CN101356954A - Preparation method of anti-stress medicine earthworm small-peptide chelated chromium - Google Patents
Preparation method of anti-stress medicine earthworm small-peptide chelated chromium Download PDFInfo
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Abstract
The invention relates to a preparation method for anti-stress drug earthworm small peptide chelated chromium, which is characterized in that the live earthworms are cleaned, added with water and triturated by a high-speed triturator; then the earthworm triturate is mixed evenly to carry out the autolysis zymohydrolysis action so as to derive an earthworm small peptide liquid; the earthworm small peptide liquid is heated and inactivated by boiling water bath, and then is centrifugated for obtaining a small peptide concentrated peptide liquid; the earthworm small peptide concentrated peptide liquid is taken to be added with chromium trichloride for the chelation reaction; after being cooled and sterilized under the room temperature, the earthworm small peptide chromium chelateed compound is obtained; the earthworm small peptide chromium chelateed compound is concentrated by vacuum, added with mycoprotein powders to be absorbed, and treated by sponging drying; finally the earthworm small peptide chromium chelateed compound is crashed so as to be prepared into the earthworm small peptide chromium acid chelated compound. The preparation method for the anti-stress drug earthworm small peptide chelated chromium can improve the immunity of fish bodies and the inhibiting ability, enhance the high-temperature resistant ability, the high-intensity resistant ability and the infection ability for resisting the infection of aeromonas hydrophila, and raise the utilization ratio of chromium, while supplementing the nutrition of small peptide and improving the palatability of the feed and the anti-stress ability of the fish bodies to promote the growth of the fish bodies.
Description
Technical field
The present invention relates to a kind of preparation method of anti-stress medicine earthworm small-peptide chelated chromium, specifically its production method of Organic Chromium element feed additive-earthworm small-peptide chromium belongs to aquatic product matched field of feed.
Background technology
Earthworm is commonly called as earthworm, Chinese medicine claims earthworm, it is a class lower animal that belongs to Annelida Oligochaeta (Olgochaeta), at present the whole world is known have 3000 surplus kind, having 13 in China belongs to, the nearly hundreds of kind of the earthworm kind of China has very high medical value, is treasure in China's medicine treasure-house.Li Shizhen (1518-1593 A.D.) had once been put down in writing the medical value of earthworm in Compendium of Material Medica, it can prepare 40 plurality of Chinese, and the prescription of preparing with wormcast has 21 kinds; Disease the having of curing the disease: the typhoid fever thermojunction, headache due to pathogenic wind-heat, infantile convulsion, renal shutdown, severe and wadering arthralgia pain, the erosion of mucous membrane in the oral cavity, the red larynx of eye are swollen, favus of the scalp scrofula etc.Main effect of getting its heat-clearing, relieving convulsion, diuresis, vein relaxing.
Discovered in recent years, earthworm contains Lumbrokinase, and angiocardiopathy is had better therapeutic effect.The earthworm enzyme can not only the activation fiber protein resolvase and thrombus, more directly solution fibrin.The clinical treatment thrombotic disease is efficient to be reached more than 80%, and the earthworm enzyme reduces blood viscosity in addition, suppresses platelet aggregation, anticoagulation, promotes effects such as blood flow is unobstructed, and apoplexy sequelae, artery sclerosis, hypertension and high blood viscosity disease etc. are had therapeutic action.Lumbrokinase has been developed patent medicine, and taking convenience, safety can not look like streptokinase, and medicines such as urea kinases cause that easily high fibrinolysin mass formed by blood stasis causes massive haemorrhage.
Earthworm is rich in protein, and fat and carbohydrate, crude protein content be up to 72%, and contains amino acid, vitamin and the trace element of needed by human body, can make the animality feed and produce medicine and cosmetics.At present, still some heavy metal that exceeds standard in the earthworm body can not be separated and limited and humanly directly eat because scientific and technological level is limit.But as the bait that holds fowl and aquatic livestock, the effect that it is brought into play may be also bigger than hormone with earthworm, and particularly its surprising reproduction speed has determined that it is the bait of high-quality, cheapness, green.
