CN114681383B - Kuh-seng fermentation liquor with exfoliating function and fermentation method - Google Patents
Kuh-seng fermentation liquor with exfoliating function and fermentation method Download PDFInfo
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Classifications
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- 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
- A61K8/97—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution from algae, fungi, lichens or plants; from derivatives thereof
- A61K8/9783—Angiosperms [Magnoliophyta]
- A61K8/9789—Magnoliopsida [dicotyledons]
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- 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
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- 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
- A61Q19/08—Anti-ageing preparations
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- 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
- A61Q19/10—Washing or bathing preparations
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
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- 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
- A61K2800/74—Biological properties of particular ingredients
- A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
- A61K2800/782—Enzyme inhibitors; Enzyme antagonists
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- 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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Abstract
The invention belongs to the technical field of microbial fermentation, and particularly relates to a kuh-seng fermentation liquid with a cutin removing function and a fermentation method. The fermentation method comprises the following steps: (1) strain activation: preparing an LB culture medium, and culturing colonies on the LB culture medium; and (2) Chinese herbal medicine treatment: radix Sophorae Flavescentis is preferably selected from various antioxidant Chinese medicines as skin conditioner; (3) fermentation: fermenting and culturing preferred strain and Chinese herbal medicine under different conditions. According to the invention, through the optimization research on the mixed fermentation conditions of the strain for producing the keratinase and the Chinese herbal medicines, the fermentation conditions with good keratinase activity are obtained. Aims at promoting the fermentation level of the strain to be further improved, reducing the fermentation cost and providing a new scientific method for the development and utilization of keratin substances.
Description
Technical Field
The invention belongs to the technical field of microbial fermentation, and particularly relates to a kuh-seng fermentation liquid with a cutin removing function and a fermentation method.
Background
Keratin is abundant in nature, and can be used as a fibrous and intractable structural protein which can be used as a protective layer to effectively resist abiotic stress and biological attack of animals. Keratin is mainly present in feathers, hair, wool, horny layer, etc.
Keratinase is a protease with a broad spectrum of substrate properties that degrades soluble and insoluble proteins. In particular, it degrades natural keratin to form soluble amino acids and polypeptides, and the reaction conditions are mild. The keratin can be hydrolyzed by the induction of the keratinase, so that the keratinase can be added into the skin care product to remove dead cells and redundant horny layer in the skin, improve the permeability of the skin, enable the skin to better enter the deep layer of the skin, act on the deep cells of the skin and achieve the effect of skin care. The skin care product can be used for removing scars and achieving the effect of beautifying. There are still many problems associated with the industrial production and use of keratinase. The extracellular expression yield of the active keratinase is still low, and the industrialization requirement is difficult to meet. Commercial keratinase is mainly used in the feed and pharmaceutical industries, and there is little research on the use of keratinase in cosmetics. In order to develop the cosmetic market and provide excellent experimental materials for the research and development of cosmetic formulas, the research and development of a cosmetic raw material containing keratinase and other active substances is urgent, and the cosmetic raw material has important theoretical value and practical significance.
The kuh-seng is the dry root of the kuh-seng of leguminous plants, has long medicament use history, mainly contains alkaloid and flavonoid components, and a plurality of researches show that the kuh-seng also has various pharmacological activities such as antioxidation, anti-inflammatory, bacteriostasis, pain relieving and the like. In recent years, the kuh-seng has increasingly wide application in cosmetics, has the effects of moisturizing, anti-inflammatory, antioxidation and the like, reduces the sensitization of the cosmetics, and brings natural skin care effect. The adoption of botanicals in cosmetic formulations is a major trend, with more and more synthetic ingredients being replaced by natural plant ingredients. The keratinase and the kuh-seng are subjected to compound fermentation, so that a fermentation liquid which is safe, mild and good in skin affinity is expected to be produced.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides the kuh-seng fermentation liquor with the exfoliating function and the fermentation method, and the invention provides a basis for biological development, popularization and application of exfoliating products by utilizing the experiment of fermenting the kuh-seng by using keratinase-producing bacteria.
In order to achieve the above purpose, the invention adopts the following technical scheme:
a fermentation method of radix Sophorae Flavescentis with cutin removing function comprises the following steps:
(1) Activating strains:
1L of LB medium is prepared, and the specific operation is as follows: 10g of Tryptone (Tryptone), 5g of Yeast extract (Yeast extract), 10g of sodium chloride (NaCl) and 800mL of distilled water were weighed and stirred uniformly, and the pH was adjusted to 7.0 with NaOH. Stirring uniformly, fixing volume to 1L, subpackaging into 100Ml conical flask, wherein one part is used as liquid culture medium, the other part is added with 0.6g of agar powder to prepare solid culture medium, sterilizing with high-pressure steam sterilizing pot, and standing at room temperature for use.
And taking out the preserved bacterial liquid from the refrigerator, thawing, and placing the bacterial liquid in an ice box for standby. Melting the solid culture medium with a microwave oven, uniformly pouring the culture medium into a sterilized culture dish on an ultra-clean workbench, and adding antibiotics. After solidification, the bacterial liquid is dipped by an inoculating loop, and streaking is carried out on the flat plate. Culturing in a 37 degree incubator overnight.
