CN104878070A - Anaerobic culture medium for culturing fusobacterium necrophorum - Google Patents

Abstract

The invention discloses an anaerobic culture medium for culturing fusobacterium necrophorum, and a preparation method of the anaerobic culture medium, and belongs to the field of culture for inspecting microorganism. The anaerobic culture medium is characterized in that the formula for preparing 1000ml of culture medium includes casein tryptone, glucose, yeast powder, sodium chloride, agar, bile salt, ethylenediaminetetraacetic acid dipotassium magnesium salt monohydrate, gamma-aminobutyric acid, valine and sterile heat-deformation calf serum; distilled water is added to reach 1000ml. Compared with the prior art, the anaerobic culture medium has the characteristics of being reliable to inspect, and high in fusobacterium necrophorum separation rate.

Description

A kind of anaerobic culture medium cultivated for actinomyces pseudonecrophorus

Technical field

The present invention relates to inspection microorganism culturing field, be specifically related to a kind of anaerobic culture medium cultivated for actinomyces pseudonecrophorus.

Background technology

Microorganism culturing is a kind of technology making microbial growth by artificial means, and the substratum that selective medium is used to promote or suppress the organism (as cell or bacterium etc.) of certain type and designs, utilize this substratum required microorganism can be separated from the microorganism mixed.The bacterium that actinomyces pseudonecrophorus (Fusobacterium necrophorum, Fn) is Fusobacterium, Gram-negative, strictly anaerobic.Pathogenic Fn bacterial strain produces multiple independent factor, as leukotoxin (leukotoxin, Lkt), hemolysin (hemolysin), hemagglutinin (hemagglutinin) and intracellular toxin (endotoxin) etc., the necrobacillosis (necrobacillosis) of multiple Mammals (comprising the mankind) and bird can be caused.Actinomyces pseudonecrophorus is the main and secondary cause of disease that various suppuration gangrenosum acne catches, and is extensively present in the natural cavities such as the oral cavity of animal and human, respiratory tract and gi tract, and in the histoorgan of suppurative necrotic lesion and infarct, belongs to conditioned pathogen.Fn generally invades body by the skin or mucous membrane that have wound, with other bacteriums as intestinal bacteria or the suppuration such as streptococcus aureus, suppurative coryneform bacteria bacterioid co-infection host cause necrobacillosis, this disease is common and harm is serious in animal, and the mankind mainly cause acute pharyngitis syndromes (Lemierre ' s), hand skin, oral cavity and lung's abscess.The typical symptom of these diseases there is abscess or gangrenosum acne focus, and foul smelling smell.In addition, what have also causes serious threat with bacteremic generation to host's life security, and clinical symptom and streptococcal pharyngitis, bacterial pneumonia symptom are similar, easy mistaken diagnosis.Therefore the correct and Rapid identification as the clinical bacteria inspection of making a definite diagnosis unique foundation is very important.

At present, actinomyces pseudonecrophorus measuring means mainly adopts with polymerase chain reaction (polymerase chain reaction, PCR) molecular biology for detection based on, the method needs to carry out DNA extraction and PCR primer design to sample, and need to be equipped with PCR related equipment, cost intensive, complicated operation, laboratories is difficult to realize.Laboratory is commonly used CDC anaerobic agar and is cultivated actinomyces pseudonecrophorus, because Clinical Laboratory In Grass Root Hospital lacks anaerobic inspection machine mostly, therefore multiplex greatly " anaerobism cylinder culture method ", so-called anaerobism cylinder is common dry cylinder, the cultivation of dry cylinder put into by flat board or the liquid nutrient medium test tube of having inoculated sample, exhaust oxygen in cylinder by physicochemical method, cause anaerobic environment.The oxygen depletion of anaerobism cylinder method needs the time, and actinomyces pseudonecrophorus is obligate anaerobic bacillus, therefore there is following defect in this kind of culture technique: the actinomyces pseudonecrophorus poor growth 1. in anaerobism cylinder on CDC anaerobic agar substratum, and positive rate is low, and within one week, qualification can be made more accurately in right and left; 2. due to non-absolute oxygen free condition, therefore in throat secretory product sample, aerophil and facultative anaerobe have life condition, and substratum separating power is poor.

