CN1077629A - Polynucleotide nutrient product and manufacture method - Google Patents

Abstract

A kind ofly do RNA and polynucleotide source with yeast, with plant pollen particularly Pollen Maydis be that polynucleotide nutrient product and manufacture method are made in DNA and deoxy polynucleotide source, be characterized in that thorough breaking cellular wall under the gentle acid-base condition of suitable height extracts RNA and DNA with yeast and pollen, the reuse enzyme hydrolysis and purify mononucleotide, make nucleotide complex and Deoxydization nucleotide complex by the human body nucleotide proportion, and and RNA, DNA together in accordance with regulations consumption be mixed with polynucleotide nutrient product, be respectively applied for high-grade polynucleotide food, beverage (containing baby-children product), flavoring agent, medicine, the production of cosmetics etc., have health invigorating and premunition, the skin of face rejuvenation, human body immunity improving power, anti-cancer and cancer-preventing, serial health effects such as anti-senility and enhancing flavoring agent delicate flavour.

Description

Polynucleotide nutrient product and manufacture method

The present invention relates to yeast and plant pollen is that raw material carries out method for processing

Nucleic acid is the carrier of life-information, serves as life quintessence's effect of storage, transmission and the expression of hereditary information.Nucleotide is the ultimate unit of forming nucleic acid, no matter be that ribonucleic acid [RNA] or DNA (deoxyribonucleic acid) [DNA] are formed by nucleotide, difference is that the nucleotide of RNA is D-ribose, and the nucleotide of DNA is the D-deoxyribose; Also has the difference of base kind, ratio and single, double chain etc. in addition.The main path of human body nucleic acid is: elder generation is purine biosynthesis and pyrimidine nucleotide in liver mainly, enters the raw material of cell as synthetic RNA by the blood transportation again; The deoxidation under the catalysis of serial enzymes of intracellular nucleotide generates Deoxydization nucleotide and as the raw material of synthetic DNA.Many and the protein bound of nucleic acid in the food is a nucleoprotein, be subjected to the effect of gastric acid and gastric enzyme to be decomposed into nucleic acid and albumen under one's belt, protein can further digest under one's belt, nucleic acid then can not, but after entering small intestinal, under the effect of pancreatic juice and intestinal juice hydrolytic enzyme, be hydrolyzed to mononucleotide and nucleoside, the two all can be absorbed and enter blood, at the same raw material that is made for synthetic RNA or DNA with synthetic nucleotide and nucleoside in the human liver.This explanation exogenous nucleic acid can be used as synthetic from the body nucleic acid raw material after human consumption absorbs.

