CN103868778B - The measuring method of herbaceous plant content of lignin - Google Patents
The measuring method of herbaceous plant content of lignin Download PDFInfo
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Abstract
The present invention relates to the measuring method of a kind of herbaceous plant content of lignin, the method comprises the following steps: (1) plant sample is dried, pulverizes, sieved, and obtains plant sample powder；(2) plant sample powder adds acetic acid, obtain the plant sample powder of fully softening；(3) it is centrifuged to obtain precipitate A；With acetic acid wash precipitate A；(4) alcohol-ether solution embathes precipitate A, is precipitated thing B；(5) sediment B is evaporated after vulcanization acid, left at room temperature after stirring；(6) add distilled water, after stirring, after cooling under boiling water bath, room temperature, sequentially add distilled water and barium chloride shakes up centrifugal, be precipitated thing C；(7), after precipitate C water rinses, add sulphuric acid, potassium dichromate successively, after reaction, be cooled to room temperature；(8) step (7) gained material is transferred in conical flask；(9) conical flask adds and after potassium iodide reacts, add starch indicator, and by sodium thiosulfate solution titrated to terminal；(10) blank assay；(11) calculate content of lignin.The present invention is easy, accurate.
Description
Technical field
The present invention relates to a kind of measuring method, particularly relate to the measuring method of herbaceous plant content of lignin.
Background technology
It is known that lignin is that in nature, content is only second to cellulosic second organic high molecular compound.Tracheary element that lignin is mainly distributed in plant xylem and fiber, prothenchyma (of wood), collenchymatous cell, particular type epidermis cell secondary wall in, three kinds of main components with cellulose, hemicellulose constitute plant skeleton, act primarily as mechanical support in plant, prevent the functions such as biodegradation, disease defence, conveying moisture.Lignin is to be polymerized by 3 class cinnamyl alcohols (single lignol): tonquinol (coumarlyalcohol), 4-hydroxy-3-methoxycinnamic alcohol (coniferlyalcohol) and sinapinic alcohol (sinapyaalcohol).Because lignin monomer is different, lignin can be divided into 3 kinds: the syringyl lignin (syringyllignin being polymerized by Syringa oblata Lindl. base oxide monomer, S-lignin), guaiacyl lignin (the guaiacyllignin being polymerized by guaiacyl oxide monomer, G-lignin) and the p-hydroxybenzene lignin (hydroxy-phenyl, H-lignin) that is polymerized by p-hydroxybenzene oxide monomer.
Different commercial production, Biomass Energy Utilization are had opposite impacts on by lignin.On the one hand, commercial production is had negative effect by lignin.In plant, lignin and cellulose, hemicellulose are tightly combined, and in the process of commercial production ethanol, can affect the transformation efficiency of cellulose, hemicellulose.The main component of the industrial wastes (black liquor) that lignin or paper industry produce in producing.On the other hand, lignin itself can carry out commercial production as the raw material of industry.Such as, with black liquor for the raw material of industry, it is synvaren by the lignin conversion in black liquor；Lignin is coprecipitated with latex, forms lignin rubber master batch for rubber industry, and lignin plays the effect of reinforcing agent wherein.Additionally, lignin has high heating value, it is possible to by chemical modification or modified be converted into reproducible bioenergy and resource (such as diesel oil, ethanol etc.)；Lignin is substantially nontoxic to human body and animal, and some lignin oligomer is likely to also have the effects such as anticancer, antitumor, can be widely used for food industry, to reduce the generation of digestive tract disease.
In plant, lignin always coexists with cellulose, hemicellulose, and cellulose and hemicellulose are all polysaccharide macromolecular compounds, in addition also has some oligosaccharide (oligosaccharide) and lignin to coexist.This is mainly due in the biosynthetic process of lignin, lignin monomer has a glycosylation process, form tonquinol glucoside, coniferin, sinapinic alcohol glucoside respectively, these monomers can synthesize at cell wall lignin and aggregate into lignin macromole on site, are therefore combined with saccharide on lignin macromole.But lignin is that physical property mixes or chemical bond is still uncertain with the Coexistence mode of these polysaccharide.There is the part-structure unit that experimental results demonstrate lignin can be chemically bound together with some glycosyl in hemicellulose at present, form lignin-saccharide complex, show that lignin and hemicellulose are chemical bond, therefore by just relatively difficult for the two separation.Additionally, can also have pyranoid form alditol acidic group, xylopyranose base, arabinofuranosidase glycosyl, Galactopyranosyl with the glycosyl of lignin condensation in plant.
