Effect of wet and dry milling on the functional properties of whole sorghum grain flour and kafirin

Abstract

The effect of wet and dry milling on the functional properties of whole sorghum grain flour (SF) and extracted kafirin were assessed. White sorghum landrace was used to prepare two SFs by wet milling (SF1) or dry milling (SF2) and to extract their respective kafirins SK1 and SK2. Protein contents of SK1 and SK2 were 90.07 and 94.23%. Wet milling of SF allowed increasing the oil binding capacity, the least gelling concentration and the instant foam capacity and decreasing the water binding capacity and foam stability. The emulsifying activity index of SF1 and SF2 were in the same range, while emulsion stability was two time higher in SF2. Functional properties of SK1 and SK2 showed an appropriate water binding capacity of 2.20 ± 0.10 and 1.82 ± 0.22 (g water/g dry mater), respectively. Both SK1 and SK2 showed higher oil binding capacity than SF1 and SF2 with no gel and foam formation. The wet milling improved water and oil binding capacities of SK1 by 17 and 5%. The emulsifying activity indexes were approximately similar for SK1 and SK2 with emulsion stability exceeding 60%. Fourier transform infrared spectroscopy of SK1 and SK2 showed that wet milling induced a decrease of α-helical structure and an increase of intermolecular β-sheet and β-turns and no change in the anti parallel β-sheet. This study showed that wet milling could allow extracting kafirin with preserving the most important functional properties of SF and kafirin and could constitute an interesting method for protein recovery and starch isolation.

Introduction

In recent years, research and interest on gluten free products have increased. Sorghum proteins are an important, renewable, safe and healthy source for food and non-food applications (Taylor et al. 2006). Sorghum (Sorghum bicolor (L.) Moench) is one of the traditional crops in the world and constitutes a major source of protein and calories in many poor income countries. It is the fifth most widely grown cereal crop in the world with a production of 67.8 million tons in 2014 (Espinosa-Ramírez and Serna-Saldívar 2016). It is more tolerant to heat and drought conditions compared to wheat, barley and maize (Day 2013). Sorghum is a safe ingredient for gluten-free products destined to people identified with celiac disease and/or other dietary intolerances to wheat, barley or rye (Day 2013; de Mesa-Stonestreet et al. 2010; Taylor et al. 2006). Kafirins are alcohol soluble and most abundant proteins in sorghum grain; they represent more than 70% of the total proteins in whole kernel (Hamaker et al. 1995). Kafirins are subdivided into four subclasses α, β, γ and δ type based on their solubility, structure and amino acid sequence (Belton et al. 2006; Mokrane et al. 2009). Their special technofunctional and biofunctional properties such as non allergenic, slow digestibility by mammalian proteases, hydrophobicity and solubility in organic solvents, have extended their uses to many applications such as; food and non-food packaging films, drug delivery application, enteric coating and more recently as microparticles (Xiao et al. 2017). Several methods were reported for kafirins extraction using ethanol, 1-propanol, tertiary-butanol and glacial acetic acid as solvent with or without addition of reducing agents (β-mercaptoethanol, dithiothreitol and sodium metabisulfite) and detergents (sodium dodecyl sulfate (SDS) and dodecyl ammonium bromide) (de Mesa-Stonestreet et al. 2010). Interest on the technofunctional properties (emulsifying, foaming and gelling properties, solubility, water binding capacity (WBC) and oil binding capacity (OBC)) of cereal flours and their extracted protein fraction increased in the two past decades (Belton et al. 2006; Espinosa-Ramírez and Serna-Saldívar 2016; Zayas 1997).

