Increase In Random Coil Research Articles

Insights into transglutaminase on structural and rheological properties of gels from adzuki bean protein pretreated by electric fields

The demand for protein is increasing as the global population continues to grow. The plant protein has been favored and plant-based food has become a research hotspot for healthy diet, environmental protection, animal ethics and other issues. Adzuki bean protein (ABP) is a quality source with complete amino acids, abundant essential amino acids and beanless flavour. ABP is rich in glutamine and lysine which are good substrates for transaminase (TGase) induced gelation. The pulsed electric field (PEF), alternating current EF (ACEF), direct current EF (DCEF) were applied to pretreat ABP aiming at improving the efficacy of TGase induced crosslinking. The results showed that the TGase decreased the free amino amount significantly and increased the molecular weight of ABP. The EF pretreatments significantly influenced the secondary and tertiary structures of ABP with a decrease in β-sheets, increase in random coils, β-turns, and α-helices, also decrease in fluorescence intensity with blueshift. The content of disulfide bond increased with the decrease of free sulfhydryl groups. The ionic bonds decreased and hydrophobic actions surpassed significantly hydrogen bonds. The hydrophobicity increased. The electric field intensity corresponded to variation of microstructure, texture and rheological behaviour of the ABP gel according to the type of the EF.

Modulation of antioxidant enzyme by light and heavy rare earth metals: A case study with catalase

The present study highlights the hazardous effect of heavy and light rare earth elements (REEs) on bovine liver catalase (BLC) using a combination of spectroscopic and computational methods. The presence of Praseodymium chloride (PrCl3) and Gadolinium chloride (GdCl3) resulted in a substantial reduction in catalytic efficiency of BLC by approximately 1.8 and 2.6 fold, respectively. The compromised activity was further accompanied by conformational rearrangements at the secondary and tertiary levels as evidenced by circular dichroism (CD) and fluorescence spectroscopy. These analyses revealed a significant decrease in α-helical content and a simultaneous increase in random coils, disrupting intramolecular hydrogen bonding. Furthermore, the zeta potential (ζ) of BLC demonstrated a reversal from negative to positive ζ values upon the addition of PrCl3 and GdCl3, indicating BLC-lanthanide complex formation. Isothermal titration calorimetry (ITC) supports spontaneous interaction with negative free energy favouring endothermic reaction. This was further supported by docking studies which revealed the binding of PrCl3 and GdCl3 within the active site of BLC thus interfering with the catalytic ability to degrade hydrogen peroxide (H2O2). Nevertheless, a significant decline in the melting temperature (Tm) of BLC was observed in the presence of lanthanides suggesting the thermal instability of the enzyme. Thus, a similar approach could be applied to evaluate the hazardous effects of lanthanides on structural and functional changes in other proteins or similar biomolecules.

The inhibitory effects of free and bound phenolics from Phyllanthus emblica Linn. on α-amylase: a comparison study.

Phyllanthus emblica Linn. (PE) is rich in polyphenols, which can be categorized into free and bound phenolics (PEFP and PEBP). This study evaluated the inhibitory effect of PEFB and PEBP on α-amylase for the first time. The mechanism of the inhibition effect of PEFP and PEBP on α-amylase was investigated by enzyme inhibition kinetics, multispectral analysis, thermodynamics, and molecular docking. Free and bound phenolics inhibited α-amylase activity effectively in a mixed type of inhibition. Fluorescence quenching and thermodynamic analyses showed that the binding of PEFP and PEBP to α-amylase occurred through a static quenching process (Kq = 6.94 × 10¹² and 5.74 × 10¹² L mol-1 s-1), which was accompanied by a redshift (λem from 343 to 347 nm), leading to a change in the microenvironment. This process was found to be a spontaneous exothermic reaction (ΔG < 0). Circular dichroism (CD) analysis confirms that the secondary structure of α-amylase was altered, in particular a decrease in α-helixes and an increase in random coils. Molecular docking studies showed that PEFP and PEBP interacted with α-amylase through hydrogen bonding and hydrophobic interactions. The present study provides valuable insights into the mechanism of action of PEFP and PEBP on α-amylase, which will provide a theoretical basis for their possible use as novel natural α-amylase inhibitors. © 2024 Society of Chemical Industry.

