CN102356879A - Functional peptide-reinforced healthcare food - Google Patents
Functional peptide-reinforced healthcare food Download PDFInfo
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- CN102356879A CN102356879A CN2011103224405A CN201110322440A CN102356879A CN 102356879 A CN102356879 A CN 102356879A CN 2011103224405 A CN2011103224405 A CN 2011103224405A CN 201110322440 A CN201110322440 A CN 201110322440A CN 102356879 A CN102356879 A CN 102356879A
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Abstract
The invention relates to a functional peptide-reinforced healthcare food which comprises, by weight, 2 to 5 parts of glutathione preparation material, 2 to 5 parts of sea cucumber or marine fish protein peptide preparation material, 0.04 to 0.1 part of R-lipoic acid, (1 to 3)X10-5 parts of folic acid, 0.2 to 0.5 part of procyanidins, 2 to 5 parts of food grade L-citrulline and 4 to 8 parts of excipient or filler. The invention provides a glutathione high-yield bacterial strain saccharomyces cerevisiae which can reduce production cost of glutathione and enables glutathione to be more widely applied nutriment. Glutathione serves as an important raw material, and a small molecular weight peptide with an important physiological function, citrulline and other physiological active materials with synergistic effect are matched with glutathione so as to provide the healthcare food which has the functions of enhanced antioxidation, aging resistance and cardiovascular protection.
Description
Technical field
The invention belongs to field of health care food, relate to especially that (Glutathione GSH) is the health food that the Functional Polypeptides of important feedstock production is strengthened with glutathione.
Background technology
(L-γ-glutamyl-L-cysteinylglycine GSH) is the tripeptide compound that is formed through the peptide bond condensation by glutamic acid (L-Glutamic acid), cysteine (L-cysteine) and glycine (Glycine) to glutathione.Extensively be present in animal, plant and the microbial cell, wherein with yeast, animal's liver equal size than horn of plenty.Glutathione has multiple important physical function in organism; It is important antioxidants in the cell; Keeping the cell reducing condition, is one of key substance of anti-damage of participation cell and metabolism adjusting, and wherein anti-oxidation function is especially noticeable with assistance transmembrane amino acid transporter function.
Need a lot of normal oxidation reactions in the vital movement, this process inevitably produces free radical; In addition, cell is at the metabolism allogenic material, and during like toxin, medicine or reply extreme environment factor (like ultraviolet ray or the like), cell also can produce free radical.These radical pair biological cells produce harm greatly, as attacking large biological molecule lipid, protein, DNA etc., cause cell membrane damage, biology enzyme inactivation and DNA to destroy and sudden change, even cause important pathology such as malignant tumour.Normal body or cell have the ability of eliminating free radical, preventing and repair damage, promptly keep the ability of intracellular redox equilibrium, guarantee the normal of vital movement.Glutathione is the important biomolecule small-molecular peptides with this ability.
The cysteine of glutathione contains sulfydryl, and (SH), this group is an active group in redox reaction, can be used as electron donor; The various free radicals that in body, produce, peroxide, hydrogen peroxide etc. provide electronics and with its deactivation; Thereby safeguard redox dynamic balance state (Valko M, Izakovic M., Mazur M. in the cell; Rhodes C.; Telser J., Role of oxygen radicals in DNA damage and cancer incidence. Molecular & Cellular Biochemistry, 2004; 266:37-56); Glutathione is participated in transmembrane amino acid transporter through the circulation of born of the same parents' intracellular metabolite; Under the assistance of ATP; With the inside of amino acid transport to the cell that combines, and the completion absorption process (Zheng Yunlang. the biological function of glutathione. the biology circular. 1995. 30 (5): 22-24).This reaction is the important step that amino acid absorbs.Protein also participated in by glutathione and DNA is synthetic, needs glutathione to paddy hydrogen reduction albumen motive power to be provided in this process.Have the bibliographical information glutathione also to have to suppress apoptotic function (Filomeni G, Rotilio G, Ciriolo M R. Cell signalling and the glutathione redox system. Biochemical Pharmacology. 2002,64,1057-1064); The deficiency of glutathione will cause the program of cell dead (Marengo, B., De Ciucis, C., Verzola; D., Pistoia, V., Raffaghello, L.; Patriarca, S., Balbis, E., Traverso; N., Cottalasso, D., Pronzato, M.A.; Marinari, U.M., Optimization of production. Acta Microbiol Pol 2008,46:105-114).
Glutathione is used aspect medicine, food.Because the glutathione production cost is high, product is expensive, thereby mainly is at present the application aspect medical.Glutathione is usually in detoxifcation, Radiation sickness control, protection liver, and antiallergy, the treatment that beauty treatment, beauty treatment, skin care, increase eyesight and ophthalmology are sick; Glutathione plays neutralization, mediates effect toxic compounds and the heavy metal that gets in the body, the clinical treatment that is used for poisonings such as acrylonitrile, fluoride, carbon monoxide, heavy metal, alcohol; Also can be used as ancillary drug anticancer, antitumor and treatment AIDS, it has very strong protective effect to the symptoms such as leucocyte minimizing that anticancer therapy causes.Glutathione also is used to treat the diabetes nerve damage.Hyperglycaemia influences glutathione metabolism, has reduced H
2O
2The elimination ability thereby cause nerve damage.With glutathione treatment diabetic neuropathy patient total effective rate 93 % (Yao Minxiu, Xu Qian, Li Ying etc. glutathione treatment diabetic neuropathy. Chinese Journal of New Drugs and Clinical Remedies. 2000. 19: 66-67).