Summary of the invention

Technical assignment of the present invention is for above the deficiencies in the prior art, provides a kind of anaerobic culture medium being suitable for actinomyces pseudonecrophorus growth.

The technical scheme that the present invention solves its technical problem is: a kind of anaerobic culture medium cultivated for actinomyces pseudonecrophorus, is characterized in that, contains in the formula of preparation 1000ml substratum:

Pancreas casein peptone 5.0 ~ 10.0g;

Glucose 5.0 ~ 10.0g;

Yeast powder 5.0 ~ 10.0g;

Sodium-chlor 5.0g;

Agar 15.0 ~ 20.0g;

Bovine bile 0.5 ~ 1.0g;

Ethylenediamine tetraacetic acid dipotassium magnesium salt salt 2 ~ 3g;

γ-aminobutyric acid 0.1 ~ 0.5g;

α-amino-isovaleric acid 0.1 ~ 0.5g;

Aseptic thermal distortion calf serum 50 ~ 100ml;

Distilled water adds to 1000ml.

Above-mentioned aseptic thermal distortion calf serum preparation method is: heat 10min at getting aseptic calf serum 80 DEG C, add sterilizing trypsin digestion 90min at 45 DEG C, and the mass ratio of enzyme-to-substrate is 1:150.

The preparation method of above-mentioned substratum is: take pancreas casein peptone; Glucose; Yeast powder; Sodium-chlor; Agar; Bovine bile; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt; γ-aminobutyric acid; α-amino-isovaleric acid is dissolved in distilled water, mixing, and 121 DEG C, sterilizing 15min, is cooled to 45 DEG C, adds the mixing of aseptic thermal distortion calf serum, packing after sterile purified water constant volume to 1000ml.

Described pancreas casein peptone contains various saccharides, amino acid, VITAMIN and trace element provides energy for actinomyces pseudonecrophorus grows; Glucose provides carbon source, the nutritive ingredients such as yeast powder rich in proteins, amino acid, polypeptide, Nucleotide, VITAMIN, somatomedin, trace element, promotes the Growth and Reproduction of actinomyces pseudonecrophorus; Sodium-chlor maintains balanced osmotic pressure; Agar is the peptizer of substratum; γ-aminobutyric acid is nutrient intensifier, promotes actinomyces pseudonecrophorus growth; α-amino-isovaleric acid is anerobe nutrition agent.

Ethylenediamine tetraacetic acid dipotassium magnesium salt salt provides the inorganic salt needed for actinomyces pseudonecrophorus growth, and the ethylenediamine tetraacetic acid dipotassium magnesium salt salt under this concentration can produce following antibacterial sterilization functions: 1. the displacement of ethylenediamine tetraacetic acid dipotassium magnesium salt salt substitutes the Yeast Nucleic Acid magnesium salts in gram-positive microorganism, cause it can not generate magnesium salts-protein complex (ribonucleoprotein, RNP), the process that the genetic information of nucleic acid encoding flows to active protein is obstructed, and produces and is directed to gram-positive microorganism killing action; 2. for the gram-positive microorganism that iso-electric point is higher, ethylenediamine tetraacetic acid dipotassium magnesium salt salt is attached to bacteria cell wall by electrical complexing action, causes cellularstructure to be out of shape, and carries out playing antibacterial sterilization functions.And outer membrane protein OMP on fusobacterium Pseudomonas cell walls and fadA adhesion protein make the cell wall area iso-electric point of actinomyces pseudonecrophorus not mate with ethylenediamine tetraacetic acid dipotassium magnesium salt salt, electrical complexing action is caused to lose efficacy, and lack Yeast Nucleic Acid magnesium salts, therefore antibacterial inefficacy due to it.By experiment respectively by common oral in throat secretory product sample

