CN107365377B - Preparation method of hemp cellulose hydrolysate conjugate protein for cultural relic detection - Google Patents
Preparation method of hemp cellulose hydrolysate conjugate protein for cultural relic detection Download PDFInfo
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- 229920002678 cellulose Polymers 0.000 title claims abstract description 22
- 239000001913 cellulose Substances 0.000 title claims abstract description 22
- 238000001514 detection method Methods 0.000 title claims abstract description 14
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 14
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 14
- 244000025254 Cannabis sativa Species 0.000 title claims abstract description 13
- 235000012766 Cannabis sativa ssp. sativa var. sativa Nutrition 0.000 title claims abstract description 13
- 235000012765 Cannabis sativa ssp. sativa var. spontanea Nutrition 0.000 title claims abstract description 13
- 235000009120 camo Nutrition 0.000 title claims abstract description 13
- 235000005607 chanvre indien Nutrition 0.000 title claims abstract description 13
- 239000011487 hemp Substances 0.000 title claims abstract description 13
- 239000000413 hydrolysate Substances 0.000 title claims abstract description 12
- 238000002360 preparation method Methods 0.000 title claims abstract description 7
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims abstract description 46
- 239000008367 deionised water Substances 0.000 claims abstract description 36
- 229910021641 deionized water Inorganic materials 0.000 claims abstract description 36
- UHOVQNZJYSORNB-UHFFFAOYSA-N Benzene Chemical compound C1=CC=CC=C1 UHOVQNZJYSORNB-UHFFFAOYSA-N 0.000 claims abstract description 33
- 238000000108 ultra-filtration Methods 0.000 claims abstract description 30
- 108090000790 Enzymes Proteins 0.000 claims abstract description 26
- 102000004190 Enzymes Human genes 0.000 claims abstract description 26
- 239000000843 powder Substances 0.000 claims abstract description 26
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims abstract description 22
- 238000001035 drying Methods 0.000 claims abstract description 18
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 claims abstract description 15
- 239000008103 glucose Substances 0.000 claims abstract description 15
- 229940092253 ovalbumin Drugs 0.000 claims abstract description 14
- 239000000706 filtrate Substances 0.000 claims abstract description 13
- 108010047754 beta-Glucosidase Proteins 0.000 claims abstract description 11
- 102000006995 beta-Glucosidase Human genes 0.000 claims abstract description 11
- 108010058846 Ovalbumin Proteins 0.000 claims abstract description 8
- IOLCXVTUBQKXJR-UHFFFAOYSA-M potassium bromide Chemical compound [K+].[Br-] IOLCXVTUBQKXJR-UHFFFAOYSA-M 0.000 claims abstract description 7
- 238000005238 degreasing Methods 0.000 claims abstract description 3
- 239000000243 solution Substances 0.000 claims description 34
- 229940088598 enzyme Drugs 0.000 claims description 25
- 238000005406 washing Methods 0.000 claims description 25
- 239000002244 precipitate Substances 0.000 claims description 24
- 239000012528 membrane Substances 0.000 claims description 22
- 238000009777 vacuum freeze-drying Methods 0.000 claims description 20
- 238000003756 stirring Methods 0.000 claims description 18
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 claims description 15
- 238000000034 method Methods 0.000 claims description 14
- 238000002791 soaking Methods 0.000 claims description 14
- 108010059892 Cellulase Proteins 0.000 claims description 11
- 229940106157 cellulase Drugs 0.000 claims description 11
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 claims description 10
- UFHFLCQGNIYNRP-UHFFFAOYSA-N Hydrogen Chemical compound [H][H] UFHFLCQGNIYNRP-UHFFFAOYSA-N 0.000 claims description 10
- MHAJPDPJQMAIIY-UHFFFAOYSA-N Hydrogen peroxide Chemical compound OO MHAJPDPJQMAIIY-UHFFFAOYSA-N 0.000 claims description 10
- 238000006243 chemical reaction Methods 0.000 claims description 10
- 230000000694 effects Effects 0.000 claims description 10
- 238000010438 heat treatment Methods 0.000 claims description 10
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 claims description 10
- 239000007788 liquid Substances 0.000 claims description 9
- 238000000967 suction filtration Methods 0.000 claims description 9
- 238000011282 treatment Methods 0.000 claims description 8
- 238000000227 grinding Methods 0.000 claims description 7
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 claims description 5