(2) And (3) treating Chinese herbal medicines:
among the various antioxidant Chinese medicines, sophora flavescens ait of Sophora of Leguminosae is preferably used as skin conditioner. On the basis of selecting high-quality medicinal materials, the Chinese medicinal materials are properly cleaned, soaked, cut, dried and the like, and then the Chinese medicinal materials with certain quality specifications are processed into Chinese medicinal material semi-finished products.
(3) Fermentation:
after 24 hours, picking single colony by using the sterilized toothpick, placing the single colony in LB liquid culture medium, shaking for 4-12 hours at 30-37 ℃ and 150-300r, and then selecting a thicker bottle of bacterial liquid.
Sucking the bacterial liquid, and inoculating the bacterial liquid into another bottle of LB liquid culture medium according to the proportion of 1-3%. And (3) shaking at 30-37 ℃ for 4-12 hours at 150-300r, and measuring an OD value until the OD value reaches about 0.4-0.6, and fermenting.
The LB culture medium is replaced by a fermentation culture medium, inoculated according to the proportion of 1-5%, and subjected to enzyme activity measurement after shaking for 36-72 hours at the temperature of 28-35 ℃ and the speed of 100-200 r.
(4) Effect test of fermentation broth
1) Exfoliating effect
Enzyme activity determination:
40. Mu.l of the diluted enzyme solution was added to 40. Mu.l of 0.5% soluble keratin substrate, reacted in a water bath at 40℃for 10 minutes, and then the reaction was terminated by adding 80. Mu.l of 0.4 mol/l TCA solution. The control group was terminated by adding 80. Mu.l of 0.4 mol/l TCA solution to 40. Mu.l of diluted enzyme solution, and after 10 min of water bath reaction at 40℃40. Mu.l of 0.5% soluble keratin substrate was added. After the reaction, the mixture was centrifuged at 10000r/min for 2min, and the supernatant was collected and absorbance was measured at 280℃and nm. The unit of keratinase activity (U) is defined as: the OD280 value per minute in the mixed reaction system was defined as 1U per liter of the control group.
2) Antioxidant capacity
Free radical (DPPH) scavenging experimental procedure:
the 10mL test tube is used for setting up a sample tube (T), a sample background (T0), a DPPH tube (C) and a solvent background (C0), 3 parallel tubes are required to be set up for each sample tube (T) of each tested concentration of each sample, and 3 parallel tubes are required to be set up for the DPPH tube (C). 1mL of the same concentration of sample solution was added to each of the sample tube (T) and the sample background (T0). All tubes (T, T, C, C0) were supplemented with solvent, water-soluble samples were made up with water, oil-soluble samples were made up with 95% ethanol, 3mL, and mixed well. 1mL of DPPH ethanol solution is added into a sample tube (T) and a DPPH tube (C), the sample background (T0) and the solvent background (C0) are replaced by 95% ethanol, the mixture is gently shaken, and the mixture is kept stand for 5 minutes at room temperature. Each reaction solution was transferred to a 1cm cuvette, and absorbance was measured at 517 nm.
3) Whitening ability
Tyrosinase activity inhibition assay:
the 10mL test tube is used for setting up a sample tube (T), a sample background (T0), an enzyme reaction tube (C) and a solvent background (C0), 3 parallel tubes are required to be set up for each sample tube (T) of each tested concentration of each sample, and 3 parallel tubes are required to be set up for the enzyme reaction tube (C). 1mL of the sample solution with the same concentration is added into the sample tube (T) and the sample background (T0), and 1mL of disodium hydrogen phosphate-citric acid buffer solution is added into the enzyme reaction tube (C) and the solvent background (C0) respectively. And 0.5mL of tyrosinase solution is respectively added into the sample tube (T) and the enzyme reaction tube (C), the sample background (T0) and the solvent background (C0) are replaced by 0.5mL of disodium hydrogen phosphate-citric acid buffer solution, the sample and the tyrosinase are fully and uniformly mixed, and the mixture is placed in a 37 ℃ water bath for incubation for 10 minutes. 2mL of levodopa solution was added to each tube in sequence, the reaction time was controlled to 5 minutes for each tube, and each tube of reaction solution was immediately transferred into a cuvette, and absorbance was measured at 475 nm.
The method is based on fermentation technology: the process of producing and accumulating large amount of metabolic products required by human fermentation is carried out by large-scale growth culture of microorganisms (or animal and plant cells) to make them chemically and physiologically changed.
Advantageous effects
The invention discloses a kuh-seng fermentation liquor with a cutin removing function and a fermentation method, and the fermentation liquor is used for optimizing and researching the mixed fermentation conditions of a strain producing keratinase and Chinese herbal medicines, so that the production conditions with better keratinase production activity are obtained. Aims at promoting the fermentation level of the strain to be further improved, reducing the production and use cost of the keratinase and providing a new scientific method for the development and utilization of keratinase substances.