Wormcast is a kind of good bio-fertilizer, contain the required trace elements of crop such as abundant nitrogen, phosphorus, potassium, humus, organic matter and magnesium, boron, nickel, manganese, be the Cheap highly effective fertilizer in fruit, flowers, vegetables and nursery, and breeding earthworm, can utilize organic waste materials (as: fowl and animal excrement, rubbish, the withered mandarin orange of plant, sugared slag, edible seedling slag, vinasse etc.) cost low, method is simple, has promoted resource circulation.Therefore earthworm has important effect, and demand heightens, and is subjected to everybody and payes attention to.
Chromium is the essential trace element of animal, its most important physiological function is active component and the insulin performance collaborative physiological function of trivalent chromium as GTF (GTF), participate in carbohydrate, lipid, protein and nucleic acid metabolism, stress, improve and special role is arranged aspect the immunity reducing the fish body, and the production performance of improvement is arranged, improve meat products matter, promote the fish bulk-growth.
Stress cause animal glycometabolism, mineral metabolism that a series of changes take place, cause glycogen degraded and gluconeogenesis to be strengthened.The reinforcement of glucose utilization can cause organizing chromium to mobilize increase also finally to excrete; replenish chromium in the case and can watch for animals to be unlikely and lose some trace element (zinc, copper, ferromanganese etc.) in response to swashing, thereby mend chromium to stressed animal and can reduce the requirement of animal important trace elements such as zinc, copper.On the one hand, animal greatly strengthens adrenal cortex secretion cortisol under stress situation, by hypothalamus-hypophysis-adrenal cortex axle, and cortisol has the effect that suppresses growth and immunologic function; The animal sympathetic activation makes adrenal medella portion discharge a large amount of catecholamines simultaneously, and the secretion of pancreas hyperglycaemia also greatly increases; On the other hand, stress suppress secretion of insulin again.Many adrenal cortexs that stress all promote discharge glucocorticoid, thereby synthesis weakens to glycogen to cause body, decomposition is strengthened, and in the acceleration adipose tissue fat oxidation Decomposition, strengthen the heteroplasia of glycogen, the result be keep blood sugar level rice higher in the blood satisfy body stress the time energy demand.Because hyperglycaemia requires more or higher bioactive pancreas islet usually resists, so the chromium that constitutes as the collaborative hormone of pancreas islet poison certainly will be mobilized.Chromium one courage is mobilized, and is not just heavily being absorbed, but is draining through dung.Therefore, stress cause the requirement of chromium to increase, chromium may have anti-stress effect.It is strong to heal, and the duration is longer, and chromium is mobilized the more in the body, and chromium is discharged also the more, more easily causes chromium deficiency; Stress the time body inner cortex hormone secretion increase, growth and immunologic function are suppressed, and cause poor growth and susceptibility to disease, thus mend chromium have prevention stress and anti-stress effect, thereby raising humoral immunity and cellular immune level, enhancing vaccine injection effect.
The chromium source comprises inorganic chromium and two kinds of forms of Organic Chromium at present, and inorganic chromium has CrCl
36H
2O, Cr
2(SO
4)
3, Cr (NO
3)
3Deng, Organic Chromium comprises high chromium yeast and chelated chromium (as nicotinic acid chromium, albumen chromium, amino acid chromium and methylpicolinic acid's chromium).The absorption of chromium and chemically bound form have very big relation, and inorganic chromium is than the difficult absorption of Organic Chromium, and animal only is 1%-3% to the absorptivity of inorganic chromium, and becomes negative correlation with feed intake, and the absorptivity of Organic Chromium is 10%-25% in the feed.Generally speaking the utilization rate of chromium is lower, has limited its utilization.The appearance of amino-acid chelate chromium, what can not only solve that inorganic salts, organic salt exist is difficult to absorb thereby waste big problem, make its absorptivity in animal body can improve 300% than inorganic salts, can also make animal take in this compound and bring two kinds of nutriments (amino acid and chromium trace element) simultaneously into, thereby chelating amino acids chromium is the desirable feed addictive that is known as animal.And the described chelate of previous patent, by chelate divalent metals such as the same respectively calcium of animal proteins such as acid hydrolysis hair, pig blood, magnesium, zinc, copper and cobalts and get, what obtain is water soluble chelate compound, its weak point is: the thing while of isolating water-soluble chelating, lose a part of non-water-soluble chelate, simultaneously separating technology equipment complexity, energy consumption height, product cost height.This research then utilizes earthworm self enzyme, behind zymyhydrolyzed protein, with the chromium chelatropic reaction, and with adsorbent mycoprotein absorption chelatropic reaction liquid, energy consumption is low, handicraft is simple, production cost is low, and raw material (earthworm) is cultured gained by debirs, easily a large amount of supplies.
