Arbaminch Agricultural research Center, Southern Agricultural Research Institute, Ethiopia
Article Type: Research Article
Manuscript ID: FNOA-1-110
Publisher: Boffin Access Limited.
Journal Type: Open Access
Copyright: © 2018 Dureto E. This is an openaccess article distributed under the terms of the Creative Commons Attribution 4.0 international License
Dureto E. Nutrient Availability in Deep Fried Flat Breads Prepared with Bran Enriched and Mineral Fortifed Whole and Refned Wheat Flour. Food Nutr OA. 2018 Aug;1(2):109
The objective was to determine nutrient availability in wheat based deep fried flat breads enriched with wheat bran (WB) and/or minerals. Products prepared either with whole (WWF) or refined wheat (RWF), enriched with WB (10 or 20%) and fortified with either iron or zinc (10 or 20 mg/100 g) were analyzed for total and in vitro digestible/ bioaccessible nutrients (protein, starch, iron, zinc, calcium). Flavor boosting ingredients prepared and mixed with cooked common bean to prepare ful( breakfast food in Ethiopia) usually prepared from faba bean and consumed with bread. There was no significant difference in sensory characteristics using a five-point hedonic scale of prepared dishes. Sensory evaluation indicated that overall acceptability decreased as common bean processed into flour due to its beany flavor.
Refining; Deep frying; Bioaccessible minerals; Digestible protein; Digestible starch; Nutritional composition
Iron deficiency anemia is recognized as public health problem throughout the developing
world with prevalence rate as high as 40% in women and children in Asian region [1,2].
While the causes of anemia are diverse, a low dietary intake and poor bioavailability are
prime causative factors. Zinc is another micromineral recognized to be deficient, especially
in wheat eating populations because of low bioavailability from wheat based diets [1-3].
Fortification of wheat flour with iron, folic acid and other micronutrients in countries where
wheat is used as a staple, has been recommended as a strategy to prevent, control and
overcome anemia by WHO [4,5]. The level of mineral salts used as well as its bioavailability
are important for the fortification to be effective.
Wheat grain is milled to flour or its fractions before consumption. The composition of flour varies greatly depending upon the way it is milled . The process of refining removes dietary fiber which is related to many health benefits and is recognized more as an essential nutrient than non-nutrient on account of its antioxidant effects [7-11]. Refining also brings down the level of many other nutrients in flour which lowers its overall nutritional quality. This can be partially corrected by addition of extra nutrients and dietary fiber to refined flours [12-14]. In addition, further processing into products can also influence the nutrient content or availability. Various thermal processing techniques such as baking, roasting, steaming, shallow frying and deep frying are used for preparing wheat products, either industrially or for domestic use. The bioavailability of iron also depends upon the food matrix, which can be altered by processing conditions. Dietary fiber and phytates are known to interfere with mineral absorption.
In Indian diets, ‘Chapatis’ (pan roasted flat breads) and ‘ Poories’, (deep fried unleavened flat bread) are two popular wheat based products used frequently as part of main meals or snacks . The objective of this study was to explore the extent of nutrient availability from Poories prepared from whole and refined wheat flour enriched with wheat bran and fortified with iron or zinc at different levels, either alone or in combination.
