Reduction of Saturated Fat in Dark Chocolate using Sacha Inchi ( Plukenetia volubilis ) Oil Oleogel

This research studied the effectiveness of Sacha Inchi Oil Oleogel (SIOO) as a partial replacement for saturated fat in dark chocolate. Sacha inchi ( Plukenetia volubilis ) oil is high in polyunsaturated fatty acids (α-linolenic and linoleic acids) and a good source of tocopherols. This study prepared oleogels using sacha inchi oil as a base oil and food-grade beeswax as an oleogelator. Different percentages of SIOO (1%, 2.5% and 5.0%) were added in the dark chocolate. Dark chocolate without SIOO was used as a control. Fatty acid profile, total polyphenols, antioxidant activity and sensory evaluation of the formulated dark chocolates with SIOO were investigated. The incorporation of SIOO significantly (p<0.05) lowered the saturated fat and increased the polyunsaturated fatty acids in dark chocolate samples. This study also showed that the total polyphenols and antioxidant activity of dark chocolates enriched with 2.5 and 5.0% SIOO were significantly higher (p<0.05) than the other chocolate samples. Sensory evaluation showed that control and dark chocolates added with SIOO (1% and 2.5%) received similar scores for all sensory attributes. However, the highest concentration of SIOO decreased significantly (p<0.05) the scores for the taste and overall acceptability of dark chocolate. Therefore, the enrichment of sacha inchi oil oleogel as a functional ingredient could reduce the saturated fat and increase the polyunsaturated fatty acids and antioxidant activity of the formulated dark chocolate, which is well-accepted by consumers.


INTRODUCTION
Dark chocolate is one of the confectionery products admired by people of all ages (Merlino et al. 2021).Dark chocolate mainly consists of cocoa liquor followed by sugar, cocoa butter and emulsifier.The high content of cocoa liquor in dark chocolate possesses antioxidant properties due to polyphenols and flavonoids, which may provide several health benefits (Shahanas et al. 2019).The market demand for dark chocolate is predicted to increase from $48.29 billion to $67.88 billion by 2029 (Market Research Report 2022).However, the incorporation of solid fats such as cocoa butter equivalents, cocoa butter substitutes and cocoa butter replacers in the dark chocolate formulation increases the content of saturated fat, which may have a negative impact on people's health such as obesity, cardiovascular diseases, high cholesterol, cancer and type II diabetes (Li & Liu 2019).For that reason, consumers demand healthy and functional chocolate products with less saturated fats by increasing the content of Polyunsaturated Fatty Acids (PUFAs) compared to conventional chocolates (Selvasekaran & Chidambaram 2021).
Edible vegetable oils (soybean, canola, sunflower and corn) are high in PUFAs, which consist of linoleic (omega-6) and α-linolenic acids (omega-3) (Loganathan & Kim-Tiu 2022).Recently, oleogels developed as the latest innovative technology for oil structuring Ishak et al. to entrap bulk vegetable oils within a thermoreversible and three-dimensional gel network by food-grade oleogelators (Perţa-Crişan et al. 2023).At the same time, vegetable oils are transformed into solid fat by lowering the content of saturated fats, increasing the amount of PUFAs and retaining the chemical composition of the oils (Manzoor et al. 2022).Various edible oleogelators are commonly used as oleogel to entrap bulk vegetable oil, including waxes, lecithin, sterols, and monoacylglycerols.Beeswax is a natural wax produced by honey bees in the bee hives.Beeswax is the most common oil structure-forming agent used for the production of vegetable oil oleogels (sunflower oil, olive oil, linseed oil and canola oil) (Frolova et al. 2022;Issara et al. 2022).
Vegetable oil oleogels are frequently used in many food products such as bakery products, filling creams and ice cream to replace saturated fat while improving the food quality and nutritional value (Jing et al. 2022;Cabrera et al. 2020;Pehlivanoglu et al. 2018).Regarding chocolate products, Li & Liu (2019) produced dark chocolate added with corn oil-monoglyceric stearate based oleogel to replace cocoa butter partially.The authors reported that incorporating oleogels lowered the saturated fats and increased the PUFAs of dark chocolate samples.Alvarez et al. (2021) also stated that the replacement of cocoa butter in the milk chocolate formulations by sunflower-oil-Hydroxypropyl Methylcellulose (HPMC) reduced the saturated fats and increased the content of PUFA and Monounsaturated Fatty Acids (MUFAs).
To the knowledge of an author, sacha inchi oleogel has not been utilised directly to reduce the saturated fats in dark chocolate.Limited information is about the impact of oleogel-based vegetable oil on the total phenolic, antioxidant activity and sensory acceptability of chocolate products.In this study, sacha inchi oil was used as a representative of vegetable oil rich in PUFAs to develop sacha inchi oil-based oleogel with beeswax for reducing the saturated fats of dark chocolate.The objective of the present study was to investigate the effects of sacha inchi oilbased oleogel at different concentrations on the fatty acid composition, total phenolic content, antioxidant activity and sensory acceptability of dark chocolate.

Design, location, and time
The research was conducted in the Cocoa Innovative and Technology Centre, Malaysian Cocoa Board, Negeri Sembilan and the Department of Food Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia, Selangor, Malaysia.The study was carried out from September 2022 to February 2023.

