Abstract

The present work was designed to prepare four different oat bar formulas (F₁, F₂, F₃ and F₄) from quick oat flakes, sunflower, chickpea and pumpkin seeds. Chemical, physical, textural, microbiological and sensory evaluation were performed. The results indicated that the substitution of oat with sunflower, chickpea and pumpkin seeds significantly enhanced the nutritional value of the resultant bars. Protein and fat contents significantly increased. Total phenols, total flavonoids, tannins and antioxidant activity significantly increased in F₂, F₃ and F₄ compared to F₁. Substitution of oat with sunflower, chickpea and pumpkin seeds has no significant effect on taste, color, flavor and overall acceptability but, it had a significant effect on appearance. Hardness, microbiological counts and peroxide values increased while water activity decreased during storage period for three months. The results clearly demonstrated the value of substituting oat with sunflower, chickpea and pumpkin seeds to produce oat bars with good nutritional value and stability up to two months. Sunflower formula was the most valuable addition with the highest acceptability followed by pumpkin formula.

1. Introduction

Recently, the consumption of fast food and snacks significantly increased, revealing a trend of change in lifestyle of the population. This is due to the facilities for the purchase of pre-prepared, frozen and ready market foods. Among these foods stand out the snacks, which are defined as small meals, light or substantial and may be related to the attributes of healthy and would be an ideal food format to deliver fruit-derived phenolic antioxidants and fiber. Because of the growing consumer demand for healthy, natural and convenient foods, attempts are being made to improve snack foods nutritional values by modifying their nutritive composition ¹. The greatest difficulty in obtaining a good cereal bar is a combination of several ingredients with specific functionality such as grains, nuts, seeds, dried fruits, raisins, thickening agents, sweeteners and flavorings, and turns them into a product with flavor, texture and decent appearance, to achieve specific nutrient goals. Cereal bars have been used for multiple purposes such as breakfast, snacks, energy and meal replacement. There is a trend towards producing natural snacks with a good fiber, calorie and protein supply ². Cereal bars have emerged as an important breakfast cereal mixture product. It is considered a ready-to-eat cereal, produced from the mixture of grain components with other ingredients such as chopped nuts and fruit pieces, through extensive processing. The principal grains used in the manufacture of cereal bars include corn, rice, wheat, oats and barley, which are overall considered healthy ingredients ³.

Oats (Avena sativa L.) have a long tradition of use in food and is widely recognized as an inexpensive and healthy food. The world production of oats reached over 22 million tonnes in 2014 and the major producers are Russia, Canada, Poland, Australia, Finland, and USA ⁴. Unlike wheat, it is usually consumed in a whole-grain form. Oat is higher in protein, calcium and essential fatty acids than are other grains. Oat is also high in dietary fiber, including soluble fiber and β-glucan, which has been shown to reduce serum cholesterol ^{5, 6}. Oat flakes are made by passing whole groats, which have been tempered or steamed, between a pair of rollers and the grains are subjected to low moisture, high shear and high temperature for a short time. The gap between the rollers determines the thickness of the flakes. The smaller flake size of quick oats require a shorter cooking time ⁷.

Sunflower (Helianthus annuus L.) is one of the most important oilseed crop grown in the world. Sunflower seed is a package of healthy unsaturated fats, protein, fiber and other important nutrients like vitamin E, selenium, copper, zinc, folate, iron and phytochemicals. Sunflower polyphenols can be used as effective antioxidants for sunflower ^{8, 9}.

Chickpea (Cicer arietinum L.) is one of the oldest and most widely consumed legume in the world due to its relatively high in protein content and wide adaptability as a food grain. It is the second most widely grown legumes in the world. The origin of the chickpeas is thought to have been Levant and Ancient Egypt ¹⁰. It is a good source of protein and carbohydrate. Its protein quality is better than other legumes such as pigeon pea, black gram and green gram ¹¹.