Thought always in the past that human body might not need to rely on supply nucleic acid such as meals, reason is that nucleotide in vivo can be synthetic by other materials.In recent years research explanation, because the aging of human body, the liver synthesis capability reduces; Baby children liver is zoon not, and synthesis capability is relatively poor; Because of factors such as liver disease synthesis capability reduction all can reduce the ability of human body nucleic acid, the anemia of pregnant woman at first will satisfy the needs of cell that fetus increases rapidly and tissue so easier shortage nucleic acid because of the raw material of synthesizing ribonucleotide.And human body lacks nucleotide, and the repairing after the synthetic and damaged of nucleic acid makes it to recover the mechanism of normal configuration and function and will seriously be obstructed, and causes the change of hereditary information, cell viability decline, senilism even take place such as serious illness such as cancer and be difficult to reverse.The test explanation: be in brephic mouse with regard to possessing the repairing ability to DNA already, facing a period of time before being born, the repairing ability is more strong; Research to the mankind also obtains similar result: folic acid is the raw material of the stage product formylpropionic acid of purine biosynthesis nucleotide, umbilical blood content generally is higher than female blood several times, illustrate that the fast fetus of growth promoter is than the big several times of parent demand nucleic acid amount, when pregnant mother has obvious folic acid deficiency, supply with in the umbilical blood of cyotrophy folate content still than female blood high and equally the woman be pregnent the later stage show in the fetal blood folate content before, increase mid-term, folic acid also plays an important role in the process of synthetic pyrimidine nucleotide.The woman has 20% pregnant mother therefore self to lack folic acid approximately at later stage of being pregnent, even being pregnent the woman of having just lack folic acid in early days, megaloblastic anemia takes place, the neutrophilic granulocyte leaflet is increased, serious even premature labor and deformity youngster.The shortage of purine nucleotides is treated indirectly with increasing the folic acid quantity delivered at present, because general daily meals, beverage such as rice, wheaten food, milk, eggs, meat are (though the intensive internal organs of cell contain a little nucleic acid, but it is too high that energy etc. are disliked again, can not eat for a long time in a large number, otherwise can attend to one thing and lose sight of another), (volume is big, 95% be water for fruit and vegerable, nucleic acid content is very little) all do not contain or seldom contain nucleic acid, can not satisfy the ingest demand of human body to exogenous nucleic acid.When human body self synthesis capability deficiency or decline, even replenish enough folic acid, the various human bodies that also are difficult to avoid the nucleic acid shortage to cause damage, " nucleic acid therapy " therefore arises at the historic moment, receive that generally endurance improves, body constitution and muscle power enhancings, skin of face rejuvenation, cardiac function strengthens and the remarkable anti-senility effect of acquisition improvements such as many kinds of senile disease such as arteriosclerosis, emphysema, glaucoma, hypertension, diabetes.Nucleic acid product therefore is described as " preserving spring meals product " abroad, rather be in great demand, WHO(World Health Organization (WHO)), FDA(FDA Food and Drug Administration FAO(FAO (Food and Agriculture Organization of the United Nation))), Japan, the European Economic Community etc. all ratify edible (multiple nucleinate-for increasing its dissolubility and stability), consumption (ADI) is not generally done particular provisions, can use by product consumption (GRAS), animal (rat) per os is acute, subacute, chronic, teratogenesis suddenlys change and all no abnormal generation of reproduction test, so be the food additive of safety non-toxic.However, be limited to the level of understanding at that time, only be flavor from nucleotide and increase bright angle and consider to augment product as seasoning nutrition, this is one sorry greatly.Even abroad adopted in recent years the anti-ageing clinical practice of nucleic acid the exceedance million people time do not see that adverse side effect is arranged, but most preparations mainly adopt in animal DNA such as the milt and to extract heat denatured dna and make raw material, this just makes the output of DNA injection and price be subjected to greatly influencing of resource and cost, and because of species differ greatly and the ratio of four kinds of nucleotide also difficulty reach the human body rational proportion, so the high physiology of difficult performance is tired, common people also can not consume.It is that so-called homologous dna improves its physiology as raw material and tires that human artificial abortion embryo DNA is arranged now, but the resource and the cost overwhelming majority are difficult to bear per capita.

Purpose of the present invention provides a kind of polynucleotide product and manufacture method for the deficiency that solves prior art exactly, promptly with the route of synthesis and the ability of human body nucleic acid, human body is a foundation to the approach that digests and absorbs of exogenous nucleic acid and the rational proportion of the various nucleotide of human body etc., extensive with resource, pure single, and easily collecting, nucleic acid content enriches and low yeast nucleic acid (RNA) and the plant pollen nucleic acid (DNA) of price is nucleic acid source, the various nucleotide proportion of balance are tired to improve product, satisfy the growing anti-senility of consumers in general with pleased acceptance of people and product of high quality and at a reasonable price, constitutional health care demand.

Polynucleotide nutrient product provided by the invention is to make by human body inner nucleotide composition (mole content) with the four kinds of deoxyribonucleotides (dAMP, dCMP, dGMP and dTMP) after RNA, DNA, four kinds of ribonucleotides of RNA (AMP, CMP, GMP, UMP) and the DNA hydrolysis, and its raw material consumption (by oven dry weight) is:

RNA 0-75 part

DNA 0-25 part

Nucleotide complex (pressing AMP24 part, CMP15 part, GMP16 part, UMP21 part) 0-75 part

Deoxydization nucleotide complex (pressing dAMP8 part, dCMP4.5 part, dGMP5 part, dTMP7.5 part) 0-25 part

Deoxydization nucleotide nutrient product and manufacture method are:

1, RNA produces: get yeast adds 2-3 times of volume under 2-3 ℃ water logging bubble 24-48 hour, thin up is to the weight ratio concentration of 5-10%, making the NaOH ultimate density at 20 ℃ of following stirring (1 revolutions per second) limits adding 40%NaOH aqueous solutions is 1%, continue to stir (1 revolutions per second) after 40 minutes, be neutralized to PH7.0 with 6NHCl, restir (acid in 10 minutes, the alkali breaking cellular wall), be heated to 90-95 ℃ and to continue 5-10 minute (hot breaking cellular wall) 10000xg centrifugal and make nature cool off (cold breaking cellular wall) rapidly, continued centrifugal 10-15 minute, the precipitation that discards is as yeast comprehensive utilization raw material or as feedstuff and food high-quality protein raw material.Centrifugal supernatant is transferred to the isoelectric point, IP PH2.5 of RNA with 6NHCl, left standstill below 10 ℃ 6-12 hour, and centrifugal 10 minutes of 10000xg, the supernatant that discards is as yeast comprehensive utilization raw material.The centrifugation of the collecting above dehydrated alcohol dehydration of food stage, dry (reclaiming ethanol reuses) gets the RNA goods.

