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
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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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