the sequencing result of the 16SrDNA amplification product in example 3;
FIG. 8 shows the result of alignment of gene sequences in example 2;
FIG. 9 is a graph showing the inhibitory effect of fermented products of Lactobacillus casei on enterococcus casseliflavus in a bacteriostatic test;
FIG. 10 is a graph showing the effect of Lactobacillus casei fermentation product on the promotion of Staphylococcus epidermidis in a bacteriostatic test;
Detailed Description
Example 1
The first aspect of this embodiment is a lactobacillus casei fermentation product obtained by fermenting an isolated strain of kimchi;
the screening method of the isolated bacterial strain in the pickle comprises the following steps: washing pickle (from supermarket food) in sterile water for 10 times to obtain sterile water containing the pickle, dipping the washed sterile water containing the pickle by using an inoculating loop, streaking on a solid MRS culture medium step by step, culturing for 24 hours at 30 ℃, and screening by using the basic physiological characteristics of colony morphology (milky, smooth and moist colony) and culture dish smell (smell of fermented yoghourt) to obtain 1 strain of lactobacillus casei candidate isolated strain; dipping a single colony in 10 mu L of sterile water by using a gun head, uniformly mixing to obtain a colony aqueous solution, taking 2 mu L of the colony aqueous solution as a template to perform PCR polymerase chain reaction and perform nucleic acid gel electrophoresis inspection, sequencing an amplification product (the amplification system is shown in table 1), further sequencing the amplification product through 16SrDNA, and introducing a sequencing result into an NCBI website to perform BLAST sequence comparison by using a sequencing result to accurately determine the strain type of the content of a fermentation liquid, wherein the sequencing result is shown in figure 1 as the sequencing result of the 16SrDNA amplification product; FIG. 2 shows the results of gene sequence comparison, which shows that the isolated strain obtained by culture screening is Lactobacillus casei, and the strain identification is completed after gene comparison, so as to obtain 1 strain of Lactobacillus casei, wherein GeneBank ID is MH 704117.1; adding the residual 8 μ L colony aqueous solution into liquid MRS culture medium for amplification culture at 30 deg.C and 200rpm for 24 hr, mixing the turbid bacterial solution with 50% (v/v) glycerol at a ratio of 1:1, and storing at-20 deg.C or-80 deg.C as frozen strain.
The fermentation mode of the isolated bacterial strain in the pickle is as follows: the isolated strain obtained by separating the lactobacillus casei strain from the pickled vegetable is inoculated into a Pierra liquid culture medium according to the inoculation amount of 5 percent of volume amount for deep fermentation shake culture under the culture conditions of 30 ℃ and 200rpm for 24 hours, the formula composition of the Pierra liquid culture medium is shown in table 2, the preparation method of the Pierra liquid culture medium is mixing, the Pierra liquid culture medium is natural pH, no additional adjustment is needed, and after the preparation of the culture medium is finished, the Pierra liquid culture medium is used after continuous high-temperature moist heat sterilization at 115 ℃ for 20 minutes.
Wherein, figure 3 is a picture of the Pierra liquid culture medium obtained by preparation, and figure 4 is a picture of the lactobacillus casei fermentation product obtained by fermentation.
According to the second aspect of the embodiment, the moisturizing water is provided, and the raw materials comprise, by mass, 0.02% of a chelating agent, 0.15% of a humectant, 0.3% of an emollient, 6.6% of a small-molecule polyol, 0.3% of a preservative, 1% of nicotinamide, 10% of a lactobacillus casei fermentation product filtrate and water which are supplemented to 100%.