The effect of processing (fermentation, malting and germination) on the dry milled sorghum flour (SF) functionality were previously investigated by many researchers (Elkhalifa et al. 2017; Elkhalifa and Bernhardt 2010; Elkhalifa et al. 2005; Ojha et al. 2018; Singh et al. 2017). The functional properties of the dry milled SF were improved. However, the effect of processing on kafirin functional properties attracted less attention than SF (Elkhalifa et al. 2016; Espinosa-Ramírez and Serna-Saldívar 2016; Musigakun and Thongngam 2007). The dry milling of the sorghum whole grain was the most used process to recover kafirin. Recently, Espinosa-Ramírez et al. (2017) compared the functional properties of kafirin extracted from dry milled SF or from the residue of starch recovery known as sorghum gluten meal treated with or without endopeptidic protease after grain decorticating.

To date, no reports have been found on the functional and structural properties of kafirin extracted directly after wet milling and before starch recovery from whole sorghum grain. Against this background, the aim of this research was first to increase the yield of kafirin recovery and secondly to assess the effect of wet or dry milling on kafirin extraction using aqueous alkaline-ethanol solvent under food compatible reducing conditions. The functional properties of the obtained SF1 and SF2 and their respective kafirins (SK1 and SK2) were investigated and compared.

Materials and methods

Materials

White sorghum landrace (Tafsout el Beida) was harvested from arid South Algerian Sahara “Ain Salah” in summer 2013. All chemicals were purchased from Sigma Aldrich and were of analytical grade.

Preparation of sorghum flours

Sorghum grains were cleaned manually to remove all foreign materials (dust, dirt, small branches and immature seeds). A modified method of Kumar et al. (2014) was used to prepare SF1 by wet milling. The sorghum grains were steeped for 48 h at 50 °C in 0.3% (w/v) sodium metabisulfite aqueous solution with a ratio sorghum grains to steeping solution of 1/2 (w/v). The obtained grains were washed with 200 mL of distilled water three times and then ground with 400 mL of distilled water in a blender (Kenwood, Germany). The formed suspension was sieved (500 µm), centrifuged (3000×g, 20 min) and then dried (40 °C, 24 h). SF2 was directly obtained after dry milling the whole sorghum grains to powder with a coffee grinder and sieving over 500 µm sieve.

Preparation of sorghum kafirin

A combined method of Kumar et al. (2014) and Emmambux and Taylor (2003) was used to extract SK1 at 50 °C for 1 h from SF1 using 70% (V/V) aqueous ethanol containing 0.1% (w/v) sodium metabisulfite and. 0.1% (w/v) sodium hydroxide. After iterative precipitation, resolubilization, centrifugation and drying, the protein extract was defatted with hexane at room temperature and air dried to obtain SK1.

The extraction of SK2 was carried out from SF2 according to Kumar et al. (2014). SK2 was then washed three times with distilled water at a ratio of 1/10 (w/v) for 15 min at 50 °C. The suspension was then centrifuged at 3000×g for 15 min and dried at 30 °C in an oven over night and then defatted. All extractions were performed in duplicate.

Protein content

Protein content of SF1, SF2, SK1 and SK2 were determined using micro-Kjeldahl method according to AACC method n^o 46-13 (AACC 2000). The protein content was estimated using a conversion factor of 6.25 (Johns and Brewster 1916). The moisture content of all samples was determined according to AACC method n^o 44-15A (AACC 2000). All measurements were performed in duplicate.

Determination of the extraction yield

The kafirin extraction yield (Y%) was defined as the weight of SK1 or SK2 per weight of protein in SF before extraction as described by Wang et al. (1999).

$$\text{Y}\%=\frac{\text{weight}\text{of}\text{kafirin}\times \text{protein}\text{content}\text{of}\text{kafirin}}{\text{weight}\text{of}\text{SF}\times \text{protein}\text{content}\text{of}\text{SF}}\times 100$$ 1

