CN1146667C - Process for extracting amino acid polypeptide with animal bone marrow and tissue - Google Patents
Process for extracting amino acid polypeptide with animal bone marrow and tissue Download PDFInfo
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- CN1146667C CN1146667C CNB011142170A CN01114217A CN1146667C CN 1146667 C CN1146667 C CN 1146667C CN B011142170 A CNB011142170 A CN B011142170A CN 01114217 A CN01114217 A CN 01114217A CN 1146667 C CN1146667 C CN 1146667C
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Abstract
The present invention discloses a technology for extracting a polypeptide of bone marrow amino acid from animal bone marrow and tissue, which comprises: taking, cleaning and crushing animal bones; deodorizing the animal bones, and removing the fishy smell of the animal bones; steaming the animal bones; carrying out the acid hydrolysis of the animal bones under the condition of pH value of 4 to 5; carrying out the enzymolysis of the animal bones via parenzymes and papain under the conditions of pH value of 3.5 to 5.5 and the temperature of 35 to 55 DEG C.; filtering and concentrating liquid to obtain liquid extract; drying the liquid extract in a spray method. The manufacturing technology is simple, and is easy to operate; the produced polypeptide of bone marrow amino acid has the effect on improving the immunological competence of a body.
Description
Technical field
The invention belongs to marrow and extract marrow amino acid polypeptide production technique, specifically with amino acid whose methods of animal bone mixed extraction marrow such as bright ox bone, bright sheep bones.
Background technology
The marrow polypeptide has the early existing document record of promoter action to the human body cell immunologic function, but at present as yet not document openly utilize the amino acid whose methods of animal bone mixed extraction marrow such as bright ox bone, bright sheep.
Summary of the invention
The object of the present invention is to provide a kind of with amino acid whose production technique of animal bone mixed extraction marrow such as bright ox bone, bright sheep bones.
The present invention's technology is as follows:
1) getting bright ox bone or bright ox bone and tissue, bright sheep bone or bright sheep bone and tissue, bright camel bone or bright camel bone and tissue cleans broken;
2) animal bone after the fragmentation is carried out deodorization, taken off the raw meat processing;
3) animal bone after the boiling fragmentation;
4) under the condition of PH4-5, carry out acid hydrolysis;
5), under temperature 35-55 ℃ the condition, carry out enzymolysis by trypsinase and papain at PH3.5-5.5;
6) cross leaching liquid and carry out the simmer down to concentrated solution;
7) concentrated solution is spray dried to powder.
When getting animal bone, can also get bright silver fox bone or bright silver fox bone and tissue and bright spotted deer bone or bright spotted deer bone and tissue, bone can cover some tissues, and the weight part of choosing each animal bone and tissue is: bright ox bone and tissue: bright sheep bone and tissue: bright camel bone and tissue: bright silver fox bone and tissue: bright spotted deer bone and tissue=70-80: 20-30: 1-2: 0-1: 0-1.
The weight ratio of Trypsin acid and papain is 2: 0.5 in technological process, and the consumption of enzyme is the 1-2 ‰ of substrate weight, and enzymolysis time is 8-10 hour.
Embodiment
Embodiment 1:
Get bright ox bone and tissue, bright sheep bone and tissue, bright camel bone and tissue, bright silver fox bone and tissue, bright spotted deer bone and tissue, weight part ratio is 75: 25: 1: 1: 1 by above technology, make dry powder, sampling utilizes high performance liquid chromatography, according to the ultraviolet absorption spectroscopy general rule, analytical test wherein marrow content of peptides is 0.38mg/mg.
Embodiment 2:
Bright ox bone and tissue, bright sheep bone and tissue, bright camel bone and tissue,-weight fraction ratio is 80: 28: 2, by above technology, makes dry powder, the sampling, utilize high performance liquid chromatography, according to ultraviolet absorption spectroscopy general rule analytical test wherein the marrow content of peptides be 0.29mg/mg.
Above product carries out inspection for food hygiene according to GB4789,2-5,10,11,15-94, and the result is as follows: arsenic (in As, mg/mg) 0.16, plumbous (in Pb, mg/kg) 0.30, total plate count (individual/g) 1.8 * 10
3, coliform (individual/100g)＜30, mould (individual/g)＜10, yeast (individual/g)＜10, pathogenic poison does not detect.Symbol GB16740-1997 health care (function) food universal standard.
