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Kafirin, the hydrophobic prolamin storage protein in sorghum grain is enriched when the grain is used for bioethanol production to give dried distillers grain with solubles (DGGS) as a by-product. There is great interest in DDGS kafirin as a new source for biomaterials. There is however a lack of fundamental understanding of how the physicochemical properties of DDGS kafirin having been exposed to the high temperature conditions during ethanol production, compare to kafirin made directly from the grain. An understanding of these properties is required to catalyse the utilisation of DDGS kafirin for biomaterial applications. The aim of this study was to extract kafirin directly from sorghum grain and from DDGS derived from the same grain and, then perform a comparative investigation of the physicochemical properties of these kafirins in terms of: polypeptide profile by sodium-dodecyl sulphate polyacrylamide gel electrophoresis; secondary structure by Fourier transform infra-red spectroscopy and x-ray diffraction, self-assembly behaviour by small-angle x-ray scattering, surface morphology by scanning electron microscopy and surface chemical properties by energy dispersive x-ray spectroscopy. DDGS kafirin was found to have very similar polypeptide profile as grain kafirin but contained altered secondary structure with increased levels of -sheets. The structure morphology showed surface fractals and surface elemental composition suggesting enhanced reactivity with possibility to endow interfacial wettability. These properties of DDGS kafirin may provide it with unique functionality and thus open up opportunities for it to be used as a novel food grade biomaterial.
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The production of alcohol from grains such as maize and sorghum for use as biofuel is a current topic of commercial interest. Increased biofuel needs are predicted to occur over the next decade as reported by the EU Regulatory Framework for Biofuels1. Dried distillers grain with solubles (DDGS) is a protein enriched by-product from this industry, that remains after fermentation and distillation by heat treatment2. At present, some DDGS may be used as an animal feed supplement, but the rest is considered waste and may be dumped in sewers and rivers3. Unlocking value from unwanted DDGS is an important step to reduce this current environmental burden. Globally, several Authorities have identified priorities for the value-added utilisation of DDGS.
The aim of this study was to extract kafirin from sorghum grain and its DDGS, then to do a comparative investigation of their physico-chemical proteins in comparison with commercial zein in terms of: polypeptide profile by electrophoresis; secondary structure by Fourier transform infra-red spectroscopy (FTIR) and x-ray diffraction (XRD); self-assembly behaviours using small-angle x-ray scattering (SAXS); morphological imaging and surface chemical composition by scanning electron microscopy (SEM) and energy dispersive x-ray spectroscopy (EDS). These physico-chemical investigations will evaluate if the sorghum DDGS kafirin may have useful techno-functionality for future biomaterial applications.
Ten kg each of whole grain sorghum and sorghum DDGS produced from the same whole grain were gifted by Dalby Bio-refinery, (Dalby, Queensland, Australia). The samples were vacuum packed and stored at 4 C before further use.
The extraction method was based on that of Lau et al.2 with minor modifications. The whole grain and the DDGS were milled into fine powder using a pin mill (Cemotec 1090 sample mill, Foss Tecator, Mulgrave, VIC, Australia) followed by blending (ZM 200 blender, Retcsh Gmbh & Co, Haan, Germany). The milled samples were passed through a 500 micron sieve with 85% recovery of the sieved fraction. Then the total sample was reconstituted.
The dried kafirin was defatted three times by washing with absolute n-hexane (40 mL/12 g) by initial manual shaking followed by standing at room temperature for 5 h. The solvent was removed by decantation, the residual solvent in the defatted kafirin was evaporated by heating for 24 h at 60 C in an oven (Memmert 854\, Schwabach, Germany).
The samples were heated in a boiling water bath with stirring for 25 min to completely dissolve the proteins. The prepared solutions (10 l) were loaded onto the gel and electrophoresis was run at 200 V for 30 min until the leading edge of the migration was close to the bottom of the gel. The gels were stained for 20 h using 0.1% Bio-safe Coomassie G250 stain (Bio-Rad Laboratories, Carlsbad, California, USA). The stained gels were de-stained using methanol/acetic acid/water at 1:1:8 (v/v/v). The de-stained gels were imaged using a Bio-Rad Universal Hood II gel imaging system (Bio-Rad Laboratories, Hercules, CA, USA).