The inhibitory activity of Flos Sophorae Immaturus extract and its major flavonoid components on pancreatic lipase

Inhibition of pancreatic lipase (PL) is a strategy to prevent obesity. The inhibitory effects of Flos Sophorae Immaturus (FSI) extract and its main flavonoid components, rutin and quercetin, on PL were investigated. The contents of rutin and quercetin in FSI extract were 44.10 ± 1.33 % and 6.07 ± 1.62 %, respectively. The IC50 values of FSI extract, rutin and quercetin on PL were 322, 258 and 71 μg/mL, respectively. Rutin and quercetin inhibited PL in a reversible and noncompetitive manner. The combination of rutin and quercetin exhibited synergistic inhibitory effects at low concentration. The binding of rutin/quercetin with PL caused the fluorescence quenching of protein. Fluorescence titration showed the binding affinity of quercetin with PL protein was stronger than that of rutin. Circular dichroism analysis showed the binding changed the secondary structure of PL with an increase in random coil and a decrease in α-Helix and β-Sheet. Molecular docking revealed that rutin and quercetin could interact with the amino acid residues around the catalytic site through multiple secondary interactions. In vivo studies showed that FSI extract can reduce fat absorption and promote fecal fat excretion through inhibition of PL activity, and the effects were mainly due to rutin and quercetin.

Multi-layered structure and physicochemical properties of reconstituted meat-based products from minced fish by physical extrusion: Impact of extrusion strength

Multi-layered structure of reconstituted meat-based products from minced fish was formed by physical extrusion, followed by an investigation into the impact of extrusion strength on structural and physicochemical properties before and after frying. Under an appropriate pressure (3–9 kPa), the air within minced fish underwent enrichment and rearrangement to form a stratified phase, promoting the formation of multi-layered structure during frying. Conversely, the lower pressure (≤1.5 kPa) was insufficient for phase separation and directional rearrangement, while the higher pressure (≥15 kPa) would cause the stratified phase to flow out of food system. Moreover, by directly increasing water mobility and meat compactness, physical extrusion indirectly caused more water loss and stronger ionic bonds during frying, which was positively correlated with multi-layered structure. However, an excessive pressure caused an increase in random coil and hydrophobic interactions during frying, which was negatively correlated with multi-layered structure. In conclusion, appropriate physical extrusion strength promoted the formation of multi-layered structure.

Effect of transglutaminase crosslinking combined with lactic fermentation on the potential allergenicity and conformational structure of soy protein.

Food allergies are a growing concern worldwide, with soy proteins being important allergens that are widely used in various food products. This study investigated the potential of transglutaminase (TGase) and lactic acid bacteria (LAB) treatments to modify the allergenicity and structural properties of soy protein isolate (SPI), aiming to develop safer soy-based food products. Treatment with TGase, LAB or their combination significantly reduced the antibody reactivity of β-conglycinin and the immunoglobulin E (IgE) binding capacity of soy protein, indicating a decrease in allergenicity. TGase treatment led to the formation of high-molecular-weight aggregates, suggesting protein crosslinking, while LAB treatment resulted in partial protein hydrolysis. These structural changes were confirmed by Fourier transform infrared spectroscopy, which showed a decrease in β-sheet content and an increase in random coil and β-turn contents. In addition, changes in intrinsic fluorescence and ultraviolet spectroscopy were also observed. The alterations in protein interaction and the reduction in free sulfhydryl groups highlighted the extensive structural modifications induced by these treatments. The synergistic application of TGase and LAB treatments effectively reduced the allergenicity of SPI through significant structural modifications. This approach not only diminished antibody reactivity of β-conglycinin and IgE binding capacity of soy protein but also altered the protein's primary, secondary and tertiary structures, suggesting a comprehensive alteration of SPI's allergenic potential. These findings provide a promising strategy for mitigating food allergy concerns and lay the foundation for future research on food-processing techniques aimed at allergen reduction. © 2024 Society of Chemical Industry.

