CN102356879B - Functional peptide-reinforced healthcare food - Google Patents
Functional peptide-reinforced healthcare food Download PDFInfo
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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, especially relate to the health food of the Functional Polypeptides strengthening of preparing taking glutathione (Glutathione, GSH) as important source material.
Background technology
Glutathione (L-γ-glutamyl-L-cysteinylglycine, GSH) is the tripeptide compound being formed by peptide bond condensation by glutamic acid (L-Glutamic acid), cysteine (L-cysteine) and glycine (Glycine).Extensively be present in animal, plant and microbial cell, wherein with yeast, animal's liver equal size compared with horn of plenty.Glutathione has multiple important physiological function in organism, it is important antioxidant in cell, maintaining cell reducing condition, is one of key substance participating in the anti-damage of cell and metabolism adjusting, and wherein anti-oxidation function and assistance transmembrane amino acid transporter function are especially noticeable.
In vital movement, need a lot of normal oxidation reactions, this process inevitably produces free radical; In addition, cell is at metabolism allogenic material, and during as toxin, medicine or reply extreme environment factor (as ultraviolet ray etc.), cell also can produce free radical.These radical pair biological cells produce harm greatly, as attacked large biological molecule lipid, protein, DNA etc., cause cell membrane damage, biology enzyme inactivation and DNA to destroy and sudden change, even cause the important pathologies such as malignant tumour.Normal body or cell have the ability of eliminating free radical, preventing and repair damage, keep the ability of intracellular redox equilibrium, ensure the normal of vital movement.Glutathione is the important biomolecule small-molecular peptides with this ability.
The cysteine of glutathione contains sulfydryl (SH), this group is active group in redox reaction, can be used as electron donor, to the various free radicals that produce in body, peroxide, hydrogen peroxide etc. provide electronics and by its deactivation, thereby safeguard Cellular Oxidation reduction dynamic balance state (Valko M, Izakovic M., Mazur M., Rhodes C., Telser J., Role of oxygen radicals in DNA damage and cancer incidence. Molecular & Cellular Biochemistry, 2004, 266:37-56), glutathione participates in transmembrane amino acid transporter by the circulation of born of the same parents' intracellular metabolite, under the assistance of ATP, combining amino acid transport is to the inside of cell, complete absorption process (Zheng Yunlang. the biological function of glutathione. biology circular. 1995. 30 (5): 22-24).This reaction is the important step that Amino Acid Absorption utilizes.Glutathione also participates in protein and DNA is synthetic, needs glutathione to provide motive power to paddy hydrogen reduction albumen in this process.There is bibliographical information glutathione also to there is function (the Filomeni G of inhibited apoptosis, 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 programmed cell death (Marengo of cell, 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 application aspect medicine, food.Because glutathione production cost is high, product costliness, thereby be mainly at present the application aspect medical.Glutathione is usually in removing toxic substances, Radiation sickness control, protection liver, and antiallergy, the treatment of beauty treatment, beauty treatment, skin care, increase eyesight and ophthalmology disease; Glutathione plays neutralization, mediates effect the toxic compounds and the heavy metal that enter in body, clinical in poisoning treatments such as acrylonitrile, fluoride, carbon monoxide, heavy metal, alcohol; Also can be used as ancillary drug anticancer, antitumor and treatment AIDS, the symptoms such as the leucocyte minimizing that it causes anticancer therapy have very strong protective effect.Glutathione is also used for the treatment of diabetes nerve damage.Hyperglycaemia affects glutathione metabolism, has reduced H
2o
2elimination ability thereby cause nerve damage.With the 93 %(Yao Min shows of glutathione treatment diabetic neuropathy patient total effective rate, 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 the 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.Prove that glutathione is all playing an important role aspect a lot of enzymatic reactions and elimination free radical, if body glutathion inside deficiency, some enzymatic reactions can not effectively be carried out, the free radical that metabolic process produces can not be eliminated in time, free radical accumulation promotes body aging, and the pathology such as induced tumor, cerebrovascular atherosclerosis, therefore glutathione has 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, although the physiological significance of glutathione is familiar with for a long time, not yet general for food, the developed countries such as Japan, the U.S. just started glutathione to be 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.