Summary of the invention
The objective of the invention is to overcome above-mentioned weak point, thereby provide a kind of and can improve immunity of fish body and oxidation resistance, improve fish body high temperature resistance, high density and opposing and have a liking for water-based Aeromonas anti-infection ability, can improve the utilization rate of chromium, and can replenish some small peptide nutrients, improve the feed palatability, improve fish body anti-stress ability, promote the preparation method of the anti-stress medicine earthworm small-peptide chelated chromium of fish bulk-growth.
According to technical scheme provided by the invention, a kind of preparation method of anti-stress medicine earthworm small-peptide chelated chromium adopts following processing step:
1, the earthworm that will live is cleaned, and allows swill in its body that as far as possible spues, takes by weighing 500～1000g earthworm and adds 1.5～3L water, smashs to pieces with the high speed bruisher, and high speed bruisher rotating speed is: 15000～20000 rev/mins, the time of smashing to pieces is: 3～5 minutes;
2, smashing earthworm to pieces material and mix, with hydrochloric acid or the NaOH adjust pH 6～8 of 0.1mol/L, is that the self-dissolving enzyme digestion reaction made earthworm small-peptide liquid in 2～4 hours under 40～55 ℃ of conditions in temperature;
3, with earthworm small-peptide liquid with 100～110 ℃ of boiling water bath heat inactivations 10～15 minutes, in room temperature, placed then 30～60 minutes, got the supernatant packing through centrifugal 10～30 minutes, sealing is standby, obtain little peptide and concentrate peptide liquid;
4, (2000～3000g) with citric acid adjust pH 2～4, is under 70～100 ℃ of conditions in temperature, adds chromium trichloride (CrCl to get concentrated peptide liquid 40～50 weight % of earthworm small-peptide
36H
2O) 5～10 weight % (100g-300g) chelatropic reactions are 20～60 minutes; Obtain the earthworm small-peptide chromium complex through 25 ℃ of cooling sterilizations of room temperature;
5, earthworm small-peptide chromic acid chelate vacuum is concentrated, vacuum is 60～90kPa, evaporates 45～55 weight % moisture content (800g-2000g), and the back adds equivalent mycoprotein powder 45～55 weight % (800g-2000g) absorption, and carry out spray drying treatment, baking temperature: 50～70 ℃; Drying time: 30～90 minutes; Moisture 8～12%;
6, at last earthworm small-peptide chromic acid chelate is pulverized, grinding particle size 150～178um makes 80～110 order earthworm small-peptide chromic acid chelate dry powder.
The 7 earthworm small-peptide chromium complexs of the present invention fish body test 56d that throws something and feeds.
Earthworm small-peptide liquid enzymolysis product molecular weight distribution determination:
According to the protein complete hydrolysis is that mean molecule quantity behind the free amino acid is about 150Da, the a-NH of this moment
2/ TN is 100% theoretically, works as a-NH
2/ TN is 50% o'clock, and mean molecule quantity then is about 300Da, obtains the mixed solution of dipeptides and tripeptides, and the back adopts calibration curve method to measure the relative molecular mass of enzymolysis sample with Waters 600 high performance liquid chromatography.Analysis condition is under 30 ℃ of TSKgel2000 SWXL300mm * 7.8mm chromatographic column, and with acetonitrile/water/trifluoroacetic acid, 45/55/0.1 (V/V) is the phase that flows, and flow velocity UV220nm measured down in 0.5ml/ minute.The used standard items of molecular weight calibration curve are: cromoci (MW12500Da), Aprotinin (MW6500Da), bacillus enzyme (MW1450Da), aminoacetic acid-aminoacetic acid-tyrosine-arginine (MW451Da), aminoacetic acid-aminoacetic acid-aminoacetic acid (MW189Da); At first according to standard protein relative molecular mass and corresponding appearance time thereof, obtaining the relative molecular mass calibration curve is: LogM=7.38-0.244 (t1-t2), use enzymolysis sample upper prop to be measured again, the record appearance time, obtain the relative molecular mass of enzymolysis sample from calibration curve, molecular weight all under 20000Da, mainly is distributed in the little peptide mixer under the 3000Da, in addition can obtain below 600 little peptide mixer.
