Proximate and sensory analysis of wheat bread supplemented with Nigella sativa oil and Nigella sativa extract

Wheat is one of the important food crops worldwide while wheat bread is the most commonly consumed form of it in different populations. As Nigella sativa is rich in several phytochemicals that possess disease preventive properties hence, the proximate and sensory analysis of the breads supplemented with Nigella sativa extract and oil have been discussed in this study. Nigella sativa is one of the famous medicinal plants that has been used for treatment of various illnesses in different parts of the world. Compositional analysis revealed that Nigella sativa extract supplemented bread contains 14.75% moisture content, 10.32% crude fat content, 4.40 % ash and 3.55 % crude fiber, 11.89% crude protein and 55.09% Nitrogen Free Extract (NFE). In comparison, analysis of Nigella sativa oil supplemented revealed that it contains 14.23%, moisture content, 10.53% crude fat content, 4.10% ash and 3.20 % crude fiber, 11.79% crude protein and 56.15% NFE. Sensory evaluation was evaluated using 9 points-hedonic scale. Maximum score for overall acceptability was recorded for Nigella sativa extract fortified bread. In the meantime, the requests for wheat-based items with value addition are developing quickly in the previous couple of decades, as customers understood that eating foods with medical advantages is superior to taking medicine. The breads fortified with Nigella sativa will not only meet the consumer demands in terms of sensory attributes but might also aid in improving their health.


Introduction
The problem of nutritional insufficiency in most of the developed countries is deteriorating with every passing day. It intensifies the need to consider strategic use of food sources that are nutritious and cheap to manage in order to control and prevent health problems. Fortifying nutritionally inadequate food items can improve their dietary properties [1]. Wheat being the most important crop of the world [2] has a special position among the bakery goods and is considered the most common and staple cereal-based food [3]. Due to its nutritional quality and sensory properties, it is consumed as the basic part of human diet from centuries. Bread is made of wheat flour [4] and can be fortified with other cereals-based flour such as sorghum, rice, maize, and barley etc. to increase its nutritional quality [5]. In countries where bread wheat is not the main domestic crop, efforts can be made to replace wheat flour with other types of available flours to overcome nutritional deficiencies [6]. Bread is a fermented food product made from wheat flour, yeast, water and salt by various methods such as mixing, kneading, shaping, fermenting, baking and finally cutting. Examination of characteristics of a loaf is crucial in order to evaluate the quality of a bread. Since formulation and production of fortified cereal-based items is increasing constantly; it is very essential to identify and explore sensory and quality characteristics of such foods. Cereals, can prevent malnutrition and related health risk by providing essential nutrients; in fact, such plants have been used for traditional remedies since long in the history of mankind as well as in modern medicines [7]. As stated by World Health Organization (WHO), around three fourth of the population in underdeveloped nations rely on therapeutic plants to provide crucial care due to the fact that more than 60% of them either don't have access or can't afford allopathic medicines [5]. At present, there in renewed interest in the use of plants as a food source and medicine has occurred [8]. Biofortification has also been suggested to improve nutritional status of marginalized societies by agronomic, breeding and transgenic modification of plants [9]. Whereas, the use of phytomedicines for the treatment of various illnesses has been elevated dramatically due to their ease of accessibility, low cost and the certainty that the natural therapies have scarcer side effects than artificially produced remedies [10]. Formulation and development of novel food products by using naturally occurring food sources is also supported and encouraged due to the fact that there exist about 300,000 species of herbs globally among which only 15 percent have been tested for their pharmacological potential [11]. Nigella sativa L. (Ranunculaceae) is historically believed to be one of the strongest and highly valuable herbs globally. Such a rich dietary value of black seeds can be associated with the presences of significant quantity of protein, fiber, minerals & vitamins [12,13]. Different sources have revealed the nutritional composition of black cumin seeds as 20-85% protein, 38.20% fat, 7-94% fiber and 31.94% total carbohydrate. Including various other identified amino acids such as glutamate, arginine, and aspartate in major while cysteine and methionine in minor quantities. The seeds of this herb constitute remarkable levels of minerals such as iron, copper, zinc, phosphorus, calcium, thiamine, niacin, pyridoxine, and folic acid as well [14]. Moreover, hundreds of phyto components have been displayed upon phytochemical analysis of Nigella sativa that mainly include alkaloids, saponins, sterols and essential oils (0.4% -2.5%) [15]. It also contains 26-34% fixed oil of which the key fatty acids are linoleic acid (64.6%) and palmitic acid (20.4%).
Including various active constituents reported so far, thymoquinone is believed to be a major biologically active component exhibiting a long list of therapeutic benefits [16,17]. Therefore, present study was planned to formulate breads containing different percentages of Nigella sativa oil or Nigella sativa extract and to examine them for their sensory attributes.

Materials and Methods
This research study was conducted at the University Institute of Diet and Nutritional Sciences (UIDNS), Faculty of Allied Health Sciences, University of Lahore, Lahore, Pakistan.

Preparation of Nigella sativa Powder
Nigella sativa seeds were bought from a local herb store in Lahore, Pakistan. The seeds were dried at room temperature and grounded to a fine powder using electrical grinder and stored until further use. Nigella sativa oil Fine quality Nigella sativa seed oil was purchased from a local store in Lahore, Pakistan.

Preparation of Nigella sativa extract
Black seeds were extracted with 60% ethanol, maintained to pH 5.5 at 50°C for a period of 3 hours. The extract was then kept in an airtight test tube and stored in refrigerator until further use.