Glutathione preparation method mainly contains four kinds of solvent extraction, chemical synthesis, fermentation method and enzyme transforming process.Extraction be mainly the animal and plant tissue and the yeast etc. that are rich in glutathione be raw material; The shortcoming of chemical synthesis process is that very complicated, reactions steps are more, the time is long, cost is high, contaminated environment, may produce racemization and affect biologically active and the 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 process costs higher.Consider each side factor, fermentation method is the method for producing at present the tool potentiality of GSH.
Fermentation method is to utilize the bacterial strain that glutathione content is higher to carry out large scale fermentation acquisition cell, therefrom extracts glutathione.But, current mechanism of fermenting its synthesizing glutathion of saccharomycete used is two-step catalysis mechanism, first step L-Glu and L cysteine generate L glutamyl cysteine through the catalysis of gamma glutamyl cysteine synthetase (γ-glutamylcysteine synthetase, γ-GCS); Second step reaction generates glutathione by glutathione synthetase (glutathione synthetase, GS) catalysis L-glutamyl cysteine and glycine reactant.The enzyme (γ-GCS) of above-mentioned first step reaction is regulated and controled by end-product glutathione feedback inhibition, and therefore, this step belongs to rate-limiting reaction.Production bacterial strain is difficult to break away from the restriction of this feedback regulation at present, and glutathione content promotes and is restricted.
(the 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 there is bifunctional Glutatione synthetase gene, be called GCL-GS, 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 insensitive of this gene pairs glutathione.Therefore, this gene is introduced and in tobacco plant, has been obtained high-yield glutathione by this article author.But have no other application reports of this gene.
Sea cucumber belongs to echinoderm, is a kind of ancient ocean software biology.The nutritive value of sea cucumber is high, its main component is collagen, mucopolysaccharide and Sea Cucumber Glycosides, collagen accounts for 74. 15%(Li Cui kingfishers of wall of sea cucumber Stichopus japonicus protein content, Dong Xiuping, Gao Yang etc. the extraction process of wall of sea cucumber Stichopus japonicus enzymatic dissolubility collagen, Dalian Polytechnic University's journal, 2008,27 (4): 1 ~ 4).Sea cucumber also has the 50 various active compositions such as chondroitin sulfate, multivitamin and each seed amino acid, is the rare high tonic that does not contain cholesterol and extremely low purine.Research shows, the physiologically actives such as that the collagen peptide that collagen hydrolysate obtains little 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 the functional characteristic such as delay senility.(Zhang Xinru. collagen food gains great popularity. Chinese health nutrient, 2003 (7): 32; Xie Yuxiang, Ding Lin. collagen and old and feeble correlation. Aged in China is learned magazine, and 2002,6 (22): 518 ~ 520) in sea cucumber, composition also has hemangiectasis, increase blood flow; The dissolving of fortifying fibre albumen, improves microcirculatory function.Sea cucumber through for the sea cucumber peptide prepared product that obtains of protease hydrolytic, the both complete distinctive nutrition of extra large stichopus japonicus that retained, again macro-molecular protein is changed into more easily absorb, small-molecular peptides with better function, more easily absorb than traditional cucumber product.In recent years prove that 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 nitric oxide production generation in vivo, therefore stimulate circulation, strengthen endotheliocyte viability (Louis J. Ignarro " No More Heart Disease ", Published 2005 by Broadway books, New York).
Soyprotein peptide is that soybean protein makes through enzyme degraded, product is rich in physiologically active small-molecular peptides and each seed amino acid, be rich in arginine and citrulling, there is anti-hypertension, suppress cholesterol, promote lipid-metabolism, improve immunity, promote function and the low antigenicity features such as profitable strain growth.
Summary of the invention
The object 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 cardiovascular protection function.
The health food of a kind of Functional Polypeptides strengthening of the present invention, 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 R-DHLA, folic acid 1 ~ 3 × 10
-5part, 0.2 ~ 0.5 part of OPC, 2 ~ 5 parts of food stage Cits; 4 ~ 8 parts of excipient or fillers.
The preparation method of health food of the present invention is as follows: glutathione, sea cucumber or ocean fish protein peptides prepared product, R-DHLA, folic acid, OPC, Cit and excipient or filler are mixed by above-mentioned part by weight, be pressed into tablet or make powdered agent with conventional medicine processing technology, dry, pack to obtain finished product.
