Physicochemical characteristics, antioxidant potential, and shelf stability of developed roselle–fig fruit bar

Abstract Fruit bars are prepared by combining different ingredients which are wholesome and nutrient rich. The current study was designed to develop roselle–fig (different proportions) fruit bars and further investigate their physicochemical characteristics and antioxidant potential. Moreover, the prepared fruit bars were scrutinized for microbial and sensory characteristics to assess the appropriateness of ingredients during storage (up to 90 days). It was observed that there was gradual increase in moisture content with the passage of time, while ash, fat, fiber, and protein contents did not change significantly during storage. The pH and total soluble solid contents of these fruit bars during storage were 3.54–4.07 and 1.71–1.86 Brix, respectively. According to the mean values for sensory evaluation, T 2 was preferred over other treatments. The bars received an acceptable sensory evaluation, demonstrating that they were suitable for 90 days of storage. Phytochemical quantities significantly increased in subsequent treatments, but decreased during storage in all the treatments. Similar trend was observed regarding total antioxidant and DPPH radical scavenging activities. The mineral contents increased significantly among the treatments. The microbial analysis of fruit bars exposed that the fruit were microbiologically safe. Hence, on the basis of the results obtained in this study, it may be concluded that the developed roselle–fig fruit bar would be acceptable and nutritious regarding physicochemical characteristics, microbiological quality, and antioxidant potential.


| INTRODUC TI ON
Changes in the daily routine of a large portion of the world's population have resulted in an increase in demand for ready-to-eat convenience products (Popkin et al., 2020). Nowadays, the interest of people in food is not only to control hunger, but also to seek the products which could be beneficial for health or may reduce the chance of development of certain illnesses (Aja et al., 2023;Roberfroid, 2000;Santos et al., 2011). Usually people enjoy eating fresh fruits of the season, but there is a lack of their availability throughout the year. Therefore, it is imperative to preserve them in order to relish their nutrition throughout the year. Since many fruits contain majority of the flavoring and nutritional components, hence these can be used to create snack foods (Ukkuru & Pandey, 2007). The snack foods are relatively growing very fast as compared to other food products (Paiva, 2008). Fruit bars (to be used as snack food) may be developed in response to market demand for high-quality nutritional food (Ryland et al., 2010). Such kinds of fruit bars are useful to provide nutrition and health benefits to the individuals of different ages.
It has been reported that fruit bars are a type of processed food that offers nutritional value in the form of vitamins, minerals, and fiber (Parekh et al., 2014). It is considered as a nutrient-dense diet (concerning bioactive compounds) which is required for the proper functioning of the body (Sun-Waterhouse et al., 2010). The shelf life of fruit bars is generally long as compared to fresh fruits (Joshipura et al., 2001). Fruit bars are not only consumed by people for energy, but also for the gratification (Silva et al., 2018). Fruit bars are gaining popularity due to their excellent shelf life, flavor, texture, attractive packaging, and portability (Parimita & Arora, 2015). The healthy fruit bars would be better replacement of junk foods.