Detailed Description
Hereinafter, the present invention will be described in detail. Before the description, it is to be understood that the terms used in this specification and the appended claims should not be construed as limited to general and dictionary meanings, but interpreted based on the meanings and concepts corresponding to technical aspects of the present invention on the basis of the principle that the inventor is allowed to define terms appropriately for the best explanation. Accordingly, the description set forth herein is merely a preferred example for the purpose of illustration and is not intended to limit the scope of the invention, so that it should be understood that other equivalents or modifications may be made thereto without departing from the spirit and scope of the invention.
The following examples are merely illustrative of embodiments of the present invention and are not intended to limit the invention in any way, and those skilled in the art will appreciate that modifications may be made without departing from the spirit and scope of the invention. Unless otherwise specified, reagents and equipment used in the following examples are commercially available products.
Example 1
A fermentation process comprising keratinase comprising the steps of:
(1) Activating strains:
1L of LB medium is prepared, and the specific operation is as follows: 10g of Tryptone (Tryptone), 5g of Yeast extract (Yeast extract), 10g of sodium chloride (NaCl) and 800mL of distilled water were weighed and stirred uniformly, and the pH was adjusted to 7.0 with NaOH. Stirring uniformly, fixing volume to 1L, subpackaging into 100Ml conical flask, wherein one part is used as liquid culture medium, the other part is added with 0.6g of agar powder to prepare solid culture medium, sterilizing with high-pressure steam sterilizing pot, and standing at room temperature for use.
And taking out the preserved bacterial liquid from the refrigerator, thawing, and placing the bacterial liquid in an ice box for standby. Melting the solid culture medium with a microwave oven, uniformly pouring the culture medium into a sterilized culture dish on an ultra-clean workbench, and adding antibiotics. After solidification, the bacterial liquid is dipped by an inoculating loop, and streaking is carried out on the flat plate. Culturing in a 37 degree incubator overnight.
(2) Fermentation:
after 24 hours, picking single colony by using the sterilized toothpick, placing the single colony in LB liquid culture medium, shaking for 4-12 hours at 30-37 ℃ and 150-300r, and then selecting a thicker bottle of bacterial liquid.
Sucking the bacterial liquid, and inoculating the bacterial liquid into another bottle of LB liquid culture medium according to the proportion of 1-3%. And (3) shaking at 30-37 ℃ for 4-12 hours at 150-300r, and measuring an OD value until the OD value reaches about 0.4-0.6, and fermenting.
The LB culture medium is replaced by a fermentation culture medium, inoculated according to the proportion of 1-5 percent, and shaken for 36-72 hours at the temperature of 28-35 ℃ and the speed of 100-200 r.
(3) Exfoliating effect:
40. Mu.l of the diluted enzyme solution was added to 40. Mu.l of 0.5% soluble keratin substrate, reacted in a water bath at 40℃for 10 minutes, and then the reaction was terminated by adding 80. Mu.l of 0.4 mol/l TCA solution. The control group was terminated by adding 80. Mu.l of 0.4 mol/l TCA solution to 40. Mu.l of diluted enzyme solution, and after 10 min of water bath reaction at 40℃40. Mu.l of 0.5% soluble keratin substrate was added. After the reaction, the mixture was centrifuged at 10000r/min for 2min, and the supernatant was collected and absorbance was measured at 280℃and nm. The unit of keratinase activity (U) is defined as: the OD280 value per minute in the mixed reaction system was defined as 1U per liter of the control group.
(4) Antioxidant capacity:
the 10mL test tube is used for setting up a sample tube (T), a sample background (T0), a DPPH tube (C) and a solvent background (C0), 3 parallel tubes are required to be set up for each sample tube (T) of each tested concentration of each sample, and 3 parallel tubes are required to be set up for the DPPH tube (C). 1mL of the same concentration of sample solution was added to each of the sample tube (T) and the sample background (T0). All tubes (T, T, C, C0) were supplemented with solvent, water-soluble samples were made up with water, oil-soluble samples were made up with 95% ethanol, 3mL, and mixed well. 1mL of DPPH ethanol solution is added into a sample tube (T) and a DPPH tube (C), the sample background (T0) and the solvent background (C0) are replaced by 95% ethanol, the mixture is gently shaken, and the mixture is kept stand for 5 minutes at room temperature. Each reaction solution was transferred to a 1cm cuvette, and absorbance was measured at 517 nm.
(5) Whitening ability:
the 10mL test tube is used for setting up a sample tube (T), a sample background (T0), an enzyme reaction tube (C) and a solvent background (C0), 3 parallel tubes are required to be set up for each sample tube (T) of each tested concentration of each sample, and 3 parallel tubes are required to be set up for the enzyme reaction tube (C). 1mL of the sample solution with the same concentration is added into the sample tube (T) and the sample background (T0), and 1mL of disodium hydrogen phosphate-citric acid buffer solution is added into the enzyme reaction tube (C) and the solvent background (C0) respectively. And 0.5mL of tyrosinase solution is respectively added into the sample tube (T) and the enzyme reaction tube (C), the sample background (T0) and the solvent background (C0) are replaced by 0.5mL of disodium hydrogen phosphate-citric acid buffer solution, the sample and the tyrosinase are fully and uniformly mixed, and the mixture is placed in a 37 ℃ water bath for incubation for 10 minutes. 2mL of levodopa solution was added to each tube in sequence, the reaction time was controlled to 5 minutes for each tube, and each tube of reaction solution was immediately transferred into a cuvette, and absorbance was measured at 475 nm.
