MONITORING CHANGES IN MOLECULAR STRUCTURES OF PROTEINS AND CARBOHYDRATES DURING FEED PROCESSING USING DSC AND DRIFT: AN OVERVIEW

In the feed industry, many forms of technological processing are applied to manipulate the site and extent of digestion in ruminants. Technological processing applied in feed industries are usually involve heat, pressure and shear forces. Such processing manipulates the site of digestion in ruminants by reducing the size of feed particles,(Goelema et al., 1996) protecting the proteins from being degraded in the rumen,(Prestløkken, 1999; Ljøkjel et al., 2003c) and making the starch granules more accessible for microbial digestion (Arieli et al., 1995; Ljøkjel et al., 2003c). Different methods have been developed to determine the extent of protein denaturation and starch gelatinization occurs due to application of feed processing. Thermal analysis such as differential scanning calorimetry (DSC) has proved to be a suitable tool to study phase transitions, such as the denaturation of proteins (Wright and Boulter, 1980) and gelatinization of starch. The technique provides the temperature of denaturation and gelatiniztion (Tp and Tg) and the enthalpy change associated with the transition (δH). The δH value represents a combination of exothermic reactions such as those associated with disruption of hydrophobic interactions and aggregation of the molecule, and of the endothermic contributions of disruption of nitrogen bonds and unfolding of the polypeptide chains (Wright and Boulter, 1980). The DSC has been successfully applied to determine to what extent proteins have been denatuarted and starch has been gelatinized when a hydro-thermal processing applied to feedstuffs. Goelema (1999) found that enthalpy change associated with the protein denaturation was highly and negatively correlated to digested undegraded protein in dairy cows in several legume seeds including faba beans, peas, lupins and soybeans. Azarfar et al. (2012; unpublished data) found that enthalpy change associated with starch gelatinization was negatively correlated to maximum fractional rate of gas production in vitro in barley, maize, peas and lupins.Beside thermal analysis, vibrational spectroscopic methods such as diffused reflectance infrared Fourier transformed spectroscopy were successfully applied to reveal molecular structural features related to proteins, carbohydrates and lipids (Yu, 2005c; Yu et al., 2011; Azarfar et al., 2013) and that the changes occur to these macromolecules when technological processes applied to feedstuffs. The technique is based on the fact that exposure to IR radiation cause vibration and rotation of molecules between different quantized discrete energy levels E0, E1 and E2 (Stuart, 2004). Whenever IR radiation transmits through a sample, energy uptake by a molecule occurs and results in transition between different energy levels (Stuart, 2004; Barth, 2007). Primarily, various IR spectra patterns result from the corresponding vibration, a kind of internal motion of the molecule. Therefore, it is possible to identify the unknown organic compounds and determine the composition of a mixture according to the frequencies, lineshapes, intensities and patterns of the characteristic peaks (Messerschmidt and Harthcock, 1988; Jackson and Mantsch, 2000b; Stuart, 2004). There can also be a slight shift of characteristic peaks resulting from diverse specimens, while the typical IR absorbance patterns do not change. Differences in chemical and structural composition, therefore, can be shown in the IR spectra. By analyzing with uni- and multi- variate analysis methods, informative differences can be probed. Various chemical compounds have various IR spectra. IR spectra are specific and characteristic, which therefore can be used as fingerprints for identifying or discriminating sample conformation (Messerschmidt and Harthcock, 1988). IR spectroscopy is widely applied in analysis of chemical composition, because it can accomplish rapid analysis with simple operation and simultaneously determine multi-nutrient composition in a nondestructive, non-pollutive manner (Barth, 2007). This technique only requires a small amount of sample. Additionally, with flexible accessories, IR spectroscopy is capable of analyzing a sample in different status (gases, liquids and solids) or with different composition (organics/inorganics, macro/micro molecules) (Wetzel et al., 1998b; Stuart, 2004). Samples are mounted on the path of IR radiation in an IR spectroscope. When a sample is exposed to IR radiation, if the molecules of the sample are active to the corresponding IR frequencies, the certain electric dipole moment in the molecule can be altered and characteristic absorption occurs. By measuring the absorption, spectroscopic information can be obtained (Stuart, 2004). IR spectroscopy is designed to characterize chemical functional groups by measuring the IR absorption in the sample. The IR spectrum is displayed as a function of frequency. All types of chemical functional groups have their own unique absorption frequency associated with energy. When the energy or frequency of IR meets any vibrational frequency of molecules in the sample, absorption occurs. A detector can record the absorption to determine the chemical function groups and examine the chemical composition of the complex matrix (Budevska, 2002). Characteristic absorption peaks can be used to analyze a large variety of compound classes. For example, in the ca. 4000-2500 cm-1 region, there are bands caused by O–H, C–H and N–H stretching. Triple-bond stretching usually exhibits absorption in the ca. 2500-2000 cm-1 region. In contrast C=C and C=O stretching absorptions are located at the ca. 2000–1500 cm-1 region (Jackson and Mantsch, 2000b; Stuart, 2004). The wavenumber range of IR from 1800 to 800 cm-1 is the so-called fingerprint region . The fingerprint region usually accounts for almost all the characteristics of biological molecules. Bands in the fingerprint region are particularly sensitive to molecular structure. However, it should be pointed out that not every band can guarantee the representation of a certain corresponding chemical structure because the vibration of chemical bonds vary in sensitively depending on the sample status and experiment circumstance (Griffiths and De Haseth, 1986; Messerschmidt and Harthcock, 1988; Yu, 2006b).Infrared spectroscopy provides comprehensive