CN101664180B - Health-care nutritional complexing agent with health effect and preparation method thereof - Google Patents

Abstract

The invention provides a nutritional complexing agent with the functions of lowering blood sugar, resisting oxidation and lowering blood fat and a preparation method thereof, in particular to a nutritional complexing agent taking Jew's-ear, hericium erinaceus and bee pollen as main raw materials and further containing Japanese stone pine kernel. By scientific matching and an extraction technique, the effective components of the raw materials are extracted and then health-care nutritional solid medicinal granules or soft capsules are prepared. Experiments show that the nutritional complexing agent has the favorable functions of lowering blood sugar, resisting oxidation and lowering blood fat.

Description

A kind of nutritional complexing agent with health-care efficacy and preparation method thereof

Technical field

The present invention relates to field of health care food, relate in particular to a kind of nutritional complexing agent with hypoglycemic, anti-oxidant, effect for reducing blood fat and preparation method thereof, be particularly related to a kind ofly take black fungus, Hericium erinaceus and Bee Pollen as primary raw material, also can comprise nutritional complexing agent of siberian dwarf pine benevolence and preparation method thereof.

Background technology

Health food refers to have the food of specific health care, and it is edible namely to be suitable for specific crowd, has the adjusting body function, not the food take the treatment disease as purpose.Function factor namely really can play the active ingredient of regulating action in food, the function factor that often contains in the health food at present comprises active polysaccharide, dietary fibre, compound sugar, active peptide, reactive protein, polyunsaturated fatty acid, lipoid, saponin, flavonoids, lactic acid bacteria, vitamin, mineral matter, allicin, choline, phytosterol, melatonin etc.

In recent years, along with improving constantly of China's living standards of the people, along with economy develops rapidly, " ciril disease " of various overnutritions appearred in people, and nutrition and health care is more and more strong requests of people.Society is to the aging development simultaneously, and people not only wish to promote longevity, and also thirsts for having the food that diseases prevention is healthy and strong, and improves the quality of living.Therefore, form worldwide " health food heat ".The health food of Japan has formed food industrialization production and selling system, and there is family more than 3000 in family more than 500 of manufacturing enterprise's eighties to the development nineties, and product reaches kind more than 20,000, and annual value of production has reached 6,000,000,000 dollars.The health food annual value of production of Germany reaches more than 50 hundred million marks, accounts for 20%～30% share at food products market.The every annual value of production of health food is with 20% speed increase after U.S. 1970, and there are 1.08 hundred million human oral nutritious supplementary pharmaceuticals in the U.S. according to estimates.China takes full advantage of theory and world medicine and the Nutritional studies latest developments of the food health of the traditional Chinese medical science, promoted the development of health food, the Chinese medicine that adopts in health food production mainly contains American Ginseng, Chinese caterpillar fungus, the Radix Astragali, Radix Angelicae Sinensis, the fruit of Chinese wolfberry, the tuber of multiflower knotweed, donkey-hide gelatin, gynostemma pentaphylla, loguat leaf etc., take the nourishing class as main.Health food accounts for 10% of the national food gross output value according to estimates.

The functional type frequency of occurrences in the existing health food is followed successively by immunological regulation, antifatigue, the element that delays senility, supplements the nutrients, regulate blood fat, beauty treatment, improvement memory, fat-reducing, anti-anoxic, enrich blood, improve gastrointestinal function, enhancing development, skin care, protect liver, improve sleep, hypoglycemic, radioresistance, anti-sudden change etc.In the health food of Ministry of Public Health's approval, have a small amount of product also to go through to have following certain function: anti-oxidation function, the increasing leukocyte function, prevention reduction of leukocyte function is improved microcirculation function, the preventing decayed tooth dental care benefits.

The health food form is take pharmaceutical formulation as main, and the suitable crowd of health food is take the unusual person of physiological function and the elderly as main.The capsule formulation health food is edible, easy to carry, under vacuum drying condition, can keep for a long time the stable of functional component, renewal along with the development of health food extraction process technology and masses' consumption idea, no longer Tablet and Capsula class health food is used as medicine and treats, the consumer that this two classes health food might be subject to grasping more healthcare knowledge likes.Health food with capsule formulation production has accounted for 25% of whole health food.

Polysaccharide is the natural macromolecular material that extensively is present in higher plant, animal cell membrane, the microorganism wall, is the important component part of all living organisms, and relevant with the necessary different physiological roles that sustains life.Be mainly manifested in the aspects such as antitumor, immunological regulation, anti-sudden change, antiviral, reducing blood lipid, hypoglycemic, anti-oxidant, anti-irradiation and antiulcer.

The contained polysaccharide body of black fungus is acid isodextran, is the polysaccharide of the about 155000D of molecular weight that separates from black fungus, and monose consists of the l-fucose, l-arabinose, d-wood sugar, d-mannose, d-glucose and glucuronic acid.Blackfungus polyhexose has multiple biologically active and pharmacological action: antiulcer, delay senility, reduce blood fat, anti-hepatitis, anti-sudden change, antitumor, Enhancin matter and nucleic acid metabolism and promote the functions such as immunity of organism, more and more be subject to pursuing nutrition as the black fungus of the important member in " black-food " family and pay attention to healthy modern.

Bee Pollen contains very abundant amino acid, carbohydrate, vitamin.In addition, also contain Flavonoid substances, trace element and enzyme in the pollen.Above-mentioned these chemical substances all can play balanced action to the various metabolism of human body.Famous Shennong's Herbal and successive dynasties book on Chinese herbal medicine have been done a lot of records to medicinal, the eating effect of pollen in ancient times.On the whole, mainly think pollen have promote longevity, conditioning skin and losing and the effect of improving sexual function.In recent years, the countries such as Russia and the U.S., day, method have formed the pollen research upsurge, and pollen medicine, food also come out one after another simultaneously.Academicly also having set up the specialism of a kind of " pollen ", explore from aspects such as biological cell, molecule, heredity.In recent years section's studies and clinical application has been affirmed fully the medical care effect of pollen.

According to data check, food industry is considered as Bee Pollen the favorite of food.Type of merchandize take pollen as raw material is various.Bee Pollen contains protein 7.5～35%, on average greater than 20%.Amino acid whose general content is 15～25%, and free amino acid accounts for 1～2%, and essential amino acid content 9.1% wherein.Fat content is lower, is 1～15%, and is average 4.8%, and contained unrighted acid is abundant.The average content of carbohydrate is 27% (15～45%).Contain the former and vitamin E of vitamin A, A in the liposoluble vitamin, indivedual pollen contain vitamin D and K.Mostly contain 7 kinds of B family vitamins, inositol and ascorbic acid.On average contain ash content 3.12% (1～5%), mainly contain phosphorus, potassium, calcium, magnesium, sodium, iron, manganese, zinc, copper, nickel, barium, boron, selenium etc., wherein the content of potassium is higher, and is average 0.58%, and the content of sodium is very low, is 0.04%.Cellulosic content majority is 10～20%.Pollen contains oxidoreducing enzyme, transferase, hydrolase, lyases, isomerase, several large classes such as enzyme and other enzymes more than totally 100 are planted enzyme continuously.Also contain flavone compound, nucleic acid, plant hormone and organic acid etc.

Bee Pollen has more comprehensive nutritional labeling; for the growth of body tissue cell and reparation provide abundant raw material; Bee Pollen contains many bioactivators such as amino acid, nucleic acid, enzyme, flavonoids, trace element simultaneously; to the various physiological functions of body, the physiological activity of each organ has adjusting, enhancing and protective effect.In a word, Bee Pollen can strengthen the metabolic capability of body, and booster immunization power is regulated endocrine, improves cardiovascular status and strengthens stress ability etc., has many-sided biological function and pharmacologically active.

In recent years, raising along with people's living standard, people's dietary structure also becomes more diverse, edible mushroom with its natural nuisance-free, be of high nutritive value, integration of drinking and medicinal herbs, unique flavor and day by day be subject to people's favor, its nutritive value, value medical health care more and more obtain people's understanding and attention, thereby have higher exploitation value and wide market prospects.

Hericium erinaceus has important medical value.Its property is flat, and it is sweet to distinguish the flavor of, useful taste, sharp the five internal organs are aid digestion.It has the effect that promotes gastric ulcer healing, can reduce the cholesterol in the blood, and neurasthenia is also had good result.With Hericium erinaceus treatment stomach and curing duodenal ulcer, total effective rate can reach 87%, and efficient to chronic gastritis (comprising atrophic gastritis) is 85%.Reach 90% to the gastrointestinal symptoms such as stomachache are efficient.Studies have shown that according to the Shanghai institute of Pharmaceutical Research, can isolate the glucoside of 5 oleanolic acids in the mycelium of Hericium erinaceus, is the active ingredient for the treatment of hepatitis.So people just have saying of " mountain delicacy hedgehog hydnum, seafood delights bird's nest " since ancient times, are good tonics, the equal edible of valetudinarian is with nourishing and fit keeping function.Feudal times, Hericium erinaceus once were used as tribute.

