Method for extracting acetylcholinesterase by use of discarded organ-chicken heads in chicken manufacturing process

A technology of acetylcholinesterase and processing, which is applied in the direction of biochemical equipment and methods, enzymes, hydrolytic enzymes, etc., can solve the problems of high price, waste of grain raw materials, and complicated preparation process, and achieve easy storage, low cost, and high realization. The effect of value

Inactive Publication Date: 2015-09-02

SOUTH CHINA UNIV OF TECH

2 Cites 1 Cited by

AI-Extracted Technical Summary

Problems solved by technology

The research on plant esterase has not been in-depth. Some studies have shown that the enzyme activity in food crops is relatively high, but for most food raw materials, the extracti...

Abstract

The invention provides a method for extracting acetylcholinesterase by use of discarded organ-chicken heads in the chicken manufacturing process. The discarded organ-chicken brain during chicken processing is used as the material source of enzyme. The method comprises the following steps: processing of chicken brains, preparation and refrigerated centrifuge of a homogenate and vacuum freeze drying of a crude extract, and through the steps, the acetylcholinesterase product can be obtained. As the acetylcholinesterase product prepared from traditional enzyme sources is high in cost and low in yield while the imported enzyme sources are high in price and relatively long in cycle, through screening of enzyme sources, the discarded organs-chicken brains in the chicken manufacturing process are fully utilized to achieve high-value utilization and reduce the cost, and through optimization and inspection, the enzyme activity is relatively high, the enzyme activity of the crude enzyme can reach 3.0-5.0 U/mL, the specific activity can reach 0.35-0.5 U/mg, and accordingly, a foundation is laid for follow-up preparation of the pure acetylcholinesterase product.

Application Domain

Hydrolases

Technology Topic

ChemistryFreeze dry +8

Examples

Experimental program(3)

Example Embodiment

[0039] Example 1:

[0040] (1) Weigh the cleaned and dried watch glass, rinse the fresh chicken heads cut vertically with normal saline, use tweezers to take the chicken brain out of the watch glass, remove the non-brain tissue, and weigh it again to calculate the chicken Brain tissue quality m;

[0041] (2) Mix the chicken brain with phosphate buffer (0.1M, pH8.0) containing 1.0% (v/v) TritonX-100 at a ratio of 1:3 (g/mL);

[0042] (3) After homogenizing the mixture obtained in the previous step with a homogenizer for 2 minutes, let it stand for 30 minutes;

[0043] (4) Centrifuge the homogenate at 8000r/min at 4°C for 20min, and take the supernatant to obtain the crude acetylcholinesterase extract. Place the obtained crude extract in a watch glass and perform vacuum freeze-drying to obtain an acetylcholinesterase product. Take 1.0 mg of the acetylcholinesterase product and dissolve it with distilled water to obtain an enzyme solution;

[0044] (5) In a 10mL colorimetric tube, add 5mL phosphate buffer (0.1M, pH7.5), then add 0.02mL enzyme solution to mix well, and keep it in a constant temperature water bath at 36.5℃ for 15min;

[0045] (6) Add 0.1mL thioacetylcholine iodide solution (20mg/mL) and 0.1mL DTNB (10mg/mL) solution to the solution obtained in the previous step and mix well;

[0046] (7) Use a cuvette in the 412nm wavelength range to compare the color, record the absorbance A = 0.9876, measure the total protein content by the Coomassie brilliant blue method, and then calculate it according to the formula of enzyme activity, specific activity and total enzyme activity, and the enzyme activity is 6.3178U/mL, the specific activity is 0.4254U/mg.

