CN115927505A - Method for preparing low molecular weight gellan gum by enzyme method - Google Patents
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- CN115927505A CN115927505A CN202211687639.2A CN202211687639A CN115927505A CN 115927505 A CN115927505 A CN 115927505A CN 202211687639 A CN202211687639 A CN 202211687639A CN 115927505 A CN115927505 A CN 115927505A
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Abstract
The invention belongs to the technical field of biochemical engineering, and relates to a method for preparing low-molecular-weight gellan gum by an enzyme method, which comprises the following steps: (1) enzymolysis: adding pectinase into the gellan gum aqueous solution for enzymolysis to obtain an enzymatic hydrolysate, wherein the pectinase at least comprises one of pectin lyase and pectinase NE 06; (2) separation and extraction: concentrating the enzymolysis liquid in a rotary evaporator to obtain a rotary evaporation product; (3) precipitation: adding the rotary evaporation product into ethanol for precipitation to obtain filaments; and (4) crushing and drying: crushing the filiform matters, and performing vacuum drying on the crushed filiform matters; (5) crushing and sieving: and (4) crushing and sieving the product prepared in the step (4). The method effectively reduces the average molecular weight and the gel strength of the prepared low-molecular-weight gellan gum by selecting the pectinase and reasonably designing the preparation steps, has higher yield, simple preparation method, mild and controllable preparation conditions, does not need complex equipment, and has universality.
Description
Technical Field
The invention belongs to the technical field of biochemical engineering, and particularly relates to a method for preparing low-molecular-weight gellan gum by an enzyme method.
Background
Gellan gum is an extracellular polysaccharide secreted by sphingolipid paucimobilis under aerobic conditions, when gellan gum is dissolved in water, molecules can automatically aggregate to form a double-helix structure, the force for stabilizing the double-helix structure is mainly intermolecular hydrogen bonds, and the double helix further aggregates to form a three-dimensional network structure, so that water molecules can be trapped conveniently to generate a gel phenomenon. The microbial exopolysaccharide is used as an industrial product for microbial fermentation, and has wide application prospects in the fields of food, chemical engineering, medicine and the like. In the food industry, gellan gum as a thickener, stabilizer, suspending agent, gelling agent, etc. can be used in foods such as jam, sausage, ice cream, salad dressing, jelly, dairy products, etc.; in the field of medicine, gellan gum can be applied to products such as eye drops, sustained-release medicines, coatings, tissue engineering scaffold materials and the like; in the chemical field, gellan gum can be used as an adhesive, toothpaste, air freshener, etc.
The molecular weight is one of basic parameters for characterizing the characteristics of the gellan gum and is an important index for quality detection in industrial production, at present, the gellan gum produced by domestic manufacturers has fewer specifications and single product variety (mostly high molecular weight gellan gum), so in order to promote the development of the gellan gum towards refinement, the existing gellan gum needs to be modified to reduce the molecular weight.
Disclosure of Invention
Aiming at the technical problems, the invention provides a method for preparing low-molecular-weight gellan gum by an enzyme method, which effectively reduces the average molecular weight of the prepared low-molecular-weight gellan gum by selecting pectinase and reasonably designing preparation steps.
Firstly, the invention provides a method for preparing low molecular weight gellan gum by an enzyme method, which comprises the following steps:
(1) Enzymolysis: adding pectinase into the gellan gum aqueous solution for enzymolysis to obtain an enzymatic hydrolysate, wherein the pectinase at least comprises one of pectin lyase and pectinase NE 06;
(2) Separation and extraction: concentrating the enzymolysis liquid in a rotary evaporator to obtain a rotary evaporation product;
(3) And (3) precipitation: adding the rotary evaporation product into ethanol for precipitation to obtain filaments;
(4) Crushing and drying: crushing the filiform matters, and performing vacuum drying on the crushed filiform matters;
(5) Crushing and sieving: and (4) crushing and sieving the product prepared in the step (4).
The invention effectively reduces the average molecular weight and the gel strength of the prepared low molecular weight gellan gum by selecting pectinase and reasonably designing the preparation steps, and has higher yield.
Preferably, the concentration of the gellan gum in the gellan gum aqueous solution in the step (1) is 0.01g/ml to 0.015g/ml.
Preferably, the addition amount of the pectin lyase and the pectinase NE06 in the step (1) is 0.05 to 0.2 percent of the gellan gum aqueous solution.