2, the hydrolysis of RNA:

Hydrolysis RNA can be with NaOH or nuclease P ₁Hydrolyze method, because NaOH method hydrolysis RNA, generate be 2 '-and 3 '-nucleotide and in the organism free exist be 5 mostly '-nucleotide, and need to consume in a large amount of perchloric acid and NaOH, when separating four kinds of nucleotide, increase the ion exchange burden and take equipment, the nuclease P that adopts Aspergillus citrimum to produce ₁Hydrolysis RNA mainly generates 5 '-nucleotide.The concrete practice is as follows: the 8% wort agar culture medium of packing in test tube was with 0.12 MPa steam pressure sterilization 15-20 minute, naturally cool to below 30 ℃ under the aseptic condition, insert the Aspergillus citrimum original seed, aseptic tampon sealing test tube mouth, 30 ℃ of incubators were cultivated after three days, transferred by aforesaid operations step and condition and carried out the seed amplification culture into the Fructus Solani melongenae bottle.With 5% glucose or sucrose, 0.5% peptone, K ₂HPO ₄And KH ₂PO ₄Each 0.05%, MgSO ₄7H ₂O and CaCl ₂2H ₂O each 0.04%, ZnSO ₄7H ₂O0.02% forms culture medium, transfer PH to 6.0,0.1 MPa steam sterilization 15-20 minute, be cooled to insert under 30 ℃ of aseptic conditions Fructus Solani melongenae bottle seed, 28-30 ℃ of feeding filtrated air ferments, and treats that the enzyme activity secretion stops fermentation when reaching maximum, put jar, pressure filter removes by filter thalline and promptly gets crude enzyme liquid, and 2-10 ℃ of following cold preservation is standby, and the several months inner enzyme vigor changes little.During hydrolysis RNA enzyme liquid that above-mentioned cold preservation is standby in advance at 65-70 ℃ of heat treatment 15-20 minute to recover and to improve nuclease P ₁Vigor, and deactivation 3 '-ribonuclease and phosphomonoesterase etc., avoid side reaction with improve 5 '-yield of nucleotide.RNA is made into 1-1.5 ℃ RNA liquid with deionized water, and transferring PH with 5%NH40H is 5.0-5.4, is warming up to 65-70 ℃, adds the Aspergillus citrimum nuclease P of 10% volume ₁63-65 ℃ of insulation degraded of crude enzyme liquid (being preheated to 45-50 ℃) 1.5-2 hour, be warming up to 95-100 ℃ and lasting 5 minutes enzyme denaturing cessation reactions rapidly, be cooled to below 10 ℃, adding hydrochloric acid accent PH after the cooling is 2.0, left standstill filtration or 10000xg centrifugal 10 minutes, tell supernatant, be the RNA enzymolysis solution that contains four kinds of nucleotide, be used for the separation of nucleotide.

3, the separation of four kinds of nucleotide:

The RNA enzymolysis solution is transferred PH to 1.5 with 2NHCl, and by cation exchange column (cation exchange column earlier with the water washing of PH1.5 till the pH value of effluent also is 1.5), upper column quantity is the 5-7% of the full exchange capacity of cation exchange resin.After upper prop is intact, with 2 times of UMP that on the water elution post of the PH1.5 of resin volume, adhere to.After finishing, the washing of PH1.5 uses the deionized water eluting instead, and first peak is 5 '-GMP, second peak is 5 '-CMP, the 3rd peak is 5 '-AMP, by collecting eluent between elution zone respectively.With GMP and the two easy mixing of CMP() to transfer PH with 2NNaOH be anion column on 4.0 (anion column should be earlier with the water balance of PH4.0) to mixed liquor, CMP(is not adsorbed) liquid.Upper prop is finished, and includes 0.004NaCl with the 0.003NHCl of 10 times of column volumes and the 0.005NHCl(of 10 times of column volumes) a spot of CMP in the eluant solution post, reuse is heated to the GMP that adsorbs on 50 ℃ 3% the NaCl eluant solution post, and the collection eluent is GMP liquid.UMP liquid is transferred PH9.0-9.5 with 2NNaOH, go up cloudy post (cloudy post is earlier with the water balance of PH9.0) after, wash when UMP begins to flow out with 0.003NHCl, use the 0.05NHCl eluting UMP that includes 3%NaCl instead and collect eluent and get UMP liquid.

