Utilization of mango seed oil as a cocoa butter replacer for the development of innovative chocolate

ABSTRACT

Chocolate is the most popular food type and flavor in this world. The key ingredient in many chocolate products is cocoa butter due to its unique fatty acid profile. Due to the expensive nature of cocoa butter, it has stimulated extensive research for fats that are cheaper and more easily available and can be used as cocoa butter substitutes. The mango (Mangifera indica) is known as the king of fruit due to its rich nutritive profile. The major parts of mango fruit are peel, pulp, and seed. Mango seed is usually discarded as waste which is a source of edible oil (7–12%). The current study was designed to produce chocolate with mango seed oil as a cocoa butter replacer. Mango seed oil was extracted using the soxhlet apparatus and its physiochemical properties were evaluated. Extracted oil was used in chocolate preparation with different proportions (0%, 30%, 70%, and 100%). Furthermore, chocolate was subjected to explore the effect of storage (21days) on product quality and sensory with 7days interval. Current results show that mango seed oil has a valuable fat profile containing palmitic acid (C16:0) 26%, stearic acid (C18:0) 36%, and oleic acid (C18:2) 33%. Moreover, innovative chocolate showed higher antioxidant activity as compared to control in different storage intervals. In addition, chocolate prepared with different proportions of mango seed oil showed higher sensory scores as compared to the control sample. The findings suggest that mango seed oil can replace cocoa butter in chocolate and reduce/manage mango seed waste to improve its antioxidant activity and nutritional value.

KEYWORDS:

• Mango seed oil
• cocoa butter
• chocolate
• compositional analysis
• antioxidant activity
• sensory

Introduction

Mango is the dominant species of the genus Mangifera and it belongs to the family Anacardiaceae. Mango fruits are widely produced in tropical regions and are considered important among the major fruits. Several types of foodstuffs can be made by utilizing the mango. Pickles, nectar, juice, jam, and leather along with slices are prepared by utilizing 20% of mango globally.^[1] The mango, “king of fruits” is the second most popular fruit in Pakistan. Mango is a well-known tropical and subtropical fresh fruit that is high in vitamins A, B, and C with high water, protein, sugar, iron, fat, and high fiber contents.^[2] Each variety has its shape, color, size, aroma, and taste.^[3] Pakistan is the third-largest exporter of mangoes in the world and ranks fifth among those that grow them.^[4] In Pakistan, around 85,000 tons of mango fruit is exported annually. The cultivating area of the mango plant is about 4.25 million acres, and yields are about 1.77 million tons yearly.^[5]

Mango has three main parts: pulp, peel, and seed. The pulp of mango fruit is consumed fresh or processed in a variety of products such as dried mango, mango juice, mango leather, and mango chutney. During mango processing, 40–60% of the fruits are discarded as waste products. The mango peel constitutes 12–15% whereas seed constitutes 15–20% of the total waste product. The mango fruits contain 10–25% of seed. In the seed, the kernel constitutes 45–75% and 20% of the total mango fruit. However, about 1 million tons of fresh mango seeds are treated as wasted. Depending on the variety, the mango seed kernel contains 6% protein, 77% carbohydrate, 11% fat 2% crude fiber, and 2% ascorbic acid.^[6] Mango seed has a valuable nutritional profile as edible oil. The oil contents of mango seed kernels are 7–15%.^[7]

Mango kernel oil is one of the few tropical fruits oil considered as a cocoa butter alternative. Due to their unique physical and chemical characteristics, in addition to their fatty acid profile which are similar to cocoa butter and shea butter.^[8] The major fatty acid is stearic (24–57%), oleic (34–56%), and palmitic acid (3–18%). Cocoa butter is an essential ingredient for the manufacturing of chocolate and other confectionery products. It is a fatty phase of chocolate and is responsible for the texture and melting behavior of chocolate. Cocoa butter is a basic fat obtained from pressing ground, roasted, and decorticated cocoa beans. The fruit of the cocoa plant is known as a cocoa bean.^[9] The fatty acid profile of cocoa butter shows that it contains 26% palmitic acid, 36% stearic acid, and 33% oleic acid.^[10] The fatty acid profile of mango seed oil shows that mango seed oil can be used as an alternative to cocoa butter in food products like chocolate. The cocoa butter alternative is not chemically similar to cocoa butter, but they are well-suited with cocoa butter.^[11] Mango seed oil/fat has a number of essential fatty acids that are not present in conventional cocoa butter replacers. One of the best advantages of mango seed fat/oil is that it has no trans-fats unlike conventional shortenings and cocoa butter replacers, and these trans-fats are linked to numerous health complications.^[8] Mango seed being the food industrial waste can be effectively utilized to obtain cocoa butter replacers, which will be economical, healthier, and environmentally friendly.

The current study was conducted to produce chocolate with mango seed oil as a cocoa butter replacer. In this study, we explored the physicochemical properties of extracted mango seed oil (MSO). Then, MSO was used to develop chocolate samples with different proportions of MSO (0%, 30%, 70%, and 100%). In addition, the physicochemical, quality, and sensorial characteristics of prepared chocolate were evaluated. Chocolate was subjected to assess the effect of storage on product quality and sensorial properties.

Material and methods

Procurement of raw material

The raw material was purchased from the local market Multan. Mango seeds were purchased from the mango processing units located at the industrial state in Multan. All the mango seeds were from fully ripe mangoes that were being processed at the processing units. All of the chemicals, and reagents were analytical grade provided by G.M. Scientific Supplies, Multan. Glasswares were made available at the Department of Food Science and Technology and Central Lab System of MNS-UAM.