More preferably, the pectin lyase and the pectinase NE06 added in the step (1) are both 0.1% of the gellan gum aqueous solution.
Preferably, the enzymolysis in step (1) is carried out in a reaction kettle, and the rotation speed of a stirring paddle in the reaction kettle is 200rpm.
Preferably, the temperature for the enzymatic hydrolysis in step (1) is 30 to 50 ℃.
Further preferably, the temperature of the enzymatic hydrolysis in the step (1) is 40 ℃.
Preferably, the enzymolysis time in the step (1) is 6 to 30 hours.
Further preferably, the time for enzymolysis in step (1) is 24 hours.
Preferably, the concentration time in the separation and extraction step in the step (2) is 10-40 min, and the parameters of a rotary evaporator are as follows: the temperature is 80 ℃ and the rotation speed is 300rpm.
Preferably, the ethanol concentration in step (3) is 95%.
Preferably, the vacuum drying conditions in step (4) are as follows: the vacuum degree is 0.07MPa, the temperature is 50 ℃, and the time is 12h.
Preferably, the step (5) comprises the steps of:
and (4) crushing the product prepared in the step (4) by using a crusher, and sieving the crushed product by using a 80-mesh sieve.
The invention further provides application of the low molecular weight gellan gum prepared by the method in the fields of food, chemical industry and medicine.
Compared with the prior art, the invention has the following beneficial effects:
(1) The average molecular weight of the prepared low molecular weight gellan gum is effectively reduced by selecting pectinase and reasonably designing the preparation steps, and the yield is higher;
(2) The preparation method provided by the invention is simple, mild and controllable in preparation conditions, free of complex equipment, universal and convenient for industrial production;
(3) The low molecular weight gellan gum prepared by the invention has stable performance and low gel strength and other gel characteristics.
Detailed Description
The invention is further described with reference to the following description and specific examples. Those skilled in the art will be able to implement the invention based on these teachings. Moreover, the embodiments of the present invention described in the following description are generally only some embodiments of the present invention, and not all embodiments. Therefore, all other embodiments obtained by a person of ordinary skill in the art based on the embodiments of the present invention without any creative effort shall fall within the protection scope of the present invention.
In the following examples, the gel strength test method was:
(1) Weighing 0.6g of sample into a weighed 250ml clean beaker, adding about 120ml of distilled water, placing the beaker into a water bath kettle at 95.5 ℃, continuously stirring to completely hydrate the sample particles (30 min is needed), and observing the dissolution condition. Adding 2ml of 2.7 percent calcium chloride while stirring, supplementing the amount of evaporated water to ensure that the net weight of the solution is 120g, and uniformly mixing;
(2) Pouring the hot glue solution into a clean cuvette, keeping the height of the liquid layer at about two thirds of the height of the cuvette, standing in a biochemical incubator at 20 ℃ for 30min, and measuring the transparency by using a spectrophotometer (the wavelength is 490nm, and distilled water is a blank reference sample). And simultaneously pouring the residual glue solution into a clean flat weighing bottle at a horizontal position, keeping the liquid layer at the height of 2-3 cm, standing for 2.5h in a biochemical incubator at the temperature of 20 ℃ and the humidity of 40-60%, measuring and recording the gel strength in a gel strength tester, and calculating the average value.
The method for detecting the molecular weight of the gellan gum comprises the following steps: pretreating a sample by using a differential detector, dissolving 0.1 percent of gellan gum in ultrapure water at 30 ℃, passing through a 0.23 mu m water film, and determining the molecular weight of the gellan gum by using high performance molecular exclusion chromatography, wherein relevant parameters are as follows: a chromatographic column: a gel column; column temperature: 30 ℃; mobile phase: ultrapure water; flow rate: 0.6ml/min; sample injection amount: 20 μ L.
In the following examples, the pectin lyase used was Ningxia Sheng industries group Limited, product batch No.: 12108021 and the enzyme activity unit: 600U/mL; the pectinase NE06 is produced by Weifang kang dien biotechnology limited company, and the enzyme activity unit is as follows: 700U/mL; foodPro CGL glucoamylase is produced by Danisco, and the enzyme activity unit is as follows: 392U/mL; maltogenase2XL maltogenic amylase, produced by Denmark Novoxil, has an enzyme activity unit: 6400U/mL.