Alternately, the health food of a kind of Functional Polypeptides strengthening of the present invention, 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 R-DHLA, folic acid 1 ~ 3 × 10
-5part, 0.2 ~ 0.5 part of OPC, 2 ~ 5 parts of food stage Cits; 4 ~ 8 parts of excipient or fillers.
The preparation method of health food of the present invention is as follows: above-mentioned glutathione prepared product, Soyprotein peptide prepared product, R-DHLA, folic acid, OPC, Cit and excipient or filler are mixed by above-mentioned part by weight, be pressed into tablet or make powdered agent with conventional medicine processing technology, dry, pack to obtain finished product.
According to the further feature of health food of the present invention, described glutathione prepared product is to extract and obtain from the cell of high-yield glutathione bacterial strain saccharomyces cerevisiae, the preparation method of this extract is as follows: described saccharomyces cerevisiae is inoculated in to YPD culture medium or contains peptone and amino acid whose culture medium, in 30 DEG C of vibrations or stir culture 48～70 hours, collect yeast cells, with ethanol or similar organic solvent extracting, centrifuging and taking supernatant, desolventizing, obtains glutathione prepared product.
According to the further feature of health food of the present invention, described high-yield glutathione bacterial strain saccharomyces cerevisiae is that preserving number is the high-yield glutathione bacterial strain saccharomyces cerevisiae BY-GSW(of CGMCC No.5153
saccharomyces cerevisiaebY-GSW).
According to the further feature of health food of the present invention, the dual-functional group that contains the responsible glutathione synthesis of sequence as shown in SEQ ID NO.1 in described high-yield glutathione bacterial strain saccharomyces cerevisiae BY-GSW is because of GSW, and 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 feature of health food of the present invention, described sea cucumber or ocean fish protein peptides prepared product are prepared by the following method: by clean fresh sea cucumber or fresh ocean fish tissue after homogenate, 98
oc insulation 10 minutes, is cooled to 65
oc, adjusts pH approximately 7.0, adds papain, and insulated and stirred approximately 3 hours, is then cooled to 50
oc, adjusts pH to 8.5, adds aminopeptidase insulation 3 hours, and enzyme digestion reaction liquid is heated to boil, and keeps 10 minutes, and spraying is dry, obtains sea cucumber or ocean fish protein peptides prepared product.
According to the further feature of health food of the present invention, described enzymatic hydrolysis of soybean protein peptides prepared product is prepared by following preparation method: soyabean protein powder suspends or is dissolved in (about 1:10) in sterilized water, is heated to 95
oc keeps 10 minutes, is cooled to 65
oc, adjusts pH approximately 7.0, adds papain, and insulated and stirred approximately 3 hours, is then cooled to 50
oc, adjusts pH to 8.5, adds aminopeptidase insulation 3 hours, and enzyme digestion reaction liquid is heated to boil, and keeps 10 minutes, and spraying is dry, obtains enzymatic hydrolysis of soybean protein peptides prepared product.
An advantage of the present invention has been to provide a kind of high-yield glutathione bacterial strain saccharomyces cerevisiae, import one to brewing yeast cell and there is bifunctional Glutatione synthetase gene sequence GSW, this sequence is similar to known GCL-GS gene height, the glutathione content of gained Engineering Yeast improves greatly, can reduce like this glutathione production cost, make it become the more generally nutriment of application.
Principle, glutathione participates in multiple metabolic response as important anti-oxidation function except itself, also there is the Amino Acid Absorption of assistance and utilize function, and in marine animal peptide prepared product and Soyprotein peptide, be rich in the important amino acids such as arginine, active peptide and other physiological activators; Glutathione is epochmaking antioxidant and has synergistic function with folic acid, lipoic acid etc., and the antioxidation of this kind of synergy strengthens the elimination ability of free radical greatly, has promoted nitric oxide production generation.Nitric oxide has inseparable relation as signaling molecule important in body with angiocardiopathy, impotence, amnesia, tumour formation etc.Therefore; the present invention is taking glutathione as important source material; be combined with the small molecular weight titanium, citrulling of important physiological function and other have synergistic physiological activator; as OPC (a kind of bioflavonoid that has special molecular structure; to remove the most effective natural of people's interior free yl) etc., health food anti-oxidant, anti-ageing and protection cardiovascular function strengthened thereby provide to have.