Extraction of oil from mango seed kernel

Washing and drying of Mango seed kernel

The seeds of mango were washed with clean running water. After washing the mango seed kernels, these were dried using conventional solar drying. About 15–18 kg of mango seeds were dried in sunlight for 4–5days and the final moisture percentage was 9.5 ± 1% on average.

Preparation and storage of Mango seed kernel powder

When the mango seeds were completely dried, these seeds were opened by a specially designed cutter to attain the seed kernels. After that, the drying kernels were manually cut into 5–6mm pieces. These pieces were converted into fine uniform powder by utilizing a grinding mill to facilitate the extraction of oil.

Dried mango seed kernels were ground using a universal grinding mill (Chenwei China), rpm: 2200 and sieve no. 40 (500µm). In the end, the collected powder was stored at 4°C within sealed packages until further use.

Extraction of oil

According to the procedures described by Association of Official Analytical Chemists, the resulting powder was utilized for the extraction of oil by two different processes. Solvent extraction and mechanical compression techniques require light heating.^[12] Oil from the mango seed kernel was extracted by both methods. For solvent extraction, firstly, the kernel separated from the mango seed was dried and converted into powder form after this, the powder of the mango kernel was filled in paper thimbles. Each thimble contains 5g of mango kernel powder or flour. A solvent such as hexane was used in the soxhlet in the amount of 450mL. The round bottom flask was filled with hexane and the temperature of the soxhlet machine at 75°C and allowed the thimbles to stay in the apparatus for about the time of 5 h throughout the removal. Afterward, fat was retrieved from the hexane using a rotary evaporator and to ensure the complete removal of hexane, the samples were placed in a hot air oven and 65°C for 60–90 min.

Analytical study of mango kernel oil

Fatty acid profile

Mango seed oil’s fatty acid composition was assessed using GC-MS (996.06) described by Bigelow et al.^[13] Methanol was used to form fatty acid methyl esters. A sample was introduced into the GC system along with an MS detector. As a carrier gas, helium was used, and the temperature range was set at 70°C to 280°C. The injection and detector temperatures were adjusted at 240°C and 250°C, respectively. Peaks on the chromatogram were identified using retention information from tested standard samples. Finally, total fatty acid contents were evaluated and calculated as percentages (%).^[14]

Free fatty acids (FFA)

The fatty acid percentage was indicated by titrating in neutralized ethanol (95%) versus NaOH solution.^[12] For the determination of free fatty acid in mango kernel fat, a sample of around 10g was brought into a cleaned, wiped conical flask together with 25mL neutralized ethanol (95%). After that, mix it correctly such that the sample is liquified in ethanol solution. When the sample was completely dissolved, add 2–3 drops of phenolphthalein as an indicator. After that, the remaining contents were agitated with 0.1 N sodium hydroxide (NaOH) solution and shaken continuously until a light pink color remained for a minimum of 35 s. The proportion of free fatty acid percentage was evaluated:

Free fatty acids% (as oleic) = $\frac{Alkaliused\left(mL\right)\times N\times 28.2}{wt.ofsample\left(g\right)}$

where is N normality of alkali.

Peroxide value

To determine the peroxide value in terms of iodine produced from the hydrogen peroxide and iodine ion reactions according to the procedures as followed in (AOAC, Method No. Cd 8b-90).^[12] To determine the peroxide value, a sample of 5g was occupied in an Erlenmeyer flask of 250mL. Afterward, glacial acetic acid which is a Chloroform solution mixture in the amount of 30mL was added. Intermingle the flask for almost 60 s to completely dissolve the oil in a solvent mixture. After that, add freshly prepared potassium iodide (KI) solution (0.5mL) into the beaker. The solution was titrated versus 0.1 N standard sodium thiosulfate (Na₂S₂O₃) solution with continuous shivering, until the yellow color completely disappeared. After that, 0.5mL solution of starch was used as an indicator and pursued the titration with active stirring unless the dark blue color vanished. Empty readings without sample runs were measured individually. The value of peroxide is measured by utilizing the formula:

Peroxide value=$\frac{\left(\mathrm{B}-\mathrm{S}\right)\times \mathrm{N}\times 1000}{wt.ofoiltaken\left(g\right)}$ $\times$ 100

where S is volume of Na₂S₂O₃ utilized for sample, B volume of Na₂S₂O₃ utilized for blank, and

N is normality of Na₂S₂O₃.

Iodine value

Iodine values are used to measure the amount of unsaturation and in chemistry, it is the mass of iodine in grams engrossed by hundred grams of oils and fats and described by AOAC^[12]; Method No. Cd 1–25. The fat extracted from the kernel of the mango seed was broken down when added in 5mL carbon tetrachloride (CCl₄) was in a flask named stoppered, along with the addition of 25mL of Wijs solution in it. Subsequently, the flask was put in a dark place for an hour distillation with 15% potassium iodide solution (20mL) along with 25mL of distilled water. The solution present in the flask was then titrated versus sodium thiosulfate (Na₂S₂O₃) mixture along with the addition of a starch indicator solution which was freshly prepared. Reading without running the sample was also noted. The number of milliliters of 0.1 N sodium thiosulfate needed to be subtracted from the mL utilized by the sample provided sodium thiosulfate equivalent of iodine value of the fat as stated:

Iodine value (g/100g)=$\frac{\left(\mathrm{B}-\mathrm{S}\right)\hspace{0.17em}\times \hspace{0.17em}12.69\hspace{0.17em}\times \mathrm{N}}{\mathrm{w}\mathrm{t}.\mathrm{o}\mathrm{f}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\left(\mathrm{g}\right)}$

where N is normality of Na₂S₂O₃, S is volume of Na₂S₂O₃ utilized for sample, B is volume of Na₂S₂O₃ utilized for empty sample.