The earthworm small-peptide chromium complex to the jian carp fish anti-stress with the experiment of growth:
A, growth experiment: jian carp is provided by fishing ground, Chinese aquatic science institute fresh water fishery research center.The jian carp of health, specification, weight basically identical is selected in test, the individual about 10g of counterpoise just.Random packet is provided with control group, establishes 3 parallel tests for every group, every parallel group 100 tail test fish.Throw something and feed 0 behind the jian carp domestication 30d, various dose earthworm small-peptide chromium complex 56d such as 12.5mg/kg, 25.0mg/kg, 50.0mg/kg, measure the influence of its growth.
B, stress test: after feeding experiment finished, the fish of choosing individual basically identical carried out stress test.Adopt high density (100g/L) stress, three parallel group of each dosage component, each parallel group is about 20 tails, putting in a suitable place to breed in same specification volume is in the 20L glass jar.
Adopt the flowing water system, 21 ℃ of water temperatures, flow velocity is 2L/ minute, and guarantees to reduce artificial disturbance by sufficient oxygen, keeps quite, and additionally stress prevent.Every 6h observes fish body death condition, observes 7d, the statistics death rate.
C, attack poison experiment: after feeding experiment finishes, three parallel group of each dosage component, each parallel group is 10 tail fishes.Pathogen employing freshwater fish bacterium-have a liking for the water-based Aeromonas is diluted the water-based Aeromonas of having a liking for after twice activation with SPSS, make final concentration be about 5 * 10
7Cells/mL.By the heavy lumbar injection bacterium of every 100g fish liquid 1.0mL, after to put in a suitable place to breed in same specification volume be in the 20L glass jar, and guarantee sufficient oxygen, observe fish body death condition every day, observe 7d, the statistics death rate.
D, result show: the earthworm small-peptide chromium complex can improve immunity of fish body and oxidation resistance, improve fish body high temperature resistance, high density and opposing and have a liking for water-based Aeromonas anti-infection ability, improved the utilization rate of chromium, and can replenish some small peptide nutrients, improve the feed palatability, improve fish body anti-stress ability, promote the fish bulk-growth.
Compared with the prior art the present invention has the following advantages:
1, adopt the complex enzyme system enzymolysis of earthworm self, acquisition main molecules amount is distributed in the little peptide mixer under the 3000KD, and cost is low, and technology is simple, easy operating.
2, the chromium chelating that passes through the proteolysis acquisition in the past mainly is an amino-acid chelate, and the present invention is the small-peptide chelated thing that earthworm small-peptide and chromium trichloride chelating obtain, and has improved the utilization rate of chromium.
3, the described albumen metallo-chelate of previous patent, by chelate divalent metals such as the same respectively calcium of animal proteins such as acid hydrolysis hair, pig blood, magnesium, zinc, copper and cobalts and get, what obtain is water soluble chelate compound, its weak point is: the thing while of isolating water-soluble chelating, lose a part of non-water-soluble chelate, simultaneously separating technology equipment complexity, energy consumption height, product cost height.
This research then utilizes earthworm self enzyme, behind zymyhydrolyzed protein, with the chromium chelatropic reaction, and absolutely utilize chelatropic reaction liquid with adsorbent, infiltration rate is fast, and efficient is higher, and energy consumption is low, handicraft is simple, production cost is low, and raw material (earthworm) is cultured gained by debirs, easily a large amount of supplies.
4, the metallic element chelatropic reaction of previous patent, main nutrition carrier adsorbent generally is wheat bran, zeolite powder etc., and what the present invention mainly adopted is that mycoprotein is a carrier, and the effect of existing carrier adsorbent has antibiotic somatotrophic effect again.
5, earthworm small-peptide chromium chelating of the present invention shows that by animal experiment earthworm small-peptide chromium chelating can improve immunity of fish body and oxidation resistance, improves fish body high temperature resistance, high density and opposing and has a liking for water-based Aeromonas anti-infection ability, promotes the fish bulk-growth.
Description of drawings
Fig. 1 is standard protein sample efficient liquid phase chromatographic analysis figure of the present invention.
Fig. 2 is enzymolysis processing condition of the present invention earthworm efficient liquid phase chromatographic analysis figure once.
Fig. 3 is two times earthworm efficient liquid phase chromatographic analysis of enzymolysis processing condition of the present invention figure.