4, the nucleoside acid solution concentrates and the finished product preparation: it is 7.0 that the isolating GMP eluent of cloudy post is transferred PH with 2NNaOH, 95% ethanol that adds 2 times of volumes, promptly produce white GMP precipitation, left standstill below 10 ℃ 8-12 hour, centrifugal 10 minutes of 5000xg, precipitation is dewatered with dehydrated alcohol, and 50-60 ℃ of oven dry (ethanol in precipitated liquid and the dehydration liquid is recycled) gets pure GMP.Will from GMP liquid that positive post obtains with 2NNaOH transfer PH be concentrating under reduced pressure below 7.0,80 ℃ to the 5-10% of original volume, add 2 times of volume 95% ethanol precipitations and go out AMP, further handle same GMP, must pure AMP.It is 8.0-8.5 that the CMP weak solution that will obtain from cloudy post is transferred PH with 2NNaOH, go up cloudy post (cloudy post is earlier with the water balance of PH8.0) again after, the post 0.05NHCl that contains 3%NaCl, be heated to 50 ℃ of eluting, collect eluent and, further handle same GMP, get pure CMP with 2 times of volume 95% ethanol precipitations.To further handle same GMP from 95% ethanol precipitation of the UMP of cloudy post washing liquid, get UMP with 2 times of volumes.

5, DNA's produces, get Pollen Maydis, bee pollen or other nontoxic plant pollen (no matter having or not vitality) under 2-3 ℃, add 1 times of volume water logging bubble 4-6 hour pollen mud, place-20 ℃ of following quick-freezings and continue and thawed rapidly with 65-75 ℃ of hot water in 40-60 minute and be diluted to the 10-15% weight concentration, making the NaOH ultimate density is 1%, 1 revolutions per second is stirred and to be warmed up to 70 ℃ and stirred 10-15 minute with 1 revolutions per second rotating speed in 40-60 minute, and speed is as cold as below 10 ℃.The centrifugal 10-20 of 10000xg minute, abandon precipitation (being used as high-grade nutrient additive etc.), supernatant is 0.14M with deionized water dilution NaCl content, precipitates 2-4 hour, abandons supernatant (reclaiming NaCl) and gets the DNP(DNA ribosome) precipitate.With the water saturated new distillation phenol of 2 times of DNP volumes and DNP vibration hydrotropy 5-10 minute, the centrifugal 10-20 of 10000xg minute, after taking out the upper strata water, Denatured protein gel layer in the middle of discarding, add lower floor's phenol liquid again and return to original volume when for the first time centrifugal, vibration hydrotropy 5-10 minute, the centrifugal 10-20 of 10000xg minute, after taking out the upper strata water, repetitive operation 5 to 8 times as stated above again.Sucking-off supernatant water add the cold dehydrated alcohol deposit D NA be equivalent to 2.5 times of water volumes and with glass rod around taking out (mother solution discharges after doing to reclaim purified treatment such as ethanol, phenol, albumen), dehydrated alcohol is cleaned dehydration, natural drying gets DNA.Making the high DNA raw material of required precision such as injecting drug use can repeatedly purify with said method, and removes salinity with 80% ethanol aqueous wash, with the dehydrated alcohol dehydration, is drying to obtain highly purified DNA under the aseptic condition at last.

6, the hydrolysis of DNA: the DNA liquid that DNA is made into 1-1.5% with deionized water, accent PH is 4.2-4.6, be warming up to 65-70 ℃, the blue or green enzyme P1 of the Fructus Citri tangerinae crude enzyme liquid that adds 10% volume that 45-50 ℃ degradation of rna uses, 63-65 ℃ of insulation degraded 1.5-2 hour, be warmed up to 95-100 ℃ rapidly, and lasting 5 minutes enzyme denaturing cessation reactions, be cooled to below 30 ℃, mistake filters out with the bonded albumen of DNA and is adding the flocculent deposit that forms under the thermal conditions, and adding hydrochloric acid accent PH is 1.8-2.2, centrifugal 10 minutes of standing over night or 10000xg, tell supernatant, be the DNA enzymolysis solution that contains four kinds of Deoxydization nucleotides, be used for the separation of Deoxydization nucleotide.