Enhancing the stability of zein pickering foams via hydrophilic reassembly with sugar alcohols and glycosides: A structural and molecular investigation

Sucralose (Suc), maltitol (Mal), mannitol (Man), and stevioside (Ste) — a group of alcohol-soluble sugar derivatives with different contents and molecular configurations of hydrophilic groups — were complexed with deaminated zein to significantly enhance the stability of zein-based Pickering foams and develop new composite coacervates to replace animal protein-based foams. Compared with Suc, Mal, and Man, Ste induced better hydrophilic reassembly in zein by forming stronger van der Waals forces and hydrogen bonds, leading to optimum foamability (+160.66%) and foam stability (+17.11%). Moreover, it increased the solubility (+0.07 mg/mL) and decreased the surface hydrophobicity index (−24.88) of zein. These enhancements could primarily be attributed to alterations in the aggregation conformation and hydrophobic interactions within Ste/ZN. Subsequently, SEM and CLSM confirmed that due to the amphiphilic structure of Ste, dispersed zein micelles could aggregate into complex stacking structures. The increase in random coils (+2.48%) and β-sheets (+2.93%) indicated the enhanced flexibility of the zein chain, thereby facilitating zein adsorption and unfolding at the air-water interface. Moreover, molecular simulation demonstrated that an average of 3.1 hydrogen bonds were formed between zein and Ste, and the average binding free energy was −14.20 kcal/mol. These findings provide novel evidence and theoretical guidance for the development of highly stable plant protein-based food foams. The enhancement of Pickering foam stability holds significant implications for the storage and development of high-performance food foams.

Alcalase-hydrolyzed insoluble soybean meal hydrolysate aggregates: Structure, bioactivity, function properties, and influences on the stability of oil-in-water emulsions

We studied the influences of hydrolysis time on the structure, functional properties, and emulsion stability of insoluble soybean meal hydrolysate aggregates (ISMHAs). We assume that the ISMHAs produced by soybean meal can be used as emulsifiers to prepare stable emulsions. The molecular weights of these ISMHAs were below 53 kDa. After hydrolysis, a decrease in α-helices and an increase in random coils indicated that the soybean meal proteins were unfolding. Moreover, the fluorescence intensity, UV absorption, and surface hydrophobicity of ISMHAs increased. These results would contribute to their antioxidant activity and functional properties. Additionally, the 90-min ISMHA sample exhibited the highest ABTS+• scavenging activity (80.02 ± 4.55 %), foaming stability (52.92 ± 8.06 %), and emulsifying properties (emulsifying activity index of 97.09 m2/g; emulsifying stability index of 371.47 min). The 90-min ISMHA emulsion exhibited the smallest particle size and excellent storage stability. Soybean meal peptide by-product emulsifier has potential for sustainable application.

Improvement of protein structural and functional properties of indica-japonica hybrid rice by radio frequency treatment

The goal of this work was to evaluate the effect of radio frequency combined with conventional hot air drying (RF-HAD) on the drying efficiency, protein structure and functional properties of indica-japonica hybrid rice. The results showed that RF-HAD significantly improved the drying efficiency. RF treatment did not affect the protein profile. However, secondary changes were observed in protein isolates with an increase in random coil and β-turn structures and a decrease in β-sheets and α-helices. The changed intrinsic fluorescence indicated alterations in tertiary structure. RF treatment noticeably improved the peak temperature and end completion temperature while decreasing the enthalpy of the protein. In addition, the functional properties in terms of solubility, oil holding capacity and emulsifying properties of protein isolates were improved after RF treatment. Thus, RF combined with conventional drying can be applied as a novel drying technology to enhance the nutritional qualities of rice.