Utilize the 835 50 AA instrument J AICP9000 of Hitachi, according to Z/NSJ001-1997 the content of elements in the product is detected, the result is as follows:the mg/kg of unit of micronutrient levels, phosphorus 415, iron 260, calcium 211, magnesium 88.1, manganese 1.50, copper 1.31, zinc 9.6, silicon 216, potassium 3356, aluminium 18.2, boron 3.40, strontium 0.07, lithium 0.61, chromium 0.42, sodium (%) 1.44. detects amino acid whose content according to GB/T14965-94, and the result is as follows: aspartic acid: 5.01% threonine: 1.76% serine: 2.47% glutamic acid: 11.68% glycine: 18.28% alanine: 8.30% cystine: 0.92% a word used in person's names propylhomoserin: 3.19% methionine: 1.46% isoleucine: 1.80% leucine: 4.00% tyrosine: 0.78% phenylalanine: 2.73% lysine: 3.43% histidine: 0.87% arginine: 7.51% proline: 10.86% gamma aminobutyric acid: 0.68% ornithine: 0.31% hydroxy-proline: 10.15%.
1, above product carries out toxicity test.
1 acute toxicity test:
1.1 experimental animal: Kunming mouse, 18-20g, each 10 of male and female.
1.2 test method: get and tried thing 30g.Be made into the even suspension of 150ml with distilled water, press the 0.3ml/10g.bw per os and irritate stomach, 16h stops eating before the filling stomach, irritated stomach once in per three hours, and gave continuously three times, accumulative total is 0.9ml/10g.bW, contain solid substance and heavily be 0.18g, be 18g/kg.bw, observe a week, and record animal toxicity symptom and death condition.The results are shown in Table 1:
Table 1 acute toxicity test in mice result
| Sex | Dosage (g/kg.bw) | Number of animals (only) | The weight of animals changes | Dead animal number (only) | Toxic reaction | |
| Initial weight | Heavy eventually | |||||
| Male | 18 | 10 | 20.5±0.8 | 25.4±0.7 | 0 | Any toxicity symptom does not appear in animal subject in one week |
| Female | 18 | 10 | 18.5±0.6 | 23.9±0.4 | 0 | |
1.3 conclusion: animal does not see obvious toxicity symptom and dead generation, LD50＞18g/kg in the week.Press the acute toxicity classification, this is tried thing and is belonged to nontoxic level class material.
2. micronucleus test:
2.1 experimental animal: Kunming mouse, 24-26g, each 30 of male and female.
2.2 test method: animal is divided into 6 groups at random by body weight, and A organizes negative control group, and the F group is the endoxan positive controls, and other establishes four test dose groups.Test group dosage is respectively 2.0,4.0,6.0,8.0g/kg.bw.Every animal per os is irritated stomach and is tried thing.Irritated stomach for twice 24 hours at interval, and irritated stomach after 6 hours for the second time, put to death animal, get the bone marrow of sternum smear, Giemsa dyeing.Every animal oil mirror is observed 1000 polychromatic erythrocytes down, and the result is as follows:
Table 2 micronucleus test observations (X ± S)
| Group | Dosage (g/kg.bw) | Number of animals (only) | Observation of cell number (individual) | Micronucleated cell number (individual) appears | Micronucleus rate of formation (‰) | ||||
| Female | Male | Female | Male | Female | Male | Female | Male | ||
| A B C D E F | 0 2.0 4.0 6.0 8.0 0.06g/kg | 5 5 5 5 5 5 | 5 5 5 5 5 5 | 5×1000 5×1000 5×1000 5×1000 5×1000 5×1000 | 5×1000 5×1000 5×1000 5×1000 5×1000 5×1000 | 10 13 10 13 12 151 | 9 8 12 13 8 146 | 2.0±0.6 2.6±1.1 2.0±0.9 2.6±1.2 2.4±0.9 30.2±6.1 | 1.8±0.8 1.6±0.5 2.4±0.9 2.6±0.9 1.6±0.4 29.2±8.9 |
2.3 conclusion: each experimental group and negative control group result relatively, difference does not have significantly must property; Show that to be tried thing bone marrow micronucleus test result negative.
3. mouse sperm deformity test:
3.1 experimental animal: Male Kunming strain mice, 23-25g, totally 30.
