Preparation method and application of natural material composite system immobilized bifunctional enzyme used for wine

A bifunctional enzyme, natural material technology, applied in the preparation of alcoholic beverages, biochemical equipment and methods, microorganism-based methods, etc., can solve the problems of few reports of EC degrading enzymes and no production scale, and reduce urea and urea. Effects of EC content, easy recovery, and high urea removal rate

Inactive Publication Date: 2014-01-22
JIANGNAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] The urease used in my country's exported rice wine is imported from abroad, and the domestic production scale has not yet formed, and there are few domestic and international reports on EC degrading enzymes

Method used

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  • Preparation method and application of natural material composite system immobilized bifunctional enzyme used for wine

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] Example 1: Weighing Weigh 3~5 g of chitosan and 1~3 g of gelatin, dissolve them in 100 mL of 1% acetic acid, place them on a magnetic stirrer and stir evenly, then ultrasonically defoam. Take 20% NaOH solution and absolute ethanol and mix them uniformly at a volume ratio of 4:1 to make 200-300 mL of coagulation solution. Drop the mixed solution of chitosan and gelatin into the coagulation solution with a syringe to form microspheres with a diameter of 3-4 mm. After coagulation for a period of time, wash the chitosan microspheres with deionized water until neutral. Under the condition of shaking in a constant temperature water bath at 30°C, chitosan microspheres were crosslinked with 0.4% genipin solution, and the ratio g / mL of chitosan microspheres: genipin solution was 1:5 , the crosslinking time was 6 hours, and the free genipin solution was removed by washing; the activated microspheres and free enzyme solution were fixed in a refrigerator at 4°C for 8 hours, and 2.5...

Embodiment 2

[0032] Example 2: Put 15 g of the immobilized bifunctional enzyme for wine obtained in Example 1 (that is, the amount of enzyme added is 37.5 U / mL) into a column with an inner diameter of 3 cm and a length of 12 cm, and adjust the flow rate of the wine sample to 1 mL / mL min, 400 mL of wine samples (with a urea content of 74.94 mg / L) flowed through the reactor, and after all the wine samples passed through the column, the total urea removal rate was measured. Circulate the treatment 4 times, and measure the urea removal rate after passing through the column each time. The immobilized bifunctional enzyme for wine could remove 50.77% of urea in the wine sample for the first time, and the same rice wine sample was repeated through the column for 4 times, the urea removal rate reached 96.92%, and the final concentration of urea was reduced to 2.31 mg / L.

Embodiment 3

[0033]Example 3: Prepare 15 mL samples of yellow rice wine and simulated wine (50% ethanol) with urea additions of 25 mg / L respectively. mL, 32°C shaker at 100 rpm for 20 h, and the removal rate of urea was determined. Under two different enzyme dosage conditions, the urea removal rates of immobilized bifunctional enzymes for rice wine were 74.16% and 93.03%, respectively, and the urea removal rates of simulated wine were 95.16% and 98.01%, respectively.

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Abstract

Belonging to the technical field of enzyme preparations and wine brewing additives, the invention relates to a preparation method and application of a natural material composite system immobilized bifunctional enzyme used for wine. The preparation method includes: subjecting the free bifunctional enzyme that is extracted from Providencia sp. JNB815 and has the activity of degrading urea and ethyl carbamate (EC) to chitosan adsorption, gelatin embedding and genipin crosslinking, thus obtaining the immobilized bifunctional enzyme. When the immobilized enzyme takes urea as the substrate, the optimum temperature is 40DEG C, and the optimum pH is 4.0; when EC is adopted as the substrate, the optimum temperature is 60DEG C, and the optimum pH is 4.5. The storage half-life periods of two enzyme activity are 54d and 45d respectively. The invention also relates to application of the immobilized bifunctional enzyme, which can reach a urea removal rate of 93.03% and an EC removal rate of 56.60%. According to the invention, the process conditions of original fermented wine cannot be changed, the wine flavor is not changed, and the immobilized bifunctional enzyme can be reused, and has more advantages in the application of removing trace urea and EC from yellow wine.

Description

technical field [0001] A preparation method and application of a natural material composite system immobilized bifunctional enzyme for wine, specifically the Providencia bacteria ( Providencia sp.) The free bifunctional enzyme extracted from JNB815 is adsorbed by chitosan, embedded in gelatin, cross-linked by genipin, and solidified to obtain an immobilized bifunctional enzyme, which belongs to the technical field of enzyme preparation and brewing additives. Background technique [0002] Ethyl carbamate (EC), a class 2A carcinogen, exists in trace amounts in most fermented foods and alcoholic beverages. Studies have found that 90% of the ethyl carbamate in rice wine is mainly produced by the chemical reaction of urea and ethanol. Adding a small amount of urease to the wine can effectively remove urea and inhibit the formation of ethyl carbamate, while the EC degrading enzyme can directly and effectively decompose the EC already produced in the finished rice wine. [0...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N11/10C12N11/04C12H1/15C12R1/01
Inventor 田亚平查小红杨广明
Owner JIANGNAN UNIV
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