The migration of the sample bands was compared with that of the molecular weight standard mixture to estimate their molecular weight and identify their subunits based on molecular weights reported in the literature2.
Crystallite size is known to affect various key properties such as solubility, stability, and molecular interactions. The crystallite size was calculated using the double-Voigt method17 where the peaks are described by a pseudo-Voigt peak and the final crystallite size extracted from its integral breadth. The fittings of the data was performed by full pattern data analysis programme, TOPAS (Bruker, Karlsruhe, Germany).
The surface morphology of DDGS kafirin, grain kafirin and zein was investigated using secondary electron (SE) imaging on a dual-beam field emission scanning electron microscope (Zeiss Neon 40EsB FEBSEM, Oberkochen, Germany). Samples were kept in a desiccator then placed onto aluminium stubs using carbon tape and coated with 6 nm of platinum using a splutter coater (208HR, Cressington, Watford, UK). A 5 kV electron beam was used20. Surface elemental composition was determined using EDS at higher kV tailored with the FEB-SEM21. Surface elemental distribution was collected over various areas and elemental identification was performed by Aztec software. (Oxford Instruments, Wiesbaden, Germany).
In 1991 the proposed nomenclature of kafirin polypeptides (subunits) was established based on their similarities with those of zein22. More recent studies have revealed greater hydrophobicity with more dominance of -helical secondary structure in grain kafirin than zein4,23. However, zein is still considered an appropriate polypeptide identification standard for kafirins and was used as an internal control in this study.
The similar electrophoretic patterns of both grain and the DDGS kafirins indicate that the kafirin polypeptides were stable to the harsh conditions used in the sorghum bioethanol production process (eg. heat and pressure) that resulted in the DDGS.
Also, our spectra agree with those seen after high temperature treatment of purified kafirin, which also indicates a propensity of the protein to produce aggregated -sheet structures at high temperatures36. Similarly, Ezeogu et al.40 reported secondary structural changes in prolamin proteins from sorghum and maize on cooking e.g., a shift in band intensities of amides. The authors presumed that during cooking some intra and intermolecular disulfide bond breakdown had led to these structural changes.
We hypothesise that this is because of during the biofuel manufacturing process the high temperature were capable enough to unravel some -helices and random coils followed by realignment and reorganisation into -sheets. The higher level of -sheets in DDGS kafirin compared to grain kafirin may make the former better suited for viscoelastic self-assembly delivery systems. It is well documented in literature that biomaterial with viscoelastic properties have high energy absorption capacity, shock absorbance behaviour and dumping response41,42,43. Thus, it become evident that DDGS kafirin might retain architecture of formulated biomaterial system in physiological environment than that of commonly used elastic biomaterials which are usually thought to be loading dependent only. The DGGS kafirin may endow interfacial wettability to biomaterials because of more aggregated -sheets, which are believed to be less hydrophobic than -helix.
Another important observation from the XRD data is that although differences in structure between grain kafirin and DDGS kafirin and are apparent, the crystallite size does not appear to have changed substantially (Table 1).
Small angle X-ray scattering (SAXS) patterns, showing the overall model and two individual unified-fit levels for zein, grain kafirin, and kafirin from sorghum dried distillers grain with solubles (DDGS).
The surface elemental composition of DDGS kafirin was different than grain kafirin and zein. In addition to C, N, O and S elements some more elements Na, Cl and K were present, Fig. 5D. It is a well-known fact that elements facing bottom left corner of periodic table such as sodium, potassium and etc. are more active suggesting increased reactive surface sites for DDGS than grain kafirin and zein.
The novelty of the DDGS kafirin is that despite presence of the additional elements on surface, atomic radius of C and N element on surface remained same and atomic radius of S and O element increased. Although, additional elemental profile gives a clear understanding that DDGS kafirin might endow interfacial wettability. Thought striking, increased atomic radius of elements (S & O), which are proven to form hydrophobic surface sites21, and additional more reactive elements suggest DDGS surface might have both hydrophobic and hydrophilic surface segments.
Thus, we hypothesise that compared to grain kafirin, DDGS kafirin if used as an active encapsulating agent might have enhanced solubility and formation of complexes with target compounds in aqueous systems. These differences in structure and functionality may also be explained by heat induced transformations in the molecular architecture of DDGS kafirin during the bioethanol production process. 152ee80cbc
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