Brief description of the drawings
Fig. 1 shows for the integration fragment of homologous recombination and builds, 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, M swimming lane is pre-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
2on the impact of transformant BY-GSW and starting strain BY growth, wherein, △ starting strain BY(is without H
2o
2); ▲: transformant BY-GSW(is without H
2o
2); ◇: starting strain BY(H
2o
2coerce); ◆ transformant BY-GSW(H
2o
2coerce).
Fig. 5 shows the impact of high temperature on transformant BY-GSW and starting strain BY growth, wherein, and zero: starting strain BY, 30 DEG C; ●: transformant BY-GSW, 30 DEG C; △: starting strain BY, 35 DEG C; ▲: transformant BY-GSW, 35 DEG C; ◇: starting strain BY, 40 DEG C; ◆: transformant BY-GSW, 40 DEG C; : starting strain BY, 45 DEG C; ■: transformant BY-GSW, 45 DEG C.
Fig. 6 shows Cu
2+on the impact 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+on the impact 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+.
Detailed description of the invention
Embodiment mono-: high-yield glutathione bacterial strain saccharomyces cerevisiae BY-GSW(
saccharomyces cerevisiaebY-GSW) structure
1, the clone of difunctional Glutatione synthetase gene GSW and yeast strain BY-GSW build
1.1 build homologous recombination fragments: synthesize and clone 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 respectively following fragment: promoter ADH1 by round pcr, terminator sequence term, marker gene KanMX4 and homologous recombination sequence Trp1, the loxP sequence that derives from P1 bacteriophage is connected in to the two ends of KanMX4 fragment, the default restriction enzyme site of application and ligase, press Fig. 1 design construction recombinant fragment, to in this fragment insertion vector pBlueScriptIISK, obtain recombinant plasmid pBlueScript-KanMX-GSW, adopt lithium acetate conversion method transformed saccharomyces cerevisiae BY, with G418(200ug/ml) screening acquisition positive colony.According to sequence GQ848551 two ends design primer, carry out pcr amplification taking positive colony subgenom DNA as template, products therefrom is through order-checking, and result sequence is SEQ ID NO.1.
1.2 transformant screening marker gene KanMX4 knock out: CRE enzyme can excise two ends the fragment of loxP sequence, to transform with the harmless carrier p416-MET of CRE enzyme gene in above-mentioned 1.1 gained GSW positive colony, if gained bacterium colony to G418 (200ug/ml) test sensitivity and in PCR detects the positive and negative object clone that is of KanMX4 gene of GSW gene band, this experiment obtains multiple object clones, 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 YPD culture medium, 30 cultivate 60 hours, centrifugal collection thalline, buffer solution suspension cell, 100 DEG C of boiling lysises, lysate is through SDS-PAGE electrophoretic separation albumen, Western blot technology for detection, proves GSW albumen successful expression.(the results are shown in Figure 2).
3, the physiological function of the horizontal transformant BY-GSW of homoglutathion test
The horizontal transformant BY-GSW of 3.1 homoglutathion is to H
2o
2the resistance of oxidative stress:
Picking yeast transformant BY-GSW and the mono-bacterium colony of starting strain BY be 30 DEG C of shaking tables (230rpm) overnight incubation in 3mlYPD, is seeded in 25ml YPD culture medium (250ml triangular flask), and regulating OD600 is 0.5, adds H
2o
2to final concentration 3mM, 30 DEG C of shaking tables (230rpm) are cultivated, in 12,24,36,48,60,72,84,96hr sampling surveys OD600, not add H
2o
2as blank.Result is presented at hydrogen peroxide oxidation pressure and coerces down, and the low glutathione horizontal cell of homoglutathion horizontal cell growth fraction is faster, and it reaches time of maximum growth amount and maximum growth amount and be not almost subject to the large impact (Fig. 4) of strong oxidizer.
The tolerance of the horizontal transformant BY-GSW of 3.2 homoglutathion to temperature:
Picking yeast transformant BY-GSW and the mono-bacterium colony of starting strain BY be 30 DEG C of shaking tables (230rpm) overnight incubation in 3mlYPD, be seeded in 25ml YPD culture medium (250ml triangular flask), regulating OD600 is 0.5, be placed in respectively 30 DEG C, 35 DEG C, 40 DEG C and 45 DEG C of cultivations, survey OD600 in sampling in 12,24,36,48,60,72,84,96 hours.Result: cultivation temperature exceedes 35 DEG C, the suppressed degree of growth of former bacterial strain is obviously serious than the transformant of homoglutathion, shows that the ability of the latter's withstand high temperatures is improved (Fig. 5).
