CN102987501B - Method for preparing nutrient (liquid) beverages and granules of healthcare food (therapy) products by using extractives of cordyceps militaris and cocoons - Google Patents
Method for preparing nutrient (liquid) beverages and granules of healthcare food (therapy) products by using extractives of cordyceps militaris and cocoons Download PDFInfo
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Abstract
The invention relates to a method for preparing nutrient (liquid) beverages and granules of healthcare food (therapy) products by using extractives of cordyceps militaris and cocoons and aims to meet market requirements by producing nutrient healthcare products with different types by using various nutrient substances extracted from waste cocoons of the cordyceps militaris. The method is characterized by comprising the following steps of: firstly extracting the nutrient substances from the cordyceps militaris and the cocoons by adopting freeze thawing, combined enzymolysis and a water-extraction alcohol-precipitation sequential extraction method, and producing nutrient liquors and granules with different functions according to different functions (requirements) and different proportions. According to the method for preparing the nutrient (liquid) beverages and the granules of the healthcare food (therapy) products by using the extractives of the cordyceps militaris and the cocoons, the synergistic effects among nutritional ingredients are adequately exerted, and the use ratios of the cordyceps militaris and the cocoons are improved.
Description
Technical field
The present invention relates to a kind of preparation method of health care food (treatment) product, exactly, disclose a kind of Cordyceps militaris, cocoon extract of utilizing and prepared keep healthy nutrition (liquid) beverage of (treatment) product of food and the method for electuary.
Background technology
There are in the world at present a lot of countries to be in fashion the food silkworm chrysalis; The American cooks chocolate cream with silkworm chrysalis, and Australia generally uses silkworm chrysalis, and the Frenchman is considered as the delicious treasure of high protein, fetch long price to silkworm chrysalis.Also there are the food silkworm chrysalis in a lot of countries in Europe, so occur multiple silkworm chrysalis food on market as silkworm chrysalis biscuit, silkworm chrysalis milk, silkworm chrysalis wine, silkworm chrysalis can etc.
Studies show that: per kilogram silkworm chrysalis dry product contains approximately 60 grams of protein, fatty 30 grams, and chitin 1 gram, 18 seed amino acids wherein have 8 kinds to be needed by human, and the ratio of content are suitable.Meet the United Nations's grain tissue (FAO) and the World Health Organization's standard, also have several mineral materials and abundant vitamin B1, B2 and E and the trace elements of selenium such as Ca, P, Fe, linoleic acid, leukotrienes etc., its nutritive value surpasses egg 14.7%, fresh milk 4%.
Silkworm chrysalis is used with Dietotherapy health with a long history in China." peace is for the emperor's inspection " record " dish unit is extremely fertile ", Ming Dynasty's Compendium of Material Medica is classified silkworm chrysalis as tonic; " Dong-eui-bo-gam " claims " silkworm chrysalis is flat, distinguishes the flavor of sweet nontoxic, controls infantile malnutrition, and labor is thin, quenches one's thirst ".Wherein " infantile malnutrition due to digestive disturbances or intestinalparasites " is equivalent to modern infantile malnutrition and the disease that causes.So-called " labor is thin " is due to malnutrition, immunologic hypofunction and cause One's spirits are drooping depressedly, and body is empty thin thin, and is weak how sick.This shows that we ancestors have had understanding to the nutritive value of silkworm chrysalis for a long time.China is a lot of local to be had the food silkworm chrysalis and it is raised pigs as feed or make the feed of animal husbandry and fishery.
Cordyceps sinensis Cordyceps Sinensis (Beck) Sacc.? find that by further investigation " China is by hair spore Hirsutella
Licc, Guo, Yu et Zong " be the real phorozoon of Cordyceps sinensis.(beam man fine jade 2003) is the distinctive species of China.With Cordyceps sinensis called after " snow silkworm ", illustrate that its flavor is extremely sweet and refreshing as far back as the outstanding Compendium of Material Medica of Li Shizhen (1518-1593 A.D.) in 1590 Christian eras.The work essentials of Matea Medica of Wang Ang in 1964, Zhao Xuemin in 1756 work supplementary Amplifications of the Compendium of Materia Medica, Wu Yi Lip river work " new compilation of materia medica " in 1757, all discuss its have strong, the function such as nourish, detoxify, reduce phlegm.Xu Queensland says in its work " compiling outside Liu Ya ", Chinese caterpillar fungus and duck boiled dinner, the large benefit.Also state in " the little knowledge in tangerine garden ": " all aftercares and deficient people often take one (Chinese caterpillar fungus) duck, can support ginseng one or two." this shows that ancients are very high to the Appraisal on Medicinal Values of Chinese caterpillar fungus.(Hu Zhaogeng 1999.2)
Modern medical knowledge research shows, contain cordycepic acid, cordycepin, amino acid, vitamin, mineral element and various bioactivators in Cordyceps sinensis, have and improve immunity, the antitumor and function such as delay senility, therefore consumption increases, even cause predatory excavating, its growth needs again special ecological environment, impels these species frequently to face extinction (Li Xianglin, yellow wingceltis small stream 2004).Therefore, seek its new product (resource), be subject to very large attention both at home and abroad.