Determination of water and oil binding capacity

WBC and OBC were determined using the method of Beuchat (1977). SF1, SF2, SK1 and SK2 were suspended in 10 mL distilled water for WBC or corn oil for OBC at protein concentration of 3% (w/v) into pre-weighed 15 ml centrifuge tubes and mixed with a vortex until full wetness. Samples were allowed to stand at room temperature for 1 h, then the suspensions were centrifuged at 3000×g for 20 min. The supernatants were discarded, while the precipitates were weighted. WBC was expressed as grams of water per gram of dry sample. OBC was expressed as grams of oil per gram of dry sample as follows:

$$\text{WBC}\left(\frac{\text{g}}{\text{g}}\right)=\frac{{\text{W}}_2-{\text{W}}_1}{{\text{W}}_0}$$ 2

and

$$\text{OBC}\left(\frac{\text{g}}{\text{g}}\right)=\frac{{\text{W}}_3-{\text{W}}_1}{{\text{W}}_0}$$ 3

where W₀ was the weight of the dry sample (g), W₁ was the weight of the tube and the dry sample (g), W₂ was the weight of the tube and residue (g) after water binding and W₃ was the weight of the tube and residue (g) after oil binding.

Determination of gelling properties

The least gelling concentration (LGC) was determined by the method of Sathe et al. (1982), using increasing SF or kafirin concentrations of 2, 4, 6, 8, 10, 12, 14, 16, 18 and 20% (w/v) in distilled water. The LGC is defined by the concentration allowing the sample to stand up without falling when the test tube was inverted.

Determination of foaming properties

The foaming properties expressed as foam capacity (FC) (%) and foam stability (FS) (%) were determined according to Coffmann and Garcia (1977) with minor modifications. SF1, SF2, SK1 or SK2 (0.2% (w/v)) were suspended in 9.0 mM sodium phosphate buffer containing 35 mM NaCl (pH 7.0). 100 ml of each suspension was placed in a graduated glass cylinder (250 mL) and whipped during 100 s at 2000 rpm at room temperature. FC is defined by the foam volume increase on whipping expressed as percentage of original liquid volume. FS was expressed as percentage of foam volume remaining after 5, 10, 15, 20 or 30 min at room temperature per the initial foam volume. FC and FS were calculated as follows:

$$\text{FC}\left(\%\right)=\frac{{\text{V}}_0-\text{V}}{\text{V}}\times 100$$ 4

and

$$\text{FS}\left(\%\right)=\frac{{\text{V}}_{\text{t}}}{{\text{V}}_0}\times 100$$ 5

where V and V₀ are respectively the foam volume before and after whipping, V_t is the foam volume remaining after 5, 10, 15, 20 or 30 min.

Determination of emulsifying properties

Emulsifying properties are defined by the emulsifying activity index (EAI) and emulsifying stability (ES).

EAI (m²/g) and ES (%) of SF1, SF2, SK1 and SK2 were measured by the turbidimetric method as described by Pearce and Kinsella (1978) with minor modifications. The emulsions were prepared by adding 7.0 mL of corn oil to 21.0 mL of protein suspension. About 0.2% (w/v) of sample was dissolved in 9.0 mM sodium phosphate buffer containing 35 mM NaCl (pH7.0). The mixture was homogenised for 1 min at room temperature, a 50 µL sample was withdrawn from the bottom of the cup and diluted in 5.0 mL of a 0.1% (w/v) SDS solution. The absorbances of the diluted emulsions were measured at 500 nm immediately and 30 min after emulsion formation. The EAI was calculated as follows:

$$\text{EAI}\left(\frac{{\text{m}}^2}{\text{g}}\right)=\frac{2\times 2.303\times \text{A}\times \text{D}}{\Phi \times \text{L}\times \text{C}\times 1000}$$ 6

where A is the absorbance at 500 nm, D is the dilution factor (D = 100), Φ is the oil volumetric fraction (Φ = 0.25), L is the curve path length (L = 0.01 m), C is the protein concentration of the sample (g/mL).

The emulsifying activities indexes were measured immediately (EAI₀), after 10 min (EAI₁₀) and after 30 min (EAI₃₀), respectively. ES is the percentage of emulsion absorbance remaining after 30 min.

$$\text{ES}=\frac{{\text{A}}_{30}}{{\text{A}}_0}\times 100$$ 7

where A₀ and A₃₀ are the absorbance at 500 nm after 0 and 30 min of emulsion formation, respectively. All samples were analyzed in duplicate.