Specific gravity

Specific gravity was measured at ambient temperature by using a pycnometer consisting of capillary bored closure by the procedures followed in AOAC.^[12] Before measuring the specific gravity of the sample, the pycnometer was calibrated to be filled with pure water and weighing the net mass of water. After that, the specific gravity of the sample at ambient temperature was deliberated as:

Specific gravity=$\frac{\mathrm{W}\mathrm{t}.\mathrm{o}\mathrm{f}\mathrm{o}\mathrm{i}\mathrm{l}\left(\mathrm{g}\right)}{\mathrm{w}\mathrm{t}.\mathrm{o}\mathrm{f}\mathrm{w}\mathrm{a}\mathrm{t}\mathrm{e}\mathrm{r}\left(\mathrm{g}\right)}$=$\frac{\left(\mathrm{C}-\mathrm{A}\right)}{\left(B-A\right)}$

here A is the weight of an empty bottle having specific gravity, B is the weight of a bottle filled with water with specific gravity, and C is the weight of a bottle filled with fats with specific gravity.

Saponification value

The saponification value is the number of milligrams of potassium hydroxide needed to fully saponify 1 g of sample containing oils or fats. Saponification of mango kernel fat was calculated as reported in the procedure followed by AOAC^[12]; Method No. Cd 3a-94. A sample of 2 g was brought in a conical flask and dissolved in 25mL of 0.5 N alcoholic potassium hydroxide solution. After that, the reaction mixture was reflowed with the help of a water condenser in a water bath for half an hour. The resulting solution was getting cold and titrated with 0.5 N HCl solution along with the addition of 1mL of phenolphthalein indicator. Empty reading was measured separately.

Saponification value=$\frac{\left(\mathrm{B}-\mathrm{S}\right)\hspace{0.17em}\times \hspace{0.17em}0.02805\hspace{0.17em}\times \hspace{0.17em}1000}{\mathrm{w}\mathrm{e}\mathrm{i}\mathrm{g}\mathrm{h}\mathrm{t}\mathrm{o}\mathrm{f}\mathrm{s}\mathrm{a}\mathrm{m}\mathrm{p}\mathrm{l}\mathrm{e}\left(\mathrm{g}\right)}$

where S is volume of KOH utilized for sample and B is volume of KOH utilized for empty.

Product development

Using laboratory-scale tools and development procedures, control chocolate and cocoa butter replaced chocolate (CBRC) with MKO were created. Using a mixer, pre-mixed cocoa powder, sugar, milk powder, and cocoa butter. An additional stage of conducting the pre-mix for at least 1 hour at 50°C was performed after it had been refined with a sieve. Once the temperature was low enough for the slurry to be used to fill small molds, manual tempering was done (food-grade plastic material). There were 12g or so for every CBRC piece. Chocolate that had been molded was stored in propylene sheets.^[7] Treatment plan for chocolate preparation was presented in Table 1.

Table 1. Treatment plan for chocolate.

Treatment No.	Cocoa butter%	Mango seed oil%
T0	100	0
T1	70	30
T2	30	70
T3	0	100

Proximate analysis of product

Moisture contents

Moisture content is the most important constituent of any product. The dry matter of food items is commonly dependent on the moisture percentage of the product which directly affects the processor and consumer’s economic status. Moisture content is commonly influencing the properties of food items. The prepared chocolate was evaluated for moisture percentage according to the method described by the Association of Official Analytical Chemists (AOAC)^[12] method No. 44-15A. The moisture contents were evaluated by drying oven (SLN-53- STD, POL-Eko-Apparatus). The sample was placed in a dried, pre-weighed china dish weighing about 2g, and the china dish was subsequently dried in an oven for 6–12hours at 105 ± 5°C.

Ash contents

The ash of prepared chocolate was evaluated according to the protocols mentioned by the Association of Official Analytical Chemists (AOAC)^[12] method No. 08–01. The contents were determined using a muffle furnace (SNOL 39/1100, Utena, Lithuania). Before depositing the sample in the muffle furnace, it was burned on a spirit lamp using around 2g of the sample in a clean, precisely weighed crucible. The muffle furnace had a temperature of 600°C. After the completion of the procedure, the muffle furnace, and left to decrease the temperature of the muffle furnace and then the crucible was placed in the desiccator for cooling.

Crude fat

The Association of Official Analytical Chemists (AOAC)^[12]; Method No. 30–25) method No. 30–25 was used to test the crude fat level of the manufactured chocolate. For the crude fat determination procedure, the sample was measured in a thimble containing roughly 30g, and n-hexane was employed as the solvent. The removal of the fats from the sample in the Soxhlet apparatus was made by adjusting the speed of 4–5 drops per second of hexane for almost 2–3hours. When 6–7 siphons back or washings were completed, the thimble was removed from the apparatus. After the removal of the thimble, it was dried in an oven for 1 h at 105°C and then weighed. After that, petri plates were cooled down in a desiccator. Weighing the plates until no decrease in weight was achieved. The below formula was acquired to measure the crude fat contents.