Cordyceps militaris (claiming again northern Chinese caterpillar Fungus, northern Chinese caterpillar fungus) Cordyceps milotaris (Lex.Fr.) Link, it is another fungi in Cordyceps.Colonize in the complex that the pupa growth of the various insects such as Lepidoptera forms, it is dispersed throughout all parts of the world, and the provinces such as China Jilin, Hebei, Shaanxi, Anhui, Guangxi, Yunnan, Gansu all are found.This Chinese caterpillar fungus was wild originally, also have medicinal nourishing effects, but output was few.Carry the function of " the Cordyceps militaris flavor is sweet, property flat, liver and kidney benefiting is arranged, mend marrow, hemostasis and phlegm " according to " Xinhua's book on Chinese herbal medicine outline "; " national Chinese herbal medicine compilation " record, the fructification of Cordyceps militaris and polypide also can be used as Cordyceps sinensis and are used as medicine.This shows, Cordyceps militaris has identical pharmacological function and clinical effectiveness with Cordyceps sinensis.
The artificial culture of Cordyceps militaris:
Experimental study both domestic and external shows, Cordyceps militaris includes active ingredient, and some has surpassed the Cordyceps sinensis of wild, and they can the endangered Cordyceps sinensis of Partial Replacement.Due to the use of Cordyceps militaris, demand increases day by day, and himself resource reduces increasingly.But in order to satisfy the demand of the growing health care of people, medicine research and development, people competitively carry out artificial test of cultivating to it.
From nineteen eighty-three, we are successively at Mt. Huang in Anhui, and the ground such as Gansu Ma Qiaoling collected wild Cordyceps militaris, through 7 years repetition tests, the Cordyceps militaris spawn that finally will pick up from Gansu Ma Qiaoling in nineteen ninety is cultivated successfully through taming and dociling to educate, and every test result all is better than the bacterial classification (strain) in other places.Enlarge on this basis and cultivate successfully, and can manufacture complete Cordyceps militaris complex, for every test, application.So far we with the bacterial classification called after Cordyceps militaris-LS901 bacterial strain of Wushao Ling, ask for an interview annex 1: bacterial classification.This bacterial strain obtains biology department of Nanjing University, the approval such as Japanese Hebei lagoon natural pond research institute in one's power of Taiwan Academia Sinica's plant research.
The success of Cordyceps militaris artificial culture for the medical-care health food industry provides vast development space, has been satisfied the demand in market.But the pupa worm cocoon body for discarded after the Cordyceps militaris industrial processes due to technical bottleneck, no longer further is used mostly.
Summary of the invention
Prepare nutrition (liquid) beverage of health care food (treatment) product and the method for electuary with Cordyceps militaris, cocoon extract, purpose is exactly the nutriment of the multiple maintenance natural activity that will extract from the Cordyceps militaris piques, be made into dissimilar nutrient and healthcare products, in order to fully improve the utilization rate of Cordyceps militaris and cocoon, about the artificial breeding technique of Cordyceps militaris, and Cordyceps militaris extract nutriment correlation technique we proposed to declare as the patent of invention technology respectively, do not give unnecessary details in the present invention, only with the active ingredient of Cordyceps militaris, CORDYCEPS as reference.
After we cultivated Cordyceps militaris successfully, department had submitted sample to coherent detection, and testing result is as follows:
One, the active ingredient of Cordyceps militaris and CORDYCEPS relatively
Table 1.SOD activity and cordycepin, the comparison of adenine
| Sample | SOD is active | Cordycepin mg.g -1 | Adenine |
| Cordyceps militaris | 57μ.ml -1 | 3.08 | 0.542 |
| Cordyceps sinensis | 14μ.ml -1 | 1.04 | 0.138 |
Table 2. constituent content relatively (PPm) detects 30 kinds of elements, now just lists part
Table 3. amino acid content compares:
Except above-listed material, still contain:
Jade-like stone peptide class: contain multiple dried meat ring dipeptides, bright ring dipeptides, figured silk fabrics ring dipeptides etc.Wherein L-§-L-bright ring dipeptides has effect anticancer and increase (by force) immunity.
Ucleosides: uracil, adenine, uridine, adenosine, cordycepin etc.
Steroid: daucosterol, campesterol, ergosterol, β-sitosterol etc.
Organic acid: aliphatic acid, oleic acid, linoleic acid, leukotrienes, palmitic acid, stearic acid, palmitic acid etc.
Alkaloid and polyamines class: nicotinic acid, niacinamide, phenoloid etc.
Two, the pharmacological action of silkworm chrysalis Cordyceps sinensis and health care thereof
A, on the impact of immunologic function
1. on the impact of monocytes/macrophages system and immune organ function
Cordyceps militaris water extraction liquid can promote the propagation of macrophage in body, and to the activate the phagocytic capacity of macrophage system, activation may be arranged, and principle may be that cordycepin (3-deoxyadenosine) promotes spleen DNA synthetic, accelerates division and the propagation of splenocyte; Show according to test, Cordyceps militaris also has radiation resistance.
2. on the impact of humoral immunity
Cordyceps militaris water extraction liquid can promote the formation of B cell proliferation and antibody.
3. on the impact of cellular immunity
Lymphocyte transformation has facilitation to Cordyceps militaris to T, and Cordyceps sinensis polysaccharide is the active ingredient of regulating immunologic function; Cordyceps militaris has significant inhibitory action to graft-rejection.
Cordyceps militaris also has inhibitory action to the existing facilitation of immune function, presents the feature of bidirectional modulation.
B, on the impact of central nervous system
1. sedation
Cordyceps militaris decocting liquid can obviously extend the length of one's sleep, and obvious sedation is arranged.
2. anticonvulsant action
Alcohol extract can obviously extend the incubation period of fainting from fear and occuring.
