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Method for splitting pectin with high esterification degree to prepare monosaccharide and oligogalacturonic acid employing pectinase

A technology of galacto-oligosaccharides and pectinase, applied in biochemical equipment and methods, methods based on microorganisms, lyase, etc., can solve the problems of low utilization efficiency and failure to obtain the degree of polymerization of monosaccharides and disaccharides , to achieve the effect of low cost, high enzyme utilization efficiency and simple process

Active Publication Date: 2015-05-06
HUBEI UNIV
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Problems solved by technology

However, the purpose of these studies was not to prepare galacturonic acid
Qian Lili reported the use of pectin lyase in her master's thesis (fermentation of pectin lyase and its application in the preparation of unsaturated galacturonic acid, master's thesis of Zhejiang University of Technology, June 2007). Preparation of oligogalacturonic acid, but the utilization efficiency of the enzyme is not high. Every 100U of enzyme can only process 2g of pectin in 24 hours, and after enzymatic hydrolysis, sugars with low polymerization degrees such as monosaccharides and disaccharides cannot be obtained.
[0004] Galacturonic acid is a monosaccharide unit of pectin, and there is currently no method to obtain this monomer by enzymatic method

Method used

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  • Method for splitting pectin with high esterification degree to prepare monosaccharide and oligogalacturonic acid employing pectinase
  • Method for splitting pectin with high esterification degree to prepare monosaccharide and oligogalacturonic acid employing pectinase

Examples

Experimental program
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Effect test

Embodiment 1

[0029] Embodiment one: the fermentation of pectinase

[0030] Prepare 50ml BMGY liquid medium (yeast extract 0.5g peptone 1.0g, yeast nitrogen source (YNB) 0.27g, ammonium sulfate 0.5g, pH6.0 phosphate buffer 5ml, glycerin 5ml, water 50ml) and 50mlBMMY liquid medium ( Yeast extract 0.5g, peptone 1.0g, yeast nitrogen source (YNB) 0.27g, ammonium sulfate 0.5g, pH 6.0 phosphate buffer 5ml, water 50ml), sterilized at 121°C, prepared 100ml solid YPD medium (glucose 2.0 g, peptone 2.0g, yeast extract 1.0g, agar 1.5g, distilled water 100ml), sterilized at 108°C and poured onto the plate. Streak the preserved GS115 / pel168s-9k strain on a solid YPD plate to activate it; select a single colony from the above YPD plate and inoculate it into a 250mL Erlenmeyer flask, which contains 50ml of liquid BMGY medium, and put it into a shaker bed, cultured at 28°C and 220rpm for 2 days, transferred the fermented bacterial solution into a sterilized sterile centrifuge tube on an aseptic operating ...

Embodiment 2

[0031] Embodiment two: the enzymolysis of 330U pectinase of 100ml substrate

[0032] Dissolve 2.0 g of the substrate in 100 ml of pH9.4 buffer, adjust the pH to about 9 with concentrated NaOH, preheat in a water bath at 50 ° C, add 3 ml of the enzyme solution prepared in Example 1 (ie: 330 U of pectin per 10 g of pectin Enzyme), react at 50°C, the reaction time is 2h, during the reaction process, use a glass rod to stir continuously, add 4.0g substrate every 5min in the next 10min and adjust the pH to about 9 with NaOH (5M). During the subsequent reaction process, the pH was also adjusted every 5 minutes, so that the pH was maintained at about 9 during the entire reaction process. Results After 2 hours of enzymolysis, the fluidity of the enzymolysis solution was good, and the hydrolysis rate of pectin was measured to be 93%.

Embodiment 3

[0033] Embodiment three: the enzymatic hydrolysis of 11U pectinase of 100ml substrate

[0034] Dissolve 2.0 g of the substrate in 100 ml of pectinase buffer at pH 9.4, adjust the pH to about 9 with NaOH (5M), preheat in a 50°C water bath, add 100 μl of the enzyme solution prepared in Example 1 (i.e., every 10 g Pectin 11U pectinase), react at 50°C, the reaction time is 2h, during the reaction, use a glass rod to stir continuously, add 4.0g of substrate every 5min in the next 10min and adjust the pH with NaOH (5M) To about 9, the pH should be adjusted every 5 minutes in the subsequent reaction process, so that the pH is maintained at about 9 during the entire reaction process. The result was that the pectin added after 2 hours was not completely dissolved, the enzymatic solution was viscous, and the substrate was not completely enzymatically hydrolyzed.

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Abstract

The invention discloses a method for splitting pectin with high esterification degree to prepare monosaccharide and oligogalacturonic acid employing pectinase. The method comprises the following steps: (1) carrying out fermenting cultivation on a GS115-pel168s-9k strain to prepare a pectin enzyme liquid; (2) dissolving 10g of pectin into a buffer solution with the pH of 9.4, adjusting the pH to be 8.8-9.2, preheating to 50 DEG C in a water bath, adding 110U of pectin enzyme liquid and reacting for two hours, so as to obtain enzymatic hydrolysate; and (3) centrifuging the enzymatic hydrolysate prepared from the step (2), evaporating and drying supernate to obtain the monosaccharide and the oligogalacturonic acid, and calculating the hydrolysis rate which can reach 93%. The method has the advantages of simple process, low cost and high enzymatic hydrolysis efficiency.

Description

technical field [0001] The invention belongs to the technical field of preparation of functional organic matter, and in particular relates to a method for producing monosaccharides and galacturonic acid oligosaccharides by using enzymes produced by pectinase-producing Pichia pastoris to crack pectin with a high degree of esterification. Background technique [0002] Pectin (pectin) is widely distributed in the roots, stems, and leaves of plant fruits in the form of protopectin and pectic acid. It has the characteristics of natural, green, nutritious, and healthy. Food ingredients that are non-toxic and have no ADI (Admissible Daily Intake) restrictions agreed by the Joint Expert Committee of the Organization (FAO / WHO). At present, pectin is widely used in the food industry as a thickener, stabilizer, gelling agent and synergist. In addition, pectin can also be used in health food and medicine. Pectin has the functions of lowering blood sugar to prevent diabetes, lowering c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12P19/14C12P19/02C12P19/00C12N9/88C12N9/26C12N9/18C12R1/84
Inventor 张桂敏龙光林马延和
Owner HUBEI UNIV
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