CN113564216B - Method for obtaining tridecapeptide through yeast fermentation and application thereof - Google Patents
Method for obtaining tridecapeptide through yeast fermentation and application thereof Download PDFInfo
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Abstract
The invention discloses a method for obtaining tridecapeptide by yeast fermentation and application thereof, comprising the following steps of S1, preparing culture medium: s11, configuring YPD culture medium; s12, configuration of BSM culture medium: mixing glycerol, phosphoric acid, trace elements and biotin, adding deionized water, stirring uniformly, adding nutrients, and regulating pH by using ammonia water to obtain a BSM culture medium; s2, fermenting and expressing; s3, treating fermentation liquor: separating the obtained product, separating by using an ultrafiltration membrane to obtain trideceth, and recovering waste liquid. The tridecapeptide is a common raw material in the field of cosmetics, is a polypeptide composed of a plurality of amino acids, can promote the generation of new cells, can repair scars on the skin surface of a human body, can promote the growth of the cells, has more stable and safer performance compared with forbidden EGF, and is an ideal substitute for EGF.
Description
Technical Field
The invention relates to the technical field of biological fermentation, in particular to a method for obtaining trideceth through yeast fermentation and application thereof.
Background
The epidermal growth factor, also called human oligopeptide-1, is an active polypeptide consisting of a plurality of amino acids, and can make skin be elastic and ruddy when used in cosmetics, but the epidermal growth factor has great side effect and can cause excessive growth of skin, so the epidermal growth factor is regulated by the national drug administration and is forbidden to be used as a raw material of cosmetics.
At present, tridecapeptide is an ideal substitute for high-activity epidermal growth factor, can be used as a raw material of cosmetics, can promote the growth of cells, reduce the generation of wrinkles, has the effects of brightening skin color and fading scar, and therefore has wide application in the field of cosmetics.
At present, the impurities contained in the product obtained by the preparation method of the tridecapeptide are excessive, which is not beneficial to large-scale industrial continuous production, so the invention of a method for obtaining the tridecapeptide by yeast fermentation and the application thereof are particularly important.
Disclosure of Invention
The invention aims to provide a method for obtaining tridecapeptide by yeast fermentation and application thereof, so as to solve the problems in the background art.
In order to solve the technical problems, the invention provides the following technical scheme: a method for obtaining tridecapeptide by yeast fermentation comprises the following steps,
s1, configuration of a culture medium:
s11, configuration of YPD medium:
(1) Dissolving yeast extract and 10-15 parts of peptone, and sterilizing to obtain solution A;
(2) Dissolving glucose, and sterilizing to obtain solution B;
(3) Mixing the solution A and the solution B, and uniformly stirring to obtain a YPD culture medium;
s12, configuration of BSM culture medium:
mixing glycerol, phosphoric acid, trace elements and biotin, adding deionized water, stirring uniformly, adding nutrients, and regulating pH by using ammonia water to obtain a BSM culture medium;
s2, fermenting and expressing:
(1) Inoculating active bacteria into YPD culture medium for culturing until colony is grown;
(2) Taking YPD culture medium, placing cultured colonies, and placing the colonies in a shaking table for culture to obtain seed liquid;
(3) Adding YPD culture medium and defoamer into a fermentation tank, and sterilizing;
(4) Inoculating the seed liquid into a fermentation tank by using a flame inoculation method for fermentation;
(5) Adding BSM culture medium and defoamer into the induction expression fermentation tank, sterilizing, and then performing high-pressure steam sterilization;
(6) Putting the seed liquid in the fermentation tank into an induced expression fermentation tank, adding methanol, starting fermentation, and obtaining fermentation liquid after fermentation;
s3, treating fermentation liquor:
separating the obtained product, separating by using an ultrafiltration membrane to obtain trideceth, and recovering waste liquid.
Further, the specific steps are as follows,
s1, configuration of a culture medium:
s11, configuration of YPD medium:
(1) Dissolving 5-7 parts of yeast extract and 10-15 parts of peptone, and sterilizing at 120-125 ℃ for 18-22min to obtain solution A;
(2) Dissolving 10-15 parts of glucose, and sterilizing at 110-115 ℃ for 13-16min to obtain a solution B;
(3) Mixing the solution A and the solution B, and uniformly stirring to obtain a YPD culture medium;
s12, configuration of BSM culture medium:
mixing 50-70 parts of glycerol, 20-30 parts of phosphoric acid, 1-2 parts of trace elements and 0.5-1 part of biotin, adding deionized water, uniformly stirring, adding nutrients, and regulating the pH to 5 by using ammonia water to obtain a BSM culture medium;
s2, fermenting and expressing:
(1) Inoculating active bacteria into YPD culture medium, and culturing at 25-30deg.C until colony is grown;
(2) Taking YPD culture medium, placing cultured colony, placing in a shaking table, and culturing at 28-30deg.C and 200r/min to obtain seed solution;
(3) Adding YPD culture medium and defoamer into a fermentation tank, and sterilizing at 120deg.C for 30min;
(4) Inoculating the seed liquid into a fermentation tank by using a flame inoculation method for fermentation, wherein the fermentation time is 10-12h;
(5) Adding BSM culture medium and defoamer into the induction expression fermentation tank, sterilizing at 120deg.C for 30min, and then performing high-pressure steam sterilization for 15min;
(6) Putting the seed liquid in the fermentation tank into an induced expression fermentation tank, adding methanol, starting fermentation, and fermenting for 72 hours to obtain fermentation liquid;
s3, treating fermentation liquor:
separating the obtained product, separating by using an ultrafiltration membrane to obtain trideceth, and recovering waste liquid.