Hericium erinaceum polysaccharide is main active material in the Hericium erinaceus, and its active component is that the main chain that β-(1 → 3) glycosidic bond connects is connected 1 → 6 with β) side chain that connects of glycosidic bond consists of glucan; Erinacine is the another kind of active ingredient of Hericium erinaceus.It can impel nerve growth factor (NGF: can prolong the N aixs cylinder, keep the N cells survival, regulate the generation of nerve cell and the regeneration of aged animal nerve cell played a role) synthetic, its activity is better than adrenaline greatly, its Main Ingredients and Appearance is Hericium erinaceus rhzomorph A, B, C, E, F, and chemical analysis is the Diterpenes material.Erinacine can be used for treating intelelectual deterioration, neurasthenia and autonomic nerve decline.Hericium erinaceum polysaccharide can also nourishing qi and blood, the righting reinforcing, and efficient to the healing of gastric and duodenal ulcer is 93%.The unrighted acid that Hericium erinaceus is contained is conducive to blood circulation, can reduce the content of Blood Cholesterol, improves immunologic function, and is of value to the growth of beard and hair.Hericium erinaceus Polysaccharides can improve the activity of NK, the generation of promotion delayed allergy (DTH), can strengthen the phagocytic function of macrophage; And it is not obvious to the proliferation function of humoral immunity and immune organ.This explanation Hericium erinaceus Polysaccharides mainly is the immunologic function of regulating body by the activity that strengthens immunocyte.Along with development and the clinical testing of modern science and technology proves repeatedly, the intension that the Hericium erinaceus nutritional medicinal value constantly makes new advances in excavation, the nutritional medicinal value of Hericium erinaceus is mainly manifested in the following aspects: 1. antiulcer and antiinflammatory action.2. antitumor action 3. liver protection function 4. anti-aging effects 5. improve body's hypoxia tolerance, increase the heart blood output quantity, accelerate the body blood circulation.6. reduce the effect of blood sugar and blood fat.

Pine nut has another name called korean pine seed, new korean pine, pine nut, pine nut, Song Yuan, is the seed of pinaceae plant Korean pine, Chinese pine, masson pine.The history in existing more than 3000 year of the edible pine nut of China." peanut " that ancients are considered as pine nut to promote longevity.Siberian dwarf pine belongs to Pinaceae, grows in the high and cold mountain area of 800 meters to 1000 meters of height above sea level, and profile is like shrub, by Daxinganling District exclusive.

Pinenut nutritional labeling and function factor are abundant, contain rich in protein, unrighted acid, vitamin, mineral matter active element.Warm in nature, flavor is sweet, have moistening the lung and relieve the cough, moisten unimpeded just, the alimentary health-care function of beautifying and anti-aging.Seed of Dwarf Siberian Pine oil contains plurality of active ingredients, unsaturated fatty acid content is more than 90% in the siberian dwarf pine grease, comprising multiple unrighted acids such as linoleic acid, leukotrienes and arachidonic acids, be the absorption of human body utilization easily, to reduction Blood Cholesterol content, prevent artery sclerosis, reducing blood lipid, hypotensively have an original effect.Contain in addition 0.75%～0.81% phospholipid composition, wherein the acyl choline accounts for 45.5%～49.1%, and phosphatidyl-ethanolamine accounts for 27.4%～28.3%, and phosphatidic acid accounts for 17%～20%.Have and improve immunologic function, Cell protection film, reduce blood fat, prevent the effect of cardiovascular and cerebrovascular disease.

But the nutritional complexing agent take the active ingredient of Blackfungus polyhexose, hericium erinaceum polysaccharide, Bee Pollen and siberian dwarf pine benevolence as main material production has no report.

Summary of the invention

The object of the present invention is to provide a kind of nutritional complexing agent hypoglycemic, anti-oxidant, effect for reducing blood fat that has, its raw material comprises black fungus, Hericium erinaceus, Bee Pollen, further also can comprise siberian dwarf pine benevolence, through science compatibility and extraction process, extract their active ingredient, healthy nutrition solid electuary or the soft capsule made.

For achieving the above object, the present invention adopts following technical scheme:

The invention provides a kind of nutritional complexing agent, its raw material comprises black fungus, Hericium erinaceus, Bee Pollen, and the weight proportion of described raw material is: black fungus 40-70 part, Hericium erinaceus 10-20 part, Bee Pollen 10-20 part.

Further, described nutritional complexing agent can also comprise siberian dwarf pine benevolence, and the parts by weight of described siberian dwarf pine benevolence are 10-20 part.

Preferably, the weight proportion of each raw material of described nutritional complexing agent is: 50 parts of black fungus, 10 parts of Hericium erinaceus, 20 parts of Bee Pollens, 20 parts of siberian dwarf pine benevolence.

Described nutritional complexing agent can be prepared into various oral formulations according to the conventional formulation method, such as electuary, soft capsule, tablet, oral liquid etc.Preferred electuary and soft capsule.

The present invention also provides a kind of nutritional complexing agent electuary, its main component comprises Blackfungus polyhexose, hericium erinaceum polysaccharide, Bee Pollen active ingredient, concrete is to comprise black fungus, hericium erinaceum polysaccharide concentrate 70-90 weight portion, Bee Pollen active ingredient concentrate 10-30 weight portion, the nutritional complexing agent powder for preparing through conventional electuary preparation method.Wherein, preferred black fungus, hericium erinaceum polysaccharide concentrate 75 weight portions, Bee Pollen active ingredient concentrate 10 weight portions.

The present invention also provides a kind of nutritional complexing agent capsule, it is comprised of content and softgel shell, the main component of its content comprises Blackfungus polyhexose, hericium erinaceum polysaccharide, Bee Pollen active ingredient and seed of Dwarf Siberian Pine oil, concrete is to comprise black fungus, hericium erinaceum polysaccharide concentrate 70-90 weight portion, Bee Pollen active ingredient concentrate 10-30 weight portion and seed of Dwarf Siberian Pine oil 10-20 weight portion; The composition of its softgel shell comprises gelatin, G ﹠ W, prepares through conventional capsule preparation method thereof.Wherein, preferred black fungus, hericium erinaceum polysaccharide concentrate 75 weight portions, Bee Pollen active ingredient concentrate 10 weight portions, seed of Dwarf Siberian Pine oil 15 weight portions.

In preferred embodiment of the present invention, described black fungus, hericium erinaceum polysaccharide concentrate are prepared by following steps:

A. extract polysaccharide solution: with black fungus 40-70 weight portion and Hericium erinaceus 10-20 weight portion through removal of impurities, drying, pulverize, sieving obtains black fungus, Hericium erinaceus dry powder; Gained dry powder is added in the buffer solution, add again cellulase and carry out the constant temperature enzymatic reaction; It is extremely neutral to regulate afterwards pH, adds neutral proteinase and carries out the constant temperature enzyme digestion reaction, and after the enzyme lixiviate was gone out in intensification rapidly, centrifugal, filtration obtained polysaccharide solution;

B. isolating protein: in polysaccharide solution, add chloroform-n-butanol mixed solution and carry out shake well, the floating preteins sex change is become insoluble substance, after centrifugation, remove protein;

C. decolouring: adopt H ₂O ₂Decoloring method decolours, after decolouring is processed again through dialysis except the little molecule pigment after the deoxidation;

D. concentrated: temperature is controlled at about 50 ℃, uses low-pressure steam, removes about 70%～80% moisture, namely gets black fungus, hericium erinaceum polysaccharide concentrate.

Wherein, described black fungus, Hericium erinaceus dry powder are by 1: the part by weight of 20-40 adds the buffer solution of pH=3.0-7.0, adds cellulase 30-150IUg ^-1, 20-60 ℃ of constant temperature enzymatic reaction 1-5h; Transfer pH to 5.0-9.0, add neutral proteinase 60-300IUg ^-1, 20-60 ℃ of constant temperature enzymolysis 1-5h is warming up to rapidly 80 ℃ of enzyme lixiviate 2h that go out, and be centrifugal, filters.

Preferably, the pH=4.5-5.5 of described buffer solution, more preferably pH=5.0; The addition of described cellulase is 60-120IUg ^-1, more preferably 90IUg ^-1Described enzymatic reaction temperature is 45-55 ℃ of reaction time 2.5-3.5h, and preferred condition is 50 ℃, 3.5h.

