CN103760132B - Chemical Pretreatment combine with FTIR analyze plant cell wall component method - Google Patents
Chemical Pretreatment combine with FTIR analyze plant cell wall component method Download PDFInfo
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Abstract
Chemical Pretreatment combines with FTIR the method analyzing plant cell wall, is utilize acidifying TGA to process plant cell wall to make cell wall material crush, and cleans free chemical reagent, then row FTIR detection and data analysis.The spectra values deducting untreated plant sample by the spectra values of chemical-treated plant sample can show the component spectra that chemical treatment is disclosed, carries out component qualification according to spectrum.The method overcome the difficult problem that some plant samples are difficult to grind, can effectively crush Plant cell wall material, can the organizational structure of actual response plant cell wall, adopt FTIR can disclose when observing during non-process observed less than component.It is particularly well-suited to observing such as the sample of fiber-like and lignifying class of Plant Secondary Materials wall material.
Description
One, technical field
The invention belongs to biochemical analysis field, for the application process of Fourier near infrared spectrum (FTIR), be specifically related to a kind of utilize Chemical Pretreatment plant cell wall sample after utilize the FTIR method analyzing wherein phenylpropyl alcohol alkyl compound component.
Two, background technology
Having a lot of plant product to derive from the cell wall of plant in nature, such as timber, fiber etc., its primary structure is secondary wall.Plant Secondary Materials wall main component have polysaccharide, glycoprotein and phenylpropyl alcohol alkyl compound (Somervilleeta1., 2004；Keegstraeta1., 2010), these component ratios, classification determine character and the product quality of cell wall.The phenylpropyl alcohol alkyl compound being formed with in plant cell wall cross-linked structure can protect the cellulose in cell wall not easily by chemistry and biodegradation (Grabbereta1.2004), can affect the growth of cell wall, intensity, morphogenesis and to the biological resistance (Boerjaneta1.2003 with abiotic stress；Bhuiyaneta1.2009).Therefore, analyzing type and the content of plant cell wall structural constituent, particularly phenylpropyl alcohol alkyl compound, the product quality tool formed for correctly evaluating plant cell wall is of great significance.
Fourier near infrared spectrum (FTIR) is a kind of method being usually used in and analyzing plant cell wall, has the following advantages: (1) can the component of simple and quick analysis plant cell wall；(2) sample size needed is little；(3) structure can be destroyed and differentiate macromole and function ingredients, and abundant structural constituent information is provided；(4) can the change that caused by the different factor (such as g and D process, variation, biological or abiotic stress etc.) of identification of cell wall fraction, and thus speculate the cross-linked structure of cell wall；(5) can combine with microscope and other analysis method identification of cell Different Organs and tissue heterogencity structure (McCannet., 2007；Alonso-Sim6neta1., 2011).
While it is true, the method also has its limitation.Such as, the cell wall sample analyzed need to wear into powdery, and some plant cell wall sample is difficult to powdered, such as Plant fiber's class sample, Typical Representative has cotton fiber, although cotton fiber only has the diameter (Kimeta1. of 11-22 μ tm, 2001), but its top layer is coated (Haiglereta1. by the primary wall of 0.2 μm, 2012), even if detecting with the sample of the produced similar powdery of cutting repeatedly, also affect the accuracy of its cotton fiber cell wall construction component detection.
The cell wall being difficult to crush is carried out physics crush, and make its component structure not suffer a loss, carry out FTIR afterwards again and analyze detection, be one of approach solving foregoing problems.This research group attempted pulverizer, cut, the method such as shred, make fiber sample reach state in small, broken bits as far as possible, not only waste time and energy, but observe with scanning electron microscope, fluorescence microscopy and have been found that the sample processed by these methods can not make secondary wall structure main in cell wall fully expose, adopt FTIR when analyzing, can not fully observe the component of plant cell wall necessary being.Additionally have employed again chemical method and process sample, such as the chemical substance treatment such as sulphuric acid, acetyl bromide, cause the destruction of cell-wall component all to a certain extent；And for example utilizing highly basic to carry out chemical treatment can only make the configuration of fiber change, and plant cell wall can not be made really to crush, and these all can affect the subsequent detection result of FTIR.