Lignin is a kind of complexity, noncrystalline, three-dimensional netted macromolecular compound, it is different from the natural polymers such as protein, polysaccharide, nucleic acid and has well-regulated structure, can represent with chemical formula, the irregular structure of lignin, structural model can only be adopted to represent.Additionally, the structure of the lignin of different plant fiber materials is different.In coniferous species timber, the construction unit of lignin is based on guaiacyl, all the other are a small amount of p-hydroxyphenyl, there is also a small amount of syringyl lignin in the lignin of some Lignum seu Ramulus Cunninghamiae Lanceolatae mature woods (lignin in Lignum seu Ramulus Cunninghamiae Lanceolatae mature wood is all different from the lignin in juvenile wood with in amount in the structure of functional group).In deciduous species timber except there is guaiacyl construction unit, there is also more Syringa oblata Lindl. based structures unit.And the lignin in herbaceous plant is made up of guaiacyl, Syringa oblata Lindl. base and three kinds of construction units of p-hydroxybenzene, wherein guaiacyl construction unit is similar to broad leaf tree with the ratio of Syringa oblata Lindl. based structures unit, but contains more p-hydroxybenzene construction unit simultaneously.Owing to the labyrinth of lignin makes it be difficult to, by chemistry or biodegradation, thus result in the mensuration content of lignin from plant quantization decided at the higher level but not officially announced highly difficult.
At present, the assay method of lignin mainly has sulfuric acid process (Klason method), acid detergent fiber (AcidDetergentFiber, it is called for short ADF) method, acetyl bromide (AcetylBromide is called for short AB) method, uv-spectrophotometric (Ultraviolet is called for short UV) method etc..These methods are broadly divided into two classes: the first kind is non-invasive analytic process, including uv-spectrophotometric (UV) method, infrared spectrum (InfraredSpectroscopy, be called for short IRS) method, near infrared spectrum (Near-InfraredSpectroscopy, be called for short NIRS) method, NMR (Nuclear Magnetic Resonance) spectrum (NuclearMagneticResonanceSpectroscopy, be called for short NMRS) method.The advantage of all these non invasive methods is the content that can measure sample lignin when not changing sample chemistry of lignin structure, but these methods all have to rely on suitable " a lignin standard sample " and rectify an instrument to carry out the mensuration of lignin.Equations of The Second Kind is to rely on the method that gravimetric method is measured.Such method is divided into again direct measuring method and Indirect Determination.Wherein, direct measuring method mainly has 72%(v/v) sulphate method (Klason method), acid detergent fiber method (ADF method) etc.；Indirect Determination mainly has acetyl bromide method (AB method), Thioglycolic acid salt algoscopy etc..
Wherein, sulfuric acid process (Klason method) is a kind of traditional method measuring content of lignin, and principle is to utilize the non-wood quality part in concentrated sulphuric acid hydrolyzation sample, and remaining residue is lignin.The shortcoming of the method is inaccurate, because there being a small amount of lignin can be dissolved in concentrated sulphuric acid, and this method is suitable only for hardwood lignin (such as Fructus Persicae heart wood, birch, the lignin of the trees such as Buxus sinica (Rehd.et Wils.)) mensuration, be not suitable for cork lignin (such as Oak Tree, cork oak, the lignin of the seeds such as cork oak), draft lignin is (such as alfalfa, switchgrass, the lignin of the plants such as Semen Maydis), and the content of lignin of annual plant is (such as Semen sojae atricolor, Semen arachidis hypogaeae, the lignin of the plants such as Herba Setariae Viridis) mensuration, because the mensuration of lignin can be produced interference by indefinite protein and mineral in these plants.Acid detergent fiber method (ADF method) is mainly used in herbaceous plant or the mensuration of annual plant lignin, and what it was mainly measured is the content of sour insoluble lignin, and in the process measured, large change can occur the chemical constitution of lignin.Ultraviolet spectroscopy is a kind of non invasive method that lignin measures, and its principle is: carry out the content of lignin in quantitative assay sample with the characteristic absorption wavelength 280nm of lignin.Although this method is simple to operate, but the acquisition for measuring standard specimen is more difficult.Acetyl bromide method (AB method) mainly uses the glacial acetic acid solution of acetyl bromide to extract lignin, content of lignin is measured again with ultraviolet spectrophotometer, this method measurement result is all ideal compared with said method, but the reaction that the method relates to has the degraded of xylan, and catabolite also has absorption at wavelength 280nm place, the accuracy of meeting interference measurement result, and when measuring for ultraviolet spectrophotometer, the acquisition of standard specimen is also relatively difficult.