7, the separation of four kinds of Deoxydization nucleotides: the deoxynucleoside acid solution that nuclease P 1 hydrolysis DNA is obtained take with the isolation of RNA hydrolyzed solution in four kinds of nucleotide similar methods separate four kinds of Deoxydization nucleotides with the condition upper prop, should note adjusting respectively in the separation process the suitable value of pH value to four kind of Deoxydization nucleotide, optimum PH is low by about 0.5 than nucleotide.

8, concentrating and the finished product preparation of deoxynucleoside acid solution: take and concentrate and the same procedure of preparation nucleotide and condition concentrate and be prepared into product deoxidation nucleotide, should note adjusting the suitable value that the PH branch is clipped to four kinds of Deoxydization nucleotides in the concentration process.

9, the preparation of nucleotide complex:

Get AMP24 part, CMP14 part, GMP16 part, UMP21 part (all by over dry substance weight than), put into rustless steel and grind body and fully ground well 5-10 minute, promptly only contained the nucleotide complex that is hydrolyzed into nucleotide monomer of four kinds of nucleotide of RNA.

10, the preparation of Deoxydization nucleotide complex:

Get dAMP8 part, dCMP4.5 part, dGMP5 part, dTMP7.5 part (all by over dry substance weight ratio), put into the rustless steel mortar and fully ground well 5-10 minute, what promptly obtain four kinds of Deoxydization nucleotides of approximate human DNA and base ratio is hydrolyzed into the monomeric Deoxydization nucleotide complex of Deoxydization nucleotide.

11, the preparation of polynucleotide nutrient product

In sterilizing room, with RNA, DNA, nucleotide complex and Deoxydization nucleotide complex, formula proportion weighing by different purposes products, be added in the stainless steel equipment that has agitator, stirred 5-10 minute with 200-400 rev/min rotating speed, fully behind the mixing, promptly get polynucleotide nutrient product.

Polynucleotide nutrient product, the main nutrient composition (calculating by the over dry material) that contains in per 100 grams is:

AMP 24 gram CMP 14 grams

GMP 16 gram UMP 21 grams

DAMP 8 gram dCMP 4.5 grams

DGMP 5 gram dTMP 7.5 grams

The purposes of polynucleotide nutrient product of the present invention is except that can be used as the nucleic acid host of producing polynucleotide tablet, oral liquid and injection, also can be used as the nucleic acid additive of varieties of food items, beverage, flavoring agent, cosmetics, be made into height that series has a nucleic acid health care tire food, beverage, flavoring agent and cosmetics, consumption all can add by the GMP standard, as various beverages, sweet food, salty food, soup stock, soy sauce, vinegar, the powerful monosodium glutamate of nucleic acid specially fresh, cold cream, skin care liquid etc.

Polynucleotide nutrient product of the present invention and manufacture method have following advantage:

1, adopt wide material sources, price is low, the nucleic acid content height, the amount of dry matter height, yeast that moisture content is low and pollen (Pollen Maydis particularly, quality is single pure, nontoxic, side effect, dna content reaches more than 1.6%, output is big, do not obtain a large amount of industrial applications always, the natural biology wasting of resources of running its course is very big) for nucleic acid source and do not require that pollen has activity easily in batches to store, so can obtain the nucleic acid source of a large amount of super quality and competitive price, factory can produce throughout the year, overcome the seasonality production that is subjected to resource limit and be beneficial to and reduce cost, enlarge the output of nucleic acid product, majority can afford to consume and be benefited, and can improve the baby, child's premunition promotes the rapid rehabilitation of patient.

2, for satisfying the needs and the ability to shoulder economically of different levels level of consumption, the present invention has designed the polynucleotide nutrient product scheme of plurality of specifications, very big difference is arranged on the cost, but physiological function is close, being convenient to consumer selects for use according to the truth of oneself, so that science and technology is numerous common people's service to greatest extent, therefore Producer also obtains better society and economic benefit.

3, adopt with a kind of hydrolase nucleic acid and same set of production equipment hydrolysis RNA and DNA, help the simplification of production technology, utilization rate of equipment and installations height, Factory Building take few, so reduced investment.The enzyme hydrolysis specificity is strong, and by-product is few, and the recovery rate height is beneficial to and improves the quality of products, and reduces cost, and reduces environmental pollution.The thorough purity of enzyme hydrolysis high and must be 5 of flavor '-nucleotide, so except that can be used for the lower food of production required precision, beverage, flavoring agent and cosmetics, also can be used for the higher medicine of production precision, be the powerful monosodium glutamate of the very competent nucleic acid of flavor as nucleotide liquor and injection and requirement, the concrete application seen embodiment.In addition, because of intravital free nucleotide be 5 '-nucleotide, tire so can improve its physiology.