The raw materials needed for the study, namely, whole wheat (Triticum aestivum) and refined sunflower oil (Helianthus annuus) were procured from local market. Wheat was purchased in a single batch and subjected to differential milling in a roller mill (plate milling-Buhler, MLV 202, Switzerland) to obtain whole wheat flour, refined wheat flour, and wheat bran separately. Wheat bran was further powdered in a grinder to reduce particle size. These were stored in airtight container under refrigeration and used for preparation of products. Sunflower oil was used for deep frying of products. All chemicals needed for the analysis were of analytical grade and procured from SD Fine Chemicals and Qualigens Chemicals India. All analysis of constituents was carried out in triplicate and bioaccessibility studies were done in duplicate samples. Glass double distilled water was used for all experiments and care was taken to avoid any direct contact of samples with metalware as samples were analyzed for micro minerals. The enzymes used for the study were pepsin (Batch No. 3-0060), pancreatin (Batch No. 0-0864), diastase (Batch No. 0695/195/270511) and papain (Batch No. 0993/493/130811). For in vitro mineral bioaccessibility studies, the dialysis tubing was procured from Sigma Aldrich Co. USA with a molecular mass cut off of 8000 kDa
The study design consisted of preparation of unleavened flat deep fried Indian bread known as ‘ Poories’ with whole (WWF) or refined wheat flour (RWF). These were enriched with wheat bran (WB) (used for replacing basic wheat flour) and fortified either with iron (ferrous sulphate) or zinc (zinc sulphate) at two levels. A total of 22 products, eleven each from WWF and RWF were prepared as detailed below. Formulation of products: For preparation of Poories, WWF or RWF along with WB and/or fortificants as required were mixed with water and kneaded well to make dough. Dough was kept aside for 10 min. small portions were rolled into thin sheet (0.2 cm thickness) and deep fried in hot sunflower oil (temperature, 170 ± 2°C) till done. These were cooled, deoiled using petroleum ether, dried in oven and used further for analysis. A record of moisture and oil uptake in fresh products was maintained for purpose of computation (Table 1). Nutritional composition of products: All products were analyzed for moisture, protein, starch, iron, zinc and calcium following standard methodologies. In brief, moisture content of the sample was determined by repeated oven drying and weighing . Total ash was estimated by incineration of sample in a muffle furnace at 600°C for 5-6 hours and weighing the residue. This was converted into solution for estimation of minerals . Total starch was estimated by degradation of starch to glucose by enzyme treatment followed by colorimetric determination of glucose [17,18]. For determination of protein, nitrogen was estimated by digestion and distillation of sample by Kjeldhal method and protein content obtained by multiplying the nitrogen value with 5.70, the conversion value used for wheat. Iron was determined colorimetrically by α-α- dipyridyl method, and zinc content was estimated through atomic absorption spectrophotometer after preliminary digestion of food sample. Calcium was analyzed by precipitation as calcium oxalate and subsequent titration with potassium permanganate [16-18]. Nutrient availability in products: Products were analyzed for digestible protein and starch and bioaccessible iron, zinc and calcium. in vitroprotein digestibility (IVPD) of samples was estimated following the technique of Akeson and Stahman  modified for digestion time . A 2.0 g sample was digested with pepsin (for 2 hours) and pancreatin enzymes (for 3 hours) to follow gastric digestion, the insoluble protein was precipitated using trichloroaceticacid and separated by centrifugation (Model R-24, Remi Sales & Engineering Works, Bangalore, India) and soluble protein was estimated through Kjeldahl distillation method as given above. in vitro starch digestibility (IVSD) was determined by a slight modification of original procedure as follows –To 100 mg sample 10 ml of water was added and pH adjusted to 6.9 with 0.2 M NaOH. A 3.0 ml α-amylase solution (4.0 mg α-amylase dissolved in 1.0 ml of phosphate buffer, pH 6.9) was added and the sample incubated (Thermotek Industries, Mysore, India) at 37°C for 2 h followed by addition of 15 ml 0.2 M glycin-HCl buffer (pH 2.0) and 15 mg pepsin and further incubation at 37°C for 2 h. At the end of the period, pH was adjusted to 6.9-7.0 with 0.2 M NaOH, 15 ml phosphate buffer (0.05 M, pH 6.9) and 15 mg pancreatin were added and incubated at 37°C for 2 h followed by adjustment of pH to 4.6 with dilute acetic acid. Then 15 ml acetate buffer (pH 4.5) and 15 mg amyloglucosidase were added and mixture was incubated at 37°C for 2 h. Finally the volume of digest was made up, amount of glucose estimated and converted to starch by multiplying the value by 0.9 [21,22]. in vitro bioaccessibility of calcium, iron, and zinc were measured by dialysis technique through determining the proportion of mineral diffused through a semi permeable membrane after digesting the samples with pepsin and pancreatin . The calcium, iron and zinc were estimated as stated earlier. Percent digestibility/ availability of nutrients was computed using the total content in respective samples.