Pumpkin (Cucurbita maxima), a member of Cucurbitaceae family has been used frequently as a functional food or medicine ¹², because its a good source of polysaccharides, protein and phenolic glycosides. Besides, it is rich in unsaturated fatty acids and tocopherols and with very high oxidative stability ^{13, 14}.

Dried fruit is a fruit has been used in baking mixes and breakfast cereals. Its consumption is rapidly increased by people due to the awareness regarding their health benefits ¹⁵.

As the formulation and development of nutritious complementary food from locally and readily available raw materials have received a lot of attention, the present work was aimed to prepare oat bars formula, enriched with different seeds and dried fruits and evaluating their physicochemical, textural, microbiological and sensorial properties to assess their nutritional quality.

2. Materials and Methods

Whole sunflower (Helianthus annuus L., Sakha 53 variety) and flaxseed seeds (Linum usitatissimum L., Sakha 2 variety) were obtained from the Field Crops Research Institute, Agricultural Research Center, Giza, Egypt. Quick oat flakes, sesame seeds, raisins, dried apricots and plums, hulled chickpea, whole pumpkin seeds, sucrose, cinnamon and glucose syrup (79.50% total soluble solids) were obtained from local markets, Cairo, Egypt. Pepsin, pancreatin enzymes, DPPH (2, 2-diphenyl-1-picrylhydrazyl) and ABTS [2,2׳-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid)] were obtained from Sigma–Aldrich Chemical Co., St. Louis, USA. All other chemicals were of analytical grade.

Sucrose was mixed with glucose syrup and water (Table 1) and the final concentration for the syrup was 68% total soluble solids (using a Laboratory Refractometer, Bellingham and Stanley Ltd, England), the temperature was 105±2°C, then cinnamon was added. The oat bars were prepared according to the method described by Silva de Paula et al. ¹⁶ with some modification as shown in Table 1. Whole sunflower, pumpkin seeds and hulled chickpea were added as a partial replacement of oat flakes (based on preliminary trials). The oat flakes and different seeds were put on a pan and heated for about two to three min at 115±2°C according to Adebiyi et al. ¹⁷. Then, dried fruits were added and mixed well with the prepared sugars syrup mixture in order to obtain a homogenous mixture. After that, sheeted in rectangular frames, glazed with a minimum amount of corn oil, manually pressed and left to cool at room temperature to formulate four oat bars (18±2 g weight, 48±2 mm length, 26±2 mm width and 14±1 mm thickness). The obtained bars were packed in polypropylene metalized bags and stored at 25±2°C for three months for subsequent analyses.

Moisture, protein, fat, crude fiber and ash contents of raw materials and oat bars were determined according to the methods of the AOAC ¹⁸. Carbohydrates were calculated by the difference method ¹⁹. Total calories of the oat bars were estimated by multiplying the crude protein, fat and carbohydrates by calculation as its basis of 4, 9 and 4 kcal/g, respectively according to the method of James ²⁰. Mineral contents (e.g. iron, zinc, copper, manganese, calcium, potassium, magnesium and sodium were determined according to the method outlined in the AOAC ¹⁸ using the Perkin Elmer (Model 300, USA) Atomic Absorption Spectrophotometer. Total phosphorus was determined by the colorimetric method of Trough and Mayer ²¹. Peroxide value (meq/kg fat) was determined according to the method of the AOAC ¹⁸.

Total and reducing sugars were determined using Somogyi–Nelson Method according to Somogy ²² and Nelson ²³. Non reducing sugars were calculated by difference between total and reducing sugars. Starch was determined according to the method described by Ranganna ²⁴.

Total phenolics content were determined using Folin-Ciocalteu method according to Singleton and Rossi ²⁵. Gallic acid was used as a standard. Total flavonoids were determined according to the method of Zhishen et al. ²⁶. Catechin was served as a standard. Tannins were determined as described by Price et al. ²⁷. Catechin was used as a standard.

Phytate content was determined according to the procedure described by Mohamed et al. ²⁸.

In vitro starch digestibility was determined using pancreatic amylase according to the method of Singh et al. ²⁹.

