Method for preparing nanocellulose by taking phellinus igniarius substrate as raw material

Abstract

The invention provides a method for preparing nanocellulose by using a phellinus igniarius substrate as a raw material, and belongs to the technical field of green biomass refinement, the method comprises the following steps: crushing and sieving the phellinus igniarius substrate, repeatedly washing with deionized water, and filtering to obtain phellinus igniarius substrate filter residues; bleaching the phellinus igniarius substrate filter residues with alkali liquor and bleaching liquor at the same time to obtain cellulose; the method comprises the following steps: carrying out enzymolysis on cellulose by utilizing an enzymolysis method, and carrying out centrifugal separation, acid flocculation, centrifugal washing and freeze drying to obtain the nano cellulose. The hydrogen peroxide is used for bleaching the cellulose, so that pigments can be effectively removed, the damage to the cellulose is smaller than that of other bleaching agents, and no chemical substance harmful to the cellulose is left after bleaching. By changing the enzymolysis conditions and the centrifugal rotating speed, three different supernates can be separated, and different nano-crystalline cellulose can be extracted from the supernates to meet the requirements of different applications. And resource utilization of the phellinus igniarius substrate is realized, and waste is turned into wealth.

Description

Technical Field

[0001] The invention relates to the technical field of green biomass refining, and in particular to a method for preparing nanocellulose by taking mulberry linterus matrix as raw material. Background Art

[0002] Phellinus igniarius is a rare medicinal fungus with anti-tumor, anti-inflammatory, hypoglycemic and antioxidant effects. Due to the scarcity of wild Phellinus igniarius resources, the current main method of artificial cultivation of Phellinus igniarius is log cultivation. However, the substrate after cultivation of Phellinus igniarius is basically piled up and not reasonably utilized, which not only wastes resources but also seriously pollutes the environment.

[0003] Different from the chemical treatment method of preparing nanocellulose by oxidizing cellulose with sulfuric acid or ammonium persulfate, the enzymatic method of preparing cellulose nanomaterials has attracted widespread attention due to its mild reaction conditions and the fact that it does not produce toxic residues. Summary of the invention

[0004] To achieve the above object, the technical solution of the present invention is as follows: A method for preparing nanocellulose using mulberry linterus matrix as raw material comprises the following steps: (1) Pretreatment: The Phellinus igniarius matrix is ​​crushed and sieved, then repeatedly washed with deionized water and filtered to obtain Phellinus igniarius matrix residue; (2) Preparation of cellulose: The Phellinus igniarius matrix residue is bleached with alkali solution and bleaching solution at the same time. The reaction solution after bleaching is centrifuged and washed until the supernatant is neutral. The solid phase collected is cellulose; (3) Preparation of nanocellulose: Enzymatic hydrolysis of cellulose followed by centrifugal separation, acid flocculation, centrifugal washing, and freeze drying to obtain nanocellulose; The alkali solution in step (2) is a 5-10wt% sodium hydroxide aqueous solution, the bleaching solution is a 10-15wt% hydrogen peroxide aqueous solution, the volume ratio of the bleaching solution to the alkali solution is (1-3):1, the total volume of the bleaching solution and the alkali solution is 100-180mL, and the material-liquid ratio is 1:(50-200); the bleaching treatment temperature is 50-90°C, and the treatment time is 50-100min.

[0005] Furthermore, in step (1), the Phellinus igniarius matrix is ​​crushed and sieved, and then soaked in deionized water for 10 to 24 hours and then repeatedly washed until the filtrate is clear.

[0006] Furthermore, in step (2), the centrifugal speed is 8000-12000 rpm, and the centrifugal time is 10-30 min; the obtained cellulose is stored at 4°C.