Further, the preparation steps of the nutrient are as follows,
(1) Adding diethyl ether into the obtained waste liquid, extracting, heating to 35 ℃, centrifuging, filtering, taking an upper-layer product, and recovering a lower-layer product;
(2) Adding tetrahydropyrrole and deionized water into the upper-layer product, controlling the pH value to be 2.5-3.5, and carrying out contact glow discharge point electrolysis reaction to obtain a product A;
(3) Mixing the product A with the lower layer product to obtain the nutrition.
Further, the absorbance (OD) of the seed solution after the seed solution is obtained is measured 600nm ) Control OD 600nm At 4-6.
Further, the mass ratio of the methanol added in the step S2 (6) to the seed solution is 5-8:1.
Further, the active bacteria are yeast and methanol-assimilating bacteria, and the ratio of the yeast to the methanol-assimilating bacteria is 5-6:1.
Further, the yeast is pichia pastoris, and the methanol-assimilating bacterium is a methylotrophic bacterium.
Further, the application of the tridecapeptide obtained by the method for obtaining the tridecapeptide through yeast fermentation in dressing.
Further, mixing the obtained tridecapeptide with anthocyanin, natural tea tree oil and honeysuckle extract, adding glycerol, carbomer and deionized water, uniformly stirring, adding triethanolamine, and stirring to obtain the dressing.
Further, the mass ratio of the trideceth to the anthocyanin and the honeysuckle extract is 2:1:1.
Compared with the prior art, the invention has the following beneficial effects: tridecapeptide is a common raw material in the field of cosmetics, is a polypeptide composed of various amino acids, can promote the generation of new cells, can repair scars on the skin surface of a human body, can promote the growth of cells, has more stable and safer performance compared with forbidden EGF, and is an ideal substitute for EGF.
The production method of the tridecapeptide mainly comprises the steps of obtaining the tridecapeptide through chemical synthesis and microbial preparation, and preparing the tridecapeptide by using a yeast fermentation method. The yeast used in the application is pichia pastoris, and the reason for selecting the pichia pastoris is that the pichia pastoris is a facultative anaerobe, can survive in aerobic and anaerobic environments, has high growth speed, is methanol nutritional yeast, and can regulate and control the gene expression by adding methanol, so that the application selects the pichia pastoris when selecting strains, and adds the methanol for regulation and control.
In the preparation process, a culture medium needs to be selected, and the culture medium can directly influence the growth, expression and gene stability of yeast, so that the selection of the culture medium is important. Glucose is selected to be used as a main carbon source when the culture medium is prepared, and nutrients such as inorganic salt, peptone and the like are added, so that the normal growth of yeast is ensured.
The application uses pichia pastoris as a main body, and controls the gene expression of the pichia pastoris by adding methanol, so that the yield of a product can be increased, but the methanol is required to be addedThe amount is controlled, the application limits that the mass ratio of the added amount of the methanol to the seed liquid is optimal in the range of 5-8:1, and the seed liquid can be prepared according to OD 600nm The value is judged and needs to be controlled to be 4-6. The normal induction expression of the pichia pastoris can be ensured by controlling the amount of the added methanol, and the yield of the product can be obviously increased.
The application passes through OD 600nm The growth condition of Pichia pastoris can be judged by the value, and the growth condition of Pichia pastoris can be judged by the OD 600nm The methanol quantity that value control was added, but this application is in order to realize large-scale continuous industrial production, and methyl alcohol is continuous to add, and methyl alcohol is added in a large number, pile up and can bring the security risk, consequently this application has added methyl alcohol assimilation bacterium on the basis of original pichia pastoris, this application has selected to add methyl acidophilus bacteria when selecting methyl alcohol assimilation bacterium, methyl acidophilic bacteria is a main carbon source of taking methyl alcohol mainly, and not regard glucose as main carbon source, consequently select to add methyl acidophilic bacteria, can decompose partial methyl alcohol as the carbon source after adding, and then reduce the content of methyl alcohol, guarantee production safe environment.
The methanotrophic bacteria can absorb methanol as a nutrient substance to grow, in order to avoid malignant competition of pichia pastoris and the methanotrophic bacteria, the ratio of the pichia pastoris to the methanotrophic bacteria is controlled to be 5-6:1, most of methanol can be ensured to be used by the pichia pastoris, and in the production process, fermentation conditions can be judged through the growth conditions of the methanotrophic bacteria.
