Commercial cocoa powder, milk powder and emulsifier purchased from a local supermarket (Cochin, Kerala, India) were used for the chocolate production. The chokita (cocoa butter equivalent) was purchased from Aak Kamani, Mumbai, India. The sweeteners, stevia (Sugar Free, Green, Zydus wellness, India) and sucralose (Sugar Free, Natura, Zydus wellness, India) were purchased online from Amazon, India. Other analytical grade chemicals used in the experiments were obtained from HiMedia® and Sigma-Aldrich® (Cochin, Kerala, India). Water used in the experiments was deionized unless specified otherwise.

Jackfruit seed powder was produced after fermentation of whole jackfruit. The seeds were placed in mud pots with jackfruit pulp and perianth and allowed to ferment anaerobically for 8 days in the presence of yeast (0.5%) and sugar (15%). The boxes were tightly closed to promote anaerobic fermentation. After anaerobic fermentation, the boxes were opened as shown in Figure 1(a), mixed thoroughly daily and subjected to aerobic fermentation for 8 days to promote oxidation (Figure 1(b)). The seeds were collected and spermoderm were removed. The cleaned seeds were dried using hot air drier (183˚C/48 h, Excalibur, US); roasted and powdered and yield was calculated [16] (Figure 1).

Chocolate base, chokita was melted until it becomes free flowing liquid (50˚C.). To this, cocoa powder, milk powder, lecithin and sweetener were added mixed thoroughly and ground using mechanical grinder to ensure uniform mixing. These were then poured in a polycarbonate set mould and refrigerated for 15 min till it solidified (Figure 2). In jackfruit seed powder incorporated chocolates, the cocoa powder was replaced with 10% jackfruit seed powder (Table 1).

The moisture (method 925.10), ash (method 923.03), protein (N × 6.25) (960.52) (KEL PLUS digestion (KES 06 R) and distillation systems (DISTYL EM S), M/s

Pelican Equipments, Tamil Nadu, India) and lipid contents (2003.05) (SOCS PLUS SCS2 R, M/s Pelican Equipments, Tamil Nadu, India) were determined according to AOAC (2005). The total dietary fiber (TDF) was determined using FIBRA PLUS FES 02 E (M/s Pelican Equipments, Tamil Nadu, India). The caloric value of the chocolates was calculated using the Atwater conversion factors, where by the major biochemical constituents were converted into calorific values using standard calorific equivalents (5.65, 9.45, and 4.20 for proteins, lipids, and carbohydrates, respectively) [17] [18].

Total carbohydrate ( g / 100 g ) = 100 − ( moisture + protein + lipid + ash )

Calorific value ( kcal / g ) = ( 4.20 × P + 9.45 × L + 4.20 × C ) / 100

where P = protein content, L = crude lipid content, C = carbohydrate content

The reducing and non-reducing sugars were determined by Lane and Eynon method [19].

The modified procedure of Giri et al. [20] was used to estimate glycemic index (GI) of chocolates. The percent available starch and starch hydrolysis index 90 (HI 90) was calculated as per the formula given by Goni et al. [21] and Holm et al. [22].

The chocolate samples (250 mg) were homogenized with 5 ml distilled water (DW) and then made up to 10 ml with DW. It was then treated with 200 µl α-amylase (heat stable) enzyme (M/s Sigma, Cochin, India) for 15 min in a boiling water bath with mixing after every 5 min. The suspension was cooled and diluted to a final volume of 25 ml with DW. The clear solution from top (1 ml) was incubated with 2 ml of sodium phosphate buffer (pH 4.75) and 1 ml of amyloglucosidase solution (5 mg amyloglucosidase dissolved in 25 ml of same buffer) (M/s Sigma, Cochin, India) at 60˚C for 30 min with gentle mixing every 5 min. It was then diluted to 10 ml with DW. Glucose was estimated by using GOD-POD kit and the percentage available starch was estimated using the equation:

Percent available starch = (μg glucose × 25 × 0.9)/sample wt. (mg, dry weight basis)

For the estimation of hydrolysis index (HI90), chocolate (1 g) was homogenized and suspended in 50 ml of 0.1 M phosphate buffer (pH 6.9); and then incubated with 5 ml of same buffer containing 500 mg pancreatin (M/s Sigma, Cochin, India) was added and the mixture was incubated at 37˚C with gentle mixing for 90 min. Clear sample solution (0.1 ml) was drawn and analyzed for maltose using the DNSA method [21]. Results were expressed as mg maltose released after 90 min of hydrolysis of 1 g sample (dry weight). Using these values and percent available starch, hydrolysis index was calculated using the formula,

HI90 = 100 × 0.9 × mg maltose / mg starch

Using the values of percent available starch and HI 90, GI was calculated using the formula as given by Goni et al. [21].

GI = 39.21 + 0.803 × HI 90

The products were packed in 50 gauge metallised polyester laminated with 220 gauge Polyethylene packets to analyze the shelf life of the product. The changes that occurred in the product during the period of storage (0, 30, 60, 90 and 120 days) were analyzed based on melting characteristics, Texture profile analysis, color analysis and sensory evaluation.

All results are expressed given as means and standard deviation. Analysis of variance was conducted using one-way ANOVA and Tukey test by pair-wise analysis with significance defined at p < 0.05.

The physio-chemical composition of chocolates is given in Table 2. The results showed that moisture content ranged between 2.7 and 4.1 g%, with control (sugar) showing the lowest moisture content, which was in accordance with earlier reports [32]. The chemical nature and the initial moisture content of the alternative sweeteners and other ingredients influence the ultimate moisture content of the chocolate; thus, affecting its sensory properties and flow characteristics of the chocolate [32]. The mineral contents ranged between 2.6 and 4.4 g%, with lowest being in jackfruit control. The fat content in the chocolates ranged between55.0 and 58.9 g%. The carbohydrate content ranged between 22.5 and 34.5 g%, with highest being in jackfruit control.

Stevia-C: Stevia chocolate, Sucralose-C: Sucralose chocolate, JF-Jackfruit.

The glycemic index and glycemic load for the chocolates are shown in Table 3. The earlier reports indicate that foods with Glycemic Index (GI) less than 55 are classified as low GI, 56 - 69 as medium GI and greater than 70 as high GI [33]. The results clearly show that the chocolates fall under the category of low glycemic Index foods. Low GI diet has therapeutic value in type 1 and type 2 diabetic patients [34]. According to Bjorck and Asp [35] GI has an effect on the nutritional quality of food. Low GI foods are recommended not only for diabetic individuals but also for normal population. The glycemic index for a commercial milk chocolate shows that it comes under high GI and thus is not suitable for diabetic patients.