[0007] Furthermore, the enzymatic hydrolysis method in step (3) is selected from one of the following methods: The first method is to use low-concentration cellulase to hydrolyze cellulose, wherein the low-concentration cellulase is 5-20u / mL, the enzymatic hydrolysis time is 40-120min, and after the enzymatic hydrolysis, the enzymatic hydrolyzate is placed in boiling water for 30min to inactivate the enzyme to obtain an enzymatic hydrolyzate; The second method is to use high-concentration cellulase to hydrolyze cellulose, wherein the high-concentration cellulase is 100-500u / mL, the hydrolysis time is 4-12h, and after the hydrolysis is completed, the hydrolyzate is placed in boiling water for 30min to inactivate the enzyme to obtain the hydrolyzate.

[0008] Furthermore, before the centrifugal separation in step (3), the pH of the enzymatic hydrolyzate needs to be adjusted to 8-10 using a sodium hydroxide aqueous solution, and the nanocellulose is separated by centrifugation after standing for 30-90 minutes.

[0009] Furthermore, the centrifugal separation in step (3) is to separate a first supernatant from an enzymatic hydrolysate produced by using a low concentration of cellulase at a speed of 500-1500 rpm for a time of 5-20 min; to separate a second supernatant at a speed of 3000-4500 rpm for a time of 5-20 min, or to separate a third supernatant from an enzymatic hydrolysate produced by using a high concentration of cellulase at a speed of 500-1500 rpm for a time of 5-20 min.

[0010] Furthermore, the acid flocculation in step (3) is performed by adjusting the pH of the supernatant to 1-3 using an aqueous hydrochloric acid solution, adding the solution dropwise while stirring until the supernatant becomes turbid and the nanocellulose begins to flocculate, and the flocculation is completed until all the nanocellulose is precipitated.

[0011] Furthermore, in step (3), the centrifugal washing is performed at a speed of 8000-12000 rpm for 5-20 min, the supernatant is discarded, and the washing is repeated by centrifugation with deionized water until the washing solution is neutral, the solid phase is collected, and freeze-dried to obtain the three purified nanocelluloses.

[0012] Compared with the prior art methods, the present invention has the following beneficial effects: The present invention uses mulberry linterus matrix as a raw material to prepare nanocellulose, which on the one hand realizes the resource utilization of the mulberry linterus matrix, and on the other hand increases the added value of the mulberry linterus matrix, is conducive to the high-value utilization process of the mulberry linterus matrix, and broadens the path of turning waste into treasure for the mulberry linterus matrix.

[0013] The present invention uses hydrogen peroxide to bleach cellulose. On the one hand, the damage to cellulose is less than that of other bleaching agents such as sodium hypochlorite, and no chemical substances harmful to cellulose remain after bleaching. On the other hand, hydrogen peroxide can effectively remove pigments, so that cellulose has a higher whiteness, good stability and is not easy to turn yellow.

[0014] The present invention uses an enzymatic hydrolysis method to prepare nanocellulose. On the one hand, by changing the enzymatic hydrolysis conditions and the centrifugal speed, energy consumption and the use of chemical reagents can be reduced. On the other hand, three different supernatants can be separated, from which different nanocelluloses can be extracted respectively to meet the needs of different applications. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 This is the Fourier transform infrared absorption spectrum of Phellinus linteus matrix and cellulose; Figure 2 Scanning electron microscopy of the first rod-shaped nanocellulose; Figure 3 This is a scanning electron micrograph of the second type of spherical nanocellulose; Figure 4 Scanning electron microscopy of the third type of spherical nanocellulose. DETAILED DESCRIPTION

[0016] The technical solution of the present invention is described in detail below in conjunction with the accompanying drawings and embodiments.

[0017] Example 1 (1) Pretreatment: The Phellinus igniarius matrix was crushed and passed through a 50-mesh sieve, then soaked in deionized water overnight and washed repeatedly until the filtrate was clear and the collected Phellinus igniarius matrix residue remained moist.