At present, the patented technology about lignin assay method is less, pertains only to a certain class or the lignin assay method of a certain plant, specific aim is relatively strong and general applicability is more weak.Such as the assay method of middle content of lignin " one grow tobacco " that: publication No. is CN103335974A, this invention is mainly used in tobacco component analysis field, it is specifically related to an assay method growing tobacco middle content of lignin, the method adopts and Nicotiana tabacum L. carries out pretreatment (using sodium hydroxide/urea liquid), make the protein in Nicotiana tabacum L., the materials such as water-soluble sugar and other ionized impurities remove from Nicotiana tabacum L., then sulfuric acid solution (low concentration) is used to process, after under cryogenic, cellulose in Nicotiana tabacum L. and hemicellulose are dissolved in sodium hydroxide/urea liquid, obtain sour insoluble lignin, again through calculating, obtain the content of sour insoluble lignin, adopt ultraviolet absorption spectroscopy to measure the content of the acid-soluble lignin of Nicotiana tabacum L. simultaneously, both are added the content that can obtain Nicotiana tabacum L. lignin.Publication No. is " in a kind of cotton fiber the determination method of content of lignin " of CN101105444A, this invention relates to the determination method of content of lignin in a kind of cotton fiber, primary operational is first choose totally by the impurity of cotton fiber, cleans soluble substance.Then digestion hydrolysis cotton fiber, dissolved lignin, the solution dilution after digestion hydrolysis, addition sodium hydroxide and oxammonium hydrochloride. are eliminated the impact of solution background, constant volume, stands, adopt ultraviolet spectrophotometer to measure absorbance at wavelength 280nm place.Publication No. is " in a kind of pear flesh the determination method of content of lignin " of CN102608054A, the determination method of content of lignin in a kind of pear flesh of this disclosure of the invention, its step include first to pear flesh to be detected after a series of chemical treatment, adopt ultraviolet spectrophotometer to measure absorbance at wavelength 280nm place, substitute into formula and calculate content of lignin.Above-mentioned patent just for a certain plant or plant fruit etc. other contain the mensuration of content of lignin of the tissue of lignin, organ, be not suitable in other plant or plant tissue organ the mensuration of lignin.Publication No. is that CN101799389A establishes the Cotton Gossypii lignin method for measuring mentioned in the method for content of lignin and fiber quality dependency relation in cotton fiber, mainly use concentrated sulphuric acid by after the material of other except lignin all dissolves in Cotton Gossypii, drying residual residue and ashing at 575 DEG C, the Ash weight weighed is Cotton Gossypii lignin measured value.Process relatively simpleization that vegetable material is processed by the method, the content of lignin error causing mensuration is excessive.Additionally, except, outside the Pass in above-mentioned several patents of invention and mensuration plant there being lignin method, being with or without other similar patents measuring lignin methods less, this illustrates that still having a lot of problem in lignin assay method needs further investigation.
Summary of the invention
The technical problem to be solved is to provide the measuring method of a kind of simplicity, accurately herbaceous plant content of lignin.