The fermentation liquor is obtained after the production is finished, and the added Methylophilus bacteria can produce protein and formic acid, wherein the formic acid has certain corrosiveness and can corrode skin to produce red swelling, so the obtained fermentation liquor is treated for multiple times, tridecapeptide products, macromolecules and formic acid can be obtained, the obtained fermentation liquor is sequentially separated and the formic acid is reprocessed, and the boiling point of the formic acid is higher, so the fermentation liquor is added with tetrahydropyrrole, the pH value is controlled to be 2.5-3.5 by adding acid liquor, and the electrolysis reaction is carried out by adopting a contact glow discharge point, so the amino acid compound can be obtained, the amino acid compound is mixed with a lower product, the lower product contains a large amount of macromolecules, the proteins contained in the amino acid compound are more, the nutrients are obtained, and glycine and proline contained in the nutrients, wherein the glycine has a certain inhibition effect on escherichia coli, and the produced proline can play a role in regulating the osmotic balance of yeasts and Methylophilus bacteria, and further the normal growth and gene expression of the yeasts and the Methylophilus bacteria can be ensured.
The trideceth prepared by the method is widely applied to the field of dressing preparation, anthocyanin, natural tea tree oil and honeysuckle extract are added in the dressing preparation process, wherein the anthocyanin is also called anthocyanidin, is a bioflavanoid substance, has certain antioxidation capability, and the added natural tea tree oil has wide antibacterial, bacteriostatic and anti-inflammatory effects, and is a natural antibacterial and anti-inflammatory product which is more effective in the market at present, but the antibacterial performance of the natural tea tree oil is reduced due to certain instability of the natural tea tree oil, so that the honeysuckle extract is additionally added in the method, and chlorogenic acid which can only be mainly contained in the honeysuckle extract has certain inhibition effect on staphylococcus, streptococcus and the like, so that the antibacterial and anti-inflammatory performances of the product are guaranteed.
Detailed Description
The following description of the embodiments of the present invention will be made clearly and completely, and it is apparent that the described embodiments are only some embodiments of the present invention, but not all embodiments. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
Example 1
A method for obtaining tridecapeptide by yeast fermentation comprises the following steps,
s1, configuration of a culture medium:
s11, configuration of YPD medium:
(1) Dissolving 5 parts of yeast extract and 10 parts of peptone, and sterilizing at 120 ℃ for 18min to obtain a solution A;
(2) Dissolving 10 parts of glucose, and sterilizing at 110 ℃ for 13min to obtain a solution B;
(3) Mixing the solution A and the solution B, and uniformly stirring to obtain a YPD culture medium;
s12, configuration of BSM culture medium:
mixing 50 parts of glycerol, 20 parts of phosphoric acid, 1 part of trace elements and 0.5 part of biotin, adding deionized water, uniformly stirring, adding nutrients, and regulating the pH to 5 by using ammonia water to obtain a BSM culture medium;
s2, fermenting and expressing:
(1) Inoculating active bacteria into YPD culture medium, and culturing at 25deg.C until colony is grown, wherein the active bacteria are yeast and methanol-assimilating bacteria, the ratio of yeast to methanol-assimilating bacteria is 5:1, the yeast is Pichia pastoris, and the methanol-assimilating bacteria are Methylophilus bacteria;
(2) Taking YPD medium, placing into cultured colony, placing into shaking table, culturing at 28deg.C under 200r/min to obtain seed solution, and testing its absorbance (OD 600nm ) Control OD 600nm At 4;
(3) Adding YPD culture medium and defoamer into a fermentation tank, and sterilizing at 120deg.C for 30min;
(4) Inoculating the seed liquid into a fermentation tank by using a flame inoculation method for fermentation, wherein the fermentation time is 10 hours;
(5) Adding BSM culture medium and defoamer into the induction expression fermentation tank, sterilizing at 120deg.C for 30min, and then performing high-pressure steam sterilization for 15min;
(6) Putting the seed liquid in the fermentation tank into an induction expression fermentation tank, adding methanol, and starting fermentation, wherein the mass ratio of the added methanol to the seed liquid is 5:1, and fermenting for 72 hours to obtain fermentation liquid;
s3, treating fermentation liquor:
separating the obtained product, separating by using an ultrafiltration membrane to obtain trideceth, and recovering waste liquid.
S4, the preparation steps of the nutrient are as follows,
(1) Adding diethyl ether into the obtained waste liquid, extracting, heating to 35 ℃, centrifuging, filtering, taking an upper-layer product, and recovering a lower-layer product;
(2) Adding tetrahydropyrrole and deionized water into the upper-layer product, controlling the pH value to be 2.5, and carrying out contact glow discharge point electrolytic reaction to obtain a product A;
(3) Mixing the product A with the lower layer product to obtain the nutrition.
Mixing the obtained tridecapeptide with anthocyanin, natural tea tree oil and honeysuckle extract, wherein the mass ratio of the tridecapeptide to the anthocyanin to the honeysuckle extract is 2:1:1, adding glycerol, carbomer and deionized water, stirring uniformly, adding triethanolamine, and stirring to obtain the dressing.
