CN102433251B - Russian olive health-care wine and preparation method thereof - Google Patents
Russian olive health-care wine and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a Russian olive health-care wine and a preparation method thereof. The Russian olive health-care wine is prepared by ageing royal jelly and Chinese medicine crops such as medlar, cistanche and Codonopsis pilosula. The preparation method comprises the following steps: picking fresh Russian olive, washing, cooking, denucleating, filtering, sulphurating, regulating sugar degree, regulating acidity and adding calcium; inoculating the Russian olive treated by the former steps with activated grape wine yeast in an inoculation amount of 4-6% for the purpose of amplifying a culture solution; fermenting for 6-10 days at the temperature of 21-25 DEG C so as to prepare a Russian olive fermentation raw wine; after main fermentation is finished, pouring, filtering, and discarding precipitate; adding the treated medlar, and the Codonopsis pilosula powder and the royal jelly in filtrate for dissolution; ageing for more than 90 days at the temperature of 16-20 DEG C, and stirring once every other 10-20 days, then gluing, and standing and clearing for 5-10 days at the temperature of 16-20 DEG C; and then separating and clearing to obtain the Russian olive health-care wine. In the invention, health-care medicines are added instantly when the main fermentation of the Russian olive fermentation raw wine is finished and then fermented with the Russian olive fermentation raw wine, in such a way, the beneficial components in the health-care products are blended into the health-care wine to exert effects as far as possible, thus the health-care effect is improved.
Description
Technical field
The invention belongs to health promoting wine, specifically a kind of Russian olive health-care wine and preparation method thereof.
Background technology
Along with going deep into of China's reform paces, people's life is day by day plentiful, yet the society people are when enjoying modern civilization to the full, also endure the ailing torment such as coronary heart disease, hypertension, diabetes, fatty liver, atherosclerosis to the fullest extent, they will go to capture these persistent ailments with various means at urgent appealing, improve people's health level, the prevention subhealth state.
Arrow-leaved oleaster (
Elaeagnus angusti foliaL.) be again Gui Xiangliu, Yin Liu, in plant taxonomy, belong to Elaeangnaceae.Arrow-leaved oleaster is machaka or dungarunga, happiness light, cold-resistant, to dust storm, saline and alkaline strong resistance, growth is fast, purposes is wide, economic worth is high.Often be born in desert area, mainly be distributed in the provinces and regions such as China northeast, North China and northwest, and with the northwest desert, Semi-desert Area be distribution center.According to preliminary investigation, NORTHWEST CHINA 5 provinces and regions arrow-leaved oleaster areas are at 130,000 hm
2Above, only just there are 2.69 ten thousand hm in the He Xizoulangsha district
2Therefore arrow-leaved oleaster has rich in natural resources in China.
Arrow-leaved oleaster is called as " Bai Baoshu ", and its edibleness and pharmaceutical use are all very high, and being developed to green food has very large potentiality.Flower of Russianolive is good nectar source; Floral leaf is the green fodder of poultry; Fruit, flower, branch, leaf all can be used as medicine again.The Elaeagnus angustifolia L. Jujubes yellowish white, particulate such as sand, little band fragrance, puckery and sour-sweet, be rich in carbohydrate, protein, fat, mineral substance and tannin, starch, flavonoid class material and the compositions such as pectin, various trace elements and organic acid, especially the content of zinc is higher, and the intelligence growth of human body is benefited.According to another mensuration, in every 100g Elaeagnus angustifolia L. Jujubes, containing total amount is 17 seed amino acids of 3804.0mg, and wherein 8 seed amino acids of needed by human account for 23.16% of total amount.The arrow-leaved oleaster fruit can be eaten raw, while or a kind of starch plant, grinding face processed can steamed sweet bun, pancake, cook noodles, it is not only white and soft that meal of elaeagnus angustifolia L. and flour mix the steamed bun that steams, and with the distinctive fragrant and sweet flavor of arrow-leaved oleaster, having the face-nourishing health-care effect, is a kind of natural green food truly.Arrow-leaved oleaster can also be widely used in and manufacture cake, make vinegar, soy sauce processed, jam processed.
Now, main arrow-leaved oleaster food has on China market: 1, former jujube.2, arrow-leaved oleaster beverage.3, narrow-leaved oleaster flower tea.
Fruit wine is to utilize the fruit that contains a certain amount of sugar and moisture through after fragmentation, squeezing, get the processing such as juice, fermentation, the various low alcohol that brew forms.China's fruit aboundresources, the fruit that can make wine has a lot, can be used for brewed fruit wine such as apple, pears, Kiwifruit, hawthorn, red bayberry etc.
Existing health promoting wine product substantially be with high wine do basic wine then several health care medicines add and wherein soak certain hour, some health-care components discharge slowly like this, might not incorporate the immersion that namely is through with in the wine, have reduced health-care effect.
Summary of the invention
The purpose of this invention is to provide a kind of Russian olive health-care wine.
Another object of the present invention provide a kind of can be in the preparation method of the Russian olive health-care wine of easy and simple to handle, technological specification.
The preparation method of Russian olive health-care wine of the present invention with the arrow-leaved oleaster fermented wine as basic wine, add together ageing and get of medicine, raw materials used weight percent is: matrimony vine 15～25%, arrow-leaved oleaster fermented wine 50～73%, Herba Cistanches 5～10%, Radix Codonopsis 5～15% and royal jelly 2～5% compositions;
The brew step of described arrow-leaved oleaster fermented wine is: in the wine yeast scale-up medium after arrow-leaved oleaster juice activates with the access of 4～6% inoculum sizes, made the arrow-leaved oleaster fermented wine in 6～10 days in 21～25 ℃ of fermentations;
With arrow-leaved oleaster fermented wine tank switching, filter, discard and precipitate to such an extent that add matrimony vine, Herba Cistanches, Radix Codonopsis powder in the arrow-leaved oleaster fermented wine filtrate, adding royal jelly dissolves, be statically placed in the wine storing jar, in 16～20 ℃ of lower ageing more than 90 days, and stirred once every 10～20 days; The Russian olive health-care wine that ferments is statically placed in the wine storing jar, adds therein mass volume ratio and be 0.2% gelatin, in 16～20 ℃ of static clarifications 5～10 days, the then Russian olive health-care wine of separating clarifying.Can remove the bitter taste in the Elaeagnus angustifolia L. Jujubes, Russian olive health-care wine that fermented, clear under cold condition, is packed in the champagne bottle with isobaric bottling machine, pack with plastic plug or stopper and get product.
Further improvement as preparation method of the present invention, described arrow-leaved oleaster juice preparation process is that the fresh arrow-leaved oleaster of arrow-leaved oleaster juice is after choosing fruit, cleaning, boiling, removal of impurities, solid-liquid ratio by arrow-leaved oleaster and water is that 1:8～1:12 adds poach system, constantly stir during this time, mash, stoning, moisturizing is to former weight ratio, soluble solid content is 5～7% arrow-leaved oleaster juice, get soluble solid content and be 5～7% arrow-leaved oleaster juice, add sucrose and adjust soluble solid content, add citric acid and adjust acidity, and add sulphur, add calcium.
As preparation method's of the present invention further improvement, the described sulphur that adds is selected and is added Na
2SO
3, Na
2SO
3Add-on with SO
2Count 30～90 mg/L, soluble solid content transfers to 18%～22% when transferring sugar, and adding citric acid during acid adjustment, to transfer to pH value be 3.5～4.5, adding CaCl when adding calcium
2Amount be 0.3～0.5g/L.
Further improvement as preparation method of the present invention, the preparation process of described wine yeast scale-up medium is: the grape wine dry yeast is placed 28～30 ℃ of water to shake up to leave standstill activation half an hour, in the 250mL triangular flask, carry out the one-level enlarged culturing after the activation, substratum is the arrow-leaved oleaster juice of soluble solid content 5～7%, culture temperature is 26～28 ℃, and incubation time is 36～48h; One-level is transferred to the triangular flask nutrient solution in the 1000mL triangular flask of the arrow-leaved oleaster juice that soluble solid content 5～7% is housed after cultivating and finishing, and carries out the secondary enlarged culturing, and it is 24～26 ℃ that secondary is cultivated culture temperature; Then carry out three grades of enlarged culturing in seeding tank, three grades of culture temperature are 24～26 ℃, and substratum is the arrow-leaved oleaster juice of soluble solid content 7%, is prepared into the wine yeast scale-up medium; Described grape wine dry yeast is bought in Angel Yeast Co.,Ltd, Angel TH grape wine high reactivity brewer's dried yeasts grape wine high reactivity brewer's dried yeasts (S. cerevisiae var.).
Development along with society, people are also more and more to the needs of healthcare products, in use there is potential toxicity in the synthetic healthcare products of tradition, that have even carcinogenesis arranged, therefore people begin the health active composition in sight trend of purchasing vegetable source natural food, healthcare products and the Chinese medicine, scientific research shows, this type of material both can effectively prevent or reduce various diseases, and have no side effect or toxic side effect very little.
The present invention considers that many special populations can't drink high wine, therefore use the arrow-leaved oleaster fermented wine as basic wine, add together ageing of health care medicine, rather than soak, innovative point of the present invention is: the one, and the high wine that replaces generally using with the arrow-leaved oleaster fermented wine of minuent is as basic wine, greatly reduce the ethanol concn of health promoting wine, make it can have more suitable crowd edible; The 2nd, finish namely to add health care medicine in the Primary Fermentation of arrow-leaved oleaster fermented wine and participate in its secondary fermentation, beneficiating ingredient in the healthcare products is dissolved into is as much as possible brought into play effect in the health promoting wine, the composition that simultaneously fermentation can make some be difficult to absorb becomes easily, has therefore improved health-care effect.
Single medicinal ingredient is difficult to preferably booster action is all played in various diseases treatment, only has multiple medicinal material synergy just can reach the desirable active effect of nutritive health-care.
Hexi Region of Gansu Province arrow-leaved oleaster aboundresources, and rich in starch, all flavors are coordinated, and are suitable for brewing fermentation wine; It is raw material that the present invention selects Gansu wild point fruit arrow-leaved oleaster, and the brew arrow-leaved oleaster fermented wine that undergoes microbial fermentation adds the peculiar wild Chinese medicinal materials matrimony vine in Gansu, Herba Cistanches, Radix Codonopsis and royal jelly again during ageing.Can act synergistically on human body at multi-level, multidigit point, the inventive method level of automation is high, easy and simple to handle, technological specification.
The present invention has the principal constituent screening of eight factors of major effect and the response surface process optimization of three major influence factors as main evaluation index, to arrow-leaved oleaster fermented wine alcoholic strength with alcoholic strength, be result through optimization of orthogonal test to raw material screening, proportioning and preparation method.
