Formulation and Evaluation of Iron-Rich Chocolate Spread from Sugarcane Syrup and Sunflower Seeds

. Anemia is widely recognized as a serious public health problem and the most common type of micronutrient defciency globally. Developing a product enriched with iron to overcome this issue has received excessive attention. For this purpose, sugarcane syrup and sunfower seeds were utilized as sugar and hazelnut substitutes in chocolate spread production. Four formulations were created and assessed for their chemical, texture, and sensory attributes, as well as their iron content by microwave plasma-atomic emission spectrometry (MP-AES) and compared with a commercial product. Fat and sugar levels were reduced by 2 − 1.3 and 1–1.5 times, respectively, in the samples. Increasing the percentage of sugarcane syrup raised the iron content 3-to 6-fold. Peroxide and free fatty acid values indicate that iron negatively afects the oil quality of the developed samples. Te samples could be recommended as novel products that are preferred as a healthy and low-cost chocolate spread.


Introduction
For many years, cocoa beans (Teobroma cacao L.) and chocolate goods have been consumed for their favor and so are polyphenols such as catechins, procyanidins, and cafeic acid, all of which have benefcial health benefts [1]. Chocolate spread (Gianduja) is a popular chocolate product among teens who use it to fll various confectionary items or spread it on bread and wafers [2]. According to the Codex (2016), chocolate spread contains a minimum total dry cocoa solid content of 32%, including a minimum dry nonfat cocoa solid content of 8%. Tis type of chocolate contains a high percentage of fat (≥40%), mostly saturated fat (palm oil), which contributes to the global increase in the prevalence of cardiovascular diseases, according to Ismail et al. [3]. One previous study [4] reduced the fat content of chocolate spread by substituting milk fat/cellulose ether emulsions for palm fat; however, this has the disadvantage of decreasing digestibility. In response to the rising consumer demand for healthier foods with good textural properties, researchers have developed a variety of these products [5][6][7][8][9]. Tey explored maltitol and grape pomace as sugar substitutes, as well as sunfower, soybean, coconut oils, and structured triacylglycerols as alternatives to palm oil. Furthermore, there have been few studies to fortify this type of chocolate with healthier ingredients or more micronutrients. Tolve et al. [8], for example, fortifed the chocolate spread with Ca +2 and vitamin D using Mg-CaCO 3 nanoparticles or by adding 166 μg of vitamin D/kg to the fnished product, respectively.
Poor dietary habits and sedentary lifestyles have contributed to an increase in diferent malnutrition-or obesityrelated diseases. Iron defciency is still one of the world's most serious public nutritional defciencies [10]. Anemia is associated with the more severe stages of iron defciency, which primarily afects children and pregnant women. Iron defciency is described as the absence of mobilizable iron stores, and the marks of a compromised iron supply to tissues, called erythron, are familiar. Te main predictors are a hemoglobin level of 110 g/l and a ferritin level of 12 g/l [11]. According to the World Health Organization [12], anemia afects 42% of children, 40% of pregnant women, and 33% of nonpregnant women worldwide, and 280 million children as well as 614 million women globally still sufer from it. In Egypt, 21% of girls and 17.5% of boys (5-19 years old) were found to be anemic. Furthermore, a quarter of women of reproductive age (15-49 years old) and 28.2% of lactating women had anemia [13]. Terefore, the consumption of foods containing iron is more efective than medications to treat this defciency. One of the excellent sources of iron is sugarcane syrup [14].
Sugarcane syrup is a dark syrup or honey produced in many sugar-producing countries, including Egypt and Portugal [15,16], and it is most likely consumed as a confectionary or with certain bakery products. It is primarily made by thermally treating fltered juice obtained by mechanically pressing fresh sugarcane stalks [17]. It contains at least 95% sugar by weight, with sucrose accounting for 30% of total sugar [16]. Sugarcane syrup has been approved to boost the levels of hemoglobin in children and pregnant women because it is an excellent source of iron, containing around 0.7% of iron by weight [14]; however, it has not been used to enrich any food with iron. On the other side, the sunfower (Helianthus annuus L.) seed is one of the world's most important oilseed crops [18]. Sunfower seed contains 35%-42% oil and is naturally high in linoleic acid (55%-70%) but low in oleic acid (20%-25%). In addition to the polyunsaturated fatty acids, sunfower seeds contain favonoids, antioxidants, and vitamins that may lower total cholesterol, particularly low-density lipoprotein (LDL) cholesterol [19]. Recently, Bascuas et al. [20] investigated the replacement of 50% of the coconut fat in chocolate spread with sunfower oil. However, sunfower seeds have not been explored as an alternative to hazelnut to reduce the cost of the fnal chocolate spread product.
Tis study aimed to increase dietary iron intake by developing a healthier chocolate spread free of palm fat. Te efects of substituting sugar for sugarcane syrup and hazelnuts for sunfower seeds on the physicochemical and nutritional properties of this form of chocolate were assessed in particular.