Crude fiber

The prepared chocolate with mango seed oil was used to evaluate the crude fiber contents according to the AOAC method No. 32–10.^[12] For this analysis, the sample (about 15g) was taken in a 250mL glass beaker. 2.5% H₂SO₄ about 200mL. The sample was heated at 90°C for 2 h for acid digestion. After the digestion, the sample was filtered with filter paper and washed the residue gradually with hot water until it was free from acid. Washed residue was poured into the 25O mL glass beaker for digestion with an alkali solution. The same process used for acid digestion was followed when an alkali solution was added to the beaker. Following the end of both digestion processes, the sample was transported to a crucible, which was then dried. The crucible was placed in a muffle furnace.

Crude protein

The protein contents of prepared chocolate were evaluated through the Kjeldahl apparatus according to the respected method recommended by the Association of Official Analytical Chemists (AOAC)^[12] method No 46–10. The Kjeldahl apparatus is composed of three steps including digestion, distillation, and titration. The 1g sample of prepared chocolate was taken in the digestion tube and added 15mL H₂SO₄ and 1g of digestion mixture were added to it, and the tube was placed in the digestion assembly for further process. After digestion, the sample solution was diluted for distillation. After distillation, ammonia gas containing boric acid was used for titration of the sample by 0.1 N HCl.

Quality analysis of product

Total phenolic content

The total phenolic contents of chocolate were determined by using the Folin-Ciocalteu reagent at prescribed intervals (0, 7, 14, and 21days) as explained by Chen et al.^[15] with slight changes. One hundred microliter of diluted sample was carefully added to measured 2.50mL (1:10) diluted Folin-Ciocalteu standard reagent, and 02mL of saturated sodium carbonate solution (75g per liter) was carefully added after 4 min. After the incubation at room temperature for calculated 120 min, the absorbance of this mixture was recorded and measured at set 760nm by using the respective standard solvent as blanks. Gallic acid was utilized as a standard to obtain a standard calibration curve. The results are precisely expressed as milligrams of Gallic acid equivalents, i.e. mg GAE per 100-g wet weight of the sample.

DPPH assay

By using the technique suggested by Kassim et al.,^[16] the calculation of free radical scavenging activity was examined using a spectrophotometer. Thoroughly mix 1.0ml solution of MeOH (methanol) with 2, 2-diphenyl-1-picryl-hydrazyl (DDPH) at a concentration of 170 uL, with 1.0ml of oil sample. All contents were continually mixed and then permitted to stand in a cool and dark place for 30 min at ambient temperature. After incubation, the reaction mixture’s color was altered, and the outcome was evaluated at 517nm. DPPH values were expressed as percent inhibition. As benchmarks, BHT, α-tocopherol, and ascorbic acid were employed.

pH determination

pH of the chocolate sample was determined by using the pH meter according to the method explained in AOAC.^[12] The sample of the product was taken, and the electrode of the pH meter was standardized with a buffer solution to set the pH at 7. The node then dipped into the sample and note the reading.

Sensory evaluation of products

The prepared chocolate was evaluated by a panel of eight judges at MNS-University of Agriculture, Multan, Department of Food Science and Technology. The chocolate was assessed by the panelist according to the hedonic scale where 9 represented extreme dislike and 1 for extremely like. Sensory evaluation of chocolate was conducted to evaluate the quality characteristics including texture, appearance, taste, flavor, and overall acceptability by following the methodology described by Lawless and Heymann.^[17]

Results and discussion

In the current research, mango seed oil was extracted through solvent extraction and mechanical extraction methods. The yield results showed that the solvent extraction method showed a higher yield as compared to the mechanical extraction method. The Desi variety showed a higher yield of 12.49 ± 0.05 followed by other varieties. The results regarding the yield of oil from different mango seeds are shown in Table 2 and Figure 1.

Figure 1. Comparison between Solvent & Mechanical.

Figure 2. Effect of treatment on hardness.

Figure 3. Effect of treatment on breaking strength.

Figure 4. Effect of treatment on cutting strength.

Table 2. Oil yield (%) from mango seed of different varieties.

Varieties	Solvent Extraction (%)	Mechanical Extraction (%)
Chaunsa	10.19 ± 0.02^a	8.63 ± 0.08^b
Fajri	9.13 ± 0.3^a	8.64 ± 0.04^b
Anwar Ratool	9.42 ± 0.05^a	8.67 ± 0.02^b
Langra	9.24 ± 0.08^a	8.62 ± 0.07^b
Desi	12.49 ± 0.05^a	9.25 ± 0.05^b
dusehri	9.47 ± 0.06^a	8.82 ± 0.08^b

Physicochemical analysis of mango seed oil

Fatty acids profiling of mango seed oil

Fatty acid composition of mango seed kernel oil was analyzed using GC-MS, which provides a comparison of its unsaturated and saturated fatty acid composition. The fatty acid composition of mango seed kernels was determined using the procedures outlined in Bigelow et al.^[13] The compositional study showed that 53.30% of the fatty acids were saturated, 44.74% were unsaturated, 43.54% were oleic acid, 36.18% were stearic acids, 7.46% were linolenic acids, 2.80% were linoleic acids, and 6.23% were palmitic acids. According to published literature^[14]oleic acid and stearic acid, with values of 43.54% and 36.18%, were found to be the major fractions in mango seed kernel oil. The fatty acids with the lowest values are the palmitic, linoleic, and linolenic acids, which had respective values of 10.06%, 6.00%, and 2.48%. These results are presented in Table 3.

Table 3. Fatty acid profile of mango seed oil.