The chelating agent is EDTA disodium; the humectant is a combination of TREMELLA FUCIFORMIS (Tremella FUCIFORMIS) fruiting body extract, sodium hyaluronate and oat (AVENA SATIVA) beta-glucan, and the weight ratio of the humectant to the TREMELLA FUCIFORMIS fruiting body extract is 1: 1: 1; the emollient is bis-PEG-18 methyl ether dimethylsilane; the micromolecular polyalcohol is a combination of butanediol and 1, 2-hexanediol, and the weight ratio of the butanediol polyol to the hexanediol polyol is 10: 1; the preservative is p-hydroxyacetophenone; filtering and sterilizing the lactobacillus casei fermentation product to obtain lactobacillus casei fermentation product filtrate, and homogenizing the lactobacillus casei fermentation product filtrate at 7000rpm for 2-10 min for use;
the preparation method of the moisturizing water comprises the following steps:
(1) weighing a chelating agent, a humectant, an emollient, micromolecular polyhydric alcohol, a preservative, nicotinamide, lactobacillus casei fermentation product filtrate and water according to mass fraction for later use, and taking butanediol in the micromolecular polyhydric alcohol according to a weight ratio of 5: 1, dividing the mixture into butanediol A and butanediol B, wherein the butanediol A is marked as micromolecular polyalcohol A, the butanediol B and the rest micromolecular polyalcohol are marked as micromolecular polyalcohol B;
(2) mixing and stirring water, a chelating agent, micromolecular polyol A and a preservative at 75 ℃ until no solid matter exists to obtain a material A;
(3) and (4) after the material A is cooled to 40 ℃, sequentially adding the rest raw materials, uniformly stirring, and discharging to obtain the moisturizing lotion.
Example 2
The first aspect of this embodiment is a lactobacillus casei fermentation product obtained by fermenting an isolated strain of yogurt;
the method for screening the isolated strains in the yoghourt comprises the following steps: diluting 1 time (by mass) of yogurt (from supermarket food) in sterile water to obtain sterile water containing yogurt, dipping the diluted sterile water containing yogurt in an inoculating loop, streaking on a solid MRS culture medium step by step, culturing at 30 ℃ for 24h, and screening by using basic physiological characteristics of colony morphology (milky, smooth and moist colony) and culture dish odor (odor of fermented yogurt) to obtain a candidate isolated strain of lactobacillus casei 1 strain; dipping single colonies in 10 mu L of sterile water by using a gun head, uniformly mixing to obtain a colony aqueous solution, taking 2 mu L of the colony aqueous solution as a template to perform PCR polymerase chain reaction and perform nucleic acid gel electrophoresis inspection, sequencing an amplification product (an amplification system is shown in table 1), further sequencing the amplification product through 16SrDNA, introducing a sequencing result into an NCBI (national center for information bus) website to perform BLAST (BLAST furnace) sequence comparison by using the sequencing result, and accurately determining the strain type of the content of a fermentation broth, wherein the sequencing result is shown in figure 5 as the sequencing result of the 16SrDNA amplification product; FIG. 6 shows the result of gene sequence comparison, which shows that the isolated strain obtained by culture screening is Lactobacillus casei, and the strain identification is completed after gene comparison, so as to obtain 1 strain of Lactobacillus casei, wherein GeneBank ID is MW 931847.1; adding the residual 8 mu L of colony aqueous solution into a liquid MRS culture medium for amplification culture under the conditions of 30 ℃ and 200rpm for 24 hours, mixing the turbid bacterial solution with 50% (v/v) glycerol according to the ratio of 1:1, and preserving the mixed solution at-20 ℃ or-80 ℃ to serve as a frozen strain.
The fermentation mode of the isolated strain in the yoghourt is as follows: the isolated strain in the yoghourt with the lactobacillus casei strain is inoculated into a Pierra liquid culture medium according to the inoculation amount of 5 percent of volume amount for deep fermentation shake culture under the culture conditions of 30 ℃ and 200rpm for 24 hours, the formula composition of the Pierra liquid culture medium is shown in table 2, the preparation method of the Pierra liquid culture medium is mixing, the Pierra liquid culture medium is natural pH, no additional adjustment is needed, and the Pierra liquid culture medium is used after being continuously sterilized by high temperature, humidity and heat at 115 ℃ for 20 minutes after being prepared.
Wherein, FIG. 3 is a picture of the prepared Pierra liquid medium, and a picture of the fermentation product of Lactobacillus casei obtained by fermentation is similar to that of FIG. 4.
According to the second aspect of the embodiment, the moisturizing lotion is provided, and the raw materials comprise, by mass, 0.02% of a chelating agent, 0.15% of a moisturizing agent, 0.3% of an emollient, 6.6% of a small molecule polyol, 0.3% of a preservative, 1% of nicotinamide, 10% of a lactobacillus casei fermentation product filtrate and water which are supplemented to 100%.