Example 2
A fermentation process comprising keratinase comprising the steps of:
(1) Activating strains:
1L of LB medium is prepared, and the specific operation is as follows: 10g of Tryptone (Tryptone), 5g of Yeast extract (Yeast extract), 10g of sodium chloride (NaCl) and 800mL of distilled water were weighed and stirred uniformly, and the pH was adjusted to 7.0 with NaOH. Stirring uniformly, fixing volume to 1L, subpackaging into 100Ml conical flask, wherein one part is used as liquid culture medium, the other part is added with 0.6g of agar powder to prepare solid culture medium, sterilizing with high-pressure steam sterilizing pot, and standing at room temperature for use.
And taking out the bacterial liquid from the refrigerator, thawing, and placing the bacterial liquid in an ice box for standby. Melting the solid culture medium with a microwave oven, uniformly pouring the culture medium into a sterilized culture dish on an ultra-clean workbench, and adding antibiotics. After solidification, the bacterial liquid is dipped by an inoculating loop, and streaking is carried out on the flat plate. Culturing in a 37 degree incubator overnight.
In the fermentation bacterial liquid, the strain is bacillus, more specifically bacillus licheniformisBacillus licheniformis). The strain preservation information is as follows, preservation unit name: industrial microbiological bacterial strain engineering research center (BNCC), deposit unit address: the storage number of the aerospace building 11 layer 47 in the science and technology park of the western third loop university in the new district of Zhengzhou, henan province is as follows: BNCC189067, BNCC139203, BNCC335814, corresponding taxonomic designations: bacillus licheniformis [ ]Bacillus licheniformis）。
In the fermentation bacterial liquid, the strain is bacillus, more specifically bacillus licheniformisBacillus licheniformis). The strain preservation information is as follows, preservation unit name: industrial microbiological bacterial strain engineering research center (BNCC), deposit unit address: the storage number of the aerospace building 11 layer 47 in the science and technology park of the western third loop university in the new district of Zhengzhou, henan province is as follows: BNCC336463, BNCC221854, BNCC137671, corresponding to class designations: bacillus licheniformis [ ]Bacillus licheniformis）。
(2) Fermentation:
after 24 hours, picking single colony by using the sterilized toothpick, placing the single colony in LB liquid culture medium, shaking for 4-12 hours at 30-37 ℃ and 150-300r, and then selecting a thicker bottle of bacterial liquid.
Sucking the bacterial liquid, and inoculating the bacterial liquid into another bottle of LB liquid culture medium according to the proportion of 1-3%. After shaking bacteria for 4-12 hours at 30-37 ℃ and 150-300r, the OD value is measured until the OD value reaches about 0.4-0.6, and fermentation is carried out, wherein one bacterial liquid number 1 in BNCC189067, BNCC139203 and BNCC335814, and one bacterial liquid number 2 in BNCC336463, BNCC221854 and BNCC137671 are carried out.
The LB culture medium is replaced by a fermentation culture medium, inoculated according to the proportion of 1-5%, and shaken for 36-72 hours at the temperature of 28-35 ℃ and the speed of 100-200r, wherein the fermentation liquor of the No. 1 bacteria is No. 3, and the fermentation liquor of the No. 2 bacteria is No. 4.
The 7 groups of flasks were numbered, ABCDEFG groups, respectively.
Adding fermentation liquor and 5% -20% of radix sophorae flavescentis powder into the group A conical flask in the bottle No. 3.
Adding bacterial liquid in a bottle No. 1, a fermentation medium and 5% -20% of radix sophorae flavescentis powder into a conical bottle B.
Adding bacterial liquid in a bottle 1, LB culture medium and 5% -20% of radix sophorae flavescentis powder into a group C conical flask.
Adding distilled water and radix Sophorae Flavescentis powder into D group conical flask
Adding fermentation liquor and 5% -20% of radix sophorae flavescentis powder into the E group conical flask.
Adding the bacterial liquid in the bottle No. 2, the fermentation medium and 5% -20% of radix sophorae flavescentis powder into the conical bottle of the group F.
Adding bacterial liquid in a bottle No. 2, LB culture medium and 5% -20% of radix sophorae flavescentis powder into a group G conical flask.
(3) Exfoliating effect:
40. Mu.l of the diluted enzyme solution was added to 40. Mu.l of 0.5% soluble keratin substrate, reacted in a water bath at 40℃for 10 minutes, and then the reaction was terminated by adding 80. Mu.l of 0.4 mol/l TCA solution. The control group was terminated by adding 80. Mu.l of 0.4 mol/l TCA solution to 40. Mu.l of diluted enzyme solution, and after 10 min of water bath reaction at 40℃40. Mu.l of 0.5% soluble keratin substrate was added. After the reaction, the mixture was centrifuged at 10000r/min for 2min, and the supernatant was collected and absorbance was measured at 280℃and nm. The unit of keratinase activity (U) is defined as: the OD280 value per minute in the mixed reaction system was defined as 1U per liter of the control group.