Bacterisation Streptococcus pyrogenes, streptococcus pneumoniae, Thrombin coagulase staphylococcus, tetanus bacillus, actinomyces pseudonecrophorus inoculation is on the ethylenediamine tetraacetic acid dipotassium magnesium salt salt CDC anaerobic selection substratum adding 3g/L, control group is common CDC anerobe Selective agar medium, in 37 DEG C, observe after cultivating 48h in anaerobic jar, the substratum of interpolation EDTA Dipotassium salt finds that there is actinomyces pseudonecrophorus bacterium colony, do not find streptococcus pyogenes, streptococcus pneumoniae, Thrombin coagulase staphylococcus bacterium colony, tetanus bacillus bacterium colony, control group substratum finds streptococcus pyogenes, streptococcus pneumoniae, Thrombin coagulase staphylococcus, tetanus bacillus and actinomyces pseudonecrophorus bacterium colony, confirm that EDTA Dipotassium salt is to streptococcus pyogenes thus, streptococcus pneumoniae, Thrombin coagulase staphylococcus, tetanus bacillus has good sterilization functions, and actinomyces pseudonecrophorus is had no significant effect.

In order to obtain miscellaneous bacteria effect of better going out, add bovine bile in formula, bovine bile can suppress Gram-positive bacteria growing, but in order to keep the electrolyte balance of appropriateness, and its effect is only the sterilizing synergy for ethylenediamine tetraacetic acid dipotassium magnesium salt salt, therefore consumption concentration is smaller.

Thermal distortion calf serum has optionally antibacterial effect, and its thermal distortion method is: heat 10min at getting aseptic calf serum 80 DEG C, adds sterilizing trypsin digestion 90min at 45 DEG C, and the mass ratio of enzyme-to-substrate is 1:150.Thermal distortion calf serum antibacterial properties is for aerophil, change aerophil cell wall structure on the one hand, cause permeability increase and dead, the redox-potential of substratum can be reduced on the other hand, promote oxygen-free environment and accelerate aerophil death, and this kind of anti-microbial effect is only directed to aerophil, then invalid for anerobe.By experiment same coli strain and actinomyces pseudonecrophorus bacterial strain are inoculated into respectively and with the addition of on the optimization CDC Selective agar medium of the thermal distortion calf serum of 80ml/L in CDC Selective agar medium and formula, cultivate at 37 DEG C, hermetically drying cylinder ring border, 24h, 48h identify, adding thermal distortion calf serum intestinal bacteria positive rate during 24h is 40%, and on the substratum not adding thermal distortion calf serum, intestinal bacteria positive rate is 70%; 48h detected result is add the substratum actinomyces pseudonecrophorus positive rate 80% of thermal distortion calf serum, actinomyces pseudonecrophorus positive rate 20% on the substratum not adding thermal distortion calf serum; Confirm that thermal distortion calf serum has good inhibition to aerophil in anaerobism cylinder thus, and promote that anerobe grows by accelerating oxygen-free environment.

L-homoarginine is activator, promotes the absorption of α-amino-isovaleric acid.

Rhizome of Grass leaf Sweelflag (formal name used at school: Acorus gramineus) belongs to Acoraceae, and be graminoid per nnial herb, medicinal effects is rhizome position.Modern medicine study shows, Rhizome of Grass leaf Sweelflag is containing compositions such as volatile oil and carbohydrate, organic acid, amino acid, inorganic elements such as α-trans-Isoasarones, there are the effects such as swelling and pain relieving, waking up the patient from unconsciousness by dissipating phlegm, ulcer mange, Rhizome of Grass leaf Sweelflag Aqueous extracts is good nutrition-fortifying agent, Rhizome of Grass leaf Sweelflag water logging agent (1:3), all has restraining effect in various degree to dermatophytess such as trichophyton, Trichophyton concentricum, star-shaped nocardias in vitro.Have report to point out, Rhizome of Grass leaf Sweelflag decoction has the effect of killing ascites cells.The research such as Masuda T finds that the α-trans-Isoasarone in Rhizome of Grass leaf Sweelflag all has restraining effect to streptococcus aureus, Candida albicans, intestinal bacteria etc.Experiment confirms, Rhizome of Grass leaf Sweelflag water cooking liquid under 200g/L concentration there is no killing action for anerobe, take anaerobic culture medium as contrast, to add 200g/L Rhizome of Grass leaf Sweelflag water cooking liquid in Anaerobic culturel based formulas for experimental group, inoculation actinomyces pseudonecrophorus bacterial strain, the positive rate of 96h is respectively 50% and 60%, and the two compares no significant difference (P>0.05).