Example 2
A method for obtaining tridecapeptide by yeast fermentation comprises the following steps,
s1, configuration of a culture medium:
s11, configuration of YPD medium:
(1) Dissolving 6 parts of yeast extract and 13 parts of peptone, and sterilizing at 123 ℃ for 20min to obtain a solution A;
(2) Dissolving 13 parts of glucose, and sterilizing at 113 ℃ for 15min to obtain a solution B;
(3) Mixing the solution A and the solution B, and uniformly stirring to obtain a YPD culture medium;
s12, configuration of BSM culture medium:
mixing 60 parts of glycerol, 25 parts of phosphoric acid, 1.5 parts of trace elements and 0.8 part of biotin, adding deionized water, uniformly stirring, adding nutrients, and regulating the pH to 5 by using ammonia water to obtain a BSM culture medium;
s2, fermenting and expressing:
(1) Inoculating active bacteria into YPD culture medium, and culturing at 27deg.C until colony is grown, wherein the active bacteria are yeast and methanol-assimilating bacteria, the ratio of yeast to methanol-assimilating bacteria is 5.5:1, the yeast is Pichia pastoris, and the methanol-assimilating bacteria is Methylophilus bacteria;
(2) Taking YPD medium, placing into cultured colony, placing into shaking table, culturing at 29 deg.C under 200r/min to obtain seed solution, and testing its absorbance (OD 600nm ) Control OD 600nm At 5;
(3) Adding YPD culture medium and defoamer into a fermentation tank, and sterilizing at 120deg.C for 30min;
(4) Inoculating the seed liquid into a fermentation tank by using a flame inoculation method for fermentation, wherein the fermentation time is 11 hours;
(5) Adding BSM culture medium and defoamer into the induction expression fermentation tank, sterilizing at 120deg.C for 30min, and then performing high-pressure steam sterilization for 15min;
(6) Putting the seed liquid in the fermentation tank into an induction expression fermentation tank, adding methanol, and starting fermentation, wherein the mass ratio of the added methanol to the seed liquid is 6:1, and fermenting for 72 hours to obtain fermentation liquid;
s3, treating fermentation liquor:
separating the obtained product, separating by using an ultrafiltration membrane to obtain trideceth, and recovering waste liquid.
S4, the preparation steps of the nutrient are as follows,
(1) Adding diethyl ether into the obtained waste liquid, extracting, heating to 35 ℃, centrifuging, filtering, taking an upper-layer product, and recovering a lower-layer product;
(2) Adding tetrahydropyrrole and deionized water into the upper-layer product, controlling the pH value to be 3.0, and carrying out contact glow discharge point electrolytic reaction to obtain a product A;
(3) Mixing the product A with the lower layer product to obtain the nutrition.
Mixing the obtained tridecapeptide with anthocyanin, natural tea tree oil and honeysuckle extract, wherein the mass ratio of the tridecapeptide to the anthocyanin to the honeysuckle extract is 2:1:1, adding glycerol, carbomer and deionized water, stirring uniformly, adding triethanolamine, and stirring to obtain the dressing.
Example 3
A method for obtaining tridecapeptide by yeast fermentation comprises the following steps,
s1, configuration of a culture medium:
s11, configuration of YPD medium:
(1) Dissolving 7 parts of yeast extract and 15 parts of peptone, and sterilizing at 125 ℃ for 22min to obtain a solution A;
(2) Dissolving 15 parts of glucose, and sterilizing at 115 ℃ for 16min to obtain a solution B;
(3) Mixing the solution A and the solution B, and uniformly stirring to obtain a YPD culture medium;
s12, configuration of BSM culture medium:
mixing 70 parts of glycerol, 30 parts of phosphoric acid, 2 parts of trace elements and 1 part of biotin, adding deionized water, uniformly stirring, adding nutrients, and regulating the pH to 5 by using ammonia water to obtain a BSM culture medium;
s2, fermenting and expressing:
(1) Inoculating active bacteria into YPD culture medium, and culturing at 30deg.C until colony grows, wherein the active bacteria are yeast and methanol-assimilating bacteria, the ratio of yeast to methanol-assimilating bacteria is 6:1, the yeast is Pichia pastoris, and the methanol-assimilating bacteria are Methylophilus bacteria;
(2) Taking YPD medium, placing into cultured colony, placing into shaking table, culturing at 30deg.C and 200r/min to obtain seed solution, and testing its absorbance (OD 600nm ) Control OD 600nm At 6;
(3) Adding YPD culture medium and defoamer into a fermentation tank, and sterilizing at 120deg.C for 30min;
(4) Inoculating the seed liquid into a fermentation tank by using a flame inoculation method for fermentation, wherein the fermentation time is 12 hours;
(5) Adding BSM culture medium and defoamer into the induction expression fermentation tank, sterilizing at 120deg.C for 30min, and then performing high-pressure steam sterilization for 15min;
(6) Putting the seed liquid in the fermentation tank into an induction expression fermentation tank, adding methanol, and starting fermentation, wherein the mass ratio of the added methanol to the seed liquid is 8:1, and fermenting for 72 hours to obtain fermentation liquid;
s3, treating fermentation liquor:
separating the obtained product, separating by using an ultrafiltration membrane to obtain trideceth, and recovering waste liquid.
S4, the preparation steps of the nutrient are as follows,
(1) Adding diethyl ether into the obtained waste liquid, extracting, heating to 35 ℃, centrifuging, filtering, taking an upper-layer product, and recovering a lower-layer product;
(2) Adding tetrahydropyrrole and deionized water into the upper-layer product, controlling the pH value to be 3.5, and carrying out contact glow discharge point electrolytic reaction to obtain a product A;
(3) Mixing the product A with the lower layer product to obtain the nutrition.
Mixing the obtained tridecapeptide with anthocyanin, natural tea tree oil and honeysuckle extract, wherein the mass ratio of the tridecapeptide to the anthocyanin to the honeysuckle extract is 2:1:1, adding glycerol, carbomer and deionized water, stirring uniformly, adding triethanolamine, and stirring to obtain the dressing.