3.2 test method: animal is divided into 5 groups at random by body weight, and A organizes negative control group, and the E group is the endoxan positive controls, and B, C, D group are test group.Test group dosage is respectively 5.0,10.0,15.0g/kg.bw.Animal continuous irrigation stomach continues to feed 30 days after five days again, puts to death animal, gets both sides epididymis smear, Yihong dyeing.Every animal high power lens is observed 1000 sperms down, record teratospermia number.The results are shown in Table 3
Table 3 mouse sperm deformity test-results (x ± s)
| Group | Dosage (g/kg.bw) | Number of animals (only) | Observe sperm count (individual) | Teratospermia number (individual) | Rate of teratosperm (%) |
| A B C D E | 0 5.0 10.0 15.0 0.04g/kg | 6 6 6 6 6 | 6×1000 6×1000 6×1000 6×1000 6×1000 | 116 148 157 155 543 | 1.9±0.5 2.4±0.5 2.6±0.3 2.6±0.6 9.0±1.2 |
3.3 conclusion: each experimental group and negative control group result compare, and difference does not have significance, and it is negative to show that this is tried thing sperm malformation test result.
4.Ames test:
4.1 test organisms pearl: employing is tested through Salmonella typhimurium histidine defect type TA97, TA98, TA100, TA102 four strain bacterial strains that authenticator closes biological requirement.
4.2 test method: adopt polychlorobiphenyl (PCB) inductive rat liver homogenate as external activation system (+S9).Tried thing and be divided into five dosage groups, every ware add-on 0.2mL contains 2,8,40,200,500ug is tried thing (in solid substance) respectively as each dosage group, 0.103Mpa, 20min sterilising treatment.Blank and positive controls are set simultaneously.Adopt flat board to mix method record secondary multiple parallel sample result.
Table 4 Salmonella reversion test result (individual/ware, x ± s)
| The change colony number is returned in agent | |||||||||
| The group amount | -S9 | +S9 | |||||||
| (ug/ ware) | TA97 | TA98 | TA100 | TA102 | TA97 | TA98 | TA100 | TA102 | |
| Become dosage group positive control from beaming back | 028 40 200 5000 annotate | 94±7 135±24 120±10 137±6 137±18 154±26 ＞6000 | 30±4 41±8 48±10 40±4 46±10 43±12 ＞6000 | 113±7 134±14 141±9 141±14 173±19 176±7 432±50 | 238±21 269±18 262±12 271±22 295±22 298±11 1086±294 | 104±7 142±8 131±14 141±30 142±12 157±19 1826±173 | 32±6.8 38±5.7 37±6.0 34±10 41±8.4 41±5.0 ＞5000 | 119±8.7 145±16.0 134±10.0 153±18.0 140±10.3 182±10.0 ＞3000 | 242±10.0 272±11.0 266±8.30 288±21.0 271±22.0 286±28 1305±277 |
Annotate :-S9 positive controls TA97,98,100 is 2,4,7-TNFone, and add-on is the 0.3ug/ ware, TA102
Positive thing is a fenaminosulf, and add-on is the 50ug/ ware.
+ S9 positive controls TA97,98,100 is 2-AF, and add-on is the 20ug/ ware, the positive thing of TA102
Be 1,8 one dihydroxyanthraquinones, add-on is the 50ug/ ware.
4.3 conclusion: each organize back and becomes the bacterium colony number average and beam back 2 times of parameters above oneself to be tried thing, and does not have dose-response relationship, so the Salmonella reversion test result is negative.
5.30 it feeding study
5.1 animal varieties: Wistar rat, totally 80, body weight 75-90g, male and female half and half.
5.2 dosage and grouping: establish three dosage groups and a control group, every group of 20 animals, male and female half and half.Recommend daily intaking amount 0.1g/kg.bw to enlarge 20,50,100 times respectively as basic, normal, high dosage group by the crowd, corresponding actual dose is 2.0,5.0,10.0g/kg.bw.Tried thing and give approach by 1.5ml/100gbw per os filling stomach, give 30 days continuously, control group gives equivalent distilled water simultaneously.Measure relevant index by test requirements document then.
5.3 observation index:
5.3.1 clinical examination: comprise general performance, behavior, poisoning and death condition, write down each treated animal body weight and food-intake weekly, calculate food utilization.
5.3.2 hematological examination: after giving 30 days continuously, measure back treated animal oxyphorase (Hb) content, red corpuscle (RBC) counting, white corpuscle (WBC) counting and classification, thrombocyte (PLT) counting, reticulocyte counts except that reticulocyte is test tube color method smear counting, all the other indexs all adopt Japan to produce SYSMEX
TMF820 type automatic hemacytometer is measured.
