CN107604022B - Method for preparing sodium carboxymethylcellulose by treating bamboo powder by alkali-enzyme coupling technology - Google Patents
Method for preparing sodium carboxymethylcellulose by treating bamboo powder by alkali-enzyme coupling technology Download PDFInfo
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Abstract
The invention relates to a method for preparing sodium carboxymethylcellulose by treating bamboo powder by an alkali-enzyme coupling technology, which is characterized in that the bamboo powder is processed into dry bamboo fibers by the alkali-enzyme coupling technology, and then the bamboo fibers are subjected to enzyme treatment, so that lignin, hemicellulose and other substances coated in the obtained bamboo cellulose can be effectively separated and removed, and the sodium carboxymethylcellulose with higher purity is obtained. The bamboo fiber with a loose structure is obtained by treating bamboo powder of a bamboo product byproduct by an alkali-enzyme coupling method, the sodium carboxymethyl cellulose is prepared by using the bamboo fiber as a raw material, the substitution degree is 0.95-1.07, the viscosity of a 1% aqueous solution at 25 ℃ can reach 81-220 mPa.s, and the technical index meets the product requirement of medium-low viscosity CMC. Because of adopting the biological-chemical coupling method, the method has less environmental pollution and relatively lower cost, and provides a new way for preparing the sodium carboxymethyl cellulose.
Description
Technical Field
The invention relates to the technical field of a preparation method of sodium carboxymethyl cellulose, in particular to a method for preparing sodium carboxymethyl cellulose by treating bamboo powder by an alkali-enzyme coupling technology.
Background
Sodium carboxymethyl cellulose (CMC) is a derivative of modified natural cellulose, is a Sodium salt of ionic, linear and water-soluble carboxymethyl cellulose ether, and is a cellulose derivative product with a wide variety of varieties, wide application fields, large production amount and high research value. CMC is known as "industrial monosodium glutamate" because of its excellent properties and wide use. CMC has been widely used in various economic fields such as paper making industry, oil field exploitation, food industry, medicine industry, daily chemical industry, ceramic manufacture, textile industry and the like. In the food processing industry, the water-soluble organic silicon dioxide mainly serves as a thickening agent, a film forming agent, a stabilizing agent, a curing agent, an extender and the like. When the preservative is used as a film forming agent, the CMC solution and the water-soluble antibacterial agent are used for treating the surface of food to generate a film with glossy surface and softness and transparency, so that the preservative effect is good; when used as a thickening agent and a stabilizing agent, the product can increase the viscosity, has the functions of stabilizing the food structure and prolonging the shelf life of the food, and is commonly used in various jams, seasoning sauce, ice cream and various dairy products; can also be used as solid agent and bulking agent for ice cream, pastry food, etc.
For a long time, the preparation of sodium carboxymethylcellulose mainly uses refined cotton linters, wood pulp and the like as raw materials, the cost of the raw materials is higher, and the market demand for CMC is more and more large, so that the development of other cellulose raw materials with abundant resources, wide sources and low price becomes a current trend. The bamboo product processing by-product is a raw material with higher research value and application prospect. The cellulose content of the product reaches 45 to 52 percent. As the world with the most abundant bamboo resources, the area of the bamboo forest in China reaches more than 520 million hectares, the area of the forest land in China is 21.87%, the total yield of the bamboo industry reaches more than 700 hundred million yuan, but most raw materials are not better utilized.
Cellulose, hemicellulose and lignin in the bamboo form lignocellulose, wherein the hemicellulose and the lignin are covalently combined to form a lignin-carbohydrate structure, and then the cellulose is wrapped, so that the combination of the hemicellulose, the lignin and the lignin is firm and difficult to completely separate. In addition, the biomass contains components such as pectin, protein, and ash. At present, basic methods for separating cellulose from lignocellulose include chemical methods such as acid hydrolysis and alkali treatment, physical methods such as steam explosion and thermal-mechanical coupling fiber opening methods, and biological methods such as enzymatic hydrolysis and microbial fermentation. By adopting a single chemical or physical method, although the efficiency is higher, the problems of equipment corrosion, environmental pollution, cellulose degradation or high energy consumption, high cost and the like are generated, and the sustainable development is not facilitated. The biological treatment method mainly based on the catalytic reaction of the compound cellulase has the advantages of mild conditions, energy conservation and environmental protection, but has the problems of long pretreatment period, low efficiency and the like. While the pure chemical method has better efficiency, but has large pollution and is not beneficial to sustainable development.