Preferably, described adjusting pH to 7.0-8.0, more preferably pH is 7.0; The addition of described neutral proteinase is 60-180IUg ^-1, more preferably 120IUg ^-140-50 ℃ of enzymolysis time 1.5-2.5h of described enzymolysis Wen Weidu, preferred condition is 45 ℃, 2.5h.

Wherein, the described albumen that removes is to add chloroform/n-butanol (4: 1) reagent, thermal agitation 30min by black fungus, the hericium erinaceum polysaccharide aqueous solution 1/4 volume, centrifugal, inclining supernatant, albuminate and lower floor's chloroform in the middle of removing, repetitive operation until the intermediate layer without albuminate.

Wherein, described decolouring is to adopt H ₂O ₂Method, with polysaccharide solution ammoniacal liquor adjust pH to 8.0,40 ℃ drip 5%H ₂O ₂, insulation 3h, dialysis gets black fungus, hericium erinaceum polysaccharide concentrate.

In preferred embodiment of the present invention, described Bee Pollen active ingredient concentrate is prepared by following steps:

A. take by weighing the Bee Pollen of 10-20 weight portion, removal of contamination adds behind the water and transfers pH to acid with acid, adds cellulase CA complex enzyme, behind the constant temperature enzyme digestion reaction, and the lixiviate behind the enzyme of going out that heats up rapidly, centrifugation obtains the Bee Pollen extract; Described cellulase CA complex enzyme is the mixture of cellulase and α-amylase;

B. the Bee Pollen enzyme is extracted slag and adds water, acid adding, boil hydrolysis after, with the alkali neutralization, centrifugation obtains the Bee Pollen extract.Extracted twice liquid merges Vacuum Concentration and namely gets Bee Pollen active ingredient concentrate.

Wherein, described Bee Pollen is by 1: the part by weight of 8-12 adds water, transfers pH to 3.0-5.0 with acid again, adds cellulase CA complex enzyme 30-150IUg ^-1, 25-65 ℃ of constant temperature enzymolysis 16-32h is warming up to rapidly 80 ℃ of enzymes that go out, lixiviate 6h, centrifugation Bee Pollen extract.

Preferably, the cellulase CA complex enzyme of adding is cellulase: the weight ratio of α-amylase is 5-7: 1 mixture, preferred 6: 1; The amount of its adding is 30-90IUg ^-1, more preferably 90IUg ^-1Described with acid accent pH to 4.0-5.0, more preferably pH is 4.0; Described hydrolysis temperature is that 40-50 ℃ of time is 24-28h, and preferred condition is 45 ℃, 28h.

The present invention also provides a kind of preparation method of described nutritional complexing agent electuary, that above-mentioned black fungus, hericium erinaceum polysaccharide concentrate 70-90 weight portion and Bee Pollen active ingredient concentrate 10-30 weight portion are mixed, add the common drugs such as dextrin, whey powder, sucrose and learn excipients or auxiliary material, stirring and dissolving, use high pressure homogenizer to carry out homogeneous, and then carry out spray-drying.Namely obtain nutritional complexing agent powder finished product after the cooling.

Described materia medica excipients or auxiliary material are maltodextrin and whey powder.

Described high-pressure homogeneous employing double-stage homogenization, one-level homogenization pressure 20Mpa, time 10min; Double-stage homogenization pressure 5Mpa, time 5min; 60 ℃ of homogenizing temperatures.

Described spray-dired feeding temperature is 40-80 ℃, and EAT is 160-240 ℃, and leaving air temp is 60-140 ℃; The preferred feedstock temperature is 40 ℃, and EAT is 180-200 ℃, and leaving air temp is 80-100 ℃.

The present invention also provides a kind of preparation method of described nutritional complexing agent capsule, comprises the steps:

A. get gelatin, the distilled water that adds 0.8 times of gelatin amount makes its imbibition, in addition in putting of the water glue tank with 0.5 times of glycerine and gelatin amount, be heated to 70～80 ℃, mix, add the gelatin that expands and stir, make and dissolve into even glue, insulation 1～2h vacuumizes the bubble of getting rid of in the glue, and it is stand-by to filter glue;

B. fill compacting: above-mentioned nutritional complexing agent powder and seed of Dwarf Siberian Pine oil 10-20 weight portion are stirred, by the hopper of the automatic rotary transformation of ownership capsule machine filling pump of flowing through, enter to roll and be packed into adhesive tape in the mould and make soft capsule through rolling mould spinning.In the automatic rotary transformation of ownership capsule machine production process, indoor temperature should be controlled at 20～24 ℃, relative air humidity 35%;

C. dry: as to fill soft capsule that compacting forms by collection chute and conveying device, enter in the drying machine after removing waste product, finalize the design, dry, whole ball, get product.

The present invention further provides described nutritional complexing agent and electuary thereof, soft capsule in the application of field of health care food, especially have hypoglycemic, anti-oxidant, the medicine of effect for reducing blood fat or the application in the health products in preparation.

Description of drawings

Fig. 1 is that the cellulase enzyme concentration is on the impact of polysaccharide extract rate;

Fig. 2 is that the cellulase action pH value is on the impact of polysaccharide extract rate;

Fig. 3 is that the cellulase degradation time is on the impact of polysaccharide extract rate;

Fig. 4 is that the cellulase degradation temperature is on the impact of polysaccharide extract rate;

Fig. 5 is that the neutral proteinase enzyme concentration is on the impact of polysaccharide extract rate;

Fig. 6 is that the neutral proteinase action pH value is on the impact of polysaccharide extract rate;

Fig. 7 is that the neutral protease enzymolysis time is on the impact of polysaccharide extract rate;

Fig. 8 is that the neutral protease enzymolysis temperature is on the impact of polysaccharide extract rate;

Fig. 9 is that spray-dired temperature is on the impact of viscosity of sludge;

Figure 10 is that extract of the present invention is on the impact of diabetic mice fasting blood-glucose;

Figure 11 is that extract of the present invention is on the impact of blood glucose in diabetic mice TG-AUC;

Figure 12 is that extract of the present invention is on the impact of diabetic mice sugar tolerance.

The specific embodiment

Nutritional complexing agent of the present invention, its raw material comprises black fungus, Hericium erinaceus, Bee Pollen, the weight proportion of described raw material is: black fungus 40-70 part, Hericium erinaceus 10-20 part, Bee Pollen 10-20 part.

Further, described nutritional complexing agent can also comprise siberian dwarf pine benevolence, and the parts by weight of described siberian dwarf pine benevolence are 10-20 part.

Preferably, the weight proportion of each raw material of described nutritional complexing agent is: 50 parts of black fungus, 10 parts of Hericium erinaceus, 20 parts of Bee Pollens, 20 parts of siberian dwarf pine benevolence.

Described nutritional complexing agent can be prepared into various oral formulations according to the conventional formulation method, such as electuary, soft capsule, tablet, oral liquid etc.Preferred electuary and soft capsule.

The invention will be further described below in conjunction with embodiment, it should be understood that these embodiment only are used for the purpose of illustration, never limit protection scope of the present invention.

The preparation of embodiment 1 black fungus, hericium erinaceum polysaccharide concentrate:

1. extraction polysaccharide solution: with black fungus and Hericium erinaceus through removal of impurities, drying, pulverize, sieving obtains black fungus, Hericium erinaceus dry powder; The part by weight of gained dry powder by 1: 30 added in the buffer solution of pH=3.0-7.0, add again cellulase 30-150IUg ^-1Carry out constant temperature enzymatic reaction 1-5h; Regulate afterwards pH to 5.0-9.0, add neutral proteinase 60-300IUg ^-1, 20-60 ℃ of constant temperature enzymolysis 1-5h is warming up to rapidly 80 ℃ of enzyme lixiviate 2h that go out, and be centrifugal, filters.

(1) cellulase is on the impact of black fungus, hericium erinaceum polysaccharide recovery rate

A. enzyme concentration is on the impact of black fungus, hericium erinaceum polysaccharide recovery rate

Cellulose is the main component of black fungus, Hericium erinaceus cell membrane, simultaneously also is the main barrier of the large molecule stripping such as intracellular polyse.Utilize cellulose hydrolyzation cell wall cellulose, be conducive to the stripping of cellular content.As seen from Figure 1, after the cellulase addition surpassed 90IUg-1, it is not obvious that polysaccharide extract rate increases trend, and the enzyme addition crosses conference and increase the polysaccharide extraction cost, and therefore, selecting the cellulase addition is to carry out orthogonal experiment about 90IUg-1.

The B.pH value is on the impact of black fungus, hericium erinaceum polysaccharide recovery rate

As seen from Figure 2, the highest at pH5 left and right sides black fungus, hericium erinaceum polysaccharide recovery rate, therefore select to carry out about pH5 orthogonal experiment.