The resistance of the horizontal transformant BY-GSW of 3.3 homoglutathion to heavy metal ion:
Picking yeast transformant BY-GSW and the mono-bacterium colony of starting strain BY be 30 DEG C of shaking tables (230rpm) overnight incubation in 3mlYPD, is seeded in 25ml YPD culture medium (250ml triangular flask), and regulating OD600 is 0.5, adds respectively 0,0.1,0.5, the Cu of 1mM
2+(CuSO
45H
2o), Cd
2+(CdCl
2) be placed in 30 DEG C of shaking tables cultivations, survey OD600 in sampling in 12,24,36,48,60,72,84,96 hours.Result shows, the transformant enduring high-concentration Cu of homoglutathion level
2+(CuSO
45H
2or Cd O)
2+(CdCl
2) ability apparently higher than starting strain (Fig. 6, Fig. 7)
Show by above-mentioned experiment, the present invention has obtained the saccharomyces cerevisiae BY-GSW(that ability is strong that coerces a strain glutathione synthetase high expressed, anti-
saccharomyces cerevisiaebY-GSW), its glutathione content approaches 20mg/ gram of cell under unoptimizable condition, has been preserved in China Committee for Culture Collection of Microorganisms's common micro-organisms center (CGMCC), and preserving number is CGMCC No.5153.
Embodiment bis-: the preparation example one of glutathione prepared product
Transformant Saccharomyces Cerevisiae in S accharomyces cerevisiae BY-GSW is inoculated in to YPD culture medium, spend the night and enter logarithmic phase in 30 DEG C of shaking flasks, access with 2% inoculum concentration as seed, in 100ml YPD culture medium (the large triangular flask of 500ml), 30 DEG C of shaking tables (230rpm) are cultivated, and respectively get 30ml bacterium liquid in 12,24,36,48,60,72 hours, and 6000rpm collects thalline for centrifugal 5 minutes, sterilized water washing 2 times is dried to constant weight in 60 DEG C of baking ovens.With the ratio re-suspended cell of 0.05g stem cell 1ml sterilized water, first put-20 DEG C 12 hours, then boiling water bath 5 minutes, centrifugal 1 minute of cooling rear 12000rpm, get supernatant by DTNB method detect glutathione content.Cultivate starting strain and detect its glutathione with similarity condition.Result shows, cultivates 60 hours, and bacterial strain BY-GSW glutathione content is 16.7mg/g (dry cell weight), than starting strain high 4 times above (Fig. 3).
Embodiment tri-: 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 in 50 liters of tanks, sterilized peptone, dusty yeast and glucose etc. form, 30 DEG C of stir culture 55 hours, centrifugation obtains cell, cell is suspended from 42% ethanol (dry cell weight: ethanol=1:8), in 25 DEG C of extractings 1.5 hours, centrifuging and taking supernatant, solvent is removed in decompression, obtains glutathione prepared product 35g.
Embodiment tetra-: the preparation example of sea cucumber protein peptides prepared product
Take the fresh sea cucumber 1000g that internal organ are clean, be cut into small pieces, add 1 liter of buffer solution, mechanical homogenate, 98 DEG C, be incubated 10 minutes, be cooled to 65 DEG C, adjust pH approximately 7.0, add papain 2 x 10
6u insulated and stirred approximately 3 hours, is then cooled to 50 DEG C, adjusts pH to 8.5, adds aminopeptidase 1.0 x 10
8u, is incubated 3 hours, and enzyme digestion reaction liquid is heated to boil, and 10 minutes, spraying was dry, obtains 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 approximately 31%, 60% is distributed in 1000 ~ 5000 Da.
Sea cucumber protein peptides prepared product of the present invention also can substitute with ocean fish protein peptides prepared product, adopts fresh ocean fish or its to organize to extract, and extracting method is with reference to the preparation method of the present embodiment.
