CN107177638A - The highly enriched fermentation process and its active product of Yunnan olive polyphenol - Google Patents
The highly enriched fermentation process and its active product of Yunnan olive polyphenol Download PDFInfo
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- CN107177638A CN107177638A CN201710355635.7A CN201710355635A CN107177638A CN 107177638 A CN107177638 A CN 107177638A CN 201710355635 A CN201710355635 A CN 201710355635A CN 107177638 A CN107177638 A CN 107177638A
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- yunnan olive
- olive polyphenol
- polyphenol
- liquid
- yunnan
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Classifications
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Abstract
The present invention provides a kind of highly enriched fermentation process and its active product of Yunnan olive polyphenol, Yunnan olive resource can be made full use of, there is provided a kind of high efficiency, low cost, purity high and easy to spread Yunnan olive polyphenol active material preparation method, and a kind of active product with anti-fatigue effect is provided.
Description
Technical field
The invention belongs to food processing technology field, and in particular to a kind of highly enriched fermentation process of Yunnan olive polyphenol and its
Active product.
Background technology
Yunnan olive also known as emblic, emblic are Euphorbiaceae phyllanthus plant emblic
（PhyllanthusemblicaL）Fruit, alias phyllanthus emblica, olive（Sichuan）, Yunnan olive（Yunnan）, Chinese olive etc..《Tang Ben
Grass》Referred to as emblic leafflower fruit, emblic,《Southern vegetation shape》" leaf is thin, like dusk is closed, and spends Huang, and food makees six like Lee, bluish yellow color, core circle for meaning
Seven ribs, the bitter first ,sweet later of food ",《Compendium of Materia Medica》Referred to as Buddhist nunnery rubs character used in proper names and in rendering some foreign names really, be loaded with " bitterness is eaten at the beginning of its taste, it is sweeter for a good while, therefore say
Emblic ".
Emblic is a kind of conventional Tibetan medicine, and with myrobalan, terminaliae billericae,fructus three is commonly referred to as " three big fruits " using frequency in Tibetan medicine
Rate is very high,《Tibetan medicine standard》In 290 kinds of contained Tibetan medicine patent medicine, there are 72 kinds containing emblic, account for the 25% of sum, the Ministry of Public Health
In the drug standards nineteen ninety-five version contained 200 kinds of patent medicine of Tibetan medicine standard, there are 59 kinds to contain emblic, account for 29%, emblic is loaded into《In
State's pharmacopeia》Version one in 1977.
The sour micro-puckery of emblic fruit, clearing heat and cooling blood, digestion-promoting spleen-invigorating promotes the production of body fluid to quench thirst.Cure mainly blood-head blood stasis, indigestion,
Abdominal distension, coughs, laryngalgia, dry and vitamin C deficiency.In Tibetan medicine, emblic cures mainly that sick Baconic, red bar disease, blood is sick, high blood
Press disease etc..Research in recent years result shows that emblic has anti-inflammatory, anti-oxidant, anti-aging, the effect such as liver protection.
Fruit (emblic)：Sweet, micro-puckery, it is cool.Clearing heat, relieving sore-throat, moistens the lung and relieve the cough.For cold, fever, sore-throat, cough, mouth
Thousand polydipsia, otalgia, Vitamin C deficiency disease.Root (emblic leafflower root)：It is pungent, tremble with fear.It is poisonous.Help digestion, Li Shui, resolving sputum, desinsection.For high blood
Press disease, stomachache, diarrhea, scrofula.Leaf (emblic leafflower leaf)：It is pungent, flat.Clearing damp diuresis.For oedema, skin eczema.Bark (phyllanthus emblica wood
Skin)：Sweet, acid, trembles with fear.Sterilize corruption of dispelling, hemostasis.For aphtha, sore, hemorrhoid, eczema of scrotum, traumatism and bleeding are treated.Insect gall (the oil of branch
Mandarin orange worm is saved)：For having a stomachache, hernia, seminal emission, infantile malnutrition, toothache.
But the current edible way to Yunnan olive is also only stayed in directly to eat or squeeze the juice and drunk in the way of fruit juice, is eaten
More dull with mode, polyphenol content is high in the olive of Yunnan, is all Yunnan olive fruit juice mostly in the olive manufacture field of Yunnan
And beverage, it is essentially all to modulate to form after squeezing the juice using fresh Yunnan olive, so as to cause Yunnan olive slag largely to waste, utilizes effect
Rate is not high.It would therefore be highly desirable to develop a kind of highly enriched fermentation process of Yunnan olive.
The content of the invention
In view of this, the present invention provides a kind of highly enriched fermentation process of Yunnan olive polyphenol, can make full use of Yunnan olive
Resource is carried there is provided the high and easy to spread Yunnan olive polyphenol active material preparation method of a kind of high efficiency, low cost, purity
It can improve immune function of human body for one kind, improve the Yunnan olive polyphenol product of immunity.