which is an underutilized plant in
Pakistan with a lot of medicinal and nutritional properties (Abbas & Ali, 2011). It is broadly dispersed in tropical as well as subtropical regions and could be found in nearly all warm countries (Qi et al., 2005). The multipurpose roselle plant has a lot of importance in traditional medicines and has been reported to cure many diseases such as sores, ulcers, intoxication, and cough (Prasongwatana et al., 2008). Roselle has also been reported to cure cardiovascular problems, inflammatory illnesses, and cancer (Nkumah, 2015).
Although its seeds, leaves, and calyces are considered valuable, the calyces offer higher nutritional and medicinal values. Roselle calyces are red in color (Cisse et al., 2009), sweet and tart in taste (Monteiro et al., 2017), and are good source of vitamin C, minerals, organic acids, fibers, and bioactive compounds like polyphenols (antioxidants), phytosterols, and organic acids (oxalic and succinic acid; Cisse et al., 2009). It has higher ascorbic acid level than orange and mango (Wong et al., 2003). It was also observed that 100 g of commercially available dried calyces contained 17 mg ascorbic acid, 150 mg calcium, 2 g protein, 10.2 g carbohydrates, and 3 mg iron (Al-Ansary et al., 2016). Moreover, roselle calyx is also rich in pectin and may be used to make jam, jelly, wines, cakes, and pudding (as fresh). It is also used in making tea, sauces, ice creams, and other desserts (Emmy, 2006;Tsai et al., 2002). Some studies concerning roselle-based drinks (Abidoye et al., 2022;Ehirim et al., 2022), fruit leathers, and jam (Fresua et al., 2022;Thonabut, 2021) have been carried out so far. Even though many other products can be prepared due to its unique taste, unfortunately it does not pique the interest of Pakistani food producers due to less awareness of its health benefits.  (Michailides, 2003), polyphenols, and amino acids with negligible quantities of fat and cholesterol (Veberic et al., 2008: Crisosto et al., 2010. It is also reported that fig fruit can be used in the treatment of many illnesses such as diarrhea, loss of appetite, colic, cardio as well as respiratory (Duke, 2002), hepatoprotective (Gond & khadabadi, 2008), immune response (Yang et al., 2009).
Due to intense competition in the consumer market, industries are constantly in search of new ingredients that can enhance nutritional quality and add value to the final product Ruiz-Ojeda et al., 2019). Owing to the health benefits of roselle calyx and figs, it would be nice to produce fruit bars with more health benefits and minimal processing. However, combining these two fruits into fruit bars could be a lucrative business idea. Therefore, this study was focused to investigate the aptness of fig and dried roselle for use in nutrient-dense fruit bars through physicochemical and sensory analyses. Moreover, shelf stability and antioxidant potential of such fruit bar were also monitored.

| Purchasing of raw materials
Dried calyces of roselle were purchased from Faisalabad (Pakistan), while the figs were purchased from the local market of Sargodha (Pakistan). Chemicals (Merck, Germany) for the analysis of final product were purchased from Merck distributor in Islamabad (Pakistan).

| Preparation of basic ingredients and treatment plan
Roselle calyx and figs were obtained in dried form and weighed according to the ratio given in treatment plan. These were then washed under running tap water to remove dust/dirt or any adhering material. Further steaming was done in steamer to decrease the microbial load and to soften the figs and dried roselle. Before washing and steaming, dried figs contain 5.98% moisture and roselle 7.64% moisture. The steaming process also facilitates figs and roselle to convert them into paste by passing through mincing machine. The moisture content of fresh fruit bars ranged from 19.60% to 29.45%. Table 1 shows the proportions of each ingredient.

| Development of roselle-fig fruit bar
Roselle-fig fruit bars were prepared by the method described by Nadeem et al. (2018) with some modifications. All the necessary machines and tools and surfaces were thoroughly cleaned with clear water and further contact surfaces were cleaned with ethanol and dried before starting any operation. Moreover, gloves were used for sheeting and cutting and handling of raw material. The steamed roselle calyces and figs were placed in mincing machine (Model: WF-3250 R; WestPoint Electronics, Bordeaux, France) to form fruit paste. The fruit paste was moved to a sheeting and cutting table so that the bar shape could be more clearly achieved. After it had been sheeted using a roller, a cutter was used to cut the sheet into 2.5 cm wide × 1 cm thick × 7.5 cm long bars. The bar weighing approximately 25 g were sealed in airtight aluminum foil. These packs were stored at refrigerated temperature (4 ± 2°C).

| Physicochemical analysis of roselle-fig fruit bar
Proximate analysis developed fruit bars, such as moisture, crude fiber (%), protein (%), fat (%), and ash (%), were scrutinized by the methods described in AOAC (2019). The pH was measured using a digital pH meter (Satorious, Germany; AOAC, 2019). Total soluble solids (TSS) were determined with the help of Atago Hand Refractometer by the method described previously (AOAC, 2019).

| Sample preparation
For the evaluation of antioxidant potential, the samples were prepared by grinding 5 g of each bar to a fine powder with the help of a grinder. Then, 50 mL of the solvent was added into each sample to make homogeneous suspension. The mixture was then transferred into polypropylene tubes and shaken for approximately 1 h at room temperature. For proper extraction, the sample was filtered twice with Whatman filter paper after centrifugation at 1509.3 g for 10 min. The extract was stored at 4°C for later use.

| Determination of total phenolic (TP) contents
The Folin-Ciocalteu reagent method as explained by Salleh et al. (2017) was used for the determination of TP contents in all the samples. For the analysis, 400 μL of diluted samples were used for the reaction of 1 mL Folin-Ciocalteu reagent (10%) and 2 mL Na 2 CO 3 (20%) in sequence. The absorbance of above mixture was measured at 760 nm using a spectrophotometer. Gallic acid was used as the standard and TP contents were expressed as mg of gallic acid equivalents (GAE) per 100 g.