The chelating agent is EDTA disodium; the humectant is a combination of TREMELLA FUCIFORMIS (Tremella FUCIFORMIS) fruiting body extract, sodium hyaluronate and oat (AVENA SATIVA) beta-glucan, and the weight ratio of the humectant to the TREMELLA FUCIFORMIS fruiting body extract is 1: 1: 1; the emollient is bis-PEG-18 methyl ether dimethylsilane; the micromolecular polyalcohol is a combination of butanediol and 1.2-hexanediol, and the weight ratio of the butanediol-hexanediol-butanediol copolymer is 10: 1; the preservative is p-hydroxyacetophenone; filtering and sterilizing the lactobacillus casei fermentation product to obtain lactobacillus casei fermentation product filtrate, and homogenizing the lactobacillus casei fermentation product filtrate at 7000rpm for 2-10 min for use;
the preparation method of the moisturizing water comprises the following steps:
(1) weighing a chelating agent, a humectant, an emollient, micromolecular polyhydric alcohol, a preservative, nicotinamide, lactobacillus casei fermentation product filtrate and water according to mass fraction for later use, and taking butanediol in the micromolecular polyhydric alcohol according to a weight ratio of 5: 1, dividing the mixture into butanediol A and butanediol B, wherein the butanediol A is marked as micromolecular polyalcohol A, the butanediol B and the rest micromolecular polyalcohol are marked as micromolecular polyalcohol B;
(2) mixing and stirring water, a chelating agent, micromolecular polyol A and a preservative at 75 ℃ until no solid matter exists to obtain a material A;
(3) and (4) after the material A is cooled to 40 ℃, sequentially adding the rest raw materials, uniformly stirring, and discharging to obtain the moisturizing lotion.
Example 3
In a first aspect of this example, a lactobacillus casei fermentation product is obtained by fermentation of an isolated strain of cheese;
the screening method of the isolated strain in cheese comprises the following steps: diluting cheese (from supermarket food) by 1 time (by mass) in sterile water to obtain sterile water containing cheese, dipping the diluted sterile water containing cheese by using an inoculating loop, streaking on a solid MRS culture medium step by step, culturing for 24h at 30 ℃, and screening by using basic physiological characteristics of colony morphology (milky, smooth and moist colony) and culture dish odor (odor of fermented yogurt) to obtain a lactobacillus casei 1 strain candidate isolate; dipping a single colony in 10 mu L of sterile water by using a gun head, uniformly mixing to obtain a colony aqueous solution, taking 2 mu L of the colony aqueous solution as a template to perform PCR polymerase chain reaction and perform nucleic acid gel electrophoresis inspection, sequencing an amplification product (the amplification system is shown in table 1), further sequencing the amplification product through 16SrDNA, and introducing a sequencing result into an NCBI website to perform BLAST sequence comparison by using a sequencing result to accurately determine the strain type of the content of a fermentation liquid, wherein the sequence result is shown in figure 7 as the sequencing result of the 16SrDNA amplification product; FIG. 8 shows the results of gene sequence comparison, which shows that the isolated strain obtained by culture screening is Lactobacillus casei, and the strain identification is completed after gene comparison, so as to obtain 1 strain of Lactobacillus casei, wherein GeneBank ID is OK 326545.1; adding the residual 8 mu L of colony aqueous solution into a liquid MRS culture medium for amplification culture under the conditions of 30 ℃ and 200rpm for 24 hours, mixing the turbid bacterial solution with 50% (v/v) glycerol according to the ratio of 1:1, and preserving the mixed solution at-20 ℃ or-80 ℃ to serve as a frozen strain.
The fermentation pattern of the isolated strain in cheese was: the isolated strain in cheese with the strain type of lactobacillus casei strain is inoculated into a Pierra liquid culture medium according to the inoculation amount of 5 percent of volume amount for deep fermentation shake culture under the culture conditions of 30 ℃ and 200rpm for 24 hours, the formula composition of the Pierra liquid culture medium is shown in table 2, the preparation method of the Pierra liquid culture medium is mixing, the Pierra liquid culture medium is natural pH, no additional adjustment is needed, and the Pierra liquid culture medium is used after being prepared and continuously sterilized by high temperature, humidity and heat for 20 minutes at 115 ℃.