Correct process sample is the premise obtaining Correct Analysis result, there is presently no and utilizes chemical method that plant cell wall is carried out pretreatment to be combined with FTIR, reaches the report of phenylpropyl alcohol alkyl compound multi-component approach in announcement plant cell wall.Detection practice in need exploitation a kind of can to the analysis method of the plant cell wall component of not easily powdered fiber sample.Cell wall should fully be crushed by this method, makes again its component structure not be damaged.
Three, summary of the invention
It is an object of the invention to provide the process for chemically pretreating of a kind of observation of plant cell-wall component that can combine with FTIR, also it is FTIR and analyzes the expansion of cell wall method, make FTIR analyze plant cell wall component time, can be observed during non-process observed less than component.
The technical thought of the present invention is to utilize chemical treatment method that plant cell wall sample is carried out pretreatment, makes cell wall fracture broken but does not cause its component structure to destroy, to realize the real structure component of application FTIR analysis cell wall.
The present invention is the method analyzing plant cell wall component that Chemical Pretreatment combines with FTIR, adopts chemical method to process and makes cell wall material crush, cleans free chemical reagent and eliminate the impact on detection, carry out FTIR detection and data analysis afterwards again.Concretely comprise the following steps:
A. cleaned plant cell wall sample is placed in serum bottle, adds after 2MHCl and add TGA, fully screw bottle cap after mixing, be placed in 98 DEG C of water-baths light and slow concussion 4h；Taking out after having reacted, ice-cold 20min, reactant liquor goes to 50mL centrifuge tube, under room temperature, 12,000rpm centrifugal 15min, abandons supernatant.
B. precipitation 10mL distilled water cleans 3 times, dries to constant weight for 45 DEG C.
C. the pulverized specimen of drying to constant weight is laid in uniformly the diamond detection window of Fourier near infrared spectrometer NicoletiS10FTIR, records the meansigma methods at 4000-650cm-1 32 spectrum of spectral region interscan.
D. deduct the spectra values of untreated plant sample by the spectra values of chemical-treated plant sample and can show the component spectra that chemical treatment is disclosed, carry out component qualification according to spectrum.
The method is analyzed the technology of phenylpropyl alcohol alkyl compound content in plant cell wall by TGA method and is enlightened, TGA method make when acidity heats the process that the sulfydryl in TGA and the benzene hydroxyl in the phenylpropyl alcohol alkyl compound in cell wall form ester bond compound carry out observation analysis, find in this process owing to defining ester bond compound, make the chemical bond rupture being cross-linked with each other between plant cell wall, cause that plant cell wall fracture is broken, secondary wall exposes, when utilizing the method detection plant cell wall of FTIR afterwards, the component that can't detect when can detect that non-process.It is difficult to wear into powdery and cannot directly apply the restriction of FTIR method thus also overcoming Plant fiber's class sample.
In fourding history of the present invention, have selected be difficult to grind, the representative that cotton fiber is fiber-like sample of typical secondary wall structure that phenylpropyl alcohol alkyl compound content is very low；Have selected the Pinus sylvestnis var. mongolica Litv. branch that degree of lignification is higher, phenylpropyl alcohol alkyl compound content is higher is degree of lignification and the representative of hardness higher material, and the effectiveness of the inventive method has been checked.
First, acidifying TGA processes plant cell wall can make cell wall crush.TGA method is a kind of for analyzing the method for phenylpropyl alcohol alkyl compound content in plant cell wall, it is when acidity heats, the sulfydryl in TGA is made to form ester bond compound with the benzene hydroxyl in the phenylpropyl alcohol alkyl compound in cell wall, this compound dissolves in alkaline solution, thus reaching the purpose (Hatfieldeta1., 2005) of quantitative test.It has been investigated that, acidifying TGA processes and causes when plant cell wall forms ester bond compound that plant cell wall fracture is broken, and Plant fiber's class sample can be overcome to be difficult to powdered shortcoming.