Effect of polysaccharides on conformational changes and functional properties of protein-polyphenol binary complexes: A comparative study

This study aimed to investigate the effect of different polysaccharides on the binding behavior and functional properties of soybean protein isolate (SPI)-quercetin (Que) complex. The binding behavior was assessed using multi-spectral technique with the Stern-Volmer equation, which confirmed the presence of static fluorescence quenching in Que and SPI. The addition of sodium alginate (SA) resulted in a reduction of the binding affinity between SPI and Que, while dextran (DX) exhibited some promoting effect. A slight blue shift was observed in amide I and amide II bands, indicating the presence of hydrophobic and electrostatic interactions. Circular dichroism spectra revealed the ordered structures transformed into a more disordered state when polysaccharides were added, leading to an increase in random coils (SA: 18.5 %, DX: 15.4 %). Docking and dynamic simulations demonstrated that SA displayed greater stability within the hydrophobic compartments of SPI than DX, increased rigidity and stability of the SPI structure in SPI-Que-SA complexes. Electrostatic forces played a significant role between SPI and SA, while van der Waals forces were the main driving forces in SPI-DX complexes. Overall, the introduction of SA led to a looser and stable structure of SPI-Que complexes, resulting in an improvement of their emulsifying, foaming, and antioxidant properties.

Determination of Sulfuric Acid Effects on Degradation and Structural Changes of Gelatin Using Fourier-Transform Infrared Spectroscopy and Peak Deconvolution Analysis

The present research was aimed to investigate the effects of sulfuric acid on the structures of gelatin polypeptides. Gelatin samples were immersed in 0.5 M sulfuric acid solution for different periods of 15, 30, 60, 120, 240, 480, 960, and 1920 s, with possible structural changes analyzed by Fourier-transform infrared spectroscopy (FT-IR). Spectra at amide I and II regions were scrutinized using the Gaussian deconvolution method for the resulting changes in the protein secondary structure. The hydrolysis process initially led to a decrease in the α-helix chain and an increase in random coil and β-sheet structures. An equilibrium was formed in degradation and these structures were sequentially turned on each other. Results revealed a correlation between the peak intensity changes of these conformations, so that the degradation process could be observed in the conversion of α-helix to random coil and β-sheet structures and vice versa, indicating the oxidation and expansion of protein structure at the onset of the degradation process.

Characterization of the improved functionality in soybean protein-proanthocyanidins conjugates prepared by the alkali treatment

This study investigated the changes in functionality, structure, and protein composition in soybean protein-proanthocyanidins (SPI-PC) conjugates prepared by the alkali method with dose-ranging PC molecular grafting (0.5, 1, 2 mg/mL). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) demonstrated that more >180 kDa polymers were formed in SPI-PC conjugates with the increase of PC levels. The structural analysis showed that conjugation to PC altered the molecular structure of SPI, appearing the protein-unfolding with PC level-dependent. Liquid chromatography tandem MS analysis (LC/MS-MS) demonstrated that the composition of protein and peptides, as well as allergen and epitopes, were changed after SPI was conjugated with PC. The antioxidant activity of SPI-PC conjugates was increased with the increase of PC levels because high PC contents caused more hydroxyl groups in polyphenols to introduce into the SPI. Emulsifying properties were increased with the increase of PC levels, probably resulting from the unfolding of proteins, increase of random coil and decrease of surface hydrophobicity. Western-bolt and enzyme-linked immunosorbent assay (ELISA) showed a reduced IgE binding capacity in SPI-PC conjugates with PC level-dependent, which was attributed to the reduction of major soybean allergens and the masking or destruction of epitopes induced by structural changes. However, the foaming properties varied from PC levels. Overall, this study demonstrated that SPI-PC conjugates formed using the alkali method have a potential application as hypoallergenic soybean products or functional ingredients in foods. In future studies, suitable reactions (e.g., PC levels) are required to be investigated in SPI-PC conjugates for balancing various functional properties. • PC increased emulsifying, foaming and antioxidant properties in SPI. • The IgE binding capacity in SPI was reduced with the increase in PC levels. • Protein modifications and structural changes in SPI varied from PC levels. • Suitable PC levels are required to balance various functionality in conjugates.