Fatty acid	Value (%)
Saturated fatty acid	53.30 ± 0.02
Unsaturated fatty acid	44.74 ± 0.03
Oleic acid	43.54 ± 0.05
Stearic acid	36.18 ± 0.07
Linolenic acid	7.46 ± 0.01
Linoleic acid	2.80 ± 0.07
Palmitic acid	6.23 ± 0.04

Free fatty acids

The FFA’s content measured the degree of extent at which the glyceride compound in the oil has deteriorated due to lipase activity. The free fatty acid is a guide to the purity as well as freshness of oil. When the free fatty acid value is higher in oil and fats it increases the darkness in color. Increased free fatty acid value also causes the decomposition of fats and oils along with rancidity. For this reason, antioxidants are used because they inhibit the chain reaction as well as oxidative stability, and quality. The high free fatty acid value may also decrease the storage life of fat and oils. Mango seed fat was analyzed, and the findings are stated in Table 3 which demonstrated that the mean value of 2.96 ± 0.01% FFA. According to the literature, free fatty acids found in edible fats and oils range between 2.92% and 3.18%.^[18] This also implies that mango seed oil/fat has natural antioxidants that slow down the production of FFAs in it.

Peroxide value

Peroxide value is a common test of lipid oxidation. It is obvious from the outcomes that the mean value for peroxide varies between 2.60 ± 0.08 meq/kg in the oil of mango kernel (Table 4). Lower amounts of peroxide present in mango demonstrated that fat of mango kernel has a greater quantity of saturated fats and can also be used in the production of edible food products. These results are identical to the study of Jahurul et al.^[8]

Table 4. Mean values of physicochemical properties of Mango Kernel Oil.

Physicochemical Test	Mean ± SD
Free Fatty acids (%)	2.96 ± 0.01
Peroxide Value (meq/kg)	2.60 ± 0.08
Iodine Value (g/100g)	40.28 ± 0.6
Specific Gravity	0.89 ± 0.02
Saponification Value (mg KOH/g)	185.45 ± 0.8
Melting Point (°C)	37.67 ± 0.5

Iodine value

The process of oxidation decreases or lowers the unsaturation rates of fats. If the resulting products consist of a higher unsaturation level, then it has increased vulnerable chances over rancidity and also shows a better iodine value. According to current research, fats-and-oils have a value of iodine varying from 40.28 to 42.69g/100g oil (Table 4). Oil from the seed kernel of mango was examined, and its findings are stated in Table 4. Mango fruit seed kernel fat has a value of iodine ranged in between (32.0 to 60.7) g/100g. The mean value of mango seed kernel fat is 40.28 ± 0.6g/100g. If the iodine value is decreased, then it expresses that the double carbon bond present in the sample of fat is little oxidized as well and if the iodine value is high, then there will be a high degree of unsaturation and oxidation in fats and oils. Mango seed kernel fat was examined, and the results are mentioned in Table 5. These results are in accordance with the results reported in the previous literature by Jahurul et al.^[8]

Specific gravity

Fat of mango kernel was subjected to demonstrate the value of specific gravity and result mentioned in Table 4. Demonstrated that the mean value of the mango is 0.89±0.02. The specific gravity of the oil found in the mango seed kernel was approximately identical to the findings referred to by Abdallah et al.^[18]

Saponification value

Saponification value has little use for the recognition of particular fats and oils with their related edible items. The utilization of saponification values is less for the reason that there is too much duplication occurring in this method. One of the important roles of saponification values is the assessment of the average molecular weight of fatty acids that exist in fats and oils. Glycerides comprised fatty acids having short-chain lengths with higher values of saponification as compared to glycerides with long-chain fatty acids. For the estimation of pure fatty acid that exists in fats and oils, the value of saponification equals the acid value. Whereas the values of the esters provide the variation between the saponification value and the acid value. The fat of mango was studied, and their outcomes are shown in Table 4. The mean for the mango is (185.45±0.8) mg KOH/g. Identical findings have also been demonstrated by Jahurul et al.^[8]

Proximate analysis of chocolate (product)

The proximate analysis of the chocolate included moisture, ash, fat, fiber, and protein indicating that the lowest value noted in the treatment T₀ was 5.23 ± 0.03 followed by the treatment T₁ 5.48 ± 0.02, T₂ 5.77 ± 0.07 and T₃ 6.27 ± 0.01, respectively, whereas the maximum value for the moisture in chocolate was seen in treatment T₃ 6.27 ± 0.01. The highest value for the ash content was measured in the treatment T₁ (3.20 ± 0.04) pursued by the treatment T₂ (3.10 ± 0.02) and T₃ (3.00 ± 0.1) and T₀ (2.96 ± 0.06). The lowest amount of the ash was measured in the treatment T₀ (0.82 ± 0.02). The highest value for the crude fat content was present in the treatment T₀ (33.09 ± 1.00) Followed by the treatments T₁ (31.27 ± 1.73), T₂ (30.42 ± 0.96) and T₃ (29.63 ± 0.99). The lowest mean value for the crude fat content was found in the treatment T₃ (29.63 ± 0.99). The outcomes from the study indicated that the highest fiber content was present in treatment T₀ (2.71 ± 0.16) and the lowest value was present in treatment T₂ (2.19 ± 0.10). By raising the value of mango kernel oil, the crude fiber content in chocolate starts increasing as the mean values for the remaining treatments are T₁ (2.34 ± 0.10), T₂ (2.19 ± 0.03), and T₃(2.61 ± 0.06). The data demonstrated that the highest protein amount was present in the treatment T₃ (9.30 ± 0.16). The least value for the protein was present in T₁ (7.45 ± 0.23). Other mean values are T0 (8.45 ± 0.23) and T₂ (8.32 ± 0.45). The proximate composition of the chocolate is shown in Table 5.

Table 5. Proximate composition of the chocolate.