Chocolate Spread Preparation.
Te components of the chocolate spread listed in Table 1 were combined to generate four chocolate spread formulae. Teir quantities were selected based on the standard for chocolate spread products [21], in which the chocolate spread must contain ≥13% hazelnut and ≥7% cacao powder. Formulation 4 contains the closest amount to the control commercial product, with 13% hazelnut, 10% milk, and >7% cocoa powder, with the remainder being sugarcane syrup. Te other formulations gradually reduce the sugarcane syrup content by increasing the other ingredients within these limits. Te formulations were prepared with some modifcations, according to Bascuas et al. [20]. Te dry ingredients, such as cocoa and milk powder, as well as the sunfower seed paste, were initially sieved separately and then combined. Te sugarcane syrup was then added and blended for 15 min at 50°C with a hand blender (ST-FP0053 D, Saturn Home Appliances, Guangdong, China). Finally, in a constant ratio, sunfower oil and lecithin were added to all formulations after cooling to room temperature (30 ± 2°C) and mixing for 5 min. All examinations were conducted after 24 h.

Total Phenolic Content Estimation.
Te samples were frst defatted twice with 10 ml of n-hexane at 30°C and then air-dried for 4 h. Te phenolic compounds were extracted from the defatted samples using 10 ml of 70% methanol [24]. Te total phenolic content (TPC) was determined as follows: 100 µl extract or blank (methanol) was mixed with 2.5 ml Folin-Ciocalteu reagent (10%) and 2.0 ml sodium carbonate (7.5%). Te mixture was mixed and incubated in the dark for 1 h, followed by measuring the absorbance at 760 nm using the specifed spectrophotometer. Total phenolic content was calculated as mg gallic acid equivalents/100 g dry sample [25].

Antioxidant Potential Activity Estimation.
Te DPPH free radical assay was applied to detect the antioxidant efect of the prepared samples. Briefy, 1 ml of the previously diluted methanolic extract was mixed with the same volume of methanolic DPPH solution (0.2 mM). After 30 min of incubation in the dark, the absorbance was measured at 517 nm with methanol as a control instead of the sample [24]. Te DPPH scavenging activity was calculated using the following equation:

Oil Quality Assessment.
Te samples were evaluated after 1 month of storage at 30°C by analyzing the peroxide value and free fatty acid content, as described by Tarakçi & Yildirim [26], with some modifcations. After mixing about 5.0 g of chocolate sample with 5.0 ml of petroleum ether for 1 min, the tubes were centrifuged at 2795 × g (Hermle, Z300, Germany) for 10 min to separate the upper phase. Te extraction was carried out twice more, and the combined extract was collected. Te ether was evaporated, and the extract was resuspended in 1 ml n-hexane. Te peroxide value of the extracted oil was determined by titration with sodium thiosulfate solution (0.002 N) in the presence of starch (1.0%) as an indicator. Te results were expressed as mg equivalent O 2 /kg of oil. Meanwhile, the acidity as free fatty acids (FFA) was determined by titration with KOH solution (0.05 N) and calculated as a percentage of oleic acid.

Iron Content Estimation.
To determine the iron content, the sample was frst digested with HNO 3 at 180°C for 8 min according to the microwave digestion system (Multiwave GO Plus, Anton Paar GmbH 208054, GRAZ, Austria). Te iron was then detected using microwave plasma atomic emission spectrometry (MP-AES 4210, Agilent Technologies, Santa Clara, CA, 95051, USA) and calculated using the following equation: where a � concentration of the metal in the test solution (ppm); b � mean concentration in the blank concentration; v � the volume of the undiluted solution in ml; df � dilution factor; and m � weight of the test sample (g) according to the AOAC method of #999.10 [22]. All measurements were performed in triplicate, with the samples being maintained at an ambient temperature of 30 ± 2°C.