- 238000001704 evaporation Methods 0.000 claims description 5
- 239000000835 fiber Substances 0.000 claims description 5
- 235000019253 formic acid Nutrition 0.000 claims description 5
- 238000004108 freeze drying Methods 0.000 claims description 5
- 239000011259 mixed solution Substances 0.000 claims description 5
- 238000002156 mixing Methods 0.000 claims description 5
- 238000005303 weighing Methods 0.000 claims description 5
- 239000003480 eluent Substances 0.000 claims description 4
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- 230000014759 maintenance of location Effects 0.000 claims description 2
- 239000013049 sediment Substances 0.000 claims 3
- 241000218236 Cannabis Species 0.000 claims 1
- 229920002488 Hemicellulose Polymers 0.000 abstract description 3
- 239000003513 alkali Substances 0.000 abstract description 3
- 238000005516 engineering process Methods 0.000 abstract description 3
- MJFMYEVFESUAQP-UHFFFAOYSA-N formic acid propan-2-one hydrate Chemical compound O.OC=O.CC(C)=O MJFMYEVFESUAQP-UHFFFAOYSA-N 0.000 abstract description 3
- 229920005610 lignin Polymers 0.000 abstract description 3
- 230000008014 freezing Effects 0.000 abstract 1
- 238000007710 freezing Methods 0.000 abstract 1
- 239000000126 substance Substances 0.000 abstract 1
- 229920002521 macromolecule Polymers 0.000 description 7
- 239000004753 textile Substances 0.000 description 6
- 239000000427 antigen Substances 0.000 description 4
- 102000036639 antigens Human genes 0.000 description 4
- 108091007433 antigens Proteins 0.000 description 4
- 239000000047 product Substances 0.000 description 4
- 241001465754 Metazoa Species 0.000 description 3
- 239000004744 fabric Substances 0.000 description 3
- 239000000463 material Substances 0.000 description 3
- 238000002965 ELISA Methods 0.000 description 2
- 108010022355 Fibroins Proteins 0.000 description 2
- 230000008878 coupling Effects 0.000 description 2
- 238000010168 coupling process Methods 0.000 description 2
- 238000005859 coupling reaction Methods 0.000 description 2
- 238000002347 injection Methods 0.000 description 2
- 239000007924 injection Substances 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000002994 raw material Substances 0.000 description 2
- 238000001262 western blot Methods 0.000 description 2
- 230000004075 alteration Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000001914 filtration Methods 0.000 description 1
- 230000001900 immune effect Effects 0.000 description 1
- 230000003053 immunization Effects 0.000 description 1
- 238000002649 immunization Methods 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 230000009870 specific binding Effects 0.000 description 1
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/76—Albumins
- C07K14/765—Serum albumin, e.g. HSA
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K19/00—Hybrid peptides, i.e. peptides covalently bound to nucleic acids, or non-covalently bound protein-protein complexes
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/02—Monosaccharides
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P19/00—Preparation of compounds containing saccharide radicals
- C12P19/14—Preparation of compounds containing saccharide radicals produced by the action of a carbohydrase (EC 3.2.x), e.g. by alpha-amylase, e.g. by cellulase, hemicellulase
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- C—CHEMISTRY; METALLURGY
- C13—SUGAR INDUSTRY
- C13K—SACCHARIDES OBTAINED FROM NATURAL SOURCES OR BY HYDROLYSIS OF NATURALLY OCCURRING DISACCHARIDES, OLIGOSACCHARIDES OR POLYSACCHARIDES
- C13K1/00—Glucose; Glucose-containing syrups
- C13K1/02—Glucose; Glucose-containing syrups obtained by saccharification of cellulosic materials
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Abstract
The invention relates to the field of cultural relics detection, and discloses a preparation method of a hemp cellulose hydrolysate coupled protein for cultural relics detection, wherein benzene and ethanol are used for degreasing hemp cellulose, a mixed system of acetone-formic acid-water is used for removing lignin, alkali liquor is used for removing hemicellulose, cellulose and β -glucosidase are used for carrying out enzymolysis on the obtained cellulose, the enzyme is recovered by using an ultrafiltration technology after the enzymolysis, filtrate is frozen and dried to obtain glucose powder, the glucose powder and ovalbumin are dissolved in deionized water and a potassium bromide solution and are placed in a constant temperature and humidity box, and then the glucose-ovalbumin coupled substance is obtained by freezing and drying.