The effect of each component of the present invention is as follows:
1. matrimony vine
The property flat flavor sweet, be the good merchantable brand of nourishing of the medicine-food two-purpose of regulation in " Ministry of Health is about the notice of further standard healthy food material management ", the traditional Chinese medical science has the saying of " medlar health preserving " very early.Compendium of Material Medica record: " matrimony vine, kidney-tonifying sperm-generating, nourishing the liver ... make eye bright and calm the nerves make us long-lived ".Modern medicine study shows, matrimony vine has obvious restraining effect to cancer cell in vitro, the immunologic function that can be used for preventing the diffusion of cancer cells and strengthen human body.In addition, matrimony vine also has lowering blood glucose, lipotropy, and can atherosclerosis.
2. Herba Cistanches
Sweet, sour, salty, warm, kidney-replenishing is arranged, benefiting essence-blood, the effect that relaxes bowel, can treat waist knee crymodynia, woman infertility, neurasthenia, dysacusis, also have reduce blood pressure, anti-ageing, anti-senile dementia, protect the liver, the functions such as defaecation, tumor aid treatment, and the hemostatic drug that can be used as urocystitis, cystorrhagia, kidney hemorrhagic disease is used.Having high pharmaceutical use, is the traditional rare traditional Chinese medicine of China, also is one of help medicine that usage frequency is the highest in the successive dynasties kidney invigorating and YANG supporting class prescription.
3. Radix Codonopsis
Be the article of regulation " can be used for protective foods " in " Ministry of Health is about the notice of further standard healthy food material management ", for China's traditional tonic commonly used, have invigorating the spleen and replenishing QI, the effect of invigorating the spleen benefit lung.Can be used for a little less than the spleen deficiency syndrome of the lung, the palpitaition of breathing hard, anorexia and loose stool, void is breathed with cough and is coughed, and interior heat is quenched one's thirst.
4. royal jelly
Function: improve nutrition, replenish mentality, improve body immunity, the prophylactic treatment cardiovascular and cerebrovascular diseases, the treatment anaemia, anti-inflammatory, pain relieving, promotion wound healing, preventing cancer, beauty treatment, anti-ageing.Be applicable to improve a poor appetite, Ginseng Extract is strengthened resistibility and strengthening immunity to disease, enhances metabolism, and accelerates rehabilitation of patient etc., therefore has the people that royal jelly is used for control various diseases, especially metabolic disease and geriatric disease.
The monarch relation is as follows: matrimony vine, Herba Cistanches are top grade medicine in " the ancient book on Chinese herbal medicine of the legendary god of farming "; The Compendium of Material Medica record: " matrimony vine, kidney-tonifying sperm-generating, nourishing the liver ... make eye bright and calm the nerves make us long-lived ", therefore matrimony vine is monarch drug in a prescription; The Herba Cistanches kidney-replenishing, benefiting essence-blood relaxes bowel, the Radix Codonopsis invigorating the spleen and replenishing QI, invigorating the spleen benefit lung, Herba Cistanches and Radix Codonopsis two medicines are ministerial drug altogether; Royal jelly improves nutrition, replenishes mentality, improves body immunity, the prophylactic treatment cardiovascular and cerebrovascular diseases, and the treatment anaemia, preventing cancer, anti-ageing, the assistant Radix Codonopsis is strengthened the invigorating the spleen and replenishing QI effect, and royal jelly is adjutant.
This health promoting wine is according to the Human Physiology characteristic, various healthy ingredients there is emphasis comprehensively, combines targetedly, therefore the various healthy ingredients in all beneficiating ingredients of Secondary Fermentation interpolation can be brought into play synergistic effect better, have invigorating the spleen and replenishing QI, invigorating the spleen benefit lung, strengthening immunity, vasodilation, step-down, improve microcirculation, strengthen hemopoietic function, thereby reach prevention or alleviate cardiovascular and cerebrovascular diseases, cancer, acquired immune deficiency syndrome (AIDS), reduce blood pressure, the purpose of the various diseases such as diabetes, anti-fatty liveranti-fatty liver and atherosclerosis, the prevention subhealth state.
The arrow-leaved oleaster fermented health-care wine of the present invention's preparation all meets GB2758-2005 fermented wine hygienic standard through the check indices, Russian olive health-care wine of the present invention has that arrow-leaved oleaster flavor is outstanding, golden yellow color, contain abundant healthy nutritive value, unique flavor, the characteristics such as tasty and refreshing, safety, with after have no side effect.
Description of drawings
Fig. 1 is the figure that affects of different feed liquid comparison arrow-leaved oleaster fermented wine alcoholic strength;
Fig. 2 is that different inoculum of dry yeast are on the figure that affects of arrow-leaved oleaster fermented wine alcoholic strength;
Fig. 3 is that different pH values are on the figure that affects of arrow-leaved oleaster fermented wine alcoholic strength;
Fig. 4 is that the different fermentations temperature is on the figure that affects of arrow-leaved oleaster fermented wine alcoholic strength;
Fig. 5 is that the different sugar addition is on the figure that affects of arrow-leaved oleaster fermented wine alcoholic strength;
Fig. 6 is different SO
2Addition is on the figure that affects of arrow-leaved oleaster fermented wine alcoholic strength;
Fig. 7 is Different Ca Cl
2Addition is on the figure that affects of arrow-leaved oleaster fermented wine alcoholic strength;
Fig. 8 is that the different fermentations time is on the figure that affects of arrow-leaved oleaster fermented wine alcoholic strength;
Fig. 9 is the response surface figure from solid-liquid ratio and sugared addition;
Figure 10 is the isogram from solid-liquid ratio and sugared addition;
Figure 11 is the response surface figure from solid-liquid ratio and fermentation time;
Figure 12 is the isogram from solid-liquid ratio and fermentation time;
Figure 13 is the response surface from sugared addition and fermentation time;
Figure 14 is the isogram from sugared addition and fermentation time.
Embodiment
Following embodiment will be easier, comprehend the present invention, but do not limit the present invention in any way.
The preparation of embodiment 1 health promoting wine 1
As basic wine, add together ageing and getting of medicine with the arrow-leaved oleaster fermented wine;
(1) preparation arrow-leaved oleaster juice
Fresh dried arrow-leaved oleaster is that 1:10 adds poach system by the material water weight ratio after choosing fruit, cleaning, removal of impurities, during constantly stir, mash, stoning, moisturizing is former weight ratio extremely, gets soluble solid content (SSC) and be 6% arrow-leaved oleaster juice;
(2) composition adjustment
Get soluble solid content (SSC) and be 6% arrow-leaved oleaster juice, add sucrose and adjust soluble solid content (SSC) to 20%, add citric acid and adjust acidity and make pH to 4.0, add Na
2SO
3(with 60mg/L SO
2Meter), add calcium (CaCl
20.3g/L);
(3) preparation wine yeast scale-up medium
The grape wine dry yeast is placed 29 ℃ of water to shake up to leave standstill activation half an hour, carries out the one-level enlarged culturing after the activation in the 250mL triangular flask, substratum is the arrow-leaved oleaster juice of soluble solid content (SSC) 7%, and culture temperature is 27 ℃, and incubation time is 42h; One-level is transferred to the triangular flask nutrient solution in the 1000mL triangular flask of the arrow-leaved oleaster juice that soluble solid content (SSC) 7% is housed after cultivating and finishing, and carries out the secondary enlarged culturing, and it is 25 ℃ that secondary is cultivated culture temperature; Then carry out three grades of enlarged culturing in seeding tank, three grades of culture temperature are 25 ℃, and substratum is the arrow-leaved oleaster juice of soluble solid content (SSC) 7%, is prepared into the wine yeast scale-up medium;
(4) preparation fermented wine
In the wine yeast scale-up medium of deployed arrow-leaved oleaster juice after with the access activation of 5% inoculum size, made the arrow-leaved oleaster fermented wine in 8 days in 23 ℃ of fermentations, the arrow-leaved oleaster fermented wine tank switching after Primary Fermentation is finished filters, and discards and precipitates to get arrow-leaved oleaster fermented wine filtrate;
(5) preparation health promoting wine
Be that 15% matrimony vine, mass percent are that 5% Herba Cistanches, mass percent are that 5% Radix Codonopsis places respectively thermostatic drying chamber in 60 ℃ of dry 3h with mass percent, pulverizer is pulverized, and crosses 80 mesh sieve powdereds; Putting into wine storing jar, to add mass percent be 73% the arrow-leaved oleaster fermented wine filtrate that has fermented, and adding mass percent is that 2% royal jelly dissolves, and in 18 ℃ of lower ageing more than 90 days, and stirred once every 15 days; The Russian olive health-care wine that ferments is statically placed in the wine storing jar, adds therein 0.2%(w/v) gelatin, in 18 ℃ of static clarifications 10 days, then the Russian olive health-care wine of separating clarifying can be removed the bitter taste in the Elaeagnus angustifolia L. Jujubes; Arrow-leaved oleaster fruit wine that fermented, clear under cold condition, is packed in the champagne bottle with isobaric bottling machine, pack with plastic plug or stopper and make health promoting wine 1.
In order to verify beneficial effect of the present invention, adopt the Russian olive health-care wine of the embodiment of the invention 1 preparation method preparation, test its golden yellow color according to standards such as GB/T 15038-1994 grape wine, fruit wine universal test method and GB 2758-2005 fermented wine hygienic standards, has obvious arrow-leaved oleaster fragrance, healthy nutritive value is abundant, unique flavor, and tasty and refreshing property is good, clear, glossy, without precipitation, microbiological indicator is qualified.
Antioxidation activity in vitro test: according to the described testing method of Blois (Blois M.S.,
Natural, 1958,26:1199.), under 70 μ g/mL concentration, the ability that health promoting wine 1 is eliminated the DPPH free radical is 88%; The described methods such as employing Yu (Yu W.,
Et al.,
Food Chem., 2004,86:525.), under 80 μ g/mL concentration, the ability that health promoting wine 1 is eliminated hydroxyl is 85%; According to the described method of Zhang (Zhang X.Y.,
Principle of Chemical Analysis.Beijing:China Science Press, 2000, pp:525.) under 1.0mg/mL concentration, the ability that health promoting wine 1 is eliminated hydrogen peroxide is 83%; The testing method that employing Liu etc. describes (Liu F.,
Et al.
Life Sci., 1997,60:763.), under 1.0mg/mL concentration, the ability that health promoting wine 1 is eliminated superoxide anion is 86%.