Physical
Analysis. Minolta (Chroma Meter, DP-301, Japan) was used to estimate the color parameters (L * , a * , and b * ) of the fnal prepared samples. L * denotes lightness, a * denotes a color range from red to blue, and b * denotes a color range from yellow to green. ΔE was calculated using the following equation: A Universal Testing Machine texture analyzer (825 University Ave, Norwood, MA, 02062-2643, USA) equipped with a cylinder probe (1 cm diameter) and a load cell of 250 lbf was used to analyze the texture profles of the tested formulae (placed in cylindrical plastic containers, 40 mm diameter) at 30 ± 2°C [27]. All parameters were calculated using TL-Pro food texture analysis software.

Sensory
Analysis. An informed panel of 50 panelists (35 women and 15 men between the ages of 18 and 30) from Cairo University's Food Science Department, Faculty of Agriculture, evaluated the prepared chocolate spread formulations. All participants gave informed consent. Parameters such as color, taste, favor, texture, spreadability, and overall acceptability were evaluated. Te samples were served at room temperature (30°C) on white plates with bread slices. Te sample was classifed using the 9 hedonic scales, with a scale of 1 indicating dislike and a scale of 9 indicating extreme liking [6].

Statistical Analysis.
A one-way analysis of variance (ANOVA) was performed on the data using CoStat statistical software. Te mean of three replicates was compared using Duncan's test [28] at a signifcant level of p < 0.05.

Te Chemical Composition of the Chocolate Spread.
According to the fndings (Table 2), all samples were within the permissible moisture limit for fungi and bacterial growth, as moisture levels may afect the shelf life of the packed product [29]. It could be realized that increasing the percentage of sugarcane syrup results in a signifcant increase in the moisture content of the spread created. Sample 1 has the lowest moisture level due to its high dry solids content (i.e., cacao and milk powder, as well as sunfower seeds), which improves the fnal product's water absorption. In accordance, the moisture content of chocolate spread created by Jeyarani et al. [5] that contains 85% soybean oil or a blend of 85% soybean oil and coconut oil (1 : 1), ranged from 5.0% to 6.3%. Moisture increased to 21.5%-24.7% when fuid skim milk was substituted for powdered skim milk . Although sugarcane syrup is high in sugar, all samples in this study exhibited a signifcant reduction (by 1-1.5 times) in sugar levels compared to the commercial control sample (p < 0.05). Te total fat content in the proposed formulations followed a similar pattern, with samples being lower than the control sample. Te frst formula had the    [30], which was higher than that reported in this study. Our fndings evidenced a reduction in the protein and fber content by increasing the sugarcane syrup, but all samples were signifcantly higher than the control (p < 0.05). Te total phenolic content (TPC) of the samples ranged from 30.01 to 117.31 mg/100 g, with signifcant diferences, and was 4.39 to 17.18 times lower than the commercial reference sample. Samples 3 and 4 were close to the control sample in cacao and milk content, but they had the lowest TPC level. Tis contradicts previous research, which found that sunfower kernels have a higher phenolic content (2938.8-4175 mg/100 g) [31] than diferent hazelnut cultivars (68.5-1704.9 mg/100 g) [32]. On the other side, Roda & Lambri [30] detected a TPC level of 31 mg GAE per 100 g hazelnut paste, which was also lower than that detected in the current study. It may be due to diferences in analytical methodologies, sample materials, or their geographic origin. Similar trends were evident in the antioxidant potential activity. Te top antioxidant potential activity was detected in sample 1, which was the most similar to the control sample (p ≥ 0.05) and had the highest TPC. Tere are no signifcant diferences between the other samples, which have DPPH values ranging from 60.73% to 74.3%. Antioxidants, as well known, protect against free radical damage by preventing radicals' formation, scavenging them, or promoting their decomposition [33]. Antioxidants naturally present in hazelnut and sunfower seeds, such as tocopherols, proanthocyanidins, and favonols, help mitigate or slow the occurrence of these degradative reactions. Sample 1 has the highest content of sunfower seeds and cacao powder, which may be responsible for its antioxidant potential. Te relative antioxidant capacity and phenolic compound content of the chocolates investigated in this study were comparable to previous reports [24], in which diferent chocolate types exhibited DPPH scavenging of 10.20-134.32 μmol Trolox/g.