| Determination of total flavonoid (TF) contents
The TF contents of roselle-fig fruit bar were assessed by the method described by Piskov et al. (2020). For analysis, 250 μL of diluted samples were taken before addition of 75 μL NaNO 2 (5%), 150 μL AlCl 3 (10%), and 500 μL NaOH (1 M) in sequence. Furthermore, 2 mL distilled water was also added into the above mixture. The absorbance was measured at a wavelength of 510 nm by using a spectrophotometer. Catechin (in ethanol) served as the standard and the TF contents were expressed as mg of (+) catechin equivalent (CE) per 100 g.

| Determination of total antioxidant activity (TAA)
The TAA of samples was calculated by applying a method described by Prieto et al. (1999). Four milliliter of the reagent (0.6 M H 2 SO 4 , 28 mM sodium phosphate, 4 mM ammonium molybdate) was added into 400 μL of each diluted sample. The absorbance was measured with a spectrophotometer at a wavelength of 695 nm. Trolox was used to create standard calibration curves. The TAA was measured in μg Trolox equivalent (TE) per gram of sample.

| Determination of DPPH radical scavenging activity
The method explained by Wang and Gao (2013)  For the experiment, 1000 μL of the diluted sample was taken. The spectrophotometer was used for taking absorbance of all prepared samples at 517 nm. Ascorbic acid was used to create standard calibration curves. In this way, the DPPH radical scavenging activity was expressed as μmol ascorbic acid equivalents (AAE) per gram.

| Mineral analysis of roselle-fig fruit bar
Mineral contents of roselle-fig fruit bar were assessed using atomic absorption spectrophotometer (AA-Shimadzu Japan) by wet digestion method as described in AACC (2000).   By using method no. 42-11 (AACC, 2000), TPC were determined.
The fruit bar sample (each 1 g) in crushed form was placed in a sterile blender jar (having diluents of 9 mL buffered phosphate). The sample was blended at slow speed for 1-2 min. One milliliter of already available sample was transferred to 9 mL for preparation of decimal dilution. To resuspend material, the dilutions were agitated. One milliliter from each dilution was taken and placed on marked duplicate Petri dishes. About 12-15 mL of cooled plate count agar was placed on plates and allowed them to solidify. The dilutions were poured onto plates. To mix agar medium and sample dilutions, the plates were rotated. The inverted Petri dishes were incubated for about 48 h at 35°C. The colonies were counted and multiplied with dilution factor. The results were expressed in log 10 CFU/g.

| Sensory evaluation
The prepared fruit bars in this study were evaluated for sensory characteristics such as appearance and color, taste, texture, and overall acceptability by a panel of 20 members including faculty and postgraduate students from the Institute of Food Science and Nutrition, University of Sargodha, by following the 9-point Hedonic scale (Santos et al., 2011).

Components Days
Treatments Total Flavonoids (mg CE/100 g) Total Phenolics (mg GAE/100 g) Treatments decreasing trend might be attributed to the oxidation of phenolics due to higher activity of naturally present polyphenol oxidases in figs. By increasing the quantities of roselle calyces in the successive treatments (fruit bars), TP contents also increased. Our results were accordant with the findings of Shin et al. (2021) who observed increased total polyphenol contents in yogurt (treatments) by the gradual increase in dried roselle calyx into their formulations.
The higher the phenolic compounds, the higher the antioxidant activity (Miladi & Damak, 2008;Zeghoud et al., 2023). The trend of total phenolic contents observed in this study were similar to that observed by Ranjha et al. (2020), who prepared date bars fortified with APE (apple peel extract) and PPE (pomegranate peel extract).
In addition, our results of this study were also very similar to the findings of Nadeem et al. (2012), who reported that the addition The TF contents were found the highest (370.26 mg CE/100 g) in freshly prepared fruit bars during T 5 treatment, whereas the lowest (241.42 mg CE/100 g) were observed in freshly prepared fruit bars during T 0 treatment. Yen et al. (2022) investigated the red dragon fruit bar and found similar results. As the concentration of passion fruit increases, so the flavonoids contents increased. The flavonoids from the passion fruit were thought to be the cause of the higher TP and TF levels in the fruit bar. Moreover, the passion fruit also had ascorbic acid, which protected the flavonoids in the fruit bar. Kolniak-Ostek et al. (2013) found that adding ascorbic acid to pasteurized cloudy apple juices had a positive effect on the polyphenol compounds. Similarly, the outcomes are consistent to the findings of Nadeem et al. (2012) who observed a gradual decrease in flavonoid content during storage of 120 days. At the day of production, the total flavonoids contents were 106.63 mg CE/g, which decreased to 44.91 mg CE/g after 120 days. Free radicals are also produced to a greater extent during storage, which may also reduce the total flavonoids contents.
Del Caro et al. (2004) prepared orange juice and stored at 4°C and showed decrease in flavonoid contents during storage.
Even though there were appreciable quantities of TF contents in T 0 (comprising 100% fig fruit), it was also observed that there was slight increment in TF contents in successive treatments. Such kind of slight variations in the treatments might be attributed to the addition of more and more roselle calyces in subsequent treatments (bars). This showed that roselle calyces also contained significant quantities of TF contents. Our results were accordant with the findings of Shin et al. (2021) who observed increased total flavonoid contents in yogurt (treatments) by the gradual increase in dried roselle calyx into their formulations.