3. cooling effect
4. on the impact of activity of monoamine oxidase in brain
Activity of monoamine oxidase is old and feeble sign, and Cordyceps militaris can suppress activity of monoamine oxidase, delays senility.
5. on the impact of cerebral blood flow
The Cordyceps militaris alcohol extract can increase the nutrient flow amount of brain tissue.
C, on the impact of cardiovascular system
1. to action of the heart
Cordyceps militaris decocting liquid can obviously resist various arrhythmia cordis and myocardial anoxia, the obvious hypoxia-bearing capability of tool.
2. the effect of hemangiectasis
Cordyceps militaris alcohol extract tool hypotensive effect.
3. antiplatelet effects
The Cordyceps militaris alcohol extract has obvious inhibitory action to platelet aggregation, is thrombus prevention and cure agent more likely.
D, on the impact of respiratory system
1. expansion bronchus
Cordyceps militaris decocting liquid can significantly strengthen the adrenergic use of doing.
2. eliminating phlegm and relieving asthma effect
3. prevent and treat pulmonary emphysema
E, on the impact of metabolism
1. glycometabolism
Cordyceps militaris decocting liquid has remarkable hypoglycemic activity, and the beta cell of pancreas islet is had certain protective effect.(prevention and treatment diabetes)
2. fat metabolism
Cordyceps militaris decocting liquid can obviously reduce cholesterol, blood plasma beta lipoprotein content.
3. energetic supersession
Cordyceps militaris decocting liquid can promote the red blood cell glycolytic pathway to generate ATP; Increase liver cell energy charge value; Activate muscle endochylema CPK active, make ADP accept the CP energy and generate ATP.
The impact of F, Endocrine
Cordyceps militaris decocting liquid shows androgen-like action.
G, to thrombopoietic effect
The intramuscular injection of Cordyceps militaris decocting liquid can make the spleen coefficient increase, and the spleen megacaryocyte increases, and in blood, blood platelet raises; And platelet counts and quality all there is certain protective effect.
H, on the impact of stress reaction
1. anti-fatigue test
2. hypoxia endurance test
The Cordyceps militaris alcohol extract has anti-normobaric hypoxia effect.
I, antiinflammatory action
The obvious antiinflammatory action of tool
J, resisting pathogenic microbes effect
Without obvious antibacterial action, certain anti-amoeba effect is arranged
K, antitumor action
Silkworm chrysalis Cordyceps sinensis has obvious antitumor action, cardinal principle is: the synthetic a kind of lead of DNA of tumor cell is the 2-deoxyadenosine, and the active component cordycepin of Cordyceps militaris (3-deoxyadenosine) can emulative combination (invertibity reaction) the DNA of tumor cell synzyme, thereby affect the breeding of tumour cell.
L, on the impact of kidney
1. to the effect of Jishengshenqiwan
Silkworm chrysalis Cordyceps sinensis has good curative effect to ephritis, particularly the early treatment curative effect is more remarkable, cardinal principle: the Cordyceps sinensis polysaccharide in silkworm chrysalis Cordyceps sinensis is regulated by humoral immunity, can disturb the original position immune complex to form, GBM negative electrical charge barrier is preserved, reduce loss or the arrangement disorder of negative electrical charge, thereby albuminuria is alleviated.
2. to the effect of experimental chronic renal failure
The silkworm chrysalis Cordyceps sinensis alcohol extract can be alleviated azotemia, alleviates albuminuria, corrects disorders of lipid metabolism, promotes liver albumen synthetic, improves anaemia, the progress of delaying chronic kidney exhaustion.Principle: can replenish the essential amino acid, particularly branched-chain amino acid that lack, correct the inside and outside disorder of amino acid metabolism of cell, suppress " the clean decomposition " of myogen, promote that the protein of liver is synthetic, upgrade thereby improve protein, alleviate azotemia.
3. Drug protection
M, health care
After the fifties in last century, Canada scientist Cuningham extracted cordycepin from Cordyceps militaris, the scholar of various countries conducts in-depth research Cordyceps militaris and inclusion thereof, it is found that Cordyceps militaris includes several amino acids, vitamin, trace element, SOD, cordycepin, cordycepic acid, the bioactivators such as Cordyceps sinensis polysaccharide, each tool function of these bioactivators.
1. cordycepin
Cordycepin is kind of a novel broad-spectrum antibiotic, not only to virus, and bacterium, fungi has obvious inhibitory action, and can reduce vascular resistence coronarius, and increase coronary flow, improve the blood supply of cardiac muscle, reduce the heart oxygen demand, anti-arrhythmia reduces cholesterolemia and low-density lipoprotein, improves HDL, alleviate Hyperlipemia and artery sclerosis, also have the insulin secretion of adjusting and fall hypoglycemic effect more.Thereby performance prevents dyslipidemia and artery sclerosis indirectly.
Cordycepin more can be blocked material---the formation of guanylic acid that tumour cell breeding institute must dependence, tumoricidal metabolism and nutrition, thereby the transfer of inhibition tumor cell and hyperplasia.As itself and antineoplastic (as endoxan) are united use, its antitumor action obviously strengthens.Cordycepin can also strengthen in brain---and first amine oxidase is active.Studies show that: after the designed for old people Cordyceps militaris, simple intelligent condition grading (MMSD) obviously improves.Test can cause that with D-galactolipin hypodermic injection mouse mouse is old and feeble fast, gives the Cordyceps militaris extract and can obviously resist this aging.