C. enzymolysis time is on the impact of black fungus, hericium erinaceum polysaccharide recovery rate

Having set 5 different action times from 1h to 5h tests, therefrom determine the suitableeest enzymolysis time range, the result as shown in Figure 3, enzymolysis 3h left and right sides polysaccharide extract rate is the highest, surpass behind the 3h prolongation along with the time, polysaccharide extract rate increases and is not obvious, therefore selects to carry out about enzymolysis time 3h orthogonal experiment.

D. hydrolysis temperature is on the impact of black fungus, hericium erinaceum polysaccharide recovery rate

As seen from Figure 4, the optimum temperature of cellulase is about 55 ℃, so hydrolysis temperature is chosen in this scope and carries out orthogonal experiment.

E. the cellulase optimal condition determines

Cellulase affects most important four factors of polysaccharide yield: enzyme concentration, enzymolysis time, hydrolysis temperature and pH value, adopt L ₉(3 ⁴) orthogonal experiment method, take the OD value as evaluation index, determine the cellulase optimal condition.The design of quadrature gauge outfit sees Table 1:

Table 1 factor level table [L ₉(3 ⁴)]

The result is carried out range analysis, determine the cellulase optimal condition, it the results are shown in Table 2.

Table 2 Orthogonal experiment results and analysis

Can be found out that by Orthogonal experiment results in selected four factors that affect the cellulase effect, its influence degree is C＞B＞A＞D, optimum combination is A ₂B ₂C ₃D ₂, i.e. enzyme dosage 90IUg ^-1, temperature 50 C, enzymolysis time 3.5 hours, pH value 5.0.

(2) neutral proteinase is on the impact of black fungus, hericium erinaceum polysaccharide recovery rate

A. enzyme concentration is on the impact of black fungus, hericium erinaceum polysaccharide recovery rate

Except containing polysaccharide, also contain the compositions such as a certain amount of protein, colloid, crude fibre and fat in black fungus, the hericium erinaceus fruiting body.The decomposition of these materials is conducive to stripping, separation and the purifying of glucide.As seen from Figure 5, the neutral proteinase addition surpasses 120IUg ^-1After, it is little that polysaccharide extract rate increases trend, and the enzyme addition crosses conference and increase the polysaccharide extraction cost, and therefore, selecting the neutral proteinase addition is 120IUg ^-1About carry out orthogonal experiment.

The B.pH value is on the impact of Blackfungus polyhexose recovery rate

In the constant situation of other condition, enzyme require acts in its optimal pH scope just can obtain preferably effect, and neutral proteinase has been adopted in this experiment, as seen from Figure 6, the Auricularia polysaccharide recovery rate is the highest in the pH7-8 left and right sides, therefore selects this scope to carry out orthogonal experiment.

C. enzymolysis time is on the impact of Blackfungus polyhexose recovery rate

Enzymolysis time is the factor that directly affects extraction effect, having set 5 different action times from 1h to 5h tests, therefrom determine an enzymolysis time the suitableeest, the result as shown in Figure 7, enzymolysis 2h left and right sides polysaccharide extract rate is the highest, and along with the prolongation of time, polysaccharide extract rate increases and is not obvious, therefore selects to carry out about enzymolysis time 2h orthogonal experiment.

D. hydrolysis temperature is on the impact of black fungus, hericium erinaceum polysaccharide recovery rate

As seen from Figure 8, the optimum temperature of neutral proteinase is 40～50 ℃, so hydrolysis temperature selects this scope to carry out orthogonal experiment.

E. the neutral proteinase optimal condition determines

Neutral proteinase affects most important four factors of polysaccharide yield: enzyme concentration, enzymolysis time, hydrolysis temperature and pH value, adopt L ₉(3 ⁴) orthogonal experiment method, take the OD value as evaluation index, determine the neutral proteinase optimal condition.The design of quadrature gauge outfit sees Table 3:

Table 3 factor level table [L ₉(3 ⁴)]

The result is carried out range analysis, determine the neutral proteinase optimal condition, it the results are shown in Table 4.

Table 4 Orthogonal experiment results and analysis

Can be found out that by Orthogonal experiment results in selected four factors that affect the neutral proteinase effect, its influence degree size is D＞C＞B＞A, optimum combination is A ₂B ₁C ₃D ₂, i.e. enzyme dosage 120IUg ^-1, temperature 45 C, enzymolysis time 2.5h, pH value 7.0.

2. isolating protein: polysaccharide extraction liquid is in the thick polysaccharide that concentrated, alcohol precipitation obtain, polyoses content is very low, protein is topmost impurity in the thick polysaccharide, its existence has increased the hygroscopicity of polysaccharide, and its electrically charged adsorbable a large amount of other impurity of institute, in removing the process of other impurity, can adsorb polysaccharide again, cause polysaccharide loss, make the grading purification difficult of polysaccharide.In addition, protein tool thermal source, the polysaccharide that contains albumen impurity can not be used for injection.So the first step of polysaccharide purification is taken off albumen exactly.The Sevage method is to remove the effective ways of floating preteins.In polysaccharide solution, add chloroform-n-butanol mixed solution and carry out shake well, the floating preteins sex change is become insoluble substance, through centrifugation, can reach the purpose of removing protein.

3. decolouring: often contain the impurity such as protein, pigment, compound sugar, monose in the thick polysaccharide, must be removed.Because the ratio of viscosities of Blackfungus polyhexose extract is larger, the decolouring difficulty is very large.The present invention has adopted activated carbon decolorizing and H ₂O ₂The two kinds of methods of decolouring.

Active carbon is the most frequently used discoloration method, and it relies on Van der Waals force that pigment is adsorbed onto activated carbon surface, and the particle of active carbon is less, and its surface area is larger, and adsorption capacity is stronger.Active carbon meeting absorbed portion polysaccharide causes polysaccharide loss.And find that through test extract can be partly dissolved active carbon and be colloidal state, and bring very large difficulty to subsequent technique, therefore, the method is not suitable for removing of Blackfungus polyhexose pigment.

Black fungus, hericium erinaceum polysaccharide are the polysaccharide of originated from fungus, owing to contain phenolic compound, so the thick polysaccharide that extracts is brown, and the alkalescence of solution is stronger, and color is darker.This class pigment is the negativity ion mostly, and the adsorbents such as use active carbon can't decolour.And this class material contains unsaturated double-bond, hydroxyl, aromatic rings etc. more, under alkali condition can and oxidant reaction, thereby reach the purpose of decolouring.Hydrogen peroxide is a kind of liquid of no color or smell, can mix with any ratio with water, and its oxidisability is stronger, acidity a little less than, it has been generally acknowledged that the bleaching principle of hydrogen peroxide is to rely on the hydroperoxy-radical ion HO that it ionizes out in the aqueous solution ₂ ^-Go the result of attack pigment.Under the weak base condition, H ₂O ₂Degree of ionization increases, and forms easily HO ₂ ^-, and HO ₂ ^-Be again a kind of nucleopilic reagent, easily cause hydrogen peroxide decomposes, produce free radical, free radical in case with the pigment effect, just can make it decolouring.For avoiding the polysaccharide degraded, bleaching temperature should be not too high, again through dialysing except the little molecule pigment after the deoxidation, black fungus, hericium erinaceum polysaccharide color shoaled after decolouring is processed.Black fungus, hericium erinaceum polysaccharide after decolouring is processed are light yellow.

4. concentrated: temperature is controlled at about 50 ℃, and uses low-pressure steam, can improve evaporation rate, prevents the heating surface hot-spot.Remove about 70%～80% moisture.

The preparation of embodiment 2 Bee Pollen active ingredient concentrates:

1. the impurity in the removing Bee Pollen adds water by 1: 10 part by weight, transfers pH to 3.0-5.0 with acid, adds cellulase CA complex enzyme (cellulase: α-amylase=6: 1) 30-150IUg ^-1, 25-65 ℃ of constant temperature enzymolysis 16-32h is warming up to rapidly 80 ℃ of enzymes that go out, lixiviate 6h, centrifugation Bee Pollen extract.

The factor that affects the pollen enzymic degradation mainly contains enzyme dosage, concentration of substrate, hydrolysis temperature, pH value and enzymolysis time.

(1) enzyme concentration determines

Bee Pollen dry powder was transferred pH to 4.5 by 1: 10, added respectively cellulase CA complex enzyme 30IUg ^-1, 60IUg ^-1, 90IUg ^-1, 120IUg ^-1, 150IUg ^-1, 45 ℃ of constant temperature enzymolysis 24h are warming up to rapidly 80 ℃ of enzymes that go out, and lixiviate 6h is centrifugal, filters constant volume.Measure protein content, the result of calculation of its thick protein recovery rate is as follows:

The different enzyme concentrations of table 5 are on the impact of thick protein recovery rate

By the experimental result of table 5 as seen, along with the increasing of enzyme dosage, the thick protein recovery rate has the trend of rising.When enzyme dosage just began to strengthen, protein degree increased comparatively obvious, was 90IUg but work as enzyme dosage ^-1When above, ascendant trend eases up.Therefore, consider economic aspect, the selection enzyme dosage is 90IUg ^-1Better.

