CN107345207A - With animal intestinal mucosa production enteric microorganism powder co-production small-molecular peptides, liquaemin, the method for alkaline phosphatase and application - Google Patents
With animal intestinal mucosa production enteric microorganism powder co-production small-molecular peptides, liquaemin, the method for alkaline phosphatase and application Download PDFInfo
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Abstract
It is a kind of to produce enteric microorganism powder co-production small-molecular peptides, liquaemin, the method for alkaline phosphatase and application with animal intestinal mucosa, it is characterised in that：It is using animal intestinal mucosa as raw material, using modern biotechnology process integration technology, in the same technological process of production, 4 kinds of enteric microorganism powder, small-molecular peptides, liquaemin, alkaline phosphomonoesterase function compositions are produced, can be widely applied to human and animal's medical treatment, prevention and health care industry.The present invention compared with prior art the characteristics of be, the separation and Extraction gut flora from intestinal mucosa, it is more safer than being extracted from excrement, transplanting gut flora, more there is integrality, and the enteric microorganism powder prepared is easy to use, intestinal flora of mammals can be transplanted and enter human body enteral, reached effect of promoting longevity, there is the huge market demand potential.The present invention uses joint production process, and workable, scale is changeable, and production cost is low, remarkable in economical benefits, is easy to implement the production of industrially scalable metaplasia.
Description
Technical field
The present invention relates to biological technical field, and in particular to a kind of to produce enteric microorganism powder by raw material of animal intestinal mucosa
Co-production small-molecular peptides, liquaemin, the method and its application of alkaline phosphomonoesterase.
Background technology
Early 20th century, Nobel's medical science and physiology encourage winner Russian scientists plum Qi Nikefu to 35 countries
Long-lived population ratio investigated, find Bulgaria long-lived population ratio highest.By being practised with circumjacent state diet
Used contrast, discovery largely drink that the Yoghourt containing lactic acid bacteria is relevant, and they drink one kilogram of Yoghourt daily per capita with Bulgarian.
1908, plum Qi Nikefu write on his discovery《It is long-lived》In one book, it is indicated that gastrointestinal bacterial flora is long-lived closely with human health
Correlation, and point out that the growth of harmful intestinal tract bacteria is the main reason for promoting people's aging.Chinese scholar Chi professors are in the world the 5th
Changshou District-Guangxi China Bama area investigation finds that the Bifidobacterium in above old man body at the age of one hundred years old is than in common old man's body
It is more more than beneficial bacterium.1974, probiotics was defined as the fungus favourable to intestinal microbial balance by Paker.
Beneficial bacteria of intestinal tract is defined as clinically to the effective viable bacteria of health, and independent of kind.People's enteron aisle
The micropopulation of kind more than 500 of inside surviving about 100,000,000,000,000, has added up football size, beats a metaphor, has 4,500,000,000 people on the earth
Mouthful, HE flora has 100,000,000 quantity, and this quantity is more than 4,500,000,000 2 ten thousand times.If saponins by human intestinal bacteria is lined up one
Row, the earth two that can detour encloses more.Thus people is not only one " people ", and it still coexists micro- with intestinal microflora composition
Environment.People and enteric microorganism are symbiosis, are coexisted, the relation of common prosperity, and the health status of the side the opposing party that changes produces therewith
Raw adverse reaction.Once gut flora balance is broken, the absorption of human nutrition composition, the excretion of harmful substance, exhale the old and inhale the new,
Human normal metabolism, physiology, biochemical function just change, and cause of disease processed holds machine and started rampantly；Malignant tumour, it is cardiovascular
Disease, hypertension, diabetes etc. disease are mutually then given birth to, and are destroyed, and damage, the physiological function for each system of human body that declines, shorten the mankind
Should be survived life-span of 200 years or so.The mankind are in aging course, the growth of the varied organisms function of gut flora by the age
Constantly reduce, the pathogenic bacteria being pernicious to people are dominant.