Embodiment five: the preparation example of Soyprotein peptide prepared product
Take soyabean protein powder 1000g, be dissolved in 10 kilograms of sterilized waters, be heated to 98 DEG C and keep 20 minutes, be cooled to 65 DEG C, adjust pH approximately 7.0, add papain 2 x 10
6u, insulated and stirred approximately 3 hours, is then cooled to 50 DEG C, adjusts pH to 8.5, adds wide spectrum aminopeptidase 1 x 10
8u, is incubated 3 hours, and enzyme digestion reaction liquid is heated to boil, and keeps 10 minutes, and spraying is dry, obtains the rich about 940g of enzymatic hydrolysis of soybean albumen prepared product.This enzymatic hydrolysis of soybean albumen prepared product 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
Formula 1: glutathione prepared product 2 g, sea cucumber or ocean fish protein peptides prepared product 2 g, R-DHLA 0.04 g, folic acid 1 ×
10 -5 g, OPC 0.2 g, Cit 2 g
,dextrin (as excipient) 4 g.
Formula 2: glutathione prepared product 3.5 g, sea cucumber or ocean fish protein peptides prepared product 3.5 g, R-DHLA 0.07 g, folic acid 2 ×
10 -5 g, OPC 0.35 g, Cit 3.5g, dextrin (as excipient) 6 g.
Formula 3: glutathione prepared product 5 g, sea cucumber or ocean fish protein peptides prepared product 5 g, R-DHLA 0.1 g, folic acid 3 ×
10 -5 g, OPC 0.5g, Cit 5g, dextrin (as excipient) 8 g.
Above-mentioned formula is taken to raw material, be pressed into tablet with conventional medicine processing technology, and make coating agent with Hydroxypropyl methylcellulose (accounting for 3%), obtain oral tablet, every agreement that contracts a film or TV play to an actor or actress 0.4g.Also can conventional medicine processing technology make powdered agent, dry, pack to obtain finished product.Raw materials used food stage or the pharmaceutical grade of being.
Finished product is as soluble in water, and solution shows light brown brown, slightly light bitter sugariness and extremely light fishlike smell.Tablet is directly swallowed, every day 6 ~ 8 of doses.
After this health food is trial-produceed successfully, many batches of testing crews are taken, and general effect is can make the physiochemical indices such as blood pressure, cholesterol, platelet aggregation keep or trend is normal, physique strengthens, the colour of skin is improved.
Embodiment seven: the preparation of health food of the present invention
Formula 1: glutathione prepared product 2 g, Soyprotein peptide prepared product 2 g, R-DHLA 0.04 g, folic acid 1 ×
10 -5 g, OPC 0.2 g, Cit 2 g
,dextrin (as excipient) 4 g.
Formula 2: glutathione prepared product 3.5 g, Soyprotein peptide prepared product 3.5 g, R-DHLA 0.07 g, folic acid 2 ×
10 -5 g, OPC 0.35 g, Cit 3.5g
,dextrin (as excipient) 6 g.
Formula 3: glutathione prepared product 5 g, Soyprotein peptide prepared product 5 g, R-DHLA 0.1 g, folic acid 3 ×
10 -5 g, OPC 0.5g, Cit 5g
,dextrin (as excipient) 8 g.
Above-mentioned formula is taken to raw material, be pressed into tablet with conventional medicine processing technology, and make coating agent with Hydroxypropyl methylcellulose (accounting for 3%), obtain oral tablet, every agreement that contracts a film or TV play to an actor or actress 0.4g.Also can conventional medicine processing technology make powdered agent, dry, pack to obtain finished product.Raw materials used food stage or the pharmaceutical grade of being.
Finished product is as soluble in water, and solution shows light brown brown, slightly light bitter sugariness.Tablet is directly swallowed, every day 6 ~ 8 of doses.
After this health food is trial-produceed successfully, many batches of testing crews are taken, and general effect is can make the physiochemical indices such as blood pressure, cholesterol, platelet aggregation keep or trend is normal, physique strengthens, the colour of skin is improved.
Sequence table
Li Si bio tech ltd of <110> Shenzhen; Zhou Shining
The health food of a <120> Functional Polypeptides strengthening
<130>
<160> 1
<170> PatentIn version 3.4
<210> 1
<211> 2265
<212> DNA
<213> is manually 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 (8)
1. a health food for Functional Polypeptides strengthening, 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 R-DHLA, folic acid 1 ~ 3 × 10
-5part, 0.2 ~ 0.5 part of OPC, 2 ~ 5 parts of food stage Cits; 4 ~ 8 parts of excipient or fillers.