The specific embodiment
Embodiment during following the present invention incites somebody to action in conjunction with the accompanying drawings is further described:
Case study on implementation one: the preparation method of a kind of anti-stress medicine earthworm small-peptide chelated chromium of the present invention adopts following processing step:
The earthworm that will live is cleaned, and allows swill in its body that as far as possible spues.Take by weighing the 500g earthworm and add 1.5L water, smash adjust pH 6 to pieces with high speed bruisher (15000 rev/mins of bruisher rotating speeds, smash to pieces 3 minutes time), the self-dissolving enzyme digestion reaction is 2 hours under 40 ℃ of conditions, 100 ℃ of boiling water bath heat inactivations 10 minutes, room temperature was placed 30 minutes, got the supernatant packing through centrifugal 10 minutes, sealing is standby, obtains the earthworm small-peptide concentrate; With citric acid adjust pH 2, is 70 ℃ conditions under in temperature with earthworm small-peptide concentrate 40 weight % (2000g), adds chromium trichloride (CrCl
36H
2O) 10 weight % (200g) chelatropic reactions are 20 minutes; Obtain the earthworm small-peptide chromium complex through 25 ℃ of cooling sterilizations of room temperature; Earthworm small-peptide chromic acid chelate vacuum is concentrated, vacuum is 60kPa, evaporate 50% left and right sides moisture (880g), the back adds equivalent mycoprotein powder 50 weight % (880g) absorption, mycoprotein powder (effluent north Shijiazhuang grand brightness protein feed Co., Ltd produces), and carry out spray drying treatment, baking temperature: 50 ℃; Drying time: 30 minutes; Moisture 8%; With earthworm small-peptide chromic acid chelate grinding particle size 178um, make the earthworm small-peptide chromic acid chelate dry powder about 80 orders at last.
The throw something and feed fish bulk-growth test of earthworm small-peptide chromium complex: jian carp is provided by fishing ground, Chinese aquatic science institute fresh water fishery research center.Jian carp 600 tails of health, specification, weight basically identical are selected in test, the individual about 10g of counterpoise just.Be divided into 2 groups at random, wherein the I group is control group, and II is a test group, establishes 3 parallel tests for every group, every parallel group 100 tail test fish.Throw something and feed 0 behind the jian carp domestication 30d, 12.5mg/kg earthworm small-peptide chromium complex 56d, measure the influence of its growth.Stress test: after above-mentioned feeding experiment finished, the fish of choosing individual basically identical carried out stress test.Adopt high density (100g/L) stress, three parallel group of each dosage component, each parallel group is about 20 tails, putting in a suitable place to breed in same specification volume is in the 20L glass jar.Adopt the flowing water system, 21 ℃ of water temperatures, flow velocity is 2L/ minute, and guarantees to reduce artificial disturbance by sufficient oxygen, keeps quite, and additionally stress prevent.Every 6h observes fish body death condition, observes 7d, the statistics death rate.Attack poison experiment: above-mentioned feeding experiment finish the back stress before and stress 24h after selection body weight basically identical fish carry out challenge test, three parallel group of each dosage component, each parallel group is 10 tail fishes.Pathogen employing freshwater fish bacterium-have a liking for the water-based Aeromonas is diluted the water-based Aeromonas of having a liking for after twice activation with SPSS, make final concentration be about 5 * 10
7Cells/mL.By the heavy lumbar injection bacterium of every 100g fish liquid 1.0mL, after to put in a suitable place to breed in same specification volume be in the 20L glass jar, and guarantee sufficient oxygen, 12.5mg/kg earthworm small-peptide chromium complex rate of body weight gain significantly (P＜0.05) has improved 9.38% respectively; High density stress 1d after the test, 12.5mg/kg earthworm small-peptide chromium complex significantly (P＜0.05) reduces blood concentration of cortisol 10.23%, blood sugar level 5.28% and concentration of malondialdehyde 15.283% have increased blood catalase 14.73%, superoxide dismutase activity 6.78%; Behind the pathogenic bacterial infection fish body, the control group death rate is 80.0%, and the 12.5mg/kg earthworm small-peptide chromium complex fish death rate is respectively 20.0%; Generally speaking, 12.5mg/kg the earthworm small-peptide chromium complex can improve immunity of fish body and oxidation resistance, improve anti-high density of fish body and opposing and have a liking for water-based Aeromonas anti-infection ability, replenished chromium in the daily ration, some small peptide nutrients have been increased, improve the feed palatability, improve fish body anti-stress ability, promote the fish bulk-growth.