With finding after repeatedly cutting the Sample Scan electron microscopic observation of like powder shape produced by cotton fiber that complete fiber is still high-visible (Fig. 1 is left), this sample obviously can affect the FTIR detection of its cell wall and analyze result.And showing as fibrous fracture after the cotton fiber sample after processing free from admixture by acidifying TGA method, clean, secondary wall exposes, and even the day growth wheel of fibrocyte secondary wall can distinguish (Fig. 1 right) dimly.The Pinus sylvestnis var. mongolica Litv. branch sample pulverized with electric crusher is strip and block (Fig. 2 is left) under scanning electron microscope, and the sample after processing by acidifying TGA method shows as bulk sample and is further separated into lamellar, the structure of cell wall is broken (Fig. 2 is right) further.Indicating the effectiveness that acidifying TGA method processes, the surface of sample cell wall construction fully can be exposed to test scanning window by it, thus ensure that the FTIR accuracy detected.
Second, this method eliminates TGA used and FTIR analyzes the impact of result.Near-infrared absorption spectrum is consulted and is shown (ThermoScientific, 2010), and the sulfydryl spectral absorption in TGA is very faint, and its spectral absorbance values is at 2560cm-1, generally without influence on the parsing of spectrum.Containing ketone group in TGA, saturated open ketone group absorption spectrum is interval at 1725-1705cm-1.Analysis result resolves, it is contemplated that this wavenumber range may be subject to the impact of free reagent.Therefore, experimental design includes the sample distilled water after by process and cleans three times, to remove the impact on experimental result of the free reagent.
3rd, through Chemical Pretreatment, make FTIR observe during non-process observed less than phenylpropyl alcohol alkyl compound component.The observation analysis of plant cell wall is successfully combined by this method by TGA chemical method pretreatment with FTIR, is effectively smashing on plant cell wall sample basis, is utilizing FTIR spectrum analysis to carry out component qualification, it is achieved that the purpose of the present invention.Wherein for cotton fiber, overcoming the problem that fiber sample is difficult to grind, the position combined at its sulfydryl is smashed, and observes thereby through FTIR and discloses the component of phenylpropyl alcohol alkyl compound in cell wall.To Pinus sylvestnis var. mongolica Litv. branch sample, although it is the sample pulverized that phenylpropyl alcohol alkyl compound content is higher, FTIR direct analysis is to obtain its component result, but through Chemical Pretreatment, make sample more broken, structural constituent containing hydroxy benzenes exposes more, it is shown that some non-chemical methods observed less than absorption crest, make FTIR analyze the component result obtained abundanter.
FTIR is utilized to have detected the cotton fiber sample of 10 Gossypium hirsutum L. (GossypiumhirsutumL.) kinds and 10 sea island cotton (GossypiumbarbadenseL.), obtain identical conclusion, have detected the sample of 3 independent Pinus sylvestnis var. mongolica Litv. branches and 3 independent cotton stalk samples simultaneously, also obtain identical conclusion, describe the repeatability of present invention application effect.
Four, accompanying drawing explanation
Cotton fiber precipitation (right side) comparison that Fig. 1 is the cotton fiber (left side) under scanning electron microscope and acidifying TGA processed is schemed.
Fig. 2 be under scanning electron microscope pulverize Pinus sylvestnis var. mongolica Litv. branch (left side) and acidifying TGA processed pulverizing Pinus sylvestnis var. mongolica Litv. branch precipitation (right side) compare scheme.
Fig. 3 is the FTIR spectrum figure of cotton fiber sample, and wherein Gh_0 represents untreated cotton fiber, and Gh_b represents acidifying TGA and processes and cleaned cotton fiber precipitation, and Gh_a represents and utilizes alkaline solution to extract the remaining residue of phenylpropyl alcohol alkyl compound.
Fig. 4 deducts, with the absorption spectrum of chemical-treated cotton fiber, the FTIR spectrum figure that the absorption spectrum of untreated cotton fiber is formed.
Fig. 5 is the FTIR spectrum figure of Pinus sylvestnis var. mongolica Litv. branch sample, wherein Pt_0. represents the Pinus sylvestnis var. mongolica Litv. branch pulverized, Pt_b represents acidifying TGA and processes and cleaned Pinus sylvestnis var. mongolica Litv. branch precipitation, and Pt_a represents the residue utilizing alkaline solution to extract phenylpropyl alcohol alkyl compound residue Pinus sylvestnis var. mongolica Litv. branch.