Aspect food, glutathione can be used for the processing of food such as Flour product, dairy products and baby food, meat, bird, fish and seafood and bread, but importantly as the application of health products.Proved that glutathione is all playing an important role aspect a lot of enzymatic reactions and the elimination free radical; If the body glutathion inside is not enough; Some enzymatic reactions can not effectively be carried out, and the free radical that metabolic process produces can not in time be eliminated, free radical accumulation promptly promotion body aging; And pathology such as induced tumor, cerebrovascular atherosclerosis; Therefore glutathione have disease-resistant anti-aging function (Sun Honggang, Lv Yizhen. the metabolism of glutathione and application. health professional education. 2004,22 (10): 126-127).
Generally speaking, though the physiological significance of glutathione is familiar with for a long time, it is not general as yet to be used for food, and developed countries such as Japan, the U.S. just began glutathione is applied to health food in last century.At home, directly the health products of interpolation glutathione are few at present.Its major reason is that glutathione is not easy to produce, and its price is very high.
The glutathione preparation method mainly contains four kinds of solvent extraction, chemical synthesis, fermentation method and enzyme transforming process.Extraction mainly is to be raw material with animal and plant tissue that is rich in glutathione and yeast etc.; The shortcoming of chemical synthesis process is that very complicated, reactions step are more, the time is long, cost is high, contaminated environment, possibly produce racemization and influence biologically active and situation such as productive rate is lower, has limited the application of the chemical synthesis process of glutathione.Enzyme transforming process needs two enzymatics (GCS and GS), and three seed amino acids are made raw material, and need ATP etc., and these factors directly cause its technology cost higher.Take all factors into consideration the each side factor, fermentation method is the method for producing the tool potentiality of GSH at present.
Fermentation method is to utilize the higher bacterial strain of glutathione content to carry out large scale fermentation to obtain cell, therefrom extracts glutathione.Yet; The mechanism of its synthesizing glutathion of saccharomycete that fermentation at present is used is two-step catalysis mechanism; First step L glutamic acid and L cysteine through gamma glutamyl cysteine synthetase (γ-glutamylcysteine synthetase, the catalysis of γ-GCS) generates the L glutamyl cysteine; (glutathione synthetase, GS) catalysis L-glutamyl cysteine and glycine reactant generate glutathione to the reaction of second step by glutathione synthetase.(γ-GCS) regulated and control by end-product glutathione feedback inhibition, therefore, this step belongs to rate-limiting reaction to the enzyme of above-mentioned first step reaction.Produce the restriction that bacterial strain is difficult to break away from this feedback regulation at present, glutathione content promotes and is restricted.
(Verena Liedschulte1 such as recent Verena Liedschulte; Andreas Wachter1; 3; An Zhigang2 and Thomas Rausch1* Exploiting plants for glutathione (GSH) production:Uncoupling GSH synthesis from cellular controls results in unprecedented GSH accumulation. Plant Biotechnology Journal, 2010,8:1 – 14) from bacterium Streptococcus thermophilus strain ATCC 19258 (DSMZ; Germany microorganism and cell culture preservation center; Braunschweig, Germany) genomic clone to has bifunctional glutathione synthetase gene, is called GCL-GS, and its sequence is numbered GQ848551 at international Genbank database; This gene outcome has above-mentioned GCS enzyme and two kinds of catalysis of GS enzyme, and the feedback inhibition of this gene pairs glutathione is insensitive.Therefore, this gene has been introduced by this article author and has been obtained high-yield glutathione in the tobacco plant.But do not see other application reports of this gene.
Sea cucumber belongs to echinoderm, is that a kind of ancient ocean software is biological.The nutritive value of sea cucumber is high; Its main component is collagen, mucopolysaccharide and sea cucumber saponin, and collagen accounts for 74. 15% (Li Cuicui, Dong Xiuping of sea cucumber body wall protein content; Gao Yang etc. the extraction process of sea cucumber body wall enzymatic dissolubility collagen; Dalian Polytechnic University's journal, 2008,27 (4): 1 ~ 4).Sea cucumber also has chondroitin sulfate, multivitamin, and 50 various active compositions such as each seed amino acid, is rare cholesterol and the high tonic that extremely hangs down purine of not containing.Research shows; Physiologically actives such as that the collagen peptide that collagen hydrolysate obtains small-molecular weight has is anti-oxidant, hypotensive, suppress platelet aggregation activity, antitumor and immunological regulation, also have simultaneously replenish the calcium, fat-reducing, beauty and skin care and functional characteristic such as delay senility.(Zhang Xinru. collagen food gains great popularity. Chinese health nutrient, 2003 (7): 32; Xie Yuxiang, the fourth beautiful jade. collagen and old and feeble correlation. Chinese gerontology magazine, 2002,6 (22): 518 ~ 520) composition also has hemangiectasis in the sea cucumber, increases blood flow; The dissolving of fortifying fibre albumen improves microcirculatory function.The sea cucumber peptide prepared product of sea cucumber through obtaining with protease hydrolytic, the both complete distinctive nutrition of extra large stichopus japonicus that kept, again macro-molecular protein is changed into more be prone to absorb, small-molecular peptides with better function, absorb more easily than traditional cucumber product.In recent years prove that the infiltration rate and the absorptivity of little molecule small peptide in human body intestinal canal is also faster than amino acid, and a lot of small peptide has physiologically active.