Fourier transform infrared spectroscopy

Spectra of protein concentrates were obtained with a Fourier Transform Infrared (FTIR) spectrometer (Spectrum two L1600301, Perkin Elmer, USA) equipped with a universal attenuated total reflectance (ATR) sampling accessory. The spectra were recorded at 1 cm⁻¹ resolution within the frequency range of 4000–450 cm⁻¹. Air in the empty crystal was used as background. The spectral data were processed by baseline correction and the peak label normalized within the amide I region (1600–1700 cm⁻¹). Fourier self-deconvolution (FSD) was carried out on the normalized amide I region with the software Spectrum 10 using 1.5 as K factor. Second derivative functions of SK1 and SK2 were obtained with PeakFit software V4.12 at smoothing points 17.9% of the FSD spectra. The quantification of protein secondary structures were obtained from the representative Gaussian curve-fitted mini-bands areas of the individual assigned bands and their fraction of the total area in the amide I regions expressed as percentages. The ratios of α-helix to intermolecular β-sheet and amide I to amide II were calculated to indicate changes in the secondary structure of the samples. All measurements were achieved in duplicate.

Results and discussion

Protein content and yield

The protein content on dry matter basis (d.m.b) of all sorghum samples SF1, SF2, SK1 and SK2 were measured and compared. Figure 1a represents the protein content of SF1, SF2, SK1 and SK2 and the yield of kafirin extraction. The dry milling of SF (SF2) allowed the extraction of higher protein content than wet milling (SF1) 13.77 ± 0.50 and 11.09 ± 0.38%, respectively. This difference could probably be due to the albumin and globulin fraction solubilisation in water during the process of wet milling. In a previous work, Mokrane et al. (2009) evaluated this fraction by Dumas protein analysis and by high performance liquid chromatography in the same sorghum landrace (Tafsout el beida), the albumins and globulins constituted almost 18.2% of the total extracted protein fraction which is mainly the same percentage of decrease in protein content in the present study.

Fig. 1.

a Protein content of wet and dry milled sorghum flour and kafirin and yields of kafirin extraction, b Water binding capacity (WBC) and oil binding capacity (OBC) of wet and dry milling sorghum flour and extracted kafirin. SF1, wet milled sorghum flour; SF2, dry milled sorghum flour; SK1, kafirin extracted from wet milled sorghum flour; SK2, kafirin extracted from dry milled sorghum flour

High protein contents on d.m.b were obtained in the two extracted kafirins SK1 and SK2. However, the dry milling allowed better kafirin extraction (94.23 ± 1.43%) than the wet milling (90.07 ± 0.44%). The highest kafirin purity achieved so far was 99.2% by Kumar et al. (2014). In the meantime, lower protein content in the kafirin extracted from whole sorghum grain was also reported by Espinosa-Ramírez and Serna-Saldívar (2016) and by Musigakun and Thongngam (2007) which were 81.5–93.3%, 88.3–88.7% and 77.48–83.13%, respectively. These differences could probably be due to the sorghum genotype and the extraction conditions. The kafirin extraction yields were 64.93 ± 4.11 and 58.68 ± 5.20% for SK1 and SK2, respectively. These results might indicate that wet milling and the ethanol purification step improved the yield of SK1 extraction. In a similar study, Espinosa-Ramírez et al. (2017) found lower yield of extraction in the wet milled sorghum flour with or without protease treatment. This is probably due to the pretreatment processes used such grain decorticating, starch recovery and Alcalase treatment before kafirin extraction which may cause the lost of almost the third part of kafirin contents.

In the light of these results, it could be assumed that both the milling and purification processes affect the kafirin purity and the yield of extraction. According to the end use, extraction of sorghum kafirin could be direct to the adequate milling process. The wet milling is particularly required for efficient starch isolation before or after protein purification.

