CN107997131A - Biological reinforced cell nutritious element is preparing the application in promoting the regenerated functional food of axoneure - Google Patents
Biological reinforced cell nutritious element is preparing the application in promoting the regenerated functional food of axoneure Download PDFInfo
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Classifications
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
- A23L7/10—Cereal-derived products
- A23L7/104—Fermentation of farinaceous cereal or cereal material; Addition of enzymes or microorganisms
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
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- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Health & Medical Sciences (AREA)
- Nutrition Science (AREA)
- Chemical & Material Sciences (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Mycology (AREA)
- Biotechnology (AREA)
- Microbiology (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
Abstract
The invention discloses biological reinforced cell nutritious element to prepare the application in promoting the regenerated functional food of axoneure.Experiment proves that biological reinforced cell nutritious element has the function that to promote embryo and the differentiation of adult central nervous mesenchymal stem cells into neurons direction, oral bio, which strengthens cell nutritious element, can strengthen differentiation of the adult brain neural precursor to mature neuron, so as to for preventing and treating nerve degenerative diseases, improving central functions and promoting the rehabilitation of injured nerve.
Description
Technical field
The present invention relates to a kind of purposes of biological reinforced cell nutritious element, exist more particularly to biological reinforced cell nutritious element
Prepare the application promoted in the regenerated functional food of axoneure
Background technology
The body of our mankind is made of upper trillion cells, these cells are the basic function units of organization of human body, carefully
The health of born of the same parents is under the regulation and control of nucleic acid, by protein, a variety of amino acid, polypeptide, polysaccharide, various trace elements, mineral matter
Deng the material with bioactivity to maintain the eubolism of cell.Cell has specific responsibility, cooperates, remains us
Health.Each organized in body by cellularity, and relevant cell tissue cluster just constitutes the organ of human body.Though
These right organs are by the different cellularity of function, and still, they need same basic nutrition component, this is support cell
The basic substance of metabolism.Cell has the service life, in theory, if cell provide suitable, enough basic nutrition into
Point, the cell of apoptosis is eliminated, it is accurate to replicate existing healthy cell, self-healing ability of the human body for disease is improved, I
Mankind's delaying sanility, keep fit.
Human body is that the mankind rely on the inherent vitality of itself, by immune system, nerve for the self-healing ability of disease
Repair mechanism based on system and internal system, repairs impaired cell, throw off one's illness keeps with one kind of sub-health state
The ability of health.It is because some human organ organization systems lack enough nutrition when the vis medicatrix naturae of human body declines
The factor, cell cannot be normally metabolized, it is impossible to correctly replicate the cell of healthy cell and the apoptosis that liquefies completely.Due to cell
Incorrect duplication so that the normal function of cell is made a discount, and the self-repairing capability of body lowers, this directly affects people
Body is for the resistivity of disease, once meeting with extraneous undesirable element, human body will be sick.
Nervous system is the system to play a leading role in body to the adjusting of physiological function activity, internal each organ, system
Function and various physiology courses be all central nervous system direct or indirect adjusting, control under carry out.However, with
The various factors such as the growth at age, cell ageing, cerebrovas-cularaccident, wound, tumour can Central nervous system cause respectively
, there is phenomena such as lesion, apoptosis of axoneure in the damage of kind various kinds, and finally causes cognitive ability to decline, even go
For the various nerve degenerative diseases such as obstacle.In view of leading position of the central nervous system in body function activity, maincenter god
Lesion through cell is the most common and most important factor died that disables, and the often life to patient and family members causes huge shadow
Ring, caused financial burden is very heavy.Unfortunately, after axoneure damage, traditional remedy measures effect is non-
Often limited, the hope of rehabilitation is very remote after morbidity or damage, therefore has great demand to new prevention and treatment method.Closely
The research in year has shown that as other body parts, there is also neural stem cell for central nervous system.Therefore, maincenter god is promoted
Break up through mesenchymal stem cells into neurons direction and then promote the regeneration of axoneure, central functions can be strengthened, prevented
Only nerve degenerative diseases and promotion injured nerve rehabilitation.For this reason, exploitation can promote cns stem cell to neuron
Direction differentiation, the enhancing regenerated food of axoneure have very big value.Compared with traditional treatment medicine, feature
Food can improve the compliance of patient, and reduction uses sense of discomfort, make that therapeutic process is free of a burden, therapeutic effect is without side-effects.
The content of the invention
The purpose of the present invention is overcome the deficiencies of the prior art and provide a kind of biological reinforced cell nutritious element in rush is prepared
Application in the regenerated functional food of pivot nerve cell.
Technical scheme is summarized as follows：
Biological reinforced cell nutritious element is preparing the application in promoting the regenerated functional food of axoneure.