Comparative example 1
A method for obtaining tridecapeptide by yeast fermentation comprises the following steps,
s1, configuration of a culture medium:
s11, configuration of YPD medium:
(1) Dissolving 7 parts of yeast extract and 15 parts of peptone, and sterilizing at 125 ℃ for 22min to obtain a solution A;
(2) Dissolving 15 parts of glucose, and sterilizing at 115 ℃ for 16min to obtain a solution B;
(3) Mixing the solution A and the solution B, and uniformly stirring to obtain a YPD culture medium;
s12, configuration of BSM culture medium:
mixing 70 parts of glycerol, 30 parts of phosphoric acid, 2 parts of trace elements and 1 part of biotin, adding deionized water, uniformly stirring, and regulating the pH to 5 by using ammonia water to obtain a BSM culture medium;
s2, fermenting and expressing:
(1) Inoculating active bacteria into YPD culture medium, and culturing at 30deg.C until colony grows, wherein the active bacteria is yeast, and the yeast is Pichia pastoris;
(2) Taking YPD medium, placing into cultured colony, placing into shaking table, culturing at 30deg.C and 200r/min to obtain seed solution, and testing its absorbance (OD 600nm ) Control OD 600nm At 6;
(3) Adding YPD culture medium and defoamer into a fermentation tank, and sterilizing at 120deg.C for 30min;
(4) Inoculating the seed liquid into a fermentation tank by using a flame inoculation method for fermentation, wherein the fermentation time is 12 hours;
(5) Adding BSM culture medium and defoamer into the induction expression fermentation tank, sterilizing at 120deg.C for 30min, and then performing high-pressure steam sterilization for 15min;
(6) Putting the seed liquid in the fermentation tank into an induction expression fermentation tank, adding methanol, and starting fermentation, wherein the mass ratio of the added methanol to the seed liquid is 8:1, and fermenting for 72 hours to obtain fermentation liquid;
s3, treating fermentation liquor:
and separating the obtained product, and separating by using an ultrafiltration membrane to obtain the trideceth.
Mixing the obtained tridecapeptide with anthocyanin, natural tea tree oil and honeysuckle extract, wherein the mass ratio of the tridecapeptide to the anthocyanin to the honeysuckle extract is 2:1:1, adding glycerol, carbomer and deionized water, stirring uniformly, adding triethanolamine, and stirring to obtain the dressing.
Comparative example 2
A method for obtaining tridecapeptide by yeast fermentation comprises the following steps,
s1, configuration of a culture medium:
s11, configuration of YPD medium:
(1) Dissolving 7 parts of yeast extract and 15 parts of peptone, and sterilizing at 125 ℃ for 22min to obtain a solution A;
(2) Dissolving 15 parts of glucose, and sterilizing at 115 ℃ for 16min to obtain a solution B;
(3) Mixing the solution A and the solution B, and uniformly stirring to obtain a YPD culture medium;
s12, configuration of BSM culture medium:
mixing 70 parts of glycerol, 30 parts of phosphoric acid, 2 parts of trace elements and 1 part of biotin, adding deionized water, uniformly stirring, adding nutrients, and regulating the pH to 5 by using ammonia water to obtain a BSM culture medium;
s2, fermenting and expressing:
(1) Inoculating active bacteria into YPD culture medium, and culturing at 30deg.C until colony grows, wherein the active bacteria are yeast and methanol-assimilating bacteria, the ratio of yeast to methanol-assimilating bacteria is 6:1, the yeast is Pichia pastoris, and the methanol-assimilating bacteria are Methylophilus bacteria;
(2) Taking YPD medium, placing into cultured colony, placing into shaking table, culturing at 30deg.C and 200r/min to obtain seed solution, and testing its absorbance (OD 600nm ) Control OD 600nm At 6;
(3) Adding YPD culture medium and defoamer into a fermentation tank, and sterilizing at 120deg.C for 30min;
(4) Inoculating the seed liquid into a fermentation tank by using a flame inoculation method for fermentation, wherein the fermentation time is 12 hours;
(5) Adding BSM culture medium and defoamer into the induction expression fermentation tank, sterilizing at 120deg.C for 30min, and then performing high-pressure steam sterilization for 15min;
(6) Putting the seed liquid in the fermentation tank into an induction expression fermentation tank, adding methanol, and starting fermentation, wherein the mass ratio of the added methanol to the seed liquid is 8:1, and fermenting for 72 hours to obtain fermentation liquid;
s3, treating fermentation liquor:
and separating the obtained product, and separating by using an ultrafiltration membrane to obtain the trideceth.
Mixing the obtained tridecapeptide with anthocyanin, natural tea tree oil and honeysuckle extract, wherein the mass ratio of the tridecapeptide to the anthocyanin to the honeysuckle extract is 2:1:1, adding glycerol, carbomer and deionized water, stirring uniformly, adding triethanolamine, and stirring to obtain the dressing.