Current research of the living nature to enteric microorganism bravely jumps very much, 20l0 March 4, world's authority's Scientific Periodicals《It is natural》
Publish an article and point out, exerted in working in concert for multinational scientist with arduous under, the mankind are it has been determined that people enteric microorganism
Gene catalogue, whether to microorganism and the understanding of disease for the mankind, or to health, particularly to lifting the mankind
Life quality, promoting longevity all has the epoch-making meaning of milestone.《Biological paddy intestinal microecology and healthy seminar》Oneself lifts
Ban Liang circle, the scholar to attend a meeting in 2016 is with expert more than thousand people；It is micro- 24-25 days in March, 2017 in Shanghai just to improve China's enteron aisle
The integral level of ecology, gut flora transplantation treatment, gut flora and nerve, tumour, metabolism, immunological diseases, and improve old
Year people living quality, reach the relevant subject under discussion such as promote longevity and carried out deeply extensive inquiring into cooperation and exchange.
The excrement of Healthy People is surgically implanted into patient's enteron aisle by rising in recent years, improves Intestinal Mucosal Injury in Patients Undergoing Tiny ecosystem to reach
Environment, beneficial, harmful, intermediate flora is set to reconfigure the physiological function for playing balance, maintaining, return to Healthy People.Washington
The biologist of university is directed to genetics research kaeberlein decades always, and research team is with mouse, and dog is as research
Object, make extension life experiment with a kind of bacterial product；The life-span of dog is -13 years 10 years, and they are tested with 24 dogs, 10 weeks
Afterwards, whether changed with the cardiac function of echocardiography dog, the dog function that as a result display has fed bacterial product obtains
Raising is arrived, the dog heart of not edible bacterial product does not change；They wish that this bacterial product is used with the mankind in the future,
Delay human longevity.The German general institute Darlo valenzano of horse are taught to prove the science of this conclusion.He is with the earth
Animal model of most short-life vertebrate Africa Qing Medaka small fish as aging, because just covering all one's life this fish some months.
Professor and his R＆D team decide to do one " excrement transplanting " experiment, and 9 weeks medium-sized medaka bubbles are being contained into antibiotic
In water, the gut flora of their own is killed.Then, these middle aged fishes are moved in sterilized water, and sprinkled in water 6 weeks big
Small fish excrement.Researcher has also made several groups of control experiments.One group of 9 weeks medium-sized middle aged fish eats fish excrement of the same age, another group 9
After sterilization, any excrement of not eating, researcher has also done the fish without any processing as control to Zhou Ban great fish.Ten
The life-span of these fishes is compared after six weeks, as a result amazing, the fish life-span for having swallowed those 9 week Ban of young small fish excrement shows
Extension, 2 1%-41% have been respectively increased, locomitivity is competing so showing similar with nonage.
Although experimental results demonstrate " excrement transplanting " can anti-aging, the active microorganism source in excrement mainly has：〈1〉
It is that intestinal mucosa does not have fix tightly；The ＞ of ＜ 2 or development go wrong；The ＞ of ＜ 3 are even beneficial and pernicious bacteria is put down in intestinal microecology
Redundance in weighing apparatus.Excrement retains in enteron aisle cry constipation for a long time, and constipation is pernicious to people, microorganism and excrement and a large amount of human bodies
Metabolic waste grows to its own together, and living environment, nutrition is all unfavorable.Sum it up, the microorganism in excrement is
The superseded product of gut flora, be discharged to together with the unwanted waste of human body it is external.And the processed place of intestine and small intestine of animal
After reason, it is the table delicacies delicacy of the mankind, does not produce harmful effect to human body, so the separation and Extraction gut flora from intestinal mucosa,
It is safe than extracting, transplanting from excrement, more there is integrality, while intestinal mucosa can also produce small-molecular peptides, liquaemin, alkali
Property phosphomonoesterase.Transplanting intestinal flora of mammals enters the elderly's enteral, can both supplement beneficial bacterium, makes beneficial bacterium with being harmful to
Bacterium is in balance in enteron aisle, has also recovered the microenvironment of enteron aisle and microorganism, allows the elderly to keep young man's intestinal physiology mechanism,
The intestinal absorption of the elderly, excretion is returned to the general level of the health, each system of body, each position nutrition is strengthened, it is not necessary to aging
Material, material are updated, and senior health and fitness obtains new life, reduce disease, are improved the quality of old people living, are reached and prolong
Lengthen one's life in year effect, is the optimal selection of Human Longevity.