5.3.3 learning, blood biochemical checks: after giving 30 days continuously, measure gpt (ALT) in each treated animal serum, glutamic-oxal(o)acetic transaminase (AST), blood urea nitrogen (BUN), glucose (Glu), creatinine (Cre), total cholesterol (T.C) content and albumins/globulins (Alb/Glo) ratio.Removing glucose adopts U.S. Johnson company to produce ONE TOUCH
TMOutside the quick paper disk method of blood sugar analyzer was measured, all the other indexs all adopted Germany to produce eppendorf
TMGive birth to the corresponding reagent box mensuration that biological high-technology company produces in ECOM-F 6124 type automated analysis instrument, Beijing.
5.3.4 take by weighing each treated animal main organs weight and calculate organ coefficient.
5.3.5 histopathologic examination: each treated animal is carried out the gross anatomy inspection, and main organs such as the heart, liver, spleen, lung, kidney are carried out histopathologic examination.
5.4 test-results:
5.4.1 clinical examination: each dosage animal does not have toxicity symptom in the test.Body weight and food utilization the results are shown in Table 5.
Visible each dosage treated animal and food utilization and control group compare in the table, and difference is analysed in credit does not by statistics have significance.
Table 5 is tried thing to the influence of rat body weight and food utilization (x ± s)
| Dosage (g/kg.bw) | Number of animals (only) | Body weight (g) | Food utilization (%) | ||||
| Initial weight | First week | Second week | The 3rd week | Heavy eventually | |||
| ♀ control group 2.0 5.0 10.0 ♂ control groups 2.0 5.0 10.0 | 10 10 10 10 10 10 10 10 | 77.2±83 75.3±8.7 83.2±4.7 78.2±6.4 87.3±3.2 78.5±4.7 87.1±3.0 86.5±3.2 | 114.7±11 110.4±8.9 120.4±3.1 107.0±11 118.0±8.7 128.0±5.5 116.0±4.5 115.3±7.0 | 155.0±23 142.0±7.2 155.0±13 131.3±18 145.0±8.1 155.3±8.7 140.4±6.5 145.6±10 | 188.0±18.4 182.0±7.9 178.0±18.4 165.4±19.7 191.0±11.0 183.0±8.8 175.0±13 181.0±13 | 199.3±15.7 214.3±10.0 206.0±13.5 195.9±14.4 229.0±8.0 217.0±7.0 227.0±16.0 227.4±23.2 | 21.8 22.4 22.0 21.9 22.6 23.5 23.9 22.4 |
5.4.2 hematological examination: each group is randomly drawed part zoometry physiochemical indice, the results are shown in Table 6.1,6.2.As can be known, every index determining result and control group compare from table, and difference is analysed in credit does not by statistics have significance.Whole blood is learned the index determining result all in normal range.
Table 6.1 is tried thing is learned index to rat blood influence (x ± s)
| Dosage (g/kg.bw) | Number of animals (only) | Hb content (g/L) | RBC counts (X10 12Individual/L) | WBC counts (X10 9Individual/L) | PLT counts (X10 9Individual/L) |
| Control group 2.0 5.0 10.0 | 10 10 10 10 | 131.0±8.1 132.6±11.3 137.3±0.3 126.2±18.0 | 8.16±0.83 8.44±0.40 8.38±0.39 8.19±0.94 | 18.2±3.6 19.9±4.6 18.0±2.2 18.0±4.7 | 895.6±187.5 916.4±241.9 921.3±16.5 876.8±184.6 |
Table 6.2 is tried thing is learned index to rat blood influence (x ± s)
| Dosage (g/kg.bw) | Number of animals (only) | WBC classify (%) | Observe RBC number (individual) | Reticulocyte counts (individual) | Net is knitted red occurrence rate (%) | |
| Lymph and monokaryon | Granulocyte | |||||
| Control group 2.0 5.0 10.0 | 10 10 10 10 | 68.2±1.10 68.0±1.05 72.1±1.3 72.5±3.5 | 31.8±1.1 32.0±1.1 27.9±1.3 27.5±3.5 | 10×1000 10×1000 10×1000 10×1000 | 280 287 290 286 | 2.80 2.87 2.90 2.86 |
Check 5.4.3 blood biochemical is learned: each group is randomly drawed part zoometry blood biochemical and is learned index, the results are shown in Table 7.1,7.2.As can be known, every index determining result and control group compare from table, and difference is analysed in credit does not by statistics have significance.