The present invention compared with prior art, has and detects the feature reliable, actinomyces pseudonecrophorus separation rate is high.

Embodiment

Below in conjunction with practical situation, the specific embodiment of the present invention is elaborated.

Embodiment 1, contains in the formula of preparation 1000ml substratum: pancreas casein peptone 10.0g; Glucose 5.0g; Yeast powder 5.0g; Sodium-chlor 5.0g; Agar 15.0g; Bovine bile 1.0g; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt 3g; γ-aminobutyric acid 0.1g; Aseptic thermal distortion calf serum 100ml; Distilled water adds to 1000ml.Preparation method: take pancreas casein peptone; Glucose; Yeast powder; Sodium-chlor; Agar; Bovine bile; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt; γ-aminobutyric acid is dissolved in distilled water, mixing, and 121 DEG C, sterilizing 15min, is cooled to 45 DEG C, adds the mixing of aseptic thermal distortion calf serum, packing after sterile purified water constant volume to 1000ml.

Described aseptic thermal distortion calf serum preparation method heats 10min at getting aseptic calf serum 80 DEG C, adds sterilizing trypsin digestion 90min at 45 DEG C, and the mass ratio of enzyme-to-substrate is 1:150.Aseptic thermal distortion calf serum preparation method in other embodiments is in the same manner as in Example 1.

Embodiment 2, contains in the formula of preparation 1000ml substratum: pancreas casein peptone 8.0g; Glucose 10.0g; Yeast powder 8.0g; Sodium-chlor 5.0g; Agar 15.0g; Bovine bile 0.5g; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt 2.5g; γ-aminobutyric acid 0.3g; α-amino-isovaleric acid 0.1g; Aseptic thermal distortion calf serum 50ml; Distilled water adds to 1000ml.Preparation method: take pancreas casein peptone; Glucose; Yeast powder; Sodium-chlor; Agar; Bovine bile; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt; γ-aminobutyric acid; α-amino-isovaleric acid, is dissolved in distilled water, mixing, and 121 DEG C, sterilizing 15min, is cooled to 45 DEG C, adds the mixing of aseptic thermal distortion calf serum, packing after sterile purified water constant volume to 1000ml.

Embodiment 3, contains in the formula of preparation 1000ml substratum: pancreas casein peptone 10.0g; Glucose 8.0g; Yeast powder 10.0g; Sodium-chlor 5.0g; Agar 20.0g; Bovine bile 0.75g; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt 3 g; γ-aminobutyric acid 0.5g; α-amino-isovaleric acid 0.5g; L-homoarginine 0.05g; Aseptic thermal distortion calf serum 80ml; Distilled water adds to 1000ml.Preparation method: take pancreas casein peptone; Glucose; Yeast powder; Sodium-chlor; Agar; Bovine bile; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt; γ-aminobutyric acid; α-amino-isovaleric acid; L-homoarginine, is dissolved in distilled water, mixing, and 121 DEG C, sterilizing 15min, is cooled to 45 DEG C, adds the mixing of aseptic thermal distortion calf serum, packing after sterile purified water constant volume to 1000ml.

Embodiment 4, contains in the formula of preparation 1000ml substratum: pancreas casein peptone 5.0g; Glucose 5.0g; Yeast powder 8.0g; Sodium-chlor 5.0g; Agar 20.0g; Bovine bile 0.75g; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt 2g; γ-aminobutyric acid 0.5g; Aseptic thermal distortion calf serum 80ml; 200g/L Rhizome of Grass leaf Sweelflag Aqueous extracts 200ml; Distilled water adds to 1000ml.Preparation method: (1) is got 40g Rhizome of Grass leaf Sweelflag and added water boil 30min, filters, gets filtrate, and adjustment volume is 200g/L to concentration, obtains Rhizome of Grass leaf Sweelflag water cooking liquid 200ml; (2) pancreas casein peptone is taken; Glucose; Yeast powder; Sodium-chlor; Agar; Bovine bile; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt; γ-aminobutyric acid, is dissolved in distilled water, mixes with the Rhizoma Acori Gramineii extract liquid of step (1) gained, and 121 DEG C of sterilizing 15min, are cooled to 45 DEG C, adds the mixing of aseptic thermal distortion calf serum, packing after sterile purified water constant volume to 1000ml.