The preparation of embodiment 2 health promoting wine 2
As basic wine, add together ageing and getting of medicine with the arrow-leaved oleaster fermented wine;
(1) preparation arrow-leaved oleaster juice
Fresh dried arrow-leaved oleaster is that 1:8 adds poach system by the material water weight ratio after choosing fruit, cleaning, removal of impurities, during constantly stir, mash, stoning, moisturizing is former weight ratio extremely, gets soluble solid content (SSC) and be 5% arrow-leaved oleaster juice;
(2) composition adjustment
Get soluble solid content (SSC) and be 5% arrow-leaved oleaster juice, add sucrose and adjust soluble solid content (SSC) to 18%, add citric acid and adjust acidity and make pH to 3.5, add Na
2SO
3(with 30mg/L SO
2Meter), add calcium (CaCl
20.4g/L);
(3) preparation wine yeast scale-up medium
The grape wine dry yeast is placed 28 ℃ of water to shake up to leave standstill activation half an hour, carries out the one-level enlarged culturing after the activation in the 250mL triangular flask, substratum is the arrow-leaved oleaster juice of soluble solid content (SSC) 5%, and culture temperature is 26 ℃, and incubation time is 36h; One-level is transferred to the triangular flask nutrient solution in the 1000mL triangular flask of the arrow-leaved oleaster juice that soluble solid content (SSC) 5% is housed after cultivating and finishing, and carries out the secondary enlarged culturing, and it is 24 ℃ that secondary is cultivated culture temperature; Then carry out three grades of enlarged culturing in seeding tank, three grades of culture temperature are 24 ℃, and substratum is the arrow-leaved oleaster juice of soluble solid content (SSC) 7%, is prepared into the wine yeast scale-up medium;
(4) preparation fermented wine
In the wine yeast scale-up medium of deployed arrow-leaved oleaster juice after with the access activation of 4% inoculum size, made the arrow-leaved oleaster fermented wine in 6 days in 21 ℃ of fermentations, the arrow-leaved oleaster fermented wine tank switching after Primary Fermentation is finished filters, and discards and precipitates to get arrow-leaved oleaster fermented wine filtrate;
(5) preparation health promoting wine
Formed by following each Chinese medicine material fermentation by mass percentage: matrimony vine 20%, Herba Cistanches 10%, Radix Codonopsis 15% oven dry are ground into the end, put into wine storing jar and add 50% the arrow-leaved oleaster fermented wine that has fermented, adding 5% royal jelly dissolves, in 16 ℃ of lower ageing more than 100 days, and stirred once every 10 days; The Russian olive health-care wine that ferments is statically placed in the wine storing jar, adds therein 0.2%(w/v) gelatin, in 16 ℃ of static clarifications 8 days, then the Russian olive health-care wine of separating clarifying can be removed the bitter taste in the Elaeagnus angustifolia L. Jujubes; Arrow-leaved oleaster fruit wine that fermented, clear under cold condition, is packed in the champagne bottle with isobaric bottling machine, pack with plastic plug or stopper and make health promoting wine 2.
In order to verify beneficial effect of the present invention, adopt the Russian olive health-care wine of the embodiment of the invention 2 preparation methods preparation, test its golden yellow color according to standards such as GB/T 15038-1994 grape wine, fruit wine universal test method and GB 2758-2005 fermented wine hygienic standards, has obvious arrow-leaved oleaster fragrance, healthy nutritive value is abundant, unique flavor, and tasty and refreshing property is good, clear, glossy, without precipitation, microbiological indicator is qualified.
Antioxidation activity in vitro test: according to the described testing method of Blois (Blois M.S.,
Natural, 1958,26:1199.), under 70 μ g/mL concentration, the ability that health promoting wine 2 is eliminated the DPPH free radical is 85%; The described methods such as employing Yu (Yu W.,
Et al.,
Food Chem., 2004,86:525.), under 80 μ g/mL concentration, the ability that health promoting wine 2 is eliminated hydroxyl is 83%; According to the described method of Zhang (Zhang X.Y.,
Principle of Chemical Analysis.Beijing:China Science Press, 2000, pp:525.) under 1.0mg/mL concentration, the ability that health promoting wine 2 is eliminated hydrogen peroxide is 83%; The testing method that employing Liu etc. describes (Liu F.,
Et al.
Life Sci., 1997,60:763.), under 1.0mg/mL concentration, the ability that health promoting wine 2 is eliminated superoxide anion is 87%.
The preparation of embodiment 3 health promoting wine 3
As basic wine, add together ageing and getting of medicine with the arrow-leaved oleaster fermented wine;
(1) preparation arrow-leaved oleaster juice
Fresh dried arrow-leaved oleaster is that 1:12 adds poach system by the material water weight ratio after choosing fruit, cleaning, removal of impurities, during constantly stir, mash, stoning, moisturizing is former weight ratio extremely, gets soluble solid content (SSC) and be 7% arrow-leaved oleaster juice;
(2) composition adjustment
Get soluble solid content (SSC) and be 7% arrow-leaved oleaster juice, add sucrose and adjust soluble solid content (SSC) to 22%, add citric acid and adjust acidity and make pH to 4.5, add Na
2SO
3(with 90mg/L SO
2Meter), add calcium (CaCl
20.5g/L);
(3) preparation wine yeast scale-up medium
The grape wine dry yeast is placed 30 ℃ of water to shake up to leave standstill activation half an hour, carries out the one-level enlarged culturing after the activation in the 250mL triangular flask, substratum is that soluble solid content (SSC) is 7% arrow-leaved oleaster juice, and culture temperature is 28 ℃, and incubation time is 48h; After one-level cultivate to finish, the triangular flask nutrient solution is transferred to is equipped with in the 1000mL triangular flask that soluble solid content (SSC) is 7% arrow-leaved oleaster juice, carry out the secondary enlarged culturing, it is 26 ℃ that secondary is cultivated culture temperature; Then carry out three grades of enlarged culturing in seeding tank, three grades of culture temperature are 26 ℃, and substratum is that soluble solid content (SSC) is 7% arrow-leaved oleaster juice, is prepared into the wine yeast scale-up medium;
(4) preparation fermented wine
In the wine yeast scale-up medium of deployed arrow-leaved oleaster juice after with the access activation of 4～6% inoculum sizes, made the arrow-leaved oleaster fermented wine in 10 days in 225 ℃ of fermentations, arrow-leaved oleaster fermented wine tank switching with after the Primary Fermentation end filters, and discards and precipitates to get arrow-leaved oleaster fermented wine filtrate;
(5) preparation health promoting wine
Formed by following each Chinese medicine material fermentation by mass percentage: matrimony vine 25%, Herba Cistanches 8%, Radix Codonopsis 10% oven dry are ground into the end, put into wine storing jar and add 53% the arrow-leaved oleaster fermented wine that has fermented, adding 4% royal jelly dissolves, in 20 ℃ of lower ageing more than 120 days, and stirred once every 20 days; The Russian olive health-care wine that ferments is statically placed in the wine storing jar, adds therein 0.2%(w/v) gelatin, in 16～20 ℃ of static clarifications 5 days, then the Russian olive health-care wine of separating clarifying can be removed the bitter taste in the Elaeagnus angustifolia L. Jujubes; Arrow-leaved oleaster fruit wine that fermented, clear under cold condition, is packed in the champagne bottle with isobaric bottling machine, pack with plastic plug or stopper and make health promoting wine 3.
In order to verify beneficial effect of the present invention, adopt the Russian olive health-care wine of the embodiment of the invention 3 preparation methods preparation, test according to standards such as GB/T 15038-1994 grape wine, fruit wine universal test method and GB 2758-2005 fermented wine hygienic standards, the pool of discoloring is golden yellow, has obvious arrow-leaved oleaster fragrance, healthy nutritive value is abundant, unique flavor, and tasty and refreshing property is good, clear, glossy, without precipitation, microbiological indicator is qualified.
The drug effect of embodiment 1 is best in the embodiment of the invention.
Antioxidation activity in vitro test: according to the described testing method of Blois (Blois M.S.,
Natural, 1958,26:1199.), under 70 μ g/mL concentration, the ability that health promoting wine 3 is eliminated the DPPH free radical is 83%; The described methods such as employing Yu (Yu W.,
Et al.,
Food Chem., 2004,86:525.), under 80 μ g/mL concentration, the ability that health promoting wine 3 is eliminated hydroxyl is 80%; According to the described method of Zhang (Zhang X.Y.,
Principle of Chemical Analysis.Beijing:China Science Press, 2000, pp:525.) under 1.0mg/mL concentration, the ability that health promoting wine 3 is eliminated hydrogen peroxide is 84%; The testing method that employing Liu etc. describes (Liu F.,
Et al.
Life Sci., 1997,60:763.), under 1.0mg/mL concentration, the ability that health promoting wine 3 is eliminated superoxide anion is 85%.
The usage of health promoting wine of the present invention and consumption: clothes once every night, each 50～100mL, long-term drinking, can play prevention or alleviate cardiovascular and cerebrovascular diseases, cancer, acquired immune deficiency syndrome (AIDS), reduce blood pressure, the effect of the various diseases such as diabetes, anti-fatty liveranti-fatty liver and atherosclerosis, with after have no side effect.
Analytical test
In order to determine best proportioning of the present invention and best processing step, the contriver has carried out a large amount of research trials in the laboratory, and various test situation are as follows:
The brew of I arrow-leaved oleaster fermented wine
Testing index: the wine degree is measured, and adopts the alcoholometer method; Soluble solid content (SSC) is measured, and adopts the hand refractometer method; Acid base titration is adopted in total acidity test; Reducing sugar test adopts Fehlings reagent; Light absorption value is measured, and adopts spectrophotometer method; Colour is measured, adopt spectrophotometer respectively at 420,520,620nm place survey light absorption value, three's sum is colour.
Fermentation strain: buy in Angel Yeast Co.,Ltd, Angel TH grape wine high reactivity brewer's dried yeasts grape wine high reactivity brewer's dried yeasts (
S. cerevisiae var.)
1, experiment of single factor
1.1 different feed liquid is than (ratio of arrow-leaved oleaster and the water) impact on arrow-leaved oleaster fermented wine alcoholic strength
Test is implemented according to design, and in Primary Fermentation end mensuration arrow-leaved oleaster fermented wine alcoholic strength, the results are shown in Figure 1, by Fig. 1 as can be known, when solid-liquid ratio is 1:4, arrow-leaved oleaster fermented wine alcoholic strength is the highest, next is solid-liquid ratio when being 1:6 and 1:10, and arrow-leaved oleaster fermented wine alcoholic strength is higher, but no difference of science of statistics between three's alcoholic strength, consider the raw materials cost problem, selecting solid-liquid ratio is that 1:10 is more reasonable; Alcoholic strength is a simple measurement index; each level of selected each factor is that a plurality of indexs are united definite in fact; although shown from alcoholic strength it is that the ratio 1:4 of arrow-leaved oleaster and water is the highest in the literary composition; but it and 1:10 and indifference in biometrics; consider to have selected 1:10 from raw materials cost so, it is 1:8-12 that therefore last the present invention selects to protect the ratio of arrow-leaved oleaster and water.