Table 7.1 is tried thing to the influence of rat blood biochemical indexes (x ± s)
| Dosage (g/kg.bw) | Number of animals (only) | ALT (U/L) | AST (U/L) | BUN (mmol/L) | T.C (mmol/L) |
| Control group 2.0 5.0 10.0 | 10 10 10 10 | 94.8±29.4 98.7±21.1 95.5±39.2 110.3±21.4 | 270.1±36.7 281.4±79.2 242.1±40.4 276.3±70.2 | 16.9±11.1 9.86±3.57 8.95±4.56 8.16±1.41 | 2.81±0.69 3.21±0.42 3.46±0.63 2.98±0.66 |
Table 7.2 is tried thing to the influence of rat blood biochemical indexes (x ± s)
| Dosage (g/kg.bw) | Number of animals (only) | Are (umol/L) | Alu (mmol/L) | Alb/Glo |
| Control group 2.0 5.0 10.0 | 10 10 10 10 | 86.8±21.6 83.5±34.6 94.6±10.1 87.5±11.6 | 2.91±0.55 2.87±0.43 2.80±0.29 2.63±0.18 | 0.78±0.19 0.75±0.06 0.55±0.38 0.70±0.08 |
5.4.4 organ weights and coefficient: take by weighing each treated animal heart, liver, spleen, lung, kidney main organs weight calculating organ coefficient of immersing oneself quietly in hard work, the results are shown in Table 8.1,8.2.As can be known, the every index determining result of each dosage group compares with control group from table, and difference is analysed in credit does not by statistics have significance.
Table 8.1 is tried the influence (X ± S) of thing Rats Organs and Tissues weight and coefficient
| Dosage (g/kg.bw) | Number of animals (only) | Heart | Liver | Food utilization | |||
| Weight (g) | Coefficient (%) | Weight (g) | Coefficient (%) | Weight (g) | Coefficient (%) | ||
| ♀ control group 2.0 5.0 10.0 ♂ control groups 2.0 5.0 10.0 | 10 10 10 10 10 10 10 10 | 0.69±0.06 0.76±0.06 0.74±0.03 0.75±0.02 0.86±0.13 0.87±0.11 0.89±0.10 0.85±0.09 | 0.35±0.26 0.35±0.09 0.36±0.06 0.38±0.04 0.38±0.07 0.40±0.09 0.39±0.06 0.37±0.04 | 7.9±1.21 8.6±1.08 7.6±0.09 8.1±0.07 9.15±0.13 10.9±0.21 10.1±0.15 11.0±0.25 | 3.94±1.18 4.03±1.05 3.68±0.08 4.13±0.06 4.00±0.15 5.02±0.13 4.50±0.11 4.84±0.15 | 0.51±0.13 0.56±0.09 0.54±0.06 0.52±0.04 0.63±0.02 0.65±0.03 0.68±0.04 0.66±0.04 | 0.25±0.83 0.26±0.05 0.26±0.03 0.27±0.03 0.28±0.05 0.30±0.04 0.30±0.02 0.29±0.07 |
Table 8.2 is tried the influence (X ± S) of thing Rats Organs and Tissues weight and coefficient
| Dosage (g/kg.bw) | Number of animals (only) | Lungs | Kidney | ||
| Weight (g) | Coefficient (%) | Weight (g) | Coefficient (%) | ||
| ♀ control group 2.0 5.0 10.0 ♂ control groups 2.0 5.0 10.0 | 10 10 10 10 10 10 10 10 | 1.43±0.15 1.54±0.32 1.26±0.19 1.40±0.26 1.48±0.23 1.68±0.15 1.56±0.09 1.56±0.08 | 0.72±0.05 0.75±0.03 0.71±0.06 0.71±0.03 0.65±0.05 0.77±0.01 0.69±0.04 0.69±0.08 | 1.48±0.25 1.67±0.08 1.61±0.05 1.62±0.03 1.79±0.02 1.81±0.11 1.73±0.07 1.81±0.06 | 0.74±0.11 0.81±0.04 0.80±0.08 0.83±0.07 0.78±0.09 0.83±0.08 0.76±0.05 0.80±0.04 |
5.4.5 the result of histopathologic examination: internal organs such as the part animal heart, liver, spleen, lung, kidney, suprarenal gland are carried out gross anatomy observe and histopathologic slide, except that a small amount of animal lung tissue presented the pneumonia of rats image, all the other internal organs did not see that obvious toxic pathology changes.