Thus, the prior art is subject to further improvement and advancement.
Disclosure of Invention
The technical problem to be solved by the invention is to provide a method for preparing sodium carboxymethyl cellulose by treating bamboo powder by an alkali-enzyme coupling technology.
The invention is realized in this way, and also provides a method for preparing sodium carboxymethyl cellulose by treating bamboo powder by an alkali-enzyme coupling technology, which comprises the following steps:
s1, pretreating bamboo powder, and processing the bamboo powder into dry bamboo fibers by using an alkali-enzyme coupling method, wherein the alkali-enzyme coupling method comprises the following steps:
s11, weighing 3g to 5g of bamboo powder, putting the bamboo powder into a conical flask, adding 35mL to 45mL of 5% sodium hydroxide solution, and putting the mixture into a constant-temperature water bath oscillator at the temperature of 45 ℃ to 60 ℃ for 0.5h to 1.5 h;
s12, rapidly cooling, filtering under reduced pressure, washing the sample with about 380-420 mL of distilled water for several times, and taking filter residues;
s13, adding 25-35 mL of 400U/mL cellulase solution, and placing in a constant-temperature water bath oscillator at 45-55 ℃ for 25-35 min;
s14, then, filtering under reduced pressure; putting the sucked and dried sample into an electric heating constant-temperature blast drying oven to be dried for 2-4 h at 125-145 ℃ to obtain dried bamboo fibers;
s2: adding 35-45 mL of 95% ethanol and 4.0-4.8 mL of 35% sodium hydroxide solution into a 250mL conical flask, fully stirring, adding 2g of the bamboo fiber prepared in the step S1, and oscillating, stirring and reacting at the temperature of 20-45 ℃ for 1.0-2.0 h;
s3, after stirring, adding 5.5-6.0 mL of 75% chloroacetic acid ethanol solution for etherification, wherein the temperature of the etherification reaction is 50-75 ℃, the temperature of 60-80 ℃ is kept for 5-15 min after the etherification reaction is carried out for 60-120 min, and then the temperature is kept for 5-15 min at 40-60 ℃;
s4, adjusting the pH value to be neutral by using 31% glacial acetic acid, filtering under reduced pressure, washing for 2 times by using 75% ethanol, and drying in vacuum at the temperature of 60-80 ℃ to obtain the sodium carboxymethyl cellulose product.
Further, the method for preparing sodium carboxymethyl cellulose by treating bamboo powder by the alkali-enzyme coupling technology also comprises a method for determining the optimum temperature test condition of the cellulase, and the method comprises the following steps:
s01, numbering four colorimetric tubes No. 1-4, and adding 6mL of 1% sodium carboxymethylcellulose solution respectively;
s02, adding 2mL of distilled water into the No. 1 tube, adding 2mL of 400U/mL cellulase solution prepared by HAc-NaAc buffer solution and having the pH value of 5.5 into the other three tubes, shaking uniformly, placing into a low-temperature constant-temperature water bath tank, controlling the temperature of the low-temperature constant-temperature water bath tank to be 37 ℃, taking out after water bath for 30 min;
s03, respectively adding 2mL of DNS reagent into each tube, heating in a boiling water bath for 8min, quickly putting into an ice water bath, cooling to room temperature, and adding distilled water to a constant volume of 10 mL;
s04, taking the No. 1 colorimetric tube test solution as a reference, and measuring the absorbance of the No. 2 test solution to the No. 4 test solution under the condition of 530nm wavelength;
s05, repeating the steps S01-S04, controlling the temperature of the low-temperature constant-temperature water bath in the step S02 to 41 ℃, 45 ℃, 49 ℃, 53 ℃, 57 ℃ and 61 ℃ respectively, continuing the test, and measuring the absorbance of the No. 2-4 test solutions respectively;
s06, comparing the measured values of absorbance obtained by the tests under different temperature conditions to obtain the temperature condition with the maximum absorbance as the optimal temperature of the enzymatic reaction of the cellulase.