2. cordycepic acid
Cordycepic acid, PEARLITOL 25C can infiltrate DNA of tumor cell, and inhibition tumor cell DNA copies.Have Green Tea Extract, anti-oxidant, hemangiectasis reduces platelet aggregation, prevents the thrombotic effect of heart and brain; And diuresis arranged, and get rid of toxin, the function such as enhance metabolism.Be used for the treatment of clinically cerebral thrombus, cerebral embolism, vasopasm, renal failure.
3. Cordyceps sinensis polysaccharide
Cordyceps sinensis polysaccharide can promote the B cell, the T lymphopoiesis, mononuclear phagocyte system and NK cell etc. there is obvious activation, can promote cell transformation, activate the immunocompetent cell of body, especially with the lymphocyte nuclear lymphokine, strengthen the ability of immunologic function and raising body immune system killing tumor cell.To senile chronic bronchitis, cor pulmonale has obvious curative effects, and can improve the detoxification ability of liver, plays liver protection effect.
Cordyceps militaris also has:
The comprehensive antioxidation of ◇; oxygen radical is a class group that produces in mankind's metabolic processes; they are very active; can destroy the even a lot of protease of human body cell film; increase along with the human body age; also fewer and feweri to the material of Green Tea Extract in body, and the SOD that Cordyceps militaris contains can be from the outside and inner ability that strengthens the human body antioxidant radical.
The regulating action of ◇ to central nervous system: the extract of Cordyceps militaris can be stablized central nervous system, has certain sedation, improves the elderly's sleep.
◇ Cordyceps militaris extract can improve the energy in the human body cell mitochondria, thereby has the raising physical efficiency, and the effect of antifatigue strengthens cold tolerance, and therefore, Cordyceps militaris has obvious anti-senescence function.
For more extensive and more deep integrated application is provided, we also give research and development application to the silk cocoon that abandons in the past etc. when cultivating the silkworm Chinese caterpillar fungus.And carry out a series of toxicity test, to eliminate edible doubt, people are familiar with to some extent again to its excellent nutrition and health care performance.
Three, theoretical research and pharmacology analysis
Experimental study shows, silk cocoon moisture 1.04%, ash content 1.14%, protein 97.31%, fiber 0.39%, ether-soluble extractive 0.26%, and silk is as long as formed by 70% fibrin (Fibroin) and 22% sericin (Soriein) bonding, and contain the formation cocoons such as a small amount of grease class, pigment, inorganic matter.
Sericin is a kind of keratin, and it forms take glycine, alanine as many.The mole number that contains glycine in the 10g silk-fibroin(s) is 578, alanine 381, valine 67, leucine 7, isoleucine 9, serine 157, threonine 12, tyrosine 67, phenylalanine 8, proline 5, tryptophan 2, lysine 4, histidine 2, arginine 6, asparatate 17, glutamic acid 13,1297 of total gram molecules.
Cocoon contains free amino acid 50mg%(in glycine), oil-containing waxy substance again, this material is by primary alconol class (C
26-C
32), solid fatty acid (C
26-C
32), paraffin (C
25-C
31) and the liquid fatty acid composition, and sitosterol or glycoside and Lupeol (Lupeol).
Cocoon is yellow orange sometimes, and is pink, taupe brown, the colors such as green.Mainly because pigment contains various carotenoid derivatives; The pigment of green cocoon is by flavonoids or chlorophyll, especially take wild silkworm as many.Someone isolates pigment and 2 kinds of yellowish-brown fluorchromes of 7 kinds of yellow-green fluorescences from green cocoon; Someone isolates 9 kinds of yellow fluorescence elements again, and they are all the glycosides of flavonoids, is worth thereby have special medical test.
Cocoon contains Mn7.06mg ∕ kg, Zn3.19mg ∕ kg, and the Mn content of yellow cocoon is 8.48mg ∕ kg, Zn3.67mg ∕ kg, and have the effect of the choline of plan.
According to the Compendium of Material Medica record, silk cocoon is sweet, and temperature is nontoxic.Be used to treatment and have blood in stool, hematuria, metrorrhagia is quenched one's thirst, gastric disorder causing nausea, the infantile malnutrition due to digestive disturbances or intestinalparasites sore, carbuncle is swollen, oral cavity anabrosis.
The international Main classification of Chinese patent application ZL00124887.1 number: C12N1 ∕ 14 discloses the method for utilizing the Cordyceps militaris powder to make capsule, but single Cordyceps militaris is only arranged in this capsule, so wherein nutrition, active material are also more single, and be directly take Cordyceps militaris as raw material.This Cordyceps militaris is not processed through the biotechnology enzyme, so the actual absorption utilization rate is lower; More there is no mutually auxiliary synergy each other between various nutrient substances.