The technical scheme is that：A kind of highly enriched fermentation process of Yunnan olive polyphenol, comprises the following steps：
（1）Pretreatment of raw material：The fresh fruit of Yunnan olive is raw material, is cleaned up with water, is put into the boiling water containing 0.1% citric acid
Middle blanching 4min, cooling crushes stoning, plus 1: 1 water mashing, pulp is vacuum dried, crushed 40 mesh sieves and obtains Yunnan olive fruit
Digested tankage；
（2）By step（1）Yunnan olive fruit digested tankage is made and presses solid-liquid ratio 1:4 are inoculated in liquid fermentation medium, in 22 DEG C of temperature
Under conditions of, then liquid fermentation and culture 3-6 days adds and splits kettle algae fermentation protein product and endophyte of plant, make to split kettle algae
The concentration of fermentation protein product is 0.15-0.85mg/mL, and the concentration of endophyte of plant is 1.9 × 106CFU/ml-4.6×
107CFU/ml then proceedes to culture 1-4 days, and separation obtains Yunnan olive fruit digested tankage zymotic fluid；
（3）From step（2）Yunnan olive polyphenol is extracted in obtained zymotic fluid；
The step（2）In liquid fermentation medium, every liter of component is as follows：
3g lactose, 2g molasses, 2g bean cake powders, 1.5g peptones, 0.1gMgSO4,0.03gCaCl2,0.1gKH2PO4,
0.1gK2HPO4；
It is described split kettle algae tunning preparation method be：
Over cleaning of learning from else's experience splits kettle algae raw material, sets enzymatic hydrolysis condition as solid-liquid ratio 1: 10（m/V）, cellulase
CellulaseACCF-4740 additions 2%, 55 DEG C of temperature, pH4.5, reaction time 1.3h, are digested, cellulose hydrolyzation
After end, boiling water bath goes out enzyme 15min, and 9000r/min centrifugations collect supernatant and obtain splitting kettle algae cellulose hydrolyzation liquid；Split kettle
Algae enzymolysis liquid goes out after enzyme activity, regulation pH value to 6.2-6.6, high pressure steam sterilization（121 DEG C, 20min）, then it is inoculated with volume fraction
The Pediococcus pentosaceus liquid of 1% MRS culture mediums activation stands, fermented to kettle algae enzymolysis liquid, 37 DEG C of constant incubators are split；Hair
After ferment terminates, 9000r/min centrifugations collect supernatant and obtain splitting kettle algae tunning；
The extracting method of the endophyte of plant is：Using tissue block method, take fresh Amaranthus retroflexus root, stem, leaf clean respectively to
There is no obvious impurity, after surface sterilization processing, it is seeded to PDA（Separate endogenetic fungus）With Gause I culture medium（In separation
Raw actinomyces）Upper culture 7-10d, then separates single bacterium colony；Picking inoculated by hypha block is fermented in equipped with 100mL from activated inclined plane
In the conical flask of culture medium, 28 DEG C, 200r/min shaking table shaken cultivations 7d are put；Fermentation culture medium remove thalline, filtrate with etc. body
Product ethyl acetate is extracted 3 times, merges organic phase, and 55 DEG C are concentrated under reduced pressure and are evaporated, as bacterium solution crude extract；Centrifuge and be filtrated to get
Thalline, with appropriate 95% ethanol condensing reflux 3 times, merges ethanol phase, is concentrated under reduced pressure and is evaporated, as thalline crude extract；Put 4 DEG C of ice
Case is saved backup.
Further, step（2）Described in separation be that 5-10min is centrifuged under the conditions of 15000r/min.
Further, the step（3）The method of middle extraction Yunnan olive polyphenol, step is as follows：
Take step（2）Obtained zymotic fluid, adds 95% ethanol of 4 times of volumes, 4 DEG C of standing 5h, 12000rpm centrifugations after mixing
10min, takes supernatant, and Yunnan olive polyphenol is made after freeze-drying.
The present invention efficiently, quickly therefrom extracts the Polyphenols of high-quality using Non-traditional Techniques using Yunnan olive as raw material
Material.It is water-soluble substances that the present invention, which takes full advantage of Yunnan olive polyphenol, first passes through and splits in kettle algae fermentation protein product and plant
Raw bacterium is fermented to fruit flesh component, fully discharges the Polyphenols active material inside the olive of Yunnan, using polyphenol it is water-soluble with
The rise of temperature and the characteristic increased, first use water blancing；Using the ethanol solution of setting concentration to polyphenol and other polysaccharides
Mutually the formed hydrogen bond of association and hydrophobic bond has a preferable blocking effect between material, and ethanol have to polyphenol it is higher
Dissolubility, the dissolubility to protein, polysaccharide etc. is relatively low, the impurity in extract is reduced, while improving extraction rate
And effect.Compared with traditional extraction technology, have the advantages that fast easy to operate, extraction rate, efficiency high, consume energy less, purity it is high,
Simultaneously in process of the present invention, the pollution to product and the decomposition of heat-sensitive substance can be also reduced.
A kind of Yunnan olive polyphenol active product, is made up of following parts by weight meter component：Yunnan olive polyphenol 40-50, potassium chloride
1-3, D-sorbite 5-8, sodium citrate 2-4, white granulated sugar 10-30, blood clam enzymolysis product 11-15, atractylodes chinensis 9-13, carbonic acid
Hydrogen sodium 2-6, kiwi flavor 0.3-0.5, calcium lactate 3-5, water 50-120.
Further, the Yunnan olive polyphenol active product, is made up of following parts by weight meter component：Yunnan olive polyphenol 43,
Potassium chloride 2, D-sorbite 6, sodium citrate 3, white granulated sugar 14, blood clam enzymolysis product 13, atractylodes chinensis 11, sodium acid carbonate 4, Mi
Monkey peach essence 0.4, calcium lactate 4, water 78.