Treatment	T0	T1	T2	T3
Moisture%	5.23 ± 0.03^c	5.48 ± 0.02^c	5.77 ± 0.07^b	6.27 ± 0.01^a
Ash%	2.96 ± 0.06^b	3.20 ± 0.04^a	3.10 ± 0.02^a	3.00 ± 0.10^b
Fat%	33.09 ± 1.00^a	31.27 ± 1.73^b	30.42 ± 0.96^b	29.63 ± 0.99^c
Fiber%	2.72 ± 0.16^a	2.34 ± 0.10^b	2.19 ± 0.03^c	2.61 ± 0.06^a
Protein%	8.63 ± 0.23^b	7.63 ± 0.23^c	7.96 ± 0.45^c	9.32 ± 0.16^a

Quality analysis of chocolate

Total Phenolic Content

Statistical results regarding Total Phenolic Content (TPC) indicated that the effects of treatment, storage, and combined effects of storage of chocolate on total phenolic contents were observed to be highly significant (Table 6). The effect of treatments (different percentages of extracts) on the TPC of chocolate indicated that the highest TPC was observed with the mean value of 29.48 ± 0.8mg GAE/100g and the lowest 28.26 ± 0.05mg GAE/100g. The interactive effect of treatments and storage times on the TPC of chocolate indicated that significantly the highest value was observed in the T₃ at storage times of 0, 7, 14, and 21days with the mean value of 29.79 ± 0.03mg GAE/100g, 29.54 ± 0.5mg GAE/100g, 29.41 ± 0.04mg GAE/100g and 29.19 ± 0.5mg GAE/100g, respectively. The results of this study show that the TPC of chocolate decreases with storage time. This similar trend was also reported by Jacimovic et al.,^[19] ranging from 10.55 to 39.82mg GAE/100g. Phenolic compounds are sensitive to higher temperatures. Higher phenolic contents are due to the fact the temperatures during the process did not exceed 70°C. With longer storage periods quantity of phenolic compounds decreases owing to oxidative agents.

Table 6. Quality analysis of chocolate.

		Storage
Parameter	Treatment	0 days	7^th days	14^th days	21^st days	Mean
TPC mg GAE/100g	T0	28.48 ± 0.02^i	28.32 ± 0.02^jf	28.19 ± 0.03^j	28.05 ± 0.03^e	28.26+0.03^A
	T1	28.73 ± 0.03^h	28.62 ± 0.05^eh	28.48 ± 0.04^a	28.29 ± 0.04^a	28.53+0.04^B
	T2	29.34 ± 0.02^ij	29.34 ± 0.04^fg	29.13 ± 0.03^e	29.03 ± 0.06^f	29.19+0.05^A
	T3	29.79 ± 0.03^kl	29.54 ± 0.02^ae	29.41 ± 0.03^c	29.19 ± 0.05^i	29.48+0.03^D
DPPH %	T0	47.12 ± 0.05^g	45.10 ± 0.08^ef	43.09 ± 0.05^r	41.47 ± 0.07^j	44.19+0.06^F
	T1	47.57 ± 0.03^d	45.31 ± 0.04^df	43.80 ± 0.05^i	41.29 ± 0.05^f	44.49+0.04^H
	T2	48.29 ± 0.06^h	46.27 ± 0.02^w	44.47 ± 0.10^a	42.66 ± 0.03^d	45.42+0.05^A
	T3	49.75 ± 0.04^f	47.62 ± 0.06^a	45.83 ± 0.03^d	42.21 ± 0.11^f	46.35+0.06^C
pH	T0	6.19 ± 0.04^g	6.29 ± 0.01^d	6.33 ± 0.01e^r	6.39 ± 0.01^c	6.30+0.02^A
	T1	6.30 ± 0.02^r	6.35 ± 0.02^ae	6.45 ± 0.01^ij	6.47 ± 0.02^b	6.39+0.03^F
	T2	6.34 ± 0.03^d	6.49 ± 0.02^h	6.58 ± 0.01^a	6.60 ± 0.02^e	6.50+0.02^B
	T3	6.20 ± 0.02^a	6.40 ± 0.02^j	6.42 ± 0.01^b	6.45 ± 0.03^a	6.37+0.04^A

Table 7. Sensory evaluation of the product.