Description
Technical Field
The invention relates to the field of cultural relic detection, in particular to a preparation method of a hemp cellulose hydrolysate conjugate protein for cultural relic detection.
Background
In recent years, China unearthes a large number of textile cultural relics, and the identification of the varieties of the textile cultural relics has great significance for the research of the historical culture of China. Several relatively precise and sensitive methods have been developed for the identification of silk fabrics, and these methods are represented by enzyme-linked immunosorbent assay, Western Blotting, and most of them are based on the basic immunological knowledge that specific binding occurs between antigen and corresponding antibody. Since silk contains a large amount of protein, the silk fibroin is used as a complete antigen to carry out immune injection on animals, antibodies capable of being specifically combined with the silk fibroin can be obtained through corresponding treatment, and whether the ancient textiles contain the silk or not can be detected by using the antibodies. However, the main component of the fibrilia is cellulose, and the fibrilia cannot be directly used as a complete antigen for animal immunization, so that the fibrilia in the ancient textile cultural relics is difficult to identify by using methods such as enzyme-linked immunosorbent assay, WesternBlotting and the like.
Disclosure of Invention
In order to solve the technical problems, the invention provides a preparation method of a hemp cellulose hydrolysate conjugate protein for cultural relic detection. The invention couples the glucose of enzymolysis product of the hemp cellulose and the biological macromolecule to form coupled macromolecule through a series of treatments. The invention couples the cellulose product with protein creatively, and provides feasibility for identifying fibrilia fabrics in ancient textile cultural relics.
The specific technical scheme of the invention is that the preparation method of the hemp cellulose hydrolysate conjugate protein for cultural relic detection comprises the following steps in g and m L:
1) grinding 9-11 g of fibrilia into powder, adding 45-55 m L benzene and 20-30 m L ethanol for soaking, degreasing the fiber, evaporating the mixed solution under reduced pressure to recover benzene and ethanol, washing the degreased fibrilia with deionized water, and drying to obtain fibrilia powder.
2) Taking 4-6g of dried fibrilia powder, adding 95-105 m L acetone and 45-55 m L deionized water, stirring and mixing uniformly, dropwise adding 8-12m L formic acid, soaking, centrifuging the solution, taking the bottom precipitate, continuously treating with the solution and centrifuging, repeating the operation for multiple times to obtain the final precipitate, washing with deionized water, adding 7-9wt% of sodium hydroxide of 180-220m L, heating in a water bath at 70-80 ℃ for 10-14h, performing suction filtration, and washing the precipitate with deionized water.
The invention uses the mixed system of acetone-formic acid-water to remove lignin basically and completely, and then uses alkali solution to remove hemicellulose so as to improve the purity of the obtained cellulose.
3) Taking the precipitate obtained in the step 2), adding 8-12wt% hydrogen peroxide solution, heating in 40-50 ℃ water bath for 4-6h, filtering, washing the precipitate with deionized water, and drying to obtain cellulose powder.
4) Taking 2-3g of cellulose powder, adding 70-80m L deionized water, weighing 0.4-0.6 g of cellulase and 0.1-0.3 g of β -glucosidase, adding into the solution, adjusting the pH to 4-5, and carrying out enzymolysis reaction for 7-9 h.
The invention can accelerate enzymolysis speed, shorten process flow, improve enzymolysis degree and increase yield by using the compound enzyme to treat cellulose.