[0018] (2) Preparation of cellulose: the alkali solution is a 5 wt % sodium hydroxide aqueous solution, the bleaching solution is a 10 wt % hydrogen peroxide aqueous solution, the volume ratio of the bleaching solution to the alkali solution is 1:1, the total volume of the bleaching solution and the alkali solution is 140 mL, the solid-liquid ratio is 1:140, the reaction temperature is 70 ° C, the reaction time is 70 min, the reaction solution after bleaching is centrifuged at a speed of 10000 rpm for 10 min, the supernatant is discarded, deionized water is added and centrifuged repeatedly until the supernatant is neutral, the collected solid phase is cellulose, freeze-dried, the yield is 42.78%, and the color difference with the mulberry linterus matrix powder is 32.5.

[0019] (3) Preparation of nanocellulose: In this embodiment, low-concentration cellulase was used to hydrolyze cellulose, the low-concentration cellulase was 10u / mL, and the hydrolysis time was 60min; after the hydrolysis, the hydrolyzate was placed in boiling water for 30min to inactivate the enzyme. The pH of the hydrolyzate was adjusted to 8 using sodium hydroxide aqueous solution, and the nanocellulose was separated by centrifugation after standing for 30min. The supernatant was separated from the hydrolyzate produced by using low-concentration cellulase at a speed of 1000rpm for 5min; The pH of the supernatant was adjusted to about 2 using aqueous hydrochloric acid solution, and stirred while adding dropwise until the supernatant was observed to become turbid, the residual cellulase was dispersed, and the nanocellulose began to flocculate until the nanocellulose was completely precipitated and flocculation was completed. The centrifugal washing speed was 10000rpm, and the time was 5min. The supernatant was discarded, and the washing was repeatedly centrifuged with deionized water until the washing solution was neutral. The solid phase was collected, and freeze-dried to obtain the purified nanocellulose. The nanocellulose obtained in this embodiment is rod-shaped nanocellulose with a length distribution of 40 to 200nm and a yield of 1.58%.

[0020] Example 2 This embodiment is different from embodiment 1 in that in step (3), a second supernatant is separated from the enzymatic hydrolyzate produced by using low-concentration cellulase at a rotation speed of 3000 rpm for 5 min. The nanocellulose obtained in this embodiment is spherical nanocellulose with a diameter distribution of 20-70 nm and a yield of 5.96%.

[0021] Example 3 This embodiment is different from embodiment 1 in that high concentration cellulase is used to hydrolyze cellulose in step (3). The high concentration cellulase is 300u / mL and the hydrolysis time is 8h. And from the hydrolyzate produced by using high concentration cellulase, the rotation speed is 500rpm and the time is 5min to separate the third supernatant. The nanocellulose in this embodiment is spherical nanocellulose with a diameter distribution of 10-30nm and a yield of 28.20%.

[0022] Example 4 This embodiment is different from embodiment 1 in that the volume ratio of the bleaching solution to the alkaline solution in step (2) is 2:1, the reaction time is 80 min, the yield is 38.22%, and the color difference with the mulberry linterus matrix powder is 31.5.

[0023] Example 5 This embodiment is different from embodiment 1 in that the volume ratio of the bleaching solution to the alkaline solution in step (2) is 1:1, the reaction time is 50 min, the yield is 43.55%, and the color difference with the mulberry linterus matrix powder is 31.8.

[0024] Example 6 This embodiment is different from embodiment 3 in that high-concentration cellulase is used to enzymolyze cellulose in step (3). The high-concentration cellulase is 100 u / mL, the enzymolysis time is 8 h, and the yield is 14.32%.

[0025] Example 7 This embodiment is different from embodiment 3 in that high-concentration cellulase is used to enzymolyze cellulose in step (3). The high-concentration cellulase is 300 u / mL, the enzymolysis time is 10 h, and the yield is 19.93%.