Learnt that by above experiment the marrow amino acid polypeptide that the present invention's explained hereafter goes out is to two kinds of sex Kunming mouse per os toxicity, it is 18g/kg.bw that three per os of accumulative total are irritated the stomach total amount, and animal does not see obvious toxicity symptom in the week, does not have animal dead yet.Press the acute toxicity classification, this is tried thing and is belonged to nontoxic level class material.The result is all negative for three mutagenicity tests (mouse polychromatic erythrocytes micronucleus test, mouse sperm deformity test, Salmonella reversion test).The feeding study result showed in 30 days: this is tried thing 2.0,5.0,10.0g/kg.bw dosage (be equivalent to crowd's day recommended intake 6g/60kg.bw 20,50,100 times), clinical examination (containing weight increase and food utilization), hematological examination, the blood biochemical of Wistar rat are learned check, main organs weight and organ coefficient and histopathologic examination all do not have the overt toxicity influence.
Two, above product carries out the immunity experiment:
1. sample gives approach: sample is made into respective concentration with sterile distilled water, and each is organized mouse per os every day and gavages corresponding dosage, gives to carry out after one month following test work continuously.The normal control group gavages ordinary tap water.
2. animal: Male Kunming strain mice, 18-22g.Hubei Province's medical experiment animal center provides.The animal approval number is Hubei Province moving doctor's pipe word 19-007.
3. grouping: recommend the crowd day to drink 6g/60kg.bw by manufacturer, enlarge 10,20,30 times, design basic, normal, high three dosage groups, promptly 1,2,3g/kg.bw.
4. test method and result:
4.1 the weight of animals record before and after the test: see Table body weight no significant difference between 1. each treated animal.
The weight of animals record before and after table 1 test (gram, X ± S)
| Group | Dosage (g/kg.bw) | Number of animals (only) | Body weight (gram) | Weightening finish (gram) | |
| Before the test | After the test | ||||
| Dosage group high dose group in the blank group low dose group | 0 1 2 3 | 10 10 10 10 | 19.2±1.9 19.5±1.4 19.4±1.7 19.3±1.4 | 39.1±2.1 39.9±1.6 39.8±1.8 39.6±1.6 | 19.9±1.0 20.4±0.8 20.4±0.5 20.3±0.4 |
4.2 influence: see Table 2, thymus gland weight ratio and spleen between each treated animal/body weight ratio no significant difference to animal lymph organ/body weight ratio.
Table 2 pair animal washes one's hair the influence (X ± S) of crust organ/body weight ratio
| Group | Dosage (g/kg.bw) | Number of animals (only) | Thymus gland/body weight ratio (X10 -3) | Spleen/body weight ratio (X10 -3) |
| Dosage group high dose group in the blank group low dose group | 0 1 2 3 | 10 10 10 10 | 2.42±0.14 2.45±0.15 2.41±0.16 2.43±0.19 | 3.26±0.13 3.28±0.11 3.26±0.11 3.29±0.13 |
4.3CouA inductive mouse spleen lymphocyte conversion test
Method: mtt assay (step congenerous experimental arrangement regulation)
The result: see Table 3, as seen from Table 3, compare with the blank group, the middle and high dosage group of this product dominance strengthens ConA inductive spleen lymphocyte proliferation ability.
Table 3 pair mouse cell Immune Function
| Group | Dosage (g/kg.bw) | ConA induces spleen lymphocyte proliferation | DNFB induces DTH | ||
| N | The OD difference | N | Left and right sides ear weight amount poor (mg) | ||
| Dosage group high dose group in the blank group low dose group | 0 1 2 3 | 10 10 10 10 | 0.012±0.003 0.019±0.006# 0.028±0.009 * 0.033±0.12 * | 10 10 10 10 | 17.6±2.6 20.9±1.9 * 21.9±2.7 * 24.1±2.8 * |
Compare with the blank group
*P＜0.01 #0.01＜P＜0.05
Annotate: get 0.1ml and measure the OD570 value
4.4 dinitrofluorobenzene (DNFB) is induced delayed allergy (DTH)
Method: ear swelling method.Behind the DNFB sensitized mice, attacked auris dextra with NDFB again on the 5th day, put to death animal behind the 24h and cut left and right sides auricular concha and take off the auricle of diameter 8mm with punch tool, weigh, represent the degree of DTH with the difference of the weight of left and right sides ear.
The result: see Table 3, as seen from Table 3: compare with the blank group, the basic, normal, high dosage group of this product significance strengthens the DTH reaction that mouse is brought out DNFB.