The yield in the following examples refers to the mass of low molecular weight gellan gum produced as a proportion of the gellan gum raw material.
Example 1
The embodiment comprises the following steps:
(1) Dissolving: slowly adding 40g of gellan gum raw material into 4L of water under stirring in a reaction kettle, and continuously stirring after the addition is finished to obtain a gellan gum dissolving solutionThe gellan gum is prepared from Zhejiang Tianwei Biotechnology corporation in J220639-3, and has molecular weight of 4.0 × 10 5 ～6.0×10 5 The gel strength is 1780;
(2) Enzymolysis: slowly adding pectin lyase and pectinase NE06 into the gellan gum dissolving solution, wherein the dosages of the pectin lyase and the pectinase NE06 are 4mL in the embodiment, uniformly stirring, and carrying out enzymolysis for 24h at the reaction kettle temperature of 40 ℃ and the stirring paddle rotating speed of 200rpm to obtain an enzymolysis solution;
(3) Separation and extraction: concentrating the enzymatic hydrolysate in a rotary evaporator for 30min, wherein the temperature of the rotary evaporator is 80 ℃, and the rotating speed is 300rpm, so as to obtain a rotary evaporation product;
(4) And (3) precipitation: adding the rotary evaporation product into 1L 95% ethanol for precipitation to obtain filaments;
(5) Crushing and drying: crushing the filiform materials, and then placing the crushed filiform materials in a vacuum drying oven for drying, wherein the drying conditions are as follows: the vacuum degree is 0.07MPa, the temperature is 50 ℃, and the time is 12h;
(6) Crushing and sieving: and (4) crushing the product prepared in the step (5) by using a micro crusher, and sieving the crushed product by using a 80-mesh sieve.
The low molecular gellan gum prepared in this example was found to have an average molecular weight of about 1.0X 10 5 Far below the lowest molecular weight (4.0X 10) of the gellan gum material 5 ) (ii) a From the gel strength of the gellan gum, the strength of the gellan gum prepared by the method is lower than that of the raw material of the gellan gum; in addition, the low molecular weight gellan gum yield of this example was as high as 85%.
Example 2: effect of different enzymolysis time on the prepared low molecular gellan gum
The present embodiment has the following differences from embodiment 1, and the rest of the same parts are not described herein again:
in the embodiment, the enzymolysis time is respectively 6h, 12h, 16h, 20h and 30h.
The low molecular gellan gum prepared in this example was examined and the following table was obtained in combination with example 1:
TABLE 1 molecular weight, gel strength and yield of low molecular weight gellan gums prepared with varying enzymolysis time
From an observation of the above table: along with the lengthening of the enzymolysis time, the molecular weight and the gel strength of the gellan gum generally show a reduction trend, and the yield is always maintained at a level higher than 80%, wherein when the enzymolysis time is 24 hours, the average molecular weight of the gellan gum reaches the lowest level, and the gel strength at the moment is the lowest, the yield is the highest, and the effect is the best.
Example 3: effect of different enzyme dosages on the Low molecular Gellan Gum produced
The difference between this example and example 1 only exists in the amount of enzyme (specifically shown in table), and the rest of the same parts are not described herein again, and the following table is obtained by examining and combining example 1:
TABLE 2 molecular weight, gel strength and yield of low molecular weight gellan gum prepared with varying enzyme dosage
The above table is observed to show that: no matter one of the pectin lyase and the pectinase NE06 is selected independently or two enzymes are simultaneously used for carrying out enzymolysis on the gellan gum raw material in a synergistic manner, the molecular weight of the prepared low-molecular-weight gellan gum is far lower than the lowest molecular weight of the gellan gum raw material, wherein the effect is best when the addition amount of the pectin lyase and the pectinase NE06 is 4ml from the aspect of molecular weight.
Example 4: influence of different enzymolysis temperatures on the prepared low-molecular gellan gum
The present embodiment has the following differences from embodiment 1, and the rest of the same parts are not described herein again:
in this example, the enzymolysis temperature was 30 ℃, 35 ℃, 45 ℃ and 50 ℃.
Upon examination and synthesis of example 1, the following table was obtained:
TABLE 3 molecular weight, gel strength and yield of low molecular weight gellan gums prepared with varying enzymolysis temperature
The above table is observed to show that: with the increase of the enzymolysis temperature, the average molecular weight and the gel strength of the gellan gum show a trend of firstly reducing and then increasing, wherein when the enzymolysis temperature is 40 ℃, the average molecular weight and the gel strength of the gellan gum reach the lowest, and the yield at the moment is higher than the yield of the gellan gum prepared at other enzymolysis temperatures.