The micropopulation of the congenital difference in functionality of kind more than 500 about 100,000,000,000,000, will keep relative stability and balance in enteron aisle, only
Have and transplanted from intestinal mucosa, just can guarantee that the overall perfect of flora, play the effect that thalline is coordinated, supplements, restricted, make the elderly
Return to the function of young man's gut flora work.Long-lived dream of the invention to realizing the mankind, people living quality is improved, reduced big
The generation of disease, solidifying difficult disease, the mankind is lacked by or mitigate the pain of disease and torment obvious benefit.The present invention was both favorable to the people
Benefit the nation again, also be compliant with national medical reform direction " anti-overweight is controlled ".
The content of the invention
It is an object of the invention to provide one kind flora co-production small-molecular peptides, liquaemin, alkalescence are transplanted from animal intestinal tract
The method and its application of phosphomonoesterase.Specifically the present invention is using animal intestinal mucosa as raw material, using modern biotechnology process
Integrated technology, in the same technological process of production, enteric microorganism powder, small-molecular peptides, liquaemin, alkaline phosphatase can be produced
4 kinds of function compositions of monoesterase, can be widely applied to human and animal's medical treatment, prevention and health care industry.Enteric microorganism powder can incite somebody to action
Intestinal flora of mammals transplanting enters human body enteral, can both supplement beneficial bacterium, beneficial bacterium is in flat in enteron aisle with harmful bacteria
Weighing apparatus, has also recovered the microenvironment of enteron aisle and microorganism, has reached effect of promoting longevity.
The purpose of the present invention is achieved through the following technical solutions：One kind produces enteric microorganism with animal intestinal mucosa
Powder co-production small-molecular peptides, liquaemin, the method for alkaline phosphatase, comprise the following steps：
（1）Animal intestinal mucosa is crushed, is beaten；
（2）Add 0.9% physiological saline solution of 2-3 times of volume；
（3）10-15min is centrifuged with centrifuge 3000-5000r/min, collects centrifugate, removes insoluble matter；
（4）By the centrifugate of collection, 1.0 μm, 0.65 μm, 0.22 μm of filter membrane are crossed successively, are collected and are not filtered on filtered fluid and film
Thing；With normal saline flushing and merge non-filtrate on film, be dried by evaporation as enteric microorganism powder；
（5）Merge the membrane filtration liquid collected, by the ultrafiltration apparatus of molecular cut off 10,000, collect permeate and retention respectively
Liquid, permeate are small-molecular peptides liquid, are oral liquid raw material after concentration；
（6）By step（5）In trapped fluid, add n-butanol to precipitate；Centrifuged with supercentrifuge, collect centrifugate and solid-state respectively
Centrifuge thing；
（7）By step（6）The centrifugate of middle collection, with isometric cold acetone precipitation, after there is sediment, in speed 3000-
6000r/min is centrifuged 5 minutes, is collected sediment, is immediately heated acetone boiling point after the centrifugation of this sediment, makes the muddy hair of acetone；
（8）By step（7）The sediment of collection adds 3-5 times and measured（W/V：g/ml）0.05 mol/L, Tris-HC1 buffering
Liquid, the buffer solution ph 7.5, magnesium chloride containing 0.01mol/L, 0.01mol/L sodium chloride, 0.01mol/L zinc chloride；It is sufficiently mixed and stirs
Mix, obtain the thick liquid of alkaline phosphomonoesterase；
（9）By step（6）The solid that supercentrifuge is received is used to produce liquaemin.
By above-mentioned steps（8）The thick liquid of alkaline phosphomonoesterase of middle gained through upper prop, elution, collection, be dried in vacuo to obtain alkali
Property phosphomonoesterase, detailed process are as follows：
A, upper DEAL-separ0se4B posts, this post pH7.5,0.05 mol/L Tris- HC1 buffer solutions are equilibrated；
B, with 0.05 mol/L of the pH7.5 containing sodium chloride, Tris-HC1 buffer solution linear gradient elutions；
C, eluting peak is detected with 280nm, collects peak height eluent；
D, it is that eluent enzymatic activity is collected in substrate detection with phosphorus benzene disodium, merges organized enzyme eluent；
E, above-mentioned organized enzyme is dried in vacuo to obtain pulvis, i.e. alkaline phosphomonoesterase with rotation thin plate evaporator at 35 DEG C.