1.2 different inoculum of dry yeast are on the impact of arrow-leaved oleaster fermented wine alcoholic strength
Test is implemented according to design, and finishes to measure arrow-leaved oleaster fermented wine alcoholic strength in Primary Fermentation, the results are shown in Figure 2, and as shown in Figure 2, when inoculum of dry yeast was 5%, arrow-leaved oleaster fermented wine alcoholic strength was the highest, was 5% therefore select the yeast optimum addition.
1.3 different pH values are on the impact of arrow-leaved oleaster fermented wine alcoholic strength
Test is implemented according to design, and in Primary Fermentation end mensuration arrow-leaved oleaster fermented wine alcoholic strength, the results are shown in Figure 3, as shown in Figure 3, the pH value is different, and impact also is not quite similar on arrow-leaved oleaster fermented wine alcoholic strength, arrow-leaved oleaster fermented wine alcoholic strength is the highest when pH=4.0, therefore the pH value is advisable with 4.0, and pH 3.5-4.5 is better.
1.4 the different fermentations temperature is on the impact of arrow-leaved oleaster fermented wine alcoholic strength
Test is implemented according to design, and in Primary Fermentation end mensuration arrow-leaved oleaster fermented wine alcoholic strength, the results are shown in Figure 4, as shown in Figure 4, leavening temperature has certain influence to arrow-leaved oleaster fermented wine alcoholic strength, when temperature is 23 ℃, arrow-leaved oleaster fermented wine alcoholic strength is the highest, so arrow-leaved oleaster fermented wine leavening temperature is final selects 23 ℃.And temperature when being 21-25 ℃ arrow-leaved oleaster fermented wine ferment effect better.
1.5 the different sugar addition is on the impact of arrow-leaved oleaster fermented wine alcoholic strength
Test is implemented according to design, and in Primary Fermentation end mensuration arrow-leaved oleaster fermented wine alcoholic strength, the results are shown in Figure 5, as shown in Figure 5, along with sugared addition increases, arrow-leaved oleaster fermented wine alcoholic strength also increases thereupon, and be that SSC is adjusted to 20% and obtains maximum in sugared addition, afterwards, add again in the arrow-leaved oleaster fermented wine sugar then alcoholic strength no longer increase and slightly descend, therefore, sugared addition is that SSC is adjusted to 20% and is advisable in the arrow-leaved oleaster fermented wine.
1.6 different sulphur additions are on the impact of arrow-leaved oleaster fermented wine alcoholic strength
Test is implemented according to design, and finishes to measure arrow-leaved oleaster fermented wine alcoholic strength in Primary Fermentation, the results are shown in Figure 6, as shown in Figure 6, the sulphur addition is different, and arrow-leaved oleaster fermented wine alcoholic strength is also slightly different, when its addition was 60g/L, arrow-leaved oleaster fermented wine alcoholic strength reached the highest, so Na
2SO
3Add-on with SO
2Meter is got 60g/L.
1.7 Different Ca Cl
2Addition is on the impact of arrow-leaved oleaster fermented wine alcoholic strength
Test is implemented according to design, and finishes to measure arrow-leaved oleaster fermented wine alcoholic strength in Primary Fermentation, the results are shown in Figure 7, as shown in Figure 7, and CaCl
2Be the parabolic shape that Open Side Down between addition and the arrow-leaved oleaster fermented wine alcoholic strength, its vertex appears at CaCl
2Addition is the 0.3g/L place.CaCl hereat
2Addition selects 0.3g/L to be advisable.
1.8 the different fermentations time is on the impact of arrow-leaved oleaster fermented wine alcoholic strength
Test is implemented according to design, and in Primary Fermentation end mensuration arrow-leaved oleaster fermented wine alcoholic strength, the results are shown in Figure 8, as shown in Figure 8, the fermentation beginning, along with fermentation time increases, arrow-leaved oleaster fermented wine alcoholic strength increased sharply, to fermentation the 8th day, arrow-leaved oleaster fermented wine alcoholic strength reaches the highest, fermentation time prolongs then that arrow-leaved oleaster fermented wine alcoholic strength no longer increases again afterwards, and therefore, fermentation time is selected 8 days.
2, principle component analysis
Test is implemented according to design, and finishes to measure arrow-leaved oleaster fermented wine alcoholic strength in Primary Fermentation, the results are shown in Table 1.Statistic analysis result sees Table 2 and is the regression model analysis of variance table, and table 3 is that factor of influence is to the anticipated impact table of alcoholic strength.
Table 1 principal constituent design and result
His-and-hers watches 1 result carries out statistical treatment, obtains alcoholic strength and tentatively expects model (Predictive Model for Y
1):
Y
1=9.95+0.391667
x 1-0.15
x 2+ 0.191667
x 3-0.3
x 4+ 0.408333
x 5+ 0.1
x 6-0.125
x 7-1.066667
x 8(equation 1)
The variance analysis of table 2 regression model
| Source of variation | Degree of freedom | Sum of squares | All square | Ratio F | Conspicuous level p | Significance |
| x 1 | 1 | 1.840833 | 1.840833 | 3.189124 | 0.1721 | |
| x 2 | 1 | 0.27 | 0.27 | 0.467757 | 0.5431 | |
| x 3 | 1 | 0.440833 | 0.440833 | 0.763715 | 0.4465 | |
| x 4 | 1 | 1.08 | 1.08 | 1.87103 | 0.2648 | |
| x 5 | 1 | 2.000833 | 2.000833 | 3.466314 | 0.1595 | |
| x 6 | 1 | 0.12 | 0.12 | 0.207892 | 0.6794 | |
| x 7 | 1 | 0.1875 | 0.1875 | 0.324832 | 0.6086 | |
| x 8 | 1 | 13.65333 | 13.65333 | 23.65351 | 0.0166 | * |
| |
8 | 19.59333 | 4.243022 | F 2= 0.1309 | ||
| |
3 | 1.731667 | 0.577222 | |||
| Summation | 11 | 21.325 |
Annotate: * *
p＜0. 01 level is extremely remarkable, *
p＜0.10 level is remarkable.
As shown in Table 2, independent variable(s)
x 8 Fermentation time the arrow-leaved oleaster fermented wine brewageed had remarkably influenced (
p＜0.10), other Effects of Factors all not significantly (
p0.10).
Table 3 factor of influence is to the anticipated impact of alcoholic strength
By as seen from Table 3, each factor is followed successively by the order of arrow-leaved oleaster fermented wine alcoholic strength impact:
x 8 x 5 x 1 x 4 x 3 x 2 x 7 x 6, i.e. fermentation time〉and sugared addition〉solid-liquid ratio〉leavening temperature〉the pH value〉inoculum of dry yeast〉CaCl
2Addition〉the sulphur addition, optimize three by Dimension Reduction Analysis accordingly the most significant factor of arrow-leaved oleaster fermented wine alcoholic strength impact is respectively: fermentation time, sugared addition and solid-liquid ratio.
2, the response surface method is optimized arrow-leaved oleaster fermented wine alcoholic strength influence factor
Table 4 three factor Box-Behnken experimental designs
His-and-hers watches 4 results carry out statistical treatment, obtain alcoholic strength and tentatively expect model:
Y
1 =7.2+0.3
x 1+0.43125
x 2-1.15625
x 3-0.15625
x 1 2+0.275
x 1 x 2+0.275
x 1 x 3
+ 0.38125
x 2 2+ 0.1375
x 2 x 3+ 0.50625
x 3 2(equation 2)
The variance analysis of table 5 Quadratic response surface regression model
Annotate: * *
p＜0. 01 level is extremely remarkable, *
p＜0.10 level is remarkable.
Simulation equation is carried out variance analysis, the results are shown in Table 5.
Can find out solid-liquid ratio from the variance analysis of table 5 regression model
x 1, sugared addition
x 2And fermentation time
x 3Once and the quadratic term of sugared addition and fermentation time on arrow-leaved oleaster fermented wine fermentative action all have remarkably influenced (
p＜0.10), wherein fermentation time once on arrow-leaved oleaster fermented wine alcoholic strength have utmost point remarkably influenced (
p＜0. 01).The impact of its dependent variable all not significantly (
p0.10), no difference of science of statistics.Regression coefficient is tested, show except fermentation time once and the quadratic term of solid-liquid ratio arrow-leaved oleaster fermented wine alcoholic strength is generated impact for the negative effect, its complementary divisor once, quadratic term and mutual item brewage function influence to the arrow-leaved oleaster fermented wine and all be positive-effect.Box-Behnken Design variance analysis has verified that further the three factor pair arrow-leaved oleaster fermented wines fermentation that the Plackett-Burman principle component analysis optimizes has the conclusion of remarkably influenced.
α=0.10 conspicuous level is rejected not significantly item, the regression equation after the simplification:
Y
1=7.2+0.3
x 1+ 0.43125
x 2-1.15625
x 3+ 0.38125
x 2 2+ 0.50625
x 3 2(equation 3)
Equation after the correction is coefficient R as calculated
2Be 90.52%, illustrate that this regression equation can explain that these 3 factors are on the impact of arrow-leaved oleaster fermented wine alcoholic strength (Y value) in 90.52% degree of confidence, testing data meets basically with the secondary mathematical model that adopts, model and practical situation fitting degree are better, can be used for the prediction of the three factor pair arrow-leaved oleaster fermented wines fermentation impact that the Plackett-Burman principle component analysis optimizes, have the practical application meaning.
Fix two factors in zero level, study the interaction between other two factors, with SAS9.2 software respond face figure and isogram (such as Fig. 9-shown in Figure 14).