Embodiment 5, contains in the formula of preparation 1000ml substratum: pancreas casein peptone 5.0g; Glucose 8.0g; Yeast powder 10.0g; Sodium-chlor 5.0g; Agar 20.0g; Bovine bile 0.5g; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt 2g; γ-aminobutyric acid 0.3g; α-amino-isovaleric acid 0.5g; Aseptic thermal distortion calf serum 80ml; The Rhizome of Grass leaf Sweelflag Aqueous extracts 250ml of concentration 200g/L; Distilled water adds to 1000ml.Preparation method: (1) is got 50g Rhizome of Grass leaf Sweelflag and added water boil 45min, filters, gets filtrate, and adjustment volume is 200g/L to concentration, obtains Rhizome of Grass leaf Sweelflag water cooking liquid 250ml; (2) pancreas casein peptone is taken; Glucose; Yeast powder; Sodium-chlor; Agar; Bovine bile; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt; γ-aminobutyric acid; α-amino-isovaleric acid, is dissolved in distilled water, mixes with the Rhizoma Acori Gramineii extract liquid of step (1) gained, and 121 DEG C of sterilizing 15min, are cooled to 45 DEG C, adds the mixing of aseptic thermal distortion calf serum, packing after sterile purified water constant volume to 1000ml.

Embodiment 6, contains in the formula of preparation 1000ml substratum: pancreas casein peptone 8.0g; Glucose 10.0g; Yeast powder 10.0g; Sodium-chlor 5.0g; Agar 20.0g; Bovine bile 1.0g; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt 3g; γ-aminobutyric acid 0.1g; α-amino-isovaleric acid 0.3g; L-homoarginine 0.01g; Aseptic thermal distortion calf serum 100ml; The Rhizome of Grass leaf Sweelflag Aqueous extracts 300ml of concentration 200g/L; Distilled water adds to 1000ml.Preparation method: preparation method: (1) is got 60g Rhizome of Grass leaf Sweelflag and added water boil 60min, filters, gets filtrate, and adjustment volume is 200g/L to concentration, obtains Rhizome of Grass leaf Sweelflag water cooking liquid 300ml; (2) pancreas casein peptone is taken; Glucose; Yeast powder; Sodium-chlor; Agar; Bovine bile; Ethylenediamine tetraacetic acid dipotassium magnesium salt salt; γ-aminobutyric acid; α-amino-isovaleric acid; L-homoarginine, is dissolved in distilled water, mixes with the Rhizoma Acori Gramineii extract liquid of step (1) gained, and 121 DEG C of sterilizing 15min, are cooled to 45 DEG C, adds the mixing of aseptic thermal distortion calf serum, packing after sterile purified water constant volume to 1000ml.

Described Rhizome of Grass leaf Sweelflag Aqueous extracts concentration 200g/L refers to often liter containing crude drug 200g.

Gained actinomyces pseudonecrophorus substratum of the present invention is used for throat secretory product sample, and have and detect the feature reliable, actinomyces pseudonecrophorus separation rate is high, be clinical data sufficient proof, pertinent data is as follows.

1 object and method.

1.1 medium preparing: experimental group establishes 6 groups altogether, are respectively embodiment 1 scheme group, embodiment 2 scheme group, embodiment 3 scheme group, embodiment 4 scheme group, embodiment 5 scheme group, embodiment 6 scheme group.Control group is common CDC anaerobic agar, and its formula is: pancreas casein peptone 15g/L, soy peptone 5g/L, yeast powder 5g/L; CYSTINE hydrochloride 0.4g/L; Agar 15g/L, sodium-chlor 5g/L, aseptic vitamin K 0.01g/L, aseptic protohemine 0.005g/L, aseptic Sheep Blood 50ml/L.

1.2 collection of specimens cultural methods: syndromic patient 27 example of height suspected acute pharyngitis, the lasting high heat that its clinical manifestation causes with oropharyngeal infections and the diffusion transfer of abscess are feature, there is abscess or gangrenosum acne focus, and foul smelling smell.Take out after gathering pharyngeal focus secretory product by sterile swab, be inoculated in each experimental group and control group substratum respectively.At 37 DEG C, anaerobism cylinder is cultivated, and carries out identification of bacteria respectively at the tiny bacterium colony of 48h, 96h picking.Each sample number censorship PCR does molecular biology identification.

