A kind of animal intestinal mucosa extract and its preparation method and application

A technology of intestinal mucosa and extract, which is applied in the field of animal intestinal mucosa extract and its preparation and application, and can solve the problem of low expression efficiency

Inactive Publication Date: 2011-12-21
FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there have been a large number of studies on the direct expression of antimicrobial peptide genes in microorganisms using genetic engineering technology, but the expression products cannot be obtained due to the strong lethality of antimicrobial peptides on host bacteria; this requires the expression of antimicrobial peptide genes in the form of fusion proteins , but there are problems such as low expression efficiency

Method used

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  • A kind of animal intestinal mucosa extract and its preparation method and application
  • A kind of animal intestinal mucosa extract and its preparation method and application
  • A kind of animal intestinal mucosa extract and its preparation method and application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] Embodiment 1, two-step enzymatic method prepares porcine intestinal mucosa extract

[0031] 1) A 50ml centrifuge tube (15.5g) was sampled and weighed (25.4g). The sample was the precipitate from the stripped inner wall of the small intestine after centrifugation. The moisture in the sample was removed, and the sample weighed 9.9g.

[0032] 2) Add 50ml of water to the above-mentioned centrifuge tube, then add 3.6g (3000-4000U) trypsin (Sigma, T1426) into the water, enzymolyze it in a water bath at 37°C with a pH value of 7.0-7.5 for 6 hours, and then extinguish it in a boiling water bath Enzyme was cooled for 5 minutes; then 0.8g (3000-5000U) of papain (Sigma, P3250) was added to the double distilled water, and the enzyme was hydrolyzed for 6 hours at 37°C with a pH value of 6.5-7.5 in a water bath, and the enzyme was inactivated in a boiling water bath for 10 minutes. Cool down; finally, centrifuge at 10,000g for 15 minutes to collect the supernatant to obtain the intes...

Embodiment 3

[0051] Embodiment 3, antibacterial activity assay

[0052] The antibacterial test was carried out by the agarose dispersion method, and the specific method was as follows:

[0053] A layer of bottom agar (10 ml) was poured onto the Petri dish. The bottom agar contains 1% agarose, 0.022% nutrient broth powder, 10mmol / L PBS (pH7.4), and Escherichia coli ATCC 25922 in logarithmic growth phase (1.0×10 6 / ml). Make a round hole with a diameter of 3mm on the bottom agar, (according to the results in Table 2, that is, calculate the volume of the crude extract added to each well according to the ratio of protein concentration, so as to ensure that the amount of protein added to each well is consistent), take out 4.4 μl and 6 μl respectively, 20 μl, 5.4 μl, add water to make up to 20 μl, add 20 μl diluted sample solution for measuring protein recovery in Example 2 to each well. After incubating at 37°C for 3 hours, cover a layer of 2× nutrient agar on the bottom agar, continue to in...

Embodiment 4

[0055] Example 4, Purification and Antibacterial Activity Determination of Pig Intestinal Mucosa Extract

[0056] The crude pig intestinal mucosa extract prepared in Example 1 was dissolved in double distilled water, ultrafiltered with an ultrafiltration tube with a molecular weight cut-off of 10KD, and the ultrafiltrate was collected. The ultrafiltrate was loaded on a Sephadex G-100 gel filtration column (Ф1.0×30.0 cm). Use 0.2M, pH7.0 sodium acetate for elution at a flow rate of 1 mL / min, collect 3 mL in each tube, and collect 20 tubes in total.

[0057] Take the volume of chromatographic effluent as the abscissa, and take the corresponding A 595nm The value is the ordinate, and the elution curve is made, see figure 1 . The antibacterial activity of the eluate was detected by the agarose dispersion method, figure 1 The elution peak indicated by the middle arrow has antibacterial activity.

[0058] The eluates with antibacterial effect were combined and freeze-dried to o...

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Abstract

The invention discloses an animal intestinal mucosa extract and a preparation method as well as application thereof. The animal intestinal mucosa extract is prepared by the method comprising the following steps: (1) taking small intestine of an in vitro animal, scraping the mucosa layer on the intestinal wall, centrifuging and collecting precipitates; (2) adding the precipitates into water, then adding trypsin into the redistilled water for carrying out enzymolysis I, and after the enzymolysis is finished, inactivating enzymes and cooling; then, adding papain into the redistilled water for carrying out enzymolysis II, and after the enzymolysis is finished, inactivating enzymes and cooling; finally, centrifuging and taking the supernatant solution to obtain an intestinal mucosa enzymolysis solution; and (3) adding an acetic acid solution of which the mass concentration is 5%-10% into the intestinal mucosa enzymolysis solution for stirring and leaching, then centrifuging the leaching solution, and collecting the supernatant solution, and regulating the pH value of the supernatant solution to be 6.0-7.0, freezing and drying to obtain the animal intestinal mucosa extract. The test proves that the intestinal mucosa extract has antibacterial activity to bacteria (such as Escherichia coli).

Description

technical field [0001] The invention relates to an animal intestinal mucosa extract, a preparation method and application thereof. Background technique [0002] my country is a big country in the production and export of casings and heparin. The intestines used in the processing of casings and heparin mainly come from pig intestines, sheep intestines and other animal digestive tract tissues. The efficiency is low, and heparin cannot be used, and it is gradually being eliminated. However, the wastewater discharge during the enzymatic hydrolysis process is extremely large. The nitrogen content in the wastewater is rich, and the chemical oxygen demand (COD value) is high, so it is not suitable for direct discharge. Therefore, it is of great significance to develop a new enzymatic hydrolysis process to improve the utilization value of discharged waste. [0003] Antibacterial peptide (Antibacterial peptide, ABP) is a kind of small molecular polypeptide with antibacterial activity...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K38/17A61K35/38
CPCY02A50/30
Inventor 周志刚赵旭民姚斌刘玉春张宇婷何夙旭曹雅男石鹏君
Owner FEED RESEARCH INSTITUTE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
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