In the present invention, the preparation method of the blood clam enzymolysis product is：S1. blood clam whole viscera is taken, it is 1 to make material-water ratio:3
(w/v) solution ph, is adjusted to 2.5 so that stomach acid or alkali environment is presented with 2mol/L hydrochloric acid, using enzyme-to-substrate concentration ratio as
1300U/g adds pepsin, 37 DEG C, 110r/min vibration enzymolysis 2h is kept in thermostatic control oscillator vibration, on this condition
Obtain the head product digested through pepsin；S2. the enzymolysis liquid pH after being digested with 2mol/L sodium hydroxide through pepsin
Value is adjusted to 8.5, keeps 37 DEG C of hydrolysis temperatures, and complex enzyme addition is trypsase and pancreas curdled milk in 5200U/g, the complex enzyme
The mass ratio of protease is 5:1, the material-water ratio 1:4.5, enzymolysis time is 3.5h, after enzymolysis terminates, in 100 DEG C of boiling water baths
Go out enzyme 10min；By the enzymolysis product in S2 by vacuum freeze drying, setting drying condition is -30 DEG C of precooling temperature, during precooling
Between 2h, since heating-up temperature reach 25 DEG C slowly heated up after 18h -10 DEG C, and remains to dry terminal, obtain blood clam enzymolysis
Product.
The preparation method of the atractylodes chinensis is：Rhizoma atractylodis are extracted respectively using organic solvent cold soaking extraction method.
Vegetable material is placed in 60 DEG C of constant temperature blast drying oven and dried to crisp, is placed in after being crushed in plant pulverizer, sieving
(0.45mm) 2 times.100g plant drymeals are weighed, are poured into 1000mL triangular flasks, 5 times of amount methanol is added, soaks 24h, every
12h is stirred 2-3 times, suction filtration after 24h.The 2nd extraction is carried out, suction filtration, the filtrate that 2 times are obtained merges, and is steamed at 65 DEG C with rotation
Instrument solvent evaporated is sent out, obtained paste i.e. Plant crude extract claims quality, is placed in sealing preserve in 4 DEG C of refrigerators, standby.
Particularly, in actual production process, blood clam enzymolysis product of the invention, atractylodes chinensis can equivalent amplification given birth to
It is prepared by production.
Raw material of the present invention are easy to buying, make simple；And rich in abundant protein, vitamin and mineral matter；Just
In carrying, instant edible quickly eliminates hunger, and mouthfeel is soft, and taste is unique；Suitable all age group crowd eats.This
Invention empirical tests, the permanent edible effect that can reach strengthen immunity, based on the active component in the present invention using simulation human body
The mode of gastro-intestinal digestion is made, and coordinates the active component extracted out of plant to be simultaneously combined into enteron aisle, on the one hand can be with
Colonized in enteron aisle, maintain the balance of intestinal microflora；On the other hand be Yunnan olive polyphenol with blood clam enzymolysis product and
Atractylodes chinensis collective effect induces intestine immunity in the immune system of host, and stimulates thymus gland, the immune organ such as spleen, promotees
Enter macrophage activity, by strengthening reactivity of B, T lymphocyte to antigenic stimulus, play specificity immuning activity, so that
Strengthen the immunologic function of body, it is easy to be absorbed by the body, the crowd's long-term taking of condition 95% is without malaise symptoms.
Embodiment
Technical scheme is clearly and completely described below in conjunction with the embodiment of the present invention, it is clear that retouched
The embodiment stated is only a part of embodiment of the invention, rather than whole embodiments.Based on the embodiment in the present invention, sheet
The every other embodiment that field those of ordinary skill is obtained under the premise of creative work is not made, belongs to the present invention
The scope of protection.
Embodiment 1
A kind of highly enriched fermentation process of Yunnan olive polyphenol, comprises the following steps：
（1）Pretreatment of raw material：The fresh fruit of Yunnan olive is raw material, is cleaned up with water, is put into the boiling water containing 0.1% citric acid
Middle blanching 4min, cooling crushes stoning, plus 1: 1 water mashing, pulp is vacuum dried, crushed 40 mesh sieves and obtains Yunnan olive fruit
Digested tankage；
（2）By step（1）Yunnan olive fruit digested tankage is made and presses solid-liquid ratio 1:4 are inoculated in liquid fermentation medium, in 22 DEG C of temperature
Under conditions of, then liquid fermentation and culture 3 days adds and splits kettle algae fermentation protein product and endophyte of plant, makes to split kettle algae hair
The concentration of ferment protein product is 0.15mg/mL, and the concentration of endophyte of plant is 4.6 × 107CFU/ml then proceedes to culture 4 days,
Separation, obtains Yunnan olive fruit digested tankage zymotic fluid；
（3）From step（2）Yunnan olive polyphenol is extracted in obtained zymotic fluid；
The step（2）In liquid fermentation medium, every liter of component is as follows：
3g lactose, 2g molasses, 2g bean cake powders, 1.5g peptones, 0.1gMgSO4,0.03gCaCl2,0.1gKH2PO4,
0.1gK2HPO4；
It is described split kettle algae tunning preparation method be：
Over cleaning of learning from else's experience splits kettle algae raw material, sets enzymatic hydrolysis condition as solid-liquid ratio 1: 10（m/V）, cellulase
CellulaseACCF-4740 additions 2%, 55 DEG C of temperature, pH4.5, reaction time 1.3h, are digested, cellulose hydrolyzation
After end, boiling water bath goes out enzyme 15min, and 9000r/min centrifugations collect supernatant and obtain splitting kettle algae cellulose hydrolyzation liquid；Split kettle
Algae enzymolysis liquid goes out after enzyme activity, regulation pH value to 6.2-6.6, high pressure steam sterilization（121 DEG C, 20min）, then it is inoculated with volume fraction
The Pediococcus pentosaceus liquid of 1% MRS culture mediums activation stands, fermented to kettle algae enzymolysis liquid, 37 DEG C of constant incubators are split；Hair
After ferment terminates, 9000r/min centrifugations collect supernatant and obtain splitting kettle algae tunning；
The extracting method of the endophyte of plant is：Using tissue block method, take fresh Amaranthus retroflexus root, stem, leaf clean respectively to
There is no obvious impurity, after surface sterilization processing, it is seeded to PDA（Separate endogenetic fungus）With Gause I culture medium（In separation
Raw actinomyces）Upper culture 7-10d, then separates single bacterium colony；Picking inoculated by hypha block is fermented in equipped with 100mL from activated inclined plane
In the conical flask of culture medium, 28 DEG C, 200r/min shaking table shaken cultivations 7d are put；Fermentation culture medium remove thalline, filtrate with etc. body
Product ethyl acetate is extracted 3 times, merges organic phase, and 55 DEG C are concentrated under reduced pressure and are evaporated, as bacterium solution crude extract；Centrifuge and be filtrated to get
Thalline, with appropriate 95% ethanol condensing reflux 3 times, merges ethanol phase, is concentrated under reduced pressure and is evaporated, as thalline crude extract；Put 4 DEG C of ice
Case is saved backup.