Above-mentioned biological reinforced cell nutritious element is made of following methods：
(1) preparation of mother liquor：
(1) it be raw material to choose cereal, after cleaning separation impurity, be placed in pulp cooking machine addition raw material gross weight 30%~
50% water, starts pulp cooking machine, rotation immersion 30~60 minutes；
(2) to stop rotating, water is put only, opens air conditioner, be passed through cold air, rotation is kept for less than -4 DEG C 60~90 minutes,
Stop rotating；
(3) air conditioner is closed, air compressor machine is opened, is passed through air at room temperature, rotates, is warming up to room temperature；
(4) air compressor machine is closed, stops rotating, is passed through steam, is rotated, 100 DEG C of holding, 30~60 minutes, steam off；
(5) stop rotating, open air compressor machine, be passed through air at room temperature, rotate, be down to 30 DEG C~50 DEG C；
(6) stop rotating, add the nourishing ingredient powder of raw material gross weight 1%~3%, rotate 30~60 minutes；The battalion
Foster dispensing is that weight ratio is 1:2:3:1 ganoderma lucidum, matrimony vine, blueberries and Chinese cassia tree；
(7) stop rotating, the complex enzyme of raw material gross weight 0.2%~0.4% is added, when rotation 1~2 is small；
(8) 0.2%~1% edible bacterium of raw material gross weight is added, rotation makes uniformly；Stop rotating, dispense to fermenter,
Ferment 8~15 days at 28~35 DEG C, it is spare up to mother liquor；
(2) preparation of sizing material：
It is (1) another that to take cereal be the second raw material, with 18~30 DEG C of water immersions 8~16 it is small when；Draining, is placed in container；
(2) water of 1~2 times of the second raw material weight is added into container, boil 1~3 it is small when；
(3) filter, go to remove water, it is spare to obtain sizing material；
(3) preparation of finished product：
(1) it is 1 by weight by mother liquor and sizing material：1~2 ratio, mixing, separation slagging-off, obtains mixed liquor；
(2) oligoisomaltose of mixed liquor weight 1%~2%, the xanthan of mixed liquor weight 0.2%~0.7% are added
Glue, the donkey-hide gelatin of mixed liquor weight 0~1%, homogeneous；Filling sterilizing, obtains biological reinforced cell nutritious element；
The complex enzyme is that mass ratio is 3:3:1:1 carbohydrase, amylase, cellulase and pectase；
The edible bacterium is Saccharomyces cerevisiae, koji yeast, lactobacillus plantarum, lactobacillus acidophilus and bulgarian milk bar
Bacterium at least two.
Preferably, raw material is black glutinous rice, yellow glutinous rice, glutinous rice, purple rice, milled glutinous broomcorn millet, maize, scented rice, sorghum rice, rice and the heart of a lotus seed
At least five kinds in rice.
The preferred black glutinous rice of second raw material, yellow glutinous rice and glutinous rice are at least one.
Advantages of the present invention：
It is demonstrated experimentally that low concentration (below 5 μ l/ml) biological reinforced cell nutritious element does not have neural stem cell toxicity, but
Have the function that remarkably promoting embryo and the differentiation of adult central nervous mesenchymal stem cells into neurons direction, oral bio strengthens thin
Born of the same parents' nutrient can strengthen differentiation of the adult brain neural precursor to mature neuron, so as to for preventing and treating
Nerve degenerative diseases, improve central functions and promote the rehabilitation of injured nerve.
Brief description of the drawings
Fig. 1 is influence of the biological reinforced cell nutritious element to mouse neuroblastoma：At biological reinforced cell nutritious element
After managing mouse neuroblastoma (Neuro-2a), nerve cell ratio and the length of neural process all significantly increase in the cell of differentiation
Add；
Fig. 2 is influence of the biological reinforced cell nutritious element to rat embryo neural stem cell:Biological reinforced cell nutritious element
After handling rat embryo neural stem cell (Embryonic Neural Stem Cell, NSC), nerve cell in the cell of differentiation
Ratio and the length of neural process all dramatically increase；
Fig. 3 is influence of the biological reinforced cell nutritious element to adult rat neural precursor：Biological reinforced cytotrophy
After element processing adult rat nerve precursor (Neural Progenitor Cell, NPC), nerve cell in the cell of differentiation
Ratio and the length of neural process all dramatically increase；
Fig. 4 is that oral bio strengthens influence of the cell nutritious element to adult mice hippocampus neural precursor：Oral life
Thing, which strengthens cell nutritious element, increases the neuron of the new hyperplasia of adult mice hippocampus.
Fig. 5 is the experiment flow explanatory drawin that oral bio strengthens cell nutritious element experiment.
Embodiment
The embodiment of the present invention is in order to enable those skilled in the art to be better understood from the present invention, but not to this
Invention imposes any restrictions.
Each food yeast bacterium of the present invention is purchased from Angel Yeast Co., Ltd (but not to the present invention
Impose any restrictions, the food yeast bacterium that the function of other company's productions is identical can be used for the present invention)；Each lactobacillus is purchased from
Hansen Corp. of Denmark (but the present invention is not imposed any restrictions, the lactobacillus that the function of other company's productions is identical can also be used
In the present invention).
With reference to specific embodiment, the present invention is further illustrated.