Case study on implementation two:
The earthworm that will live is cleaned, and allows swill in its body that as far as possible spues.Take by weighing the 750g earthworm and add 2.25L water, smash adjust pH 7 to pieces with high speed bruisher (17500 rev/mins of bruisher rotating speeds, smash to pieces 4 minutes time), the self-dissolving enzyme digestion reaction is 3 hours under 50 ℃ of conditions, 100 ℃ of boiling water bath heat inactivations 10 minutes, room temperature was placed 45 minutes, got the supernatant packing through centrifugal 20 minutes, sealing is standby, obtains the earthworm small-peptide concentrate; With citric acid adjust pH 3, is 85 ℃ conditions under in temperature with earthworm small-peptide concentrate (45 weight %) 2500g, adds chromium trichloride (CrCl
36H
2O) 8 weight % (200g) chelatropic reactions are 40 minutes; Obtain the earthworm small-peptide chromium complex through 25 ℃ of cooling sterilizations of room temperature; Earthworm small-peptide chromic acid chelate vacuum is concentrated, vacuum is 75kPa, evaporate 47% moisture (1350g), the back adds equivalent mycoprotein powder 47 weight % (1350g) absorption, mycoprotein powder (effluent north Shijiazhuang grand brightness protein feed Co., Ltd produces), and carry out spray drying treatment, baking temperature: 60 ℃; Drying time: 60 minutes; Moisture 10%; With earthworm small-peptide chromic acid chelate grinding particle size 150um, make the earthworm small-peptide chromic acid chelate dry powder about 100 orders at last.
The throw something and feed fish bulk-growth test of earthworm small-peptide chromium complex: jian carp is provided by fishing ground, Chinese aquatic science institute fresh water fishery research center.Jian carp 600 tails of health, specification, weight basically identical are selected in test, the individual about 10g of counterpoise just.Be divided into 2 groups at random, wherein the I group is control group, and II is a test group, establishes 3 parallel tests for every group, every parallel group 100 tail test fish.Throw something and feed 0 behind the jian carp domestication 30d, 25.0mg/kg earthworm small-peptide chromium complex 56d, measure the influence of its growth.Stress test: after above-mentioned feeding experiment finished, the fish of choosing individual basically identical carried out stress test.Adopt high density (100g/kg), three parallel group of each dosage component, each parallel group is about 20 tails, and putting in a suitable place to breed in same specification volume is in the 20L glass jar.Adopt the flowing water system, 21 ℃ of water temperatures, flow velocity is 2L/ minute, and guarantees to reduce artificial disturbance by sufficient oxygen, keeps quite, and additionally stress prevent.Every 6h observes fish body death condition, observes 7d, the statistics death rate.Attack poison experiment: above-mentioned feeding experiment finish the back stress before and stress 24h after selection body weight basically identical fish carry out challenge test, three parallel group of each dosage component, each parallel group is 10 tail fishes.Pathogen employing freshwater fish bacterium-have a liking for the water-based Aeromonas is diluted the water-based Aeromonas of having a liking for after twice activation with SPSS, make final concentration be about 5 * 10
7Cells/ml.By the heavy lumbar injection bacterium of every 100g fish liquid 1.0ml, after to put in a suitable place to breed in same specification volume be in the 20L glass jar, and guarantee sufficient oxygen, observe fish body death condition every day, observe 7d, the statistics death rate.The result shows: compare with control group, 25.0mg/kg earthworm small-peptide chromium complex rate of body weight gain significantly (P＜0.05) has improved 12.56% respectively; High density stress 1d after the test, 25.0mg/kg g earthworm small-peptide chromium complex significantly (P＜0.05) reduces blood concentration of cortisol 12.58%, blood sugar level 6.38% and concentration of malondialdehyde 10.27% have increased blood hydrogen peroxidase 12 .68%, superoxide dismutase activity 8.44%; Behind the pathogenic bacterial infection fish body, the control group death rate is 85.0%, and the 1.0mg/kg earthworm small-peptide chromium complex fish death rate is respectively 14.0%; Generally speaking, 25.0mg/kg the earthworm small-peptide chromium complex can improve immunity of fish body and oxidation resistance, improve anti-high density of fish body and opposing and have a liking for water-based Aeromonas anti-infection ability, replenished chromium in the daily ration, some small peptide nutrients have been increased, improve the feed palatability, improve fish body anti-stress ability, promote the fish bulk-growth.