Mean and standard deviation for all values was calculated. Data is presented individually for products prepared with either whole wheat (WWF) or refined wheat flour (RWF) with statistical significance in comparison to respective controls. The significant differences between total and bioaccessible nutrients from different products were determined using Student’s ‘T’ test. Comparison was also done between whole and refined flour products to understand the effect of refining.
to exclude the variability of moisture, which differs in products. The
control Poori had 11.91% protein and 70.63% starch. Inclusion of
wheat bran altered the nutritional composition of products. There
was an increase in protein content of all bran added products to
varying extent, and the differences were significant for all. An
opposite trend was seen for the starch content, wherein there was a
reduction in starch content of all bran added products. No effect was
seen in products fortified only with minerals for protein and starch.
The iron, zinc and calcium content of WB added products increased
and were marginally significant in comparison to control. The
changes in iron and zinc content of products with externally added
iron and zinc were significant for both levels of addition and increase
was concurrent with the added amounts as given in Table 2, the
values are self explanatory. The calcium content of control products
and variations with added mineral salts did not change significantly.
These differences reflect the distribution of these macro
nutrients in grain wherein the outer layer has more of protein and
cellulosic fractions and the inner part has lesser proportions. As bran
portion of the grain is concentrated source of minerals, its addition
to products at both levels increased their contents. The reduction in
starch content of Poori is related to high fiber content of bran as well
as the processing used for Poori preparation. It has been reported
that processing can affect the starch profile of foods and alter the
ratio between rapidly digestible starch, slowly digestible starch and
resistant starch .
The refined wheat flour represents a different matrix due to removal of fiber and components associated with fiber in comparison to whole wheat flour. In an earlier study, the dietary fiber content from differentially milled components of wheat grain from same batch were reported to be 13.0, 3.7 and 46.9% for whole wheat flour, refined wheat flour and wheat bran respectively . The nutritional composition of Poories prepared with RWF complied in Table 3 shows that control product had 10.67% protein which was lower than the product prepared with WWF. In contrast, the starch content was much higher at 79.17% indicating that process of refining retained more of inner starchy portion of the grain. All the minerals analyzed viz., iron, zinc and calcium were significantly lesser in RWF Poori in comparison to WWF Poori.
The protein content of all variations, both bran enriched and mineral fortified Poories (10.81-12.14%) were not significantly different from control, though addition of bran increased the protein content slightly. Starch content was lesser in WB added products, with the decrease being significant at 20% addition of bran. Mineral fortification alone did not influence starch content of products. Addition of bran increased the iron and zinc content significantly in all products. Mineral fortified products had significantly enhanced mineral content in proportion to the amount of iron or zinc added to the products. A marginally significant increase in calcium content of wheat bran added products was also seen.
The reported results are supported by many studies which indicate that bran is a richer source of many nutrients and the incorporation of bran can alter the composition of products. Oghbaei and Prakash  reported that bran obtained from differential milling of wheat had a much higher content of protein (19.45% dwb), minerals, vitamins and lesser content of starch, whereas RWF had higher content of starch and lower content of minerals, vitamins and dietary fiber. Bilgicli et al.  reported that addition of wheat bran to Tarhana (soup based on yoghurt and wheat flour) increased the mineral and protein content significantly. It has also been reported that the extent of compositional change in WB added products would depend on the level of replacement .
The results of in vitro digestibility/accessibility of nutrients in
Poories made with WWF are presented in Table 4. The digestible
protein in products ranged from 5.28-5.81% (dwb) and was not
significantly different among variations. Replacement of wheat
flour with bran decreased starch digestibility to varying extent
in all bran added products. Higher level of bran resulted in larger
reduction in digestibility (24.16% in comparison to control value of
38.97%). Addition of iron or zinc salts alone did not influence starch
digestibility. This effect could have been mediated because of high
fiber content of bran, which is a well known phenomenon. Dietary
fiber lowers starch digestion and thereby controls blood sugar levels.
Another reason could also be deep frying of the product as it is
known to reduce starch digestibility . Many factors are reported
to affect content and extent of starch digestibility, the most important
ones are food processing, retrogradation, storage time and botanical
origin of the food.