Can find out from Fig. 9 and Figure 10, the two interaction is very remarkable, in the poor situation of sugared addition, raises rapidly along with the solid-liquid ratio level reduces alcoholic strength, and the two is negative correlation; In the poor situation of solid-liquid ratio, along with sugared addition level rising alcoholic strength reduces rapidly, the two also is negative correlation; Wherein when solid-liquid ratio is in minimum level-1.0000, and sugared addition is in highest level+1.0000 o'clock, and arrow-leaved oleaster fermented wine alcoholic strength is obtained maximum value; Can find out from isogram, process is more responsive to the variation of the variation comparison solid-liquid ratio of sugared addition; Can find out from Figure 11 and Figure 12, the two interaction is very remarkable, in the poor situation of fermentation time, slowly reduces along with the solid-liquid ratio level reduces alcoholic strength, and the two is proportionate; And in the poor situation of solid-liquid ratio, along with fermentation time level rising alcoholic strength reduces rapidly, the two also is negative correlation; Wherein very fermentation time is in minimum level-1.0000 place to optimum point close to solid-liquid ratio is in highest level+1.0000, and obtains maximum value near these 2; Can find out from isogram, process is more responsive to the variation of the variation comparison solid-liquid ratio of fermentation time; Can obviously find out from Figure 13 and Figure 14, its interaction is very remarkable, in the poor situation of fermentation time, raises slightly again after alcoholic strength slowly reduces at the beginning along with sugared addition level reduces, and the two non-single linear is relevant; And in the poor situation of sugared addition, along with fermentation time level rising alcoholic strength reduces rapidly, the two is negative correlation; Wherein very fermentation time is in minimum level-1.0000 place to optimum point close to sugared addition is in highest level+1.0000, and obtains maximum value near these 2; Can find out from isogram, the variation that process is compared sugared addition to the variation of fermentation time is responsive a little.
Above Box-Behnken Design response surface is drawn and further to have been verified " Plackett-Burman design Dimension Reduction Analysis obtains preferably that impact sequentially is followed successively by fermentation time on arrow-leaved oleaster fermented wine alcoholic strength〉sugared addition〉solid-liquid ratio.Different technical parameters is not quite similar on arrow-leaved oleaster fermented wine fermentative action impact, wherein solid-liquid ratio, sugared addition and fermentation time once and the quadratic term of sugared addition and fermentation time on arrow-leaved oleaster fermented wine fermentative action all have remarkably influenced (
p＜0.10), and fermentation time once on arrow-leaved oleaster fermented wine alcoholic strength have utmost point remarkably influenced (
p＜0. 01).The impact of its dependent variable all not significantly (
p0.10), no difference of science of statistics." conclusion.
Figure it can also be seen that from response surface, the saddle face appears in the quadratic response face of solid-liquid ratio, sugared addition and fermentation time, without the existence of extreme value, therefore can not directly find out best influence factor parameter from quadratic response face, need further make Ridge analysis (ridge analysis).
3, three factors see Table 6 to the Ridge analysis of arrow-leaved oleaster fermented wine alcoholic strength impact
Table 6 Ridge analysis
| The coding radius | Response value | Standard error | x 1 | x 2 | x 3 |
| 0.0 | 7.20000 | 0.18808 | 0.00000 | 0.00000 | 0.00000 |
| 0.1 | 7.33082 | 0.18750 | 0.02060 | 0.03338 | -0.09199 |
| 0.2 | 7.46954 | 0.18583 | 0.03581 | 0.06517 | -0.18566 |
| 0.3 | 7.61648 | 0.18329 | 0.04651 | 0.09482 | -0.28080 |
| 0.4 | 7.77192 | 0.18029 | 0.05336 | 0.12193 | -0.37721 |
| 0.5 | 7.93611 | 0.17747 | 0.05689 | 0.14620 | -0.47475 |
| 0.6 | 8.10926 | 0.17568 | 0.05754 | 0.16745 | -0.57328 |
| 0.7 | 8.29157 | 0.17604 | 0.05566 | 0.18555 | -0.67266 |
| 0.8 | 8.48322 | 0.17977 | 0.05153 | 0.20042 | -0.77277 |
| 0.9 | 8.68438 | 0.18807 | 0.04541 | 0.21207 | -0.87348 |
| 1.0 | 8.89521 | 0.20185 | 0.03750 | 0.22052 | -0.97466 |
The result can find out from table 6 Ridge analysis, and response value Y is maximum when the coding radius is 1.0, is about 8.89521, and solid-liquid ratio this moment (w/v) is 1:8.2, and sugared addition (%) is 22.2%, and fermentation time is 6d.
Anti-oxidant test
1 experimentation on animals
1.1 laboratory animal
Select 8-12 monthly age aged mouse.Single sex, 15 every group of mouse.
1.2 dosage grouping and given the test agent give the time
Three dosage groups and a model control group are established in experiment, and take 5 times of human body recommended amounts as one of them dosage group, other establishes two dosage groups, establishes in case of necessity positive controls, blank group.Given the test agent gives 30 days time, can extend to 60 days in case of necessity.
1.3 experimental technique
1.3.1 aged animal
Select 8-12 monthly age mouse.Press MDA level grouping in the blood, be divided at random I aged control group and 3 given the test agent dosage groups.3 dosage groups give the different concns given the test agent, and control group gives with the volume solvent.Put to death animal when experiment finishes and survey LPO, antioxidase activity.
1.3.2 peroxide injury model
1.3.2.1 D one semi-lactosi model
1.3.2.1.1 principle
It is excessive that the D-semi-lactosi is supplied with, extraordinary generation active oxygen.Break the active oxygen that is controlled by hereditary pattern and produced and the equilibrium state of eliminating, caused the peroxidation effect.
1.3.2.1.2 modeling method
Select 25-30g healthy adult mouse, except the blank group, all the other animals D-semi-lactosi 40mg-1.2g/kg BW nape section's subcutaneous injection or abdominal injection modeling, injection volume is 0.1 mL/10g, every day 1 time, continuously 6 weeks of modeling, get blood and survey MDA, press the grouping of MDA level.Be divided at random 1 model control group and 3 given the test agent dosage groups, 3 dosage group per os give the different concns given the test agent, model control group gives with the volume solvent, when giving given the test agent, model control group and each dosage group continue to give same dose D-semi-lactosi nape section subcutaneous or abdominal injection, and experiment finishes to put to death animal and surveys LPO and antioxidase activity.
1.3.2.2 irradiation model
1.3.2.2.1 principle
Ionizing rays is by unsaturated fatty acids in the direct disrupting biofilm and indirectly produce free radical by the water radiolysis, causes lipid peroxidation.
1.3.2.2.2 modeling method
Select 18-22g healthy adult mouse, divide at random 5 groups, 1 blank group, 1 model control group and 3 given the test agent dosage groups, 3 dosage groups give the different concns given the test agent.Control group gives with the volume solvent, after 30 days, gets blood and surveys antioxidase activity, and after this, except the blank group, each group gives 5-8Gy
60The irradiation of 1 property of Co gamma-rays whole body was put to death each treated animal on the 3rd, 4 day after the irradiation, got hepatic tissue (or put to death animal on the 9th, 10 day, get testis tissue) and surveyed LPO and antioxidase activity.
1.3.2.3 bromobenzene model
1.3.2.3.1 principle
The stupid Mouse Liver that causes of bromo is poisoned, and causes liver lipid peroxidation..
1.3.2.3.2 modeling method
Select 18-22g healthy adult mouse, random minute 5 groups, 1 blank group, 1 model control group and 3 given the test agent dosage groups, 3 dosage groups give the different concns given the test agent, control group gives with the volume solvent, after 30 days, get blood and survey antioxidase activity, after this animal hunger is spent the night, give after given the test agent 0.5-1 hour, except the blank group, each organizes gavage 0.16-0.47mg/kgBW bromobenzene oil, gavage amount 0.2mL/20g, approximately put to death animal after 18-22 hour, get hepatic tissue and survey LPO and antioxidase activity.
1.3.3 lipid peroxide (LPO) assay
Lipid peroxidation can form mda, ethane, conjugated diolefine, fluorescence-causing substance and can produce chemiluminescent material.If the content of these products in body fluid and tissue increases, show that then the body lipid peroxidation strengthens.
13.3.1 lipid peroxide degraded product mda (MDA) assay in the blood.
Lipid peroxide degraded product mda (MDA) content adopts colorimetric method for determining in the blood.
1.3.3.1.1 colorimetry principle
MDA (malondiadehycle) is one of snperoxiaized end product of Cell membrane lipids, but surveys the degree of its content indirect Estimation lipid peroxidation.1 mda (MDA) molecule and 2 thiobarbituricacidα-s (TBA) molecule heat altogether under acidic conditions forms pink.This material has very big absorption peak at wavelength 532nm.Available light splitting light method is measured.
1.3.3.1.2 instrument and reagent
Instrument 721 spectrophotometers, micro sample adding appliance, thermostat water bath, generic centrifuge, DL device, tool plug centrifuge tube.
Reagent 0.2M acetate buffer pH3.5
0.2M acetic acid solution 185mL
0.2M sodium acetate solution 15ML
1 mmol/L tetraethoxypropane (stock solution was preserved 3 months for 4 ℃) is diluted with water to 40nmol/mL before use
8.1% sodium lauryl sulphate SDS
0.8% thiobarbituricacidα-TBA
0.2M phosphate buffered saline buffer pH7.4
0.2M Sodium phosphate dibasic 1920mL
0.2M potassium primary phosphate 480mL
1.3.3.1.2.3 experimental procedure
1.3.3.1.2.3.1 sample preparation
Hemolysate sample: get blood 20 μ L adding 0.98mL distilled water and make 2% hemolysate.
1.3.3.1.2.3.2 sample determination
Annotate: as use test kit, can be undertaken by the operational requirement of test kit.
If * use serum, sample hose 0.1mL, standard pipe 0.1mL.
1.3.3.1.2.3.3 calculate
LPO (nmol/mL2% hemolysate)=
LPO (nmol/mL serum)=
A: blank tube absorbancy
B: sample absorbancy
F: tetraethoxypropane absorbancy
C: tetraethoxypropane concentration (40nmol/mL)
K: extension rate
1.3.3.1.2.4 data processing and result judge
General employing variance analysis, but need carry out first homogeneity test of variance by the program of variance analysis, variance is neat, calculates the F value, F value＜F
0.05Conclusion: each organizes no significant difference between mean: F value 〉=F
0.05P≤0.05, in twos comparative approach with mean between a plurality of experimental group and control group is added up: the data of abnormal or heterogeneity of variance are carried out suitable variable conversion, after satisfying the neat requirement of normal state or variance, add up with the data after the conversion: if do not reach yet normal state or the neat purpose of variance after the variable conversion, use rank test instead and add up.
Given the test agent group and model (or aged) control group compares, and LPO reduces statistical significance, judges that this given the test agent has the reduction lipid peroxidation, and experimental result is positive.