Further, step（2）Described in separation be that 5-10min is centrifuged under the conditions of 15000r/min.
Further, the step（3）The method of middle extraction Yunnan olive polyphenol, step is as follows：
Take step（2）Obtained zymotic fluid, adds 95% ethanol of 4 times of volumes, 4 DEG C of standing 5h, 12000rpm centrifugations after mixing
10min, takes supernatant, and Yunnan olive polyphenol is made after freeze-drying.
The present invention efficiently, quickly therefrom extracts the Polyphenols of high-quality using Non-traditional Techniques using Yunnan olive as raw material
Material.It is water-soluble substances that the present invention, which takes full advantage of Yunnan olive polyphenol, the characteristic that its water solubility increases with the rise of temperature,
First use water blancing；Using the ethanol solution of setting concentration to mutually associating what is formed between polyphenol and other polysaccharose substances
Hydrogen bond and hydrophobic bond have preferable blocking effect, and ethanol has higher dissolubility to polyphenol, to protein, polysaccharide etc.
Dissolubility it is relatively low, the impurity in extract is reduced, while improving extraction rate and effect.With traditional extraction technology phase
Than, have the advantages that fast easy to operate, extraction rate, efficiency high, consume energy less, purity it is high, while in process of the present invention, and also
The pollution to product and the decomposition of heat-sensitive substance can be reduced.
Embodiment 2
A kind of highly enriched fermentation process of Yunnan olive polyphenol, it is characterised in that comprise the following steps：
（1）Pretreatment of raw material：The fresh fruit of Yunnan olive is raw material, is cleaned up with water, is put into the boiling water containing 0.1% citric acid
Middle blanching 4min, cooling crushes stoning, plus 1: 1 water mashing, pulp is vacuum dried, crushed 40 mesh sieves and obtains Yunnan olive fruit
Digested tankage；
（2）By step（1）Yunnan olive fruit digested tankage is made and presses solid-liquid ratio 1:4 are inoculated in liquid fermentation medium, in 22 DEG C of temperature
Under conditions of, then liquid fermentation and culture 6 days adds and splits kettle algae fermentation protein product and endophyte of plant, makes to split kettle algae hair
The concentration of ferment protein product is 0.15mg/mL, and the concentration of endophyte of plant is 1.9 × 106CFU/mll then proceedes to culture 1 day,
Separation, obtains Yunnan olive fruit digested tankage zymotic fluid；
（3）From step（2）Yunnan olive polyphenol is extracted in obtained zymotic fluid.
Embodiment 3
A kind of highly enriched fermentation process of Yunnan olive polyphenol, it is characterised in that comprise the following steps：
（1）Pretreatment of raw material：The fresh fruit of Yunnan olive is raw material, is cleaned up with water, is put into the boiling water containing 0.1% citric acid
Middle blanching 4min, cooling crushes stoning, plus 1: 1 water mashing, pulp is vacuum dried, crushed 40 mesh sieves and obtains Yunnan olive fruit
Digested tankage；
（2）By step（1）Yunnan olive fruit digested tankage is made and presses solid-liquid ratio 1:4 are inoculated in liquid fermentation medium, in 22 DEG C of temperature
Under conditions of, then liquid fermentation and culture 4 days adds and splits kettle algae fermentation protein product and endophyte of plant, makes to split kettle algae hair
The concentration of ferment protein product is 0.3mg/mL, and the concentration of endophyte of plant is 7.5 × 106CFU/ml then proceedes to culture 4 days, point
From obtaining Yunnan olive fruit digested tankage zymotic fluid；
（3）From step（2）Yunnan olive polyphenol is extracted in obtained zymotic fluid.
Embodiment 4
A kind of highly enriched fermentation process of Yunnan olive polyphenol, it is characterised in that comprise the following steps：
（1）Pretreatment of raw material：The fresh fruit of Yunnan olive is raw material, is cleaned up with water, is put into the boiling water containing 0.1% citric acid
Middle blanching 4min, cooling crushes stoning, plus 1: 1 water mashing, pulp is vacuum dried, crushed 40 mesh sieves and obtains Yunnan olive fruit
Digested tankage；
（2）By step（1）Yunnan olive fruit digested tankage is made and presses solid-liquid ratio 1:4 are inoculated in liquid fermentation medium, in 22 DEG C of temperature
Under conditions of, then liquid fermentation and culture 3-6 days adds and splits kettle algae fermentation protein product and endophyte of plant, make to split kettle algae
The concentration of fermentation protein product is 0.15-0.85mg/mL, and the concentration of endophyte of plant is 2.4 × 107CFU/ml then proceedes to training
Support 2 days, separation obtains Yunnan olive fruit digested tankage zymotic fluid；
（3）From step（2）Yunnan olive polyphenol is extracted in obtained zymotic fluid.