Fig. 6 deducts, with the absorption spectrum of chemical-treated Pinus sylvestnis var. mongolica Litv. branch, the FTIR spectrum figure that the absorption spectrum of untreated Pinus sylvestnis var. mongolica Litv. branch is formed.
Five, detailed description of the invention
(1) detection of embodiment 1------cotton fiber is analyzed
1. instrument and reagent
Fourier near infrared spectrometer (NicoletiS10FTIR)；DKZ-2 type electric heating constant temperature concussion tank (the upper grand experimental facilities company limited of Nereid)；CR20B2 type High speed refrigerated centrifuge (Hitachi, Ltd)；EYELANDO-700 constant temperature thermostatic drying chamber (Shanghai)；Small-sized electric pulverizer.
TGA (Sigma-Aldrich)；Tris-HCl (Beijing Xin Jingke Bioisystech Co., Ltd)；Triton x-100 is analytical pure (Beijing Xin Jingke Bioisystech Co., Ltd)；NaCl is analytical pure (Xi'an chemical reagent factory)；Acetone is analytical pure (Zhengzhou Dezhong Chemical Reagent Factory)；HCl (Beijing Chemical Plant).
Homogeneous buffer (50mMTris-HCl, 1% (v/v) triton x-100,1M sodium chloride, pH8.3).
2. cotton fiber sample treatment and detection
(1) sample clean
First choose the non-cotton fiber blade being mixed in cotton fiber and plant the impurity such as shell fragment, weigh 1.0g cotton sample, with homogenization buffer solution for cleaning cotton fiber twice, then clean twice with 80% acetone respectively and pure acetone cleans once to avoid the soluble substance interference to analyzing.Clean every time all vortex, repeatedly extrude, finally cotton fiber is placed on the liquid extruding residual in metal clip Bulbus Allii device, 45 DEG C of dry for standby.
(2) chemical treatment of sample
1. weigh and clean and the cotton fiber 0.1g dried, be placed in 30mL serum bottle, after adding 5mL2MHCl, add 0.75mL TGA, fully screw bottle cap after mixing, be placed in 98 DEG C of water-baths light and slow concussion 4h；Take out cooled on ice 20min after having reacted, reactant liquor is gone to 50mL centrifuge tube, 12,000rpm centrifugal 15min under room temperature, abandon supernatant, precipitation is cleaned 3 times with 10mL distilled water respectively, 45 DEG C of dry for standby.
2. the fiber residue after phenylpropyl alcohol alkyl compound is extracted with alkaline solution
With acidifying TGA method processing procedure with above-mentioned 1., the precipitation obtained uses 5mL0.5M respectively
NaOH extracts phenylpropyl alcohol alkyl compound 2 times, and after 2MNaOH extracts 1 time, fiber residue cleans 3 times with 10mL distilled water respectively, 45 DEG C of dry for standby.
Repeatedly cut cleaned cotton fiber as there is no chemically treated comparison with sharp blade, extract the fiber residue after phenylpropyl alcohol alkyl compound with alkaline solution after processing with acidifying TGA and compare as second.
(3) the FTIR detection of sample
The pulverized specimen of 45 DEG C of drying to constant weight is laid in uniformly the diamond detection window of Fourier near infrared spectrometer (NicoletiS10FTIR), rotational pressure knob is until the pressure of instruction is thus ensureing the sample light line dropout that make incidence unlikely with the close contact of test surfaces window, environmental condition is relative humidity 65 ± 2%, temperature is 24 ± 1 DEG C, the FTIR spectrum of sample is that the SmartiTRBasic record equipped by this instrument obtains, and records the meansigma methods at 4000-650crn-1 32 spectrum of spectral region interscan.The background scans of blank clean diamond must be done before making sample.The spectroscopic data of all acquisitions is used that the provisioned software OMNIC of NicoletiS10FTIR spectrogrph does baseline correction and homogenization processes, and then calculates spectral differences.The testing result spectrogram of sample is shown in Fig. 3, Fig. 4.The present embodiment spectroscopic data processes through Baseline wander and homogenization, and spectrogram data are from the meansigma methods (n=30) of 10 cotton varieties of independent analysis, each kind result in triplicate.