On the other hand; Sea cucumber is one of the abundantest animal of natural L-arginine content; Nobel laureate L.J.Ignarro points out that arginine and citrulling can increase NO production in vivo; Therefore stimulate circulation, strengthen endothelial cell vigor (Louis J. Ignarro " No More Heart Disease ", Published 2005 by Broadway books, New York).
Soyprotein peptide is that soybean protein makes through the enzyme degraded; Product is rich in physiologically active small-molecular peptides and each seed amino acid; Be rich in arginine and citrulling, have function and low antigenicity characteristics such as anti-hypertension, inhibition cholesterol, promotion lipid-metabolism, raising immunity, the growth of promotion profitable strain.
Summary of the invention
The purpose of this invention is to provide a kind of novel healthy food; It is rich in small molecular weight titanium, citrulling, the arginine of important physiological function and has synergistic physiological activator, and their combination will be strengthened anti-oxidant, anti-ageing and the cardiovascular protection function.
The health food that a kind of Functional Polypeptides of the present invention is strengthened, the weight proportion of each composition is: 2 ~ 5 parts of glutathione prepared products, 2 ~ 5 parts of sea cucumber or ocean fish protein peptides prepared products, 0.04 ~ 0.1 part of dextrorotation lipoic acid, folic acid 1 ~ 3 * 10
-5Part, 0.2 ~ 0.5 part of OPC, 2 ~ 5 parts of food-grade L-citrulling; 4 ~ 8 parts of excipient or fillers.
The preparation method of health food according to the invention is following: glutathione, sea cucumber or ocean fish protein peptides prepared product, dextrorotation lipoic acid, folic acid, OPC, L-citrulling and excipient or filler are pressed above-mentioned part by weight mixing; Be pressed into tablet or process powdered agent with the conventional medicine processing technology; Drying, pack finished product.
Alternately, the health food that a kind of Functional Polypeptides of the present invention is strengthened, the weight proportion of each composition is: 2 ~ 5 parts of glutathione prepared products, 2 ~ 5 parts of Soyprotein peptide prepared products, 0.04 ~ 0.1 part of dextrorotation lipoic acid, folic acid 1 ~ 3 * 10
-5Part, 0.2 ~ 0.5 part of OPC, 2 ~ 5 parts of food-grade L-citrulling; 4 ~ 8 parts of excipient or fillers.
The preparation method of health food according to the invention is following: above-mentioned glutathione prepared product, Soyprotein peptide prepared product, dextrorotation lipoic acid, folic acid, OPC, L-citrulling and excipient or filler are pressed above-mentioned part by weight mixing; Be pressed into tablet or process powdered agent with the conventional medicine processing technology; Drying, pack finished product.
According to the further characteristic of health food of the present invention, described glutathione prepared product is from the cell of high-yield glutathione bacterial strain saccharomyces cerevisiae, to extract to obtain, and this preparation method of extract is following: said saccharomyces cerevisiae is inoculated in the YPD culture medium or contains peptone and amino acid whose culture medium; In 30 ℃ of vibrations or stir culture 48～70 hours; Collect yeast cells, with ethanol or similar organic solvent extracting, centrifuging and taking supernatant; Desolvate, obtain the glutathione prepared product.
According to the further characteristic of health food of the present invention, described high-yield glutathione bacterial strain saccharomyces cerevisiae be preserving number be CGMCC No.5153 high-yield glutathione bacterial strain saccharomyces cerevisiae BY-GSW (
Saccharomyces cerevisiaeBY-GSW).
Further characteristic according to health food of the present invention; Contain the synthetic dual-functional group of the responsible glutathione of sequence shown in SEQ ID NO.1 among the described high-yield glutathione bacterial strain saccharomyces cerevisiae BY-GSW because of GSW, one of this gene code has the difunctional synzyme of gamma glutamyl cysteine synthetase function and γ-glutathione synthetase function simultaneously.
According to the further characteristic of health food of the present invention, described sea cucumber or ocean fish protein peptides prepared product prepare through following method: with clean bright sea cucumber or bright ocean fish tissue after homogenate, 98
o C insulation 10 minutes is cooled to 65
oC transfers pH about 7.0, adds papain, and about 3 hours of insulated and stirred is cooled to 50 then
oC, adjustment pH to 8.5 adds aminopeptidase insulation 3 hours, and enzyme digestion reaction liquid is heated to and boils, and keeps 10 minutes, and spray-drying obtains sea cucumber or ocean fish protein peptides prepared product.
According to the further characteristic of health food of the present invention, described enzymatic hydrolysis of soybean protein peptides prepared product prepares through following preparation method: soyabean protein powder suspends or is dissolved in (about 1:10) in the sterilized water, is heated to 95
oC kept 10 minutes, was cooled to 65
oC transfers pH about 7.0, adds papain, and about 3 hours of insulated and stirred is cooled to 50 then
oC, adjustment pH to 8.5 adds aminopeptidase insulation 3 hours, and enzyme digestion reaction liquid is heated to and boils, and keeps 10 minutes, and spray-drying obtains enzymatic hydrolysis of soybean protein peptides prepared product.