(2) the pH value is on the impact of Bee Pollen effective component extraction rate

Bee Pollen dry powder transferred respectively pH respectively to 3.0,3.5,4.0,4.5,5.0 by 1: 10, added CA complex enzyme 60IUg ^-1, 45 ℃ of constant temperature enzymolysis 24h are warming up to rapidly 80 ℃ of enzymes that go out, and lixiviate 6h is centrifugal, filters constant volume.Measure protein content, the result of calculation of its thick protein recovery rate is as follows:

The different pH values of table 6 are on the impact of thick protein recovery rate

By the experimental result of table 6 as seen, the pH value is more important on the impact of mmp reaction speed.In general, a kind of enzyme only just has the highest vigor in narrow pH scope.Be that 4.5 o'clock effects are better by the visible pH value of result of the test.

(3) enzymolysis time is on the impact of Bee Pollen effective component extraction rate

Bee Pollen dry powder was transferred pH to 4.5 by 1: 10, added CA complex enzyme 60IUg ^-1, 45 ℃ of constant temperature enzymolysis 16h, 20h, 24h, 28h, 32h are warming up to rapidly 80 ℃ of enzymes that go out, and lixiviate 6h is centrifugal, filters constant volume.Measure protein content, the result of calculation of its thick protein recovery rate is as follows:

The different enzymolysis times of table 7 are on the impact of thick protein recovery rate

By table 7 experimental result as seen, along with the prolongation of hydrolysis time, degree of hydrolysis increases.But simultaneously along with the prolongation of hydrolysis time, the corresponding reduction of hydrolysis rate in the unit interval.Therefore, considering the selection hydrolysis time is that 24h is better.

(4) hydrolysis temperature is on the impact of Bee Pollen effective component extraction rate

Bee Pollen dry powder was transferred pH to 4.5 by 1: 10, added CA complex enzyme 60IUg ^-1, at 25 ℃, 35 ℃, 45 ℃, 55 ℃, 65 ℃ constant temperature enzymolysis 24h, be warming up to rapidly 80 ℃ of enzymes that go out respectively, lixiviate 6h, centrifugal, filter constant volume.Measure protein content, the result of calculation of its thick protein recovery rate is as follows:

The different hydrolysis temperatures of table 8 are on the impact of thick protein recovery rate

By table 8 experimental result as seen, along with temperature begins to rise, protein degree is also along with rising, and when temperature reached 45 ℃, protein degree was maximum, and temperature raises again, and protein degree descends on the contrary, therefore, select hydrolysis temperature be 45 ℃ better.

(5) cellulase CA complex enzyme optimal condition determines

By above-mentioned experiment of single factor result, determine that cellulase CA complex enzyme affects most important four factors of Bee Pollen effective component extraction rate: enzyme concentration, enzymolysis time, hydrolysis temperature and pH value, adopt L ₉(3 ⁴) orthogonal experiment method, take the thick protein recovery rate as evaluation index, determine the suitableeest hydrolysising condition of cellulase CA complex enzyme.The design of quadrature gauge outfit sees Table 9:

Table 9 factor level table [L ₉(3 ⁴)]

Measure protein content, the result of calculation of its thick protein recovery rate is as follows:

Table 10 Bee Pollen enzymolysis optimised process orthogonal test table

According to the size of extreme difference, can get each factor and on the primary and secondary of the impact of protein hydrolysis degree sequentially be: B (enzyme dosage)＞A (enzymolysis time)＞C (pH value)＞D (temperature), best enzymolysis process condition is A ₃B ₃C ₁D ₂, i.e. enzyme dosage (E/S) 90IUg ^-1, enzymolysis time 28h, the pH value is 4.0, hydrolysis temperature is 45 ℃.

2. the Bee Pollen enzyme is extracted slag and adds water, acid adding, boil hydrolysis after, with the alkali neutralization, centrifugation obtains the Bee Pollen extract.Extracted twice liquid merges Vacuum Concentration and namely gets the Bee Pollen concentrate.

In the concentration process, temperature is controlled at about 50 ℃, and uses low-pressure steam, can improve evaporation rate, prevents the heating surface hot-spot.Remove about 70%～80% moisture.

The preparation of embodiment 3 nutritional complexing agent electuaries:

Bee Pollen active ingredient concentrate 10～30 weight portions of black fungus, hericium erinaceum polysaccharide concentrate 70～90 weight portions and the embodiment 2 of embodiment 1 are mixed, add the common drugs such as dextrin, whey powder, sucrose and learn excipients or auxiliary material, stirring and dissolving, use high pressure homogenizer to carry out homogeneous, and then carry out spray-drying.Be the powder finished product after the cooling.

Described materia medica excipients or auxiliary material are maltodextrin and whey powder.When spray-drying, dextrin and whey powder can replace the hydrone that loses and stability when making skeletal substance keep similar hydrated state.

High-pressure homogeneous general employing double-stage homogenization, namely material is made immediately the lower second level homogeneous of pressure and is processed after the higher one-level homogeneous of pressure.Emulsifying agent is distributed on the contact interface better, emulsion stability is improved greatly.Final definite homogeneous parameter is: one-level homogenization pressure 20MPa, time 10min; Double-stage homogenization pressure 5MPa, time 5min, 60 ℃ of homogenizing temperatures.

Determining of best spray-drying condition:

(1) feeding temperature determines

The viscosity of solution of the present invention is subjected to the impact of temperature larger, at this moment suitably improves feeding temperature, can reduce charging viscosity, thereby improves input concentration, simultaneously on product quality also to some extent impact, as shown in Figure 9.Therefore, the temperature that improves feed liquid just can have been ignored the impact of product quality.Can increase thermal energy consumption and improve feed temperature, thereby increase cost.Therefore, this experiment has determined that feeding temperature is 40 ℃.

(2) EAT determines

As shown in Table 11: the best EAT of emulsion spray drying is 180～200 ℃.Improve EAT to a certain degree, moisture is evaporated rapidly, guaranteed the flowability that product is good.But EAT is high, but the oxidation deterioration of expedite product.

Table 11 EAT is on the spray-dired impact of electuary of the present invention

Only have EAT is controlled between 180～200 ℃, just can make at the bottom of the tower without wet pulp, reduce the wet-milling wall sticking phenomenon.

(3) leaving air temp determines

Leaving air temp should be complementary with suitable EAT.Leaving air temp is crossed and low moisture can not fully be evaporated, and causes the wet pulp in the tower, and the steam in the tower can be condensed into water and be adsorbed on the dry surface of microcapsule, sticks together and affects the quality of product.So leaving air temp should be in optimum range.As shown in table 12.

Table 12 leaving air temp is on the spray-dired impact of electuary of the present invention

As seen from the above table, the best leaving air temp during spray-drying is 80～100 ℃.

The quality analysis of electuary is measured

(1) mensuration of main chemical

Table 13 main chemical

(2) sanitary index

1. total number of bacteria: 23000cfu/g.

2. coliform:＜30cfu/100g.

The preparation of embodiment 4 nutritional complexing agent capsules of the present invention:

Content comprises black fungus, the hericium erinaceum polysaccharide concentrate 70-90 weight portion of embodiment 1, the Bee Pollen active ingredient concentrate 10-30 weight portion of embodiment 2, and seed of Dwarf Siberian Pine oil (food-grade) 10-20 weight portion.It is for subsequent use at first black fungus, hericium erinaceum polysaccharide concentrate and Bee Pollen active ingredient concentrate to be prepared the powder finished product according to the method for embodiment 3.

Auxiliary Liquid Material is corn oil and surfactant Tween-80 and suspending agent yellow wax.Plasticizer is glycerine, both mixtures of sorbierite, and ratio is 5: 1; Plasticizer: dry gelatin=0.6: 1.0, water is 1: 1 with the ratio of dry gelatin; Smoke agent for shielding titanium dioxide, its consumption are every kg gelatin 4g; The anticorrisive agent mixture of methyl p-hydroxybenzoate (1.6%) and propylparaben (0.04%).30～35 ℃ of baking temperatures, forced air drying under relative air humidity 20% condition.

(1) preparation glue

Get gelatin, the distilled water that adds 0.8 times of gelatin amount makes its imbibition, in addition in putting of the water glue tank with 0.5 times of glycerine and gelatin amount, be heated to 70～80 ℃, mix, add the gelatin that expands and stir, make and dissolve into even glue, insulation 1～2h vacuumizes the bubble of getting rid of in the glue, and it is stand-by to filter glue.