Water and oil binding capacity

During food preparation, proteins interact with water and oil and deeply affect the food texture and flavor. WBC and OBC are functional properties that evaluate the ability of a protein to bind water or oil (Zayas 1997). High values of WBC are desirable for enhancing viscosity and thickening without protein dissolution. Figure 1b shows the WBC and OBC values of SF1, SF2, SK1 and SK2. WBC values of the SF1 and SF2 were 1.37 ± 0.01 and 1.42 ± 0.15 g water/g, respectively on d.m.b. Wet milling slightly decreased the WBC of SF. The obtained WBC values of SF were in the range but slightly higher than those obtained on untreated SF by Elkhalifa and Bernhardt (2010) and more recently by Ojha et al. (2018) which were 1.31 and 1.27 g water/g, respectively on d.m.b. WBC values of SK1 and SK2 were higher than those of SFs and were 2.20 ± 0.10 and 1.82 ± 0.22 g water/g, respectively on d.m.b. Accordingly, SK1 and SK2 seem to have a better ability to bind water compared to SF1 and SF2 (Fig. 1b). Moreover, the extraction method of SK1 improved the ability of kafirins to bind water. In a similar study, Elkhalifa et al. (2016) obtained comparable WBC results. However, higher values of kafirin WBC were obtained by Espinosa-Ramírez and Serna-Saldívar (2016) and Musigakun and Thongngam (2007) 1.99–2.48 and 3.5 g water/g, respectively on d.m.b and more recently by Espinosa-Ramírez et al. (2017) with 2.82 ± 0.10 g water/g d.m.b for kafirin extracted from sorghum gluten meal of decorticated grains. One possible reason for the lower WBC values of SK1 and SK2 could be their high protein purity obtained in the present study and their high hydrophobocity.

As shown in Fig. 1b, wet milling increased the OBC values of SFs by about 31%. The obtained capacity to bind corn oil of both dry and wet milled SF were higher than those obtained by Ojha et al. (2018) recently for whole, malted or fermented dry milled SF. The OBC values of SK1 and SK2 were 1.41 ± 0.01 and 1.34 ± 0.03 g oil/g protein, respectively. In addition, the extraction method of SK1 seemed to improve kafirin OBC values by 5%. The obtained OBC values of kafirin in the present study were lower than those reported previously (Elkhalifa et al. 2016; Espinosa-Ramírez and Serna-Saldívar 2016; Musigakun and Thongngam 2007). SK1 and SK2 exhibited higher OBC than SF1 and SF2. The higher oil content in SFs may lead to a lower tendency for oil binding, through the possible blocking of the hydrophobic sites. The obtained WBC and OBC values of both SF and their respective extracted kafirins were comparable to those obtained for lupin seed flour and its protein concentrate (Sathe et al. 1982). However, they were lower than those obtained by Makeri et al. (2017) for soybean and winged bean protein concentrates and by Chandi and Sogi (2007) for rice bran protein and casein.

Gelling properties

Good gelling properties are requisite for many food formulations. Higher gelling concentrations are desirable for infants weaning foods (Singh et al. 2017). While lower gelling concentrations are researched to be used as ingredient to form a curd or as an additive to other gel forming materials in food products (Chandra et al. 2015). The gelling properties are related to the type of protein as well as to the contents of non-protein components (Elbaloula et al. 2014). The ability of SF1, SF2, SK1 and SK2 to form a gel was measured and the LGC was marked as (100%) in Table 1. The obtained LGC value for SF1 was 6%, while it was 16% for SF2. These results were similar to those obtained by Elbaloula et al. (2014) and by Elkhalifa et al. (2005) for non germinated or unfermented SF. In the present study, the wet milling increased the gelling ability of SF 2.67 times while Elkhalifa et al. (2005) stated that fermentation increased the gelling properties only 2.25 times. The good gelling properties of the wet milled SF might be related to its particular composition characterized by the absence of the albumin and globulin fraction. However, no gelling was observed for the two sorghum kafirins SK1 and SK2.