5) And after the enzymolysis reaction is finished, performing ultrafiltration on the enzymolysis liquid by using an ultrafiltration membrane, stirring and stirring the filtrate, collecting the filtrate, and performing vacuum freeze drying to obtain glucose powder.
6) And (3) taking down the ultrafiltration membrane, washing the ultrafiltration membrane to elute the enzyme on the membrane, and carrying out vacuum freeze drying on the eluent to obtain the recovered enzyme.
The invention utilizes ultrafiltration technology to recover the enzyme, the activity of the recovered enzyme is more than 92 percent, and the production cost is saved.
7) Dissolving 0.15-0.25 g of ovalbumin and 0.8-1.2 g of glucose powder in 18-22m L deionized water, uniformly stirring, performing vacuum freeze drying, dissolving the powder obtained after freeze drying in 25-35 m L saturated potassium bromide solution, and placing in a constant temperature and humidity box with the relative humidity of 60-70% at 55-65 ℃ for 6-8 days.
By the method, the enzymolysis product glucose of cellulose and ovalbumin can be coupled and produced into new coupled macromolecules, and the obtained coupled macromolecules are stable.
8) And (3) carrying out vacuum freeze drying on the solution obtained in the step 7) to finally obtain the glucose-ovalbumin conjugate.
Preferably, in the step 1), the grinding time is 3-5min, the soaking time is 6-10h, and the drying temperature is 45-55 ℃.
Preferably, in the step 2), the soaking time is 1.5-2.5h, and the centrifugation rate is 900-1100 r/min.
Preferably, in the step 3), the mass ratio of the precipitate to the feed liquid of the hydrogen peroxide solution is 1:25-35, and the drying temperature is 45-55 ℃.
Preferably, in the step 4), the enzyme activities of the cellulase and the β -glucosidase are 10000 u/g.
Preferably, in the step 5), the retention amount of the ultrafiltration membrane is 30kDa, the temperature during ultrafiltration is controlled to be 18-22 ℃, the pressure is 0.15-0.25MPa, and the stirring speed of the filtrate is 180-.
Compared with the prior art, the invention has the beneficial effects that:
1. the method provided by the invention can be used for coupling and producing new coupled macromolecules by coupling the enzymolysis products of cellulose, namely glucose and ovalbumin, and the obtained coupled macromolecules are relatively stable, and the coupled macromolecules are taken as complete antigens to carry out immune injection on animals, so that corresponding specific antibodies can be obtained, and feasibility is provided for identifying hemp fabrics in ancient textile cultural relics.
2. The invention uses the mixed system of acetone-formic acid-water to remove lignin basically and completely, and then uses alkali solution to remove hemicellulose so as to improve the purity of the obtained cellulose.
3. The method can accelerate the enzymolysis by using the complex enzyme to treat the cellulose, shorten the experimental process, improve the enzymolysis degree and increase the yield, and then recover the enzyme by using the ultrafiltration technology, so that the activity of the recovered enzyme is more than 92 percent, and the production cost is saved.
Detailed Description
The present invention will be further described with reference to the following examples.
Example 1
1) Grinding 10 g of fibrilia for 3 minutes, placing the fibrilia in a beaker, adding 45 m of L benzene and 20m of L ethanol, soaking for 6 hours to degrease the fibers, performing reduced pressure evaporation on the mixed solution after the treatment is finished to recover the benzene and the ethanol, washing the degreased fibrilia with deionized water for 5 times, and drying in an oven at 50 ℃.
2) Taking 5 g of dried fibrilia powder, adding 100 m L acetone and 50 m L deionized water, stirring and mixing uniformly, then dropwise adding 10 m L formic acid, soaking for 2 h, centrifuging the solution at the rotating speed of 1000 r/min, taking the bottom precipitate, continuously treating with the solution and centrifuging, repeating the operation for 4 times to obtain the final precipitate, washing with deionized water for 5 times, adding 200 m L of 7% sodium hydroxide, heating in a 75 ℃ water bath for 12 h, then carrying out suction filtration, and washing the precipitate with deionized water for 5 times.