Further, the method for preparing sodium carboxymethyl cellulose by treating bamboo powder by the alkali-enzyme coupling technology also comprises a method for determining the most suitable pH value test condition of the cellulase, and the method comprises the following steps:
t01, taking two colorimetric tubes, and adding 6mL of 1% sodium carboxymethylcellulose solution into each colorimetric tube;
t02, adding 2mL of HAc-NaAc buffer solution with the pH value of 4.0 respectively to prepare 1mg/mL of cellulase solution, shaking up, putting into a low-temperature constant-temperature water bath tank, controlling the temperature of the low-temperature constant-temperature water bath tank to be 49 ℃, carrying out water bath for 30min, and taking out;
t03, respectively adding 2mL of DNS reagent into each tube, heating in boiling water bath for 5min, rapidly placing in ice water bath, cooling to room temperature, and adding distilled water to constant volume of 10 mL;
t04, measuring the absorbance of the test solutions of the two colorimetric tubes respectively under the condition of 540nm wavelength, and taking the average value to obtain the average value of the absorbance of the cellulase mixed solution with the pH value of 4.0;
t05, repeating the steps T01-T04, controlling the pH values of the cellulase solution in the step T02 to be 4.5, 5.0, 5.5 and 6.0 respectively, continuing the test, and measuring the absorbance of the test solutions of the two colorimetric tubes respectively;
t06, comparing the absorbance average values obtained by the above tests, and obtaining the pH value condition with the highest absorbance of the cellulase solution in the HAc-NaAc buffer solution system as the optimum pH value of the cellulase enzymatic reaction.
Compared with the prior art, the method for preparing the sodium carboxymethyl cellulose by treating the bamboo powder by the alkali-enzyme coupling technology takes the bamboo powder of the bamboo product byproduct as a raw material, firstly performs primary treatment on a bamboo powder sample by a chemical method, and then performs enzymatic treatment, so that substances such as lignin, hemicellulose and the like wrapping cellulose in lignocellulose can be effectively separated and removed, the cellulose with higher purity is obtained, and the sodium carboxymethyl cellulose is prepared. The substitution degree of the sodium carboxymethylcellulose prepared by the method is 0.95-1.07, the viscosity of 1% aqueous solution at 25 ℃ can reach 81-220 mPa.s, and the technical index of the sodium carboxymethylcellulose meets the product requirement of medium-low viscosity CMC. The project adopts a biological-chemical coupling method in raw material pretreatment, has little pollution to the environment and relatively low cost, promotes the development of green economy, and also provides a new way for preparing the sodium carboxymethyl cellulose.
Drawings
FIG. 1 is a schematic diagram of the content of alpha-cellulose in bamboo fiber products obtained by 7 bamboo powder pretreatment methods of the present invention;
FIG. 2 shows a: bamboo powder, b: pretreating bamboo powder by an alkali-enzyme coupling method, and c: CMC product, d: infrared spectrum schematic of commercially available food grade CMC;
FIG. 3 shows a: bamboo powder, b: bamboo fiber after alkaline hydrolysis, c: bamboo fiber after enzymolysis, d: bamboo fiber treated by alkali-enzyme coupling method and e: electron microscopy scanning of CMC product;
FIG. 4 is a graph showing the effect of temperature on cellulase activity;
FIG. 5 is a graph showing the effect of pH on cellulase activity.
Detailed Description
In order to make the technical problems, technical solutions and advantageous effects to be solved by the present invention more clearly apparent, the present invention is further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative of the invention and are not intended to limit the invention.
Example 1:
the method for preparing sodium carboxymethyl cellulose by treating bamboo powder by using the alkali-enzyme coupling technology comprises the following steps:
s1, pretreating bamboo powder, and processing the bamboo powder into dry bamboo fibers by using an alkali-enzyme coupling method, wherein the alkali-enzyme coupling method comprises the following steps:
s11, weighing 4g of bamboo powder, putting the bamboo powder into a conical flask, adding 40mL of 5% sodium hydroxide (NaOH) solution, and putting the mixture into a constant-temperature water bath oscillator at the temperature of 50 ℃ for 1.0 h;
s12, rapidly cooling, filtering under reduced pressure, washing the sample by 400mL of distilled water for several times, and taking filter residues;
s13, adding 30mL of 400U/mL cellulase solution, and placing in a constant-temperature water bath oscillator at the temperature of 49 ℃ for 30 min;
s14, then, filtering under reduced pressure; and (3) drying the sucked and dried sample in an electric heating constant-temperature air blast drying oven for 3 hours at 135 ℃ to obtain the dried bamboo fiber.