Textural modification of Chinese traditional stewed pig trotter: Effect of acid or alkaline-induced degradation of collagen fibers.

This study aimed to investigate the effects of acid or alkaline treatments on the textural properties of Chinese traditional stewed pig trotter in relation to the degradation of collagen fibers. Pig trotters were subjected to different pHs of 4, 5, 6, 7, and 8 and then stewed at 95°C for 60 min. Textural parameters (springiness, chewiness, hardness, and gumminess) of pig trotters and Raman spectroscopy, cross-links, decorin, and glycosaminoglycans contents of collagen fibers were assessed. The acid or alkaline treatments at pH 4, 5, 6, and 8 improved the textural properties evidenced by lower chewiness, hardness, and gumminess, and promoted the unfolding of the secondary structure evidenced by a loss of α-helix paralleled with an increase of random coil, as well as induced a breakage to the covalent cross-links evidenced by the reduction of cross-links, decorin, and glycosaminoglycans. This study thus concluded positive effects of acid or alkaline treatments on the textural modification of Chinese traditional stewed pig trotter in relation to the induced degradation of the collagen fibers.

Changes in structure and emulsifying properties of coconut globulin after the atmospheric pressure cold plasma treatment

Atmospheric pressure cold plasma (ACP) treatment, a non-thermal sterilization technology with great potential in coconut milk, can maximize the retention of the taste and flavour. However, it is unavoidable to have an impact on the coconut milk components. In this study, coconut globulin (CG), a component important for coconut milk stability, was selected to explore the effect of the ACP treatment on the structure and emulsifying properties of CG. After the ACP treatment, the structure of CG unfolded, which was the increase in random coils and reduction in intrinsic fluorescence intensity. In addition, the carbonyl and S–S bond content increased, while the free –SH content decreased. Compared with untreated CG (440.57 nm), the particle size of CG after the ACP treatment decreased, especially at 60 kV 60 s (207.63 nm) and 60 kV 90 s (204.20 nm). Simultaneously, poor modification and excessive modification of CG have similar surface hydrophobicity and interfacial tension to untreated CG. Notably, moderate modification (60 kV 60 s and 60 kV 90 s) resulted in the unfolding of CG molecules and the exposure of hydrophobic groups, increasing their adsorption at the oil-water interface. As expected, the emulsifying ability and emulsifying stability of CG were significantly improved after the ACP treatment at 60 kV 60 s and 60 kV 90 s. In conclusion, moderate ACP treatment could improve the emulsifying properties of CG by modifying its structure. This study provides new insights into the effect of ACP on food components during liquid food sterilization.

Preparation of high-solubility rice protein using an ultrasound-assisted glycation reaction

To date, the poor solubility of rice protein (RP) has limited its industrial application in food products. This study aimed to prepare high-solubility RP conjugates using ultrasound-assisted glycation between RP and dextran. The conditions of glycation, treatment at 82 °C for 22 min under 600 W ultrasonic power, were optimized using single-factor experiments and response surface analysis. The solubility of the conjugates reached 90.6 %. Compared with traditional wet-heating glycation, ultrasound-assisted treatment brought more high-molecular-weight components, higher degree of graft, and less contents of arginine and lysine, indicating that ultrasound accelerated the glycation reaction. Ultrasound also resulted in a looser and more flexible structure of RP, manifesting in a 10 % reduction in β-sheets and 10.6 % increase in random coils of conjugates. The analysis of fluorescence and surface hydrophobicity indicated that ultrasound increased the exposure of hydrophobic residues towards aqueous environment. Besides solubility, the foaming and emulsifying properties of RP were improved through conformational changes. In summary, ultrasound-assisted glycation was found to be an efficient and green method for preparing high-solubility RP conjugates.