Mineral bioaccessibility in products with 10% and 20% WB was in same range as their control and changes were insignificant. Calcium bioaccessibility was also not affected by fortification with other minerals but most of variations showed slightly lower values, which could be due to interaction among minerals.
Bioaccessible iron increased significantly to 1.162 and 2.042 mg/100 g in Poories on fortification. Higher level of iron reduced zinc bioaccessibility, which can be attributed to competitive inhibition among minerals. Bioaccessible zinc in Poori fortified with 10 mg zinc was much higher (0.770 and 0.549 mg) than control. The addition of 20 mg zinc increased it further to 1.866 and 1.259 mg, which was significant. The bioaccessible iron in Poori variation with zinc reduced very slightly. Addition of zinc is known to lower iron absorption. However, it depends on the level of zinc added, as smaller level of addition may not have adverse effects. The calcium bioaccessibility was not influenced either by addition of WB or minerals for any products indicating that the added levels did not affect calcium absorption.
Phytate chelates metal ions, especially zinc, iron and calcium in the gastrointestinal tract, making them unavailable for absorption . Since WB has a higher phytate content, its incorporation would increase the phytate: zinc molar ratio altering the amount of bioaccessible zinc from products. In our earlier study, we found the phytate content of WB to be 3396 mg/100 g in comparison to whole wheat flour which was 604 mg/100 g .
The digestible/bioaccessible nutrients in Poori based on RWF are presented in Table 5. For control Poori, IVPD and IVSD were 5.65, and 57.21% respectively. Bioaccessible iron, zinc and calcium were lower than WWF Poori. Protein digestibility of all experimental variations was similar to control with no significant differences (5.46-5.89%). Starch digestibility reduced significantly on addition of WB, which was highest with 20% WB. Iron bioaccessibility was much higher in iron fortified products in comparison to control. On addition of WB, this decreased marginally. WB enrichment alone did not affect iron bioaccessibility. Similar effect was seen for zinc fortified products, where addition of WB and iron did not affect zinc bioaccessibility and it was highest in zinc fortified products. Available calcium was also not affected by addition of minerals, though, a reduction was observed on addition of WB.
Food matrix is considered a very powerful determinant of nutrient availability. Food processing techniques can change the bioaccessibility of nutrients, due to breakage of nutrient–matrix complexes, softening of cell walls, and production of new complexes with other available nutrients and activation or deactivation of inhibitors. Thermal and physical processing, mastication, and the process of digestion release nutrients from the food matrix making them available for absorption in the intestine . In our earlier studies on rice based products fortified with iron and rice bran, noodles (steamed product) exhibited a much higher nutrient availability in comparison to wafers (deep fried in oil) indicating that both compositional alteration of flour and processing techniques (streaming versus frying) influenced the nutrient bioavailability . Similar effect was also seen in finger millet based products, where vermicelli (steamed, wet matrix) exhibited better bioaccessibility of calcium, iron and zinc than wafers (deep fried, dry matrix). Sieving of flour (similar to refining) resulted in lower nutrient content but higher bioaccessibility due to reduced anti-nutrient content .
The percent in vitro digestibility/bioaccessibility of selected
nutrients for both WWF and RWF products are presented in Figure 1
(a-e). The percent IVPD of Poori variations ranged from 37.3 to 45.4
for WWF products (Figure 1a). For all RWF products it was higher
(45.97-54.54%). Between the variations, addition of WB brought
down the IVPD in some products, whereas for others, differences were
not significant. Percent starch digestibility was also higher in all RWF
products (range, 64.77-72.26%) and between variations; addition of
WB lowered it significantly for all products (Figure 1b). The average
reduction in percent IVSD of all WWF Poori variations with 10% WB
(with added iron/zinc) was 5.9% which was significantly different
from control and 20% WB variation (9.8%). Percent IVSD of fortified
variations, with minerals alone was not significantly different from
control for both set of products. Among RWF Poori variations,
percent IVPD and IVSD reduced significantly in presence of WB with
least values in products with 20% WB.