S2: 40mL of 95% ethanol and 4.4mL of 35% sodium hydroxide (NaOH) solution are added into a 250mL conical flask, the mixture is fully stirred, 2g of the bamboo fiber prepared in the step S1 is added, and the mixture is stirred and reacted for 2.0h at the temperature of 35 ℃.
And S3, after stirring, adding 5.9mL of 75% chloroacetic acid ethanol solution for etherification, wherein the etherification reaction temperature is 70 ℃, keeping the temperature of 70 ℃ for 10min after 120min of etherification reaction, and keeping the temperature of 50 ℃ for 10 min.
S4, adjusting the pH value to be neutral by using 31% glacial acetic acid, filtering under reduced pressure, washing for 2 times by using 75% ethanol, and drying in vacuum at 70 ℃ to obtain a sodium carboxymethylcellulose (CMC) product.
Wherein the bamboo powder is prepared by peeling bamboo or bamboo waste, processing into chips, washing with water, oven drying, pulverizing with a pulverizer, sieving with 60 mesh sieve, and detecting that its alpha-cellulose content is 43.2% (dry weight), hemicellulose content is 25.1%, and lignin content is 15.2%.
Example 2:
the method for preparing sodium carboxymethyl cellulose by treating bamboo powder by using the alkali-enzyme coupling technology comprises the following steps:
s1, pretreating bamboo powder, and processing the bamboo powder into dry bamboo fibers by using an alkali-enzyme coupling method, wherein the alkali-enzyme coupling method comprises the following steps:
s11, weighing 3g of bamboo powder, putting into a conical flask, adding 35mL of 5% sodium hydroxide solution, and putting into a constant-temperature water bath oscillator at 45 ℃ for 0.5 h;
s12, rapidly cooling, filtering under reduced pressure, washing the sample with about 380mL of distilled water for several times, and taking filter residues;
s13, adding 25mL of 400U/mL cellulase solution, and placing in a constant-temperature water bath oscillator at 45 ℃ for 25 min;
s14, then, filtering under reduced pressure; and (3) drying the sucked and dried sample in an electric heating constant-temperature air blast drying oven for 2 hours at 125 ℃ to obtain the dried bamboo fiber.
S2: 35mL of 95% ethanol and 4.0mL of 35% sodium hydroxide solution were added to a 250mL conical flask, and 2g of the bamboo fiber prepared in step S1 was added after sufficient stirring, followed by reaction at 20 ℃ for 1.0 hour with shaking and stirring.
And S3, after stirring, adding 5.5mL of 75% chloroacetic acid ethanol solution for etherification, wherein the etherification reaction temperature is 50 ℃, keeping the temperature at 60 ℃ for 5min after the etherification reaction is carried out for 60min, and keeping the temperature at 40 ℃ for 5 min.
S4, adjusting the pH value to be neutral by using 31% glacial acetic acid, filtering under reduced pressure, washing for 2 times by using 75% ethanol, and drying in vacuum at 60 ℃ to obtain the sodium carboxymethyl cellulose product.
Example 3
A method for preparing sodium carboxymethylcellulose by treating bamboo powder by an alkali-enzyme coupling technology is characterized by comprising the following steps:
s1, pretreating bamboo powder, and processing the bamboo powder into dry bamboo fibers by using an alkali-enzyme coupling method, wherein the alkali-enzyme coupling method comprises the following steps:
s11, weighing 5g of bamboo powder, putting the bamboo powder into a conical flask, adding 45mL of 5% sodium hydroxide solution, and putting the conical flask into a constant-temperature water bath oscillator at the temperature of 60 ℃ for 1.5 h;
s12, rapidly cooling, filtering under reduced pressure, washing the sample with about 420mL of distilled water for several times, and taking filter residues;
s13, adding 35mL of 400U/mL cellulase solution, placing in a constant-temperature water bath oscillator, and carrying out temperature regulation at 55 ℃ for 35 min:
s14, then, filtering under reduced pressure; and (3) drying the sucked and dried sample in an electric heating constant-temperature air-blast drying oven for 4 hours at 145 ℃ to obtain the dried bamboo fiber.
S2: adding 45mL of 95% ethanol and 4.8mL of 35% sodium hydroxide solution into a 250mL conical flask, fully stirring, adding 2g of the bamboo fiber prepared in the step S1, and reacting at 45 ℃ for 2.0h under shaking and stirring.