An advantage of the present invention has provided a kind of high-yield glutathione bacterial strain saccharomyces cerevisiae; Import one to brewing yeast cell and had bifunctional glutathione synthetase gene order GSW; This sequence is similar with known GCL-GS gene height; The glutathione content of gained engineering yeast improves greatly, can reduce the glutathione production cost like this, makes it become the more nutriment of widespread usage.
On principle; Glutathione is participated in the multiple metabolic response as important anti-oxidation function except that itself; Also have and assist amino acid to absorb function, and be rich in important amino acid, active peptide and other physiological activators such as arginine in marine animal peptide prepared product and the Soyprotein peptide; Glutathione is epochmaking anti-oxidant and has synergistic function with folic acid, lipoic acid etc., and the antioxidation of this kind synergy strengthens the elimination ability of free radical greatly, has promoted nitric oxide production generation.Nitric oxide all has inseparable relation as signaling molecule important in the body with angiocardiopathy, impotence, amnesia, tumour formation etc.Therefore; The present invention is important source material with the glutathione; The small molecular weight titanium, citrulling and other that are combined with important physiological function have synergistic physiological activator; Like OPC (a kind of bioflavonoid that special molecular structure is arranged is to remove in the human body the most effectively natural of free radical) etc., strengthen health food anti-oxidant, anti-ageing and the protection cardiovascular function thereby provide to have.
Description of drawings
Fig. 1 shows that the integration fragment be used for homologous recombination makes up, wherein, and Trp=homologous recombination sequence, loxp-KanMX4=selection markers and knock out system, ADH=promoter, gene X=GSW gene, term=terminator sequence.
Fig. 2 is the SDS-PAGE collection of illustrative plates of GSW expression product, and wherein, the M swimming lane is preparatory dsred protein Marker III, and 1 swimming lane is GSW product (~ 86 kDa).
Fig. 3 shows that yeast transformant BY-GSW and starting strain BY glutathione content change, wherein, and: starting strain BY; ■: transformant BY-GSW.
Fig. 4 shows H
2O
2To the influence of transformant BY-GSW and starting strain BY growth, wherein, △ starting strain BY (no H
2O
2); ▲: transformant BY-GSW (no H
2O
2); ◇: starting strain BY (H
2O
2Coerce); ◆ transformant BY-GSW (H
2O
2Coerce).
Fig. 5 shows the influence of high temperature to transformant BY-GSW and starting strain BY growth, wherein, and zero: starting strain BY, 30 ℃; ●: transformant BY-GSW, 30 ℃; △: starting strain BY, 35 ℃; ▲: transformant BY-GSW, 35 ℃; ◇: starting strain BY, 40 ℃; ◆: transformant BY-GSW, 40 ℃; : starting strain BY, 45 ℃; ■: transformant BY-GSW, 45 ℃.
Fig. 6 shows Cu
2+To the influence of transformant BY-GSW and starting strain BY growth, wherein, zero: starting strain BY4741,0mM Cu
2+●: transformant BY-G, 0mM Cu
2+△: starting strain BY4741,0.1mM Cu
2+▲: transformant BY-G, 0.1mM Cu
2+◇: starting strain BY4741,0.5mM Cu
2+◆: transformant BY-G, 0.5mM Cu
2+: starting strain BY4741,1mM Cu
2+■: transformant BY-G, 1mM Cu
2+
Fig. 7 shows Cd
2+To the influence of transformant BY-GSW and starting strain BY growth, wherein, zero: starting strain BY4741,0mM Cd
2+●: transformant BY-G, 0mM Cd
2+△: starting strain BY4741,0.1mM Cd
2+▲: transformant BY-G, 0.1mM Cd
2+◇: starting strain BY4741,0.5mM Cd
2+◆: transformant BY-G, 0.5mM Cd
2+: starting strain BY4741,1mM Cd
2+■: transformant BY-G, 1mM Cd
2+
The specific embodiment
Embodiment one: high-yield glutathione bacterial strain saccharomyces cerevisiae BY-GSW (
Saccharomyces cerevisiaeBY-GSW) structure
1, the clone of difunctional glutathione synthetase gene GSW and yeast strain BY-GSW make up
1.1 make up the homologous recombination fragment: synthetic and clone's acquisition GSW sequence (seeing sequence table SEQ ID NO.1) according to Genbank database sequence GQ848551; From the Yeast Deletion Clones series of products of Invitrogen company, obtain DNA as masterplate; Obtain following fragment: promoter ADH1, terminator sequence term, marker gene KanMX4 and homologous recombination sequence Trp1 respectively through round pcr; Be connected in the loxP sequence that derives from the P1 bacteriophage at the two ends of KanMX4 fragment; Use preset restriction enzyme site and ligase, press Fig. 1 design construction recombinant fragment, this fragment is inserted among the carrier pBlueScriptIISK obtained recombinant plasmid pBlueScript-KanMX-GSW; Adopt lithium acetate conversion method transformed saccharomyces cerevisiae BY, obtain positive colony with G418 (200ug/ml) screening.According to sequence GQ848551 two ends design primer, be that template is carried out pcr amplification with the sub-genomic DNA of positive colony, products therefrom is through order-checking, and sequence is SEQ ID NO.1 as a result.