Embodiment 1
Biological reinforced cell nutritious element is made of following methods：
(1) preparation of mother liquor：
(1) it is 1 to choose part by weight:1:1:2:Five kinds of 2 black glutinous rice, yellow glutinous rice, purple rice, milled glutinous broomcorn millet and rice cereal are original
Material, after cleaning separates impurity, is placed in pulp cooking machine and adds the water of raw material gross weight 30%, start pulp cooking machine, rotation
Immersion 30 minutes；The purpose of immersion is raw material is fully absorbed moisture content；
(2) stop rotating, water is put only, opens air conditioner, be passed through cold air, rotation is kept for less than -4 DEG C 60 minutes, is stopped
Rotation；The purpose let cool is the raw material expansion made after water suction；
(3) air conditioner is closed, air compressor machine is opened, is passed through air at room temperature, rotates, is warming up to room temperature；The purpose preheated in advance
It is to save the energy for the temperature rise before leading to steam in next step；
(4) air compressor machine is closed, stops rotating, is passed through steam, is rotated, 100 DEG C of holding, 60 minutes, steam off；Its purpose
It is to cure and sterilize；
(5) stop rotating, open air compressor machine, be passed through air at room temperature, rotate, be down to 30 DEG C；Its purpose is that follow-up add battalion
Support dispensing, complex enzyme and strain and suitable temperature is provided；
(6) stop rotating, add the nourishing ingredient powder of raw material gross weight 2%, rotate 30 minutes；The nourishing ingredient is attached most importance to
Amount is than being 1:2:3:1 ganoderma lucidum, matrimony vine, blueberries and Chinese cassia tree；
(7) stop rotating, the complex enzyme of raw material gross weight 0.2% is added, when rotation 1 is small；Complex enzyme is that mass ratio is 3:
3:1:1 carbohydrase, amylase, cellulase and pectase；
(8) 0.2% edible bacterium of raw material gross weight is added, rotation makes uniformly；Stop rotating, dispense to fermenter, 28
DEG C fermentation 15 days, it is spare up to mother liquor；Edible bacterium is that weight ratio is 3:1 bread microzyme and lactobacillus bulgaricus；
(2) preparation of sizing material：
(1) it is another to take weight ratio as 1:1 yellow glutinous rice and glutinous rice is the second raw material, with 18 DEG C of water soak 16 it is small when；Draining,
Rice after draining is placed in container；
(2) water of 1 times of the second raw material weight is added into container, boil 1 it is small when；
(3) filter, go to remove water, it is spare to obtain sizing material；
(3) preparation of finished product：
(1) it is 1 by weight by mother liquor and sizing material：1 ratio, mixing, separation slagging-off, obtains mixed liquor；
(2) oligoisomaltose of mixed liquor weight 1%, the xanthans of mixed liquor weight 0.2%, homogeneous are added；It is filling
Sterilizing, obtains biological reinforced cell nutritious element.
Rotary rpm in the present embodiment is 15 revs/min.
Embodiment 2
Biological reinforced cell nutritious element is made of following methods：
(1) preparation of mother liquor：
(1) it is 1 to choose weight ratio:1:2:1:1:2:1 black glutinous rice, purple rice, milled glutinous broomcorn millet, glutinous rice, maize, sorghum rice and big
Seven kinds of cereal of rice are raw material, after cleaning separation impurity, are placed in pulp cooking machine and add the water of raw material gross weight 40%, start paper
Starch precooker, rotation immersion 40 minutes；
(2) stop rotating, water is put only, opens air conditioner, be passed through cold air, rotation is kept for less than -4 DEG C 80 minutes, is stopped
Rotation；
(3) air conditioner is closed, air compressor machine is opened, is passed through air at room temperature, rotates, is warming up to room temperature；
(4) air compressor machine is closed, stops rotating, is passed through steam, is rotated, 100 DEG C of holding, 50 minutes, steam off；
(5) stop rotating, open air compressor machine, be passed through air at room temperature, rotate, be down to 40 DEG C；
(6) stop rotating, add the nourishing ingredient powder of raw material gross weight 1%, rotate 60 minutes；The nourishing ingredient is attached most importance to
Amount is than being 1:2:3:1 ganoderma lucidum, matrimony vine, blueberries and Chinese cassia tree；
(7) stop rotating, add the complex enzyme of raw material gross weight 0.3%, when rotation 1.5 is small, complex enzyme is that mass ratio is
3:3:1:1 carbohydrase, amylase, cellulase and pectase；
(8) 0.5% edible bacterium of raw material gross weight is added, rotation makes uniformly；Stop rotating, dispense to fermenter, 32
DEG C fermentation 12 days, it is spare up to mother liquor；Edible bacterium is that weight ratio is 2:1:1 Saccharomyces cerevisiae, koji yeast and plant breast
Bacillus；
(2) preparation of sizing material：
(1) it is another to take weight ratio as 1：1 yellow glutinous rice and glutinous rice is the second raw material, with 30 DEG C of water soak 8 it is small when；Draining,
It is placed in container；
(2) water of 1.5 times of the second raw material weight is added into container, boil 2 it is small when；
(3) filter, go to remove water, it is spare to obtain sizing material；
(3) preparation of finished product：
(1) it is 1 by weight by mother liquor and sizing material：1.5 ratio, mixing, separation slagging-off, obtains mixed liquor；
(2) oligoisomaltose of mixed liquor weight 2%, the xanthans of mixed liquor weight 0.4%, mixed liquor weight are added
0.1% donkey-hide gelatin, homogeneous；Filling sterilizing, obtains biological reinforced cell nutritious element.