Oil Quality.
Te primary lipid oxidation product, hydroperoxide, was determined in diferent samples after 1 month of storage. Te peroxide values of the samples varied signifcantly (Table 2), with sample 4 having the highest peroxide value (1.33 mg equivalent O 2 /kg). Sample 1 had the lowest peroxide value that was comparable to the reference sample (p ≥ 0.05). Fresh fats have a peroxide value of less than one, and when the PV exceeds ten, unwanted sensorial properties (rancidity) occur [34]. Nonetheless, all of the tested chocolate samples reached the threshold of rancid oil.
Te most serious mode of failure in nut-containing chocolate products is oxidative rancidity, which produces free fatty acids with of-favors. Te primary quality attribute of edible-grade oil or fat is free fatty acids (FFA). Te maximum acceptable limit of FFA in chocolate is estimated to be 3% [35]. Referring to the results in Table 2, the samples have diferent acidity values (p < 0.05). Te reference sample had the lowest value, while sample 4 had the highest. Te FFA values in samples 3 and 4, which contain the highest percentages of sugarcane syrup, exceeded the acceptable limits. Due to inactivation, the action of oxidation enzymes such as lipoxygenase, which catalyzes the oxidation of linoleic acid (the main unsaturated fatty acid in sunfower seeds), had no efect in our samples because we used roasted sunfower seeds. Rancidity is classifed into two types: hydrolytic and oxidative. Te oxidative reaction requires the presence of oxygen and is accelerated by heat, light, moisture, and metal catalysts such as iron. Because the main abundant ingredient in the samples is sugarcane syrup, which is a rich source of iron [14], we hypothesize that metal iron is the main cause of oxidation in our samples. Iron is a known prooxidant because it can decompose lipid hydroperoxide into free radicals via a redox cycling pathway [36]. Iron-rich foods, as a result, require chelating agents such as EDTA to prevent oxidation and extend their shelf life. Although fortifed chocolate spread with added iron or from natural sources has been created in some previous studies [8,37], there is no data on its efect on the oxidative quality of its fat.

Iron Content.
Iron is a necessary element that performs critical functions such as oxygen transport, muscle metabolism, and DNA synthesis [12]. Because iron is present in cacao [38] and sugarcane syrup [14], determining the iron content of the proposed samples is essential. Figure 1 compares the iron content of the suggested formulations with the control sample and the RDA for adults, male and female, as well as children aged 4-9 years. According to the data, increasing the amount of sugarcane syrup nonetheless the sunfower seed content increased the iron content by three to six times over the control. All formulations provide iron above the RDA requirement for male adults and children because they include 11.00 to 20.34 mg/100 g of product. Remarkably, formulation #4, with 20.34 mg of iron/ 100 g, surpasses the RDA for both male and female adults and children. An earlier study [37] increased the iron content of prepared chocolate spread to 4.13 mg/100 g by adding carob four, which was lower than that achieved in the current study.

Te Color.
Te color of a product is the most important physical property that infuences its appeal. Color diferences in the chocolate spread can be attributed to the formulation (ingredients) variations, roasting degree, and processing parameters during production [39]. Table 3 shows the color parameters of the chocolate spread samples. Based on these data, replacing sugar with sugarcane syrup and hazelnut with sunfower seeds has no signifcant efect (p ≥ 0.05) on the L * value, indicating that it does not reduce lightness compared to the control sample. Te redness index Journal of Food Quality (a * ) and greenish (b * ) indices difered signifcantly between all formulations and the control sample. Color changes could be seen as light blue and brown with positive lower a * values and negative b * values, respectively. a * values of the formulations ranged from 0.85 to 0.97, with no statistically signifcant diferences (p ≥ 0.05). b * values increased in a negative direction as the sugarcane syrup content increased due to the sugarcane syrup color (dark brown). Furthermore, the color diference factor (ΔE) varied from 0.94 to 1.14, showing that the substitution of sugar and hazelnut with sugarcane syrup and sunfower seeds results in a substantial shift in the instrumental color values compared to the control. In contrast to the control sample, the results also reveal that formula (3) had the greatest color diference (ΔE), followed by formula (1). Tis is probably due to the high proportion of roasted sunfower seeds (23%). Our results are consistent with those of Acan et al. [7], as increasing grape pomace in prepared chocolate spreads resulted in negative b * and h°values with no signifcant diferences in L * values, which were attributed to the bright brown color of the pomace.

