The objectives of this study were to reveal protein structures of feed tissues affected by heat processing at a cellular level, using the synchrotron-based Fourier transform infrared microspectroscopy as a novel approach, and quantify protein structure in relation to protein digestive kinetics and nutritive value in the rumen and intestine in dairy cattle. The parameters assessed included 1) protein structure α-helix to β-sheet ratio; 2) protein subfractions profiles; 3) protein degradation kinetics and effective degradability; 4) predicted nutrient supply using the intestinally absorbed protein supply (DVE)/degraded protein balance (OEB) system for dairy cattle. In this study, Vimy flaxseed protein was used as a model feed protein and was autoclave-heated at 120°C for 20, 40, and 60 min in treatments T1, T2, and T3, respectively. The results showed that using the synchrotron-based Fourier transform infrared microspectroscopy revealed and identified the heat-induced protein structure changes. Heating at 120°C for 40 and 60 min increased the protein structure α-helix to β-sheet ratio. There were linear effects of heating time on the ratio. The heating also changed chemical profiles, which showed soluble CP decreased upon heating with concomitant increases in nonprotein nitrogen, neutral, and acid detergent insoluble nitrogen. The protein subfractions with the greatest changes were PB1, which showed a dramatic reduction, and PB2, which showed a dramatic increase, demonstrating a decrease in overall protein degradability. In situ results showed a reduction in rumen-degradable protein and in rumen-degradable dry matter without differences between the treatments. Intestinal digestibility, determined using a 3-step in vitro procedure, showed no changes to rumen undegradable protein. Modeling results showed that heating increased total intestinally absorbable protein (feed DVE value) and decreased degraded protein balance (feed OEB value), but there were no differences between the treatments. There was a linear effect of heating time on the DVE and a cubic effect on the OEB value. Our results showed that heating changed chemical profiles, protein structure α-helix to β-sheet ratio, and protein subfractions; decreased rumen-degradable protein and rumen-degradable dry matter; and increased potential nutrient supply to dairy cattle. The protein structure α-helix to β-sheet ratio had a significant positive correlation with total intestinally absorbed protein supply and negative correlation with degraded protein balance.

Wheat differs from corn in biodegradation kinetics and fermentation characteristics. Wheat exhibits a relatively high rate (23% h(-1)) and extent (78% DM) of biodegradation, which can lead to metabolic problems such as acidosis and bloat in ruminants. The objective of this study was to rapidly characterize the molecular chemistry of the internal structure of wheat (cv. AC Barrie) and reveal both its structural chemical make-up and nutrient component matrix by analyzing the intensity and spatial distribution of molecular functional groups within the intact seed using advanced synchrotron-powered Fourier transform infrared (FTIR) microspectroscopy. The experiment was performed at the U2B station of the National Synchrotron Light Source at Brookhaven National Laboratory, New York, USA. The wheat tissue was imaged systematically from the pericarp, seed coat, aleurone layer and endosperm under the peaks at approximately 1732 (carbonyl C=O ester), 1515 (aromatic compound of lignin), 1650 (amide I), 1025 (non-structural CHO), 1550 (amide II), 1246 (cellulosic material), 1160, 1150, 1080, 930, 860 (all CHO), 3350 (OH and NH stretching), 2928 (CH(2) stretching band) and 2885 cm(-1) (CH(3) stretching band). Hierarchical cluster analysis and principal component analysis were applied to analyze the molecular FTIR spectra obtained from the different inherent structures within the intact wheat tissues. The results showed that, with synchrotron-powered FTIR microspectroscopy, images of the molecular chemistry of wheat could be generated at an ultra-spatial resolution. The features of aromatic lignin, structural and non-structural carbohydrates, as well as nutrient make-up and interactions in the seeds, could be revealed. Both principal component analysis and hierarchical cluster analysis methods are conclusive in showing that they can discriminate and classify the different inherent structures within the seed tissue. The wheat exhibited distinguishable differences in the structural and nutrient make-up among the pericarp, seed coat, aleurone layer and endosperm. Such information on the molecular chemistry can be used for grain-breeding programs for selecting a superior variety of wheat targeted for food and feed purposes and for predicting wheat quality and nutritive value in humans and animals. Thus advanced synchrotron-powered FTIR technology can provide a greater understanding of the plant-animal interface.