Table 1.

Gelation properties of sorghum flours and sorghum kafirins

Concentration (%) (w/v)	2 (%)	4 (%)	6 (%)	8 (%)	10 (%)	12 (%)	14 (%)	16 (%)	18 (%)	20 (%)
SF1	< 50	> 50	100	100	100	100	100	100	100	100
SF2	0	< 50	< 50	< 50	> 50	> 50	> 50	100	100	100
SK1	0	0	0	0	0	0	0	0	0	0
SK2	0	0	0	0	0	0	0	0	0	0

0%: Not gelled, < 50%: Gelled less than 50%, > 50%: Gelled more than 50%, 100%: Gelled

From the above results, it could be concluded that kafirin extraction method did not seem to affect its ability to form a gel. Some proteins such kafirin did not exhibit gelling properties but they might be used as gelling enhancers when they are added. For example, bovine serum albumin (BSA) is the gel-forming protein in cheese whey, its gelling capacity increased when α-lactalbumin was added (Zayas 1997). Wet milling of SF is favorable to its application in weaning foods. However, dry milling seems to be more appropriate to be used as gelling enhancer additive.

Foaming properties

According to Zayas (1997), during foam formation food palatability and smoothness along with flavor volatilization are improved by the protein distribution in the air bubbles. Hence, the foaming properties of protein concentrates are always researched. In the present study, SF1 formed instable foam with an instant high FC value of 26.5% and no FS. While in a previous work, Elkhalifa and Bernhardt (2010) stated that no FC was detected on Sudanese wet milled SF and after 2 days of steeping only a low FC value of 2.25% was detected.

In this study, in contrast to wet milling, the dry milling allowed forming stable foam in SF2 achieving FS value of 77% after 30 min with a lower FC value of 14% as shown in Fig. 2. These results might indicate that the wet milling increased the water surface tension and consequently reduced the foam formation as previously described by Elkhalifa et al. (2005). According to Day (2013), good foaming properties were found in plant protein fractions rich in albumins. Thus, the differences in foaming properties between SF1 and SF2 shown in the present study may probably be due to that wet milling led to a high decrease in albumin fraction in SF1 and consequently led to a decrease in foaming properties.

Fig. 2.

Foaming capacity (FC) and foam stability (FS) of SF2 the dry milled sorghum flour

In contrast, the extracted sorghum kafirins SK1 and SK2 formed negligible foam which disappeared once the mixing process stopped. In a similar study, Elkhalifa et al. (2016) found a low FC of 2.75% and no FS in kafirin extracted from high tannins sorghum. The low solubility of kafirin in aqueous solutions is probably the main reason for its low FC and FS. The obtained FC values may be due to the remaining non protein components in the kafirin extract (Kato and Nakai 1980).

Emulsifying properties

Good protein emulsifying properties are desired during food manufacturing of milk beverage and meat analogs (Zayas 1997).

The EAI values at 0, 10 and 30 min and ES for SF1, SF2, SK1 and SK2 are illustrated in Fig. 3. SF1 exhibited slightly higher EAI₀ values than SF2 14.79 and 13.17 m²/g, respectively. Whereas in term of stability, SF2 showed the highest ES (74.60%) and SF1 the least one (33.88%). After 30 min, EAI₃₀ values of SF1 became 2.2 times lower than that of SF2. Consequently, it can be assumed that wet milling highly decreased the stability of the sorghum flour emulsifying properties; this is probably due to the starch volume increase during SF wet milling.

Fig. 3.