1.3.3.2 lipid peroxide degraded product mda (MDA) assay in the tissue
1.3.3.2.1 principle
See 1.3.3.1.2.1
1.3.3.2.2 instrument and reagent
Instrument 721 spectrophotometers, micro sample adding appliance, thermostat water bath, generic centrifuge, DL device, tool plug centrifuge tube, tissue homogenizer
Reagent is seen 1.3.3.1.2.2
1.3.3.2.3 experimental procedure
1.3.3.2.3.1 sample preparation
Tissue homogenate sample: get a certain amount of required internal organs, normal saline flushing, wipe away dried, weigh, shred, put in the homogenizer, add the 0.2M phosphate buffered saline buffer, with 20000r/min homogenate 10s, 30s intermittently, repeatedly carry out 3 times, make 10% tissue homogenate (W/V), the centrifugal 5～10min of 3000r/min gets supernatant liquor to be measured.
1.3.3.2.3.2 sample determination
Annotate: as use test kit, can be undertaken by the operational requirement of test kit
1.3.3.2.3.3 calculate
LPO (nmol/mg tissue)=
A: blank tube absorbancy
B: the glimmering absorbancy of sample
F: tetraethoxypropane absorbancy
C: tetraethoxypropane concentration (40nmol/mL)
K: extension rate
1.3.3.2.3.4 data processing and result judge
General employing variance analysis, but need carry out first homogeneity test of variance by the program of variance analysis, variance is neat, calculates the F value, F value＜F
0.05, conclusion: each organizes no significant difference between mean: F value 〉=F
0.05, P≤0.05.In twos comparative approach with mean between a plurality of experimental group and control group is added up: the data of abnormal or heterogeneity of variance are carried out suitable variable conversion, after satisfying that normal state or variance are neat and requiring, add up with the data after changing; If do not reach yet normal state or the neat purpose of variance after the variable conversion, use rank test instead and add up.
Given the test agent group and model (or aged) control group compares, and LPO reduces statistical significance, judges that this given the test agent has the reduction lipid peroxidation, and experimental result is positive.
1.3.3.3 lipofuscin (Lipofasci) assay in the tissue
1.3.3.3.1 principle
Lipofuscin is the crosslinked and compound with fluorescence that generates of the material (such as phosphoric acid acyl thanomin, protein and nucleic acid) of mda and free amine group, be shill alkali, can make extraction agent with the mixed solution of chloroform and methyl alcohol, it is extracted and measure from tissue, measure the content of shill alkali, know cell by the degree of radical damage, indirectly antimer inner lipid levels of peroxide.
1.3.3.3.2 instrument and reagent
Instrument: spectrophotofluorometer, tissue homogenizer, whizzer, ultraviolet lamp, thermostat water bath
Reagent Chloroform (AR)
Methyl alcohol (AR)
Chloroform one methyl alcohol mixed liquor (2:1 v/v) (fresh preparation)
0.1mol/L sulfuric acid
Quinine Sulphate Di HC reference liquid (0.1 μ g/mL 0.1mol/L sulfuric acid)
Above used glassware all needs behind 50% nitric acid dousing 24h, again through distilled water, distilled water drip washing drying.
1.3.3.3.3 experimental procedure
Get and organize 200mg, add 2:1(v/v) chloroform methanol mixed solution 4mL (w/v=1:20).With homogenizer 2500r/min homogenize 1min in 45 ℃ of water-baths, make 5% homogenate take the chloroform methanol mixed solution as medium.Add subsequently 4mL distilled water, fully mix 1min with 2000r/min (homogenizer), remove the flavine chaff interference, sample is divided into 3 layers behind the centrifugal 10min of 3000r/min, and the upper strata is water, and the middle level is tissue, and lower floor is the chloroform methanol phase.Carefully suck water layer, pass the middle level along tube wall, lower floor chloroform methanol liquid is taken out, water can not be sneaked in the extracting solution, if water is sneaked in the extracting solution centrifugal removal water again.Add methyl alcohol 0.2mL in the chloroform methanol extracting solution, the mixing that vibrates gently makes it as clear as crystal, puts under the ultraviolet lamp and shines 30s, pours in the quartz curette, measures fluorescence intensity.
Take Quinine Sulphate Di HC (0.1 μ g/mL 0.1mol/L sulfuric acid) as standard control, at slit 4.4, sensitivity 3.6, excitation wavelength 360nm, emission wavelength 450nm, its fluorescence intensity is 55-60U, working sample fluorescence intensity under this condition.The chloroform methanol mixed solution is blank.
Calculation formula:
Lipofuscin content (μ g/g tissue)=
1.3.3.3.4 data processing and result judge
General employing variance analysis, but need carry out first homogeneity test of variance by the program of variance analysis, variance is neat, calculates the F value, F value＜F
0.05, conclusion: each organizes no significant difference between mean: F value 〉=F
0.05, P≤0.05 is added up with the in twos comparative approach of mean between a plurality of experimental group and control group: the data of abnormal or heterogeneity of variance are carried out suitable variable conversion, after satisfying that normal state or variance are neat and requiring, add up with the data after changing; If do not reach yet normal state or the neat purpose of variance after the variable conversion, use rank test instead and add up.
Given the test agent group and model (or aged) control group compares, and the lipofuscin content has statistical significance, judges that this given the test agent has the reduction lipid peroxidation, and experimental result is positive.
1.3.3.3.5 precaution:
1.3.3.3.5.1 want extreme care when drawing chloroform layer, in order to avoid bring tissue particles and water into, affect fluorometric assay.
1.3.3.3.5.2 whole chemical property and the characteristics of fluorescent chemicals are unclear, some normal biochemical material also has the fluorescence spectrum of similar fluorescence-causing substance such as retinene and flavin compound.The flavin material is water-soluble substances.Washing chloroform methanol mixed solution, just can remove: retinene is fat-soluble, rapidly degraded after uviolizing in chloroform, some other conjugated polyene compound also can be removed with uviolizing.
1.3.3.3.5.3 the crosslinking reaction of mda and free amino group is comparatively slow, the formation of Schiff alkali is a long process, can not reflect immediately the variation of radical damage reaction.Therefore, the time that gives given the test agent will be grown, and general 2-3 month, the elder can reach more than 6 months.
1.3.4 antioxidase activity is measured
SOD catalysis ultra-oxygen anion free radical (O
2 -) generation H
2O
2, by other antioxidase such as Selenoperoxidase (GSH-PX) and catalase effect generation water, can remove O so again
2 -Toxic action to cell.SOD, the GSH-Px content in some organ of animal and human body erythrocyte all has significantly to increase and changes age, and the growth of enzymic activity and biological age is inversely proportional to.The ability of eliminating free radical is directly proportional with enzymic activity.
1.3.4.1 superoxide-dismutase (SOD) vitality test in the Xue ∕ tissue
1.3.4.1.1 principle
O
2 -The final product of oxidation hydroxyl is nitrite, and the latter presents red-purple under Sulphanilic Acid and methyl naphthylamine effect, at wavelength 530nm place very big absorption peak is arranged, and available spectrophotometry is measured.When SOD eliminates O
2 -The nitrite of rear formation reduces.
1.3.4.1.2 instrument and reagent
Instrument 721 spectrophotometers, whizzer, thermostat water bath, homogenizer
Reagent 65mM phosphate buffered saline buffer (PBS) pH 7.8
The 10mmo/L oxammonium hydrochloride
Oxammonium hydrochloride 6.95mg adds PBS to 10mL
7.5mmoL/L xanthine
Xanthine 11.41mg adds 0.1M NaOH 2.5mL dissolving, adds PBS to 10mL
0.2g/L XOD
Get 10g/L XOD 0.2mL and add ice-cold PBS 9.8mL to 10mL
0.1% methyl naphthylamine
Get 0.2g α one methyl naphthylamine and be dissolved in the 40mL distilled water that boils, coolly add the 50mL Glacial acetic acid to room temperature, add again the cool distilled water of 110mL to 200mL
0.33% Sulphanilic Acid
Get the 0.66g Sulphanilic Acid and be dissolved in 150mL temperature distilled water, add the 50mL Glacial acetic acid to 200mL
The SOD standard substance
Trichloromethane
(v ∕ v) for 95% ethanol
0.9% physiological saline
1.3.4.1.3 experimental procedure
The preparation of red corpuscle extract: 10 μ l whole bloods pour 0.5mL physiological saline, and the centrifugal 3min of 2000r/min abandons supernatant, add ice-cold distilled water 0.2mL mixing, add 95% ethanol 0.1mL, vibration 30s, add trichloromethane 0.1mL, put flash mixer extracting 1min, the centrifugal 3min of 4000r/min, layering, the upper strata is the SOD extract, the middle level is the oxyphorase throw out, and lower floor is trichloromethane, and record supernatant liquor volume is to be measured.
The preparation of tissue homogenate: a certain amount of required internal organs of clip, normal saline flushing, wipe away dried, weigh, shred, to glass homogenizer, add cold saline 20000r/min homogenate 10s, 30s intermittently, repeatedly carry out three times, make 1% tissue homogenate (the most handy ultrasonic generator is processed 30s), plastosome is shaken brokenly, shaken broken with the green B dyeing of toluylene red one Zhan Na Shi proof plastosome.With the centrifugal 5min of 4000r/min, get supernatant liquor 20 μ l to be measured.
The SOD standard suppresses curve is mixed with the SOD standard substance 750U/mL with phosphate buffered saline buffer solution, redilution to 50 times, be that the SOD amount is 15U/mL(1.5 μ g/mL), measure the percent inhibition of the SOD reference liquid of different amounts with this law, take percent inhibition as ordinate zou, take SOD unit of activity U/mL as X-coordinate drawing standard curve.
The SOD percent inhibition=
Calculate
Corresponding SOD amount was a unit when SOD inhibiting rate reached 50% in every mL reaction solution
SOD vigor (U/mL)=
Also available enzyme specific activity method is namely found corresponding SOD U/mL with the percent inhibition of every pipe sample from the SOD typical curve, multiply by extension rate (1mL/ sampling amount).
If sample is tissue homogenate, according to homogenate concentration or tissue protein content, be U/g tissue or U/mg albumen with unit conversion.If sample is the red corpuscle extract, according to content of hemoglobin, can be scaled U/g Hb.
The sample determination step:
EDTA 0.5g
NaCl 280g
Adding distil water is used common filter paper filtering, room temperature preservation to 1000mL.
0.32mol/L Na
2HPO
4Solution:
Na
2HPO
422.7g adding distil water is to 500mL, room temperature preservation.
The DTNB nitrite ion
DTNB 40mg
Trisodium citrate 1.0g
Adding distil water is to 100mL, and 4 ℃ kept in Dark Place 1 month.
0.2M phosphoric acid buffer pH7.4
0.9% physiological saline
1.3.4.2.3 experimental procedure
1.3.4.2.3.1 sample preparation
Hemolysate: get mouse blood 10 μ l and join in the 1mL distilled water, shake well makes it whole haemolysis 1:100 to be measured, measures enzyme activity in the 4h.If had little time the same day to measure, with the anticoagulant heparin whole blood put-20 ℃ frozen, measure in the 3d, if. deposit for 4 ℃, must survey in the 28h.Taking out the sample room temperature before surveying thaws naturally.