In vitro protein digestibility was determined according to the method of Akeson and Stahmann ³⁰. After enzymatic digestion of samples with pepsin and pancreatin, the protein in the resultant supernatant was estimated using the Kjeldahl method ¹⁸. The percentage of protein digestibility was calculated by the ratio of protein in the supernatant to protein in the sample as the following equation:

The antioxidant activity was determined using the radical scavenging activity in reacting with DPPH free radical according to Brand-Williams et al. ³¹ and the ABTS assay which was carried out according to Re et al. ³². The radical scavenging percentage was calculated using the following equation:

A₀= Absorbance of the control reaction (containing all reagents except the test compounds). A₁= Absorbance in the presence of the tested extracts after 30 min.

The oat bar samples were organolyptically evaluated for its some sensory characteristics, i.e., appearance, flavor, taste, texture and overall acceptability by ten panelists according to the method of Stone and Sidel ³³.

External color of the oat bars was measured by a hand-held Tristimulus reflectance colorimeter Minolta Chromameter (model CR-400, Konica Minolta, Japan). Results recorded in lightness with L* = (100 for lightness, and zero for darkness), a* [chromaticity on a green (-) to red (+)] and b* [chromaticity on a blue (-) to yellow (+)]. Values reported are the means of triplicate determinations.

The water activity (a_w) of the oat bars was measured using Rotronic Hygrolab3 CH-8303, Switzerland as mentioned by Cadden ³⁴.

The hardness of oat bar samples was measured by Universal Testing Machine (Cometech, B type, Taiwan) provided with software as described by Bourne ³⁵. Three replicates of each oat bars formula were cut using a flat ended probe (2.50 mm thickness) with a cross-head speed of 1 mm/s at a 20% level of compression. The hardness was recorded by Newton (N).

Total plate count (TPC) and yeasts and moulds count of samples were determined according to the APHA ³⁶. The results of the microbiological analysis were expressed as log cfu/g.

The obtained data were analyzed using Costat statistical software and were statistically analyzed for means values and standard deviations according to Steel and Torrie ³⁷. The data were subjected to one-way analysis of variance (ANOVA) at P<0.05 followed by Duncan's new multiple range tests to assess differences between samples mean.

3. Results and Discussion

The chemical composition of selected raw materials (oat flakes, sunflower, chickpea and pumpkin seeds) is presented in Table 2. The results pointed out that sunflower seeds had a high content of ash and crude fiber. While, pumpkin seeds had a high contents of protein and fats (33.48 and 45.02%, respectively). Oat flakes and chickpea had the lowest values of all proximate analysis except for total carbohydrates. The results are close with those found by Brahma et al. ³⁸; Alexandrino et al. ³⁹; Sharma et al. ⁴⁰ and Hassan et al. ⁴¹ who analyzed oat, sunflower, chickpea and pumpkin seeds, respectively.

Table 3 displays the chemical composition of four oat bars [oat flakes (F₁), sunflower (F₂), chickpea (F₃) and pumpkin seeds formula (F₄)]. As estimated, substitution with sunflower, chickpea and pumpkin seeds significantly increased protein, ash and fat contents of the oat bars. Data showed that F₄ had the highest contents in protein (10.30%) and fat (6.91%) followed by F₂ (9.92 and 6.31%, respectively). On the other hand, F₄ had the lowest total carbohydrates content (80.89%). Whereas, crude fiber content insignificantly affected by the replacement of oat with different seeds.

The present findings are in accordance with Silva et al. ⁴² who mentioned that the combination between oats and pumpkin increased the contents of crude protein in cereal bars. However, it is possible to notice an increasing trend in the content of fats due to the addition of pumpkin seeds, which has higher content of total fat than that of the oats. Also, it increases the contents of dietary fiber, which are essential to maintain health and reduce the risk of several diseases.