Embodiment 5
A kind of Yunnan olive polyphenol active product, is made up of following parts by weight meter component：Yunnan olive polyphenol 43, potassium chloride 2, sorbose
Alcohol 6, sodium citrate 3, white granulated sugar 14, blood clam enzymolysis product 13, atractylodes chinensis 11, sodium acid carbonate 4, kiwi flavor 0.4, breast
Sour calcium 4, water 78.
In the present invention, the preparation method of the blood clam enzymolysis product is：S1. blood clam whole viscera is taken, it is 1 to make material-water ratio:3
(w/v) solution ph, is adjusted to 2.5 so that stomach acid or alkali environment is presented with 2mol/L hydrochloric acid, using enzyme-to-substrate concentration ratio as
1300U/g adds pepsin, 37 DEG C, 110r/min vibration enzymolysis 2h is kept in thermostatic control oscillator vibration, on this condition
Obtain the head product digested through pepsin；S2. the enzymolysis liquid pH after being digested with 2mol/L sodium hydroxide through pepsin
Value is adjusted to 8.5, keeps 37 DEG C of hydrolysis temperatures, and complex enzyme addition is trypsase and pancreas curdled milk in 5200U/g, the complex enzyme
The mass ratio of protease is 5:1, the material-water ratio 1:4.5, enzymolysis time is 3.5h, after enzymolysis terminates, in 100 DEG C of boiling water baths
Go out enzyme 10min；By the enzymolysis product in S2 by vacuum freeze drying, setting drying condition is -30 DEG C of precooling temperature, during precooling
Between 2h, since heating-up temperature reach 25 DEG C slowly heated up after 18h -10 DEG C, and remains to dry terminal, obtain blood clam enzymolysis
Product.
The preparation method of the atractylodes chinensis is：Rhizoma atractylodis are extracted respectively using organic solvent cold soaking extraction method.
Vegetable material is placed in 60 DEG C of constant temperature blast drying oven and dried to crisp, is placed in after being crushed in plant pulverizer, sieving
(0.45mm) 2 times.100g plant drymeals are weighed, are poured into 1000mL triangular flasks, 5 times of amount methanol is added, soaks 24h, every
12h is stirred 2-3 times, suction filtration after 24h.The 2nd extraction is carried out, suction filtration, the filtrate that 2 times are obtained merges, and is steamed at 65 DEG C with rotation
Instrument solvent evaporated is sent out, obtained paste i.e. Plant crude extract claims quality, is placed in sealing preserve in 4 DEG C of refrigerators, standby.
Particularly, in actual production process, blood clam enzymolysis product of the invention, atractylodes chinensis can equivalent amplification given birth to
It is prepared by production.
Raw material of the present invention are easy to buying, make simple；And rich in abundant protein, vitamin and mineral matter；Just
In carrying, instant edible quickly eliminates hunger, and mouthfeel is soft, and taste is unique；Suitable all age group crowd eats.This
Invention empirical tests, the permanent edible effect that can reach strengthen immunity, based on the active component in the present invention using simulation human body
The mode of gastro-intestinal digestion is made, and coordinates the active component extracted out of plant to be simultaneously combined into enteron aisle, on the one hand can be with
Colonized in enteron aisle, maintain the balance of intestinal microflora；On the other hand be Yunnan olive polyphenol with blood clam enzymolysis product and
Atractylodes chinensis collective effect induces intestine immunity in the immune system of host, and stimulates thymus gland, the immune organ such as spleen, promotees
Enter macrophage activity, by strengthening reactivity of B, T lymphocyte to antigenic stimulus, play specificity immuning activity, so that
Strengthen the immunologic function of body, it is easy to be absorbed by the body, the crowd's long-term taking of condition 95% is without malaise symptoms.
Embodiment 6
The present embodiment provides a kind of Yunnan olive polyphenol active product, is made up of following parts by weight meter component：Yunnan olive polyphenol 40,
Potassium chloride 2, D-sorbite 6, sodium citrate 3, white granulated sugar 14, blood clam enzymolysis product 11, atractylodes chinensis 9, sodium acid carbonate 4,
Kiwi flavor 0.4, calcium lactate 4, water 78.Yunnan olive polyphenol, blood clam enzymolysis product, the composition of atractylodes chinensis in the present embodiment
And preparation method is consistent with embodiment 5.
Embodiment 7
The present embodiment provides a kind of Yunnan olive polyphenol active product, is made up of following parts by weight meter component：Yunnan olive polyphenol 50,
Potassium chloride 2, D-sorbite 6, sodium citrate 3, white granulated sugar 14, blood clam enzymolysis product 15, atractylodes chinensis 13, sodium acid carbonate 4,
Kiwi flavor 0.4, calcium lactate 4, water 78.Yunnan olive polyphenol, blood clam enzymolysis product, the composition of atractylodes chinensis in the present embodiment
And preparation method is consistent with embodiment 5.
Embodiment 8
The present embodiment provides a kind of Yunnan olive polyphenol active product, is made up of following parts by weight meter component：Yunnan olive polyphenol 50,
Potassium chloride 2, D-sorbite 6, sodium citrate 3, white granulated sugar 14, blood clam enzymolysis product 15, atractylodes chinensis 9, sodium acid carbonate 4,
Kiwi flavor 0.4, calcium lactate 4, water 78.