3. Taking the finally obtained precipitate in the step 2, adding 8% hydrogen peroxide with the material liquid mass ratio of 1:30, heating in a water bath at 45 ℃ for 5 hours, then carrying out suction filtration, washing the precipitate with deionized water for 5 times, and drying in a drying oven at 50 ℃ to obtain cellulose powder.
4) And (3) adding 75 m of L deionized water into 2.5 g of the cellulose powder obtained in the step (3), accurately weighing 0.4 g of cellulase with the enzyme activity of 10000 u/g and 0.1 g of β -glucosidase with the enzyme activity of 10000 u/g, adding the cellulase and the β -glucosidase into the solution, adjusting the pH value to 4, and carrying out enzymolysis reaction for 8 hours.
5.) after the enzymolysis reaction is finished, carrying out ultrafiltration on the enzymolysis liquid by adopting an ultrafiltration membrane with the interception amount of 30kDa, controlling the temperature at 20 ℃ and the pressure at 0.2 Mpa during ultrafiltration, stirring the filtrate at the rotating speed of 200 r/min, collecting the filtrate, and carrying out vacuum freeze drying to obtain glucose powder.
6.) removing the ultrafiltration membrane, washing the ultrafiltration membrane to elute the enzyme on the membrane, and performing vacuum freeze drying on the eluate to obtain the recovered enzyme.
7) And (3) dissolving 0.2 g of ovalbumin and 1 g of glucose powder obtained in the step (5) in 20m L of deionized water, uniformly stirring, carrying out vacuum freeze drying on the solution, dissolving the powder obtained after freeze drying in 30 m L of saturated potassium bromide solution, and placing the solution in a constant temperature and humidity box at 60 ℃ and with the relative humidity of 65% for 7 days.
8) And (4) carrying out vacuum freeze drying on the solution obtained in the step (7) to finally obtain the glucose-ovalbumin conjugate.
Example 2
1) Grinding 10 g of fibrilia for 4 minutes, placing the fibrilia in a beaker, adding 50 m of L benzene and 25m of L ethanol, soaking for 8 hours to degrease the fibers, performing reduced pressure evaporation on the mixed solution after the treatment is finished to recover the benzene and the ethanol, washing the degreased fibrilia with deionized water for 5 times, and drying in an oven at 50 ℃.
2) Taking 5 g of dried fibrilia powder, adding 100 m L acetone and 50 m L deionized water, stirring and mixing uniformly, then dropwise adding 10 m L formic acid, soaking for 2 h, centrifuging the solution at the rotating speed of 1000 r/min, taking the bottom precipitate, continuously treating with the solution and centrifuging, repeating the operation for 4 times to obtain the final precipitate, washing with deionized water for 5 times, adding 200 m L% sodium hydroxide, heating in a 75 ℃ water bath for 12 h, then carrying out suction filtration, and washing the precipitate with deionized water for 5 times.
3) And (3) taking the precipitate finally obtained in the step (2), adding 10% hydrogen peroxide with the material liquid mass ratio of 1:30, heating in a water bath at 45 ℃ for 5 hours, then carrying out suction filtration, washing the precipitate with deionized water for 5 times, and drying in a drying oven at 50 ℃ to obtain cellulose powder.
4) And (3) adding 75 m of L deionized water into 2.5 g of the cellulose powder obtained in the step (3), accurately weighing 0.5 g of cellulase with the enzyme activity of 10000 u/g and 0.2 g of β -glucosidase with the enzyme activity of 10000 u/g, adding the cellulase and the β -glucosidase into the solution, adjusting the pH value to 4.5, and carrying out enzymolysis reaction for 8 hours.
5) And after the enzymolysis reaction is finished, performing ultrafiltration on the enzymolysis liquid by adopting an ultrafiltration membrane with the cutoff amount of 30kDa, controlling the temperature at 20 ℃ and the pressure at 0.2 Mpa during ultrafiltration, stirring the filtrate at the rotating speed of 200 r/min, collecting the filtrate, and performing vacuum freeze drying to obtain glucose powder.