Percent bioacessibility of all minerals were higher in RWF products. In WWF Poories iron bioaccessibility ranged from 4.27 to 7.59% (Figure 1c). The control Poori showed 5.78% bioaccessibility which was significantly higher than variations with added WB and zinc (range 4.27-5.26%). Iron fortification markedly improved percent bioaccessibility to 7.03 and 7.59. In RWF Poories, iron bioaccessibility was highest in fortified products (8.28 and 10.93%) followed by variation made with WB+iron. Addition of Zn+WB also showed a reduction in bioaccessible iron.
Percent bioaccessible zinc in WWF control (5.68%) increased significantly due to addition of zinc (8.11 and 9.45%). The interaction of iron+zinc and inhibitory role of WB components significantly reduced percent bioaccessibility of zinc to 3.02, 4.03 and 3.71% in variation with 20% WB and those with 10% WB+10 and 20 mg/100 g iron respectively (Figure 1d). In RWF control product percent bioaccessible zinc was 9.39 and increased significantly on addition of zinc. Addition of WB and WB+iron reduced percent bioaccessibility. Reduction was highest in WB+iron added products. This can be explained on the basis of high phytate content of bran. Phytate, which is present in staple foods like cereals, corn and rice, has a strong negative effect on zinc absorption from composite meals. Inositol hexaphosphates and pentaphosphates are the phytate forms that exert these negative effects, whereas the lower phosphates have no or little effect on zinc absorption. Iron can have a negative effect on zinc absorption, if given together in a supplement, whereas no effect is observed when the same amounts are present in a meal as fortificants . De Romana et al.  reported that flour iron and zinc fortification increased their availability but co-fortification of zinc sulphate showed detrimental effect on iron absorption.
In Poories percent calcium bioaccessibility was markedly higher than iron and zinc (Figure 1e). Highest bioaccessible calcium was in control Poori (38.5%). All variation enriched with WB showed significantly lesser calcium bioaccessibility with minimum value for product with WB+iron (25.30%). The reduction in calcium bioaccessibility in fortified variations was not significant. RWF control Poori showed highest percent calcium bioaccessibility (57.45%) and least was seen with 20% WB (22.71%). Addition of WB and higher level of minerals reduced percent calcium bioaccessibility. Some studies state that in cereals especially in wheat, calcium/ phosphorous ratio below 0·5–0·8 is recommended for a satisfactory calcium use by the body. Whole-grain wheat also contains calcium absorption enhancers such as fructans and/or resistant starch, which increase the apparent absorption of calcium from 20 to 50% in rats. Similarly, insulin increased calcium absorption by about 12% in human subjects [33,34].
Figure 1a-e. Percent in vitro digestible/bioaccessible nutrients in whole and refined wheat flour Poories
Overall results indicate that though absolute amount of available nutrients may be higher in WWF products, in terms of percent availability, RWF was better. This is obviously due to lower antinutritional factors in RWF which prevent binding of nutrients and help in absorption.
Data were further analyzed to evaluate the effect of refining on nutritional quality of Poories and the results are compiled in Table 6. In general, refining decreased the protein and mineral contents in products, and increased starch content. In products, differences were significant for some of the variations. Iron was significantly lower in most of the products except with 20% WB. Zinc also showed similar pattern, though all zinc added products had comparable values to WWF Poories. Calcium was significantly lower in all RWF products. The IVPD was higher in RWF products, though differences for most were not significant. Starch showed a significantly higher digestibility for all products. Iron and zinc bioaccessibility was significantly lower in many RWF products in comparison to WWF Poories, however zinc fortified products exhibited higher bioaccessibility. These results show that refining did alter the composition as well as the nutrient availability in wheat four products to a significant extent.
The study explored the nutrient availability from deep fried products prepared with bran enriched and mineral fortified whole and refined wheat flours. Bran enrichment and mineral fortification resulted in improved nutritional quality of both whole and refined wheat flour products. Externally added iron and zinc exhibited enhanced bioaccessibility even in presence of added wheat bran. Hence, both bran and minerals can be added together to flour as a preventive measure to combat mineral deficiencies as well as for health benefits of fiber. This is especially useful for refined flour as it is devoid of natural fiber and has lower mineral content.