Rotary rpm in the present embodiment is 20 revs/min.
Embodiment 3
Biological reinforced cell nutritious element is made of following methods：
(1) preparation of mother liquor：
(1) it is 1 to choose weight ratio:1:1:2:1:0.5:1:1:0.5 black glutinous rice, glutinous rice, purple rice, milled glutinous broomcorn millet, maize, perfume (or spice)
Nine kinds of rice, sorghum rice, rice and Semen Coicis cereal are raw material, and after cleaning separation impurity, it is total to be placed in addition raw material in pulp cooking machine
The water of weight 50%, starts pulp cooking machine, rotation immersion 60 minutes；
(2) stop rotating, water is put only, opens air conditioner, be passed through cold air, rotation is kept for less than -4 DEG C 90 minutes, is stopped
Rotation；
(3) air conditioner is closed, air compressor machine is opened, is passed through air at room temperature, rotates, is warming up to room temperature；
(4) air compressor machine is closed, stops rotating, is passed through steam, is rotated, 100 DEG C of holding, 30 minutes, steam off；
(5) stop rotating, open air compressor machine, be passed through air at room temperature, rotate, be down to 50 DEG C；
(6) stop rotating, add the nourishing ingredient powder of raw material gross weight 3%, rotate 30 minutes；Nourishing ingredient is weight ratio
For 1:2:3:1 ganoderma lucidum, matrimony vine, blueberries and Chinese cassia tree；
(7) stop rotating, add the complex enzyme of raw material gross weight 0.4%, when rotation 2 is small, complex enzyme is that mass ratio is 3:
3:1:1 carbohydrase, amylase, cellulase and pectase；
(8) 1% edible bacterium of raw material gross weight is added, rotation makes uniformly；Stop rotating, dispense to fermenter, at 35 DEG C
Fermentation 8 days, it is spare up to mother liquor；Edible bacterium is that weight ratio is 2:1:1 Saccharomyces cerevisiae, koji yeast and acidophilus breast bar
Bacterium；
(2) preparation of sizing material：
It is (1) another that to take black glutinous rice be the second raw material, with 25 DEG C of water immersions 10 it is small when；Draining, is placed in container；
(2) water of 2 times of the second raw material weight is added into container, boil 3 it is small when；
(3) filter, go to remove water, it is spare to obtain sizing material；
(3) preparation of finished product：
(1) it is 1 by weight by mother liquor and sizing material：2 ratio, mixing, separation slagging-off, obtains mixed liquor；
(2) oligoisomaltose of mixed liquor weight 2%, the xanthans of mixed liquor weight 0.7%, mixed liquor weight are added
1% donkey-hide gelatin, homogeneous；Filling sterilizing, obtains biological reinforced cell nutritious element.
Rotating speed in each embodiment is 15-20 revs/min, is not intended to limit, reaches the purpose is to mix material, but to rotating speed
It can also be used to the other rotating speeds for mixing purpose.
It is subject to the drying technologies such as microwave, pneumatic conveying drying, spraying again on the basis of embodiment 1-3, the present invention can be made
Powder shaped food, alleviates the weight of filling liquid, convenient to carry out, instant when eating；
The additional proportion of xanthans is improved in above-mentioned finished product process again on the basis of embodiment 1-3, is added natural
Pectin, may be made as similar g., jelly-like food, or is added in the food such as chocolate, the extract of malt and milk, biscuit, bread and eats；Also may be used
Oral liquid is refined into take.Solid custard shape food is can be made into again, in addition, the present invention may also be fabricated which that biscuit, candy, dessert etc. are eaten
Product, facilitate each types of populations to eat.
Embodiment 4
Biological reinforced cell nutritious element (prepared by embodiment 1) promotes mouse neuroblastoma cell to neuron direction point
Change：
Experimental procedure：
(1) plating cells：When mouse neuroblastoma cell (Neuro-2a) (is purchased from U.S.'s ATCC living resources
The heart) growth when be in logarithmic phase, got off cell dissociation with 2ml pancreatin, with containing 10%FBS's (hyclone) in equal volume
MEM complete mediums terminate digestion, then the mixed liquor of pancreatin and cell suspension is drawn into 15 milliliters of centrifuge tube, from
In centrifuging 5min with 1035rpm in scheming, supernatant is abandoned, the fresh MEM culture mediums of 2ml suspension cell again is added, carries out cell
Count, cell concentration is suitably diluted, according to 1x105The density in/hole is inoculated in 24 orifice plates (200 μ l/ holes).
(2) drug-treated：The cell being newly inoculated with is at 37 DEG C, 5%CO2After cultivating 12h in incubator, culture medium is sucked, is added
Enter the new culture medium of the biological reinforced cell nutritious element prepared containing 0,5,10,20 μ l embodiments 1, at 37 DEG C, 5%CO2Culture
Continue culture in case to the time of setting.Each concentration sets three multiple holes.