Parameter	Treatment	Storage				Mean
		0 days	7^th days	14^th days	21^st days
Appearance	T0	6.38 ± 0.74a	6.50 ± 0.53ed	6.50 ± 0.76e	6.63 ± 0.52e	6.50 ± 0.64C
	T1	6.50 ± 0.76se	6.50 ± 0.53a	6.50 ± 0.53d	6.63 ± 0.52r	6.53 ± 0.59B
	T2	6.63 ± 0.52w	6.63 ± 0.52b	6.75 ± 0.46r	6.75 ± 0.46i	6.69 ± 0.49A
	T3	6.75 ± 0.46i	6.75 ± 0.71n	6.75 ± 0.71d	6.88 ± 0.64a	6.78 ± 0.63D
Taste	T0	6.50 ± 0.93ij	6.63 ± 0.74f	6.88 ± 0.64h	7.00 ± 0.53b	6.75 ± 0.71A
	T1	6.75 ± 0.71a	7.00 ± 0.76a	7.13 ± 0.83j	7.25 ± 0.71d	7.03 ± 0.75F
	T2	7.00 ± 0.53b	7.00 ± 0.76ea	7.50 ± 0.53e	7.50 ± 0.53e	7.25 ± 0.59H
	T3	7.25 ± 0.71d	7.25 ± 0.46s	7.25 ± 0.71r	7.50 ± 0.53c	7.31 ± 0.60C
Flavor	T0	6.00 ± 1.51c	6.50 ± 0.53r	6.88 ± 0.64f	6.88 ± 0.64d	6.56 ± 0.83D
	T1	6.50 ± 0.53ed	6.63 ± 0.74d	8.00 ± 0.64i	7.13 ± 0.83h	7.06 ± 0.69D
	T2	6.50 ± 0.53m	6.63 ± 0.74rf	7.00 ± 0.00h	7.13 ± 0.64i	6.81 ± 0.48B
	T3	6.75 ± 0.71er	6.88 ± 0.64t	7.75 ± 0.46j	7.25 ± 0.71j	7.03 ± 0.63C
Texture	T0	6.63 ± 0.52d	6.75 ± 0.46y	6.75 ± 0.46a	6.75 ± 0.71f	6.72 ± 0.54D
	T1	6.75 ± 0.71f	6.75 ± 0.71i	6.75 ± 0.71d	6.88 ± 0.35a	6.78 ± 0.62C
	T2	6.75 ± 0.71a	6.88 ± 0.83gi	6.88 ± 0.64f	7.00 ± 0.00c	6.88 ± 0.55B
	T3	6.88 ± 0.64q	6.88 ± 0.64f	6.88 ± 0.83e	7.13 ± 0.64b	6.94 ± 0.69D
Overall acceptability	T0	6.38 ± 0.92ef	6.88 ± 0.64e	7.13 ± 0.64e	7.25 ± 0.46s	6.91 ± 0.67C
	T1	6.88 ± 0.64ei	7.00 ± 0.53it	7.13 ± 0.64h	7.38 ± 0.52i	7.09 ± 0.58A
	T2	7.00 ± 0.76f	7.00 ± 0.76h	7.38 ± 0.52b	7.63 ± 0.52v	7.25 ± 0.64B
	T3	7.13 ± 0.64g	7.25 ± 0.71j	7.38 ± 0.52n	7.75 ± 0.46d	7.38 ± 0.58A

DPPH Assay

The percentage of DPPH in all samples was determined at the intervals of 0, 7, 14, and 21days. The influence of both storage and treatment on the DPPH content of chocolate is shown in Table 5. Table 5 shows that the maximum DPPH content was observed at 0days in T₃ (49.75 ± 0.04%), whereas a minimum DPPH was observed in T₁ (41.29 ± 0.05%) at 21days of storage. Means of DPPH in the controlled treatment T₀ were detected as 47.12 ± 0.05%, 45.10 ± 0.08%, 43.09 ± 0.05%, and 41.47 ± 0.0.07% at 0, 7, 14, and 21days of storage, respectively. The lowest percentage of DPPH was observed at 21days, whereas the highest percentage was observed at 0days. In T₁, mean values of DPPH content were observed as 47.57 ± 0.03%, 45.31 ± 0.04%, 43.80 ± 0.05%, and 41.29 ± 0.05% at 0, 7, 14, and 21days of storage, respectively. The highest DPPH was shown at 0days. whereas the lowest value was seen at 21days during storage. In T₂, the mean value was observed as 48.29 ± 0.06%, 46.27 ± 0.02%, 44.47 ± 0.10%, and 42.66 ± 0.03% at 0, 7, 14 and 21days of storage, respectively, at 0days of storage. The highest DPPH was seen at 0days, whereas the lowest DPPH was observed at 21days of storage. In T₃. DPPH contents were observed as 49.75 ± 0.04%, 47.62 ± 0.06%, 45.83 ± 0.03%, and 42.21 ± 0.11% at 0, 7, 14, and 21days of storage, respectively. On 21days of storage, a maximum DPPH content was detected at 0days, while at 21days, a minimum percentage of DPPH was observed. Momeny et al.^[20] reported similar results where the radical scavenging potential was 41% to 43%. The current study is also in line with Jacimovic et al.^[19] The antioxidant activity (expressed as percent inhibition of oxidation) ranged from 45% to 55%. Antioxidant capacity is directly linked with the amount of phenolic compounds in the product. As time passes the phenolic compounds degrade owing to oxidative agents and hence the reduction of percent inhibition.

pH

The percentage of pH in all samples was determined with the help of a pH meter apparatus at the interval of 0, 7, 14 and 21days. The influence of both storage and treatment on the pH of chocolate is shown in Figure 3. This mean figure shows that the maximum pH was observed at 21days in T₃ (6.60 ± 0.02), whereas a minimum pH was observed in T₀ (6.19 ± 0.01) at 0days of storage. Means of pH in controlled treatment (T₀) were detected as 6.19 ± 0.02, 6.29 ± 0.01, 6.33 ± 0.01, and 6.39 ± 0.01 at 0, 7, 14, and 21days of storage, respectively. The lowest percentage of pH was observed at 0days, whereas the highest percentage was observed at 21days. In T₁, the mean values of pH were observed as 6.30 ± 0.01, 6.35 ± 0.01, 6.45 ± 0.01, and 6.47 ± 0.01 at 0, 7, 14, and 21days of storage, respectively. The highest pH was observed at 21days, whereas the lowest value was observed at 0days during storage. In T₂, the mean value was observed as 6.34 ± 0.02, 6.49 ± 0.0.3, 6.58 ± 0.01, and 6.60 ± 0.04 at 0, 7, 14, and 21days of storage, respectively. At 21days of storage, maximum pH was observed, whereas the lowest pH was observed at 0 day of storage. In T₃. pH was observed as 6.20 ± 0.01, 6.40 ± 0.03, 6.42 ± 0.04, and 6.45 ± 0.02 at 0, 7, 14, and 21days of storage, respectively. At 21days, maximum pH was observed, while at 0-day, minimum percentage of pH was observed. The obtained results are also approximately equal to the values found by Rao and Tamber.^[21] An increase in pH may be attributed to free fatty acids being released with an increase in storage time.