Te Texture.
While favor is usually deemed important in product identifcation, chocolate texture and appearance are key qualities in customer choice and acceptance [40]. Te hardness (frmness) of this product infuences its spreadability, which decreases as the hardness value increases [37]. Te control sample had a work shear value of 313.20 N, and the hardness of the tested samples decreased as the concentration of sugarcane syrup increased, indicating better spreadability (Table 4). Because it contains many solids, such as milk, cocoa powder, and sunfower seeds, sample 1 had the highest signifcant hardness value (371.30 N) compared to the control and the other samples. Tis fnding fts well with that of Fayaz et al. [41], who revealed that adding cocoa powder and sugar to chocolate spreads increased the solids, leading to a stronger network. Compared with the control, formula 4 had the lowest hardness value, which could be explained by considering its fat content (15.11 vs. 30.96%) and moisture content (7.37 vs. 2.57%). Acan et al. [7] evidenced a negative correlation between sample spreadability and its water activity, which matches the behavior of our samples. A similar pattern was observed in adhesion scores, where all samples substantially (p < 0.05) exceeded the control sample. In contrast, the springiness of samples 1 and  [37]. †Diferent letters show the signifcance between samples (p < 0.05). All experiments were performed in triplicate and the data are presented as means ± S.D (one-way ANOVA). 2 was similar to the control (p ≥ 0.05), which could be attributed to their high content of milk and cacao powder. As evidenced by their moisture content (Table 2), these additives may absorb moisture from the fnished product, resulting in more springiness. Te ultimate result of hardness and cohesiveness, gumminess, is an essential component of all semisolid food items. Formula (3), which had the highest gumminess score (206.00 N), also had the lowest degree of springiness. It was followed by formula #1, despite having a low concentration of sugarcane syrup. Te rheology behavior of molten chocolate is afected by many interconnected factors, including moisture level, ingredients (fat content), and processing strategy [8]. As a result of these observations, sample 1 is likely to necessitate excessive spreading efort, resulting in technological failures such as pumping, piping, and flling. Processing time during the ball mill and rotational speed should be considered for this purpose. When Aydemir & Atalar [42] used sunfower oil instead of palm oil, lower spreadability values were detected than those of the control. Meanwhile, the level of stickiness in chocolate containing coconut butter and hydrogenated palm oil was higher than in the control sample. Acan et al. [7] also found that higher solids in grape pomace decreased the spreadability of that chocolate due to its fber content. Figure 2 and its data (Supplementary 1) depict the sensory results for six parameters of the proposed chocolate spread formulations. All samples received varying ratings for color, taste, favor, texture, spreadability, and overall acceptability. In terms of texture, favor, and spreadability, sample 4 was the closest preferred formula to the control, which may be attributed to the presence of a high percentage of sugarcane syrup in relation to the solid components. In contrast, formulae 1 and 3 scored poorly in most characteristics, particularly spreadability and texture. Tese fndings corroborated the texture profle study results, in which formula 1 revealed the greatest hardness value (Table 4). Spreadability, or how easily a sample can be uniformly distributed across a surface, is a critical characteristic of this type of chocolate. Te highsolid ingredients, such as cacao powder and sunfower seeds, .20 e ± 0.14 * Diferent superscript letters within rows show the signifcance between samples (p < 0.05). All experiments were performed in triplicate and the data is presented as means ± S.D (one-way ANOVA). Journal of Food Quality increase the hardness, which infuences the spreadability and overall product acceptance. Te proportion of sunfower seeds also afected the acceptability of the formulations, with lower percentages of the latter being more acceptable. Tat indicates that sunfower paste at 13% was preferred over 23% for replacing hazelnuts. Color diferences have also been discerned by the panelists. Te color scores of the created formulae declined in the following order: formula 4 > 2 > 1 > 3, with formula 3 scoring the lowest and having the greatest ΔE value (Table 3).

Te Sensory Attributes.
Te study's weakness was that the chocolate formulae were evaluated blindly. Te participants had no information about the chocolates' composition, particularly their iron content, and were unaware of the reduced amount of fat and sugar in the designed chocolates. Accordingly, if the product's benefts are revealed to consumers, acceptance may signifcantly increase [40]. Consequently, it was concluded that the supplement of up to 63% sugarcane syrup did not adversely afect the product quality in terms of sensorial aspects.

Conclusions
Te study's main goal of developing acceptable spreads devoid of hydrogenated palm oil and with greater iron content than conventional chocolate spreads was successfully achieved. Te suggested formulae #4 can assist with anemia to meet the daily recommended dietary intake, especially for the most vulnerable individuals, such as children and adult females, because 100 g of this iron-rich source provides ≈20.5 mg iron. Finally, future investigations are encouraged to support our fndings by examining the iron bioavailability and digestibility of the proposed formulations.

Data Availability
Te data used to support the fndings of this study are included in the article.

Conflicts of Interest
Te author declares that there are no conficts of interest.