And S3, after stirring, adding 6.0mL of 75% chloroacetic acid ethanol solution for etherification, wherein the etherification reaction temperature is 75 ℃, after the etherification reaction is carried out for 120min, the temperature is kept at 80 ℃ for 15min, and then the temperature is kept at 60 ℃ for 15 min.
S4, adjusting the pH value to be neutral by using 31% glacial acetic acid, filtering under reduced pressure, washing for 2 times by using 75% ethanol, and drying in vacuum at 80 ℃ to obtain the sodium carboxymethyl cellulose product.
For the pretreatment of the bamboo powder, the bamboo powder can be processed into dry bamboo fibers by the following methods, specifically comprising a water boiling method, a weak acid method, an alkaline solution method, an enzymolysis method, a weak acid-enzymolysis coupling method and a water boiling-enzymolysis coupling method.
The water boiling method for bamboo powder pretreatment comprises the following steps: weighing 3 parts of 4g of bamboo powder, respectively adding 40mL of distilled water, placing in a constant temperature water bath oscillator, reacting at 50 ℃ for 1h, rapidly cooling, filtering under reduced pressure, washing the sample with 400mL of distilled water in several times, and drying the filter residue in an electric heating constant temperature air blast drying oven at 135 ℃ for 3 h. And putting the dried product into a sealing bag to be stored in dark.
The weak acid method for bamboo powder pretreatment comprises the following steps: weighing 3 parts of 4g of bamboo powder, respectively adding 0.1mol/L hydrochloric acid solution, placing in a constant temperature water bath oscillator, reacting at 50 ℃ for 1h, rapidly cooling, filtering under reduced pressure, washing the sample with 400mL of distilled water in several times, and drying the filter residue in an electric heating constant temperature air blast drying oven at 135 ℃ for 3 h. And putting the dried product into a sealing bag to be stored in dark.
The alkali liquor method for bamboo powder pretreatment comprises the following steps: weighing 3 parts of 4g of bamboo powder, respectively adding 5% NaOH solution, placing in a constant-temperature water bath oscillator, reacting at 50 ℃ for 1h, rapidly cooling, filtering under reduced pressure, washing the sample with 400mL of distilled water in several times, and drying the filter residue in an electric heating constant-temperature air-blast drying oven at 135 ℃ for 3 h. And putting the dried product into a sealing bag to be stored in dark.
The enzymolysis method for bamboo powder pretreatment comprises the following steps: weighing 4g of bamboo powder, adding 40mL of 400U/mL cellulase solution, placing in a constant temperature water bath oscillator, reacting at 49 ℃ for 30min, rapidly cooling, filtering under reduced pressure, washing the sample with 400mL of distilled water in several times, and drying the filter residue in an electric heating constant temperature air-blast drying oven at 135 ℃ for 3 h. And putting the dried product into a sealing bag to be stored in dark.
The weak acid-enzymolysis coupling method for bamboo powder pretreatment comprises the following steps: weighing 3 parts of 4g of bamboo powder, respectively adding 40mL of 0.1mol/L hydrochloric acid solution, placing the bamboo powder in a constant-temperature water bath oscillator, reacting at 50 ℃ for 1h, quickly cooling, filtering under reduced pressure, washing the sample with 400mL of distilled water for several times, taking filter residue, adding 30mL of 400U/mL cellulase solution, placing the filter residue in the constant-temperature water bath oscillator, reacting at 49 ℃ for 30min, filtering under reduced pressure, taking the filter residue, drying in a 135 ℃ electric heating constant-temperature air-blowing drying oven for 3h, and placing the dried product in a sealed bag for light-tight storage.
The water boiling-enzymolysis coupling method for bamboo powder pretreatment comprises the following steps: weighing 3 parts of 4g of bamboo powder, respectively adding distilled water, placing in a constant-temperature water bath oscillator, reacting at 50 ℃ for 1h, rapidly cooling, filtering under reduced pressure, washing the sample with 400mL of distilled water in batches, taking filter residue, adding 30mL of 400U/mL cellulase solution, placing in the constant-temperature water bath oscillator, reacting at 49 ℃ for 30min, filtering under reduced pressure, taking the filter residue, drying in a 135 ℃ electric heating constant-temperature air blast drying box for 3h, and placing the dried product in a sealed bag for light-tight storage.