Textural attributes of chocolate

Hardness (N)

Statistical results for the hardness of chocolate prepared with mango seed oil indicated that observed to be highly significant, while treatment was observed to be non-significant. Effect of treatment on hardness were shown in Figure 2. The effect of varieties indicated that the highest hardness observed in chocolate prepared by mango seed oil in T3 was 12.36 ± 0.02, whereas the lowest hardness value was observed in T3 at 11.68 ± 0.04 N. The results also follow the results reported by Mantihal and fellow researchers,^[22] who found that the hardness of chocolate ranges from 10.25 to 13.64 N.

Breaking strength (N)

tatistical results for breaking strength of chocolate prepared with mango seed oil showed that to be highly significant. The effect of treatment indicated that the highest breaking strength values were observed in chocolate prepared with mango seed oil in T3 with a mean value of 19.09 ± 0.05 N, Whereas the lowest breaking strength value was noticed in T0 with the mean value of 16.31 ± 0.17 N as revealed in (Fig.6). The breaking strength of chocolate was obtained according to the respective protocol followed by Nurhayati et al.^[16]

Cutting strength (N)

Statistical results for cutting strength of chocolate prepared with mango seed oil indicated that observed to be highly significant. The effect of treatment indicated that the highest cutting strength values were observed in chocolate prepared with mango seed oil in T3 followed by the mean value of 29.30 ± 0.17 N, Whereas the lowest cutting strength was observed in T0 with the mean value of 28.30 ± 0.07 N as revealed in (Figure 7). The results of this study are also similar to the results of Lasta and colleagues,^[23] who found that the cutting strength of the chocolate ranged from 25 to 32 N. Effect of treatment on cutting strength were shown in Figure 4.

Sensory evaluation of chocolate

Sensory scores for each parmeter of the product were presented in Table 7. The highest mean value of appearance was found in T₃ at 0 day which was 7.63 in chocolate. The lowest value of appearance was noticed in the treatment of T₁ which is 5.50 at 14days. The highest mean value of taste was found in T₂ at 0 day which was 7.75 in chocolate. The lowest value of taste was noticed in the treatment of T₁ which is 4.75 at 21days. The highest mean value of flavor was found in T₀ at 0 day which was 8.50 in chocolate. The lowest value of flavor was noticed in the treatment of T₀, which is 6.25 at 14days. The highest mean value of texture was found in T₂ at 0 day which was 7.63 in chocolate. The lowest value of texture was noticed in the treatment of T₀ which is 6.50 at 21days. The highest mean values of overall acceptability were found in T₁ at 0 day which was 7.75 in chocolate. The lowest value for the overall acceptability was observed in the treatment of T₀ (6.13) at 14days.^[17]

Conclusion

In the present study, the solvent extraction method showed a higher yield of mango seed oil as compared to mechanical extraction. Mango seed oil is a good source of fatty acids and is safe for edible uses. Current findings showed that mango seed oil was used in the production of chocolate. The results of this research are proximate analysis of the chocolate prepared with mango seed oil are moisture 6.27 ± 0.01%, ash 3.00 ± 0.10%, fat 29.63 ± 0.99%, fiber 2.61 ± 0.06%, protein 9.32 ± 0.16%, and NFE 49.16 ± 0.04%. The results of chocolate made with mango seed oil, TPC was 29.79 ± 0.03, 29.54 ± 0.02, 29.41 ± 0.03, and 29.19 ± 0.05 at 0, 7, 14, and 21, respectively. Antioxidants that can delay, inhibit, or prevent the oxidation of oxidizable materials by scavenging free radicals and diminishing oxidative stress. The antioxidant activity of the chocolate shows the quality of the products. The obtained results were significant that was 48.18 ± 0.04, 46.07 ± 0.05, 44.30 ± 0.06, and 41.91 ± 0.06 at 0, 7, 14, and 21days, respectively. The lowest value was 41.29 ± 0.05 in T₂ at 21days, and the highest value was 48.18 ± 0.04 in T₃ at 0 day. The highest pH was observed in T₂ which was 6.60 ± 0.02 at 21days and the lowest value observed in T₃ which was 6.20 ± 0.02 at 0 day. At 0days, T3 had the highest mean appearance value of 7.63 in chocolate, and T1 had the lowest at 14.50 at 14days. Chocolate taste has the highest mean value at 0 day, 7.75, and the lowest value is 4.75 at 21days in T1. At 0days, T0 had the highest mean value of flavor: 8.50 in chocolate. At 14days, T0 had the lowest mean value of flavor: 6.25. At 0days, T2 had the highest mean value of texture of 7.63, while T0 had the lowest mean value of texture of 6.50. At 0days, T2 had the highest mean value of texture of 7.63, while T0 had the lowest mean value of texture of 6.50.

Acknowledgement

The Author(s) are thankful to the Department of Food Science and Technology, MNS University Multan Pakistan and the Agricultural Extension Directorate, MAAR, Damascus, Syria for providing lab facilities.

Supplementary Material

Supplemental data for this article can be accessed online at https://doi.org/10.1080/10942912.2023.2267784

Disclosure statement

No potential conflict of interest was reported by the author(s).