(3) immunofluorescent staining：Map-2 immunofluorescence dyeings experiment be used for Neuro-2a cell differentiations situation into
Row identification.In Neuro-2a cells after biological reinforced cell nutritious element handles 24h and 48h, old culture medium in culture plate is siphoned away,
PBS (phosphate buffer) is cleaned twice, is fixed 30min with 4%PFA (paraformaldehyde), is then used 0.3%TritonX-100
Permeable membrane handles 20min, and permeable membrane processing is made at closing after terminating with the PBST (X-100 containing 0.3%Triton) containing 3%BSA
Manage 20min, be incubated primary antibody (Rabbit anti-mouse Map-2,1:500), 12h is incubated in 4 DEG C.After incubation terminates, use
PBS cleaning 3 times, adds secondary antibody (1:1000, Goat anti-rabbit) room temperature be incubated 2h, PBS cleaning twice, with DAPI dye liquors
Nucleus is redyed, in fluorescence microscopy Microscopic observation, is taken pictures.
(4) cellular morphology observation and statistical analysis：Neuro-2a cells through biological reinforced cell nutritious element processing 24h and
After 48h, in fluorescence microscopy Microscopic observation cellular morphology, ImageJ softwares are used after taking pictures to being sought through the biological reinforced cell of various concentrations
The differentiation situation for supporting the later Neuro-2a cells of element processing different time carries out quantitative analysis, and quantifying project includes noble cells
Ratio and neurite lengths, quantized data carry out statistical analysis with 5 softwares of GraphPad Prism.
Test result indicates that biological reinforced cell nutritious element prepared by embodiment 1 can remarkably promote Neuro-2a cells
Differentiation becomes the cell (Figure 1A, 20 μ l/ml) for having many projections, and the overwhelming majority is in the cell that immunofluorescence dyeing proves to break up
The neuron (Figure 1B) of the Map-2 positives.After the bright biological reinforced cell nutritious element processing mouse neuroblastoma of statistical analysis table,
Nerve cell ratio (Fig. 1 C) and the length (Fig. 1 D) of neural process all dramatically increase in the cell of differentiation, and biological reinforced cell is sought
Obvious dosage and time dependence (Fig. 1 C-D) is presented in the effect for supporting element.This results show, biology prepared by embodiment 1
Strengthening cell nutritious element can promote mouse neuroblastoma Neuro-2a cells to break up to neuron direction.
It is demonstrated experimentally that biological reinforced cell nutritious element prepared by embodiment 2, embodiment 3 promotes Neuro-2a cells Godwards
Activity through the differentiation of first direction is active similar to embodiment 3.
Experimental example 5
Biological reinforced cell nutritious element (prepared by embodiment 1) promotes rat embryo Neural Stem Cells direction point
Change：
Experimental procedure：
(1) rat embryo neural molecular biology in vitro culture：Taking the Wistar rat embryos of pregnant 18 days, (pregnant rats are purchased from
This experimental animal technology Co., Ltd of Changchun hundred million) 3, whole brain is cut with operating scissors, the D-Hanks for being placed in precooling is put down
Weigh in liquid (Invitrogen), on aseptic working platform, separate the basis culture that embryonic forebrain is put in fresh DMEM/F12
In base, embryonic forebrain tissue shear is broken into fritter with eye scissors, then is blown and beaten repeatedly with pipette tips, until becoming cell suspension.Collect
Filter 23 is secondary in cell screen clothes (NEST) after cell suspension, so as to obtain single cell suspension.Single cell suspension is collected to 15-
In ml centrifuge tubes, refrigerated centrifuge 1032rpm centrifugation 5min, abandon supernatant, with the DMEM/F12 complete mediums that 3ml is fresh
(DMEM/F12, EGF 20mg/L, FGF-210mg/L, Glutamine 2mmol/L, B272%, 1%P-S) suspends thin again
Born of the same parents, are uniformly inoculated in Tissue Culture Dish or orifice plate.37 DEG C are placed in cell incubator, 5%CO2Culture 3-8 days, at this moment can be with
See homogeneous, round and smooth full nerve ball is formed.
(2) neural stem cell adhere-wall culture and differentiation：When neurosphere was by the 6th day, Neural Stem Cells ' Growth is in
Vigorous multiplicative stage, the neural molecular biology at this moment formed are transparent and uniform.Suspension nerve ball is collected into sterile 15-ml
In EP pipes, 1035rpm centrifugation 5min, abandon supernatant, add 1ml trypsase, be placed in 37 DEG C of constant incubators and digest 5min, so
2ml 10%DMEM/F12 culture mediums are added afterwards and terminate digestion, are blown and beaten to form cell suspension repeatedly with cell pipettor, 1035rpm
Supernatant is removed in centrifugation, then with the DMEM/12 culture mediums of 6ml serum-frees again suspension cell, is filtered through 40 μm of nylon wires (NEST), from
And obtain single cell suspension.Cell count is carried out, then according to 5x104The cell number of/ml be seeded in PDL it is pretreated 24
In orifice plate.