Four, technical matters of the present invention:
The method for preparing health care food (treatment) product-nutrition (liquid) beverage and electuary with Cordyceps militaris, cocoon extract, it is characterized in that first by freeze thawing combination enzymolysis, and water extracting alcohol precipitation substep extraction method, extract the nutriment in Cordyceps militaris and cocoon, its extraction process is as follows:
A. get and soak in distilled water after Cordyceps militaris powder and cocoon powder mix, Cordyceps militaris/cocoon/water is 1:0.5:1.5, spends the night under room temperature, and under-20 ℃ of environment, multigelation is 2-4 time, and centrifugal (6000g ∕ 15 minutes) gets supernatant;
B. make up enzymolysis and extraction, 5 ~ 10 times of the Cordyceps militaris slag after freeze thawing and cocoon slag adding distil waters add lysozyme and pectase hydrolysis 2-3 hour, add respectively again amylase, papain, pepsin, trypsase, bromelain, carbohydrase, monascus, cellulase, each is hydrolyzed 0.5 ~ 1.0 hour, the centrifuging and taking supernatant;
C. the Cordyceps militaris slag after enzymolysis and cocoon slag adding distil water are 5 ~ 10 times, stir at 80 ℃ and extract 2 times, centrifugal merging supernatant;
D. the supernatant with above-listed (A.B.C) merges in rearmounted stainless steel or glass container, and big fire is boiled, and little fire is kept 3-4 hour → filtration → filtrate and continued 1 ∕ 4～1 ∕ 5 that heating is concentrated into volume, pours metering in clean container into, and is cooling, precipitation;
E. get cooling rear concentrate, will not precipitate residue and bring into, slowly add ethyl alcoh(ol);
Alcohol addition (ml)=75 * concentrate (ml)
When in mixed liquor, alcohol concentration was about 75%, polysaccharide became floccule to sink;
F. after hold over night, when the things such as polysaccharide all are sink to the bottom, first remove the part supernatant with siphon, precipitated liquid centrifugal (3500g ∕ 15 minutes) obtains being coffee brown look block → pulverizing after thickness paste → vacuum drying, i.e. the nutrition extract of Cordyceps militaris cocoon.
Enzyme in flow process B generally is used in conjunction with 2 kinds of enzymes, and its hydrolysis temperature is respectively: the hydrolysis temperature of papain is 45 ℃ ± 2 ℃, lysozyme, and pectase, amylase, pepsin, tryptic hydrolysis temperature are 37 ± 2 ℃; Bromelain, carbohydrase, monascus, the hydrolysis temperature of cellulase are 35 ± 2 ℃; The consumption of enzyme is: during 1 kilogram of Cordyceps militaris cocoon powder of every enzymolysis, and lysozyme 1 gram that adds food to use, amylase, papain, pepsin, each 2 grams of trypsase, bromelain, carbohydrase, monascus, each 3 grams of cellulase, pectase 4 grams.
The beneficial effect of this technique mainly contains:
1. owing to having taked the substep extractive technique, make the nutritional labeling in Cordyceps militaris, cocoon be fully extracted out;
2. when extracting with enzymatic isolation method, use different enzyme combination enzymolysis, both improved extraction effect, make again protein fully be converted into free amino acid;
In leaching process without post processings such as bronsted lowry acids and bases bronsted lowry and any organic solvents, this had both kept natural activity, and was free from environmental pollution again;
4. according to the processing of rear product and the difference of quality thereof, each goes on foot extract and both can use respectively, can be used in conjunction with again, thereby the quality of rear product is further improved, and effect is more outstanding.
5. technique is simple, and easy operating can be used for suitability for industrialized production.
The specific embodiment
According to above-mentioned technological process, after Cordyceps militaris and cocoon are mixed pulverizing, process through bioengineered enzyme again and (separate, extraction, synthetic) extraction active ingredient wherein, through chemically examining its main component be: free amino acid, active polysaccharide cordycepin, protein etc., carry out the clinical pathology experiment with the said extracted thing.
Clinical testing and interpretation of result
One, toxicity test
1. acute toxicity test in mice:
Select 40 male and female half and half of body weight 18 ~ 22g healthy mice.Get the Cordyceps militaris powder by gavage of dosage per os empty stomach of 20g ∕ kg.bw, observed for two weeks, record poisoning symptom and death condition.
Result within the observation period of 14 days, there are no any intoxicating phenomenon, none dead mouse.Show that this Cordyceps militaris cocoon powder is to male and female its mouse oral LD
50All greater than 10g ∕ kg.bw.Press the true border of acute toxicity dose grading standard nontoxic level material.
2. 30 days feeding trials of rat
1. establish three dosage groups, be respectively 0.63g ∕ kg.bw, 1.25g ∕ kg.bw, 2.5g ∕ kg.bw, maximum dose level have been equivalent to the adult and have used 150g ∕ D, have reached the drinkable water product of general vegetables.
2. feeding patterns: with 10% intake as every day of rat body weight, be made into the mash feed of different Cordyceps militaris cocoon powder contents by metering, control group is freely taken in to basal feed.
3. test method: select 40 of 55 ~ 70g ablactation rats, male and female half and half are divided into 4 groups at random by body weight, i.e. control group and 3 dosage groups.Cage is fed 30d continuously, freely ingests and drinks water, and every day, observed and recorded, claimed weekly body weight one time.30d gets tail blood and surveys the shallow lake hemoglobin, the hematological indices such as red, white blood cell count(WBC) and leukocyte differential count; Fasting 16h, 31d broken end get blood, survey GPT, GOT, BUN, CRE, GLUC, TRIG, CHOL, TPRO, and the biochemical indicator such as protein; Dissect rat and core, liver, spleen, kidney, the internal organs such as testis (ovary), observation changes and weighs, and calculates dirty body ratio; Main organs is carried out pathologic finding.