Relationship between protein native conformation and ultrasound efficiency: For improving the physicochemical stability of water–in–oil emulsions

To reveal the relationship between the native conformation of proteins and ultrasound efficiency, the effects of ultrasound power (0–500 W) on the structures and properties of different types of proteins (sodium caseinate, CAS; whey protein isolate, WPI; chickpea protein isolation, CPI; and pea protein isolation, PPI) were investigated. The results showed that the extent of structural changes of proteins depended on their native conformation rather than their origin. Irregular coil proteins (CAS) were more sensitive to ultrasound treatment and showed intense changes in secondary structure (e.g., 26.1% decrease in α–helix and 39.4% increase in random coil). In globular proteins, the smallest particle size (108 nm) of CPI limited the efficiency of ultrasound. The improvement in functional properties of proteins depended on the extent of structural modifications. The enhancement of functional properties improved the physicochemical stability of water–in–oil (W/O) emulsions (e.g., the turbiscan stability index of CAS–added emulsion was reduced by 2.33; the primary and secondary oxidation products of lipid oxidation were reduced by 16.96% and 17.31%). However, the effects of ultrasound–treated CPI on the physicochemical stability of W/O emulsions was not significant. For proteins with small particle size, future research could consider the application of chemical/enzymatic means in combination with ultrasound to enhance their performance.

The inhibition mechanisms between asparagus polyphenols after hydrothermal treatment and tyrosinase: A circular dichroism spectrum, fluorescence, and molecular docking study

Asparagus contains high quantity of polyphenols, including gallic acid, coumaric acid, chlorogenic acid, quercetin, p-hydroxybenzoic acid, rutin, trans-4-hydroxycinnamic acid, and ferulic acid. An understanding of the antityrosinase capacity and polyphenols changes during hydrothermal treatments was crucial for the development of asparagus. The results show that hydrothermal treatments (HT) enhance the total polyphenols in asparagus, and HT-160 has the highest total polyphenol contents (5.321 mg/g·dw) and tyrosinase inhibitory rate (95.87%). Rutin content reach 1754.01 μg/mL at 140°C. Increased polyphenols caused 7.5%, 4.8%, 2% decrease in α-helix, β-sheet, β-turn and a 7.4% increase in random coil using circular dichroism spectra. The fluorescence quenching of tyrosinase by HT samples (HT-80, HT-100, HT-120, HT-140, and HT-160) was accompanied by bigger ranges of red shift. Molecule docking analysis indicated that antityrosinase capacity was attributed to the capacity of polyphenols to bind to protein molecules and change the microenvironment of tryptophan or tyrosine residues. Conventional hydrogen bond, van der Waals, π-π stacked, π-σ interaction, and π-alkyl interactions were generated between asparagus polyphenols (ASP) and residues. As a promising inhibitor of tyrosinase, ASP could be employed as a potential agent to reduce food browning.

Effect of Graphite Oxide on the Catalytic Behavior of (S)-Selective Amine Transaminases