Above-mentioned steps（9）The specific method of middle production liquaemin is as follows：
A, by solid triplication（W/V：g/ml）0.9% physiological saline solution；
B, pH to 8.0-9.0 is adjusted with 40%NaOH solution, heating stirring is incubated two hours to 60 DEG C；
C, it is proportionally added into 0.20-0.25%（W/V：g/ml）Solid-state trypsase, digest three hours, temperature is maintained at 37 DEG C,
PH is between 8.0-9.0；
D, filter while hot, collect filtered fluid, adjust pH5.5-6.0 with 10%HCl, be warming up to 95 DEG C immediately, under agitation in proportion
5%（W/V：g/ml）The sodium chloride of addition；
E, protein is made to be separated with liquaemin；
F, it is incubated, declines when having sediment, filter residue is received in filtering immediately, makees animal protein feed after processing；Filtrate with
6000-10000 r/min are centrifuged, and collect centrifugate, centrifugate is cooled into 5-8 DEG C；
G, it is slow to cross D-254 resin columns, coutroi velocity, make liquaemin fully by resin adsorption；D-254 resins dress post is prior
It is good with acid, soda balance；
H, the pillar stopped is adsorbed, is washed with 3% sodium chloride solution, removes non-heparin sodium material；
I, pillar is eluted with 5% sodium chloride solution again, collects eluent；The eluent of collection is added into ethanol, makes solution alcohol whole
Concentration reaches 43-45%, stands 6 hours, collects sediment；
J, sediment is dehydrated with absolute ethyl alcohol, then is dehydrated with acetone, and the liquaemin of dehydration is dried in vacuo at 50 DEG C produces crude product liver
Plain sodium.
Enteric microorganism powder can make the enteron aisle of separation and Extraction in intestinal mucosa in product of the present invention after being eaten under physician guidance
Flora enters the elderly's enteral, can both supplement beneficial bacterium, beneficial bacterium is in balance in enteron aisle with harmful bacteria, has also recovered enteron aisle
With the microenvironment of microorganism, allow the elderly to keep young man's intestinal physiology mechanism, return to the intestinal absorption of the elderly, excretion
The general level of the health, each system of body, each position nutrition are strengthened, it is not necessary to aging material, material be updated, senior health and fitness obtains
To new life, disease is reduced, the quality of old people living is improved, reaches effect of promoting longevity, is the optimal choosing of Human Longevity
Select.Liquaemin can treat concurrent disseminated intravascular coagulation (DIC) early stage, prevention and treatment artery and vein thrombus and the lung bolt of various diseases
Plug, unstable angina pectoris (mitigate symptom, prevention miocardial infarction), acute myocardial infarction (prevent early stage from blocking again and infarct
Extend, reduce case fatality rate), in artificial heart-lung, peritoneal dialysis or haemodialysis as anticoagulation medicine, for transfusing blood when prevent
Blood clotting and blood bank preserve the external anti-coagulants such as blood.Small-molecular peptides can be easily directly entered into the cell, and
Through skin barrier, through blood-brain barrier, through placental barrier, through gastrointestinal mucosa barrier, efficiently, completely, thoroughly by machine
Body absorbs, converts and utilized.Small-molecular peptides participate in the synthesis of enzyme, excite the activity of enzyme, strengthen the function of enzyme, maintain the steady of enzyme
It is fixed；Small-molecular peptides improve intermediate supersession film (gastrointestinal mucosa, capillary wall, alveolar, meninges, red blood cell wall, glomerulus substrate
Film）Permeability, can effectively absorb nutrition, Edera Extract, and the invasion of pathogen can be defendd.Alkaline phosphomonoesterase is
One of the most frequently used marker enzyme of immune diagnostic reagent product at present, is widely used in molecular biology and enzyme is exempted from analysis.
Main innovation point is as follows compared with prior art by the present invention：
1st, the present invention looks for another way, the separation and Extraction gut flora from intestinal mucosa, than being extracted from excrement, transplanting gut flora more
Safety, more there is integrality, and the enteric microorganism powder prepared is easy to use, and excrement transplanting is compared and need not planted by performing the operation
Enter.