(3) drug-treated：Experimental group and control group be set, and experimental group is respectively with 5 μ l/ml, 10 μ l/ml, 20 μ after cell attachment
Tri- concentration gradient biological reinforced cell nutritious element processing cells of l/ml, control group is the cell normally cultivated, and all experiments are all
If three multiple holes.
(4) immunofluorescent staining：The experiment of Map-2 and TuJ-1 immunofluorescence dyeings is used for neural stem cell (NSC)
Differentiation situation is identified.In NSC cells after biological reinforced cell nutritious element handles 72h, old culture medium in culture plate is siphoned away,
PBS cleaning fixes 30min twice, with 4%PFA, then handles 20min, permeable membrane processing knot with 0.3%Triton X-100 permeable membranes
PBSTs (containing 0.3%Triton X-100) of the Shu Yihou containing 3%BSA makees Seal treatment 20min, is incubated primary antibody (Rabbit
anti-mouse Map-2,1:500 or Rabbit anti-mouse TuJ-1,1:400), 12h is incubated in 4 DEG C.Incubation terminates
After, with PBS cleaning 3 times, add secondary antibody (1:1000, Goat anti-rabbit) room temperature be incubated 2h, PBS cleaning twice, use
DAPI dye liquors redye nucleus, in fluorescence microscopy Microscopic observation, take pictures.
(5) cellular morphology observation and statistical analysis：NSC cells through biological reinforced cell nutritious element handle different time after,
In fluorescence microscopy Microscopic observation cellular morphology, with ImageJ softwares at through various concentrations biological reinforced cell nutritious element after taking pictures
The differentiation situation for managing the later cell of different time carries out quantitative analysis, and quantifying project includes noble cells ratio and neural process length
Degree, quantized data carry out statistical analysis with 5 softwares of GraphPad Prism.
Experimental result is shown, starts to grow neural process (Fig. 2A, control) after NSC cell attachments 24h, biological reinforced
After cell nutritious element processing, discovery has more many cells to grow neural process, and neural process substantially extends and has a large amount of branch shapes
Into the further interconnection of neural process, forms abundant neutral net connection structure (Fig. 2A, 5 μ l/ml, 10 μ l/ml, 20 μ l/
), ml obvious dosage and time dependence (Fig. 2 B is presented in the effect of biological reinforced cell nutritious element：Noble cells ratio and god
Through projection length；2C：Map-2 and TuJ-1 positive cells ratio).This is it is demonstrated experimentally that biological reinforced cell prepared by embodiment 1
Nutrient can promote differentiation of the rat embryo neural stem cell to mature neuron direction.
It is demonstrated experimentally that biological reinforced cell nutritious element prepared by embodiment 2, embodiment 3 is promoting rat embryo nerve cord
The activity that cell NSC breaks up to neuron direction is active close with embodiment 1.
Experimental example 6
Biological reinforced cell nutritious element (abbreviation ZGE) (prepared by embodiment 1) promotes adult rat neural precursor Godwards
Break up through first direction：
Experimental procedure：
(1) acquisition of hippocampus of adult rat neural precursor：With Wistar rats, purchased from Changchun hundred million, this experiment is dynamic for experiment
Thing technology Co., Ltd.First rat anesthesia, 75% alcohol disinfecting are placed on ultra-clean aseptic working platform, are cut with operating scissors
Whole brain down, is placed in the D-Hanks equilibrium liquids (Invitrogen) of precooling, on aseptic working platform, peels off whole brain
It is placed in D-Hanks equilibrium liquids, carefully separates its hippocampus position, be put into the basal medium of fresh DMEM/F12, and
Hippocampal tissue is shredded into fritter with eye scissors, then is blown and beaten repeatedly with pipette tips, until becoming cell suspension.Collect cell suspension with
The filter 23 in cell screen clothes (NEST) afterwards, so as to obtain single cell suspension.Single cell suspension is collected into 15-ml centrifuge tubes,
In 4 DEG C, 1032rpm centrifugation 5min, abandon supernatant, with DMEM/F12 complete mediums (DMEM/F12, the EGF 20mg/ that 3ml is fresh
L, FGF-210mg/L, Glutamine 2mmol/L, B272%, 1%P-S) suspension cell again, uniformly it is inoculated in cell culture
In ware or orifice plate.37 DEG C are placed in cell incubator, 5%CO2Culture 6-8 days, it is at this moment it can be seen that homogeneous, it is round and smooth full
Nerve ball formed.
(2) neural stem cell adhere-wall culture and differentiation：When nerve cell ball is grown 6-8 days, cell growth is in vigorous
Multiplicative stage, the nerve ball at this moment formed is transparent and uniform, digested by the stage nerve ball formed individual cells division it is fast
Speed, growth conditions are excellent.The suspension nerve ball in this stage is collected into sterile 15-ml EP pipes, 1035rpm centrifugation 5min,
Supernatant is abandoned, 1ml trypsase is added, is placed in 37 DEG C of constant incubators and digests 5min, then adds 2ml 10%DMEM/F12
Culture medium terminates digestion, is blown and beaten to form cell suspension repeatedly with cell pipettor, and supernatant is removed in 1035rpm centrifugations, then with 6ml without blood
Clear DMEM/12 culture mediums suspension cell again, is filtered through 40 μm of nylon wires (NEST), so as to obtain single cell suspension.Carry out
Cell count, then according to 5x104The cell number of/ml is seeded in in 24 pretreated PDL orifice plates.