TABLE 1 enzyme activity measurement results for different fermentation groups
Example 3
A fermentation process comprising keratinase comprising the steps of:
(1) Activating strains:
1L of LB medium is prepared, and the specific operation is as follows: 10g of Tryptone (Tryptone), 5g of Yeast extract (Yeast extract), 10g of sodium chloride (NaCl) and 800mL of distilled water were weighed and stirred uniformly, and the pH was adjusted to 7.0 with NaOH. Stirring uniformly, fixing volume to 1L, subpackaging into 100Ml conical flask, wherein one part is used as liquid culture medium, the other part is added with 0.6g of agar powder to prepare solid culture medium, sterilizing with high-pressure steam sterilizing pot, and standing at room temperature for use.
And taking out the preserved bacterial liquid from the refrigerator, thawing, and placing the bacterial liquid in an ice box for standby. Melting the solid culture medium with a microwave oven, uniformly pouring the culture medium into a sterilized culture dish on an ultra-clean workbench, and adding antibiotics. After solidification, the bacterial liquid is dipped by an inoculating loop, and streaking is carried out on the flat plate. Culturing in a 37 degree incubator overnight.
And taking out the bacterial liquid from the refrigerator, thawing, and placing the bacterial liquid in an ice box for standby. Melting the solid culture medium with a microwave oven, uniformly pouring the culture medium into a sterilized culture dish on an ultra-clean workbench, and adding antibiotics. After solidification, the bacterial liquid is dipped by an inoculating loop, and streaking is carried out on the flat plate. Culturing in a 37 degree incubator overnight.
In the fermentation bacterial liquid, the strain is bacillus, more specifically bacillus licheniformisBacillus licheniformis). The strain preservation information is as follows, preservation unit name: industrial microbiological bacterial strain engineering research center (BNCC), deposit unit address: the middle-day aviation building 11 layer 47 of the science and technology park of the western three-loop university of the new district of Zhengzhou city of Henan province is given the following preservation number: BNCC189067, BNCC139203, BNCC335814, corresponding taxonomic designations: bacillus licheniformis [ ]Bacillus licheniformis）。
In the fermentation bacterial liquid, the strain is bacillus, more specifically bacillus licheniformisBacillus licheniformis). The strain preservation information is as follows, preservation unit name: industrial microbiological bacterial strain engineering research center (BNCC), deposit unit address: the middle-day aviation building 11 layer 47 of the science and technology park of the western three-loop university of the new district of Zhengzhou city of Henan province is given the following preservation number: BNCC336463, BNCC221854, BNCC137671, corresponding to class designations: bacillus licheniformis [ ]Bacillus licheniformis）。
(2) Fermentation:
after 24 hours, picking single colony by using the sterilized toothpick, placing the single colony in LB liquid culture medium, shaking for 4-12 hours at 30-37 ℃ and 150-300r, and then selecting a thicker bottle of bacterial liquid.
Sucking the bacterial liquid, and inoculating the bacterial liquid into another bottle of LB liquid culture medium according to the proportion of 1-3%. After shaking bacteria for 4-12 hours at 30-37 ℃ and 150-300r, the OD value is measured until the OD value reaches about 0.4-0.6, and fermentation is carried out, wherein one bacterial liquid number 1 in BNCC189067, BNCC139203 and BNCC335814, and one bacterial liquid number 2 in BNCC336463, BNCC221854 and BNCC137671 are carried out.
The LB culture medium is replaced by a fermentation culture medium, inoculated according to the proportion of 1-5%, and shaken for 36-72 hours at the temperature of 28-35 ℃ and the speed of 100-200r, wherein the fermentation liquor of the No. 1 bacteria is No. 3, and the fermentation liquor of the No. 2 bacteria is No. 4.
The 7 groups of flasks were numbered, ABCDEFG groups, respectively.
Adding fermentation liquor and 5% -20% of radix sophorae flavescentis powder into the group A conical flask in the bottle No. 3.
Adding bacterial liquid in a bottle No. 1, a fermentation medium and 5% -20% of radix sophorae flavescentis powder into a conical bottle B.
Adding bacterial liquid in a bottle 1, LB culture medium and 5% -20% of radix sophorae flavescentis powder into a group C conical flask.
Adding distilled water and radix Sophorae Flavescentis powder into D group conical flask
Adding fermentation liquor and 5% -20% of radix sophorae flavescentis powder into the E group conical flask.
Adding the bacterial liquid in the bottle No. 2, the fermentation medium and 5% -20% of radix sophorae flavescentis powder into the conical bottle of the group F.
Adding bacterial liquid in a bottle No. 2, LB culture medium and 5% -20% of radix sophorae flavescentis powder into a group G conical flask.