Graphite oxide (GO) has been used for the immobilization of several classes of enzymes, exhibiting very interesting properties as an immobilization matrix. However, the effect the nanomaterial has on the enzyme cannot be predicted. Herein, the effect GO has on the catalytic behavior of several (S)-selective amine transaminases [(S)-ATAs] has been investigated. These enzymes were the focus of this work as they are homodimers with pyridoxal 5′-phosphate in their active site, significantly more complex systems than other enzymes previously studied. Addition of GO (up to 0.1 mg/ml) in the reaction medium leads to activation (up to 50% improved activity) for most enzymes studied, while they maintain their temperature profile (they perform better between 40 and 45°C) and their stability. However, the effect is not universal and there are enzymes that are negatively influenced by the presence of the nanomaterial. More profound is the effect on the (S)-ATA from Chromobacterium violaceum which loses almost 50% of its activity in the presence of 0.1 mg/ml GO, while the stability was significantly decreased, losing its activity after 2 h incubation at 40°C, in the presence of 25 μg/ml GO. This negative effect seems to rise from minor secondary structure alterations; namely, a loss of α-helices and subsequent increase in random coil (∼3% in the presence of 25 μg/ml GO). We hypothesize that the effect the GO has on (S)-ATAs is correlated to the surface chemistry of the enzymes; the less negatively-charged enzymes are deactivated from the interaction with GO. This insight will aid the rationalization of ATA immobilization onto carbon-based nanomaterials.

L-Arginine inhibits the activity of α-amylase: Rapid kinetics, interaction and functional implications

In this work, the rapid unfolding kinetics of pancreas α-amylase (PPA) induced by l-arginine and the interaction mechanism were investigated. The unfolding followed a first-level reaction kinetics equation, without intermediates. l-arginine interacted with PPA though diffusion-controlled process rather than complexion. The interaction between l-arginine and PPA resulted in a pronounced decrease in β-sheet and a significant increase in random coil, and thereby the enzyme activity decreased. However, the unfolding of PPA could be compensated and the second structure change could be recovered to some extent by the macromolecular crowded medium of Pluronics. Further insight into the mechanism disclosed that the broken H-bond network of water may contribute to PPA unfolding. This work provides a new perspective on the interaction of l-arginine with digestive enzyme. The unfolding mechanism of enzymes by may help to understand the effects of other structurally similar drugs, which is of concern in food-drug interactions.

8 - Dentine Conditioners: Effect on Collagen Probed By Amide-III Peak Analysis

To assess the extent of demineralization and changes in the collagen type I structure of dentine conditioned with various acids, by using the amide-III peak for analysis, which is not affected by the presence of water, as opposed to the commonly used Amide-I peak. Coronal dentin specimens (n=10/group) were analyzed by ATR-FTIR spectroscopy before and after conditioning (15 s for all) with 32% phosphoric acid (PA), 3% nitric acid (NA), 20% phytic acid (PT), 20% citric acid (CT) and 17% neutral EDTA (ED). The extent of demineralization (DM%) was measured from the PO 4 (1185-885 cm -1 ) / Amide-III (1370-1150 cm -1 ) peak area ratios, normalized against the native dentine specimens. The Amide-III peak was curve-fitted in four components assigned to α-helix (1285-1275 cm -1 ), β-turns (1255-1245 cm -1 ), random coils (1220-1210 cm -1 ) and β-sheets (1210-1200 cm -1 ) and the percentage subpeak areas were calculated relative to the total Amide-III peak. Statistical analysis was performed by one-way ANOVA (DM%) and t-test/Wilcoxon signed rank test for the curve-fitted components (α=0.05) The results of DM% were (mean/sd): PA: 89(3), NA: 95(2), PT:71(7), CT: 58 (8), ED: 25 (9) with a ranking of significant differences NA,PA>PT,CT>ED. Curve-fitting of the Amide- III peak revealed the following significant differences: Reduction in -helices and β-turns (PA); reduction in α-helices, β-turns and increase in β-sheets (NA); reduction in α-helices, β-turns and increase in random coils (PT); reduction in β-turns and an increase in β-sheets and random coils (CT) and increase in β-sheets and α-helices (ED) Amide-III peak may provide important information for the extent of demineralization and the collagen type I structure of acid etched dentine, without the water interferences considered in Amide-I analysis. The inorganic and phytic acids, with the highest demineralization, disorganized the α-helix and β-turn structures, citric acid adversely affected only b-turns, whereas ED was the least aggressive treatment.