Total dietary protein content and Osborne protein fractionation

In document Compositional and nutritional properties of tef and tef-based food products (Page 168-173)

CHAPTER 5: TEF PROTEIN: SOLUBILITY CHARACTERIZATION, IN VITRO DIGESTIBILITY AND ITS SUITABILITY AS A GLUTEN-FREE INGREDIENT

5.4.1 Total dietary protein content and Osborne protein fractionation

The total protein content (Table 5.1) of the tef varieties varied from 8.5-9.4 g/100 g dm flour. Total protein contents in the range of 10-11.6 g/100 g were reported for different tef varieties (Adebowale et al., 2011; Bultosa, 2007), using a 6.25 nitrogen to protein conversion factor unlike to the 5.4 used in our case. The difference in the conversion factor could be the reason for the slight variation in the results. The nitrogen to protein conversion factor for cereals ranged from 5.3 to 5.8, while 5.4 is considered as the most appropriate and accepted value for cereals (Mariotti et al., 2008). From a nutritional point of view, tef could be seen as a good protein source as its total protein content is either fairly higher or equal to the other common cereals (Gebremariam et al., 2012). Although not analyzed in our study, tef also contains high amounts of some essential amino acids such as lysine, methionine and valine (Gebremariam et al., 2012), which are limited in many cereals.

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Figure 5.1 shows the SDS–PAGE protein molecular weight distribution profile of whole tef, gluten-free and white wheat flour protein extracts. The major protein bands of all the tef varieties were observed between a molecular weight of 14.4-66.2 kDa. Similarly, major bands of tef protein extracts were seen in the region of 17-60 kDa (Moroni et al., 2010). Unlike the tef varieties which have diverse low molecular weight (LMW) and high molecular weight (HMW) proteins, the wheat used as a comparison showed its major protein bands between 66.2-116.2 kDa representing proteins with HMW.

Figure 5.1 SDS–PAGE of whole tef, gluten-free and white wheat flour protein extracts

(1,7 and 12: Molecular weight marker; 2: Boset; 3: Dega; 4: Quncho; 5: Simada; 6: Tsedey; 8: Zagurey; 9: Zezew; 10: Wheat; 11: Gluten-free flour).

The Osborne solubility fractions of different tef varieties are shown in Table 5.1. The contents of storage proteins: albumin, globulin, prolamin and glutelin ranged from 5.4 to 8.7, 9.6 to 13, 2 to 2.5 and 0.3 to 0.6 g/100 g dm, respectively among the varieties. The total recovery of total protein content after the Osborne fractionation was very low, ranging from 18 to 25% among the different varieties. These recoveries are low, especially when compared to the used reference (commercial white wheat flour), having a total protein recovery of 85.7%. Albumin and globulin contributed to 86-90% of the recovered total proteins in tef whereas the major contribution (78%) of wheat proteins is from prolamin and glutelin.

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Table 5.1 Proteina, glutenb and storage protein of tef, wheat and gluten-free flours

Varieties Protein Gluten Tef storage proteins (g/100 g total protein)

Albumin Globulin Prolamin Glutelin Recovery%

Boset 9.4±0.1f 14.3±0.2b 6.1 ± 0.9a (25.3) 9.6 ± 1.0a (29.0) 2.1 ± 0.2 (12.1) 0.59 ± 0.23b (3.03) 18 ± 2a Dega 8.7±0.1b 8.3±0.9a 8.7 ± 0.4b (24.2) 12.9 ± 0.8b (48.1) 2.5 ± 0.3 (9.63) 0.42 ± 0.12ab (2.09) 25 ± 2b Quncho 8.9±0.01c 7.6±1.1a 5.4 ± 0.5a (12.5) 11.3 ± 1.9ab (28.5) 2.1 ± 0.8 (12.1) 0.27 ± 0.01a (0.59) 19 ± 0.1a Simada 8.8±0.01bc 7.4±1.5a 6.2 ± 0.8a (25.3) 11.8 ± 0.6ab (34.4) 2.3 ± 0.2 (8.60) 0.25 ± 0.14a (1.04) 21 ± 2ab Tsedey 9.1±0.1d 8.7±0.7a 6.4 ± 0.5ab (15.1) 9.6 ± 1.2a (28.8) 2.1 ± 0.3 (7.03) 0.34 ± 0.01ab (0.75) 18 ± 2a Zagurey 8.5±0.1a 12.6±2b 7.3 ± 1.5ab (22.8) 10.8 ± 1.1ab (26.2) 2.1 ± 0.3 (11.1) 0.51 ± 0.01ab (2.20) 21 ± 3ab Zezew 9.3±0.01e 14.5±1.1b 7.2 ± 0.5ab (25.0) 11.9 ± 0.6ab (48.2) 2.0 ± 0.7 (8.64) 0.27 ± 0.10a (1.87) 22 ± 1ab p-value 0.021 < 0.001 0.010 0.019 0.784 0.004 0.026 Wheat 9.32±0.17 > 100 14.43 ± 1.1 (29.0) 4.07 ± 0.2 (90.8) 28.63 ± 4 (83.2) 38.57 ± 0.5 (22.4) 85.70 ± 6 GF < 4

a,b,c,d,e,f Values within a column with different superscripts are significantly different (p < 0.05). Values in brackets indicate the purity of the storage protein extracts (g storage

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There are only a few but conflicting reports regarding Osborne solubility fractions of tef proteins. Adebowale et al. (2011) has reported 10-12% albumin + globulin, 38-43% prolamin and 20-24% glutelin in three tef varieties. On the other hand, Mulugeta 1978, as cited in Ketema (1997) has reported 37% albumin, 7% globulin, 12% prolamin and 45% glutelin as fractions present in tef.

