INTRODUCTIONToday, nearly all levels of society consume biscuit as snacks or weaning for infants. It has pleasant aroma, various taste, low cost, and high stability during storage at ambient temperature [1]. Recently, the consumption of biscuit has increased, which encourages manufacturers to develop new formulations of biscuit with non-traditional raw materials to enrich the flavor of biscuit [2].In addition, the increasing demand for functional and nutraceutical food products makes it possible to increase consumption of bakery products such as biscuit by fortifying it with protein, fiber and other essential constituents from various sources.The fortification process provides several advantages, namely it can predict heart, coronary and diabetic diseases, as well as improve body functions. Generally, the main ingredients used in biscuit production include wheat flour, fat, sugar, eggs, emulsifiers, salt, etc. The chemical composition of these ingredients lacks for vitamins and protein (8–10%),which reduces the nutritional properties of bakery products [3–5]. Therefore, manufacturers have made efforts to replace the main ingredients of biscuit with more nutritional and healthy ones.Egyptian kishk is characterized by high nutritive value and good stability during all year seasons. The chemical composition of Egyptian kishk includes a high content of essential fatty acids, especially oleic and linoleic, as well as a high concentration of essential amino acids, especially lysine, tyrosine, and proline [6]. The main ingredients of kishk are parboiled wheat and milk, which make kishk an available and rich source for vitamins, minerals and fiber [7, 8].We found no research on improvement of the nutritional properties and volatile content of snacks like biscuit by replacing wheat flour with Egyptian kishk. Therefore, the current study aimed to reveal effects of various proportions of Egyptian kishk on the nutritional composition, sensory characteristics, antioxidant activity, and volatile content of biscuit.STUDY OBJECTS AND METHODSThe objectives of the study were biscuit without kishk (control samples) and biscuit with 10–50% of kishk (experimental samples). Kishk, wheat flour (72% extraction), sugar, shortening, and baking powder were purchased from the local market (Dokki, Egypt). Folin-Ciocalteu’s reagent, ABST and DPPH were purchased from Sigma-Aldrich Canada (Ontario, Canada). All chemicals were of analytical grade.The biscuit formulation was developed at the National Research Center (Dokki, Egypt) as described by Hussein et al. [9]. Control and experimental formulations are shown in Table 1.Proximate composition. The biscuit samples and Egyptian kishk were analyzed for moisture, ash, crude fat, and crude protein contents using Association of Official Analytical Chemist’ Approved Methods 925.10, 920.87, 920.85, 923.03, and 963.09, respectively [10]. The analyses were conducted in triplicate.Rheological properties. Rheological properties of dough samples with different amounts of kashk were evaluated using a farinograph according to [11].Physical characteristics of fortified biscuits. The volume and weight of three biscuit samples of each treatment were recorded. Specific volume was calculated by dividing of the volume to weight. The diameter, height and spread ratio (diameter/height) of the biscuits were determined according to the method described in [11]. The thickness and diameter of the biscuits were directly measured using STECO (Germany) as mentioned by Mc-Watters et al. [12]. The spread ratio was calculated as quotient of diameter and thickness. The biscuits were weighed (Precisa 105 A, Swiss) and the weight was expressed in grams. A total of ten biscuits were used for the measurement.Color parameters. Surface color characteristics of biscuit (intact) were measured in terms of Hunter color parameters, namely L*, a*, and b* values as mentioned by Dauda et al. For this, we used a spectrocolorimeter (Tristimulus Color Machine) with the CIE Lab color scale (Hunter, Lab Scan XE-Reston VA, USA) in the reflection mode [13]. The instrument was standardized with the white tile of Hunter Lab Color Standard(LX No. 16379): X = 72.26, Y = 81.94, and Z = 88.14(L* = 92.46; a* = –0.86; b* = –0.16). Color difference (ΔE) was calculated from a*, b* and L* parameters using the Hunter-Scotfield’s equation: ΔE = (Δa2 +Δb2 + ΔL2)0.5. A total of ten biscuits were involved in the experiment.Texture analysis. The texture of the baked biscuit samples was determined by a texturometer (Brookfield, CT3-10 kg, USA) equipped with a cylinder probe(TA-AACC36). Texture profile analysis was conducted to determine hardness. The analyzer was set to perform two cycle measurements to give a two bite texture profile curve. Trigger load and test speed were 9.00 N and2.5 mm/sec, respectively.Sensory evaluation. The sensory properties were evaluated using the 9-point Hedonic scale where9 = like extremely, 8 = like very much, 7 = like moderately, 6 = like slightly, 5 = neither like nor dislike, 4 = dislike slightly, 3 = dislike moderately, 2 = dislike very much, and 1 = dislike extremely [14]. The analysis was