| Total antioxidant activity (TAA)
The This showed that roselle calyces contained enormous quantities of TP and TF contents as reported by a study who observed considerable quantities of total polyphenols (740 mg/100 g), flavonoids (350 mg/100 g), and anthocyanins (1653 mg/100 g) in roselle extract (Pacôme et al., 2014). During storage, phenolic as well as flavonoid contents might be degraded, resulting in a reduction of total antioxidant activity of fruit bars.
These outcomes were in agreement to the findings of Agrahari this is because of the synergistic effects of these antioxidants, which has a high capacity to donate hydrogen atoms and thus neutralize the free radicals produced during the thermally induced oxidation and auto-oxidation processes (Yen et al., 2022). Similar results were stated by Sakhale et al. (2015) in fig jam. They observed that at the day of production, the DPPH content were 43.70, which reduced to 28.90 after 60 days of storage at low temperature. Nevertheless, it may be assumed that the oxidative reaction and prolonged storage may affect hydrolysis of compounds and lead to gradual reduction. Ascorbic acid is extremely susceptible to heat. Therefore, thermal deterioration of ascorbic acid during manufacturing and oxidation may contribute to that decrease (Mutkule et al., 2014).
Even though there was notable values of DPPH radical scavenging activity in T 0 (comprising 100% fig fruit), it was also observed that there was slight increment in DPPH radical scavenging activity in successive treatments. Such kind of slight variations in the treatments might be attributed to the addition of more and more roselle calyces in subsequent treatments (bars). This showed that roselle calyces contained enormous quantities of TP and TF contents which might have shown DPPH radical scavenging activity. During storage, phenolic as well as flavonoids might be degraded, thereby resulting in a reduction of DPPH radical scavenging activity of fruit bars.

| Mineral contents of roselle-fig fruit bar
The mean values regarding mineral composition of fruit bar are presented in Figure 3 Our results showed that mineral contents of fruit bars differed significantly (p ≤ .05). All the treatments (T 0 -T 5 ; fruit bars) showed increasing trend of minerals as most of the minerals in dried roselle were found in higher concentrations. Among the major minerals, K was   values (log 10 CFU/g) of TPC of T 0 were the highest at all stages of storage period as compared to the counts of all other treatments, whereas the lowest counts were found in T 5 . It was observed that by increasing the concentrations of roselle calyces in the treatments, TPC gradually decreased as roselle had antimicrobial properties.
During a 3-month storage period at refrigerated temperatures, TPC grows significantly, but remains within tolerable limits. This rise in TPC may be attributable to the availability of nutrients in fruit bars.
The observations are in conformance with those obtained by Munir et al. (2019), who reported the same decreasing tendency among the treatment of apricot-based bars, as the mean values varied from 3.0 log 10 CFU/g (T 0 ) to 2.2 log 10 CFU/g (T 3 ). The findings of this study were also supported by the earlier findings of Al-Hooti, Sidhu, Al-Otaibi, Al-Ameeri, and Al-Qabazard (1997), who discovered that TPC in date bar samples ranged from 1.00 to 2.18 log 10 CFU/g. The change in TPC during storage for different fruit bars are in accordance to the results of Akhtar et al. (2014), who reported that TPC increased significantly in apple-date fruit bar during storage of 90 days from 0.5 log 10 CFU/g to 3.21 log 10 CFU/g. Similarly increase in TPC was observed by Nadeem et al. (2022) in water chestnut-supplemented date bars. The increase in TPC during storage of 90 days ranged from 1.64 log 10 CFU/g to 2.31 log 10 CFU/g.