Yunnan olive polyphenol, blood clam enzymolysis product, the composition of atractylodes chinensis and preparation method and embodiment 5 in the present embodiment
Unanimously.
Embodiment 9
The present embodiment provides a kind of Yunnan olive polyphenol active product, is made up of following parts by weight meter component：Yunnan olive polyphenol 40,
Potassium chloride 2, D-sorbite 6, sodium citrate 3, white granulated sugar 14, blood clam enzymolysis product 11, atractylodes chinensis 13, sodium acid carbonate 4,
Kiwi flavor 0.4, calcium lactate 4, water 78.
Yunnan olive polyphenol, blood clam enzymolysis product, the composition of atractylodes chinensis and preparation method and embodiment 5 in the present embodiment
Unanimously.
Effect example
1. strengthen immunity effect experiment
It will be dried by active product thickening made from embodiment 5, and be prepared into powdered, and be used as test sample.
Reference substance：Its vigorous and graceful board Soybean Peptide albumen powder；Source：Beijing Tongrentang Health Pharmaceutical Co., Ltd.；Adult
Clinic recommends consumption：0.167g.kg-1.d-1。
Animal subject strain and rank：BALB/c mouse, Hartley cavys, SPF grades；Quantity and sex：BALB/c is small
Mouse 60, male；Hartley cavys 6,.Buy body weight BALB/c mouse 17-19g, Hartley cavy 250-
350g.There is provided by Guangdong Medical Lab Animal Center, experimental animal uses credit number：SYXK（Guangdong）2013-0002.Experiment
Period, mouse ad lib drinking-water observes mouse state daily.
Dose design is carried out according to the basic condition of sample, sample it is basic, normal, high by adult 5 times of quantity, 10 times,
20 times of design dosage, respectively 0.15g.kg-1.d-1、 0.30g.kg-1.d-1 、0.60g.kg-1.d-1；Positive controls dosage
It is 0.835g.kg by 5 times of designs of adult's recommended dose-1.d-1.60 male mices are respectively divided into 5 groups at random（Negative control
The basic, normal, high dosage group of group, positive controls, given the test agent）, every group 12, wherein 2 are only used as standby animal.Each group mouse is every
It presses 0.1mL/10g body weight gavage respective sample liquid, and negative control group gives equivalent pure water, 1 time a day, successive administration 30
My god.On-test, off-test are weighed in 1 time, are weighed in weekly during experiment 1 time.
1.1 mouse lymphocyte transformation experiments
Last dose 1h, it is sterile to take spleen to be placed in the culture dish for filling RPMI-1640 nutrient solutions, and 200 eye mesh screens are placed, with note
Emitter piston is ground above it is made splenocyte suspension, packing splenocyte suspension to the centrifuge tube added with lymphocyte separation medium
In.400g, centrifuges 20min at 20 DEG C, centrifugation is finished, and takes out centrifuge tube medium size lymphocyte layer centrifuge tube, adds RPMI-1640 trainings
Nutrient solution is washed for several times, 250g, and 10min is centrifuged at 4 DEG C.After washing is finished, abandoning supernatant adds complete medium, and piping and druming is equal
It is even, expect that blue dyeing counting ensures more than 95% living cell rate with platform, cell concentration is adjusted to 3 × 106Individual/ml.
Every part of splenocyte suspension point holes is added in 48 well culture plates, per hole 0.5ml, a hole adds 25 ul ConA liquid
（Equivalent to 5ug/ml）, another hole is placed in 5%CO as control2,37℃CO2Every hole after 48h is cultivated in incubator and gently sucks supernatant
The ml of liquid 0.3, adds 0.3 mlRPMI-1640 nutrient solutions, while adding the ul/ holes of 8 reagents of CCK 50, continues to cultivate 2 h.Knot
After beam culture, piping and druming is uniform, is then dispensed into 96 well culture plates, makees 3 parallel laboratory tests per hole, and 450 nm ripples are surveyed with ELIASA
OD value under long.The multiplication capacity of lymphocyte is by the OD value that adds the OD value in ConA holes Yu be not added with ConA holes
Difference represent.
1.2 mouse humoral immune functions（Serum hemolysin）Influence
Administration 25 days, the sheep blood 3 times of fiber has been taken off with brine (2000r/min, 10min), in hematocrit SRBC plus
Enter physiological saline and be made into 2%（v/v）Cell suspension, every mouse peritoneal injection 0.2ml, at the 30th day, administration 1h after, weigh,
All mouse extract eyeball and take blood 0.8ml in centrifuge tube, stand 1h, centrifuge 2000r/min, 10min, collect serum.Cervical vertebra takes off
Mortar puts to death mouse.The serum for diluting 300 times with SA buffer solutions takes 1ml to put in test tube, and 10% is added again（v/v）
SRBC0.5ml, with SA buffer solutions 9 times of complement 1ml is diluted, control tube is set（Serum is replaced with SA）, 37 DEG C of waters bath with thermostatic control
20min, then ice bath, are then centrifuged for 2000r/min, 10min.Supernatant 1ml is taken in new test tube, plus 3.75ml Dou Shi reagents,
0.25ml10%（v/v）SRBC, is fully mixed, and is stood 10min, using control tube as blank, is determined each Guan Guangmi at 540nm respectively
Angle value, the amount of hemolysin is with half hemolytic value（HC50）Represent.