1.2 transformant screening marker gene KanMX4 knocks out: there is the fragment of loxP sequence at CRE endonuclease capable excision two ends; The harmless carrier p416-MET that will have CRE enzyme gene transforms in above-mentioned 1.1 gained GSW positive colony; If the gained bacterium colony to G418 (200ug/ml) test sensitivity and in PCR detects positive and negative purpose clone that is of KanMX4 gene of GSW gene band; This experiment obtains a plurality of purpose clone, one of them called after Saccharomyces cerevisiae BY-GSW.
2, the expression of difunctional glutathione synthetase in yeast
Get Saccharomyces cerevisiae BY-GSW and be inoculated in the YPD culture medium, 30 cultivated centrifugal collection thalline 60 hours; The buffer solution suspension cell, 100 ℃ of boiling lysises, lysate is through SDS-PAGE electrophoretic separation albumen; Western blot technology for detection proves GSW albumen successful expression.(result sees Fig. 2).
3, the physiological function of the horizontal transformant BY-GSW of homoglutathion test
3.1 the horizontal transformant BY-GSW of homoglutathion is to H
2O
2The resistance of oxidative stress:
The single bacterium colony of picking yeast transformant BY-GSW and starting strain BY 30 ℃ of shaking tables (230rpm) overnight incubation in 3mlYPD is seeded in the 25ml YPD culture medium (250ml triangular flask), and regulating OD600 is 0.5, adds H
2O
2To final concentration 3mM, 30 ℃ of shaking tables (230rpm) are cultivated, in 12,24,36,48,60,72,84, the 96hr sampling surveys OD600, not add H
2O
2As blank.The result is presented at hydrogen peroxide oxidation pressure and coerces down, and the low glutathione horizontal cell of homoglutathion horizontal cell growth fraction is much fast, and it reaches the time of maximum growth amount and the big influence (Fig. 4) that the maximum growth amount does not almost receive strong oxidizer.
3.2 the horizontal transformant BY-GSW of homoglutathion is to the tolerance of temperature:
The single bacterium colony of picking yeast transformant BY-GSW and starting strain BY 30 ℃ of shaking tables (230rpm) overnight incubation in 3mlYPD; Be seeded in the 25ml YPD culture medium (250ml triangular flask); Regulating OD600 is 0.5; Place 30 ℃, 35 ℃, 40 ℃ and 45 ℃ of cultivations respectively, survey OD600 in sampling in 12,24,36,48,60,72,84,96 hours.The result: cultivation temperature surpasses 35 ℃, and the growth of former bacterial strain receives the inhibition degree obviously serious than the transformant of homoglutathion, shows that the ability of latter's withstand high temperatures is improved (Fig. 5).
3.3 the horizontal transformant BY-GSW of homoglutathion is to the resistance of heavy metal ion:
The single bacterium colony of picking yeast transformant BY-GSW and starting strain BY 30 ℃ of shaking tables (230rpm) overnight incubation in 3mlYPD is seeded in the 25ml YPD culture medium (250ml triangular flask), and regulating OD600 is 0.5, adds 0,0.1,0.5 respectively, the Cu of 1mM
2+(CuSO
45H
2O), Cd
2+(CdCl
2) place 30 ℃ of shaking tables to cultivate, survey OD600 in sampling in 12,24,36,48,60,72,84,96 hours.The result shows, the transformant tolerance high concentration Cu of homoglutathion level
2+(CuSO
45H
2O) or Cd
2+(CdCl
2) ability apparently higher than starting strain (Fig. 6, Fig. 7)
Show through above-mentioned experiment, the present invention obtained a strain glutathione synthetase high expressed, anti-coerce the strong saccharomyces cerevisiae BY-GSW of ability (
Saccharomyces cerevisiaeBY-GSW), its glutathione content near 20mg/ gram cell, has been preserved in China Committee for Culture Collection of Microorganisms common micro-organisms center (CGMCC) under the unoptimizable condition, and preserving number is CGMCC No.5153.
Embodiment two: the preparation example one of glutathione prepared product
Transformant Saccharomyces Cerevisiae in S accharomyces cerevisiae BY-GSW is inoculated in the YPD culture medium, shakes the bottle entering logarithmic phase of spending the night in 30 ℃, insert with 2% inoculum concentration as seed; In the 100ml YPD culture medium (the big triangular flask of 500ml); 30 ℃ of shaking tables (230rpm) were cultivated, and respectively got 30ml bacterium liquid in 12,24,36,48,60,72 hours, and 6000rpm collected thalline in centrifugal 5 minutes; Sterilized water washing 2 times is dried to constant weight in 60 ℃ of baking ovens.With the ratio re-suspended cell of 0.05g stem cell 1ml sterilized water, put earlier-20 ℃ 12 hours, boiling water bath is 5 minutes then, centrifugal 1 minute of cooling back 12000rpm gets supernatant and detects glutathione content by the DTNB method.Cultivate starting strain and detect its glutathione with similarity condition.The result shows, cultivates 60 hours, and bacterial strain BY-GSW glutathione content is 16.7mg/g (dry cell weight), and is higher more than 4 times (Fig. 3) than starting strain.