The antioxidants present in both fig and roselle calyx may have ef-
fect on the shelf stability of prepared bars in this study.

| Sensory characteristics of roselle-fig fruit bar
The fruit bars prepared in this study were assessed for appearance and color, taste, texture, and overall acceptability during storage period (0-90 days; Figure 4). The findings of this study revealed that the scores regarding appearance and color attribute among different treatments varied significantly (p < .05). Among the treatments, freshly prepared T 5 showed the highest score (7.5), whereas freshly prepared T 0 showed the lowest score (6.1). The of color score of soy-enriched apple bar from 4.9 to 3.8 during period of 6 months storage. Similarly, Al-Hooti, Sidhu, Al-Otaibi, Al-Ameeri, and Al-Qabazard (1997) also found the decrease in color of date bars from 6.8 to 6.2 during storage period up to 6 months. Ahmed and Ramaswamy (2005) also observed that color of papaya fruit bars was affected significantly by storage periods. They explained that score of color varied from 6.6 to 6.2 in 6 months of storage period. Nadeem et al. (2018)  It was observed that taste score varied significantly (p < .05) due to more addition of roselle calyces. The highest taste score Taste is perceived by the taste buds of the tongue, while the composition of food, flavor, and texture can modify it. A food is generally accepted or rejected by taste. The taste score was declined during storage because of development of bitterness with increased storage time (Sharma et al., 2013). Similar trend of taste score was observed in wood apple bar (Bhatt & Jha, 2015), choco-quinoa nutri bar (Padmashree et al., 2018), nutri bar (Athira et al., 2018), defatted soy flour cereal bar (Yadav & Bhatnagar, 2016), peach-soy fruit leather (Anju et al., 2014). Nadeem et al. (2012) found that the taste of date bars remained acceptable for the storage period up to 90 days, however, the score for taste decreased with increase in storage period. Prasad (2009) concluded that scores for taste of mango bars increased with the increase in quantity of mango fruit powder up to 30% and decreased thereafter. Nadeem et al. (2022) prepared a fruit bar supplemented with water chestnut and observed similar results. The highest taste score 7.72 was noticed at the production day, while the lowest taste score 5.79 was noticed after 90 days of storage.
Regarding texture, it was observed that the score varied significantly (p < .05) due to more addition of roselle calyces. The highest texture score (7.7) was obtained by freshly prepared T 3 (roselle- Texture is the most significant property which defines the consumer acceptability and overall quality of a food product. Texture can be scored by touch and visually. Texture is the perception of the rheological and structural characteristics of a product that can be judged by mechanical, tactile, visual, and auditory receivers. Jan et al. (2012) described a gradual reduction in the texture score (8.00 to 7.10) of nutri bars for lactating women. Silva et al. (2016) showed that texture values nonsignificantly decreased from 7.14 to 7.11 in bars made with Jeriva fruit flour in Brazil.  found that the texture score of date bars decreased from 7.0 to 5.9 during 1 to 180 days of storage. The changes in texture score of various treatments prepared in this study was also in accordance to the findings of Nadeem et al. (2022), who observed the highest texture score (7.53) in fresh water chestnut-supplemented date bars, which gradually decreased Consequently, it might have a lot of sales potential due to its high nutritional value.

| CON CLUS ION AND RECOMMENDATIONS
The results revealed that there was significant effect on the ash,

ACK N OWLED G M ENTS
The authors are thankful to the Library Department, University of Sargodha, Sargodha and IT Department, Higher Education Commission (HEC, Islamabad) for access to journals, books, and valuable databases. The authors are also grateful to Kampala International University, Kampala, Uganda for its support.

FU N D I N G I N FO R M ATI O N
No funding was received for this study.

CO N FLI C T O F I NTER E S T S TATEM ENT
All authors have no conflict of interest.

DATA AVA I L A B I L I T Y S TAT E M E N T
Even though adequate data have been given in the form of tables, all authors declare that if more data are required then the data will be provided on request basis.

CO N S ENT FO R PU B LI C ATI O N
All authors agreed for publication of this manuscript.