carried out by 10 trained panelists for four sensory descriptors, namely appearance, color, odor, taste, and texture.Total phenolic and antioxidant activity. Total phenolic content. The biscuit and Egyptian kishk samples were finely powdered and extracted with petroleum ether (40–60°C) to remove fats and resinous materials. The residues were thoroughly separately extracted with 500 mL of 70% methanol. The extract was filtrated through Whatman No. 1 filter papers and the filtrates were evaporated to dryness under reduced pressure on a rotary evaporator at 40°C.Total phenols were evaluated using the Folin-Ciocalteu method described in [15]. The results were expressed as milligram gallic equivalents (GAE) per gram of biscuit.Antioxidant activity. The methanol extract (25, 50, and 75 uL) was used for antioxidant activity determination. DPPH-radical scavenging activity and ABST assay were determined as described by [16, 17], respectively.Volatile compound analysis. Extraction of volatile compounds. Volatile compounds of the biscuits under study were isolated by headspace solid phase microextraction (HS-SPME) sampling and analyzed by gas chromatography coupled with mass spectrometry (GC/MS) [18]. Ten grams of grinded biscuits were put into a 20-mL headspace vial fitted with a Teflon-lined septum. Volatiles were sampled for 30 min at 50°C from the headspace of the vial using DVB/CAR/PDMS fiber (Divinylbenzene/Carboxene/Polydimethylsiloxane, Supelco, Bellefonte, Pa., U.S.A.). The fiber was then immediately inserted into the injection port of the gas chromatograph for 5 min at 260°C.Chromatographic conditions. A Hewlett-Packard HP5890II gas chromatograph coupled to a 5971 MSD quadrupole mass spectrometer was used. Chromatograph was equipped with a MDN-5(60 m×0.25 mm×0.25 μm, Supelco) column. Operating conditions for GC/MS were the following: helium flow 0.6 mL/min, initial oven temperature 40°C (3 min), then 8°C/min to 200°C, and 20°C/min to 280°C (3 min).Identification of volatile compounds. Volatile compounds were identified by comparison of their retention indices and mass spectra with standards, or, in some cases tentatively only by NBS 75K mass spectra library search and retention indices. Retention indices were calculated for each compound using homologous series of C6–C22 n-alkanes [19].Statistical analysis. Statistical analysis was conducted using SPSS software version 16 (SPSS Inc., Chicago, USA). All analyses were performed in triplicate and data reported as means ± standard deviation (SD). Data were subjected to analysis of variance (ANOVA). The confidence limits used in this study were based on 95% (P < 0.05) [20].RESULTS AND DISCUSSIONChemical composition of biscuit. Table 2 demonstrates the proximate chemical composition of biscuits without Egyptian kishk (control sample) and biscuits with 10–50% of Egyptian kishk (experimental samples). An increased amount of kishk from 10 to 50% resulted in an increased content of protein, fat, ash as well as fiber. The 50% kishk biscuit had the highest content of protein (9.38%) compared to the control sample (8.29%). A similar trend was found by Eneche, who revealed that biscuit fortified with pigeon pea flour showed an increase in protein content [21].Values are the mean ± SD of three replicates. Mean followed by different letters in the same column differs significantly (P ≤ 0.05)In general, the increase in fat, fiber and ash of kishk supplemented biscuit can be explained by the high content of those ingredients in kishk. Similar findings were also obtained by Nazni et al. and Pastuszkaet al. [22, 23]. On the other hand, total carbohydrates decreased with an increase in the amount of Egyptian kishk (Table 2).In the current investigation, we also determined the total phenolic content of methanolic extract in kishk, as well as in the control and experimental biscuits. The obtained data were expressed as mg GAE/g. The results showed that the biscuits with kishk had increased total phenolic content (9.56–12.04 mg GAE/g) compared to kishk and the control biscuit (1.57 and 8.23 mg GAE/g, respectively). The highest phenolic content was revealed in the 50% kishk biscuits (12.04 mg GAE/g).Rheological properties. Table 3 shows the rheological properties of wheat flour (control) and its mixtures with Egyptian kishk in different amounts (10, 20, 30, 40, and 50%). The water absorption, arrival time, dough development time (DDT) and weakening of dough increased with an amount of kishk. That may be due to high fiber and protein amounts containing in kishk, while fibers in wheat flour tend to bind more water. On the other hand, interaction of kishk fibers with wheat flour constituents and the added water may cause a decreased stability of dough.In this context, Kim et al. reported that an increased proportion of rice grain dietary fibers in the recipe of bread increased the water absorption of dough [24]. Other researchers reported that an increasing amount of barley flour caused a progressive increase in water absorption, arrival time, and dough stability, with addition of gelatinized corn flour to wheat flour [24–26]. Whereas in our work, dough