Embodiment three: the preparation example two of glutathione prepared product
With the bacterial classification that spends the night of transformant Saccharomyces Cerevisiae in S accharomyces cerevisiae BY-GSW, be inoculated in 30 liters of culture mediums that sterilized peptone, dusty yeast and glucose in 50 liters of jars etc. form 30 ℃ of stir culture 55 hours; Centrifugalize cell; Cell be suspended from 42% ethanol (dry cell weight: ethanol=1:8), in 25 ℃ of extractings 1.5 hours, centrifuging and taking supernatant; Solvent is removed in decompression, obtains glutathione prepared product 35g.
Embodiment four: the preparation example of sea cucumber protein peptides prepared product
Take by weighing the clean bright sea cucumber 1000g of internal organ, be cut into small pieces, add 1 liter of buffer solution, mechanical homogenate, 98 ℃, be incubated 10 minutes, be cooled to 65 ℃, transfer pH about 7.0, adding papain 2 x 10
6About 3 hours of U insulated and stirred is cooled to 50 ℃ then, and adjustment pH to 8.5 adds aminopeptidase 1.0 x 10
8U is incubated 3 hours, and enzyme digestion reaction liquid is heated to and boils, and 10 minutes, spray-drying obtained the about 198g of sea cucumber protein peptides prepared product.This sea cucumber protein peptides prepared product is rich in holothurian collagen peptide and amino acid, and the peptide molecule of peptide yield about 31%, 60% is distributed in 1000 ~ 5000 Da.
The also available ocean fish protein peptides of the sea cucumber protein peptides prepared product prepared product that the present invention adopted substitutes, and adopts bright ocean fish or its to organize and extracts, and method for distilling is with reference to the preparation method of present embodiment.
Embodiment five: the preparation example of Soyprotein peptide prepared product
Take by weighing soyabean protein powder 1000g, be dissolved in 10 kilograms of sterilized waters, be heated to 98 ℃ and kept 20 minutes, be cooled to 65 ℃, transfer pH about 7.0, add papain 2 x 10
6U, about 3 hours of insulated and stirred is cooled to 50 ℃ then, and adjustment pH to 8.5 adds wide spectrum aminopeptidase 1 x 10
8U is incubated 3 hours, and enzyme digestion reaction liquid is heated to and boils, and keeps 10 minutes, and spray-drying obtains the rich about 940g of enzymatic hydrolysis of soybean protein Preparation thing.This enzymatic hydrolysis of soybean protein Preparation thing is rich in small-molecular peptides and amino acid, peptide content>70%, peptide mean molecule quantity < 3000.
Embodiment six: the preparation of health food of the present invention
Prescription 1: glutathione prepared product 2 g, sea cucumber or ocean fish protein peptides prepared product 2 g, dextrorotation lipoic acid 0.04 g, folic acid 1 *
10 -5 G, OPC 0.2 g, L-citrulling 2 g
,Dextrin (as excipient) 4 g.
Prescription 2: glutathione prepared product 3.5 g, sea cucumber or ocean fish protein peptides prepared product 3.5 g, dextrorotation lipoic acid 0.07 g, folic acid 2 *
10 -5 G, OPC 0.35 g, L-citrulling 3.5g, dextrin (as excipient) 6 g.
Prescription 3: glutathione prepared product 5 g, sea cucumber or ocean fish protein peptides prepared product 5 g, dextrorotation lipoic acid 0.1 g, folic acid 3 *
10 -5 G, OPC 0.5g, L-citrulling 5g, dextrin (as excipient) 8 g.
Above-mentioned prescription is taken by weighing raw material, be pressed into tablet, and make coating agent, get oral, every agreement that contracts a film or TV play to an actor or actress 0.4g with Hydroxypropyl methylcellulose (accounting for 3%) with the conventional medicine processing technology.Also can the conventional medicine processing technology process powdered agent, drying, pack finished product.Raw materials used food-grade or the pharmaceutical grade of being.
Finished product such as soluble in water, solution shows light brown brown, has a little light bitter sugariness and extremely light fishlike smell.Tablet is directly swallowed, and every day, dose was 6 ~ 8.
After this health food was trial-produceed successfully, many batches of testing crews were taken, and general effect is physiochemical indices such as blood pressure, cholesterol, platelet aggregation power are kept or trend is normal, physique strengthens, the colour of skin is improved.
Embodiment seven: the preparation of health food of the present invention
Prescription 1: glutathione prepared product 2 g, Soyprotein peptide prepared product 2 g, dextrorotation lipoic acid 0.04 g, folic acid 1 *
10 -5 G, OPC 0.2 g, L-citrulling 2 g
,Dextrin (as excipient) 4 g.
Prescription 2: glutathione prepared product 3.5 g, Soyprotein peptide prepared product 3.5 g, dextrorotation lipoic acid 0.07 g, folic acid 2 *
10 -5 G, OPC 0.35 g, L-citrulling 3.5g
,Dextrin (as excipient) 6 g.
Prescription 3: glutathione prepared product 5 g, Soyprotein peptide prepared product 5 g, dextrorotation lipoic acid 0.1 g, folic acid 3 *
10 -5 G, OPC 0.5g, L-citrulling 5g
,Dextrin (as excipient) 8 g.