That 4, among NaOH that uses in the extraction of DNA and RNA and hydrolysis and the HCl and back produces is NaCl, it is all to allow in food and the medicine (containing injection) to use and residual material, soluble in water and easily remove, harmless, this helps reducing refining number of times and loss and does not influence quality.

5, the final chemical drugs that uses is edible ethanol and easily thoroughly volatilization at room temperature, and the recyclable ethanol of mother solution is reused, and is beneficial to reduce cost, and drying effect is good, and environmental pollution is little.

6, pollen and yeast breaking cellular wall extract in DNA and the RNA process, adopt high and low temperature, alkali and the acid treatment cell wall of suitable intensity simultaneously, can not only make sporoderm-broken rate (the double-deck breaking cellular wall of plant pollen) reach 100%, and can prevent to greatest extent that RNA and DNA are dissociated into nucleotide and other material, thereby improve the nucleic acid yield.

7, according to main body base A, T, G, C, the U general just proportioning and the different principle that puts in order in the organism, above-mentioned main base contents is very near the ratio in the human body in the polynucleotide nutrient of the present invention; Thereby effectively overcome exogenous nucleic acid because of the species difference base ratio difference far away not high shortcoming of tiring greatly, also overcome artificial abortion embryo DNA resource and reached the high shortcoming of price less, have high-quality, at a low price, serial significant advantage such as the source is wide.

Embodiment:

1, gets nucleotide complex 40 grams, Deoxydization nucleotide complex 13 grams, add 26.5 liters of fully dissolvings of injection water, get the polynucleotide injection or the oral liquid product of 2% concentration, become whole finished product by injection 5ml/ peace bottle or oral liquid 10ml/ peace bottle sterile packaged, also can encapsulate various volumetrical products on demand.

2, get RNA75 gram, DNA25 gram, in the rustless steel mortar, fully grind well, put into the stainless steel equipment that has agitator, add 10 kilograms of sucrose or glucoses, 200-400 rev/min was stirred 5-10 minute, abundant mixing, concentration is 0.1% polynucleotide raw sugar material, be used to make polynucleotide food and beverage, consumption can be by GMP.

3, get nucleotide complex 75 grams, Deoxydization nucleotide complex 25 grams, be dissolved in water and be polynucleotide saturated solution (concentration about 30%), be used to make all kinds of high order nucleic acid skin protection cosmeticss, can directly absorb in the use by skin and root of hair cell, and the raising cell viability, therefore rejuvenation and having no side effect of skin of face.Use amount can be by GMP.

4, get RNA40 gram, DNA13 gram, nucleotide complex 47 grams, in the rustless steel mortar, fully grind well the polynucleotide nutrient product that must be used to make health care and increase bright all kinds of flavoring agent.Adding monosodium glutamate by 5% consumption is in the sodium glutamate, and mixing (crystal gourmet powder made should be pulverized earlier and be the end) promptly gets the specially fresh polynucleotide powerful monosodium glutamate that keeps healthy; Add by the consumption of 0.005-0.01% in the flavouring agents such as soy sauce that daily life uses, vinegar, seasoning salt, promptly get flavouring agents such as specially fresh polynucleotide health promoting soy sauce, vinegar, seasoning salt.Because RNA and DNA, nucleotide all are soluble in dense saline solution, so it is very good to be bright effect.

5, get nucleotide complex 10 grams, Deoxydization nucleotide complex 3 grams, DNA6 gram, RNA20 gram, putting into the rustless steel mortar ground well 3-5 minute, must be used for the nucleotide in milk, the additional Lac Bovis seu Bubali of baby children powder infant and child foods such as (as Chinese invention patent application number 93110889.6-breast milk baby children powder and manufacture methods), and induce the nuclease vigor, to strengthen the resistivity of infant to disease such as bacillary.Consumption 0.005-0.01%.