stability decreased with increasing Egyptian kishk content (Table 3).Physical characteristics of biscuit. The studied physical characteristics (weight, volume, specific volume, diameter, thickness, and spread ratio) exhibited a significant effect (P < 0.05) of Egyptian kishk on the quality of biscuits (Table 4).The weight and volume of the experimental biscuits were significantly lower than those of the control sample (P < 0.05). This effect may be due to higher fiber and protein contents in kishk. Moreover, kishk also affected the diameter, thickness, and spread ratio of the biscuit. The results obtained by Brito et al. [27] showed a decrease of volume in cookies with higher amount of quinoa flour compared to that in cookies fromcorn starch.In our work, due to the decrease in diameter in biscuit with Egyptian kishk, its surface also decreased compared to control. As we can see from Table 4, high amounts of Egyptian kishk showed a remarkable correlation with the decrease in physical properties of the biscuits. In other study carried out by Mofasser et al., the authors found an increase in diameter of gluten-free biscuit compared to traditional biscuit [28].Color values and hardness evaluation. The main criteria for the consumer to purchase the food product are its appearance and color. Table 5 demonstrates the color characteristics of the biscuits under study,namely L*, a*, b*, and ∆E.The lightness values (L*) in the biscuits fortified with kishk were higher (60.80–64.39) than that in the control sample (60.16). Yellowness values (b*) were found to be lower in the samples with Egyptian kishk (29.60–31.85) than in the control biscuit (32.44). A similar trend for color values in both surface and back of biscuit was observed. Redness (a*) showed no significant difference between the control and fortified sample with 10% of kishk, as well as between 40 and 50% kishk samples in the surface analysis (Table 5). An opposite trend was found in back analysis, which had significant variation between the samples.The main color characteristics of biscuit depends on the degree of caramelization, or Maillard reaction, and are affected by various factors such as protein, carbohydrates, water activity, pH, etc. Therefore, the processing conditions during baking should be controlled [29, 30].We also analyzed the effect of kishk in biscuit on its textural properties, namely hardness (Table 5). The obtained results indicated that the increasing amount of kishk increased the hardness of biscuit from 27.43 to 47.76 g (for the 10 and for 50% kishk samples, respectively). The same trend was observed by Drisya et al. who added dehydrated Murraya koenigii powder to cookies [31]. Our results are also consistent with Parul et al., who found that spirulina and sorghum flours incorporated in dough increased the hardness ofbiscuit [32].The highest hardness of the 50% kishk biscuit compared to the other samples may be explained by its high protein content [33]. A gradual increase in hardness of biscuits with increasing the Egyptian kishk amount may be due to water absorption by kishk. Therefore, it is important to control baking conditions during addition of kishk with the view to replace wheat flour. Nandeesh et al. also reported that 30% of differently treated wheat brans increased the hardness of biscuit dough and decreased its cohesiveness, springiness, and adhesiveness [34].Sensory evaluation. Table 6 demonstrates the effect of replacing wheat flour with Egyptian kishk in biscuit on its sensory properties. We evaluated appearance, color, aroma, taste, and texture. All the attributes showed a lower score in the fortified samples compared to control. Our results are similar to those reported by Drisya et al. who used Murraya koenigii leaf powder in the amount of 10% to fortify cookies, and this amount was acceptable [31]. Bajerska et al., who used green tea extract in bread, mentioned its adverse effect on the sensory properties of the bread [35]. These data may contrast due to significant differences in processing conditions for biscuit and bread.The obtained results are in agreement with those reported by Patel and Rao, who fortified biscuit with black gram flour [4]. Another study carried out by Ezeagu et al., who substituted wheat flour with 15% of Muccuna seed flour in biscuit, supported ourfindings [36]. The results of sensory evaluation were in parallel with the color metric measurements.Antioxidant activity. Figure 1 shows antioxidant activity in the control and fortified biscuits determined by DPPH and ABST assays. Both assays revealed a pronounced increase in antioxidant activity with the increase of kishk amount in the dough. However, the DPPH assay showed better results compared to ABST. Our results are in good agreement with Galla et al., who supplemented biscuit with spinach leaves extract in the amount of 5–15% [37].The correlation coefficient (R2) between total phenolic and antioxidant activity by DPPH and ABST methods were 0.973 and 0.984, respectively, which revealed that phenolic compounds are responsible for antioxidant activity in the biscuit samples. Phenolic compounds form