Result such as following table 1,2,3,4
The impact of table 1. Cordyceps militaris cocoon powder on the rat blood index
| Sex | Dosage (g ∕ kg) | Number of animals (only) | Lactoferrin (g ∕ L) | Red blood cell number (x10 12∕L） | Leukocyte count (x10 9∕L） | Lymphocyte (%) | Neutrophil leucocyte (%) |
| Female | Contrast | 10 | 147.3±10.6 | 7.5±0.9 | 17.2±2.8 | 79.8±3.0 | 18.1±2.5 |
| Female | 0.63 | 10 | 139.6±8.8 | 6.7±0.8 | 19.2±3.7 | 81.3±2.9 | 17.6±2.6 |
| Female | 1.25 | 10 | 142.9±8.1 | 6.9±0.8 | 19.2±5.5 | 82.1±3.4 | 16.4±3.3 |
| Female | 2.50 | 10 | 141.7±7.2 | 6.6±0.8 | 19.4±3.9 | 80.8±5.8 | 18.0±5.3 |
| ? | P value | ? | 0.2649 | 0.1081 | 0.6020 | 0.6300 | 0.7058 |
| Male | Contrast | 10 | 148.2±9.8 | 7.3±0.8 | 16.2±3.4 | 79.0±5.1 | 18.5±4.7 |
| Male | 0.63 | 10 | 151.0±10.8 | 6.6±0.5 | 18.2±2.9 | 78.9±5.5 | 19.5±4.7 |
| Male | 1.25 | 10 | 148.3±11.8 | 7.0±0.7 | 18,7±4.0 | 80.0±5.0 | 18.0±4.0 |
| Male | 2.50 | 10 | 148.5±7.2 | 6.7±0.7 | 19.0±3.5 | 81.2±6.8 | 17.8±6.2 |
| ? | P value | ? | 0.9092 | 0.1083 | 0.2646 | 0.7807 | 0.8511 |
The impact (1) of table 2. Cordyceps militaris cocoon powder on the rat biochemical indicator
| Sex | Dosage (g ∕ kg) | Number of animals (only) | GPT（u∕L） | GOT(u∕L) | BUN（mmol∕L） | CRE（μmol∕L） | GLUC（mmol∕L） |
| Female | Contrast | 10 | 45.87±11.14 | 104.79±8.21 | 6.57±1.86 | 93.88±9.93 | 4.02±0.72 |
| Female | 0.63 | 10 | 46.21±21.55 | 112.16±14.73 | 6.36±1.96 | 95.93±14.00 | 4.55±1.00 |
| Female | 1.25 | 10 | 45.64±10.51 | 102.89±8.65 | 6.93±1.82 | 92.65±10.93 | 3.99±0.68 |
| Female | 2.50 | 10 | 55.80±6.63 | 110.54±10.35 | 6.78±1.79 | 92.24±15.02 | 5.00±1.33 |
| ? | P value | ? | 0.2828 | 0.1857 | 0.9100 | 0.9143 | 0.0752 |
| Male | Contrast | 10 | 60.45±4.99 | 99.26±16.06 | 6.77±2.17 | 97.17±12.40 | 4.31±0.42 |
| Male | 0.63 | 10 | 55.97±12.81 | 108.24±19.73 | 6.52±1.54 | 103.95±16.69 | 5.17±0.84 |
| Male | 1.25 | 10 | 58.15±11.17 | 111.37±15.51 | 6.75±1.51 | 78.58±28.83 | 4.78±1.22 |
| Male | 2.50 | 10 | 55.91±15.27 | 109.90±19.76 | 6.57±1.37 | 91.83±14.50 | 4.86±0.72 |
| ? | P value | ? | 0.7970 | 0.4383 | 0.9814 | 0.1271 | 0.0767 |
The impact (2) of table 3. Cordyceps militaris cocoon powder on the rat biochemical indicator
| Sex | Dosage (g ∕ kg) | Number of animals (only) | TRIG（mmol∕L） | CHOL（mmol∕L） | TPRO（g∕L） | ALB（g∕L） | A∕G |
| Female | Contrast | 10 | 0.73±0.15 | 1.76±0.41 | 73.98±8.77 | 35.52±2.69 | 1.04±0.30 |
| Female | 0.63 | 10 | 0.63±0.12 | 1.44±0.36 | 77.82±4.57 | 37.73±3.23 | 0.95±0.15 |
| Female | 1.25 | 10 | 0.59±0.16 | 1.76±0.27 | 71.80±5.84 | 36.31±2.64 | 1.12±0.23 |
| Female | 2.50 | 10 | 0.72±0.18 | 1.57±0.24 | 77.66±9.29 | 38.51±2.78 | 1.04±0.30 |
| ? | P value | ? | 0.1498 | 0.0893 | 0.2079 | 0.0995 | 0.5206 |
| Male | Contrast | 10 | 0.73±0.23 | 1.87±0.34 | 76．20±4.17 | 36.85±2.63 | 1.03±0.17 |
| Male | 0.63 | 10 | 0.64±0.32 | 1.45±0.45 | 79.73±3.35 | 38.20±3.16 | 0.93±0.17 |
| Male | 1.25 | 10 | 0.71±0.19 | 1.54±0.29 | 77.87±3.17 | 72±2.67 | 0.97±0.25 |
| Male | 2.50 | 10 | 0.64±0.24 | 1.72±0.39 | 79.46±4.08 | 38.73±3.48 | 0.98±0.23 |
| ? | P value | ? | 0.8135 | 0.0860 | 0.1429 | 0.5579 | 0.8018 |
Interpretation of result as seen from table, hematology between each dosage group and control group and each dosage group and blood parameters show that all without significant difference this Cordyceps militaris cocoon powder all has no adverse effects to red blood cell number, leukocyte count and classification, content of hemoglobin and liver, renal function, fat metabolism, protein metabolism, the glycometabolism of rat.
Organ weights and histopathologic examination the results are shown in Table 5.