Wherein, FIG. 3 is a picture of the prepared Pierra liquid medium, and a picture of the fermentation product of Lactobacillus casei obtained by fermentation is similar to that of FIG. 4.
According to the second aspect of the embodiment, the moisturizing water is provided, and the raw materials comprise, by mass, 0.02% of a chelating agent, 0.15% of a humectant, 0.3% of an emollient, 6.6% of a small-molecule polyol, 0.3% of a preservative, 1% of nicotinamide, 10% of a lactobacillus casei fermentation product filtrate and water which are supplemented to 100%.
The chelating agent is EDTA disodium; the humectant is a combination of TREMELLA FUCIFORMIS (Tremella FUCIFORMIS) fruiting body extract, sodium hyaluronate and oat (AVENA SATIVA) beta-glucan, and the weight ratio of the humectant to the TREMELLA FUCIFORMIS fruiting body extract is 1: 1: 1; the emollient is bis-PEG-18 methyl ether dimethylsilane; the micromolecular polyalcohol is a combination of butanediol and 1.2-hexanediol, and the weight ratio of the butanediol-hexanediol-butanediol copolymer is 10: 1; the preservative is p-hydroxyacetophenone; filtering and sterilizing the lactobacillus casei fermentation product to obtain lactobacillus casei fermentation product filtrate, and homogenizing the lactobacillus casei fermentation product filtrate at 7000rpm for 2-10 min for use;
the preparation method of the moisturizing water comprises the following steps:
(1) weighing a chelating agent, a humectant, an emollient, micromolecular polyhydric alcohol, a preservative, nicotinamide, lactobacillus casei fermentation product filtrate and water according to mass fraction for later use, and taking butanediol in the micromolecular polyhydric alcohol according to a weight ratio of 5: 1, dividing the mixture into butanediol A and butanediol B, wherein the butanediol A is marked as micromolecular polyalcohol A, the butanediol B and the rest micromolecular polyalcohol are marked as micromolecular polyalcohol B;
(2) mixing and stirring water, a chelating agent, micromolecular polyol A and a preservative at 75 ℃ until no solid matter exists to obtain a material A;
(3) and (4) after the material A is cooled to 40 ℃, sequentially adding the rest raw materials, uniformly stirring, and discharging to obtain the moisturizing lotion.
TABLE 1
TABLE 2
Performance testing
1. Experiment for inhibiting bacteria
Table 3 shows the common flora of the skin.
The experimental steps are as follows: the 3 lactobacillus casei fermentation products separated and screened from example 1 (sauerkraut), example 2 (yogurt) and example 3 (cheese) were all crushed by centrifugation, supernatant removal, 20 μ LPBS addition and ultrasonication (power 400W, ultrasonication 2s, 7s rest, total duration 15 min). After treatment, the crushing liquid is changed from granular turbid liquid to clear homogeneous liquid. Adding the crushed mixed solution into 2-3 mL of LBS liquid culture medium, inoculating 5mL of BS liquid culture medium without adding fermented crushed mixed solution into the same amount of 10 mu L of seed solution of skin bacteria (staphylococcus epidermidis, staphylococcus aureus, streptococcus pneumoniae, streptococcus hemolyticus B and enterococcus casseliflavus) respectively, and culturing for 12h, and then comparing the biomass of the culture solution by measuring the absorbance value of the culture solution at 600 nm.
FIG. 9 is a graph showing the inhibitory effect of a fermentation product of Lactobacillus casei on enterococcus casseliflavus in a bacteriostatic test; FIG. 10 is a graph showing the effect of fermentation products of Lactobacillus casei on the promotion of Staphylococcus epidermidis in bacteriostatic experiments. In FIGS. 9 to 10, the kimchi corresponds to the fermentation product of Lactobacillus casei of example 1; the yoghurt corresponds to the lactobacillus casei fermentation product in example 2; cheese corresponds to the lactobacillus casei fermentation product of example 3.
The results show that: the results of fig. 9-10 show that the lactobacillus casei fermentation product has a significant effect on regulating the skin micro-ecology.