in bakery products as a result of Maillard reaction and caramelization [38, 39].With increasing amounts of kishk in biscuit, its antioxidant activity rose (Fig. 1). Biscuit fortified with marine collagen peptide, as reported by Kumar et al., demonstrated a weak antioxidant activity due to low content of phenolic compounds, which is responsible for antioxidant activity [40]. Therefore, the improvement in the antioxidant activity of the biscuit fortified with Egyptian kishk in the present study may be due to formation of Maillard reaction products during bakery processing as reported by Krystyjan et al. and Antoniewska et al. [41, 42]. The authors mentioned that antioxidant activity in biscuit and muffins was due to Maillard reaction products rather than polyphenols compounds. Therefore, a more detailed investigation on isolation and identification of Maillard reaction products formed in the fortified biscuit is needed.Volatile compounds. Table 7 shows volatile constituents detected in the headspace of the biscuit samples fortified with Egyptian kishk. A total of 28 volatile compounds were identified and quantified. They were alcohols (4), aldehydes (9), ketones (2), carboxylic acids (2), esters (2) terpenes (1), and sulfur-containing compounds (8).Aldehydes were the main group of volatile compounds in the biscuit samples, especially in the control biscuits, followed by sulfur-containing compounds and alcohols. The predominant aldehyde was benzldehyde (16.52%). The volatile compounds differed significantly among the control and fortified biscuits. The kishk-fortified biscuits showed higher levels of aldehydes, especially hexanal, heptanal and nonanal, which exhibited about a 2–10-fold increase compared to the control sample due to their presence in the raw material.Iranmanesh et al. analyzed volatile compounds in dried kishk obtained from different animals. The authors detected aldehydes in all samples, with significant amounts in those samples containing flour during preparation [43]. Another explanation for the high levels of aldehydes in the fortified samples is thermal treatment of food products [44]. Among Strecker aldehydes forming via Maillard reaction, 2-Methylbutanal and 3-Methylbutanal were present both in the control and the fortified biscuit samples. However, significant amounts of hexanal, octanal, and heptanal were determined in the fortified samples compared to the control biscuit. This fact clarifies why the sensory properties the biscuits with high amounts of kishk had low scores. All the biscuit samples included alcohol. These compounds were identified in biscuit with purple wheat byPasqualone et al. [45].The furan compounds were mostly represented by furfural, a caramel-like odorant deriving by Maillard reaction, significantly more abundant in the control and 5% kishk samples, compared to the other fortified samples. Furfural is typically present in biscuits, which was reported in several studies [45, 47, 48]. Little amounts of pyrazines, produced by Maillard reaction, were also detected in the current research.Mohsen et al. found three Strecker aldehydes in wheat cookies: 2-methylbutanal, 3-methylbutanal, and benzaldehyde. Those could have been derived from some amino acids, e.g. leucine, isoleucine or phenylalanine [45, 47]. These aldehydes are in good correlation with biscuit flavor which formed during preparation from Maillard reaction especially Strecker degradation [49–51]. In our study, we found that the biscuit fortified with kishk contained low amounts of these aldehydes (Table 7), which explains its low score of sensory evaluation. On the other hand, pentanal, 2-hexenal and alcohols, such as 1-pentanol, have an adverse effect on biscuit flavor due to linoleic acid oxidation [47, 52]. Low amounts of these aldehydes and alcohol make off-flavor with the increasing of kishk in biscuit (Table 7).CONCLUSIONThe current study found that fortification of biscuit with Egyptian kishk (10–50%) improved its nutritional composition such as protein, fiber, etc. The water absorption, arrival time, dough development time, and weakening of dough increased in the kishk-fortified biscuit. The fortified biscuit, especially the sample with 50% of kishk, also had higher lightnessvalues (L*) compared to the control sample. In addition, both the control and fortified with kishk biscuit contained an increased amount of total phenolic compounds. The antioxidant activity increased with increasing kishk amounts.However, kishk had a negative effect on the sensory characteristics, physical properties, as well as volatile compounds of the biscuit. The kishk-fortified biscuits showed higher amounts of aldehydes, especially hexanal, heptanal, and nonanal. Further studies are needed to reveal effects of storage conditions on the physicochemical attributes and volatile compounds of kishk biscuits and to isolate and identify Maillard reaction products formed in the biscuits.CONTRIBUTIONAll the authors equally contributed to the study and are equal responsible for plagiarism.CONFLICT OF INTERESTThe authors declare no conflict of interests related to the publication of this article.