Mid-IR techniques were used to characterize any changes that occurred on a molecular level in flaxseed that had been heated using an autoclave. The objectives were to investigate the effects of autoclave heating on differences in diffuse reflectance infrared Fourier transform (DRIFT) and synchrotron-based Fourier transform infrared microspectroscopy (SFTIRM) based measurements of the protein α-helix to β-sheet ratio for flaxseed (Linum usitatissimum), cv. Vimy. Hierarchical cluster analysis (CLA) and principal components analysis (PCA) were also conducted to identify molecular differences in the DRIFT spectra. Flaxseed samples were kept raw for the control or autoclaved in batches at 120 • C for 20, 40 or 60 min for treatments 1, 2 and 3, respectively. DRIFT analysis of protein secondary structure ratios showed a decrease (P < 0.05) in the α-helix to β-sheet ratio for the whole seed while the results from synchrotron SFTIRM spot data from the endosperm tissue in flaxseed showed autoclaving had the opposite effect (P > 0.05). CLA and PCA were successfully used to make distinctions between the different treatment spectra and showed enhanced sensitivity upon selection of a smaller spectral window to include only the amide I and II portion of the IR spectrum. Our results indicated that autoclaving had a great enough effect on the mid-IR spectrum of flaxseed to identify the altered α-helix to β-sheet ratio and subsequently differentiated between the treatments using PCA and CLA suggesting greater sensitivity of mid-IR spectral methods in identifying the effect of heat treatment on protein secondary structure. Future study is needed to quantify the relationship between protein secondary structure and protein functionality. K.J. Doiron et al. / Detecting molecular changes to Vimy flaxseed protein structure DRIFT: Diffuse reflectance infrared Fourier transform NPN: Non-protein nitrogen PCA: Principal components analysis SCP: Soluble crude protein SFTIRM: Synchrotron radiation-based Fourier Transform Mid-IR Microspectroscopy IntroductionCurrent methods of food/feed evaluation, particularly for protein quality, are typically slow and cumbersome processes that require the destruction of a sample for any given measurement during "wet" chemical analysis. The information gleaned from such procedures is also not totally in context; that is to say some pertinent information is lost due to the process by which the measurements are taken. Part of the reason for this is that these standard methods destroy any spatial information as well as the distribution of those elements of interest [3]. These elements may be linked to degradability and digestibility in human/animals.Vibrational spectroscopy or mid-Infrared spectroscopy has the capacity to overcome some of the issues with modern food/feed characterization methods. The results from mid-IR spectroscopy are complex in nature. The information contained within each spectra is not only related to the chemical bonds in each sample but interrelations between these chemical bonds [10]. S...

The objective of this study was to use advanced synchrotron-sourced FTIR microspectroscopy (SFTIRM) as a novel approach to identify the differences in protein and carbohydrate molecular structure (chemical makeup) between these two varieties of barley and illustrate the exact causes for their significantly different degradation kinetics. Items assessed included (1) molecular structural differences in protein amide I to amide II intensities and their ratio within cellular dimensions, (2) molecular structural differences in protein secondary structure profile and their ratios, and (3) molecular structural differences in carbohydrate component peak profile. Our hypothesis was that molecular structure (chemical makeup) affects barley quality, fermentation, and degradation behavior in both humans and animals. Using SFTIRM, the protein and carbohydrate molecular structural chemical makeup of barley was revealed and identified. The protein molecular structural chemical makeup differed significantly between the two varieties of barleys. No difference in carbohydrate molecular structural chemical makeup was detected. Harrington was lower than Valier in protein amide I, amide II, and protein amide I to amide II ratio, while Harrington was relatively higher in model-fitted protein alpha-helix and beta-sheet, but lower in the others (beta-turn and random coil). These results indicated that it is the molecular structure of protein (chemical makeup) that may play a major role in the different degradation kinetics between the two varieties of barleys (not the molecular structure of carbohydrate). It is believed that use of the advanced synchrotron technology will make a significant step and an important contribution to research in examining the molecular structure (chemical makeup) of plant, feed, and seeds.

Conventional “wet” chemical analyses rely heavily on the use of harsh chemicals and derivatization, thereby altering native seed structures leaving them unable to detect any original inherent structures within an intact tissue sample. A synchrotron is a giant particle accelerator that turns electrons into light (million times brighter than sunlight) which can be used to study the structure of materials at the molecular level. Synchrotron radiation-based Fourier transform IR microspectroscopy (SR-FTIRM) has been developed as a rapid, direct, non-destructive and bioanalytical technique. This technique, taking advantage of the brightness of synchrotron light and a small effective source size, is capable of exploring the molecular chemistry within the microstructures of a biological tissue without the destruction of inherent structures at ultraspatial resolutions within cellular dimensions. This is in contrast to traditional ‘wet’ chemical methods, which, during processing for analysis, often result in the destruction of the intrinsic structures of feeds. To date there has been very little application of this technique to the study of plant seed tissue in relation to nutrient utilization. The objective of this study was to use novel synchrotron radiation-based technology (SR-FTIRM) to identify the differences in the molecular chemistry and conformation of carbohydrate and protein in various plant seed endosperms within intact tissues at cellular and subcellular level from grains with different biodegradation kinetics. Barley grain (cv. Harrington) with a high rate (31.3%/h) and extent (78%), corn grain (cv. Pioneer) with a low rate (9.6%/h) and extent of (57%), and wheat grain (cv. AC Barrie) with an intermediate rate (23%/h) and extent (72%) of ruminal DM degradation were selected for evaluation. SR-FTIRM evaluations were performed at the National Synchrotron Light Source at the Brookhaven National Laboratory (Brookhaven, NY). The molecular structure spectral analysis involved the fingerprint regions of ca. 1720–1485 cm−1 (attributed to protein amide I C=O and C—N stretching; amide II N—H bending and C—N stretching), ca. 1650–950 cm−1 (non-structural CHO starch in endosperms), and ca. 1185–800 cm−1 (attributed to total CHO C—O stretching vibrations) together with agglomerative hierarchical cluster and principal component analyses. Analyses involving the protein amide I features consistently identified differences between all three grains. Other analyses involving carbohydrate features were able to differentiate between wheat and barley but failed however to differentiate between wheat and corn. These results suggest that SR-FTIRM plus the multivariate analyses can be used to identify spectral features associated with the molecular structure of endosperm from grains with different biodegradation kinetics, especially in relation to protein structure. The Novel synchrotron radiation-based bioanalytical technique provides a new approach for plant seed structural molecular studies at ultraspatial resolution and within i...