Organize supernatant liquor: the animal overnight fasting, after the execution, take out immediately required internal organs, put into the floating blood of cold saline flush away, reject fat and reticular tissue, after filter paper blots, be cut into fragment at ice bath, take by weighing an amount of tissue, add cold 0.2M phosphoric acid buffer, with 20000r/min homogenate 10s, 30s intermittently, repeatedly make 5% tissue homogenate 3 times, operate in the ice bath and carry out, homogenate is with the centrifugal 10min of 12500 * g (low-temperature and high-speed whizzer), to be precipitated as broken cell, cell debris, nuclear and plastosome, supernatant liquor is in order to survey the enzyme activity in the cytosol, preferably surveyed the same day, be sub-packed in plastics tubing otherwise add 20% (v/v) glycerine, placed-20 ~-80 ℃, can preserve several weeks, and enzyme activity does not subtract.
1.3.4.2.3.2 the making of GSH typical curve:
Get 1.0mmol/LGSH solution 0,0.2,0.4,0.6,0.8,1.0mL, place respectively 10mL small vessels bottle, each adds metaphosphoric acid precipitation agent 8mL, is diluted to the 10mL scale with distilled water, and namely obtaining concentration is the GSH reference liquid of 0,20,40,60,80,100 μ mol/L.
Get above-mentioned each 2mL of different concns reference liquid, put into test tube, add 0.32mol/L N
A2HPO
42.5mL, adding DTNB nitrite ion 0.5mL optical path 1cm cup before the colorimetric, the inherent visible light 423nm of 5min wavelength is surveyed the OD value, with the distilled water zeroising.
Take GSH content (μ mol/L) as X-coordinate, OD
423Value is ordinate zou, drawing standard curve.
1.3.4.2.3.3 determination step:
In 423nm wavelength (1cm optical path), read the OD value, accuracy of reading within the 5min behind the color reaction 1min.
* sample is when organizing supernatant liquor, and non-enzyme pipe changes the supernatant liquor of organizing that heating makes enzyme deactivation into.
* hemolysate 0.1-0.4mL
Organize supernatant liquor 1:20 dilution, get diluent 0.4mL
1.3.4.2.3.4 calculate
Mouse GSH-Px activity unit stipulates every 1mL whole blood, per minute, the log[GSH of deduction non-enzyme reaction] reduce after, make log[GSH] to reduce by 1 be an enzyme activity unit.
Tissue GSH-Px is than every milligram of protein of unit of activity regulation, and per minute is deducted non-enzyme reaction, and making GSH concentration reduce by 1 μ mol/L is an enzyme activity unit.
1.3.4.2.4 data processing and result judge
General employing variance analysis, but need carry out first homogeneity test of variance by the program of variance analysis, variance is neat, calculates the F value, F value＜F
0.05, conclusion: each organizes no significant difference between mean; F value 〉=F
0.05, P≤0.05 is added up with the in twos comparative approach of mean between a plurality of experimental group and control group; The data of abnormal or heterogeneity of variance are carried out changing in the suitable change, after satisfying the neat requirement of normal state or variance, add up with the data after the conversion; If do not reach yet normal state or the neat purpose of variance after the variable conversion, use rank test instead and add up.
Given the test agent group and model (or aged/normal) control group relatively, the GSH-Px vigor raises statistical significance, judges that this given the test agent has rising GSH-Px effect, the experimental result positive.
1.3.4.2.5 precaution
1.3.4.2.5.1 because H
2O
2Easily decompose and cause the concentration change, face the time spent and get stock solution with its concentration of spectrophotometric instrumentation, get stock solution 3mL, measure the OD of the 240nm place value of 1cm optical path.
Concentration (mmol/L)=
If the OD value is 0.45, then show H
2O
2Concentration is 12.5mmol/L.
1.3.4.2.5.2 the colour developing of 5-sulfo-2-nitrobenzoyl acid anion is not only relevant with hydrogen ion concentration in the whole reaction system, limited by the reaction times.After adding developer, reaction system pH is 6.5 o'clock, and 11 min begin colour developing, the interior accuracy of reading of colorimetric 5min this moment.
Data processing and result judge
General employing variance analysis, but need carry out first homoscedasticity inspection inspection by the program of variance analysis, variance is neat, calculates the F value, F value＜F
0.05, conclusion: each organizes no significant difference between mean; F value 〉=F
0.05, P≤0.05 is added up with the in twos comparative approach of mean between a plurality of experimental group and control group; The data of abnormal or heterogeneity of variance are carried out suitable variable conversion, after satisfying the neat requirement of normal state or variance, add up with the data after the conversion.If change does not reach normal state or the neat purpose of variance after changing most yet, use rank test instead and add up.
Given the test agent group and model (or aged) control group compares, and LPO reduces statistical significance, judges that this given the test agent has the reduction lipid peroxidation, and real face result is positive.
Given the test agent group and model (or aged/normal) control group relatively, antioxidase activity raises statistical significance, judges that this given the test agent has the effect of rising antioxidase activity, experimental result is positive.
The result judges
In the LPO in arbitrary index and the anti-oxidant function enzymic activity arbitrary index all positive, can judge that the anti-oxidant results of animal of this given the test agent is positive.
Human feeding trial
2.1 experimenter's inclusive criteria
Select the age in 45-65 year, physical integrity is good, without obviously brain, the heart, liver, lung, kidney, Hematological Diseases, without the Long-term taking medicine history, volunteers the crowd that tested assurance cooperates.
Get rid of experimenter's standard
2.2.1 gestation or lactating women are to the protective foods allergy sufferers.
2.2.2 merge to have the inclination, the serious disease patients such as liver, kidney and hemopoietic system.
2.2.3 take in a short time the article relevant with tested function, have influence on the judgement person to the result.
2.2.4 do not meet inclusive criteria, eat in accordance with regulations given the test agent, can't judge effect or data not umbra sound effect or security judgement person.
Experimenter's grouping
The experimenter is divided into test-meal group and control group at random by MDA, SOD, GSH-Px level.Consider as far as possible affect result's principal element such as age, sex, Diet lifestyle etc., carry out harmony to check, with the comparability between the assurance group.Every group of experimenter is no less than 50 examples.
Test method
Adopt two kinds of control design between self and group, test group is subjected to trial product by recommending instructions of taking, dose to take every day, and control group can be taken placebo or adopt negative control.Given the test agent gives 3 months time, can extend to 6 months in case of necessity.Duration of test control group and the former life of test-meal group, diet are constant.
Observation index
Indices respectively detects 1 time when on-test and end.
2.5.1 safety indexes
2.5.1.1 general status comprises spirit, sleep, diet, stool and urine, blood pressure etc.
2.2.1.2 blood, urine, change routine inspection
2.5.1.3 liver, kidney function test
2.5.1.4 Chest X-rays, electrocardiogram(ECG, Abdominal B type ultrasonography inspection
2.5.2 effect index
2.5.2.1 variation and the MDA decline percentage of MDA before and after the LPO experimental observation.(measuring method is seen 1.3.3.1)
MDA decline percentage=
2.5.2.2 variation and the SOD rising percentage of SOD before and after the superoxide-dismutase viewing test.(measuring method is seen 1.3.4.1)
SOD rising percentage=
2.5.2.3 variation and the GSH-PX rising percentage of GSH-PX before and after the Selenoperoxidase viewing test.(measuring method sees attached 1)
GSH-PX rising percentage=
2.6 data processing and result judge
All own control data can adopt paired t-test, two groups of means relatively adopt in groups t check, and the latter need carry out homogeneity test of variance, and the data of skewed distribution or heterogeneity of variance are carried out suitable variable conversion, wait satisfy the normal state variance neat after, carry out t with the data of conversion and check; If translation data still can not satisfy the neat requirement of normal state variance, use t ' check or rank test instead; But the variation coefficient too data of large (such as CV＞50%) is used rank test.Under the prerequisite of comparing difference without significance, can test between rear group and compare between before test, organizing.
Between group statistical significance is arranged more all after each functional observation index Test front and back self comparison and the test-meal, can judge that this index is positive.
Each experimental result is positive in LPO, superoxide-dismutase, three experiments of Selenoperoxidase, can judge that this given the test agent has the anti-oxidant function effect.
Blood Glutathione Peroxidase (GSH-Px) vitality test
2.7.1 principle
Selenoperoxidase (GSH-Px) is that a kind of selenium that contains that exists in the body is removed free radical and the system that suppresses free radical reaction.To preventing that interior free yl from causing membrane lipid peroxidation particularly important, its vigor is with the speed of response of catalysis GSH oxidation, and the amount that GSH reduces in the unit time represents, GSH and 5, the reaction of 5 '-two sulphur p-nitrobenzoic acids (DTNB) can generate yellow 5-sulfo-2-nitrobenzoyl acid anion under GSH-Px catalysis, in the 423nm wavelength maximum absorption band is arranged, measure this ionic concn, can calculate the amount that GSH reduces, because GSH can carry out the non-enzyme reaction oxidation, during all last calculating enzyme activities, must reduce by the caused GSH of deduction non-enzyme reaction.
Reagent and instrument
Instrument 721 spectrophotometers, thermostat water bath, micro sample adding appliance, whizzer
Reagent sodium azide phosphoric acid buffer pH7.0
NaN
316.25mg final concentration 2.5mmol/L
EDTA-Na
27.44mg final concentration 0.2mmol/L
Na
2HPO
41.732g final concentration 0.2mol/L
NaH
2PO
41.076g final concentration 0.2mol/L
Adding distil water is transferred pH7.0,4 ℃ of preservations to 100mL with a small amount of HCl, NaOH.
1mmol/L gsh (reduced form GSH) solution
GSH 30.7mg adds the sodium azide phosphoric acid buffer to 100mL, before use preparation, stored frozen 1-2 day.
1.25-1.5mmol/L H
2O
2Solution
Get 30% H
2O
20.15-0.17mL, be diluted to 100mL with distilled water, as stock solution, 4 ℃ keep in Dark Place, and before use stock solution are got final product with 10 times of distilled water dilutions.
The metaphosphoric acid precipitated liquid
HPO
316.7g(use first dissolved in distilled water)
EDTA 0.5g
NaCl 280g
Adding distil water is used common filter paper filtering, room temperature preservation to 1000mL.