Case study on implementation three:
The earthworm that will live is cleaned, and allows swill in its body that as far as possible spues.Take by weighing the 1000g earthworm and add 3.0L water, smash adjust pH 8.0 to pieces with high speed bruisher (20000 rev/mins of bruisher rotating speeds, smash to pieces 5 minutes time), the self-dissolving enzyme digestion reaction is 4 hours under 55 ℃ of conditions, 100 ℃ of boiling water bath heat inactivations 10 minutes, room temperature was placed 60 minutes, got the supernatant packing through centrifugal 60 minutes, sealing is standby, obtains the earthworm small-peptide concentrate; With citric acid adjust pH 4, is 100 ℃ conditions under in temperature with earthworm small-peptide concentrate 50 weight % (3000g), adds chromium trichloride (CrCl
36H
2O) 5 weight % (150g) chelatropic reactions are 60 minutes; Obtain the earthworm small-peptide chromium complex through 25 ℃ of cooling sterilizations of room temperature; Earthworm small-peptide chromic acid chelate vacuum is concentrated, and vacuum 90kPa evaporates 45% moisture (1900g), after add equivalent mycoprotein powder 45 weight % (1900g) absorption, and carry out spray drying treatment, baking temperature: 70 ℃; Drying time: 90 minutes; Moisture 12%; With earthworm small-peptide chromic acid chelate grinding particle size 124um, make the earthworm small-peptide chromic acid chelate dry powder about 120 orders at last.
The throw something and feed fish bulk-growth test of earthworm small-peptide chromium complex: jian carp is provided by fishing ground, Chinese aquatic science institute fresh water fishery research center.Jian carp 600 tails of health, specification, weight basically identical are selected in test, the individual about 11g of counterpoise just.Be divided into 2 groups at random, wherein the I group is control group, and II is a test group, establishes 3 parallel tests for every group, every parallel group 100 tail test fish.Throw something and feed 0 behind the jian carp domestication 30d, 50.0mg/kg earthworm small-peptide chromium complex 56d, measure the influence of its growth.Stress test: after above-mentioned feeding experiment finished, the fish of choosing individual basically identical carried out stress test.Adopt high density (100g/L), three parallel group of each dosage component, each parallel group is about 20 tails, and putting in a suitable place to breed in same specification volume is in the 20L glass jar.Adopt the flowing water system, 21 ℃ of water temperatures, flow velocity is 2L/ minute, and guarantees to reduce artificial disturbance by sufficient oxygen, keeps quite, and additionally stress prevent.Every 6h observes fish body death condition, observes 7d, the statistics death rate.Attack poison experiment: above-mentioned feeding experiment finish the back stress before and stress 24h after selection body weight basically identical fish carry out challenge test, three parallel group of each dosage component, each parallel group is 10 tail fishes.Pathogen employing freshwater fish bacterium-have a liking for the water-based Aeromonas is diluted the water-based Aeromonas of having a liking for after twice activation with SPSS, make final concentration be about 5 * 10
7Cells/mL.By the heavy lumbar injection bacterium of every 100g fish liquid 1.0mL, after to put in a suitable place to breed in same specification volume be in the 20L glass jar, and guarantee sufficient oxygen, observe fish body death condition every day, observe 7d, the statistics death rate.The result shows: compare with control group, 50.0mg/kg earthworm small-peptide chromium complex rate of body weight gain significantly (P＜0.05) has improved 13.14% respectively; High density stress 1d after the test, 2.0mg/kg earthworm small-peptide chromium complex significantly (P＜0.05) reduces blood concentration of cortisol 11.44%, blood sugar level 5.78% and concentration of malondialdehyde 9.87% have increased blood catalase 14.66%, superoxide dismutase activity 9.78%; Behind the pathogenic bacterial infection fish body, the control group death rate is 79.0%, and the 50.0mg/kg earthworm small-peptide chromium complex fish death rate is respectively 10.0%; Generally speaking, 50.0mg/kg the earthworm small-peptide chromium complex can improve immunity of fish body and oxidation resistance, improve anti-high density of fish body and opposing and have a liking for water-based Aeromonas anti-infection ability, replenished chromium in the daily ration, some small peptide nutrients have been increased, improve the feed palatability, improve fish body anti-stress ability, promote the fish bulk-growth.