The biomass of staphylococcus epidermidis in the BS culture medium added with the fermentation liquid crushed mixed liquid is improved by 9.12 percent compared with the biomass of the BS culture medium without the fermentation crushed mixed liquid, which shows that the addition of the lactobacillus casei fermentation product can promote the growth of skin resident bacteria staphylococcus epidermidis. On the contrary, the addition of the lactobacillus casei fermentation product can inhibit the growth of the skin pathogenic bacteria enterococcus casseliflavus, and the biomass of the enterococcus casseliflavus in the BS culture medium added with the fermentation liquid crushed mixed liquid is reduced by 28.18% compared with the biomass of the BS culture medium not added with the fermentation crushed mixed liquid, so that the lactobacillus casei fermentation product can enable skin flora to be more stable and healthy.
TABLE 3
2. The stability was tested as follows:
(1) and (3) testing thermal stability: respectively putting the moisturizing water obtained in the embodiments 1-3 into a constant-temperature incubator at 50 +/-2 ℃ for 24 hours, taking out, recovering the room temperature, and then changing the color, layering and precipitation into qualified products, or else, obtaining the qualified products;
(2) cold storage property test: the moisturizing water in the embodiments 1-3 is respectively put into a refrigerator with the temperature of minus 5 to minus 10) DEG C plus or minus 1 ℃ for 24 hours, and the moisturizing water is qualified after being recovered to the room temperature without color change, layering and precipitation change, otherwise, the moisturizing water is unqualified.
(3) Mechanical stability: and (3) respectively placing the moisturizing water samples in the embodiments 1-3 in a centrifuge, testing at the rotating speed of 3500rpm for 30min, and observing whether the samples are not separated and whether the layering condition is qualified or not.
3. Odor was judged by laboratory related developers.
The test results are shown in table 4:
TABLE 4
Claims (10)
1. A Lactobacillus casei fermentation product, wherein the Lactobacillus casei fermentation product is obtained by fermenting an isolated strain of food source.
2. The lactobacillus casei fermentation product of claim 1, wherein the screening of the isolated strain of food source comprises: inoculating sterilized water containing food into solid culture medium by plate streaking method, culturing, and screening.
3. A lactobacillus casei fermentation product according to claim 1 or 2 wherein the food product is selected from yoghurt, cheese, sauerkraut.
4. The lactobacillus casei fermentation product according to claim 2, wherein the temperature for cultivation is 28-37 ℃ and the time for cultivation is 12-48 h.
5. The lactobacillus casei fermentation product of claim 2 wherein the screening of the isolated strain of food product further comprises identification of the species.
6. A Lactobacillus casei fermentation product according to claim 5, wherein the identification of the species comprises the steps of: dipping a gun head into a single colony, uniformly mixing the single colony in 10 mu L of sterile water to obtain a colony aqueous solution, taking the colony aqueous solution as a template to perform PCR polymerase chain reaction and nucleic acid gel electrophoresis inspection, sequencing an amplified product, introducing a sequencing result into an NCBI website to perform BLAST sequence comparison, and accurately determining the strain type of the isolated strain, wherein the strain type is the reservation of lactobacillus casei strains.
7. A fermented product of Lactobacillus casei according to claim 6, wherein the amplification system in PCR polymerase chain reaction is as follows: template DNA 1. mu.L, Forward primer 1. mu.L, Reverse primer 1. mu. L, dNTP 0.2. mu. L, Buffer 5. mu. L, Phanta 0.2.2. mu. L, ddH2O4. mu.L, a total of 12.4. mu.L.
8. A lactobacillus casei fermentation product according to claim 1 wherein the isolated strain of the food product is fermented by: and inoculating the isolated strain with the lactobacillus casei strain in a Pitera liquid culture medium according to the inoculation amount of 3-10% by volume for submerged fermentation shake culture.
9. A Lactobacillus casei fermentation product according to claim 8, wherein the Pierra broth comprises skimmed milk 10.0g/L, yeast extract 1.0g/L, glucose 6.0 g/L.
10. A skin care product containing a Lactobacillus casei fermentation product, which is characterized by comprising a Lactobacillus casei fermentation product filtrate obtained by filtering and sterilizing the Lactobacillus casei fermentation product according to any one of claims 1 to 9.
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| CN117298032A (en) * | 2023-11-17 | 2023-12-29 | 天津中耀博研生物医药有限公司 | Metaplasia mask for changing skin microecology and preparation method thereof |
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