Example 5: effect of different concentration times on Low molecular Gellan Gum produced
The present embodiment has the following differences from embodiment 1, and the rest of the same parts are not described herein again:
in this embodiment, the concentration time in the separation and extraction step is 10min, 20min, and 40min, respectively.
Upon examination and synthesis of example 1, the following table was obtained:
TABLE 4 molecular weight, gel strength and yield of low molecular weight gellan gum prepared with varying concentration time
The above table is observed to show that: with the increase of the concentration time, the average molecular weight and the gel strength of the gellan gum show a trend of firstly decreasing and then increasing, wherein when the separation and extraction time is 30min, the average molecular weight and the gel strength of the gellan gum reach the optimal levels, and the yield is highest at the moment.
Example 6
The present embodiment has the following differences from embodiment 1, and the rest of the same parts are not described herein again:
in the dissolving process in the step (1), the mass of the gellan gum raw material is 60g in the present example.
Comparative example 1
The comparative example only has the following differences from example 1, and the rest of the same parts are not described herein again:
in the enzymolysis process in step (2) of the comparative example, danisch FoodPro CGL saccharifying enzyme is used instead of pectin lyase and pectinase NE06.
Through detection, the average molecular weight of the gellan gum prepared by the comparative example is not obviously changed, the gel strength reaches 1734, and the difference with the raw material gellan gum strength 1780 is not large, so that the danish gram FoodPro CGL saccharifying enzyme cannot effectively realize enzymolysis on the gellan gum.
Comparative example 2
The comparative example only has the following differences from example 1, and the rest of the same parts are not described herein again:
in the enzymolysis process in the step (2) of the comparative example, novacin Maltogenase2XL maltogenic amylase is used to replace pectin lyase and pectinase NE06.
Through detection, the average molecular weight of the gellan gum prepared by the comparative example is not obviously changed, the gel strength reaches 1767, and the difference from the raw material gellan gum strength 1780 is not large, so that the enzymolysis of the gellan gum cannot be effectively realized by using the Novit Maltogenase2XL maltogenic amylase.
Claims (10)
1. A method for preparing low molecular weight gellan gum by an enzyme method is characterized by comprising the following steps:
(1) Enzymolysis: adding pectinase into the gellan gum aqueous solution for enzymolysis to obtain an enzymatic hydrolysate, wherein the pectinase at least comprises one of pectin lyase and pectinase NE 06;
(2) Separation and extraction: concentrating the enzymolysis liquid in a rotary evaporator to obtain a rotary evaporation product;
(3) And (3) precipitation: adding the rotary evaporation product into ethanol for precipitation to obtain filaments;
(4) Crushing and drying: crushing the filiform matters, and performing vacuum drying on the crushed filiform matters;
(5) Crushing and sieving: and (4) crushing and sieving the product prepared in the step (4).
2. The method of claim 1, the concentration of the gellan gum in the gellan gum aqueous solution in the step (1) is 0.01 g/ml-0.015 g/ml.
3. The method according to claim 2, wherein the pectin lyase and the pectinase NE06 added in step (1) are both 0.05-0.2% of the gellan gum aqueous solution.
4. The method according to claim 1, wherein the enzymatic hydrolysis in step (1) is carried out in a reaction vessel having a paddle rotation speed of 200rpm.
5. The method according to any one of claims 1 or 4, wherein the temperature of the enzymatic hydrolysis in step (1) is 30 to 50 ℃.
6. The method according to any one of claims 1 or 4, wherein the time for enzymolysis in step (1) is 6 to 30 hours.
7. The method according to claim 1, wherein the concentration time in the separation and extraction step in the step (2) is 10-40 min, and the parameters of the rotary evaporator are as follows: the temperature is 80 ℃ and the rotation speed is 300rpm.
8. The method according to claim 1, wherein the ethanol concentration in step (3) is 95%.
9. The method according to claim 1, wherein the vacuum drying conditions in step (4) are as follows: the vacuum degree is 0.07MPa, the temperature is 50 ℃, and the time is 12h.
10. The low molecular weight gellan gum prepared by the preparation method of claims 1 to 9 is used in the fields of food, chemical industry, and medicine.
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