2nd, enteric microorganism powder prepared by the present invention can make the gut flora of separation and Extraction in intestinal mucosa enter the elderly's intestines
It is interior, beneficial bacterium can be both supplemented, beneficial bacterium is in balance in enteron aisle with harmful bacteria, has also recovered the micro-loop of enteron aisle and microorganism
Border, the intestinal absorption of the elderly, excretion is returned to the general level of the health, improve the quality of old people living, reach effect of promoting longevity
Fruit, it is the optimal selection of Human Longevity.
3rd, production of the present invention uses joint production process, using modern biotechnology process integration technology, in same production technology stream
Cheng Zhong, can produce 4 kinds of enteric microorganism powder, small-molecular peptides, liquaemin, alkaline phosphomonoesterase function compositions, and the technique is adopted
It is combined with traditional handicraft with modern crafts, simple machine is combined with modern comfort, workable, and scale is changeable,
Production cost is low, remarkable in economical benefits, is easy to implement the production of industrially scalable metaplasia.
4th, present invention process mainly using enteric microorganism powder as core active ingredient, co-production small-molecular peptides, liquaemin,
3 kinds of active ingredients such as alkaline phosphomonoesterase, authorized with 2010《Using pig intestinal mucosa as raw material coproduction alkaline phosphatase and
The technique of liquaemin》Compare, more functional components have not only been produced in same production technology, improve animal intestinal mucosa
Resource utilization, be also greatly reduced the production cost of itself of liquaemin and alkaline phosphomonoesterase, be produced into both
This reduces 63% compared with former technique, more with economic benefit, social benefit and market efficiency.
5th, liquaemin, small-molecular peptides, alkaline phosphomonoesterase can be widely applied to human and animal doctor in product of the present invention
Medicine treatment, prevention and health care industry, make full use of intestinal mucosa raw material, create more values.
6th, the long-lived dream of the invention to realizing the mankind, improves people living quality, reduces serious disease, solidifying difficult sick generation, makes one
The pain of disease and torment are lacked by or mitigated to class obvious benefit, has the huge market demand potential.
Embodiment
With reference to specific embodiment, the present invention is further illustrated.
Embodiment 1
4 kinds of enteric microorganism powder, small-molecular peptides, liquaemin, alkaline phosphomonoesterase function composition joint production process include following step
Suddenly：
（1）Animal intestinal mucosa is crushed, is beaten；
（2）Add 0.9% physiological saline solution of 3 times of volumes；
（3）15min is centrifuged with centrifuge 5000r/min, collects centrifugate, removes insoluble matter；
（4）Centrifugate will be collected, crosses 1.0 μm of filter membrane, collects non-filtrate on filtered fluid and film.
A, filter membrane of the filtered fluid of 1.0 μm of films after 0.65 μm will be crossed, collects non-filtrate on filtered fluid and film.
B, filter membrane of the filtered fluid of 0.65 μm of film after 0.22 μm will be crossed, collects non-filtrate on filtered fluid and film.
C, 1.0 μm of films were merged, 0.65 μm, the filtered fluid of 0.22 μm of film.To extract alkaline phosphomonoesterase.
D, merge end and cross 1.0 μm, 0.65 μm, the non-filtrate of 0.22 μm of film, prepare for production enteric microorganism.
E, 1.0 μm are not crossed more than, 0.65 μm, the non-filtrate of 0.22 μm of film, is collected with physiological saline.
F, will not 1.0 μm excessively, 0.65 μm, the mixture that 0.22 μm of film is collected with physiological saline, with rotation thin plate evaporator
Below 35 DEG C of Yu is dried in vacuo, and produces enteric microorganism powder.
H, enteric microorganism powder is filled in enteric glue.Eaten under physician guidance.
（5）Combining step（4）The membrane filtration liquid of middle collection, produce small-molecular peptides.
Centrifugate above is passed through to the ultrafiltration apparatus of molecular cut off 10,000, permeate is collected respectively with physiological saline A,
It is oral liquid raw material and trapped fluid, permeate are small-molecular peptides liquid, after concentration.
B, small-molecular peptides liquid adds xylitol, and the spices of different taste is edible health beverage.
C, small-molecular peptides can improve the immunologic function of people, particularly common cold initial stage.