Bread making
Three types of breads were made; a control bread (Bread A), one with Nigella sativa extract (Bread B), and one with Nigella sativa oil (Bread C). Refined wheat flour (900gm) was used for all types of bread formulations while breads supplemented with Nigella sativa oil or Nigella sativa extract were prepared by replacing corresponding amount of wheat flour in the formulation (0.3% of flour for black seed powder extract enriched bread) and 0.875% of flour for black seed oil enriched bread). To each bread formulation water, salt and yeast were added to make respective dough(s). The dough was mixed using a spiral mixture for 7 minutes and stored for fermentation. Small batches of dough (300gm) were baked in preheated oven at 180 °C for 35 minutes and then cool down for 24 hours at room temperature.

Screening of participants
Participants were screened using a questionnaire along and practical test in asking participants to identify the five basic tastes. An inclusion criterion was given as: -Probability of the nature of product -The participant are the ones who consume the product regularly -The participant could distinguish between five basic tastes; sweet, bitter, sour, salty, umami and could detect a neutral sample Finally, 7 participants were included in the sensory evaluation panel.

Training of Panel
The participants were invited to the test about a week before the session. The invitations contained instructions that needed to be followed before the test i.e.
-Try not to smoke, consume coffee, tea or spicy food close to the test session. -Try not to wear strong scented perfumes or hairsprays. -Try not to be too full or too hungry during the test session. -Before and after testing each sample, rinse mouth with water Proximate analysis The analysis of crude protein, crude fiber, crude fat and moisture content of three types of bread formulations was carried out by following appropriate various methods. Three repetitions were performed for each type of test in this study.

Moisture content
The moisture content of the breads sample was carried out as given by the Association of Official Analytical Chemists [18].Weighed 5g of the bread formulations was kept in an air-oven at 100 o C overnight for drying to carry out the procedure for determining the moisture content.

Fat content
Weighed 10g of bread samples were dried overnight in the oven first to remove the moisture. After the removal of moisture, the oil was extracted in a Soxhlet apparatus for about 5 to 6 hours using n-hexane as solvent [18]. Protein content For the estimation of protein content in the breads 5g of the sample was treated by the Kjeldahl method. In the end of the procedure the protein was calculated with the help of a nitrogen factor i.e. 6.25 which was given by [18]. Ash content Weighed 10g sample of each of the respective bread was oven dried overnight to remove moisture. Then the sample was ignited in order to remove carbon and placed in a muffle furnace at 550oC for about 4 -5 hours [18]. The result was presented as percentage of dry matter.

Nitrogen free extract (NFE)
The nitrogen free extract of the bread samples was obtained by subtracting the sum of total moisture, fat, ash, protein and fiber from 100.

Results and Discussion Sensory analysis of breads
Sensory scores were noted for five different bread attributes from the trained panel. The (Table 1) shows the result of sensory evaluation with mean and standard deviation differences of three breads made with plain flour, Nigella sativa extract and Nigella sativa oil. The differences in the composition of Nigella sativa extract and Nigella sativa oil made breads different in taste, texture, appearance, odour as well as overall acceptability. A clustered bar chart is also presented in (Fig. 1) showing the overall distribution of means on the basis of all the attributes of evaluation by the panellist for three types of bread formulations. Sensory assessment uncovered that the breads enhanced with Nigella sativa extract demonstrated most noteworthy scores for general acceptability (8.43 ± 0.53) whereas the plain bread has shown the lowest mean value (6.57 ± 0.53) in terms of overall acceptability. On the other hand, Nigella sativa oil supplemented bread have shown an intermediate mean score (7.00 ± 0.82) for overall acceptability by the panel, In the current study bread B (8.14±0.90) showed the best in taste followed by bread C (6.86±0.90) and the lowest taste values was found in the control bread group (5.71±0.76).Textures determine the quality of the product. The results indicated that bread B (8±0.63) is ranked as the best in comparison with other samples bread C (6.67±0.52) and bread A (5.67±0.82) in terms of texture by the panelists. Appearance is the most important parameter as it is the first thing human eye attracts the most. In present study the appearance scores for bread B (8.29±0.76) was much good followed by bread C (7.00±0.82). The lowest values for appearance was shown in bread A (5.71±0.95). The odour of bread was affected with increase in flour amount used to make bread. The bread B (8.29±0.76) shows the highest score for the aroma followed by bread C (6.57±0.53) and the lowest value of odor was shown in bread A (5.57±0.53). The bread supplemented of with Nigella sativa oil or Nigella sativa concentrate improves its nutritional properties when compared with the control bread. Another similar study was conducted on the sensory evaluation of Nigella sativa supplemented breads and found that substitution of 5% of flour in bread with Nigella sativa depicted satisfactory consumer acceptability in terms of flavor, texture, aroma and overall acceptability [21]. Ligarnasari et al. (2018) reported that addition of Nigella sativa oil in brownies improved their sensory attributes [22]. Melapa (2015) have reported that soft texture of the product is influenced by its water content, fat as well as protein content and the use of baking ingredient [23]. Black seed oil has been reported to contain oleic acid, lauric acid, limonene (2.57%), linoleic acid (57.24%), jupine (0.83%), palmitic acid (3.11%), and thymoquinone (13.80%) and these constituents have potential to improve texture of the food product [24]. The substitution of extract and oil didn't reduce the moisture content of the bread and hence fortified breads maintained the softness that was comparable with the plain bread. According to Cauvain et al. (2006) the water content of a product can improve its softness [25] as the high moisture content in the material will cause the texture of the product to become softer.