Comparative example 3
A method for obtaining tridecapeptide by yeast fermentation comprises the following steps,
s1, configuration of a culture medium:
s11, configuration of YPD medium:
(1) Dissolving 7 parts of yeast extract and 15 parts of peptone, and sterilizing at 125 ℃ for 22min to obtain a solution A;
(2) Dissolving 15 parts of glucose, and sterilizing at 115 ℃ for 16min to obtain a solution B;
(3) Mixing the solution A and the solution B, and uniformly stirring to obtain a YPD culture medium;
s12, configuration of BSM culture medium:
mixing 70 parts of glycerol, 30 parts of phosphoric acid, 2 parts of trace elements and 1 part of biotin, adding deionized water, uniformly stirring, adding nutrients, and regulating the pH to 5 by using ammonia water to obtain a BSM culture medium;
s2, fermenting and expressing:
(1) Inoculating active bacteria in YPD culture medium, and culturing at 30deg.C until colony is grown, wherein the active bacteria are yeast and methanol-assimilating bacteria, the ratio of yeast to methanol-assimilating bacteria is 6:1, and the methanol-assimilating bacteria are Methylophilus bacteria;
(2) Taking YPD medium, placing into cultured colony, placing into shaking table, culturing at 30deg.C and 200r/min to obtain seed solution, and testing its absorbance (OD 600nm ) Control OD 600nm At 6;
(3) Adding YPD culture medium and defoamer into a fermentation tank, and sterilizing at 120deg.C for 30min;
(4) Inoculating the seed liquid into a fermentation tank by using a flame inoculation method for fermentation, wherein the fermentation time is 12 hours;
(5) Adding BSM culture medium and defoamer into the induction expression fermentation tank, sterilizing at 120deg.C for 30min, and then performing high-pressure steam sterilization for 15min;
(6) Putting the seed liquid in the fermentation tank into an induction expression fermentation tank, adding methanol, and starting fermentation, wherein the mass ratio of the added methanol to the seed liquid is 8:1, and fermenting for 72 hours to obtain fermentation liquid;
s3, treating fermentation liquor:
separating the obtained product, separating by using an ultrafiltration membrane to obtain trideceth, and recovering waste liquid.
S4, the preparation steps of the nutrient are as follows,
(1) Adding diethyl ether into the obtained waste liquid, extracting, heating to 35 ℃, centrifuging, filtering, taking an upper-layer product, and recovering a lower-layer product;
(2) Adding tetrahydropyrrole and deionized water into the upper-layer product, controlling the pH value to be 3.5, and carrying out contact glow discharge point electrolytic reaction to obtain a product A;
(3) Mixing the product A with the lower layer product to obtain the nutrition.
Mixing the obtained tridecapeptide with anthocyanin, natural tea tree oil and honeysuckle extract, wherein the mass ratio of the tridecapeptide to the anthocyanin to the honeysuckle extract is 2:1:1, adding glycerol, carbomer and deionized water, stirring uniformly, adding triethanolamine, and stirring to obtain the dressing.
Comparative example 4
A method for obtaining tridecapeptide by yeast fermentation comprises the following steps,
s1, configuration of a culture medium:
s11, configuration of YPD medium:
(1) Dissolving 7 parts of yeast extract and 15 parts of peptone, and sterilizing at 125 ℃ for 22min to obtain a solution A;
(2) Dissolving 15 parts of glucose, and sterilizing at 115 ℃ for 16min to obtain a solution B;
(3) Mixing the solution A and the solution B, and uniformly stirring to obtain a YPD culture medium;
s12, configuration of BSM culture medium:
mixing 70 parts of glycerol, 30 parts of phosphoric acid, 2 parts of trace elements and 1 part of biotin, adding deionized water, uniformly stirring, adding nutrients, and regulating the pH to 5 by using ammonia water to obtain a BSM culture medium;
s2, fermenting and expressing:
(1) Inoculating active bacteria in YPD culture medium, and culturing at 30deg.C until colony is grown, wherein the active bacteria are yeast and methanol-assimilating bacteria, the ratio of yeast to methanol-assimilating bacteria is 6:1, and the methanol-assimilating bacteria are Methylophilus bacteria;
(2) Taking YPD medium, placing into cultured colony, placing into shaking table, culturing at 30deg.C and 200r/min to obtain seed solution, and testing its absorbance (OD 600nm ) Control OD 600nm At 6;
(3) Adding YPD culture medium and defoamer into a fermentation tank, and sterilizing at 120deg.C for 30min;
(4) Inoculating the seed liquid into a fermentation tank by using a flame inoculation method for fermentation, wherein the fermentation time is 12 hours;
(5) Adding BSM culture medium and defoamer into the induction expression fermentation tank, sterilizing at 120deg.C for 30min, and then performing high-pressure steam sterilization for 15min;
(6) Putting the seed liquid in the fermentation tank into an induction expression fermentation tank, starting fermentation, and fermenting for 72 hours to obtain fermentation liquid;
s3, treating fermentation liquor:
separating the obtained product, separating by using an ultrafiltration membrane to obtain trideceth, and recovering waste liquid.
S4, the preparation steps of the nutrient are as follows,
(1) Adding diethyl ether into the obtained waste liquid, extracting, heating to 35 ℃, centrifuging, filtering, taking an upper-layer product, and recovering a lower-layer product;
(2) Adding tetrahydropyrrole and deionized water into the upper-layer product, controlling the pH value to be 3.5, and carrying out contact glow discharge point electrolytic reaction to obtain a product A;
(3) Mixing the product A with the lower layer product to obtain the nutrition.