(3) Antioxidant capacity:
the 10mL test tube is used for setting up a sample tube (T), a sample background (T0), a DPPH tube (C) and a solvent background (C0), 3 parallel tubes are required to be set up for each sample tube (T) of each tested concentration of each sample, and 3 parallel tubes are required to be set up for the DPPH tube (C). 1mL of the same concentration of sample solution was added to each of the sample tube (T) and the sample background (T0). All tubes (T, T, C, C0) were supplemented with solvent, water-soluble samples were made up with water, oil-soluble samples were made up with 95% ethanol, 3mL, and mixed well. 1mL of DPPH ethanol solution is added into a sample tube (T) and a DPPH tube (C), the sample background (T0) and the solvent background (C0) are replaced by 95% ethanol, the mixture is gently shaken, and the mixture is kept stand for 5 minutes at room temperature. Each reaction solution was transferred to a 1cm cuvette, and absorbance was measured at 517 nm.
TABLE 2 determination of radical scavengers for different fermentation groups
Analysis of the results in table 2:
radical scavenging rate indicated:
the effect is best when one bacterial liquid of bacillus licheniformis BNCC189067, BNCC139203 and BNCC335814 is used in LB culture medium added with kuh-seng.
The fermentation medium added with the kuh-seng has the best effect when one bacterial liquid of the bacillus licheniformis BNCC336463, BNCC221854 and BNCC137671 is used.
Example 4
A fermentation process comprising keratinase comprising the steps of:
(1) Activating strains:
1L of LB medium is prepared, and the specific operation is as follows: 10g of Tryptone (Tryptone), 5g of Yeast extract (Yeast extract), 10g of sodium chloride (NaCl) and 800mL of distilled water were weighed and stirred uniformly, and the pH was adjusted to 7.0 with NaOH. Stirring uniformly, fixing volume to 1L, subpackaging into 100Ml conical flask, wherein one part is used as liquid culture medium, the other part is added with 0.6g of agar powder to prepare solid culture medium, sterilizing with high-pressure steam sterilizing pot, and standing at room temperature for use.
And taking out the preserved bacterial liquid from the refrigerator, thawing, and placing the bacterial liquid in an ice box for standby. Melting the solid culture medium with a microwave oven, uniformly pouring the culture medium into a sterilized culture dish on an ultra-clean workbench, and adding antibiotics. After solidification, the bacterial liquid is dipped by an inoculating loop, and streaking is carried out on the flat plate. Culturing in a 37 degree incubator overnight.
And taking out the bacterial liquid from the refrigerator, thawing, and placing the bacterial liquid in an ice box for standby. Melting the solid culture medium with a microwave oven, uniformly pouring the culture medium into a sterilized culture dish on an ultra-clean workbench, and adding antibiotics. After solidification, the bacterial liquid is dipped by an inoculating loop, and streaking is carried out on the flat plate. Culturing in a 37 degree incubator overnight.
In the fermentation bacterial liquid, the strain is bacillus, more specifically bacillus licheniformisBacillus licheniformis). The strain preservation information is as follows, preservation unit name: industrial microbiological bacterial strain engineering research center (BNCC), deposit unit address: the storage number of the aerospace building 11 layer 47 in the science and technology park of the western third loop university in the new district of Zhengzhou, henan province is as follows: BNCC189067, BNCC139203, BNCC335814, corresponding taxonomic designations: bacillus licheniformis [ ]Bacillus licheniformis）。
In the fermentation bacterial liquid, the strain is bacillus, more specifically bacillus licheniformisBacillus licheniformis). The strain preservation information is as follows, preservation unit name: industrial microbiological bacterial strain engineering research center (BNCC), deposit unit address: the storage number of the aerospace building 11 layer 47 in the science and technology park of the western third loop university in the new district of Zhengzhou, henan province is as follows: BNCC336463, BNCC221854, BNCC137671, corresponding to class designations: bacillus licheniformis [ ]Bacillus licheniformis）。
(2) Fermentation:
after 24 hours, picking single colony by using the sterilized toothpick, placing the single colony in LB liquid culture medium, shaking for 4-12 hours at 30-37 ℃ and 150-300r, and then selecting a thicker bottle of bacterial liquid.
Sucking the bacterial liquid, and inoculating the bacterial liquid into another bottle of LB liquid culture medium according to the proportion of 1-3%. After shaking bacteria for 4-12 hours at 30-37 ℃ and 150-300r, the OD value is measured until the OD value reaches about 0.4-0.6, and fermentation is carried out, wherein one bacterial liquid number 1 in BNCC189067, BNCC139203 and BNCC335814, and one bacterial liquid number 2 in BNCC336463, BNCC221854 and BNCC137671 are carried out.
The LB culture medium is replaced by a fermentation culture medium, inoculated according to the proportion of 1-5%, and shaken for 36-72 hours at the temperature of 28-35 ℃ and the speed of 100-200r, wherein the fermentation liquor of the No. 1 bacteria is No. 3, and the fermentation liquor of the No. 2 bacteria is No. 4.
The 7 groups of flasks were numbered, ABCDEFG groups, respectively.
Adding fermentation liquor and 5% -20% of radix sophorae flavescentis powder into the group A conical flask in the bottle No. 3.