(2) adhesive tape manufacturing

Glue is made adhesive tape by glue box and drum automatically in automatic rotary transformation of ownership capsule machine, the thickness of adhesive tape is controlled by the gap of regulating between oily axle and the drum.

(3) fill compacting

Above-mentioned powder finished product and seed of Dwarf Siberian Pine oil are stirred, by the hopper of the automatic rotary transformation of ownership capsule machine filling pump of flowing through, enter to roll and be packed into adhesive tape in the mould and make soft capsule through rolling mould spinning.In the automatic rotary transformation of ownership capsule machine production process, indoor temperature should be controlled at 20～24 ℃, relative air humidity 35%.Every weight of soft capsule (420 ± 42) mg that requires compacting to form, capsule 's content net weight (280 ± 14) mg.

(4) drying

The soft capsule that the filling compacting forms is by collection chute and conveying device, enter in the drying machine after removing waste product, finalize the design and preliminarily dried, drying machine is comprised of 4 joint rotating cages and 2 opposed blower fans, the about 4h of forced air drying under 30～35 ℃ of baking temperatures, relative air humidity 20% condition makes the moisture of soft capsule shell reach 6%～8% scope.The scrap rubber that produces in compacting capsule process simultaneously also enters the scrap rubber bucket by conveying device and reclaims.

(5) shampooing ball

Owing to adopt in the production process of soft capsules vegetable oil to carry out the lubricated of capsule machine, at capsule outer surface some vegetable oil of adhesive tape inevitably, it also is that capsule is polished in clean that this operation is wiped examination with adhesive tape at capsule outer surface edible oil.Solution flush away capsule surface oil reservoir in washing pill wiping machine with V (ethanol): V (acetone)=5: 1 dries up washing lotion.

(6) final dry

Final drying claims again whole ball, in further dry to soft capsule through the long period, control higher relative air humidity and impel soft capsule smooth surface, full, making in the capsule drying not can surperficial dehydration, shrivelled, and impels the shape of capsule and size further to keep evenly.Capsule is moved into basin, 21～24 ℃ of temperature, dry 30h in the hothouse of relative humidity 30%～40%.

(7) check, count

Dried capsule separates substandard product with shaking screen, then rejects the undesirable product of color and luster with the color and luster grader, carries out Auto-counting with electronic counter at last, and packing is sealed up for safekeeping and got product automatically.20～24 ℃ of the storage requirement temperature of soft capsule, relative humidity 50%.

Target level of product quality

A. sense index

The health-care efficacy research of experimental example 1 nutritional complexing agent soft capsule of the present invention

The employing Kunming mouse (26 ± 2g), male, provided by animal housing of province tumour hospital; The Wistar rat (200 ± 20g), male, provided by animal housing of province tumour hospital.

Adopt nutritional complexing agent soft capsule of the present invention (below be called extract of the present invention); High lipid food prescription: yolk powder 10%, lard 8%, pig cholate 0.5%, basal feed 81.5%.

1, the research of extract hypoglycemic activity of the present invention

(1) dosage grouping and given the test agent give the time

Three dosage groups and a model control group are established in experiment, establish simultaneously intact animal group and a blank group of giving extract sample high dose of the present invention, 10 every group.Given the test agent gives 30 days time.

(2) reduce the fasting blood-glucose experiment

1. hyperglycemia model animal

Behind the Male Kunming strain mice fasting 24h, lumbar injection alloxan (180mg/kg body weight).The tail point is got the hematometry fasting blood-glucose after 7 days, and blood glucose value 10～25mmol/L is hyperglycemia model success animal.

Select the hyperglycemia model animal to be divided at random model control group and three dosage groups (the poor 1.1mmol/L that is not more than between group) by fasting blood-glucose, every group 10, press 5 of human body recommended amounts, 10,20 times are made as respectively low, in, a Senior Three dosage group, low dose group gives the 100mg/kg body weight extract of the present invention, middle dosage group gives the 200mg/kg body weight extract of the present invention, high dose group gives the 400mg/kg body weight extract of the present invention, take every day the gavage amount extract of the present invention is mixed with the solution of respective concentration as 2% of Mouse Weight, model control group gives respective volume distilled water.Gavage is 30 days continuously, surveys fasting blood sugar, relatively each treated animal blood glucose value and blood sugar decline percentage.

Blood glucose value * 100% before blood sugar decline percentage=(blood glucose value after blood glucose value before the experiment-experiment)/experiment.

(2) intact animal

Male Kunming strain mice is by the fasting blood-glucose random packet, selects at random tested group of 1 control group and 1 high dose extract of the present invention, and all the other operate with the hyperglycemia model animal.

(3) sugar tolerance experiment

The hyperglycemia model animal is divided into model control group and three dosage groups at random by fasting blood-glucose, every group 10, press 5 of human body recommended amounts, 10,20 times are made as respectively low, in, a Senior Three dosage group, low dose group gives the 100mg/kg body weight extract of the present invention, middle dosage group gives the 200mg/kg body weight extract of the present invention, high dose group gives the 400mg/kg body weight extract of the present invention, model control group gives respective volume distilled water, gave glucose 2.0g/kg by mouth in 15-20 minute, measure behind the glucose 0,0.5,2 hours blood glucose value, observe model control group and given the test agent group to glucose after the variation of each time point Area under the curve of blood glucose.

Area under the curve of blood glucose=1/2 * (0 hours blood glucose value+0.5 hours blood glucose value) * 0.5+1/2 * (2 hours blood glucose values+0.5 hours blood glucose value) * 1.5=0.25 * (0 hours blood glucose value+4 * 0.5 hours blood glucose values+3 * 2 hours blood glucose values)

2, the research of extract antioxidation of the present invention

(1) dosage grouping and given the test agent give the time

Animal used as test is that (26 ± 2g), three dosage groups and a blank group, 10 every group are established in experiment to Male Kunming strain mice.Low dose group gives the 100mg/kg body weight extract of the present invention, middle dosage group gives the 200mg/kg body weight extract of the present invention, high dose group gives the 400mg/kg body weight extract of the present invention, take every day the gavage amount extract of the present invention is mixed with the solution of respective concentration as 2% of Mouse Weight, the blank group gives respective volume distilled water.Given the test agent gives 30 days time.After feeding end, take a blood sample behind the mouse fasting 12h, anticoagulant heparin is measured every Antioxidant Indexes.

(2) mensuration of TAC

1. assay method: many polyphenoils are arranged in the body, can make Fe ³⁺Be reduced into Fe ²⁺, the latter can form stable complex compound with luxuriant and rich with fragrance quinoline class material, can measure the height of its oxidation resistance by colorimetric.

2. operating procedure: see Table 14, after reagent three is used liquid and added, with the abundant mixing of eddy blending machine, put 37 ℃ of water-bath 30min, with the abundant mixing of eddy blending machine, place 10min behind adding reagent four and the sample, the distilled water zeroing, the 1cm optical path, the 520nm place surveys respectively manages light absorption value.

Table 14 TAC assay method

3. the calculating of TAC in the blood plasma: in the time of 37 ℃, when every milliliter of blood plasma of per minute makes the every increase by 0.01 of light absorption value (OD) of reaction system, be a TAC unit.Computing formula is as follows:

(3) superoxide dismutase is measured

1. assay method: adopt xanthine oxidase

2. operating procedure: see Table 15, reagent and sample with the abundant mixing of eddy blending machine, are put 37 ℃ of water-baths 40 minutes.Add developer, colorimetric.

Table 15SOD assay method

3. calculate the calculating of SOD vigor in the blood plasma: corresponding SOD amount was a unit of activity (U) when the SOD inhibiting rate reached 50% in every milliliter of reactant liquor.Computing formula:

(4) MDA (MDA) is measured

1. method of testing: adopt thiobarbituricacidα-(TBA) colorimetric method.

2. operating procedure: see Table 16, with the abundant mixing of eddy blending machine, the test tube mouth is tightened with preservative film, stings an aperture with syringe needle, 95 ℃ of heating water baths (or uncap boil with pot) 40 minutes, flowing water cooling after taking out, then the centrifugal 10min of 3500～4000r/min gets supernatant, the 532mm place, the 1cm optical path, the distilled water zeroing is surveyed and is respectively managed light absorption value.

Table 16MDA assay method

3. MDA computing formula:

(4) glutathione peroxidase is measured

1. assay method: two sulfo-dinitrobenzoic acid (DTNB) colorimetric methods

2. operating procedure: see Table 17.