（6）By step（5）In trapped fluid, add n-butanol to precipitate；Centrifuged with supercentrifuge, respectively collect centrifugate and
Solid-state centrifuges thing；
（7）By step（6）The centrifugate of middle collection, with isometric cold acetone precipitation, after there is sediment, in speed 3000-
6000r/min is centrifuged 5 minutes, is collected sediment, is immediately heated acetone boiling point after the centrifugation of this sediment, makes the muddy hair of acetone；
（8）By step（7）The sediment of collection, extract alkaline phosphomonoesterase.
A, sediment is collected more than and adds 3 times of amounts（W/V）0.05 mol/L, Tris-HC1 buffer solutions, the buffer solution
PH7.5, magnesium chloride containing 0.01mol/L, 0.01mol/L sodium chloride, 0.01mol/L zinc chloride；
B, stirring is sufficiently mixed, obtains the thick liquid of alkaline phosphomonoesterase；
C, upper DEAL-separ0se4B posts, this post pH7.5,0.05 mol/L Tris- HC1 buffer solutions are equilibrated；
D, with 0.05 mol/L of the pH7.5 containing sodium chloride, Tris-HC1 buffer solution linear gradient elutions；
E, eluting peak is detected with 280nm, collects peak height eluent；
F, it is that eluent enzymatic activity is collected in substrate detection with phosphorus benzene disodium, merges organized enzyme eluent；
G, above-mentioned organized enzyme is dried in vacuo to obtain pulvis, i.e. alkaline phosphomonoesterase with rotation thin plate evaporator at 35 DEG C.
H, alkaline phosphomonoesterase can be used as cosmetic material.
（9）By step（6）The solid that supercentrifuge is received is used to produce liquaemin.
A, solid is measured with 3 times（W/V）0.9% physiological saline solution；
B, pH to 8.5 is adjusted with 40%NaOH solution, heating stirring is incubated two hours to 60 DEG C；
C, it is proportionally added into 0.25%（W/V）Solid-state trypsase, digest three hours, temperature is maintained at 37 DEG C, and pH is in 8.0-
Between 9.0；
D, filter while hot, collect filtered fluid, adjust pH6.0 with 10% HC1, be warming up to 95 DEG C immediately, under agitation in proportion 5%
（W/V）Add sodium chloride；
E, protein is made to be separated with liquaemin；
F, it is incubated, declines when having sediment, filter residue is received in filtering immediately, makees animal protein feed after processing；Filtrate with
6000r/min is centrifuged, and collects centrifugate, centrifugate is cooled into 5-8 DEG C；
G, it is slow to cross D-254 resin columns, coutroi velocity, make liquaemin fully by resin adsorption；D-254 resins dress post is prior
It is good with acid, soda balance；
H, the pillar stopped is adsorbed, is washed with 3% sodium chloride solution, removes non-heparin sodium material；
I, pillar is eluted with 5% sodium chloride solution again, collects eluent；The eluent of collection is added into ethanol, makes solution alcohol whole
Concentration reaches 45%, stands 6 hours, collects sediment；
J, sediment is dehydrated with absolute ethyl alcohol, then is dehydrated with acetone, and the liquaemin of dehydration is dried in vacuo at 50 DEG C produces crude product liver
Plain sodium.