2. health food according to claim 1, it is characterized in that, the preparation method of described health food is as follows: glutathione, sea cucumber or ocean fish protein peptides prepared product, R-DHLA, folic acid, OPC, Cit and excipient or filler are mixed by above-mentioned part by weight, be pressed into tablet or make powdered agent with conventional medicine processing technology, dry, pack to obtain finished product.
3. a health food for Functional Polypeptides strengthening, 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 R-DHLA, folic acid 1 ~ 3 × 10
-5part, 0.2 ~ 0.5 part of OPC, 2 ~ 5 parts of food stage Cits; 4 ~ 8 parts of excipient or fillers.
4. health food according to claim 3, it is characterized in that, the preparation method of described health food is as follows: above-mentioned glutathione prepared product, Soyprotein peptide prepared product, R-DHLA, folic acid, OPC, Cit and excipient or filler are mixed by above-mentioned part by weight, be pressed into tablet or make powdered agent with conventional medicine processing technology, dry, pack to obtain finished product.
5. according to the health food described in claim 1 or 3, it is characterized in that: described glutathione prepared product is to extract and obtain from the cell of high-yield glutathione bacterial strain saccharomyces cerevisiae, the preparation method of this extract is as follows: described saccharomyces cerevisiae is inoculated in to YPD culture medium or contains peptone and amino acid whose culture medium, in 30 DEG C of vibrations or stir culture 48～70 hours, collect yeast cells, use ethanol extracting, centrifuging and taking supernatant, desolventizing, obtains glutathione prepared product.
6. health food according to claim 1, is characterized in that: described sea cucumber or ocean fish protein peptides prepared product are prepared by the following method: by clean fresh sea cucumber or fresh ocean fish tissue after homogenate, 98
oc insulation 10 minutes, is cooled to 65
oc, adjusts pH approximately 7.0, adds papain, and insulated and stirred approximately 3 hours, is then cooled to 50
oc, adjusts pH to 8.5, adds aminopeptidase insulation 3 hours, and enzyme digestion reaction liquid is heated to boil, and keeps 10 minutes, and spraying is dry, obtains sea cucumber or ocean fish protein peptides prepared product.
7. health food according to claim 3, is characterized in that: described Soyprotein peptide prepared product is prepared by following preparation method: soyabean protein powder suspends or is dissolved in sterilized water, is heated to 95
oc keeps 10 minutes, is cooled to 65
oc, adjusts pH approximately 7.0, adds papain, and insulated and stirred approximately 3 hours, is then cooled to 50
oc, adjusts pH to 8.5, adds aminopeptidase insulation 3 hours, and enzyme digestion reaction liquid is heated to boil, and keeps 10 minutes, and spraying is dry, obtains Soyprotein peptide prepared product.
8. health food according to claim 7, is characterized in that: described preparation method is in soyabean protein powder: the ratio that sterilized water is 1:10 suspends soyabean protein powder or be dissolved in sterilized water.
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| CN201110322440.5A CN102356879B (en) | 2011-10-21 | 2011-10-21 | Functional peptide-reinforced healthcare food |
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| EP2815760B1 (en) * | 2012-02-15 | 2019-10-09 | Kyowa Hakko Bio Co., Ltd. | Agent for preventing or ameliorating vascular endothelial malfunction |
| JP5899008B2 (en) * | 2012-03-05 | 2016-04-06 | アークレイ株式会社 | Hazardous metal excretion promoter and method for evaluating the amount of harmful metal using the same |
| CN103054040B (en) * | 2013-01-13 | 2014-05-21 | 浙江奥默生物医药有限公司 | Pharmaceutical composition for increasing hypoxia tolerance |
| 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 |
| 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 |
| KR102056338B1 (en) * | 2019-08-09 | 2019-12-16 | 주식회사 에브릿 | A health functional composition having effect of improving blood circulation containing ginsenoside ingredient of red ginseng and glutathione ingredient derived from microorganism fermentation of garlic |
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Effective date of registration: 20170307 Address after: 201802 Shanghai, Jiading District silver Xiang Road, room 655, No. 603 Patentee after: Shanghai, Li Weikang Biotechnology Co. Ltd. Patentee after: Zhou Shining Address before: 518052 Guangdong city of Shenzhen province Nanshan District Dingtai Fenghua Bruce, before the sea seven No. 202 Patentee before: Shenzhen Lisi Biological Technology Co., Ltd. Patentee before: Zhou Shining |
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