4.5 the mensuration of serum hemolysin
Method: blood clotting method.According to the functional experiment procedure operation, the level calculation of condensing degree according to serum goes out the antibody product.
The result: as seen from Table 4, but the product high dose group significance rising serum hemolysin content that the present invention produces.
The influence of table 4 pair mouse humoral immune function
| Group | Dosage (g/kg.bw) | Number of animals (only) | Serum hemolysin (antibody product) |
| Dosage group high dose group in the blank group low dose group | 0 1 2 3 | 10 10 10 10 | 72.6±16.7 36.0±26.7 99.0±26.4# 115.8±24.2 * |
Compare with the blank group
*P＜0.01 #0.01＜P＜0.05
4.6 peritoneal macrophage is engulfed the chicken red blood cell test
Method: half intracorporal method.The chicken erythrocyte suspension of system blood 20%; Every this suspension of mouse abdominal injection 1mL is put to death animal behind the 30min, it is faced upward upright being fixed on the mouse plate, open abdomen, inject physiological saline 2mL through the abdominal cavity, rotate mouse 1min, then, sucking-off abdominal cavity washing lotion 1mL, average mark drip on 2 slide glasss, and 37 ℃ of incubation 30min educate to finish and use the physiological saline rinsing, airing, acetone methanol solution with 1: 1 is fixed, and 4%Giemsa-phosphoric acid buffer dyeing 3min dries with the distilled water rinsing again.The oil mirror is 100 scavenger cells of counting down, are calculated as follows phagocytic rate and phagocytic index:
The result: see Table 5, as seen from Table 5, the low middle and high dosage of this product is engulfed percentage ratio, phagocytic index all apparently higher than the blank group.
The influence of table 5 pair Turnover of Mouse Peritoneal Macrophages phagocytic function
| Group | Dosage (g/kg.bw) | Number of animals (only) | Phagocytic percentage (%) | Phagocytic index |
| Dosage group high dose group in the blank group low dose group | 0 1 2 3 | 10 10 10 10 | 38.3±3.7 44.3±3.0 * 49.5±2.2 * 52.0±2.5 * | 0.413±0.04 0.482±0.03 * 0.529±0.02 * 0.558±0.03 * |
Compare with the blank group
*P＜0.01
4.7 mouse carbon clearance test
Method: according to the method for procedure stipulation.According to blood carbon concentration (survey light absorption value) behind injection prepared Chinese ink 2min, the 10min, calculate the phagocytic index of respectively organizing mouse.
Result: see Table 6, as seen from Table 6, clean up phagocytic index but the basic, normal, high dosage group of the product significance that the present invention produces improves mouse carbon.
The table 6 pair influence that mouse carbon is cleaned up function
| Group | Dosage (g/kg.bw) | Number of animals (only) | Phagocytic index |
| Dosage group high dose group in the blank group low dose group | 0 1 2 3 | 10 10 10 10 | 4.088±0.2 4.708±0.3 * 5.053±0.4 * 5.255±0.6 * |
Compare with the blank group
*P＜0.01
4.8 antibody-producting cell detects
Method: improvement Jerne slide method (summary).
The result: see Table 7, as seen from Table 7, middle and high dosage group is all apparently higher than the normal control group, and analyzing difference by statistics has significance (p＜0.01).
The influence of table 7 antagonist founder cell function
| Group | Dosage (g/kg.bw) | Number of animals (only) | Hemolysis plaque number (/ 10 2Splenocyte) |
| Dosage group high dose group in the blank group low dose group | 0 1 2 3 | 10 10 10 10 | 441±33.6 478±29.3# 517±12.7 * 534±28.44 * |
Above test-results shows: with the blank group relatively, 1) middle and high dosage group can obviously strengthen ConA inductive mice spleen lymphocytes proliferation ability; 2) basic, normal, high dosage group can obviously strengthen the delayed allergy of dinitrofluorobenzene inductive mouse; 3) the middle and high dosage group mice serum hemolysin content that can obviously raise; 4) basic, normal, high dosage group can obviously strengthen Turnover of Mouse Peritoneal Macrophages and engulfs the chicken red blood cell function; 5) basic, normal, high dosage group can obviously strengthen mouse carbon and cleans up ability; 6) middle and high dosage group can obviously strengthen the antibody-producting cell ability.According to immunoregulation effect assessment process regulation, this is tried thing and is had immunoregulation effect.