1.3 detect positive criteria: take out culture dish, and according to " clinical microbiology diagnosis and diagram ", observe colony growth situation on substratum, positive thalline is shaft-like, without gemma, without pod membrane, bacterium colony is translucent or opaque, rough, edge is in fan sample or lose shape, intermediate projections.Net result gold standard is PCR qualification.

2 results: 27 parts of acute pharyngitis syndrome patients throat secretory product samples, through PCR be finally accredited as the actinomyces pseudonecrophorus positive be 25 example.Each group of 48h detects the positive, false positive, feminine gender, false negative bacterial strain number of cases see the following form,

Grouping	Sum	PCR is positive	Positive	False positive	Negative	False negative
							Embodiment 1	27	25	15	2	12	12
Embodiment 2	27	25	16	1	11	10
							Embodiment 3	27	25	17	0	10	8
Embodiment 4	27	25	17	1	10	9
							Embodiment 5	27	25	18	1	9	8
Embodiment 6	27	25	18	0	9	7
							Control group (CDC)	27	25	5	1	22	21

The above results can be found out, cultivate after 48 hours, each embodiment group and control group positive rate more all have pole notable difference (P<0.005).

Each group of 96h detects the positive, false positive, feminine gender, false negative bacterial strain number of cases see the following form,

Grouping	Sum	PCR is positive	Positive	False positive	Negative	False negative
							Embodiment 1	27	25	22	1	5	4
Embodiment 2	27	25	22	1	5	4
							Embodiment 3	27	25	23	0	4	2
Embodiment 4	27	25	23	0	4	2
							Embodiment 5	27	25	24	1	3	2
Embodiment 6	27	25	25	0	2	0
							Control group (CDC)	27	25	13	1	14	13

The above results can be found out, cultivate after 96 hours, the positive rate of control group (CDC) increases, but each embodiment group compares with control group positive rate, all has notable difference (P<0.05).Each embodiment group compares with PCR detected result, positive rate no significant difference (P>0.05), and control group positive rate is then starkly lower than PCR detected result (P<0.001).

Above result shows, sample is after cultivating in the dry cylinder of embodiment of the present invention substratum, occur during 48h rising appreciably, separation rate is apparently higher than existing substratum (CDC), after 96 hours, each embodiment and control group positive rate difference reduce, but difference still has statistical significance, each embodiment true positives recall rate detects with PCR and compares, no difference of science of statistics, and in common CDC anaerobic agar substratum anaerobism cylinder, cultivate under-nutrition, miscellaneous bacteria breeding rapidly, actinomyces pseudonecrophorus poor growth, although there is obvious bacterial strain Growth positive during 96h, but positive rate is still starkly lower than PCR detected result, do not play diagnostic effect.Substratum of the present invention is used for cultivating actinomyces pseudonecrophorus under dry cylinder ring border, and detect reliable, separation rate is high, thus the time is made a definite diagnosis in shortening.

Claims (1)

1., for the anaerobic culture medium that actinomyces pseudonecrophorus is cultivated, it is characterized in that containing in the formula of preparation 1000ml substratum:

Pancreas casein peptone 5.0 ~ 10.0g;

Glucose 2.0 ~ 5.0g;

Yeast powder 5.0 ~ 10.0g;

Sodium-chlor 5.0g;

Agar 15.0 ~ 20.0g;

Bovine bile 0.5 ~ 1.0g;

Ethylenediamine tetraacetic acid dipotassium magnesium salt salt 2 ~ 3g;

γ-aminobutyric acid 0.1 ~ 0.5g;

α-amino-isovaleric acid 0.1 ~ 0.5g;

Aseptic thermal distortion calf serum 50 ~ 100ml;

Distilled water adds to 1000ml;

Wherein said aseptic thermal distortion calf serum preparation method is: heat 10min at getting aseptic calf serum 80 DEG C, add sterilizing trypsin digestion 90min at 45 DEG C, and the mass ratio of enzyme-to-substrate is 1:150.