6) And (3) taking down the ultrafiltration membrane, washing the ultrafiltration membrane to elute the enzyme on the membrane, and carrying out vacuum freeze drying on the eluent to obtain the recovered enzyme.
7) And (3) dissolving 0.2 g of ovalbumin and 1 g of glucose powder obtained in the step (5) in 20m L of deionized water, uniformly stirring, carrying out vacuum freeze drying on the solution, dissolving the powder obtained after freeze drying in 30 m L of saturated potassium bromide solution, and placing the solution in a constant temperature and humidity box at 60 ℃ and with the relative humidity of 65% for 7 days.
8) And (4) carrying out vacuum freeze drying on the solution obtained in the step (7) to finally obtain the glucose-ovalbumin conjugate.
Example 3
1) Grinding 10 g of fibrilia for 5 minutes, placing the fibrilia in a beaker, adding 55 m L benzene and 30 m L ethanol, soaking for 10 hours to degrease the fiber, performing reduced pressure evaporation on the mixed solution after the treatment is finished to recover the benzene and the ethanol, washing the degreased fibrilia with deionized water for 5 times, and drying in an oven at 50 ℃.
2) Taking 5 g of dried fibrilia powder, adding 100 m L acetone and 50 m L deionized water, stirring and mixing uniformly, then dropwise adding 10 m L formic acid, soaking for 2 h, centrifuging the solution at the rotating speed of 1000 r/min, taking the bottom precipitate, continuously treating with the solution and centrifuging, repeating the operation for 4 times to obtain the final precipitate, washing with deionized water for 5 times, adding 200 m L% sodium hydroxide, heating in a 75 ℃ water bath for 12 h, then carrying out suction filtration, and washing the precipitate with deionized water for 5 times.
3) And (3) taking the precipitate finally obtained in the step (2), adding 12% hydrogen peroxide with the material liquid mass ratio of 1:30, heating in a water bath at 45 ℃ for 5 hours, then carrying out suction filtration, washing the precipitate with deionized water for 5 times, and drying in a drying oven at 50 ℃ to obtain cellulose powder.
4) And (3) adding 75 m L deionized water into 2.5 g of the cellulose powder obtained in the step (3), accurately weighing 0.6 g of cellulase with the enzyme activity of 10000 u/g and 0.3 g of β -glucosidase with the enzyme activity of 10000 u/g, adding the cellulase and the β -glucosidase into the solution, adjusting the pH value to 5, and performing enzymolysis reaction for 8 hours.
5) And after the enzymolysis reaction is finished, performing ultrafiltration on the enzymolysis liquid by adopting an ultrafiltration membrane with the cutoff amount of 30kDa, controlling the temperature at 20 ℃ and the pressure at 0.2 Mpa during ultrafiltration, stirring the filtrate at the rotating speed of 200 r/min, collecting the filtrate, and performing vacuum freeze drying to obtain glucose powder.
6) And (3) taking down the ultrafiltration membrane, washing the ultrafiltration membrane to elute the enzyme on the membrane, and carrying out vacuum freeze drying on the eluent to obtain the recovered enzyme.
7) And (3) dissolving 0.2 g of ovalbumin and 1 g of glucose powder obtained in the step (5) in 20m L of deionized water, uniformly stirring, carrying out vacuum freeze drying on the solution, dissolving the powder obtained after freeze drying in 30 m L of saturated potassium bromide solution, and placing the solution in a constant temperature and humidity box at 60 ℃ and with the relative humidity of 65% for 7 days.
8) And (4) carrying out vacuum freeze drying on the solution obtained in the step (7) to finally obtain the glucose-ovalbumin conjugate.
The raw materials and equipment used in the invention are common raw materials and equipment in the field if not specified; the methods used in the present invention are conventional in the art unless otherwise specified.
The above description is only a preferred embodiment of the present invention, and is not intended to limit the present invention, and all simple modifications, alterations and equivalents of the above embodiments according to the technical spirit of the present invention are still within the protection scope of the technical solution of the present invention.