Regarding to Dietary Reference Intakes (DRI) ⁴³ of protein, each 100g oat bar provide 18.30, 21.56, 20.52 and 22.39% from daily intake of protein for female and male aged 19-50y with 55kg body weight (based on 46 g protein/day, respectively) from F₁, F₂, F₃ and F₄, respectively. And 15.04, 17.71, 16.86 and 18.39% from daily intake of protein for male aged 19-50y with 65kg body weight (based on 56g protein/day) from F₁, F₂, F₃ and F₄, respectively.

Each 100g oat bar provide 18.43, 21.03, 18.93 and 23.03% from daily intake of fat (based on 30g fat/ day) from F₁, F₂, F₃ and F_4, respectively according to DRI ⁴³ of daily total fat.

Total sugars significantly increased in different formula. F₂ and F₄ had the lowest content of reducing sugars (10.68 and 10.78%) and starch content (52.81 and 52.48%) compared to other formulas.

Accordingly, the use of pumpkin and sunflower seeds in the formulation of oat bars enhance the nutritional quality which could be used as breakfast, snacks, energy foods.

Table 4 exhibit the minerals content of the oat bars. Data indicated that substitution of oat with sunflower, chickpea and pumpkin seeds significantly affected the mineral contents in all oat bars. F₁ had the highest content of magnesium, manganese, copper and zinc. While, F₂ had the highest content of calcium and phosphorus. F₃ recorded the highest values of iron and potassium. The differences in minerals are probably due to the type of seeds and ingredients used in the formulation of the oat bars. Similar results were found by Paiva et al. ⁴⁴, who found that cereal bars contain rice, soy and pineapple had higher levels of phosphorus, potassium, magnesium, copper, manganese, zinc and iron. Roy et al. ⁴⁵ mentioned that chickpea is an excellent source of minerals, especially calcium, phosphorous, iron, and magnesium.

The reference daily intakes for iron (18 mg or female and 8 mg for male aged from 19-50y) and zinc (8 mg for female and 11 mg for male male aged from 19-50y) according to DRI ⁴⁶. Therefore, each 100 g of oat bars provides 23.50 and 52.87% for F₁, 23.06 and 51.87% for F₂, 23.55 and 53.00% for F₃ and 22.22 and 50.00% for F₄ from daily intake of iron for female and male, respectively. Each 100 g of oat bars provides 35.37 and 25.73% for F₁, 32.75 and 23.82% for F₂, 32.25 and 23.45% for F₃ and 31.75 and 23.09% for F₄ from daily intake of zinc for female and male, respectively.

From the above mentioned data about the nutritional characteristics, it could be demonstrated that the oat bars had reasonable amounts of Fe and Zn.

Table 5 represents the phytochemicals (e.g., total phenol, flavonoids, tannins and phytic acid) and antioxidant activity. Data showed that total phenols, flavonoids and tannins significantly increased in all oat bars. F₂ had the higest total phenols content (123.36 mg/100g) compared to all formula. While, F₄ followed by F₃ recorded the highest contents in total flavonoids and tannins (70.26 and 40.58 mg/100g), respectively. Sharma et al. ⁴⁰ reported that whole chickpea seeds contain from 70.0 to 220.0 mg/100g tannins.

Regarding phytate content, data revealed that phytate content ranged from 483.23-570.18 mg/100g. Phytate content significantly increased compared to F₁, due to its high content in sunflower, chickpea and pumpkin seeds.

Antioxidant activity as DPPH and ABTS significantly increased in all formulas compared to F₁. F₂ had the highest antioxidant percentages as DPPH and ABTS (23.02% and 29.18%). Cereal bar products contain healthy and natural ingredients (cereals, nuts and fruits) that can contribute to a balanced, rich and healthy diet ³.

Generally, substitution of oat with sunflower, chickpea and pumpkin seeds significantly increased phytochemicals and antioxidant activity compared to the F₁.