Claims (4)
1, a kind of preparation method of anti-stress medicine earthworm small-peptide chelated chromium is characterized in that: adopt following processing step:
(1), the earthworm that will live cleans, and allows swill in its body that as far as possible spues, adds entry then in earthworm, smashs to pieces with the high speed bruisher, high speed bruisher rotating speed is: 15000～20000 rev/mins, the time of smashing to pieces is: 3～5 minutes;
(2), smash earthworm to pieces material and mix, adjust pH 6～8 is that the self-dissolving enzyme digestion reaction made earthworm small-peptide liquid in 2～4 hours under 40～55 ℃ of conditions in temperature;
(3), with earthworm small-peptide liquid with 100～110 ℃ of boiling water bath heat inactivations 10～15 minutes, in room temperature, placed then 30～60 minutes, got the supernatant packing through centrifugal 10～30 minutes, sealing is standby, obtain little peptide and concentrate peptide liquid;
(4), get earthworm small-peptide and concentrate peptide liquid 40～50 weight %, adjust pH 2～4 is under 70～100 ℃ of conditions in temperature, adds chromium trichloride 5～10 weight %, chelatropic reaction 20～60 minutes; Obtain the earthworm small-peptide chromium complex through room temperature cooling sterilization;
(5), earthworm small-peptide chromic acid chelate vacuum is concentrated moisture content 45～55 weight %, add equivalent mycoprotein powder 45～55 weight % absorption again, and carry out spray drying treatment, baking temperature: 50～70 ℃; Drying time: 30～90 minutes; Moisture 8～12%;
(6), at last earthworm small-peptide chromic acid chelate is pulverized, make 80～110 order earthworm small-peptide chromic acid chelate dry powder.
2, the preparation method of a kind of anti-stress medicine earthworm small-peptide chelated chromium according to claim 1 is characterized in that described grinding particle size is 150～178um.
3, the preparation method of a kind of anti-stress medicine earthworm small-peptide chelated chromium according to claim 1 is characterized in that hydrochloric acid or NaOH or the citric acid of described adjust pH with 0.1mol/L.
4, the preparation method of a kind of anti-stress medicine earthworm small-peptide chelated chromium according to claim 1 is characterized in that described vacuum is 60～90kPa.
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| CN101972449A (en) * | 2010-10-22 | 2011-02-16 | 杨孟臻 | Chinese medicinal material earthworm enzyme and peptide compound preparation and preparation method thereof |
| CN102188743A (en) * | 2011-05-13 | 2011-09-21 | 黄鹰 | Antibacterial and anti-inflammatory medical skin healing membrane and preparation method thereof |
| CN105661554A (en) * | 2016-01-14 | 2016-06-15 | 安徽生物肽产业研究院有限公司 | Silkworm chrysalis small molecule peptide chelate for improving immunity and preparation method thereof |
| CN105815569A (en) * | 2016-03-17 | 2016-08-03 | 上海源耀生物股份有限公司 | Polypeptide chromium chelate prepared from soybean protein isolate taken as substrate and preparation method thereof |
| CN107974478A (en) * | 2017-10-31 | 2018-05-01 | 海盐县凌特生物科技有限公司 | A kind of preparation method of earthworm polypeptide |
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| CN101972449A (en) * | 2010-10-22 | 2011-02-16 | 杨孟臻 | Chinese medicinal material earthworm enzyme and peptide compound preparation and preparation method thereof |
| CN101972449B (en) * | 2010-10-22 | 2012-03-28 | 杨孟臻 | Chinese medicinal material earthworm enzyme and peptide compound preparation and preparation method thereof |
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| CN112220049A (en) * | 2020-10-23 | 2021-01-15 | 黑龙江省野生动物研究所 | Earthworm protein peptide zinc chelate |
| CN114403456A (en) * | 2022-01-27 | 2022-04-29 | 华南理工大学 | Herbal whole grain instant staple food with specific effects of reducing blood sugar and improving diabetic complications and industrial production method thereof |
| CN115669924A (en) * | 2022-10-13 | 2023-02-03 | 及古(广州)生物科技有限公司 | Earthworm producing area processing method and application thereof |
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