Mixing the obtained tridecapeptide with anthocyanin, natural tea tree oil and honeysuckle extract, wherein the mass ratio of the tridecapeptide to the anthocyanin to the honeysuckle extract is 2:1:1, adding glycerol, carbomer and deionized water, stirring uniformly, adding triethanolamine, and stirring to obtain the dressing.
Experiment
With example 3 as a control, comparative examples 1, 2 and 3 were set, wherein the seed inoculum sizes of example 1, example 2, example 3, comparative example 1, comparative example 2 and comparative example 3 were the same, and a control experiment was performed in which no methanol-assimilating bacteria were added in comparative example 1, no formic acid was treated in comparative example 2, common yeast was used in comparative example 3, and no methanol was added in comparative example 4.
The fermentation broths obtained in example 1, example 2, example 3, comparative example 1, comparative example 2, comparative example 3, comparative example 4 were analyzed, and the results were as follows,
| experimental group | Peptides | Proteins | Formic acid |
| Example 1 | Detection of | Detection of | Detection of |
| Example 2 | Detection of | Detection of | Detection of |
| Example 3 | Detection of | Detection of | Detection of |
| Comparative example 1 | Detection of | Detection of | Not detected |
| Comparative example 2 | Detection of | Detection of | Detection of |
| Comparative example 3 | Detection of | Detection of | Detection of |
| Comparative example 4 | Detection of | Detection of | Detection of |
List one
The dressing obtained in example 1, example 2, example 3, comparative example 1, comparative example 2, comparative example 3, comparative example 4 was analyzed for formic acid content, and the results were as follows,
| experimental group | Example 1 | Example 2 | Example 3 | Comparative example 1 | Comparative example 2 | Comparative example 3 | Comparative example 4 |
| Formic acid | Not detected | Not detected | Not detected | Not detected | Detection of | Not detected | Not detected |
Watch II
The nutrients of example 1, example 2, example 3, comparative example 1, comparative example 2, comparative example 3, comparative example 4 were analyzed, and the results were as follows,
| experimental group | Formic acid | Tetrahydropyrrole | Glycine (Gly) | Proline (proline) |
| Example 1 | Not detected | Not detected | Detection of | Detection of |
| Example 2 | Not detected | Not detected | Detection of | Detection of |
| Example 3 | Not detected | Not detected | Detection of | Detection of |
| Comparative example 1 | --- | --- | --- | --- |
| Comparative example 2 | --- | --- | --- | --- |
| Comparative example 3 | Not detected | Not detected | Detection of | Detection of |
| Comparative example 4 | Not detected | Not detected | Detection of | Detection of |
Watch III
The dressings obtained in example 1, example 2, example 3, comparative example 1, comparative example 2, comparative example 3, and comparative example 4 were subjected to an escherichia coli antibacterial property test, and the results were as follows,
| experimental group | Example 1 | Example 2 | Example 3 | Comparative example 1 | Comparative example 2 | Comparative example 3 | Comparative example 4 |
| Antibacterial rate% | 94 | 95 | 94 | 91 | 94 | 93 | 90 |
Table four
As can be seen from Table I, the fermentation broths of examples 1, 2, 3 and 4 contain a certain amount of formic acid, because methanol is used as a main carbon source by the methanol-assimilating bacteria added thereto to produce formic acid, and therefore if the fermentation broths are not treated, the products contain formic acid, which causes damage to human skin.
As can be seen in table two, the dressing of example 1, example 2, example 3, comparative example 1, comparative example 3, comparative example 4 did not contain formic acid, whereas formic acid could be detected in comparative example 2, thus demonstrating that the dressing prepared in example 1, example 2, example 3 had higher safety performance.
In Table III, it can be seen that in the nutrients of examples 1, 2, 3 and 4, formic acid and tetrahydropyrrole could not be detected, glycine and proline could be detected, and glycine and proline could promote the growth of microorganisms, thereby achieving the purpose of improving the yield.
As can be seen in table four, the antibacterial rates of example 1, example 2, example 3, comparative example 1, comparative example 2, comparative example 3, and comparative example 4 against escherichia coli were all above 90%, which indicates that the dressing prepared in this application has excellent antibacterial performance, but the antibacterial rate of comparative example 4 is lower than that of example 1, example 2, and example 3, whereas no methanol was added in comparative example 4, but a slightly small content could be detected in the detection of glycine and proline, which indicates that the growth rate of the methanol-assimilating bacteria was the lowest in the environment containing no methanol, i.e., the methanol-assimilating bacteria grew slowly or even did not grow in the case of containing only a glucose carbon source, which indicates the accuracy of selecting the methanol-assimilating bacteria to be added in this application.