For solving the problems referred to above, the measuring method of herbaceous plant content of lignin of the present invention, comprise the following steps:
(1) the plant sample collected is dried under 80 DEG C of conditions 48 ~ 72h, adopts microphyte pulverizer to be pulverized by plant sample, cross 30 mesh sieves after pulverizing, collect plant sample powder；
(2) weigh plant sample powder described in 0.05 ~ 0.10g, and concrete numerical value is designated as n；Then described plant sample powder is put in 50mL centrifuge tube, add the acetum 10mL that mass concentration is 0.1%, place 20 ~ 30min after stirring with Glass rod, obtain the plant sample powder of fully softening；
(3) by the centrifuge tube cover lid of the plant sample powder of built-in described abundant softening, put in centrifuge, with the centrifugal 5min of the rotating speed of 3500rpm, respectively obtain supernatant A and precipitate A；Outwell described supernatant A, and clean described precipitate A with the acetum 5mL that mass concentration is 0.1%；
(4) embathe the precipitate A3 ~ 5min of described step (3) gained with alcohol-ether solution 5mL, respectively obtain supernatant B and sediment B；Discard described supernatant B；Repeat to embathe 3 times；
(5) the centrifuge tube of built-in described sediment B is put into when water temperature is 80 DEG C water-bath or directly heats until this centrifugal liquid in pipe is evaporated in cold water；Then in this centrifuge tube, add the sulfuric acid solution 3mL that mass concentration is 72%, stir with Glass rod, stand 16h at ambient temperature, make cellulose fully be dissolved in sulphuric acid；
(6) in the centrifuge tube of described step (5) gained, add 10mL distilled water, after stirring, it is placed into 5min in 95 ~ 100 DEG C of boiling water baths, cool down under room temperature after taking out this centrifuge tube, sequentially add 5mL distilled water and barium chloride solution 0.5mL that mass concentration is 10% and shake up, the centrifugal 5min when 4000rpm；Respectively obtain supernatant C and precipitate C；Discard described supernatant C；
(7) after described precipitate C distilled water flushing 2 ~ 3 times, being initially charged the sulfuric acid solution 10mL that mass concentration is 10%, add the potassium bichromate solution 10mL of 0.1mol/L, stir, then in 95 ~ 100 DEG C of boiling water baths, stirring reaction 15min is sufficiently complete to reaction；Centrifuge tube is taken out from boiling water bath, stands and wait that managing interior solution is cooled to room temperature；
(8) all substances in the centrifuge tube of described step (7) gained are transferred in 150mL conical flask, with 15 ~ 20mL distilled water, this centrifuge tube are cleaned 2 ~ 3 times, and cleanout fluid is transferred completely in described conical flask；
(9) in the solution containing residue of described step (8) gained, add the liquor kalii iodide 5mL that mass concentration is 20%, shake described conical flask and make it fully react；It is subsequently adding the starch indicator 1mL that mass concentration is 0.5%, by the sodium thiosulfate solution titrated configured, treats that solution colour becomes bright green, and in 30s, solution colour is titration end-point when not changing；Record the consumed hypo solution consumption of titration, and it is designated as b；
(10) blank assay: add mass concentration in 150mL conical flask and be sulfuric acid solution 10mL and the 0.1mol/L potassium bichromate solution 10mL of 10% and mix, with described step (9) in the sodium thiosulfate solution titrated that configured, the hypo solution volume consumed is designated as a, as blank result；
(11) it is calculated as follows content of lignin:
X=K(a-b)/(n*48)
In formula: 48 is 1molC11H12O4Be equivalent to the equivalents of sodium thiosulfate；
K is concentration of sodium thiosulfate, mol/L；
A is consumed sodium thiosulfate volume, mL by blank titration；
B is consumed sodium thiosulfate volume, mL by volumetric soiutions；
N is taken quality, g by sample.
Described step (4) in alcohol-ether solution refer to ethanol mix homogeneously with ether equal-volume after the mixed liquor of gained.
The present invention compared with prior art has the advantage that
1, the present invention uses the experimental provision and instrument and equipment that laboratory is conventional, there is no special special equipment, experimental drug is conventional general reagent, cheap, therefore, not only workload is little, method is simple, processing ease, and analyzes that result is correct, analyze speed, favorable reproducibility, and the present invention can effectively overcome the deficiency of prior art system, provide a kind of effective method for herbal fibre structure composition and quality evaluation.
2, the present invention is that herbal lignin measures and sets up a set of comparatively complete, easy, the technical system that quick, popularization is wide, is beneficial to carry out good forage (such as alfalfa, Trifolium repense, rye grass etc.) and carries out breed breeding and quality evaluation.Additionally, assessment and raising yield to some excellent biomass energy plant (such as switchgrass, Miscanthus etc.) herbal industrial production efficiency have important value, it is possible to popularization and application in each laboratory and related factories.
