Znse crystal

The protein molecular spectral profiles were collected using a JASCO FT/IR-ATR-4200 spectroscope (JASCO Corp., Tokyo, Japan) at the molecular spectroscopy lab (University of Saskatchewan, Saskatoon, Canada). The spectroscope was equipped with a MIRacle ATR accessory module, a ZnSe crystal and pressure clamp (Pike Technologies, Madison, WI). Spectra were collected from the mid-infrared range from ca. 4000–700 cm^-1 with 256 scans per spectrum at 4 cm^-1 resolution. The protein related molecular spectral peak bands were identified according to the published literature [24 ] including: amide I (region ca. 1723~1588 cm^-1; peak ca. 1644 cm^-1), amide II (region ca. 1588~1482 cm^-1; peak ca. 1539 cm^-1), α-helix (ca. 1650 cm^-1) and β-sheet (ca. 1637 cm^-1). Different peak area and height intensity ratios were also calculated in this study. The OMNIC 7.3 (Thermo Electron Corp., Madison, WI, US) was used for quantification of absorption peaks areas and heights of spectral bands related to protein primary and secondary structures. The principle component analysis (PCA) was performed on the overall spectral data related to protein structures (ca. 1723~1482) to visualize the overall difference in protein molecular structures between raw barley and moist-heated barley. The detailed information regarding PCA for ATR-FTIR spectral data are described in Yu (2007) [24 ].

Raman spectroscopy was carried out with a Thermo Scientific DXR micro-Raman spectrometer (Thermo Scientific, Waltham, MA, USA) with 532-nm laser excitation at a spectral resolution of 2 cm⁻¹ and a spatial resolution of 10 μm. Fourier transform infrared (FTIR) spectroscopy was conducted using a Magna Model 560 instrument (Nicolet Instrument Corporation, Madison, WI, USA) attached to an attenuated total reflectance (ATR) accessory with a single reflection ZnSe crystal (Pike Technologies, Madison, WI, USA). X-ray diffraction (XRD) measurements were carried out on a Philips PW3040 X-ray Diffractometer in the range from 10° to 90° with CuKα radiation (λ = 1.54 Å) with a step size of 0.02°.
TEM imaging and selected area electron diffraction were conducted using a Philips field emission CM20 TEM/STEM system coupled to a low temperature sampling attachment with a Gatan 792 Multi-scan CCD (1 K × 1 K) camera, Schottky field emitter and TWIN pole pieces, objective lens: 0.282 nm point resolution, 0.144 nm line resolution and 0.180 nm information limit at 200 KV (TEM mode).

Infrared spectroscopic measurements of LEV-ARG mixtures were performed on a MB3000 Fourier transform infrared spectrometer (ABB, Saint-Laurent, QC, Canada) equipped with a MIRacle ATR sampling accessory with a ZnSe crystal (PIKE Technologies, Madison, WI, USA). The infrared spectra were collected with Horizon MB software (version 3.2.5.2, ABB, Saint-Laurent, QC, Canada). Spectra were collected from 600 to 4000 cm⁻¹ and calculated as a mean of 64 spectra with a resolution of 16 cm⁻¹. Reference spectra of the surrounding atmosphere were obtained as a mean of 64 spectra with a resolution of 16 cm⁻¹.