Table 6 represents the in vitro protein digestibility (IVPD) and starch digestibility (IVSD) of different oat bars formula. Substitution of oat with sunflower, chickpea and pumpkin seeds insignificantly affected the IVPD percentages compared to the F₁. The IVPD values ranged from 68.00 to 70.17%, and F₁ had the highest value. The IVPD ranged from 63.14-77.09% and 67.11-80.23% for raw and dehulled chickpea, respectively ⁴⁷.

The in vitro starch digestibility of the oat bars are also shown in the same Table. It could be noticed that F₃ exhibited the highest in vitro starch digestibility (69.88%) followed by F₂ (69.15%). However, F₄ had the lowest value (67.49%) compared to all formulas.

Legumes are an important source of dietary protein and starch for human, but their acceptability and utilization has been limited due to some antinutritional substances such as trypsin inhibitors, phytate and tannins etc. ⁴⁸. However, digestibility of starch can be improved through heat treatments e.g. cooking, roasting and autoclaving ⁴⁹.

Color measurements of the oat bars are demonstrated in Table 7. Data indicated that substitution of oat with sunflower, chickpea and pumpkin seeds insignificantly decreased the lightness (L*) values of the oat bars comparing with F₁. Lightness of F₃ significantly increased comparing with F₂ and this may be due to the color of the different seeds. Regarding redness (a*) and yellowness (b*) values, there were non significant differences amoung all formulas. Where, F₃ recorded the highest b* value (26.77) and F₁ recorded the lowest ones (25.03) compared with other formula.

Silva et al. ⁴² mentiond that the b* values characterize the predominance of yellow, and there was no difference between all cereal bars.

Sensory evaluation of oat bars formula are presented in Table 8. Data reveal that the substitution of oat with sunflower, chickpea and pumpkin seeds had partially significantly affects appearance attributes of all oat bars. But, it had no significant effects on flavor, taste, texture and overall acceptability. Sunflower formula (F₂) recorded the highest scores of all tested attributes followed by F₄ which recorded the highest scores for overall acceptability comparing with other formula.

In the sensory acceptance test (a 9-point hedonic scale), all cereal bar samples presented acceptable scores (>5) which were considered a good score and the high acceptance rate obtained by the cereal bars reflects in a large potential purchase of such product ⁵⁰. Using of pumpkin in the preparation of the cereal bars led to improve the sensory attributes (e.g. texture, flavor and overall appearance), and consumer preference. These products can be classified as light products and an alternative to consumers as healthy and functional food ⁴².

Water activity gives information about the safety and quality of food. It characterizes the different states in which water can be found, which includes how much water is "bound" in the food, how much water is available to participate in chemical or biochemical reactions, and how much water is available to help the growth of microorganisms ⁵¹. Figure 1 represents the a_w values of the oat bars during storage. All the given values of the a_w were measured at 22±1°C and the data demonstrated that using of sunflower, chickpea and pumpkin seeds had insignificantly affected the a_w values of the oat bars at zero time. F₁ recorded the lowest value of a_w (0.683). The a_w significantly decreased during storage for three months as found by Estévez et al. ⁵². The water activity values of cereal bars containing pineapple peel ranged from 0.66 to 0.72 ⁵³.

Figure 2 represents the hardness of the oat bars during storage period for three months. Data show that substitution of oat with sunflower, chickpea and pumpkin seeds (F₂, F₃ and F₄) had significantly decreased of hardness being 6.57, 5.37 and 4.11 N, respectively compared to F₁ (7.01 N) at zero time. From the above mentioned data, it could be stated that F₁ had the highest hardness and F₄ had the lowest ones. This may be due to the higher fat content of F₄ than F₁. After storage for three months, hardness values significantly increased gradually in all formula compared to zero time.

The values of hardness influence in the acceptance of the cereal bars ⁵². The addition of pumpkin made cereal bar more compact, which increased its strength to cut and hardness. In the case of the total replacement of oats with pumpkin in the preparation of cereal bar, it is possible to observe a decrease in the strength to cut and hardness ⁴².