Claims (3)

1, a kind of extraction yeast rna and plant pollen DNA and hydrolyzate thereof are done the polynucleotide source and are made polynucleotide nutrient product and manufacture method, it is characterized in that the raw material consumption (by oven dry weight) of this polynucleotide nutrient is:

RNA0-75 part

DNA0-25 part

Nucleotide complex (pressing AMP24 part, CMP14 part, GMP16 part, UMP21 part) 0-75 part

Deoxydization nucleotide complex (pressing dAMP8 part, dCMP4.5 part, dGMP5 part, dTMP7.5 part) 0-25 part

2, according to the manufacture method of the described polynucleotide nutrient product of claim 1, it is characterized in that:

(1), the preparation of RNA

Get yeast adds 2-3 times of volume under 2-3 ℃ water logging bubble 24-48 hour, thin up is to the weight ratio concentration of 5-10%, add the 40%NaOH aqueous solution while stirring with 1 revolutions per second rotating speed under 20 ℃ and reach 1% to the NaOH ultimate density, continue to stir after 40 minutes, be neutralized to PH7.0 with 6NHCl, restir 10 minutes is heated to 90-95 ℃ and lasting 5-10 minute, and 10000xg is centrifugal and it is cooled off rapidly naturally, continue to discard precipitation after centrifugal 10-15 minute, it is cold putting 6-12 hour below 2.5,10 ℃ that supernatant is transferred PH with 6NHCl, centrifugal 10 minutes of 10000xg, abandoning supernatant, collecting precipitation with the above dehydrated alcohol dehydrate of food stage, gets the RNA goods.

(2), the preparation of DNA

Get Pollen Maydis, bee pollen or other nontoxic plant pollen (no matter have not vitality) add 1 times of volume under 2-3 ℃ water logging bubble 4-6 hour, placed-20 ℃ of following quick-freezing 40-60 minutes, thaw rapidly with 65-75 ℃ of hot water, be diluted to the 10-15% weight concentration, add 40%NaOH liquid, make the NaOH final concentration be 1%, 1 revolutions per second and stirred 40-60 minute, be warmed up to 70 ℃ and stirred 5-10 minute with 1 revolutions per second rotating speed, speed is as cold as below 10 ℃.The centrifugal 10-20 of 10000xg minute, abandon precipitation, supernatant is 0.14M with deionized water dilution NaCl content, precipitates 2-4 hour, abandons supernatant and gets DNP.With 2 times after the water saturated new distillation phenol of DNP volume and DNP shook molten 5-10 minute, the centrifugal 10-20 of 10000xg minute, remove middle Denatured protein gel layer after taking out the upper strata water, adding the volume that lower floor's phenol liquid returns to when for the first time centrifugal again shook molten 5-10 minute, the centrifugal 10-20 of 10000xg minute, take out behind the water of upper strata again repetitive operation as stated above 5-8 time, till the intermediate interface place no longer protein gel occurs, the sucking-off supernatant with 2.5 times of food stage above dehydrated alcohol precipitation dehydration DNA and with glass rod around getting DNA, natural drying gets the DNA finished product.