Detailed description of the invention
The measuring method of herbaceous plant content of lignin, comprises the following steps:
(1) the plant sample collected is dried under 80 DEG C of conditions 48 ~ 72h, adopts microphyte pulverizer to be pulverized by plant sample, cross 30 mesh sieves after pulverizing, collect plant sample powder.
(2) weigh 0.05 ~ 0.10g plant sample powder, and concrete numerical value is designated as n；Then plant sample powder is put in 50mL centrifuge tube, add the acetum 10mL that mass concentration is 0.1%, place 20 ~ 30min after stirring with Glass rod, obtain the plant sample powder of fully softening.
(3) by the centrifuge tube cover lid of the plant sample powder of built-in abundant softening, put in centrifuge, with the centrifugal 5min of the rotating speed of 3500rpm, respectively obtain supernatant A and precipitate A；Outwell supernatant A, and with the acetum 5mL washing and precipitating thing A that mass concentration is 0.1%.
(4) embathe the precipitate A3 ~ 5min of step (3) gained with alcohol-ether solution 5mL, respectively obtain supernatant B and sediment B；Abandoning supernatant B；Repeat to embathe 3 times.
Wherein: alcohol-ether solution refer to ethanol mix homogeneously with ether equal-volume after the mixed liquor of gained.
(5) the centrifuge tube of built-in sediment B is put into when water temperature is 80 DEG C water-bath or directly heats until this centrifugal liquid in pipe is evaporated in cold water；Then in this centrifuge tube, add the sulfuric acid solution 3mL that mass concentration is 72%, stir with Glass rod, stand 16h at ambient temperature, make cellulose fully be dissolved in sulphuric acid.
(6) in the centrifuge tube of step (5) gained, add 10mL distilled water, after stirring, it is placed into 5min in 95 ~ 100 DEG C of boiling water baths, cool down under room temperature after taking out this centrifuge tube, sequentially add 5mL distilled water and barium chloride solution 0.5mL that mass concentration is 10% and shake up, the centrifugal 5min when 4000rpm；Respectively obtain supernatant C and precipitate C；Abandoning supernatant C.
(7) precipitate C is with, after distilled water flushing 2 ~ 3 times, being initially charged the sulfuric acid solution 10mL that mass concentration is 10%, add the potassium bichromate solution 10mL of 0.1mol/L, stir, and then in 95 ~ 100 DEG C of boiling water baths, stirring reaction 15min is sufficiently complete to reaction；Centrifuge tube is taken out from boiling water bath, stands and wait that managing interior solution is cooled to room temperature.
(8) all substances in the centrifuge tube of step (7) gained are transferred in 150mL conical flask, with 15 ~ 20mL distilled water, this centrifuge tube are cleaned 2 ~ 3 times, and cleanout fluid is transferred completely in conical flask.
(9) adding the liquor kalii iodide 5mL that mass concentration is 20% in the solution containing residue of step (8) gained, shake conical flask makes it fully react；It is subsequently adding the starch indicator 1mL that mass concentration is 0.5%, by the sodium thiosulfate solution titrated configured, treats that solution colour becomes bright green, and in 30s, solution colour is titration end-point when not changing；Record the consumed hypo solution consumption of titration, and it is designated as b.
(10) blank assay: add mass concentration in 150mL conical flask and be sulfuric acid solution 10mL and the 0.1mol/L potassium bichromate solution 10mL of 10% and mix, with step (9) in the sodium thiosulfate solution titrated that configured, the hypo solution volume consumed is designated as a, as blank result；
(11) it is calculated as follows content of lignin:
X=K(a-b)/(n*48)
In formula: 48 is 1molC11H12O4Be equivalent to the equivalents of sodium thiosulfate；
K is concentration of sodium thiosulfate, mol/L；
A is consumed sodium thiosulfate volume, mL by blank titration；
B is consumed sodium thiosulfate volume, mL by volumetric soiutions；
N is taken quality, g by sample.