The impact of table 4. Cordyceps militaris cocoon powder on the dirty body ratio of rat
| Sex | Dosage (g ∕ kg) | Number of animals (only) | Xin ∕ body (%) | Gan ∕ body (%) | Pi ∕ body (%) | Shen ∕ body (%) | Ovary (testis) ∕ body (%) |
| Female | Contrast | 10 | 0.36±0.05 | 3.42±0.21 | 0.28±0.05 | 0.74±0.04 | 0.058±0.015 |
| Female | 0.63 | 10 | 0.37±0.03 | 3.26±0.27 | 0.30±0.04 | 0.72±0.04 | 0.058±0.006 |
| Female | 1.25 | 10 | 0.33±0.04 | 3.20±0.31 | 0.28±0.08 | 0.71±0.07 | 0.059±0.007 |
| Female | 2.50 | 10 | 0.34±0.05 | 3.21±0.26 | 0.27±0.07 | 0.72±0.14 | 0.058±0.007 |
| ? | P value | ? | 0.1400 | 0.2300 | 0.8101 | 0.8998 | 0.9895 |
| Male | Contrast | 10 | 0.30±0.07 | 3.21±0.22 | 0.26±0.05 | 0.67±0.04 | 1.24±0.15 |
| Male | 0.63 | 10 | 0.33±0.05 | 3.31±0.19 | 0.29±0.03 | 0.71±0.06 | 1.27±0.11 |
| Male | 1.25 | 10 | 0.31±0.05 | 3.36±0.22 | 0.27±0.05 | 0.68±0.09 | 1.26±0.10 |
| Male | 2.50 | 10 | 0.32±0.05 | 3.33±0.28 | 0.28±0.05 | 0.71±0.09 | 1.29±0.12 |
| ? | P value | ? | 0.5061 | 0.4889 | 0.6879 | 0.4131 | 0.8289 |
As can be seen from Table 4, the dirty body between each amount group and control group and each dosage group is than the difference without conspicuousness.Show that this Cordyceps militaris cocoon powder has no significant effect growing of each internal organs of animal.Pathologic finding is showed no the specificity pathological change.
3. Teratogenicity test: select 12 all rats of not mating, male and female are 2:1, same cage, and check cloudy bolt and sperm inferior morning, finds that cloudy bolt and sperm person are decided to be to become pregnant 0 day.Pregnant mouse is divided into 4 groups at random, wherein feeds respectively Cordyceps militaris cocoon powder 1.25g ∕ kg.bw for 3 groups, 0.63g ∕ kg.bw, 0.32g ∕ kg.bw, control group are to distilled water, and the drinking-water of freely ingesting adds a peanut every day to improve and to have additional nutrients.The 7-16 days per os gavage Cordyceps militaris cocoon powder of becoming pregnant.Claim its body weight in the 0th, 7,12,16,20 day that becomes pregnant, and observed pregnant mouse growing state, adjust measuring to feeding of Cordyceps militaris cocoon powder depending on changes of weight.Pregnancy 20 days, the cervical vertebra dislocation is put to death, and takes out the uterus and weighs, and recognizes and record embryo nidation, the tire of living, stillborn foetus and absorption tire number.The tire mouse that lives is weighed one by one, measures body long, check outward appearance deformity situation, the results are shown in following table 5,6:
Table 5. Cordyceps militaris cocoon powder is on the impact of pregnant mouse body weight (X ± SD)
| Dosage (g ∕ kg) | Pregnant mouse number (only) | 0 day (g) | 7 days (g) | 12 days (g) | 16 days (g) | 20 days (g) |
| Negative group | 11 | 269.3 ± 17.3 | 292.6 ± 19.8 | 314.7 ± 22.2 | 340.7 ± 21.6 | 382.8 ± 26.8 |
| 0.32 | 10 | 265.8 ± 12.8 | 281.3 ± 12.5 | 304.9 ± 12.5 | 332.1 ± 12.8 | 384.7 ± 13.8 |
| 0.63 | 10 | 264.7 ± 19.2 | 282.6 ± 17.3 | 307.9 ± 19.9 | 342.4 ± 21.1 | 390.2 ± 20.6 |
| 1.25 | 12 | 265.5 ± 27.1 | 293.9 ± 23.5 | 317.6 ± 22.0 | 344.6 ± 22.6 | 389.9 ± 26.7 |
| P value | 0.5705 | 0.2685 | 0.4022 | 0.4830 | 0.8287 |
Through check, each dosage group and control group compare, and no significant difference shows that this Cordyceps militaris cocoon powder is to pregnant mouse free of toxic effects.
The impact that table 6. Cordyceps militaris cocoon powder forms rat embryo and the tire mouse grows
| Dosage (g ∕ kg) | Fertilization mouse (one) | Litter size (only) | Pregnant mouse number (one) | Equal tire young (one) | Absorb tire (one) | Stillborn foetus (one) | The young all body weight (g) of tire | The young all heights (cm) of tire |
| Negative group | 13 | 11 | 140 | 12.73 | 0 | 0 | 3.60 ± 0.25 | 3.79 ± 0.40 |
| 0.32 | 13 | 10 | 131 | 13.10 | 1 | 0 | 3,68 ± 0.24 | 3.80 ± 0.11 |
| 0.63 | 13 | 10 | 102 | 10.20 | 0 | 1 | 3.67 ± 0.30 | 3.77 ± 0.13 |
| 1.25 | 13 | 12 | 143 | 11.92 | 2 | 0 | 3.67 ± 0.36 | 3.79 ± 0.19 |
| P value | 0.9830 | 0.8484 |
This shows, each dosage group and control group be no significant difference relatively, shows the tire mouse is formed and grows without obviously harmful effect.In addition, all no abnormal through the inspection of outward appearance deformity, internal organ inspection, skeletal survey.