Emulsifying activity index (EAI) and emulsifying stability (ES) of sorghum flours and sorghum kafirins treated by wet or dry milling. SF1, wet milled sorghum flour; SF2, dry milled sorghum flour; SK1, kafirin extracted from wet milled sorghum flour; SK2, kafirin extracted from dry milled sorghum flour

As shown in Fig. 3, SK1 and SK2 showed comparable EAI₀, EAI₁₀ and EAI₃₀ values slightly higher for the kafirin extracted from dry milled SF. Results obtained in the present study were much higher than those reported by Espinosa-Ramírez and Serna-Saldívar (2016) for kafirin-rich protein extracts from different sorghum genotypes which varied from 1.08 to 1.73 m²/g, with no stability. While ES values of the two kafirin extracts SK1 and SK2 were high and exceeded 60%. These interesting emulsifying properties could probably be due to the kafirin hydrophobicity and the higher protein purities obtained in this study. The type of oil used and the genotype could also be a reason for this difference (Belton et al. 2006; de Mesa-Stonestreet et al. 2010; Espinosa-Ramírez and Serna-Saldívar 2016; Mokrane et al. 2009). A strong correlation between emulsifying capacity and hydrophobicity of proteins was observed by Kato and Nakai (1980) wich could explain our findings. The wet milling decreased the emulsifying properties of SF compared to the dry milling and the two extraction method of kafirin seemed having a slight effect on EAI and ES.

FTIR

One of the major applications of the FTIR technique is the estimation of protein secondary structure. Figure 4 represents the FTIR spectra in the amide I region of kafirin extracted from SF treated by wet or dry milling.

Fig. 4.

Analysis of the FT-IR spectra in the amide I region of kafirin extracted from sorghum flour treated by wet or dry milling. a SK1, kafirin extracted from wet milled sorghum flour; b SK2, kafirin extracted from dry milled sorghum flour

The FTIR spectra of SK1 and SK2 were similar with a small displacement in wavelengths. The characteristic absorption bands of a protein are the amide A, B, I, II, III, IV and VI. The most important differences were detected in two regions Amide I and II. Amide I appeared between 1600 and 1700 cm⁻¹ resulting primarily from C=O stretching vibrations and possibly from C–N stretching and C(C–) deformation, while Amide II appeared between 1480 and 1575 cm⁻¹ corresponding to C–N stretching and N–H bending (Kong and Yu 2007).

The interesting sensitivity of the amide I band to small variations in molecular geometry makes its exclusivity for the estimation of the protein secondary structure (Kong and Yu 2007). The absorption peaks of SK1 and SK2 appeared at 1633 and 1624 cm⁻¹, respectively. In the amide I region, four major structures were identified; intermolecular β-sheet (anti parallel β-sheet), random coil, α-helix and β-turn which were represented by the components centered in the regions of 1625–1635 cm⁻¹, 1638–1640 cm⁻¹, 1648–1659 cm⁻¹ and 1678–1682 cm⁻¹, respectively (Xiao et al. 2014). The analysis of FTIR spectra in the amide I region for SK1 and SK2 are shown in Fig. 4 and the percentages of their different secondary structures based on Gaussian curve fitting are reported in Table 2. SK1 had amide I absorption at 1654.92 and 1642.81 cm⁻¹ indicating α-helix and random coil absorption with 22.10 and 17.25% of the total area, respectively. The band absorption at 1628.72 cm⁻¹ due to intermolecular β-sheet constituted 23.92% of the SK1 secondary structure. The β-turns absorption region showed three bands at 1665.97, 1676.49 and 1686.41 cm⁻¹ with a total of 31.21% of the protein structure. As shown in Table 2, dry milling and purification of SK2 with water revealed higher contents of the random coil and α-helix structure of 51.94% corresponding to 25.49% of α-helix and 26.45% of random coils, while wet milling exhibited only 39.35%. However, wet milling induced an increase in the intermolecular β-sheet and β-turns content from 21.32 to 23.92% and from 22.26 to 31.21%, respectively. These detected decreases in wet milled samples could be due to the changes in conformation from random coil and α-helix conformation (The predominant conformation in SK1 and SK2) into intermolecular β-sheet and β-turns conformation. The random coil and α-helix percentage of SK2 were higher than those obtained by Espinosa-Ramírez et al. (2017), Xiao et al. (2014) and Georget et al. (2012). This probably means that the kafirin obtained after dry milling was more related to a native form of kafirin. In the present study, SK1 and SK2 ratio of α-helix to intermolecular β-sheet were 1.00 and 1.25, respectively. These results were slightly higher than those observed by Espinosa-Ramírez et al. (2017) which ranged from 0.96 to 1.07 (Table 2) for kafirin extracted from dry milled decorticated sorghum flour and 0.87 for kafirin extracted from decorticated sorghum gluten meal. According to Gao et al. (2005), the high level of α-helix may confer to the kafirin extract more heat stability and a good ability to film formation. In contrast, the high level of β-sheet might indicate a high protein denaturation and a tendency to aggregation.