The fiber sample of 10 cotton varieties is analyzed respectively by FTIR method, including the cotton fiber (Fig. 3 repeatedly cut, Gh_0), cotton fiber precipitation (Fig. 3 that acidifying TGA processes and uses distilled water cleaned, Gh_b), acidifying TGA method utilizes alkaline solution to extract the remaining residue of phenylpropyl alcohol alkyl compound (Fig. 3, Gh_a) after processing.Analyze by contrast, untreated fiber and extracted the fiber of phenylpropyl alcohol alkyl compound at 1500-1100cm-1The approximate overlap of wavenumber range, and the cotton fiber processed is deposited in 1730cm-1, 1150-1350cm-1Wavenumber range is significantly higher than untreated cotton fiber.Illustrate that primary wall is coated the accuracy that have impact on the detection of cotton fiber cell wall construction component, also illustrate that the degree of depth of FTIR Scanning Detction cell wall is limited.The absorption spectrum deducting untreated cotton fiber with the absorption spectrum of chemical-treated cotton fiber can show the component spectra (Fig. 4) that chemical treatment is disclosed.
The corresponding relation of absworption peak and component, 1730cm in plant cell wall component result is analyzed according to existing document FTIR-1It is accredited as phenolic ester (SilversteinandWebster, 1998), additionally the sulfydryl from the know-why aspect TGA of this method forms ester bond compound with the benzene hydroxyl in the phenylpropyl alcohol alkyl compound in cell wall, and second comparison in extract after residue this crest disappear (Fig. 3, Gh_a), the existence having demonstrate,proved phenol ester is also reflected further.1270cm-1Being accredited as monomer 4-hydroxy-3-methoxycinnamic alcohol (Ranaeta1., 2010), this crest can with 1730cm-1Echo mutually, without being processed the impact of reagent.At 1150-1350cm-1Wavenumber range also has the unidentified absworption peak that some chemical treatments are disclosed, provides clue for analyzing its component of announcement further.Observation indicate that, this chemical treatment method can effectively smash fiber sample, overcome the problem that fiber sample is difficult to grind, the position combined according to its sulfydryl benzene hydroxyl in cell wall, also it is that it smashes the position of sample, is so conducive to FTIR to observe and discloses the component of phenylpropyl alcohol alkyl compound in cell wall.
(2) detection of embodiment 2------Pinus sylvestnis var. mongolica Litv. branch is analyzed
The present embodiment instrument and material medicine are identical with embodiment 1.
(1) cleaning of the preparation of sample and soluble substance
The Ramulus Pini bar blade of dry Pinus sylvestnis var. mongolica Litv. is scraped debark, the marrow of cambium layer and inside, only leave xylem, and be cut into segment, pulverize with small-sized electric pulverizer, the cleaning process of soluble substance is with the cleaning of fiber-like sample.The solution every time cleaned all with centrifuge at room temperature 12,000rpm centrifugal 15min, adds cleanout fluid and is carried out after abandoning supernatant.Thoroughly rear 45 DEG C of dry for standby are cleaned.
(2) chemical treatment of sample
The chemical treatment of Pinus sylvestnis var. mongolica Litv. branch sample is with the chemical treatment of cotton fiber sample in embodiment 1.
By the Pinus sylvestnis var. mongolica Litv. branch powder of the cleaned soluble substance pulverized as there is no chemically treated comparison, extract the Pinus sylvestnis var. mongolica Litv. bar residue after phenylpropyl alcohol alkyl compound with alkaline solution after processing by acidifying TGA method and compare as second.
(3) the FTIR detection of sample
The FTIR detecting step of sample is with the detection of embodiment 1 cotton fiber class sample.Testing result spectrogram is shown in Fig. 5, Fig. 6.
Fig. 5 is the FTIR spectrum figure of Pinus sylvestnis var. mongolica Litv. branch sample, wherein Pt_0 is the Pinus sylvestnis var. mongolica Litv. branch pulverized, Pt_b is that acidifying TGA processes and cleaned Pinus sylvestnis var. mongolica Litv. branch precipitation, and Pt_a is the residue utilizing alkaline solution to extract phenylpropyl alcohol alkyl compound residue Pinus sylvestnis var. mongolica Litv. branch.