Above-mentioned prescription is taken by weighing raw material, be pressed into tablet, and make coating agent, get oral, every agreement that contracts a film or TV play to an actor or actress 0.4g with Hydroxypropyl methylcellulose (accounting for 3%) with the conventional medicine processing technology.Also can the conventional medicine processing technology process powdered agent, drying, pack finished product.Raw materials used food-grade or the pharmaceutical grade of being.
Finished product such as soluble in water, solution shows light brown brown, has a little light bitter sugariness.Tablet is directly swallowed, and every day, dose was 6 ~ 8.
After this health food was trial-produceed successfully, many batches of testing crews were taken, and general effect is physiochemical indices such as blood pressure, cholesterol, platelet aggregation power are kept or trend is normal, physique strengthens, the colour of skin is improved.
Sequence table
< 110>pari Si of Shenzhen bio tech ltd; Zhou Shining
< 120>a kind of health food of Functional Polypeptides reinforcement
<130>
<160> 1
<170> PatentIn?version?3.4
<210> 1
<211> 2265
<212> DNA
< 213>artificial synthetic
<400> 1
atgacattaa?accaactgct?tcaaaaactg?gaagctacca?gccctattct?ccaagctaat 60
tttggaatcg?agcgcgagag?tctacgtgtc?gataggcaag?gacaactggt?gcatacacct 120
cacccatcct?gtctaggagc?tcgtagtttc?cacccctata?ttcagactga?tttttgcgag 180
tttcagatgg?aactcatcac?accagttgcc?aaatctacta?ctgaggctcg?ccgatttctg 240
ggagctatta?ctgatgtagc?tggccgctct?attgctacag?acgaggttct?ctggccttta 300
tccatgccac?ctcgtctaaa?ggcagaggag?attcaagttg?ctcaactgga?aaatgacttc 360
gaacgccatt?atcgtaacta?tttggctgaa?aaatacggaa?ctaaactaca?agctatctca 420
ggtatccact?ataatatgga?actgggtaaa?gatttagttg?aggccttgtt?ccaagaaagt 480
gatcagaccg?atatgattgc?cttcaaaaac?gccctctatc?ttaagctggc?tcagaactac 540
ttgcgctacc?gttgggtgat?tacctatctc?tttggggcct?cacccatcgc?cgaacaaggt 600
ttctttgacc?aggaagttcc?agaacctgtg?cgttccttcc?gtaacagtga?ccacggctat 660
gtcaataagg?aagagattca?agtatccttt?gtaagtctag?aagattatgt?ctcagccatt 720
gaaacctata?tcgaacaagg?agatttgaat?gcagagaaag?aattttactc?agctgttcgt 780
ttccgtggac?aaaaggttaa?tcgttccttc?cttgacaaag?gaatcaccta?cctagagttc 840
cgtaatttcg?accttaaccc?ttttgagcgt?atcggtatta?gtcagactac?tatggacact 900
gtgcacttac?tcattttagc?cttcctttgg?cttgatagcc?ctgaaaatgt?cgaccaagct 960
cttgcacaag?gccacgcgtt?aaatgagaaa?attgccctct?ctcatcctct?agaacctcta 1020
ccttcggagg?ctaaaactca?ggacattgta?actgccctag?accaactggt?gcaacacttt 1080
ggacttggtg?actatcatca?agatctggtt?aagcaagtta?aggcagcctt?tgcggatcca 1140
aatcaaacgc?tctctgccca?gctcttaccc?tatatcaaag?acaaatctct?agccgaattt 1200
gctttaaaca?aggctcttgc?ctatcatgat?tacgactgga?ctgcccacta?tgctctcaag 1260
ggctatgaag?agatggaact?ctccacccag?atgttgctct?ttgatgccat?ccaaaagggg 1320
attcactttg?aaatattgga?tgagcaagat?caattcctaa?aactttggca?ccaagaccat 1380
gttgaatacg?tcaaaaacgg?taacatgacc?tcaaaagaca?actacgtggt?tccccttgct 1440
atggctaata?agaccgtaac?caagaagatt?ctagcagatg?ctggctttcc?agttccttca 1500
ggagacgaat?ttaccagtct?tgaggaagga?cttgcctact?accctcttat?caaggataag 1560
caaattgttg?tcaaacccaa?gtcaactaac?tttggtctgg?gaatttccat?tttccaagaa 1620
cctgccagtc?ttgacaacta?tcaaaaagcc?cttgaaattg?ctttcgcaga?agatacctct 1680
gtccttgttg?aagaatttat?tccaggaacc?gaataccgtt?tcttcatctt?ggatgggcgt 1740
tgtgaggctg?ttcttctgcg?tgtcgctgcc?aatgttattg?gtgatggcaa?acacaccatt 1800
cgtgaactag?tcgctcagaa?aaatgctaat?ccattgcgtg?gccgtgatca?ccggtcacct 1860
ctggaaatca?ttgagctagg?agacatcgaa?caactaatgt?tagctcaaca?gggttacaca 1920
cctgatgata?ttctcccaga?aggaaaaaag?gtcaatctgc?gtcgtaattc?caacatctct 1980
acaggtggtg?actctattga?tatcactgag?accatggatt?cctcttacca?agaattagcc 2040
gcagccatgg?caactagcat?gggcgcctgg?gcttgcgggg?ttgatctgat?aattccagat 2100
gaaactcaaa?ttgccaccaa?ggaaaatcct?cattgcacct?gcattgagct?caactttaac 2160
ccttcgatgt?atatgcacac?ctactgtgct?gagggtcctg?gccaagctat?cactactaaa 2220
atcctagata?aactttttcc?agaaatagtg?gctggtcaaa?cttaa 2265
Claims (9)
1. the health food that Functional Polypeptides is strengthened is characterized in that the weight proportion of each composition is: 2 ~ 5 parts of glutathione prepared products, 2 ~ 5 parts of sea cucumber or ocean fish protein peptides prepared products, 0.04 ~ 0.1 part of dextrorotation lipoic acid, folic acid 1 ~ 3 * 10
-5Part, 0.2 ~ 0.5 part of OPC, 2 ~ 5 parts of food-grade L-citrulling; 4 ~ 8 parts of excipient or fillers.