Example Embodiment

[0047] Example 2:

[0048] (1) Weigh the cleaned and dried watch glass, rinse the fresh chicken heads cut vertically with normal saline, use tweezers to take the chicken brain out of the watch glass, remove the non-brain tissue, and weigh it again to calculate the chicken Brain tissue quality m;

[0049] (2) Mix the chicken brain with phosphate buffer (0.1M, pH8.0) containing 1.0% (v/v) TritonX-100 in a ratio of 1:4 (g/mL);

[0050] (3) After homogenizing the mixture obtained in the previous step with a homogenizer for 2 minutes, let it stand for 30 minutes;

[0051] (4) Centrifuge the homogenate at 8000r/min at 4°C for 20min, and take the supernatant to obtain the crude acetylcholinesterase extract. Place the obtained crude extract in a watch glass and perform vacuum freeze-drying to obtain an acetylcholinesterase product. Take 1.0 mg of the acetylcholinesterase product and dissolve it with distilled water to obtain an enzyme solution;

[0052] (5) In a 10 mL colorimetric tube, add 5 mL of phosphate buffer (0.1M, pH 7.5), then add 0.02 mL of enzyme solution to mix, and keep it in a constant temperature water bath at 36.5°C for 15 minutes;

[0053] (6) Add 0.1mL thioacetylcholine iodide solution (20mg/mL) and 0.1mL DTNB (10mg/mL) solution to the solution obtained in the previous step and mix well;

[0054] (7) Use a cuvette to compare the color in the 412nm wavelength range, record the absorbance A = 0.9606, and measure the total protein content by the Coomassie brilliant blue method, and then calculate it according to the formula of enzyme activity, specific activity and total enzyme activity, according to enzyme activity Sum specific activity formula calculates that the enzyme activity is 6.1450 U/mL and the specific activity is 0.4836 U/mg.

Example Embodiment

[0055] Example 3:

[0056] (1) Weigh the cleaned and dried watch glass, rinse the fresh chicken heads cut vertically with normal saline, use tweezers to take the chicken brain out of the watch glass, remove the non-brain tissue, and weigh it again to calculate the chicken Brain tissue quality m;

[0057] (2) Mix chicken brain with phosphate buffer (0.1M, pH8.0) containing 0.5% (v/v) TritonX-100 in a ratio of 1:4 (g/mL);

[0058] (3) After homogenizing the mixture obtained in the previous step with a homogenizer for 2 minutes, let it stand for 30 minutes;

[0059] (4) Centrifuge the homogenate at 8000r/min at 4°C for 20min, and take the supernatant to obtain the crude acetylcholinesterase extract. Place the obtained crude extract in a watch glass and perform vacuum freeze-drying to obtain an acetylcholinesterase product. Take 1.0 mg of the acetylcholinesterase product and dissolve it in distilled water to obtain an enzyme solution;

[0060] (5) Place the supernatant in a watch glass, freeze-dry it in a vacuum with a liquid thickness of 0.6 cm, and collect the acetylcholinesterase product.

[0061] (6) In a 10mL colorimetric tube, add 5mL phosphate buffer (0.1M, pH7.5), then add 0.02mL enzyme solution to mix well, and keep it in a constant temperature water bath at 37°C for 15min;

[0062] (7) Add 0.1mL thioacetylcholine iodide solution (20mg/mL) and 0.1mL DTNB (10mg/mL) solution to the solution obtained in the previous step and mix well;

[0063] (8) Use a cuvette to compare the color in the 412nm wavelength range, record the absorbance A = 0.7119, calculate the enzyme activity to 4.5758 U/mL and the specific activity to 0.3965 U/mg according to the formula of enzyme activity and specific activity.

PUM

Property	Measurement	Unit
Enzyme activity	6.3178	U/ml
Specific vitality	0.4254	U/mg
Specific vitality	0.4836	U/mg

Description & Claims & Application Information

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Method for extracting acetylcholinesterase by use of discarded organ-chicken heads in chicken manufacturing process

AI-Extracted Technical Summary

Problems solved by technology

Abstract

Examples

Example Embodiment

Example Embodiment

Example Embodiment

PUM

Description & Claims & Application Information

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