The content of alpha-cellulose is used as a judgment standard, and the content of alpha-cellulose of the dried bamboo fiber products obtained by various bamboo powder pretreatment methods is measured and compared, so that the content of alpha-cellulose of the bamboo fiber pretreated by various bamboo powder pretreatment methods can reach more than or equal to 70 percent, please refer to fig. 1. Wherein, the content of alpha-cellulose of the bamboo fiber product obtained by adopting the alkali-enzyme coupling method is obviously higher than that of other treatment methods, reaches 87.7-90.6 percent, is respectively 16.2-20.0 percent higher than that of the alpha-cellulose (75.5 percent) of the bamboo fiber product obtained by a single enzymolysis method, and is 12.7-16.5 percent higher than that of the alpha-cellulose (77.8 percent) of the bamboo fiber product obtained by single alkali liquor treatment. Thus, the alkali-enzyme coupling method of the present invention has obvious advantages. The graph shows that the alpha-cellulose content of the product obtained by the alkali-enzyme coupling method is significantly different from that obtained by other methods (p < 0.05).
The invention relates to a method for measuring alpha-cellulose content, which comprises the following steps: weighing 30mL of 17.5% sodium hydroxide, adding the sodium hydroxide into a porcelain cup, weighing 2.00g of the prepared bamboo fiber sample, putting the bamboo fiber sample into the porcelain cup, uniformly stirring, and covering with a cover; standing for 30 min. Stirring every 10min for about 1 min. After 30min, 30mL of distilled water is added, the mixture is stirred and filtered under reduced pressure, and filter residues are washed for multiple times. Adding 30mL of 10% glacial acetic acid solution, soaking for 5min, filtering under reduced pressure, and washing with hot distilled water to neutrality. And (3) drying the cleaned filter residue in a 135 ℃ oven for 3 hours to obtain a dried bamboo fiber sample to be detected.
The characteristic peak of sodium carboxymethylcellulose (CMC) in the infrared spectrum is used for identifying the prepared CMC product. The infrared spectrum detection method comprises the following steps: firstly, mixing some potassium bromide and a CMC sample to be detected, putting the mixture into a grinder for grinding, putting the mixture into a dryer for drying after grinding to a certain degree, taking the mixture out after 5min, preparing tablets, and putting the tablets into an infrared spectrometer for detection.
The infrared spectra of samples a and b before and after bamboo powder pretreatment, and CMC product c and food-grade CMC product d available in the market as standard are shown in FIG. 2. Wherein, the CMC product c is prepared by the method for preparing sodium carboxymethyl cellulose by treating bamboo powder by the alkali-enzyme coupling technology. The reported sodium carboxymethylcellulose (CMC) is 1423cm-1And 1620cm-1Has characteristic peaks. The characteristic peak of the sodium carboxymethylcellulose (CMC) can be found out from the CMC product prepared by the invention and accords with the characteristic, such as the part indicated by a small arrow in figure 2. It can be concluded that the product prepared above meets the properties of sodium carboxymethylcellulose (CMC) products.
Viscosity determination method of CMC product: weighing 2g of CMC product, putting the CMC product into a beaker, adding 98mL of distilled water, stirring the CMC product for 2.5 hours by using a magnetic stirrer, filtering the CMC product under reduced pressure, and drying the CMC product to obtain the mass M of filter residue1The pure CMC content in the obtained CMC product is W, and M is weighed according to the proportion and the required 2g of pure CMC2CMC product, repeat the above dissolution step, and measure viscosity.
In the formula: w is the pure CMC content (%) of the CMC product, M1Mass (g) of undissolved impurities in the CMC product, M2Is the quality of the CMC product.
The viscosity of the 1% water solution of the CMC product prepared by the method reaches 81-220 mPa.s at 25 ℃ and is higher than that of the CMC prepared by other processing method raw materials, which accords with the technical index of the CMC with medium and low viscosity. pH7.2, and the degree of substitution is between 0.95 and 1.07.
The method for detecting CMC by using a scanning electron microscope comprises the following steps: and (4) carrying out conductive treatment on the gold-sprayed powder of the CMC sample to be detected by using a TM-1000 scanning electron microscope.