Adding bacterial liquid in a bottle No. 1, a fermentation medium and 5% -20% of radix sophorae flavescentis powder into a conical bottle B.
Adding bacterial liquid in a bottle 1, LB culture medium and 5% -20% of radix sophorae flavescentis powder into a group C conical flask.
Adding distilled water and radix Sophorae Flavescentis powder into D group conical flask
Adding fermentation liquor and 5% -20% of radix sophorae flavescentis powder into the E group conical flask.
Adding the bacterial liquid in the bottle No. 2, the fermentation medium and 5% -20% of radix sophorae flavescentis powder into the conical bottle of the group F.
Adding bacterial liquid in a bottle No. 2, LB culture medium and 5% -20% of radix sophorae flavescentis powder into a group G conical flask.
(3) Whitening ability:
the 10mL test tube is used for setting up a sample tube (T), a sample background (T0), an enzyme reaction tube (C) and a solvent background (C0), 3 parallel tubes are required to be set up for each sample tube (T) of each tested concentration of each sample, and 3 parallel tubes are required to be set up for the enzyme reaction tube (C). 1mL of the sample solution with the same concentration is added into the sample tube (T) and the sample background (T0), and 1mL of disodium hydrogen phosphate-citric acid buffer solution is added into the enzyme reaction tube (C) and the solvent background (C0) respectively. And 0.5mL of tyrosinase solution is respectively added into the sample tube (T) and the enzyme reaction tube (C), the sample background (T0) and the solvent background (C0) are replaced by 0.5mL of disodium hydrogen phosphate-citric acid buffer solution, the sample and the tyrosinase are fully and uniformly mixed, and the mixture is placed in a 37 ℃ water bath for incubation for 10 minutes. 2mL of levodopa solution was added to each tube in sequence, the reaction time was controlled to 5 minutes for each tube, and each tube of reaction solution was immediately transferred into a cuvette, and absorbance was measured at 475 nm.
TABLE 3 measurement of tyrosinase inhibition rate for different fermentation groups
The tyrosinase inhibition rate reflects one bacterial liquid in bacillus licheniformis BNCC336463, BNCC221854 and BNCC137671, and the whitening effect is optimal.
From the three groups, the fermentation effect of the group F is optimal, and the fermentation result of one bacterial liquid of bacillus licheniformis BNCC336463, BNCC221854 and BNCC137671 in a fermentation culture medium added with kuh-seng powder is obtained.
The above embodiments are only for illustrating the technical solution of the present invention, and are not limiting; although the invention has been described in detail with reference to the foregoing embodiments, it will be apparent to one skilled in the art that modifications may be made to the technical solutions described in the foregoing embodiments, or equivalents may be substituted for some of the technical features thereof; such modifications and substitutions do not depart from the spirit and scope of the corresponding technical solutions.
Claims (7)
1. A fermentation method of a kuh-seng fermentation broth with a cutin removing function, which is characterized by comprising the following steps:
step 1: activating strains: preparing an LB culture medium, and culturing colonies on the LB culture medium;
the strain is bacillus licheniformis, and the bacillus licheniformis is one of bacillus licheniformis with the preservation numbers of BNCC336463, BNCC221854 and BNCC137671 or one of bacillus licheniformis with the preservation numbers of BNCC189067, BNCC139203 and BNCC 335814;
the preparation of the LB culture medium comprises the following steps: weighing the following raw materials in parts by weight: 10 parts of tryptone, 5 parts of yeast extract and 10 parts of sodium chloride are added with distilled water, stirred uniformly, pH is regulated to 7.0, volume is fixed after stirring uniformly, and split charging is carried out, wherein one part is used as a liquid culture medium, the other part is added with agar powder to prepare a solid culture medium, and the solid culture medium is sterilized by a high-pressure steam sterilizing pot and then is placed at room temperature for standby;
step 2: and (3) treating Chinese herbal medicines: treating radix Sophorae Flavescentis to obtain semi-finished product of radix Sophorae Flavescentis;
step 3: fermentation: step 1, culturing preferred strains in bacterial colonies, and carrying out fermentation culture on the preferred strains and the semi-finished product of the kushen obtained in the step 2 to obtain fermentation liquor;
the specific method for the preferred strain is as follows: culturing the bacterial colony in the step 1 for 20-36 hours, picking a single bacterial colony by using a sterilized toothpick, placing the single bacterial colony in an LB liquid culture medium, shaking the bacterial colony at 30-37 ℃ for 150-300r for 4-12 hours, and then selecting a thicker bottle of bacterial liquid for fermentation;
the specific method for fermentation comprises the following steps:
sucking the selected bacterial liquid, inoculating the bacterial liquid into another bottle of LB liquid culture medium according to the proportion of 1-3%, shaking bacteria for 4-12 hours at the temperature of 30-37 ℃ and 150-300r, and measuring an OD value until the OD value reaches 0.4-0.6, thus obtaining the bacterial liquid for fermentation;
replacing LB culture medium with fermentation culture medium, inoculating fermentation broth according to 1-5%, adding water, LB culture medium or fermentation culture medium according to 5-20% of radix Sophorae Flavescentis, shaking bacteria at 28-35deg.C for 36-72 hr, measuring enzyme activity, and detecting tyrosinase inhibition rate and DPPH clearance rate;
step 4: and (3) effect verification: and (3) performing effect verification on the fermentation broth obtained in the step (3), wherein the method comprises the following steps of: and (5) verifying exfoliating effect, whitening effect and antioxidation capability.