Claims (5)
1. a kind of method that enteric microorganism powder co-production small-molecular peptides, liquaemin, alkaline phosphatase are produced with animal intestinal mucosa,
It is characterized in that：It is using animal intestinal mucosa as raw material, using modern biotechnology process integration technology, in the same technological process of production
In, 4 kinds of enteric microorganism powder, small-molecular peptides, liquaemin, alkaline phosphomonoesterase function compositions are produced, are comprised the following steps：
（1）Animal intestinal mucosa is crushed, is beaten；
（2）Add 0.9% physiological saline solution of 2-3 times of volume；
（3）10-15min is centrifuged with centrifuge 3000-5000 r/min, collects centrifugate, removes insoluble matter；
（4）By the centrifugate of collection, 1.0 μm, 0.65 μm, 0.22 μm of filter membrane are crossed successively, are collected and are not filtered on filtered fluid and film
Thing；Merge non-filtrate on film, be dried by evaporation as enteric microorganism powder；
（5）Merge the membrane filtration liquid collected, by the ultrafiltration apparatus of molecular cut off 10,000, collect permeate and retention respectively
Liquid, permeate are small-molecular peptides liquid, are oral liquid raw material after concentration；
（6）By step（5）In trapped fluid, add n-butanol to precipitate；Centrifuged with supercentrifuge, collect centrifugate and solid-state respectively
Centrifuge thing；
（7）By step（6）The centrifugate of middle collection, with isometric cold acetone precipitation, after there is sediment, in speed 3000-
6000r/min is centrifuged 5 minutes, is collected sediment, is immediately heated acetone boiling point after the centrifugation of this sediment, makes the muddy hair of acetone；
（8）By step（7）The sediment of collection adds 3-5 times of 0.05 mol/L measured, Tris-HC1 buffer solutions, the buffer solution
PH7.5, magnesium chloride containing 0.01mol/L, 0.01mol/L sodium chloride, 0.01mol/L zinc chloride；Stirring is sufficiently mixed, obtains alkaline phosphorus
The thick liquid of acid monoester enzyme；
（9）By step（6）The solid that supercentrifuge is received is used to produce liquaemin.
2. according to the method for claim 1, it is characterised in that：By step（8）The thick liquid of alkaline phosphomonoesterase of middle gained
Through upper prop, elution, collection, alkaline phosphomonoesterase is dried in vacuo to obtain, detailed process is as follows：
A, upper DEAL-separ0se4B posts, this post pH7.5,0.05 mol/L Tris-HC1 buffer solutions are equilibrated；
B, with 0.05 mol/L of the pH7.5 containing sodium chloride, Tris-HC1 buffer solution linear gradient elutions；
C, eluting peak is detected with 280nm, collects peak height eluent；
D, it is that eluent enzymatic activity is collected in substrate detection with phosphorus benzene disodium, merges organized enzyme eluent；
E, above-mentioned organized enzyme is dried in vacuo to obtain pulvis, i.e. alkaline phosphomonoesterase with rotation thin plate evaporator at 35 DEG C.
3. according to the method for claim 1, it is characterised in that：Step（9）The specific method of middle production liquaemin is as follows：
A, by 0.9% physiological saline solution of solid triplication；
B, pH to 8.0-9.0 is adjusted with 40%NaOH solution, heating stirring is incubated two hours to 60 DEG C；
C, 0.20-0.25% solid-state trypsase is proportionally added into, is digested three hours, temperature is maintained at 37 DEG C, and pH is in 8.0-
Between 9.0；
D, filter while hot, collect filtered fluid, adjust pH5.5-6.0 with 10%HCl, be warming up to 95 DEG C immediately, under agitation in proportion
5% adds sodium chloride；
E, protein is made to be separated with liquaemin；
F, it is incubated, declines when having sediment, filter residue is received in filtering immediately, makees animal protein feed after processing；Filtrate with
6000-10000 r/min are centrifuged, and collect centrifugate, centrifugate is cooled into 5-8 DEG C；
G, it is slow to cross D-254 resin columns, coutroi velocity, make liquaemin fully by resin adsorption；D-254 resins dress post in advance will
It is good with acid, soda balance；
H, the pillar stopped is adsorbed, is washed with 3% sodium chloride solution, removes non-heparin sodium material；
I, pillar is eluted with 5% sodium chloride solution again, collects eluent；The eluent of collection is added into ethanol, makes solution alcohol whole
Concentration reaches 43-45%, stands 6 hours, collects sediment；
J, sediment is dehydrated with absolute ethyl alcohol, then is dehydrated with acetone, and the liquaemin of dehydration is dried in vacuo at 50 DEG C produces crude product liver
Plain sodium.
4. according to the method for claim 1, it is characterised in that：In step（4）In, using physiological saline collect filtered fluid and
Non- filtrate on film.
5. a kind of enteric microorganism powder transplanting by the production of claim 1 methods described into the application of human body enteral, can both mend
Filled with beneficial bacteria, beneficial bacterium is in balance in enteron aisle with harmful bacteria, also recovered the microenvironment of enteron aisle and microorganism, can reach and prolong
The effect that year lengthens one's life.
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| TWI796957B (en) * | 2022-02-18 | 2023-03-21 | 蒂美生技股份有限公司 | Method for extracting heparin |
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