Claims

1. A method for preparing nanocellulose using Phellinus linteus matrix as raw material, characterized in that: The method comprises the following steps: (1) Pretreatment: The Phellinus igniarius matrix is ​​crushed and sieved, then repeatedly washed with deionized water and filtered to obtain Phellinus igniarius matrix residue; (2) Preparation of cellulose: The Phellinus igniarius matrix residue is bleached with alkali solution and bleaching solution at the same time. The reaction solution after bleaching is centrifuged and washed until the supernatant is neutral. The solid phase collected is cellulose; (3) Preparation of nanocellulose: Enzymatic hydrolysis of cellulose followed by centrifugal separation, acid flocculation, centrifugal washing, and freeze drying to obtain nanocellulose; The alkali solution in step (2) is a 5-10wt% sodium hydroxide aqueous solution, the bleaching solution is a 10-15wt% hydrogen peroxide aqueous solution, the volume ratio of the bleaching solution to the alkali solution is (1-3):1, the total volume of the bleaching solution and the alkali solution is 100-180mL, and the material-liquid ratio is 1:(50-200); the bleaching treatment temperature is 50-90°C, and the treatment time is 50-100min.

2. The method for preparing nanocellulose using Phellinus linteus matrix as raw material according to claim 1, characterized in that: In step (1), the Phellinus igniarius matrix is ​​crushed and sieved, then soaked in deionized water for 10 to 24 hours and then repeatedly washed.

3. The method for preparing nanocellulose using Phellinus linteus matrix as raw material according to claim 1, characterized in that: In step (2), the centrifugal speed is 8000-12000 rpm, and the centrifugal time is 10-30 min; the obtained cellulose is stored at 4°C.

4. The method for preparing nanocellulose using Phellinus linteus matrix as raw material according to claim 1, characterized in that: The enzymatic hydrolysis method in step (3) is selected from one of the following methods: The first method is to use low-concentration cellulase to hydrolyze cellulose, wherein the low-concentration cellulase is 5-20u / mL, the enzymatic hydrolysis time is 40-120min, and after the enzymatic hydrolysis, the enzymatic hydrolyzate is placed in boiling water for 30min to inactivate the enzyme to obtain an enzymatic hydrolyzate; The second method is to use high-concentration cellulase to hydrolyze cellulose, wherein the high-concentration cellulase is 100-500u / mL, the hydrolysis time is 4-12h, and after the hydrolysis is completed, the hydrolyzate is placed in boiling water for 30min to inactivate the enzyme to obtain the hydrolyzate.

5. The method for preparing nanocellulose using Phellinus linteus matrix as raw material according to claim 1, characterized in that: Before the centrifugal separation in step (3), the pH of the enzymatic hydrolyzate needs to be adjusted to 8-10 using a sodium hydroxide aqueous solution, and the nanocellulose is separated by centrifugation after standing for 30-90 minutes.

6. The method for preparing nanocellulose using Phellinus linteus matrix as raw material according to claim 5, characterized in that: In step (3), the first supernatant is separated from the enzymatic hydrolyzate produced by using low-concentration cellulase at a speed of 500 to 1500 rpm for 5 to 20 minutes. A second supernatant is separated from an enzymatic hydrolyzate produced by using a low concentration of cellulase at a rotation speed of 3000-4500 rpm for 5-20 min; or a third supernatant is separated from an enzymatic hydrolyzate produced by using a high concentration of cellulase at a rotation speed of 500-1500 rpm for 5-20 min.

7. The method for preparing nanocellulose using Phellinus linteus matrix as raw material according to claim 6, characterized in that: The acid flocculation in step (3) is to adjust the pH of the supernatant to 1-3 using an aqueous hydrochloric acid solution, and add the solution dropwise while stirring until the supernatant becomes turbid and the nanocellulose begins to flocculate, and the flocculation is completed until all the nanocellulose is precipitated.

8. The method for preparing nanocellulose using Phellinus linteus matrix as raw material according to claim 7, characterized in that: The centrifugal washing in step (3) is carried out at a speed of 8000-12000 rpm for 5-20 min, the supernatant is discarded, and the washing is repeated by centrifugation with deionized water until the washing solution is neutral, the solid phase is collected, and freeze-dried to obtain purified nanocellulose.

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