2. The method for fermenting a fermentation broth of kuh-seng with exfoliating function according to claim 1, wherein in the step 1, the specific method for culturing the colony is as follows: and taking out the preserved bacterial liquid from the refrigerator, thawing, placing in an ice box for standby, melting the solid culture medium by a microwave oven, uniformly pouring the culture medium into a sterilized culture dish on an ultra-clean workbench, adding antibiotics, dipping the bacterial liquid by an inoculating loop after solidification, scribing on a flat plate, and placing in a 37 ℃ incubator for overnight culture.
3. The method for fermenting a fermentation broth of kuh-seng with exfoliating function according to claim 1, wherein in step 4, the method for verifying exfoliating effect is as follows: the activity of keratinase is measured by taking self-extracted soluble keratinase in fermentation broth as a substrate.
4. The method for fermenting a fermentation broth of kuh-seng with exfoliating function according to claim 3, wherein the method for measuring the activity of keratinase by using self-extracted soluble keratins as a substrate comprises the following steps: adding 40 mu l of 0.5% soluble keratin substrate into 40 mu l of diluted soluble keratin enzyme solution, reacting for 10 min in a water bath at 40 ℃, and adding 80 mu l of 0.4 mol/l TCA solution to terminate the reaction; taking 40 mu l of diluted enzyme solution from a control group, adding 80 mu l of 0.4 mol/l TCA solution to terminate the reaction, and adding 40 mu l of 0.5% soluble keratin substrate after water bath reaction at 40 ℃ for 10 min; after the reaction is finished, centrifuging for 2min at 10000r/min, sucking the supernatant, and measuring absorbance at 280 nm wavelength; the unit of keratinase activity (U) is defined as: the OD280 value per minute in the mixed reaction system was defined as 1U per liter of the control group.
5. The method for fermenting a fermentation broth of kuh-seng with exfoliating function according to claim 1, wherein in step 4, the method for verifying the whitening effect is as follows: the specific method for detecting the tyrosinase inhibition capacity and the tyrosinase activity inhibition experiment comprises the following steps: a 10mL test tube is used for setting up a sample tube (T), a sample background (T0), an enzyme reaction tube (C) and a solvent background (C0), 3 parallel tubes are required to be set up for each sample tube (T) with tested concentration of each sample, and 3 parallel tubes are required to be set up for the enzyme reaction tube (C); 1mL of sample solution with the same concentration is respectively added into a sample tube (T) and a sample background (T0), and 1mL of disodium hydrogen phosphate-citric acid buffer solution is respectively added into an enzyme reaction tube (C) and a solvent background (C0); adding 0.5mL of tyrosinase solution into a sample tube (T) and an enzyme reaction tube (C), replacing a sample background (T0) and a solvent background (C0) by 0.5mL of disodium hydrogen phosphate-citric acid buffer solution, fully and uniformly mixing the sample and tyrosinase, and incubating for 10 minutes in a water bath at 37 ℃; 2mL of levodopa solution was added to each tube in sequence, the reaction time was controlled to 5 minutes for each tube, and each tube of reaction solution was immediately transferred into a cuvette, and absorbance was measured at 475 nm.
6. The method for fermenting a fermentation broth of kuh-seng with exfoliating function according to claim 1, wherein in step 4, the method for verifying the antioxidant capacity is as follows: detecting free radical scavenging efficiency, and a free radical scavenging experimental method: a 10mL test tube is used for setting up a sample tube (T), a sample background (T0), a DPPH tube (C) and a solvent background (C0), 3 parallel tubes are required to be set up for each sample tube (T) with tested concentration of each sample, and 3 parallel tubes are required to be set up for the DPPH tube (C); 1mL of the sample solution with the same concentration is added into each of the sample tube (T) and the sample background (T0); supplementing solvent in all test tubes (T, T0, C, C0), adding water-soluble sample and oil-soluble sample with 95% ethanol, supplementing 3mL, and mixing; 1mL of DPPH ethanol solution is added into a sample tube (T) and a DPPH tube (C), the sample background (T0) and the solvent background (C0) are replaced by 95% ethanol, the mixture is gently shaken, and the mixture is kept stand for 5 minutes at room temperature; each reaction solution was transferred to a 1cm cuvette, and absorbance was measured at 517 nm.
7. A kuh-seng fermentation broth with a cutin removing function, which is characterized by being prepared by the fermentation method of any one of claims 1-6.
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| KR20160025053A (en) * | 2014-08-25 | 2016-03-08 | 인코스(주) | Culture Medium Including Protease And Cosmetic Composition Including The Same |
| CN109908060A (en) * | 2019-04-25 | 2019-06-21 | 武汉骏安生物科技有限公司 | A kind of raspberry fermentation magma and its preparation method and application |
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