2. health food according to claim 1; It is characterized in that; The preparation method of said health food is following: glutathione, sea cucumber or ocean fish protein peptides prepared product, dextrorotation lipoic acid, folic acid, OPC, L-citrulling and excipient or filler are pressed above-mentioned part by weight mixing; Be pressed into tablet or process powdered agent with the conventional medicine processing technology, drying, pack finished product.
3. the health food that Functional Polypeptides is strengthened is characterized in that the weight proportion of each composition is: 2 ~ 5 parts of glutathione prepared products, 2 ~ 5 parts of Soyprotein peptide prepared products, 0.04 ~ 0.1 part of dextrorotation lipoic acid, folic acid 1 ~ 3 * 10
-5Part, 0.2 ~ 0.5 part of OPC, 2 ~ 5 parts of food-grade L-citrulling; 4 ~ 8 parts of excipient or fillers.
4. health food according to claim 1; It is characterized in that; The preparation method of said health food is following: above-mentioned glutathione prepared product, Soyprotein peptide prepared product, dextrorotation lipoic acid, folic acid, OPC, L-citrulling and excipient or filler are pressed above-mentioned part by weight mixing; Be pressed into tablet or process powdered agent with the conventional medicine processing technology, drying, pack finished product.
5. according to claim 1 or 3 described health foods, it is characterized in that: described glutathione prepared product is from the cell of high-yield glutathione bacterial strain saccharomyces cerevisiae, to extract to obtain, and this preparation method of extract is following: said saccharomyces cerevisiae is inoculated in the YPD culture medium or contains peptone and amino acid whose culture medium; In 30 ℃ of vibrations or stir culture 48～70 hours; Collect yeast cells, with ethanol or similar organic solvent extracting, centrifuging and taking supernatant; Desolvate, obtain the glutathione prepared product.
6. health food according to claim 5 is characterized in that: described high-yield glutathione bacterial strain saccharomyces cerevisiae be preserving number be CGMCC No.5153 high-yield glutathione bacterial strain saccharomyces cerevisiae BY-GSW (
Saccharomyces cerevisiaeBY-GSW).
7. health food according to claim 6; It is characterized in that: contain the synthetic dual-functional group of the responsible glutathione of sequence shown in SEQ ID NO.1 among the described high-yield glutathione bacterial strain saccharomyces cerevisiae BY-GSW because of GSW, one of this gene code has the difunctional synzyme of gamma glutamyl cysteine synthetase function and γ-glutathione synthetase function simultaneously.
8. health food according to claim 1 is characterized in that: described sea cucumber or ocean fish protein peptides prepared product prepare through following method: with clean bright sea cucumber or bright ocean fish tissue after homogenate, 98
oC insulation 10 minutes is cooled to 65
oC transfers pH about 7.0, adds papain, and about 3 hours of insulated and stirred is cooled to 50 then
oC, adjustment pH to 8.5 adds aminopeptidase insulation 3 hours, and enzyme digestion reaction liquid is heated to and boils, and keeps 10 minutes, and spray-drying obtains sea cucumber or ocean fish protein peptides prepared product.
9. health food according to claim 3 is characterized in that: described enzymatic hydrolysis of soybean protein peptides prepared product prepares through following preparation method: soyabean protein powder suspends or is dissolved in (about 1:10) in the sterilized water, is heated to 95
oC kept 10 minutes, was cooled to 65
oC transfers pH about 7.0, adds papain, and about 3 hours of insulated and stirred is cooled to 50 then
oC, adjustment pH to 8.5 adds aminopeptidase insulation 3 hours, and enzyme digestion reaction liquid is heated to and boils, and keeps 10 minutes, and spray-drying obtains enzymatic hydrolysis of soybean protein peptides prepared product.
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| CN104146142A (en) * | 2014-07-28 | 2014-11-19 | 四川安益生物科技有限公司 | Composite health food and preparation method thereof |
| JPWO2013122188A1 (en) * | 2012-02-15 | 2015-05-18 | 協和発酵バイオ株式会社 | Preventive or ameliorating agent for vascular endothelial function decline |
| CN106072659A (en) * | 2016-06-29 | 2016-11-09 | 中国人民解放军第三军医大学第附属医院 | Multielement organism antioxidants compositions and application thereof |
| CN112335875A (en) * | 2019-08-09 | 2021-02-09 | 株式会社艾福瑞特 | Health functional composition with blood circulation improving effect |
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|---|---|
| CN102356879B (en) | 2014-08-06 |
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