Respectively detecting a sample a before bamboo powder pretreatment, a sample b after bamboo powder is subjected to alkaline hydrolysis pretreatment, a sample c after bamboo powder is subjected to enzymatic hydrolysis pretreatment, a sample d after bamboo powder is subjected to alkaline-enzyme coupling pretreatment and a CMC product e by using a scanning electron microscope to obtain an electron microscope scanning image shown in figure 3. Wherein the CMC product is prepared by the process of the present invention. As can be seen from the figure, the fibers of the bamboo chips are bundled, the surface is smooth, and the structure is dense, as shown in figure 3-a. After pretreatment by alkaline hydrolysis or enzymatic hydrolysis, the cellulose is broken down into small bundles with a loose structure as shown in FIGS. 3-b and 3-c. The cellulose obtained by the alkali-enzyme coupling method has a looser structure, and is beneficial to the next treatment, as shown in figure 3-d. The surface of the CMC product is destroyed and the structure becomes more loose and shows the characteristics of a salt, as shown in fig. 3-e.
In the present invention, cellulase is used, and in order to maximize the enzymatic activity of cellulase during the process, the cellulase is used under the most suitable conditions of temperature and pH. The cellulose can hydrolyze sodium carboxymethylcellulose under certain conditions to generate reducing sugar, and the reducing sugar generated by the reaction is determined by using a DNS method, so that the optimal reaction conditions of the cellulose are obtained. The cellulose polymerization degree can be reduced by utilizing proper enzymolysis of the cellulase, the lignocellulose structure is loosened, the cellulose is dissociated from lignin and hemicellulose, and a cellulose product with higher purity is obtained.
Since too high or too low a pH may result in a change in the higher order structure of the enzyme, the enzyme is inactivated, also known as acid-or alkali-denatured. Further, since enzymes have many dissociable groups, and the dissociable states of these groups are different and different in charge in different pH environments, the dissociable states of these groups have an important role in the binding ability of the enzyme to a substrate and the catalytic ability of the enzyme, and thus a change in the pH of a solution can affect the activity of the enzyme by affecting the dissociable states of these groups. Furthermore, pH influences the enzymatic reaction rate by influencing the dissociation state of the substrate and of the intermediate complex ES. If other conditions are not changed, the enzyme can only exhibit catalytic activity within a certain pH range, and the enzymatic reaction rate is maximized at a certain pH, which is referred to as the optimum pH of the enzyme. The cellulase of the present invention is preferably prepared under the following conditions.
The invention also discloses a temperature measuring method in the optimum temperature test condition of the cellulase, which comprises the following steps:
s01, numbering four colorimetric tubes No. 1-4, and adding 6mL of 1% sodium carboxymethylcellulose (CMC) solution respectively.
S02, adding 2mL of distilled water into the tube No. 1, adding 2mL of 400U/mL cellulase solution prepared by HAc-NaAc buffer solution and having pH of 5.5 into the other three tubes, shaking uniformly, placing into a low-temperature constant-temperature water bath, controlling the temperature of the low-temperature constant-temperature water bath to be 37 ℃, carrying out water bath for 30min, and taking out.
S03, respectively adding 2mL of DNS reagent into each tube, heating in boiling water bath for 8min, rapidly placing in ice water bath, cooling to room temperature, and adding distilled water to a constant volume of 10 mL.
S04, measuring the absorbance of the No. 2-4 colorimetric tube test solution under the condition of 530nm wavelength by using the No. 1 colorimetric tube test solution as a control.
S05, repeating the above steps S01-S04, controlling the temperature of the low temperature constant temperature water bath in the step S02 at 41 ℃, 45 ℃, 49 ℃, 53 ℃, 57 ℃ and 61 ℃ respectively, continuing the test, and measuring the absorbance of the No. 2-4 test solutions respectively.
S06, comparing the measured values of absorbance obtained by the tests under different temperature conditions to obtain the temperature condition with the maximum absorbance as the optimal temperature of the enzymatic reaction of the cellulase.
The above experiment resulted in the temperature-versus enzyme activity curve shown in FIG. 4. When the temperature is lower than 49 ℃, the activity of the cellulase is increased along with the increase of the temperature; when the temperature is higher than 49 ℃, the cellulase activity decreases with decreasing temperature.
The invention also discloses a pH value determination method for the cellulase under the condition most suitable for pH value test, which comprises the following steps:
t01, two cuvettes were each charged with 6mL of 1% sodium carboxymethylcellulose (CMC) solution.