Fig. 6 deducts, with the absorption spectrum of chemical-treated Pinus sylvestnis var. mongolica Litv. branch, the FTIR spectrum figure that the absorption spectrum of untreated Pinus sylvestnis var. mongolica Litv. branch is formed.
Pinus sylvestnis var. mongolica Litv. branch phenylpropyl alcohol alkyl compound content is higher, and the Pinus sylvestnis var. mongolica Litv. branch of pulverizing is at 1500-1100cm-1Wavenumber range has shown more absworption peak, the Pinus sylvestnis var. mongolica Litv. branch sample that acidifying TGA processed shows more rich absorption crest, and extracted crest relevant to phenylpropyl alcohol alkyl compound in the Ramulus Pini bar residue spectrum of phenylpropyl alcohol alkyl compound and disappear, it was demonstrated that these wavenumber range are relevant to phenylpropyl alcohol alkyl compound.The absorption spectrum deducting untreated Pinus sylvestnis var. mongolica Litv. branch with the absorption spectrum of chemical-treated Pinus sylvestnis var. mongolica Litv. branch can show the component spectra that chemical treatment is disclosed.The present embodiment spectroscopic data processes through Baseline wander and homogenization.Spectroscopic data is from the meansigma methods (n=9) of the sample of 3 Pinus sylvestnis var. mongolica Litv. branches of independent analysis, each sample result in triplicate.
The corresponding relation of absworption peak and component, 1730cm in plant cell wall component result is analyzed according to existing document FTIR-1It is accredited as phenolic ester；1510cm-1It is accredited as lignin；1465、1270、1140cm-1It is accredited as monomer 4-hydroxy-3-methoxycinnamic alcohol, 1330cm-1Be accredited as monomer sinapinic alcohol (SilversteinandWebster, 1998；Ranaeta1., 2010).
The corresponding relation list of references catalog of absworption peak and component
Claims (1)
1. Chemical Pretreatment combines with FTIR the method analyzing plant cell wall component, it is characterized in that adopting chemical method to process makes cell wall material crush, and cleans free chemical reagent and eliminates the impact on detection, carries out FTIR detection and data analysis afterwards again；It concretely comprises the following steps:
A. cleaned plant cell wall sample is placed in serum bottle, adds after 2MHCl and add TGA, fully screw bottle cap after mixing, be placed in 98 DEG C of water-baths light and slow concussion 4h；Taking out after having reacted, ice-cold 20min, reactant liquor goes to 50mL centrifuge tube, under room temperature, 12,000rpm centrifugal 15min, abandons supernatant；
B. precipitation 10mL distilled water cleans 3 times, dries to constant weight for 45 DEG C；
C., pulverized specimen after drying is laid in the diamond detection window of Fourier near infrared spectrometer NicoletiS10FTIR uniformly, and record is at 4000-650cm-1The meansigma methods of 32 spectrum of spectral region interscan；
D. deduct the spectra values of untreated plant sample by the spectra values of chemical-treated plant sample and can show the component spectra that chemical treatment is disclosed, carry out component qualification according to spectrum.
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| CN103364516A (en) * | 2013-06-28 | 2013-10-23 | 新疆农业科学院核技术生物技术研究所 | Detection method of phenylpropane structure monomer contained in cotton fiber |
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Non-Patent Citations (3)
| Title |
|---|
| Elicitation of Lignin Biosynthesis and Isoperoxidase Activity by Pectic Fragments in Suspension Cultures of Castor Bean;Robert J. Bruce and Charles A. West;《Plant Physiol》;19891031;全文 * |
| Molecular and Biochemical Evidence for Phenylpropanoid Synthesis and Presence of Wall-linked Phenolics in Cotton Fibers;Ling Fan et，al.;《Journal of Integrative Plant Biology》;20091231;全文 * |
| 不同浓度NaOH 处理对棉花纤维的影响;胡文冉等;《华北农学报》;20111231;全文 * |
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