Claims (6)
1. A preparation method of a hemp cellulose hydrolysate conjugate protein for cultural relic detection is characterized by comprising the following steps of (g and m L):
1) grinding 9-11 g of fibrilia into powder, adding 45-55 m L benzene and 20-30 m L ethanol for soaking, degreasing the fiber, performing reduced pressure evaporation on the mixed solution after the treatment to recover benzene and ethanol, washing the degreased fibrilia with deionized water, and drying to obtain fibrilia powder;
2) taking 4-6g of the dried fibrilia powder, adding 95-105 m L acetone and 45-55 m L deionized water, stirring and mixing uniformly, then dropwise adding 8-12m L formic acid, soaking, then carrying out centrifugal treatment on the solution, taking bottom sediment, continuously using the solution for treatment and centrifuging, repeating the operation for multiple times to obtain final sediment, washing with deionized water, adding 7-9wt% of sodium hydroxide of 180-220m L, heating in a water bath at 70-80 ℃ for 10-14h, then carrying out suction filtration, and washing the sediment with deionized water;
3) taking the precipitate obtained in the step 2), adding 8-12wt% hydrogen peroxide solution, heating in a water bath at 40-50 ℃ for 4-6h, then carrying out suction filtration, washing the precipitate with deionized water, and drying to obtain cellulose powder;
4) taking 2-3g of the cellulose powder obtained in the step 3), adding 70-80m L deionized water, weighing 0.4-0.6 g of cellulase and 0.1-0.3 g of β -glucosidase, adding into the solution, adjusting the pH to 4-5, and carrying out enzymolysis reaction for 7-9 h;
5) after the enzymolysis reaction is finished, performing ultrafiltration on the enzymolysis liquid by using an ultrafiltration membrane, stirring the filtrate, collecting the filtrate, and performing vacuum freeze drying to obtain glucose powder;
6) taking down the ultrafiltration membrane, washing the ultrafiltration membrane to elute the enzyme on the membrane, and carrying out vacuum freeze drying on the eluent to obtain the recovered enzyme;
7) dissolving 0.15-0.25 g of ovalbumin and 0.8-1.2 g of glucose powder obtained in the step 5) in 18-22m L deionized water, uniformly stirring, carrying out vacuum freeze drying, dissolving the powder obtained after freeze drying in 25-35 m L saturated potassium bromide solution, and placing in a constant temperature and humidity cabinet with the relative humidity of 60-70% for 6-8 days at 55-65 ℃;
8) and (3) carrying out vacuum freeze drying on the solution obtained in the step 7) to finally obtain the glucose-ovalbumin conjugate.
2. The method for preparing the hemp cellulose hydrolysate conjugated protein for cultural relic detection according to claim 1, wherein in the step 1), the grinding time is 3-5min, the soaking time is 6-10h, and the drying temperature is 45-55 ℃.
3. The method for preparing the conjugate protein of the cellulase hydrolysate of the cannabis for cultural relics detection as claimed in claim 1, wherein the soaking time in the step 2) is 1.5-2.5h, and the centrifugation rate is 900-1100 r/min.
4. The method for preparing the hemp cellulose hydrolysate conjugated protein for cultural relic detection according to claim 1, wherein in the step 3), the mass ratio of the precipitate to the hydrogen peroxide solution is 1:25-35, and the drying temperature is 45-55 ℃.
5. The method for preparing the hemp cellulose hydrolysate conjugate protein for cultural relic detection according to claim 1, wherein the enzyme activities of the cellulase and β -glucosidase in the step 4) are 10000 u/g.
6. The method for preparing the fibrilia enzyme hydrolysate conjugate protein for cultural relics detection as claimed in claim 1, wherein in the step 5), the retention amount of the ultrafiltration membrane is 30kDa, the temperature during ultrafiltration is controlled to be 18-22 ℃, the pressure is 0.15-0.25MPa, and the stirring rate of the filtrate is 180-220 r/min.
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