And T02, adding 2mL of HAc-NaAc buffer solution with the pH value of 4.0 respectively to prepare 1mg/mL of cellulase solution, shaking uniformly, putting into a low-temperature constant-temperature water bath tank, controlling the temperature of the low-temperature constant-temperature water bath tank to be 49 ℃, carrying out water bath for 30min, and taking out.
T03, respectively adding 2mL of DNS reagent into each tube, heating in boiling water bath for 5min, rapidly placing in ice water bath, cooling to room temperature, and adding distilled water to constant volume of 10 mL.
And T04, measuring the absorbance of the test solutions of the two colorimetric tubes under the condition of the wavelength of 530nm, and taking the average value to obtain the average value of the absorbance of the cellulase mixed solution with the pH value of 4.0.
And T05, repeating the steps T01-T04, controlling the pH values of the cellulase solution in the step T02 to be 4.5, 5.0, 5.5 and 6.0 respectively, continuing the test, and measuring the absorbance of the test solutions of the two colorimetric tubes respectively.
T06, comparing the absorbance average values obtained by the above tests, and obtaining the pH value condition with the highest absorbance of the cellulase solution in the HAc-NaAc buffer solution system as the optimum pH value of the cellulase enzymatic reaction.
The above tests gave the following test data:
TABLE 1 absorbance measurement data and average values of cellulase solutions at different pH values
Taking pH as abscissa and OD540nm as ordinate, a pH-OD value curve can be obtained, and the optimum pH value of the cellulase can be determined from the OD value which is in direct proportion to the activity of the cellulase. If the maximum OD value is defined as 100% of the relative enzyme activity, the ordinate value can be converted into the relative enzyme activity, as shown in fig. 5. When the pH value is less than 5.5, the activity of the cellulase is increased along with the increase of the pH value; at pH values above 5.5, cellulase activity decreases with increasing pH.
According to the experimental results, the optimum reaction temperature of the cellulase is 49 ℃, and the pH value is 5.5.
The above description is only for the purpose of illustrating the preferred embodiments of the present invention and is not to be construed as limiting the invention, and any modifications, equivalents and improvements made within the spirit and principles of the present invention are intended to be included within the scope of the present invention.
Claims (1)
1. A method for preparing sodium carboxymethylcellulose by treating bamboo powder by an alkali-enzyme coupling technology is characterized by comprising the following steps:
s1, pretreating bamboo powder, and processing the bamboo powder into dry bamboo fibers by using an alkali-enzyme coupling method, wherein the alkali-enzyme coupling method comprises the following steps:
s11, weighing 3g to 5g of bamboo powder, putting the bamboo powder into a conical flask, adding 35mL to 45mL of 5% sodium hydroxide solution, and putting the mixture into a constant-temperature water bath oscillator at the temperature of 45 ℃ to 60 ℃ for 0.5h to 1.5 h;
s12, rapidly cooling, filtering under reduced pressure, washing the sample by 380-420 mL of distilled water in batches, and taking filter residues;
s13, adding 25-35 mL of 400U/mL cellulase solution, and placing in a constant-temperature water bath oscillator at the temperature of 45-55 ℃ for 25-35 min;
s14, then, filtering under reduced pressure; putting the sucked and dried sample into an electric heating constant-temperature air blast drying oven to be dried for 2-4 h at 125-145 ℃ to obtain dried bamboo fibers, wherein the content of alpha-cellulose of the dried bamboo fibers reaches 87.7-90.6%;
s2, adding 35-45 mL of 95% ethanol and 4.0-4.8 mL of 35% sodium hydroxide solution into a 250mL conical flask, fully stirring, adding 2g of the bamboo fiber prepared in the step S1, and reacting at 20-45 ℃ for 1.0-2.0 h by oscillating and stirring;
s3, after stirring, adding 5.5-6.0 mL of 75% chloroacetic acid ethanol solution for etherification, wherein the etherification reaction temperature is 50-75 ℃, the temperature is 60-80 ℃ after the etherification reaction is carried out for 60-120 min, the temperature is kept for 5-15 min, and then the temperature is kept for 5-15 min at 40-60 ℃;
s4, adjusting the pH value to be neutral by using 31% glacial acetic acid, filtering under reduced pressure, washing for 2 times by using 75% ethanol, and drying in vacuum at the temperature of 60-80 ℃ to obtain a sodium carboxymethyl cellulose product with the substitution degree of 0.95-1.07.
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