Table 17MDA assay method

Enzymatic reaction:

Mixing, 3500～4000r/min centrifugal 10 minutes, gets supernatant 1.5ml and makes chromogenic reaction.

Chromogenic reaction:

Mixing, after 15 minutes, the 412nm place, 1cm optical path cuvette, the distilled water zeroing is surveyed and is respectively managed the OD value.

3. the calculating of GSH-PX enzyme activity: stipulate that every 0.1ml serum 37 ℃ of reactions 5 minutes, deducts non-enzymatic reaction effect, making in the reaction system GSH concentration reduce by 1 μ mol/L is an enzyme activity unit.

GSH-Px vigor computing formula:

3, the research of extract effect for reducing blood fat of the present invention

(1) dosage grouping and given the test agent give the time

Select healthy Wistar rat, experiment divides six groups: Normal group, high fat control group, low dosage extract group of the present invention, middle dosage extract group of the present invention, high dose extract group of the present invention and positive controls (Zhibituo), except the basal feed group, other each group all adopts high lipid food.Wherein the dosage group is made as respectively basic, normal, high three dosage groups by 2.5,5,10 times of human body recommended amounts, take every day the gavage amount extract of the present invention and Zhibituo be mixed with the solution of respective concentration as 2% of rat body weight, low dose group gives the 50mg/kg body weight extract of the present invention, middle dosage group gives the 100mg/kg body weight extract of the present invention, high dose group gives the 200mg/kg body weight extract of the present invention, basal feed control group and high lipid food control group give respective volume distilled water, positive controls gives the 70mg/kg Zhibituo, and experiment periods is 30 days.After feeding end, rat fasting 12h gets blood, the centrifugal 15min of 3000r/min, and separation of serum is to be measured.Experimental project is body weight, serum total cholesterol, triglycerides, HDL-C and LDL-C.

(2) assay method of TC, TG, HDL-C, LDL-C in the serum

1. the mensuration of T-CHOL (TC)

Assay method: enzymic colorimetric (CHOD-PAP method)

Operating procedure: get one bottle of R1 of 10ml R2 redissolution, be working solution after the dissolving.

Table 18 T-CHOL (TC) assay method

After preparing solution with the method in the table, mixing respectively, 37 ℃ of insulations 6 minutes, in 500nm place, 1cm optical path cuvette, zero with reagent blank pipe school, with 722 spectrophotometric determination A _StandardAnd A _Sample

The total cholesterol concentration computing formula:

Wherein: normal concentration is 5.17mmol/L

2. the mensuration of triglycerides (TG)

Assay method: enzymic colorimetric (GPO-PAP method)

Operating procedure: take out R1 and R2 from kit, the R1 that gets 10ml adds among one bottle of R2, is working solution after the dissolving, and working solution is incubated in advance to probe temperature.

After preparing solution with the method in the table, respectively solution is mixed, 37 ℃ of reaction temperatures insulation 10 minutes, in 500nm place, 1cm optical path cuvette, zero with reagent blank pipe school, with 722 its A of spectrophotometric instrumentation _StandardAnd A _Sample

Table 19 triglycerides (TG) assay method

The triglyceride concentration computing formula:

Wherein: normal concentration is 2.26mmol/L

3. the mensuration of HDL-C (HDL-C)

Assay method: phosphoric acid tungsten-magnesium precipitate method

Operating procedure: get one bottle of R1 of 10ml R2 redissolution, be working solution after the dissolving.Serum is mixed with 1: 1 with precipitating reagent R3, and room temperature was placed after 10 minutes, and centrifugal 15 minutes of 3000r/min gets supernatant and does cholesterol determination, must finish in 4 hours.

Table 20 HDL-C (HDL-C) assay method

After preparing solution with the method in the table, mix respectively, 37 ℃ of insulations 6 minutes, in 500nm place, 1cm optical path cuvette, zero with reagent blank pipe school, read A _StandardAnd A _Sample

HDL-C concentration computing formula:

Wherein: normal concentration is 1.47mmol/L

*---serum diluting multiple

4. the mensuration of LDL-C (LDL-C)

Assay method: polyvinyel sulfate

Operating procedure: first with R ₃5.25ml with R ₄The thorough mixing of 1ml is the LDL-C precipitating reagent.Serum is mixed with 2: 1 with precipitating reagent, and room temperature was placed after 15 minutes, and centrifugal 15 minutes of 3000r/min gets supernatant and makes cholesterol determination, and after serum and the precipitant mix, standing time must not be above 1 hour.Then get 10mlR ₂Redissolve 1 bottle of R ₁, be working solution after the dissolving.

Table 21 LDL-C (LDL-C) assay method

Mix respectively, 37 ℃ of insulations 6 minutes, in the 500nm place, 1cm optical path cuvette with reagent blank pipe school zero, read A _StandardAnd A _Sample

The concentration of low density lipoprotein cholesterol computing formula:

LDL-C concentration=T-CHOL-supernatant cholesterol (mmol/L or mg/dl)

Wherein: normal concentration is 2.56mmol/L

4, data are processed

All (x ± s), statistics adopts SPSS11.0 software to carry out variance analysis and t check to all data, and P＜0.05 represents significant difference, and P＜0.01 expression difference is extremely remarkable with (mean ± standard deviation) expression.

5, results and analysis

5.3.1 the research of extract hypoglycemic activity of the present invention

(1) reduces the fasting blood-glucose experimental result

Can be found out by table 22 and Figure 10, during the experiment beginning, blood glucose value between model control group and each dosage group is all without significant difference (P＞0.05), after testing 30 days, model control group and low dose group blood glucose value have by a relatively large margin rising, compare remarkable rising (P＜0.01) during with this group experiment beginning, middle dosage group and high dose group blood sugar descend when beginning than the experiment of this group, middle dosage group blood sugar decline percentage is 1.0%, high dose group blood sugar decline percentage is 44.1%, the low dose group there was no significant difference of comparing with model control group, middle dosage group and high dose group all are starkly lower than model control group (P＜0.01).

Table 22 extract of the present invention is on the impact of diabetic mice fasting blood-glucose

Compare with the contemporaneity model control group a:P＜0.05; Compare with the contemporaneity model control group b:P＜0.01

As shown in Table 23, when blood glucose value was with this treated animal experiment beginning when the intact animal high dose group finished in experiment and the intact animal control group difference (P＞0.05) of comparing that there are no significant, visible extract high dose group of the present invention is to the intact animal fasting blood-glucose and have no significant effect.Therefore, can judge that the fasting blood-glucose experimental result positive falls in extract of the present invention.

Table 23 extract of the present invention is on the impact of normal mouse fasting blood-glucose

(2) sugar tolerance experimental result

Can be found out that by table 24 and Figure 11, Figure 12 the glucose tolerance curve of extract doses group tissue of experimental diabetic mice of the present invention obviously moves down than model control group, and obviously improve the sugar tolerance of day part point.Area under the curve of blood glucose was starkly lower than model control group (P＜0.01) after wherein dosage group and high dose group gave glucose in the extract of the present invention, compare with model control group, the low dose group Area under the curve of blood glucose reduces by 20.9% (P＜0.05), middle dosage group reduces by 34.1% (P＜0.01), and high dose group reduces by 54.3% (P＜0.01).Therefore, can judge that extract sugar tolerance experimental result of the present invention is positive.

Table 24 extract of the present invention is on the impact of diabetic mice sugar tolerance

Compare with model control group a:P＜0.05; Compare with model control group b:P＜0.01

(3) discuss

The present invention selects intact animal and alloxan to cause the diabetes model animal as point of penetration, extract of the present invention is regulated the function of blood sugar and is estimated.Alloxan (Alloxan) is a kind of β cytotoxic agent, and the beta Cell of islet that it can optionally damage many animals causes insulin secretion low, causes the test-type diabetes, and diuresis appears in animal, drink more, become thin and blood sugar significantly raises.Experimental result shows that extract of the present invention has no obvious effect to normal animal blood glucose level, may have no significant effect normal glycometabolism process; The alloxan diabetes mouse is had hypoglycemic activity, and can obviously improve the sugar tolerance of diabetic mice, this shows that extract of the present invention may weaken alloxan to the damage of beta Cell of islet or improve the function of the β cell of damaged.