0.32mol/LNa
2HPO
4Solution;
Na
2HPO
422.7g adding distil water is to 500mL, room temperature preservation
The DTNB nitrite ion
DTNB 40mg
Trisodium citrate 1.0g
Adding distil water is to 100ml, and 4 ℃ kept in Dark Place 1 month,
2.7.3 experimental procedure
2.7.3.1 sample preparation
Hemolysate is got blood 20 μ l and is joined in the 1mL distilled water, and shake well makes it whole haemolysis 1:100 to be measured, measures enzyme activity in 4 hours.If had little time the same day to measure, with the anticoagulant heparin whole blood put-20 ℃ frozen, measure in the 3d, if 4 ℃ are deposited, must survey in the 28h.Taking out the sample room temperature before surveying thaws naturally.
The making of typical curve
Get 1.0mmol/LGSH solution 0,0.2,0.4,0.6,0.8,1.0mL, place respectively 10mL small vessels bottle, each adds metaphosphoric acid precipitation agent 8mL, is diluted to the 10mL scale with distilled water, and namely obtaining concentration is the GSH reference liquid of 0,20,40,60,80,100 μ mol/L.
Get above-mentioned each 2mL of different concns reference liquid, put into test tube, add 0.32mol/L Na
2HPO
42.5mL, adding DTNB nitrite ion 0.5mL optical path 1cm cup before the colorimetric, the inherent visible light 423nm of 5min wavelength is surveyed the OD value, with the distilled water zeroising.
Take GSH content (μ mol/L) as X-coordinate, OD
423Value is ordinate zou, drawing standard curve.
Determination step:
In 423nm wavelength (1cm optical path), read the OD value, accuracy of reading within the 5min behind the color reaction 1min.
Calculate
GSH-Px activity unit stipulates per 8 μ l whole bloods, and at 37 ℃ of reaction 5min, behind the deduction non-enzyme reaction, making GSH concentration reduce by 1 μ mol/L concentration is an enzyme activity unit.
GSH-Px activity U/mL blood=(non-enzyme pipe OD-sample hose OD) * A
** 5**
*A=
When * 5 is converted in the 1mL reaction solution GSH concentration, need multiply by extension rate 5
2.7.3.5 precaution
2.7.3.5.1 because H
2O
2Easily decompose and cause the concentration change, face the time spent and get stock solution with its concentration of spectrophotometric instrumentation, get stock solution 3mL, measure the OD of the 240nm place value of 1cm optical path.
Concentration (mmol/L)=
If the OD value is 0.45, then show H
2O
2Concentration is 12.5mmol/L.
2.7.3.5.2 the colour developing of 5-sulfo-2-nitrobenzoyl acid anion is not only relevant with hydrogen ion concentration in the whole reaction system, limited by the reaction times.After adding developer, reaction system pH is 6.5 o'clock, and 11min begins colour developing, the interior accuracy of reading of colorimetric 5min this moment.
Clinical trial
1, generalized case:
Patients with Cardiovascular/Cerebrovascular Diseases 120 examples, hyperpietic's 50 examples, atherosclerotic's 70 examples, diabetic subject's 40 examples are selected respectively in outpatient service, wherein male 152 examples, women 128 examples, age 42-68 year, average 58 years old, medical history 3-20, average 12 years.
Excluded cases standard (comprising inadaptation or rejecting standard)
(1) age is more than 70 years old, gestation or lactating women, allergic constitution or to the alcohol allergy sufferers.
(2) be associated with the serious primary disease such as cardiovascular, the cerebrovascular, hypertension and arterial thrombosis, mental patient.
(3) do not meet inclusive criteria, not in accordance with regulations medication can't be judged that curative effect or data are not congruent to affect the treatment or security judgement person.
2, methods for the treatment of:
Take every night the health promoting wine of the present invention preparation, each 50～100mL is a course for the treatment of all around.
Viewing duration all medicines of stopping using, and prohibit and take sweet mung bean soup, have a blood test simultaneously pressure, routine urinalysis, electrocardiogram(ECG, Color doppler ultrasound, blood sugar and blood fat etc., the above index of check after 4th week finishes.
3, curative effect judging standard:
(1) clinical recovery: corresponding clinical symptom all disappears.
(2) produce effects: corresponding clinical symptom disappearance is more than 70%.
(3) effective: corresponding clinical symptom disappearance is more than 30%.
(4) invalid: corresponding clinical symptom disappearance is below 30%.
4, observation of curative effect: see the following form:
| Disease type | Total example sum | Produce effects | Effectively | Invalid | Efficient |
| Cardiovascular and |
120 | 8 | 98 | 14 | 88.33 |
| Hypertension | |||||
| 50 | 5 | 36 | 9 | 82% | |
| Atherosclerosis | 70 | 7 | 59 | 4 | 94.29% |
| Diabetes | 40 | 4 | 28 | 8 | 80% |
| Add up to | 280 | 24 | 221 | 35 | 87.5% |
As can be seen from the table: the prepared health promoting wine of the embodiment of the invention is all effective in cure for above illness, obvious effective rate 8.57%, efficient 78.93%, total effective rate is 87.5%, and is wherein efficient the highest for atherosclerosis, reaches 94.29%, efficient secondly to cardiovascular and cerebrovascular diseases, reach 88.33%, efficient slightly poor to hypertension and diabetes, reach respectively 82% and 80%.
(1) recurrence rate: clinical produce effects 24 examples, with access 6 months, 3 example recurrences, recurrence rate is 12.5%, again drinks still effective.
(2) untoward reaction: period in a medicine has the symptoms such as alcohol anaphylaxis, asthma, palpitating speed, can cut out.
Claims (2)
1. the preparation method of a Russian olive health-care wine, it is characterized in that: it with the arrow-leaved oleaster fermented wine as basic wine, add together ageing and get of medicine, raw materials used weight percent is: matrimony vine 15～25%, arrow-leaved oleaster fermented wine 50～73%, Herba Cistanches 5～10%, Radix Codonopsis 5～15% and royal jelly 2～5% compositions;
The brew step of described arrow-leaved oleaster fermented wine is: in the wine yeast scale-up medium after arrow-leaved oleaster juice activates with the access of 4～6% inoculum sizes, made the arrow-leaved oleaster fermented wine in 6～10 days in 21～25 ℃ of fermentations;
Described arrow-leaved oleaster juice preparation process is: fresh arrow-leaved oleaster is after choosing fruit, cleaning, boiling, removal of impurities, solid-liquid ratio by arrow-leaved oleaster and water is that 1:8～1:12 adds poach system, constantly stir during this time, mash, stoning, moisturizing is to former weight ratio, soluble solid content is 5～7% arrow-leaved oleaster juice, get soluble solid content and be 5～7% arrow-leaved oleaster juice, add sucrose and adjust soluble solid content, add citric acid and adjust acidity, and add sulphur, add calcium;
The described sulphur that adds is selected and is added Na
2SO
3, Na
2SO
3Add-on with SO
2Count 30～90 mg/L, soluble solid content transfers to 18%～22% when transferring sugar, and adding citric acid during acid adjustment, to transfer to pH value be 3.5～4.5, adding CaCl when adding calcium
2Amount be 0.3～0.5g/L;
The preparation process of described wine yeast scale-up medium is: the grape wine dry yeast is placed 28～30 ℃ of water to shake up to leave standstill activation half an hour, in the 250mL triangular flask, carry out the one-level enlarged culturing after the activation, substratum is the arrow-leaved oleaster juice of soluble solid content 5～7%, culture temperature is 26～28 ℃, and incubation time is 36～48h; One-level is transferred to the triangular flask nutrient solution in the 1000mL triangular flask of the arrow-leaved oleaster juice that soluble solid content 5～7% is housed after cultivating and finishing, and carries out the secondary enlarged culturing, and it is 24～26 ℃ that secondary is cultivated culture temperature; Then carry out three grades of enlarged culturing in seeding tank, three grades of culture temperature are 24～26 ℃, and substratum is the arrow-leaved oleaster juice of soluble solid content 7%, is prepared into the wine yeast scale-up medium;
With arrow-leaved oleaster fermented wine tank switching, filter, discard and precipitate to get arrow-leaved oleaster fermented wine filtrate, in arrow-leaved oleaster fermented wine filtrate, add matrimony vine, Herba Cistanches, Radix Codonopsis powder, adding royal jelly dissolves, be statically placed in the wine storing jar, in 16～20 ℃ of lower ageing more than 90 days, and stirred once every 10～20 days; The Russian olive health-care wine that ferments is statically placed in the wine storing jar, adds therein mass volume ratio and be 0.2% gelatin, in 16～20 ℃ of static clarifications 5～10 days, the then Russian olive health-care wine of separating clarifying.
2. the Russian olive health-care wine that makes of described preparation method according to claim 1.
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| CN105176723B (en) * | 2015-07-13 | 2017-06-23 | 甘肃有色冶金职业技术学院 | A kind of health grape wine and preparation method thereof |
| CN107802695A (en) * | 2016-09-09 | 2018-03-16 | 江苏康缘药业股份有限公司 | A kind of health composition of the strengthen immunity containing saline cistanche and preparation method thereof |
| CN109136020A (en) * | 2018-09-29 | 2019-01-04 | 江安金兰农业科技有限公司 | A kind of plum wine and its brewing method |
| CN109829133A (en) * | 2019-01-24 | 2019-05-31 | 重庆苞谷珠科技有限公司 | A kind of production method and its optimization method of puffed rice |
| CN112500968A (en) * | 2021-01-15 | 2021-03-16 | 甘肃滨河食品工业（集团）有限责任公司 | Elaeagnus angustifolia flower flavoring wine and preparation method thereof |
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|---|---|---|---|---|
| CN1108488A (en) * | 1994-03-18 | 1995-09-20 | 丁乾俊 | Narrow-leaved oleasater series food |
| CN1399914A (en) * | 2001-08-07 | 2003-03-05 | 吴多强 | Body-building and face-nourishing nutrient solution |
| CN101637500A (en) * | 2009-09-03 | 2010-02-03 | 天津科技大学 | Milk veteh, oleaster, walnut green husk and worm grass-containing health wine and effect thereof |
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2012
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1108488A (en) * | 1994-03-18 | 1995-09-20 | 丁乾俊 | Narrow-leaved oleasater series food |
| CN1399914A (en) * | 2001-08-07 | 2003-03-05 | 吴多强 | Body-building and face-nourishing nutrient solution |
| CN101637500A (en) * | 2009-09-03 | 2010-02-03 | 天津科技大学 | Milk veteh, oleaster, walnut green husk and worm grass-containing health wine and effect thereof |
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| Title |
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| 天晓菊 等.沙枣果酒加工工艺研究.《安徽农业科学》.2011,第39卷(第2期),832-833，834. |
| 沙枣果酒加工工艺研究;天晓菊 等;《安徽农业科学》;20111231;第39卷(第2期);832-833，834 * |
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