1.3 mouse humoral immune（Antibody-producting cell）Influence
Administration 25 days, the sheep blood 3 times of fiber has been taken off with brine, 2000r/min, 10min have been centrifuged every time, in hematocrit
Physiological saline is added in SRBC and is made into 2%（v/v）Cell suspension, every mouse peritoneal injection 0.2ml, at the 30th day, be administered 1h
Afterwards, weigh.Cervical dislocation is put to death after mouse, is taken out spleen, is placed in and is placed with Hank ' s liquid plates, by 200 mesh sieve net filtrations
Afterwards, then with Hank ' s liquid wash twice, be added in 5mL RPMI-1640 nutrient solutions and cell suspends, count cell, and adjustment
Cell concentration is 5 × 106Individual/mL.
After the top layer culture medium heating for dissolving for adding distilled water to be formed to 100mL 1g agaroses, 40-45 DEG C of water-bath is incubated
Mix, shake up in equal volume with PH7.2-7.4,2 times of Hank ' s liquid, often pipe 0.5mL dispenses different test tubes, continue to add to every pipe
With SA buffers into 10%SRBC (v/v）50ul and splenocyte suspension 20ul, it is quick to mix, it is poured into and scribbles one layer
On the thin slice of very thin agarose, parallel plate is done, when after agarose solidification, wafer level button being placed on horse, two are put into
After carbonoxide incubator culture 1-1.5h, the complement that 9 times are diluted with SA buffer solutions is added in the groove of glass frame, continues to train
1-1.5h is supported, hemolysis plaque number is then counted.
1.4 mouse monokaryons-macrophage phagocytic function and the measure of immune organ influence
Last dose 1h, by body weight from tail vein injection physiological saline 1:The india ink of 3 dilutions（10mL/kg）, treat prepared Chinese ink
Injection, immediately timing.2,10min after prepared Chinese ink is injected, respectively from orbital venous plexus 20 μ L of blood sampling, and 2mL 0.1% is added to immediately
Na2CO3In solution, with Na2CO3Solution makees blank control, and OD value is determined at 600nm wavelength with spectrophotometer（OD）,
Calculate phagocytic index a.
Dislocate and put to death after mouse blood sampling, solution takes liver, spleen and thymus gland, and filter paper is blotted after internal organs peripheral blood, it is accurate
Weigh, calculate internal organs/body weight ratio.
1.5 NK cytoactive detections
Last dose 1h, it is sterile to take spleen, it is sterile to take spleen to be placed in the culture dish for filling RPMI-1640 nutrient solutions, and place 200 mesh
Screen cloth, is ground and splenocyte suspension is made above it with syringe piston, and packing splenocyte suspension is to added with separation of lymphocytes
In the centrifuge tube of liquid.400g, centrifuges 20min at 20 DEG C, centrifugation is finished, and takes out centrifuge tube medium size lymphocyte layer centrifuge tube, adds
RPMI-1640 nutrient solutions are washed for several times, 250g, and 10min is centrifuged at 4 DEG C.After washing is finished, abandoning supernatant adds training completely
Base is supported, piping and druming is uniform, and blue dyeing counting viable count is expected with platform（Should be more than 95%）, finally with the complete culture solutions of RPMI 1640
It is 2 × 10 to adjust cell concentration7Individual/mL.
In proportion by effector cell and target cell（50:1）Each 100ul adds U-shaped 96 orifice plate；Target cell and nutrient solution are each
100ul adds target cell Spontaneous release hole；Target cell and each 100ul of 0.25%Triton add the maximum release aperture of target cell.Every group
It is all provided with 3 parallel holes, 37 DEG C, after 5%CO2 incubator cultures 4h, centrifuges 1500rpm, 5min.Per hole Aspirate supernatant 100ul in
Flat 96 orifice plate, adds LDH matrix liquid 100ul/ holes, reacts 3-10min, adds 1mol/ml HCL 30ul/ holes, enzyme mark
490nm determines OD value.
All data carry out statistical analysis using the softwares of SPSS 16.0, and test result is as shown in the table.
The influence that the embodiment sample of table 1 is converted with control group to mouse lymphocyte
Note：Statistical analysis is carried out using variance analysis method, compared with negative control group, " a ": p<0.05;With positive controls
Compare, " b ": p<0.01
The embodiment sample of table 2 is with control group to mouse antibodies level（Serum hemolysin）Influence
Note：Statistical analysis is carried out using variance analysis method, compared with negative control group, " a ": p<0.05;With positive controls
Compare, " b ": p<The embodiment sample of 0.01 table 3 and influence of the control group to mouse hemolysis plaque number
Note：Statistical analysis is carried out using variance analysis method, compared with negative control group, " a ": p<0.05;With positive controls
Compare, " b ": p<0.01 .
The embodiment sample of table 4 and influence of the control group to mouse phagocytic index
Note：Statistical analysis is carried out using variance analysis method, compared with negative control group, " a ": p<0.05;With positive controls
Compare, " b ": p<0.01 .
Influence of the embodiment sample of table 6 with control group to NK cells in mice activity
Note：Statistical analysis is carried out using variance analysis method, compared with negative control group, " a ": p<0.05;With positive controls
Compare, " b ": p<0.01 .
It is obvious to a person skilled in the art that the invention is not restricted to the details of above-mentioned one exemplary embodiment, Er Qie
In the case of without departing substantially from spirit or essential attributes of the invention, the present invention can be realized in other specific forms.Therefore, no matter
From the point of view of which point, embodiment all should be regarded as exemplary, and be nonrestrictive, the scope of the present invention is by appended power
Profit is required rather than described above is limited, it is intended that all in the implication and scope of the equivalency of claim by falling
Change is included in the present invention.