Peroxide value is an indication for lipid oxidation with subsequent formation of peroxides and aldehydes, ketones, alcohols, hydrocarbons, esters, furans and lactones. These compounds negatively affect physical, chemical and sensory properties of food ^{54, 55}.

Data of peroxide value (PV) of oils extracted from oat bars formula during storage at room temperature (25±2°C) for 3 months were illustrated in Table 9. At zero time the PV was 0.87, 1.03, 0.85 and 0.99 meq/kg for oil extracted from F₁, F₂, F₃ and F₄, respectively. The PV values increased with increasing time during storage for one and two months and ranged from 1.46-1.97 and 3.41-4.27 meq/kg, respectively but it was within the acceptable range. The PV gradually increased up to the end of the storage periods in all samples. After storage for 3 months, the PV values in all bar samples increased and ranged from 4.06 to 5.76 meq/kg oil compared to all formulas; however oat bars substituted with pumpkin seeds (F₄) had the highest values (5.76 meq/kg oil). Oat bars could be stored up to two months with safety peroxide values.

Rezig et al. ⁵⁶ indicated that phenolic compounds play a determinant role due to their attributes such as flavor, shelf life and resistance against oil oxidation.

O’Brien ⁵⁷ stated that a product with peroxide value between 1 and 5 meq/kg is classified at low oxidation state; that between 5 and 10 meq/kg at moderate oxidation and above 10 meq/kg is classified as a high oxidation state. The maximum level of peroxide in virgin oils and cold pressed fats and oils up to 15 milliequivalents of active oxygen/kg oil and other fats and oils up to 10 milliequivalents of active oxygen/kg oil ⁵⁸.

Figure 3 and Figure 4 present total plate count (TPC) and yeasts and moulds of oat bars supplemented with sunflower, chickpea pumpkin seeds during storage periods. Data show that the total bacterial count of oat bars affected by replacing of oat with sunflower, chickpea and pumpkin seeds. The results indicated that the addition of sunflower, chickpea and pumpkin seeds showed a decrease in total bacterial count in oat bars at zero time (0.80-0.91 log cfu/g in F₂ to F₄) compared to F₁ (0.92 log cfu/g) which had the highest bactreial count. Total bacterial count in all formula slightly increased after storage for three months (1.95-2.16 Log cfu/g).

The changes in yeasts and moulds counts at zero time and during storage period are illustrated in Figure 4. The yeasts and moulds counts were not detected at zero time and increased with progressing of the studied storage periods (1.92-2.07 Log cfu/g). Also, it could be seen that the highest counts of yeasts and moulds were recorded for oat bars (F₁). Decreasing in total bacterial count and yeasts and moulds in oat bars containing sunflower, chickpea and pumpkin seeds could be due to the presence of phytochemicals in those seeds. Phenolic compounds acting as an antioxidant and antimicrobial agents ⁵⁹.

Luh and Woodroof ⁶⁰ found that when the moisture content of food is below 8%, microorganisms do not grow while when its content is above 18% some microorganisms may be gradually reproduced. Cooksey ⁶¹ stated that food products with water activities between 0.30 and 0.85 have some water available in the product that could allow some microorganisms to grow. According to Egyptian Standards ⁶², total plate count must not be more than 1000 cfu/g and yeasts and moulds must not exceed 10 cells/g in cereal-based foods.

Generally, the microbial load was acceptable for all formula till two months of storage.

4. Conclusion

Finally, it could be clearly concluded that substituting oat bars with sunflower, chickpea and pumpkin seeds enhances the nutritional characteristics and quality of the oat bars. Where, they are a reasonable source of protein, bioactive components, Fe and Zn with good protein digestibility and stability. Besides, it could be stored up to 2 months at room temperature. Along overall sensory quality of the oat bar samples, it had a high acceptable sensory characteristics. The highest acceptability formula was the sunflower followed by pumpkin formula.

Acknowledgements

The authors would like to thank the Food Technology Research Institute, Agricultural Research Center for continuing cooperation to support research by intramural funding and provided facilities required to carry out the most wanted objectives of the research work.

References