Two, conclusion: in sum, it is safe that the pure fructification of the Cordyceps militaris of artificial culture and cocoon are used for human body as food, can be widely used in health food, health diet and other nourishing based food fully.
Embodiment prepares nutrition (liquid) beverage of health care food (treatment) product and the method for electuary with Cordyceps militaris, cocoon extract:
According to the difference of its function (requirements), its formula (ratio is by weight) any (comply with its function and different)
A, Cordyceps militaris, cocoon nutrition extract 85%, auxiliary material 15%
B, Cordyceps militaris, cocoon nutrition extract 95%, auxiliary material 5%
C, Cordyceps militaris, cocoon nutrition extract 98%, auxiliary material 2%
Auxiliary material can be selected starch, β dextrin etc.; The knack workman that these auxiliary materials commonly used are all this areas knows.
Cordyceps militaris is chosen respectively the extract of above-mentioned A or B or C, or single preparation or mutually mix adapted (by weight):
A. nutrient solution
It is even 86.5% that each portion of extract (being 1:1:1) of getting three groups of A, B, C mixes, white granulated sugar 4%, and honey 9%, citric acid 0.3%, stabilizing agent (CMC-Na and sodium alginate 1:1 mix) 0.2% is through (1.47 ~ 1.96) x10
6Handkerchief is homogeneous → sterilization (115 ℃ 5 seconds) → finished product.
B. electuary
After extract (A1.2, B0.5, C0.3) mixing 78%, white granulated sugar 10%, amylodextrin 12% → pulverize → sieve → packing → sterilization → finished product.
Claims (2)
1. prepare the method for health food with Cordyceps militaris cocoon extract, described health food is beverage or electuary, it is characterized in that:
The first step extracts the nutriment in Cordyceps militaris and pupa worm cocoon, and its extraction process is as follows:
A. freeze thawing is extracted: spend the night under the immersion room temperature in distilled water after getting the mixing of Cordyceps militaris powder and pupa worm cocoon powder, then multigelation 2-4 time under-20 ℃ of environment, 6000g subsequently, got supernatant, wherein Cordyceps militaris in centrifugal 15 minutes: pupa worm cocoon: the water consumption ratio is 1:0.5:1.5;
B. make up enzymolysis and extraction: Cordyceps militaris and the pupa worm cocoon slag of freeze thawing after centrifugal enters 5 ~ 10 times of distilled water in adding in A step, add lysozyme and pectase hydrolysis 2-3 hour, then add respectively amylase, papain, pepsin, trypsase, bromelain, carbohydrase, monascus, cellulase, each is hydrolyzed 0.5 ~ 1.0 hour, the centrifuging and taking supernatant; Wherein, every 1 kilogram of Cordyceps militaris and pupa worm cocoon powder, the consumption of each enzyme is: lysozyme 1 gram, pectase 4 grams, amylase, papain, pepsin, each 2 grams of trypsase, bromelain, carbohydrase, monascus, each 3 grams of cellulase;
C. add 5 ~ 10 times of distilled water in the Cordyceps militaris after the enzymolysis and pupa worm cocoon slag, stir at 80 ℃ and extract 2 times, centrifugal merging supernatant;
D. after the supernatant that above-listed A, B and the C step is obtained merges, be placed in stainless steel or glass container, big fire is boiled, after little fire is kept 3-4 hour, filter, the filtrate of filtration is continued 1 ∕ 4～1 ∕ 5 that heating is concentrated into the substance accumulated amount, pour metering in clean container into, cooling, precipitation;
E. get cooling rear concentrate, will not precipitate residue and bring into, slowly add ethyl alcoh(ol);
F. after hold over night, when the things such as polysaccharide all are sink to the bottom, first remove the part supernatant with siphon, precipitated liquid 3500g obtained the thickness paste in centrifugal 15 minutes, was coffee brown look block after vacuum drying, pulverized the nutrition extract that namely gets the Cordyceps militaris cocoon;
Second step, the preparation health food, Cordyceps militaris cocoon nutrition extract 85% by weight, auxiliary material 15%, or Cordyceps militaris cocoon nutrition extract 95%, auxiliary material 5%, or Cordyceps militaris cocoon nutrition extract 98%, auxiliary material 2% is mixed with respectively raw material one, two and three;
When the preparation beverage, prepare by following raw material: get raw material one, raw material two and raw material three and mix by 1:1:1 weight, account for 86.5% of raw material weight percentage, white granulated sugar accounts for 4%, honey accounts for 9%, citric acid accounts for 0.3%, and stabilizing agent CMC-Na and sodium alginate 1:1 mixing account for 0.2%, through 1.47 ~ 1.96x10
6Handkerchief is homogeneous, gets finished product after sterilization;
When the preparation electuary, prepare by following raw material: get raw material one, raw material two and raw material three and mix by 1.2:0.5:0.3 weight, account for 78% of raw material weight percentage, white granulated sugar accounts for 10%, and amylodextrin accounts for 12%, pulverizes after mixing, sieve, get product by packing and sterilizing.
2. the method for preparing health food with Cordyceps militaris cocoon extract according to claim 1, it is characterized in that: described auxiliary material is selected starch, β dextrin.
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