(3), the preparation of nucleotide complex

In test tube, add 8% beerwort-agar culture medium, with 0.12 MPa vapour pressure sterilization 15-20 minute, naturally cool under the aseptic condition and insert the mould original seed of Fructus Citri tangerinae poison below 30 ℃, 30 ℃ of incubators were cultivated 3 days, changed the Fructus Solani melongenae bottle over to and carried out the seed amplification culture by aforesaid operations step and condition.The production culture medium is by 5% glucose or sucrose, 0.5% peptone, 0.05%k ₂HPO ₄, 0.05KH ₂PO ₄, MgSO ₄7H ₂O and CaCl ₂H ₂O each 0.04%, ZnSO ₄7H ₂O 0.02% supply nutrition, and accent PH is 6.0,0.1 MPa steam sterilization 15-20 minute, be cooled to insert below 30 ℃ Fructus Solani melongenae bottle seed, 25-30 ℃ of feeding filtrated air carries out aerobic fermentation, treats to stop fermentation when the enzyme activity secretion reaches maximum, puts jar, filter pressing is removed thalline and is got crude enzyme liquid, and cold preservation is standby below 10 ℃.RNA is made into the water liquid of 1-1.5%, 5%NH ₄It is 5.0-5.4 that OH transfers PH, be warmed up to 65-70 ℃, the crude enzyme liquid (temperature is at 45-50 ℃) that improves enzyme activity in advance through 65-70 ℃ of heat treatment that adds 10% volume, degraded 1.5-2 hour for 63-65 ℃, be warming up to 95-100 ℃ rapidly and continue 5 minutes enzyme denaturing, be cooled to below 10 ℃, adding HCl accent PH is 2.0, centrifugal 10 minutes of standing over night or 10000xg tell supernatant, and reuse 2NHCl transfers PH to 1.5, last cation exchange column is finished, UMP liquid, finish with 2 times of UMP that on the water elution post of the PH1.5 of resin volume, adhere to, use instead deionized water wash post successively must GMP, CMP and AMP washing liquid.Is that anion column is finished on 4.0 with GMP and CMP mixed liquor with 2NNaOH accent PH, get CMP liquid, use the 0.003NHCl of 10 times of column volumes and the 0.005NHCl(of 10 times of column volumes instead and include 0.004NNaCl) remaining CMP in the eluant solution post, the 3%NaCl liquid that reuse is 50 ℃ is washed post and is got the GMP washing liquid.After transferring PH9.0-9.5 to go up cloudy post with 2NNaOH the UMP liquid, wash post when UMP begins to flow out, use the 0.05NHCl that includes 3%NaCl instead and wash post and get UMP liquid with 0.003NHCl.It is 7.0 that the isolating GMP washing liquid of cloudy post is transferred PH with 2NNaCl, adds 95% ethanol of 2 times of volumes, left standstill below 10 ℃ 8-12 hour, centrifugal 10 minutes of 5000xg, precipitation is with the dehydrated alcohol dehydration, 50-60 ℃ dry GMP.The AMP dilute concentration washing liquid that positive post is obtained with 2NNaOH transfer PH be concentrating under reduced pressure below 7.0,80 ℃ to the 5-10% of original volume, add 95% ethanol precipitation AMP of 2 times of volumes, further handle same GMP, must AMP.It is 8.0-8.5 that the CMP weak solution that will obtain from cloudy post is transferred PH with 2NNaOH, go up cloudy post again after, post is heated to 50 ℃ of eluting with the 0.05NHCl that contains 3%NaCl, collects washing liquid and with 2 times of volume 95% ethanol precipitations, further handles same GMP, gets CMP.To further handle same GMP from 95% ethanol precipitation of the UMP of cloudy post washing liquid, get UMP with 2 times of volumes.

Get AMP24 part, CMP14 part, GMP16 part, UMP21 part (all by over dry substance weight ratio), put into the rustless steel mortar fully to grind well 5-10 minute, get nucleotide complex.

(4) preparation of Deoxydization nucleotide complex: the DNA liquid that DNA is made into 1-1.5% weight ratio concentration with deionized water, accent PH is 4.2-4.6, be warmed up to 65-70 ℃, add the Aspergillus citrimum nuclease P 1 crude enzyme liquid that 10% volume degradation of rna is used, 63-65 ℃ of insulation degraded was warmed up to 95-100 ℃ of enzyme denaturing 5 minutes in 1.5-2 hour rapidly, be cooled to below 30 ℃, the cotton-shaped albumen precipitation of filtering, it is 1.8-2.2 that HCl transfers PH, the centrifugal 10-15 of standing over night or 10000xg minute, get supernatant and carry out the separation of Deoxydization nucleotide, concentrate and the finished product preparation, four kinds of nucleotide of method and step and preparation similar, just the optimum PH of Deoxydization nucleotide is low by about 0.5, takes and prepares four kinds of Deoxydization nucleotides of four kinds of nucleotide similar methods preparations.

Get dAMP8 part, dCMP4.5 part, dGMP5 part, dTMP7.5 part (all by the oven dry weight ratio) puts into the rustless steel mortar fully to grind well 5-10 minute, gets the Deoxydization nucleotide complex.

(5) preparation of polynucleotide nutrient: in sterilizing room, with RNA, DNA, nucleotide complex and Deoxydization nucleotide complex, formula for a product ratio weighing in different purposes, be added in the stainless steel equipment of belt stirrer, stir with 200-400 rev/min rotating speed and promptly got polynucleotide nutrient product in 5-10 minute.

3, according to the purposes of the described polynucleotide nutrient of claim 1, it is characterized in that this product except that can be used as the polynucleotide tablet, outside the nucleic acid host of oral liquid and injection, also can be used as the nucleic acid additive of varieties of food items, beverage, flavoring agent and cosmetics, be made into height that series has a nucleic acid health care tire food, beverage, flavoring agent and cosmetics, consumption can add by GMP, as various beverages, sweet food, salty food, soup stock, soy sauce, vinegar, the powerful monosodium glutamate of nucleic acid specially fresh, cold cream, skin care liquid etc.