Both of these reports considerably disagree to each other and to our results. This could be attributed to the use of different solvents. Tert-butanol 60% (v/v) with DTT as reducing agent was used to extract prolamin in the work of Adebowale et al. (2011) while 60% and 70% of ethanol was used (Mulugeta, 1978) as cited in Ketema (1997) and in our study, respectively. Osborne fractions from proteins of different cereals will have a different amino acid composition which certainly affects the efficiency of the extracting solvents used. This is evidenced in our study that the 70% (v/v) ethanol and 0.02 M acetic acid were fairly efficient in extracting relatively very high prolamin and glutelin, respectively from wheat while this amount was very low in the case of tef samples.

Higher efficiency of prolamin extraction in tef was attributed to the use of tert-butanol solvent assisted by DTT (Adebowale et al., 2011) a less polar solvent than ethanol. The fact that ethanol is used in our experiment and was able to extract high amount of prolamin from wheat but not from tef, may indicate that tef prolamin is less polar than that of wheat prolamin. However, there is no evidence that prolamin in gluten-free cereals or in tef has less polar properties than those in gluten containing cereals. Therefore, the reason why ethanol (70%) is efficient in extracting wheat prolamin but failed to extract tef prolamin is unclear. It has been stated that the content of the Osborne fractions are not clear-cut and varies considerably and depends on genotype and growing conditions of the starting materials and experimental conditions leading to different and sometimes contradictory results (Koehler and Wieser, 2013). This could be consolidated by significantly varied Osborne solubility within the same and among different cereals as reviewed by Janssen et al. (2016).

Globulin showed higher recovery rate compared to the other three storage protein which can be justified as follows: The tef flour was milled as whole which makes it to be rich in aleurone layer and germ, besides it was very fine in that 66-77% of the flour was able to pass through a sieve of 150 µm pore size. Both these factors could lead to a higher solubility of globulins and its overall higher proportion in the tef protein.

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The SDS-PAGE of Osborne fractions of two white (Quncho and Tsedey), two brown (Zagurey and Zezew) tef varieties and a white wheat flour are shown in Fig. 5.2A, 5.2B and 5.2C, respectively. There was a visible difference among the lanes of the extracts of the white wheat flour protein and each of the Osborne fractions of tef varieties. There was also a clear difference among the lanes of the four Osborne fractions within a variety and among the tef varieties.

Figure 5.2 SDS-PAGE of storage proteins of tef and wheat

A: SDS-PAGE Osborne fractions Quncho and Tsedey (1,7: Molecular weight marker; 2: Quncho whole flour; 3: Quncho albumin; 4: Quncho globulin; 5: Quncho prolamin; 6: Quncho glutelin; 8: Tsedey whole flour; 9: Tsedey albumin; 10: Tsedey globulin; 11: Tsedey prolamin; 12: Tsedey glutelin), B: SDS-PAGE Osborne fractions Zagurey and Zezew (1,7: Molecular weight marker; 2: Zagurey whole flour; 3: Zagurey albumin; 4: Zagurey globulin; 5: Zagurey prolamin; 6: Zagurey glutelin; 8: Zezew whole flour; 9: Zezew albumin; 10: Zezew globulin; 11: Zezew prolamin; 12: Zezew glutelin), C: SDS-PAGE Osborne fractions wheat (1,7: Molecular weight marker; 2: Wheat whole flour; 3: Wheat albumin; 4: Wheat globulin; 5: Wheat prolamin; 6: Wheat glutelin).

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The major bands of each of the corresponding Osborne fractions showed a close similarity across all the tef varieties. Albumin fractions of each of the tef varieties showed bands with the highest molecular weight range between 14.4-97.4 kDa. Next to the albumin fraction, globulin showed the highest range of major bands between 14.4-66.2 kDa in all the varieties with an approximate MW of 15.4, 15.8, 16.2, 33 46 and 51.3 kDa. The major bands of prolamin fractions are situated between 16.4 and 46.3 kDa in all the tef varieties. Unlike to the other fractions, glutelin showed less major bands and even with no visible band appeared in the case of Zezew variety. The SDS-PAGE of a white wheat flour whole protein extract and Osborne fractions are used for comparison.

There is scarcity of literature on the SDS-PAGE molecular characterization of total protein and Osborne fractions of tef. The literature so far only focused on prolamin fraction and showed that major proteins of prolamins in different tef varieties have approximate MW of 20.3 and 22.8 kDa (Adebowale et al., 2011) and 22.5 and 25.0 kDa (Tatham et al., 1996). These results are in close agreement with some of the major bands of the prolamin fraction in our study.

In document Compositional and nutritional properties of tef and tef-based food products (Page 168-173)