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Preparation method of solid enzyme complex for measuring sodium fructose diphosphate content

A technology of sodium fructose diphosphate and complexes, which is applied in biochemical equipment and methods, microbial measurement/inspection, enzymes, etc., can solve the problems of reduced final yield, incomplete cell lysis, and sticky meat paste, and achieve the goal of improving the final product quality. yield effect

Inactive Publication Date: 2014-10-01
吴海春 +1
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Problems solved by technology

However, the method of extracting enzymes in muscle with water mentioned in this patent will inevitably result in incomplete cell lysis, low extraction efficiency, and ultimately lead to insufficient yield
The gauze filtration method is difficult to filter the enzyme extract in the minced meat to the greatest extent, which will also reduce the final yield; and the minced meat is extremely viscous, and the efficiency of manual extrusion of gauze is difficult to standardize, which affects the subsequent extraction process
No specific data is given in the ammonium sulfate precipitation method, which is not conducive to the stability of mass production; and the precipitation conditions cannot effectively increase the unit enzyme activity. After the ammonium sulfate saturation reaches more than 70%, the precipitate is mostly miscellaneous proteins
Therefore, the original technical solution has low production efficiency and high cost

Method used

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  • Preparation method of solid enzyme complex for measuring sodium fructose diphosphate content
  • Preparation method of solid enzyme complex for measuring sodium fructose diphosphate content
  • Preparation method of solid enzyme complex for measuring sodium fructose diphosphate content

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Embodiment 1

[0028] One was taken from the house, fixed, and after sudden death, the back and thigh muscles were peeled off, placed on crushed ice, and cut into pieces with scissors at the same time. Use a meat grinder pre-cooled in a refrigerator at -20°C to grind the minced meat into a meat puree and weigh it. Put it in a beaker, add 0.05M KOH solution according to the amount of 1mL / g minced meat, stir evenly for 15 minutes, and centrifuge at 7000rpm in a refrigerated centrifuge at 4°C for 10 minutes. Transfer the supernatant to a new beaker, and then use half the amount of 0.05M KOH to extract and centrifuge in the same way as the ground meat, then combine the supernatants of the two centrifuges. The above operations are all performed on crushed ice or at 4°C. Add saturated ammonium sulfate solution (706g / L, 4°C) slowly, stir until the final 40% saturation, adjust the pH to 5.0, and place on crushed ice for 30 minutes. Then refrigerate and centrifuge at 8000 rpm for 15 minutes at 4°C, ...

Embodiment 2

[0034] Method for measuring the activity of special solid compound enzyme:

[0035] National Pharmacopoeia Huafa [2009] No. 12 Document (Draft for Sodium Fructose Diphosphate and Its Preparation Quality Standards): Solid compound enzyme reagent in the detection method of sodium fructose diphosphate (FDP). The experimental results are attached image 3 , using two methods to extract the solid enzyme complex in rabbit exercise muscle, weigh it after freeze-drying and measure the unit enzyme activity. In the figure, the yield of the solid enzyme complex prepared in Example 1 was significantly increased by about 2.0 times, and the unit enzyme activity was also significantly increased by about 1.9 times, *P<0.001.

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Abstract

The preparation method of the solid enzyme complex for determining the content of sodium fructose diphosphate uses rabbit meat as raw material, and extracts aldolase (ALD) and triose phosphate isomerase ( TIM) and glycerol phosphate dehydrogenase (GDH) complex, the cleavage is alkaline lysis, and an alkaline solution with a concentration range of 0.01-0.1M is added to each 1 mL / g of the raw material. By adopting the technical scheme, the final yield, unit enzyme activity and batch production stability of the solid enzyme complex can be effectively improved.

Description

technical field [0001] The invention relates to a preparation method of a solid enzyme complex for measuring the content of sodium fructose diphosphate. Background technique [0002] The determination methods of sodium fructose diphosphate mainly include high performance liquid phase (HPLC) method, ultraviolet (UV) absorption method, gas chromatography (GC) method, various chemical colorimetric methods and enzymatic method. Among them, the enzymatic method is highly specific, accurate and rapid. However, aldolase (ALD), triose phosphate isomerase (TIM) and glycerol phosphate dehydrogenase (GDH) involved in the enzymatic determination are expensive, resulting in high cost of determination. In order to solve this problem, there is a patent 200910091934.X at this stage, which extracts a solid enzyme complex from animal muscle to replace the above three enzymes, and has achieved certain results. However, the method for extracting enzymes in muscle with water mentioned in this ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/00C12N9/88C12N9/90C12N9/04C12Q1/533C12Q1/527C12Q1/32
Inventor 吴海春
Owner 吴海春
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