Claims (13)

1. nutritional complexing agent with health-care efficacy, it is characterized in that, its main component comprises Blackfungus polyhexose, hericium erinaceum polysaccharide, Bee Pollen active ingredient, concrete is to comprise black fungus, hericium erinaceum polysaccharide concentrate 70-90 weight portion, Bee Pollen active ingredient concentrate 10-30 weight portion, the nutritional complexing agent powder for preparing through conventional electuary preparation method;

Described black fungus, hericium erinaceum polysaccharide concentrate are prepared by following steps:

A. extract polysaccharide solution: with black fungus 40-70 weight portion and Hericium erinaceus 10-20 weight portion through removal of impurities, drying, pulverize, sieving obtains black fungus, Hericium erinaceus dry powder; Gained dry powder is added in the buffer solution, add again cellulase and carry out the constant temperature enzymatic reaction; Regulate afterwards pH to 5.0-9.0, add neutral proteinase and carry out the constant temperature enzyme digestion reaction, after the enzyme lixiviate was gone out in intensification rapidly, centrifugal, filtration obtained polysaccharide solution;

B. isolating protein: in polysaccharide solution, add chloroform-n-butanol mixed solution and carry out shake well, the floating preteins sex change is become insoluble substance, after centrifugation, remove protein;

C. decolouring: adopt the H2O2 decoloring method to decolour, after decolouring is processed again through dialysis except the little molecule pigment after the deoxidation;

D. concentrated: temperature is controlled at about 50 ℃, uses low-pressure steam, removes about 70%～80% moisture, namely gets black fungus, hericium erinaceum polysaccharide concentrate;

Wherein, in the steps A, described black fungus, Hericium erinaceus dry powder are the buffer solutions that adds pH=3.0-7.0 by the part by weight of 1:20-40, add cellulase 30-150IUg ^-1, 20-60 ℃ of constant temperature enzymatic reaction 1-5h; Add neutral proteinase 60-300IUg ^-1, 20-60 ℃ of constant temperature enzymolysis 1-5h is warming up to rapidly 80 ℃ of enzyme lixiviate 2h that go out, and be centrifugal, filters;

Wherein, among the step B, described except albumen be to add the chloroform of 4:1/n-butanol reagent by black fungus, the hericium erinaceum polysaccharide aqueous solution 1/4 volume, thermal agitation 30min, centrifugal, inclining supernatant, albuminate and lower floor's chloroform in the middle of removing, repetitive operation until the intermediate layer without albuminate;

Wherein, among the step C, described decolouring is to adopt H ₂O ₂Method, with polysaccharide solution ammoniacal liquor adjust pH to 8.0,40 ℃ drip 5%H ₂O ₂, insulation 3h, dialysis gets black fungus, hericium erinaceum polysaccharide concentrate;

Described Bee Pollen active ingredient concentrate is prepared by following steps:

A. take by weighing the Bee Pollen of 10-20 weight portion, removal of contamination adds behind the water and transfers pH to acid with acid, adds cellulase CA complex enzyme, behind the constant temperature enzyme digestion reaction, and the lixiviate behind the enzyme of going out that heats up rapidly, centrifugation obtains the Bee Pollen extract; Described cellulase CA complex enzyme is the mixture of cellulase and α-amylase;

B. the Bee Pollen enzyme is extracted slag and adds water, acid adding, boil hydrolysis after, with the alkali neutralization, centrifugation obtains the Bee Pollen extract, extracted twice liquid merges Vacuum Concentration and namely gets Bee Pollen active ingredient concentrate;

Wherein, in the steps A, the part by weight that described Bee Pollen is pressed 1:8-12 adds water, transfers pH to 3.0-5.0 with acid again, adds cellulase CA complex enzyme 30-150IUg ^-1, 25-65 ℃ of constant temperature enzymolysis 16-32h is warming up to rapidly 80 ℃ of enzymes that go out, lixiviate 6h, centrifugation Bee Pollen extract.

2. nutritional complexing agent according to claim 1 is characterized in that, wherein said black fungus, hericium erinaceum polysaccharide concentrate 75 weight portions, Bee Pollen active ingredient concentrate 10 weight portions.

3. nutritional complexing agent with health-care efficacy, it is characterized in that, formed by content and softgel shell, the main component of its content comprises Blackfungus polyhexose, hericium erinaceum polysaccharide, Bee Pollen active ingredient and seed of Dwarf Siberian Pine oil, concrete is to comprise black fungus, hericium erinaceum polysaccharide concentrate 70-90 weight portion, Bee Pollen active ingredient concentrate 10-30 weight portion and seed of Dwarf Siberian Pine oil 10-20 weight portion; The composition of its softgel shell comprises gelatin, G ﹠ W, prepares through conventional capsule preparation method thereof; Wherein said black fungus, hericium erinaceum polysaccharide concentrate and Bee Pollen active ingredient concentrate are prepared by method claimed in claim 1.

4. nutritional complexing agent according to claim 3 is characterized in that, wherein said black fungus, hericium erinaceum polysaccharide concentrate 75 weight portions, Bee Pollen active ingredient concentrate 10 weight portions, seed of Dwarf Siberian Pine oil 15 weight portions.

5. the nutritional complexing agent with health-care efficacy according to claim 1 is characterized in that, among the preparation process A of described black fungus, hericium erinaceum polysaccharide concentrate, and the pH=4.5-5.5 of buffer solution; The addition of described cellulase is 60-120IUg ^-1Described enzymatic reaction temperature is 45-55 ℃ of reaction time 2.5-3.5h;

Described adjusting pH to 7.0-8.0; The addition of described neutral proteinase is 60-180IUg ^-1Described hydrolysis temperature 40-50 ℃ enzymolysis time 1.5-2.5h.

6. the nutritional complexing agent with health-care efficacy according to claim 5 is characterized in that, in the described steps A, and the pH=5.0 of buffer solution, the addition of described cellulase is 90IUg ^-1, described enzymatic reaction temperature is 50 ℃ of reaction time 3.5h;

Described adjusting pH to 7.0, the addition of described neutral proteinase is 120IUg ^-1, 45 ℃ of enzymolysis time 2.5h of described hydrolysis temperature.

7. the nutritional complexing agent with health-care efficacy according to claim 1, it is characterized in that, among the preparation process A of described Bee Pollen active ingredient concentrate, the cellulase CA complex enzyme of adding is cellulase: the weight ratio of α-amylase is the mixture of 5-7:1; The amount of its adding is 30-90IUg ^-1, described with acid accent pH to 4.0-5.0; Described hydrolysis temperature is that 40-50 ℃ of time is 24-28h.

8. the nutritional complexing agent with health-care efficacy according to claim 7 is characterized in that, in the described steps A, the cellulase CA complex enzyme of adding is cellulase: the weight ratio of α-amylase is the mixture of 6:1, and the amount of its adding is 90IUg ^-1, described is 4.0 with acid accent pH, described hydrolysis temperature is that 45 ℃ of times are 28h.

9. method for preparing nutritional complexing agent claimed in claim 1, it is characterized in that, that above-mentioned black fungus, hericium erinaceum polysaccharide concentrate 70-90 weight portion and Bee Pollen active ingredient concentrate 10-30 weight portion are mixed, add dextrin, whey powder, sucrose common drug excipients, stirring and dissolving, use high pressure homogenizer to carry out homogeneous, and then carry out spray-drying, namely obtain nutritional complexing agent powder finished product after the cooling;

Described high-pressure homogeneous employing double-stage homogenization, one-level homogenization pressure 20Mpa, time 10min; Double-stage homogenization pressure 5Mpa, time 5min; 60 ℃ of homogenizing temperatures;

Described spray-dired feeding temperature is 40-80 ℃, and EAT is 160-240 ℃, and leaving air temp is 60-140 ℃.

10. the preparation method of nutritional complexing agent according to claim 9 is characterized in that, described feeding temperature is 40 ℃, and EAT is 180-200 ℃, and leaving air temp is 80-100 ℃.

11. a method for preparing nutritional complexing agent claimed in claim 3 is characterized in that, comprises the steps:

A. get gelatin, the distilled water that adds 0.8 times of gelatin amount makes its imbibition, in addition in putting of the water glue tank with 0.5 times of glycerine and gelatin amount, be heated to 70～80 ℃, mix, add the gelatin that expands and stir, make and dissolve into even glue, insulation 1～2h vacuumizes the bubble of getting rid of in the glue, and it is stand-by to filter glue;

B. fill compacting: above-mentioned nutritional complexing agent powder and seed of Dwarf Siberian Pine oil 10-20 weight portion are stirred, by the hopper of the automatic rotary transformation of ownership capsule machine filling pump of flowing through, enter to roll and be packed into adhesive tape in the mould and make soft capsule through rolling mould spinning, in the automatic rotary transformation of ownership capsule machine production process, indoor temperature should be controlled at 20～24 ℃, relative air humidity 35%;

C. dry: as to fill soft capsule that compacting forms by collection chute and conveying device, enter in the drying machine after removing waste product, finalize the design, dry, whole ball, get product.

12. claim 1 or 3 described nutritional complexing agents are in the application of preparation field of health care food.

13. application according to claim 12 is characterized in that, the application in the health food of, effect for reducing blood fat hypoglycemic, anti-oxidant for having in preparation.

Images