Moreover, it will be appreciated that although the present specification is described in terms of embodiments, not each embodiment is only wrapped
Containing an independent technical scheme, this narrating mode of specification is only that for clarity, those skilled in the art should
Using specification as an entirety, the technical solutions in the various embodiments may also be suitably combined, forms those skilled in the art
It may be appreciated other embodiment.The ins and outs not being described in detail in the present invention, can pass through any in this area
Prior art is realized.Particularly, all technical characterstics not being described in detail can be realized by any prior art in the present invention.
Claims (5)
1. a kind of highly enriched fermentation process of Yunnan olive polyphenol, it is characterised in that comprise the following steps：
（1）Pretreatment of raw material：The fresh fruit of Yunnan olive is raw material, is cleaned up with water, is put into the boiling water containing 0.1% citric acid
Middle blanching 4min, cooling crushes stoning, plus 1: 1 water mashing, pulp is vacuum dried, crushed 40 mesh sieves and obtains Yunnan olive fruit
Digested tankage；
（2）By step（1）Yunnan olive fruit digested tankage is made and presses solid-liquid ratio 1:4 are inoculated in liquid fermentation medium, in 22 DEG C of temperature
Under conditions of, then liquid fermentation and culture 3-6 days adds and splits kettle algae fermentation protein product and endophyte of plant, make to split kettle algae
The concentration of fermentation protein product is 0.15-0.85mg/mL, and the concentration of endophyte of plant is 1.9 × 106CFU/ml-4.6×
107CFU/ml then proceedes to culture 1-4 days, and separation obtains Yunnan olive fruit digested tankage zymotic fluid；
（3）From step（2）Yunnan olive polyphenol is extracted in obtained zymotic fluid；
The step（2）In liquid fermentation medium, every liter of component is as follows：
3g lactose, 2g molasses, 2g bean cake powders, 1.5g peptones, 0.1gMgSO4,0.03gCaCl2,0.1gKH2PO4,
0.1gK2HPO4；
It is described split kettle algae tunning preparation method be：
Over cleaning of learning from else's experience splits kettle algae raw material, sets enzymatic hydrolysis condition as solid-liquid ratio 1: 10（m/V）, cellulase
CellulaseACCF-4740 additions 2%, 55 DEG C of temperature, pH4.5, reaction time 1.3h, are digested, cellulose hydrolyzation
After end, boiling water bath goes out enzyme 15min, and 9000r/min centrifugations collect supernatant and obtain splitting kettle algae cellulose hydrolyzation liquid；Split kettle
Algae enzymolysis liquid goes out after enzyme activity, regulation pH value to 6.2-6.6, high pressure steam sterilization（121 DEG C, 20min）, then it is inoculated with volume fraction
The Pediococcus pentosaceus liquid of 1% MRS culture mediums activation stands, fermented to kettle algae enzymolysis liquid, 37 DEG C of constant incubators are split；Hair
After ferment terminates, 9000r/min centrifugations collect supernatant and obtain splitting kettle algae tunning；
The extracting method of the endophyte of plant is：Using tissue block method, take fresh Amaranthus retroflexus root, stem, leaf clean respectively to
There is no obvious impurity, after surface sterilization processing, it is seeded to PDA（Separate endogenetic fungus）With Gause I culture medium（In separation
Raw actinomyces）Upper culture 7-10d, then separates single bacterium colony；Picking inoculated by hypha block is fermented in equipped with 100mL from activated inclined plane
In the conical flask of culture medium, 28 DEG C, 200r/min shaking table shaken cultivations 7d are put；Fermentation culture medium remove thalline, filtrate with etc. body
Product ethyl acetate is extracted 3 times, merges organic phase, and 55 DEG C are concentrated under reduced pressure and are evaporated, as bacterium solution crude extract；Centrifuge and be filtrated to get
Thalline, with appropriate 95% ethanol condensing reflux 3 times, merges ethanol phase, is concentrated under reduced pressure and is evaporated, as thalline crude extract；Put 4 DEG C of ice
Case is saved backup.
2. the highly enriched fermentation process of Yunnan olive polyphenol as claimed in claim 1, it is characterised in that step（2）Described in
Separation is that 5-10min is centrifuged under the conditions of 15000r/min.
3. the highly enriched fermentation process of Yunnan olive polyphenol as claimed in claim 1, it is characterised in that the step（3）In carry
The method for taking Yunnan olive polyphenol, step is as follows：
Take step（2）Obtained zymotic fluid, adds 95% ethanol of 4 times of volumes, 4 DEG C of standing 5h, 12000rpm centrifugations after mixing
10min, takes supernatant, and Yunnan olive polyphenol is made after freeze-drying.
4. a kind of Yunnan olive polyphenol active product, it is characterised in that be made up of following parts by weight meter component：Yunnan olive polyphenol 40-
50, potassium chloride 1-3, D-sorbite 5-8, sodium citrate 2-4, white granulated sugar 10-30, blood clam enzymolysis product 11-15, atractylodes chinensis
9-13, sodium acid carbonate 2-6, kiwi flavor 0.3-0.5, calcium lactate 3-5, water 50-120.
5. a kind of Yunnan olive polyphenol active product as claimed in claim 4, it is characterised in that by following parts by weight meter component group
Into：Yunnan olive polyphenol 43, potassium chloride 2, D-sorbite 6, sodium citrate 3, white granulated sugar 14, blood clam enzymolysis product 13, rhizoma atractylodis are extracted
Thing 11, sodium acid carbonate 4, kiwi flavor 0.4, calcium lactate 4, water 78.
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