Claims (6)
1, a kind of animal bone and tissue extraction marrow amino acid polypeptide production technique, its feature process is:
1) getting bright ox bone or bright ox bone and tissue, bright sheep bone or bright sheep bone and tissue, bright camel bone or bright camel bone and tissue cleans broken;
2) animal bone after the fragmentation is carried out deodorization, taken off the raw meat processing;
3) animal bone after the boiling fragmentation;
4) under the condition of PH4-5, carry out acid hydrolysis;
5), under temperature 35-55 ℃ the condition, carry out enzymolysis by trypsinase and papain at PH3.5-5.5;
6) cross leaching liquid and carry out the simmer down to concentrated solution;
7) with the concentrated solution spraying drying.
2, animal bone and tissue extraction marrow amino acid polypeptide production technique according to claim 1 is characterized in that obtaining animal bone and also have bright silver fox bone or bright silver fox bone and tissue; Bright spotted deer bone or bright spotted deer bone and tissue.
3, animal bone and tissue extraction marrow amino acid polypeptide production technique as claimed in claim 1 or 2, it is characterized in that covering on the animal bone tissue, the weight part of each animal bone and tissue is: bright ox bone and tissue: bright sheep bone and tissue: bright camel bone and tissue: bright silver fox bone and tissue: bright spotted deer bone and tissue=70-80: 20-30: 1-2: 0-1: 0-1.
4, animal bone and tissue extraction marrow amino acid polypeptide production technique according to claim 1, the weight ratio that it is characterized in that trypsinase and papain is 2: 0.5.
5, as animal bone and tissue extraction marrow amino acid polypeptide production technique as described in the claim 4, the consumption that it is characterized in that enzyme is the 1-2 ‰ of substrate weight.
6, as animal bone and tissue extraction marrow amino acid polypeptide production technique as described in claim 1 or 4 or 5, it is characterized in that enzymolysis time is 8-10 hour.
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Families Citing this family (15)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN1319471C (en) * | 2004-05-26 | 2007-06-06 | 内蒙古草原兴发股份有限公司 | Yak marrow powder health care product and its making method |
| CN100334225C (en) * | 2005-04-22 | 2007-08-29 | 马相林 | I-type protein small peptide and its preparing method |
| CN102669405A (en) * | 2012-05-22 | 2012-09-19 | 新沂市嘉禾鸭业有限公司 | Preparation method of duck bone protein peptide |
| CN104003766B (en) * | 2014-06-23 | 2016-01-27 | 蒋常德 | The waste water of livestock and poultry harmless treatment and meat meal tankage is utilized to produce the method for amino acid fertilizer |
| CN104561210A (en) * | 2015-01-27 | 2015-04-29 | 张恒 | Extraction method of deer bone peptide |
| CN105886583A (en) * | 2015-01-28 | 2016-08-24 | 北京汉合盛世国际能源贸易有限公司 | Bovine bone marrow peptide extraction method |
| CN105876442A (en) * | 2015-01-28 | 2016-08-24 | 北京汉合盛世国际能源贸易有限公司 | Extraction method of sheep bone marrow peptides |
| CN104621338A (en) * | 2015-03-03 | 2015-05-20 | 浙江小二黑食品有限公司 | Method for preparing novel silkie bone polypeptides |
| CN105166957A (en) * | 2015-07-30 | 2015-12-23 | 王之宝 | Bovine bone marrow powder production process and application thereof |
| CN105919094A (en) * | 2016-05-11 | 2016-09-07 | 安徽中森生物技术有限公司 | Immunity improving and fatigue preventing composition and preparation method thereof |
| CN106854232B (en) * | 2017-03-07 | 2020-10-09 | 中国科学院新疆理化技术研究所 | Preparation method and application of bone marrow protein |
| CN107287268A (en) * | 2017-06-16 | 2017-10-24 | 南京佰泰克生物技术有限公司 | A kind of adjuvant of the nucleic acid vaccine immunity originality of enhancing HIV 1 |
| CN107981233A (en) * | 2017-11-29 | 2018-05-04 | 银川伊百盛清真食品有限公司 | Ox, sheep bone biological enzymolysis and peptide, calcium separating and extracting process |
| CN108671221A (en) * | 2018-07-26 | 2018-10-19 | 广东羲准生物科技有限公司 | Gugua polypeptide composition and its application |
| CN109349613A (en) * | 2018-12-21 | 2019-02-19 | 内蒙古神元康肽生物工程有限公司 | A kind of camel bone collagen peptide oral liquid and preparation method thereof |
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