(3) drug-treated：Experimental group and control group are set, and experimental group is respectively with 5 μ l/ml, 10 μ l/ml, 20 μ l/ml tri-
Concentration gradient biological reinforced cell nutritious element handles cell, and control group is the unicellular NPC normally cultivated.
(4) immunofluorescent staining：The experiment of Map-2 and TuJ-1 immunofluorescence dyeings is used for NPC cell differentiation situations
Identified.In NPC cells after biological reinforced cell nutritious element handles 72h, it is clear to siphon away old culture medium, PBS in culture plate
Wash twice, 30min fixed with 4%PFA, then with 0.3%Triton X-100 permeable membranes handle 20min, permeable membrane processing terminate with
Make Seal treatment 20min with the PBST (X-100 containing 0.3%Triton) containing 3%BSA afterwards, be incubated primary antibody (Rabbitanti-
mouse Map-2,1:500 or Rabbit anti-mouse TuJ-1,1:400), 12h is incubated in 4 DEG C.After incubation terminates,
With PBS cleaning 3 times, secondary antibody (1 is added:1000, Goat anti-rabbit) room temperature is incubated 2h, and PBS cleaning twice, contaminates with DAPI
Liquid redyes nucleus, in fluorescence microscopy Microscopic observation, takes pictures.
(5) cellular morphology observation and statistical analysis：NPC cells through biological reinforced cell nutritious element handle different time after,
In fluorescence microscopy Microscopic observation cellular morphology, with ImageJ softwares at through various concentrations biological reinforced cell nutritious element after taking pictures
The differentiation situation for managing the later cell of different time carries out quantitative analysis, and quantifying project includes noble cells ratio and neural process length
Degree, quantized data carry out statistical analysis with 5 softwares of GraphPad Prism.
Experimental result is shown, starts to grow neural process after NPC cell attachments 24h, at biological reinforced cell nutritious element (ZGE)
After processing, there are more many cells to grow neural process compared with control group.Immunofluorescence dyeing proves the cell and fiber of differentiation
It is most for the Map-2 and TuJ-1 positives (Fig. 3 A) in projection.After statistical analysis shows ZGE processing adult rats NPC, point
Nerve cell ratio (Fig. 3 B) and the length (Fig. 3 C) of neural process all dramatically increase in the cell of change, and the effect presentation of ZGE is bright
Aobvious dosage and time dependence (Fig. 3 A-C).This is it is demonstrated experimentally that biological reinforced cell nutritious element (ZGE) prepared by embodiment 1
It can promote differentiation of the adult rat neural precursor NPC to mature neuron direction.
It is demonstrated experimentally that biological reinforced cell nutritious element prepared by embodiment 2, embodiment 3 is before adult rat nerve is promoted
The activity that body cell NPC breaks up to neuron direction is active close with embodiment 3.
Experimental example 7
Oral bio strengthens cell nutritious element (prepared by embodiment 1) and promotes adult mice hippocampus neural precursor Godwards
Break up through first direction：
Experimental procedure：
(1) intragastric administration on mice：6-8 week old C57B6L mouse are purchased from Beijing Vital River Experimental Animals Technology Co., Ltd..It is real
Test group and gavage is carried out with biological reinforced cell nutritious element (ZGE) to it daily, gavage volume is 300 μ l, and control group uses PBS daily
Same processing is carried out, is carried out continuously 30 days, initial 7 days after experiment starts carry out BrdU intraperitoneal injections (50mg/ to mouse
BrdU is disabled after 7 days kg, once a day), continues oral bio and strengthens cell nutritious element or physiological saline to the 29th day, such as Fig. 5
It is shown：
(2) tissue block embedding is fixed：At night, mouse is anaesthetized, cardiac perfusion, Ran Houyong are carried out with PFA within 29th day
The whole brain of operating scissors separating mouse, is placed in making in 40% sucrose solution sedimentation overnight, second day with OCT investing tissues, bag
Bury and pour into isopentane outside box, then add dry ice, make OCT quick solidifications.Tissue storage preserves in -80 DEG C of low temperature refrigerator.
(3) cut into slices：Before section, tissue is first placed in rewarming 30min in -20 DEG C of refrigerators, is then placed in freezing-microtome
Coronal section is carried out, slice thickness starts to collect first section when being switched to hippocampus for 30 μm of, continuously cuts 5 and be affixed on 5
On different glass slides, then the another location on glass slide is pasted ensuing five and is organized, tissue on every glass slide
Number is 4, until hippocampus is collected completely.Section is stored in -80 DEG C of refrigerators.