Finally, it should be noted that: the foregoing description is only a preferred embodiment of the present invention, and the present invention is not limited thereto, but it is to be understood that modifications and equivalents of some of the technical features described in the foregoing embodiments may be made by those skilled in the art, although the present invention has been described in detail with reference to the foregoing embodiments. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (4)
1. A method for obtaining tridecapeptide by yeast fermentation, characterized in that: the steps are as follows,
s1, configuration of a culture medium:
s11, configuration of YPD medium:
(1) Dissolving yeast extract and peptone, and sterilizing to obtain solution A;
(2) Dissolving glucose, and sterilizing to obtain solution B;
(3) Mixing the solution A and the solution B, and uniformly stirring to obtain a YPD culture medium;
s12, configuration of BSM culture medium:
mixing glycerol, phosphoric acid, trace elements and biotin, adding deionized water, stirring uniformly, adding nutrients, and regulating pH by using ammonia water to obtain a BSM culture medium;
s2, fermenting and expressing:
(1) Inoculating active bacteria into YPD culture medium for culturing until colony is grown;
(2) Taking YPD culture medium, placing cultured colonies, and placing the colonies in a shaking table for culture to obtain seed liquid;
(3) Adding YPD culture medium and defoamer into a fermentation tank, and sterilizing;
(4) Inoculating the seed liquid into a fermentation tank by using a flame inoculation method for fermentation;
(5) Adding BSM culture medium and defoamer into the induction expression fermentation tank, sterilizing, and then performing high-pressure steam sterilization;
(6) Putting the seed liquid in the fermentation tank into an induced expression fermentation tank, adding methanol, starting fermentation, and obtaining fermentation liquid after fermentation;
s3, treating fermentation liquor:
separating the obtained product, separating by using an ultrafiltration membrane to obtain trideceth, and recovering waste liquid;
the preparation steps of the nutrient are as follows,
(1) Adding diethyl ether into the obtained waste liquid, extracting, heating to 35 ℃, centrifuging, filtering, taking an upper-layer product, and recovering a lower-layer product;
(2) Adding tetrahydropyrrole and deionized water into the upper-layer product, controlling the pH value to be 2.5-3.5, and carrying out contact glow discharge point electrolysis reaction to obtain a product A;
(3) Mixing the product A with the lower-layer product to obtain a nutrient;
the active bacteria are yeast and methanol-assimilating bacteria, and the ratio of the yeast to the methanol-assimilating bacteria is 5-6:1;
the methanol-assimilating bacterium is a Methylophilus bacterium.
2. A method for obtaining tridecapeptide by means of yeast fermentation according to claim 1, characterized in that: the specific steps are as follows,
s1, configuration of a culture medium:
s11, configuration of YPD medium:
(1) Dissolving 5-7 parts of yeast extract and 10-15 parts of peptone, and sterilizing at 120-125 ℃ for 18-22min to obtain solution A;
(2) Dissolving 10-15 parts of glucose, and sterilizing at 110-115 ℃ for 13-16min to obtain a solution B;
(3) Mixing the solution A and the solution B, and uniformly stirring to obtain a YPD culture medium;
s12, configuration of BSM culture medium:
mixing 50-70 parts of glycerol, 20-30 parts of phosphoric acid, 1-2 parts of trace elements and 0.5-1 part of biotin, adding deionized water, uniformly stirring, adding nutrients, and regulating the pH to 5 by using ammonia water to obtain a BSM culture medium;
s2, fermenting and expressing:
(1) Inoculating active bacteria into YPD culture medium, and culturing at 25-30deg.C until colony is grown;
(2) Taking YPD culture medium, placing cultured colony, placing in a shaking table, and culturing at 28-30deg.C and 200r/min to obtain seed solution;
(3) Adding YPD culture medium and defoamer into a fermentation tank, and sterilizing at 120deg.C for 30min;
(4) Inoculating the seed liquid into a fermentation tank by using a flame inoculation method for fermentation, wherein the fermentation time is 10-12h;
(5) Adding BSM culture medium and defoamer into the induction expression fermentation tank, sterilizing at 120deg.C for 30min, and then performing high-pressure steam sterilization for 15min;
(6) Putting the seed liquid in the fermentation tank into an induced expression fermentation tank, adding methanol, starting fermentation, and fermenting for 72 hours to obtain fermentation liquid;
s3, treating fermentation liquor:
separating the obtained product, separating by using an ultrafiltration membrane to obtain trideceth, and recovering waste liquid;
the preparation steps of the nutrient are as follows,
(1) Adding diethyl ether into the obtained waste liquid, extracting, heating to 35 ℃, centrifuging, filtering, taking an upper-layer product, and recovering a lower-layer product;
(2) Adding tetrahydropyrrole and deionized water into the upper-layer product, controlling the pH value to be 2.5-3.5, and carrying out contact glow discharge point electrolysis reaction to obtain a product A;
(3) Mixing the product A with the lower-layer product to obtain a nutrient;
the active bacteria are yeast and methanol-assimilating bacteria, and the ratio of the yeast to the methanol-assimilating bacteria is 5-6:1.
3. A method for obtaining tridecapeptide by means of yeast fermentation according to claim 2, characterized in that: after obtaining seed liquid, the absorbance OD thereof needs to be tested 600nm Control OD 600nm At 4-6.
4. A method for obtaining tridecapeptide by means of yeast fermentation according to claim 2, characterized in that: the mass ratio of the methanol added in the step S2 (6) to the seed liquid is 5-8:1.
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| CN110343677A (en) * | 2019-07-26 | 2019-10-18 | 中农新科(苏州)有机循环研究院有限公司 | The method for improving Pichia pastoris catalase expression quantity |
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| CN110343677A (en) * | 2019-07-26 | 2019-10-18 | 中农新科(苏州)有机循环研究院有限公司 | The method for improving Pichia pastoris catalase expression quantity |
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