Table 2.

Percentages of the different secondary structures of SK1 and SK2 based on Gaussian curve fitting

Structural properties	Present studya		Gao et al. (2005)a	Espinosa-Ramírez et al. (2017)a		Xiao et al. (2014)b	Georget et al. (2012)a
	SK1	SK2		I	II
Intermolecular β-sheet (%)	23.92 ± 1.65	21.32 ± 1.81	–	36	42	24	40
α-helix (%)	22.10 ± 3.28	25.49 ± 2.93	–	–	–	–	–
Random coil (%)	17.25 ± 8.84	26.45 ± 4.67
Random coil and α-helix (%)	39.35 ± 5.55	51.94 ± 7.60	–	35	33	49	39
β-turns (%)	31.21 ± 1.66	22.26 ± 5.37	–	28	25	27	22
α-helix/intermolecular β-sheet ratio	1.00 ± 0.04	1.25 ± 0.23	0.96	0.96–1.07	0.87	–	–
amide I/amide II ratio	1.16 ± 0.08	1.21 ± 0.01	–	–	–	–	–

I: kafirin extract from decorticated sorghum flour (dry milling) and II: kafirin extract from decorticated sorghum gluten meal (wet milling)

^aEthanol extraction

^bUltrasound-assisted tert-butanol extraction

The ratio of Amide I/Amide II is related to the change in the kafirin secondary and tertiary structures. For SK1 and SK2, these ratios were 1.16 and 1.21, respectively. Hence, dry milling affected the global secondary and tertiary structures of kafirin more than that of wet milling. However, this ratio was lower than those observed by Espinosa-Ramírez and Serna-Saldívar (2016) on kafirin extracted from several sorghum genotypes which ranged from 1.42 to 1.46. This is probably due to the differences in the isolation procedures.

In the light of our findings, dry milling of SF seemed preserving the native form of kafirin compared to wet milling. The difference between SK1 and SK2 may be due to the structural transition from native to aggregated proteins (Espinosa-Ramírez and Serna-Saldívar 2016).

Conclusion

In the present study, the effect of wet and dry milling on the functional properties of SFs and on their respective extracted kafirin was investigated and compared to previous reports. Wet milling decreased the SF protein content, the WBC and LGC while it increased the OBC and the FC but in the meantime decreased the FS and ES. The dry milling allowed obtaining better kafirin extractability exceeding 94% in terms of purity and higher ES. No foam or gelling properties were obtained in the two extracted kafirins from wet or dry milled SFs. Kafirin FTIR analysis showed that dry milling preserved α-helix structure and then the native form of the extracted proteins. In contrast wet milling increased the intermolecular β-sheet and β-turns content allowing forming more aggregated proteins.

The functional properties of SF and extracted kafirin might be a starting point to their applications in many food and non-food products, particularly for vegan communities and celiac patients. According to the end use, the SF processing could be oriented to the wet or dry milling depending on the desired outcome. As wet milling allows extracting kafirin and starch in the same time without affecting the most important kafirin functional properties, it would be more appropriate to be used in the future.