Measure Numerical examples such as table 1:
3 kind sample content of lignin testing result examples of table 1. alfalfa
Claims (1)
1. the measuring method of herbaceous plant content of lignin, comprises the following steps:
(1) the plant sample collected is dried under 80 DEG C of conditions 48 ~ 72h, adopts microphyte pulverizer to be pulverized by plant sample, cross 30 mesh sieves after pulverizing, collect plant sample powder；
(2) weigh plant sample powder described in 0.05 ~ 0.10g, and concrete numerical value is designated as n；Then described plant sample powder is put in 50mL centrifuge tube, add the acetum 10mL that mass concentration is 0.1%, place 20 ~ 30min after stirring with Glass rod, obtain the plant sample powder of fully softening；
(3) by the centrifuge tube cover lid of the plant sample powder of built-in described abundant softening, put in centrifuge, with the centrifugal 5min of the rotating speed of 3500rpm, respectively obtain supernatant A and precipitate A；Outwell described supernatant A, and clean described precipitate A with the acetum 5mL that mass concentration is 0.1%；
(4) embathe the precipitate A3 ~ 5min of described step (3) gained with alcohol-ether solution 5mL, respectively obtain supernatant B and sediment B；Discard described supernatant B；Repeat to embathe 3 times；Described alcohol-ether solution refer to ethanol mix homogeneously with ether equal-volume after the mixed liquor of gained；
(5) the centrifuge tube of built-in described sediment B is put into when water temperature is 80 DEG C water-bath or directly heats until this centrifugal liquid in pipe is evaporated in cold water；Then in this centrifuge tube, add the sulfuric acid solution 3mL that mass concentration is 72%, stir with Glass rod, stand 16h at ambient temperature, make cellulose fully be dissolved in sulphuric acid；
(6) in the centrifuge tube of described step (5) gained, add 10mL distilled water, after stirring, it is placed into 5min in 95 ~ 100 DEG C of boiling water baths, cool down under room temperature after taking out this centrifuge tube, sequentially add 5mL distilled water and barium chloride solution 0.5mL that mass concentration is 10% and shake up, the centrifugal 5min when 4000rpm；Respectively obtain supernatant C and precipitate C；Discard described supernatant C；
(7) after described precipitate C distilled water flushing 2 ~ 3 times, being initially charged the sulfuric acid solution 10mL that mass concentration is 10%, add the potassium bichromate solution 10mL of 0.1mol/L, stir, then in 95 ~ 100 DEG C of boiling water baths, stirring reaction 15min is sufficiently complete to reaction；Centrifuge tube is taken out from boiling water bath, stands and wait that managing interior solution is cooled to room temperature；
(8) all substances in the centrifuge tube of described step (7) gained are transferred in 150mL conical flask, with 15 ~ 20mL distilled water, this centrifuge tube are cleaned 2 ~ 3 times, and cleanout fluid is transferred completely in described conical flask；
(9) in the solution containing residue of described step (8) gained, add the liquor kalii iodide 5mL that mass concentration is 20%, shake described conical flask and make it fully react；It is subsequently adding the starch indicator 1mL that mass concentration is 0.5%, by the sodium thiosulfate solution titrated configured, treats that solution colour becomes bright green, and in 30s, solution colour is titration end-point when not changing；Record the consumed hypo solution consumption of titration, and it is designated as b；
(10) blank assay: add mass concentration in 150mL conical flask and be sulfuric acid solution 10mL and the 0.1mol/L potassium bichromate solution 10mL of 10% and mix, with described step (9) in the sodium thiosulfate solution titrated that configured, the hypo solution volume consumed is designated as a, as blank result；
(11) it is calculated as follows content of lignin:
X=K(a-b)/(n*48)
In formula: 48 is 1molC11H12O4Be equivalent to the equivalents of sodium thiosulfate；
K is concentration of sodium thiosulfate, mol/L；
A is consumed sodium thiosulfate volume, mL by blank titration；
B is consumed sodium thiosulfate volume, mL by volumetric soiutions；
N is taken quality, g by sample.
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| CN104237453A (en) * | 2014-09-30 | 2014-12-24 | 云南瑞升烟草技术（集团）有限公司 | Method for measuring content of cellulose in tobacco |
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| CN108872118B (en) * | 2017-10-13 | 2021-01-22 | 济宁明升新材料有限公司 | Method for detecting lignin content of papermaking pre-cooking liquid |
| CN108872119B (en) * | 2017-10-13 | 2021-01-22 | 济宁明升新材料有限公司 | Method for detecting lignin content in papermaking black liquor |
| CN109738610A (en) * | 2018-12-21 | 2019-05-10 | 广东省生态环境技术研究所 | A kind of method of Fast Evaluation lignocellulose castoff compost rotten degree |
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