(4) immunohistochemical staining：Section, room temperature rewarming are taken out from -80,4%PFA fixes 20min, and 3%BSA (is used
PBST dilutes) Seal treatment 15min, then it is incubated primary antibody (mouse anti-BrdU 1:200, Rabbit anti-NeuN 1:
500), stay overnight for 4 DEG C, be then incubated secondary antibody (Donkey anti-mouse 1:1000 and Goat anti-rabbit 1:1000),
DAPI mountings, micro- Microscopic observation are taken pictures.
(5)Brdu+/NeuN+Cell number is analyzed：Since first glass slide, per in 5 adjacent glass slide pieces with
Machine takes out one (it is n/5 to take out number altogether), carries out immunofluorescence dyeing experiment, counts and cut in a organized way on this glass slide
Piece Brdu+/NeuN+ cell numbers, are denoted as M1, and so on, a piece of contaminated is taken out at random in ensuing five glass slides
Color, it is M2 ... to write down its number, then the hippocampus of mice area Brdu+/NeuN+ cell numbers are about M1*5+M2*5+ ...+M
(n/5)*5.The hippocampal tissue for randomly selecting 5 mouse in experimental group and control group respectively carries out slice analysis, uses
Hippocampus of mice DG area Brdu in GraphpadPrism statistical analyses experimental group and control group+/NeuN+Cell number, experimental result
As shown in Figure 4 A, figure Green is BrdU, and red is NeuN.Statistics gained cell number is carried out with Graphpa Prism softwares
Analysis.The result is shown in Fig. 4 B.
This experiment proves that biological reinforced cell nutritious element (ZGE) prepared by oral embodiment 1 can promote adult mice extra large
Horse area NPC breaks up to neuron direction, makes the neuron of the new hyperplasia of adult mice hippocampus increase.
It is demonstrated experimentally that biological reinforced cell nutritious element prepared by embodiment 2, embodiment 3 promotes hippocampus of mice area NPC Godwards
Activity through the differentiation of first direction is active close with embodiment 1.
Claims (4)
1. biological reinforced cell nutritious element is preparing the application in promoting the regenerated functional food of axoneure.
2. application according to claim 1, it is characterized in that the biological reinforced cell nutritious element is made of following methods：
(1) preparation of mother liquor：
(1) it is raw material to choose cereal, after cleaning separation impurity, is placed in addition raw material gross weight 30%~50% in pulp cooking machine
Water, start pulp cooking machine, rotation immersion 30~60 minutes；
(2) stop rotating, water is put only, opens air conditioner, be passed through cold air, rotation is kept for less than -4 DEG C 60~90 minutes, is stopped
Rotation；
(3) air conditioner is closed, air compressor machine is opened, is passed through air at room temperature, rotates, is warming up to room temperature；
(4) air compressor machine is closed, stops rotating, is passed through steam, is rotated, 100 DEG C of holding, 30~60 minutes, steam off；
(5) stop rotating, open air compressor machine, be passed through air at room temperature, rotate, be down to 30 DEG C~50 DEG C；
(6) stop rotating, add the nourishing ingredient powder of raw material gross weight 1%~3%, rotate 30~60 minutes；The nutrition is matched somebody with somebody
Expect that for weight ratio be 1:2:3:1 ganoderma lucidum, matrimony vine, blueberries and Chinese cassia tree；
(7) stop rotating, the complex enzyme of raw material gross weight 0.2%~0.4% is added, when rotation 1~2 is small；
(8) 0.2%~1% edible bacterium of raw material gross weight is added, rotation makes uniformly；Stop rotating, dispense to fermenter, 28
~35 DEG C ferment 8~15 days, spare up to mother liquor；
(2) preparation of sizing material：
It is (1) another that to take cereal be the second raw material, with 18~30 DEG C of water immersions 8~16 it is small when；Draining, is placed in container；
(2) water of 1~2 times of the second raw material weight is added into container, boil 1~3 it is small when；
(3) filter, go to remove water, it is spare to obtain sizing material；
(3) preparation of finished product：
(1) it is 1 by weight by mother liquor and sizing material：1~2 ratio, mixing, separation slagging-off, obtains mixed liquor；
(2) oligoisomaltose of mixed liquor weight 1%~2% is added, the xanthans of mixed liquor weight 0.2%~0.7%, is mixed
Close the donkey-hide gelatin of liquid weight 0~1%, homogeneous；Filling sterilizing, obtains biological reinforced cell nutritious element；
The complex enzyme is that mass ratio is 3:3:1:1 carbohydrase, amylase, cellulase and pectase；
The edible bacterium is Saccharomyces cerevisiae, and koji yeast, lactobacillus plantarum, lactobacillus acidophilus and lactobacillus bulgaricus are extremely
It is two kinds few.
3. application according to claim 2, it is characterized in that the raw material is black glutinous rice, yellow glutinous rice, glutinous rice, purple rice, broomcorn millet
At least five kinds in rice, maize, scented rice, sorghum rice, rice and Semen Coicis.
4. application according to claim 2, it is characterized in that second raw material for black glutinous rice, yellow glutinous rice and glutinous rice at least
It is a kind of.
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