Materials and tools
Cold-pressed sacha inchi oil (Khatijah Herbs, Malaysia) and natural yellow beeswax (Personal Formula Resources, Malaysia) were used as materials to prepare Sacha Inchi Oil Oleogel (SIOO).Meanwhile, ingredients used in the production of dark chocolate were cocoa liquor, cocoa butter, icing sugar, and soy lecithin as emulsifier from Malaysian Cocoa Board, Nilai, Negeri Sembilan.Chemicals used in this study were: methanolic potassium hydroxide (KOH), n-hexane and sodium carbonate (Na 2 CO 3 ) from R & M Chemicals, Malaysia.Meanwhile, gallic acid, Folin-Ciocalteu and 2,2-diphenyl-1picrylhydrazyl (DPPH) reagents were obtained from Sigma-Aldrich, United States.

Procedures
Preparation of sacha inchi oil oleogel.SIOO was prepared according to the method of Calligaris et al. (2020) with slight modification.Firstly, Sacha Inchi Oil (SIO) and 10% beeswax were heated by stirring them in a dark room at not more than the maximum melting temperature of beeswax (65°C) for 30 min.The heating process is completed when the beeswax is fully soluble Dark chocolate with reduced saturated fat in the SIO.SIOO was cooled overnight at room temperature (27°C) and stored at 20°C for analysis and usage in dark chocolate formulations.

Development of dark chocolate added with different percentages of sacha inchi oil oleogel.
The addition of SIOO at different percentages (1.0%, 2.5% and 5.0%) in dark chocolate was conducted.Four dark chocolate formulations were prepared as follow: dark chocolate without SIOO was used as a control sample (F1); dark chocolate with 1.0% SIOO (F2); dark chocolate with 2.5% SIOO (F3) and dark chocolate with 5.0% SIOO (F4).Dark chocolate was produced according to the method described by Biswas et al. (2017).Dark chocolate ingredients (cocoa liquor, sugar and a quarter of melted cocoa butter) were mixed in a concher (Pascal Engineering, England) at 45°C for 5 min.After that, the particle sizes of the chocolate mixtures were reduced three times to less than 30 μm using a triple roller mill (Pascal Engineering, England).After refining, the chocolate mixtures and the remaining melted cocoa butter were mixed in concher for 6 h at 45°C.Two hours before the completion of the conching process, SIOO and soy lecithin were added to the chocolate mixtures.Afterwards, the liquid dark chocolate was tempered manually on the marble slab by reducing the temperature of the liquid chocolate from 45°C to 27°C to obtain the most stable form of fatty acid crystals of cocoa butter.The tempered dark chocolate was poured into a polycarbonate mould and cooled at 13±1°C for 60 min to solidify the chocolate.The dark chocolate was then removed from the mould and stored in a plastic container at room temperature for further analysis (fatty acid composition, total phenolic content, antioxidant activity and sensory acceptability).
Determination of fatty acid profile.The fatty acid composition of the dark chocolate added with different percentages of SIOO was determined as described by Md Ali & Dimick (1994).Gas Chromatography (GC) (Hewlett-Packard 6890, Agilent Technologies, Palo Alto, CA, USA) with a flame ionisation detector was used to analyse the fatty acid methyl esters (FAMEs) of chocolate samples.Fatty acid profiles of the chocolate sample were measured according to the chromatogram peak of the FAMEs.
Determination of total polyphenol content.The total polyphenol content of dark chocolate added with different percentages of SIOO was determined according to de Camargo et al. (2015).The total polyphenol content was expressed as gallic acid equivalents in milligrams per gram of sample (mg GAE/g dark chocolate) with the following formulation: Where C is the concentration determined from the standard curve (mg/L), and M is the mass of the extracted sample (g).
Determination of antioxidant activity.The 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging activity of the dark chocolate added with different percentages of SIOO was measured according to Urbańska & Kowalska (2019).DPPH free radical scavenging activity of the chocolate sample was calculated using the following formula: Where A1 is the absorbance of the control sample, and A2 is the absorbance of the chocolate sample.The free radical scavenging activity is recorded in percentage.
Sensory evaluation.Hedonic scale rating test was carried out to evaluate the sensory acceptability of dark chocolate added with different percentages of SIOO (da Silva et al. 2013).Fifty untrained panelists consist of staff (academic and laboratory) and students aged 20-55 from the Department of Food Sciences, Faculty of Science and Technology, Universiti Kebangsaan Malaysia.The 7-point hedonic scale was made by an unstructured 10 cm horizontal linear ruler, with both ends marked with a legend.The panels indicate the intensity of each sensory attribute for chocolate sample on the 10 cm unstructured scale (where 1="least liked" and 7="most liked").

Data analysis
Total polyphenol content and antioxidant activity of dark chocolate added with different percentages of SIOO were carried out in three replications (n=3), while fatty acid profile and sensory evaluation of dark chocolate added with different percentages of SIOO were conducted in two replications (n=2).All data obtained are analysed using one-way Analysis of Variance (ANOVA) and Tukey's post-hoc test to determine if there was a significant difference between

Ishak et al.
samples.A confidence level of 95% (p<0.05) was used to see significant differences between samples.All data are expressed as mean±standard deviation.

RESULTS AND DISCUSSION
The fatty acid composition of dark chocolate added with different percentages of SIOO is showed in Table 1.Palmitic and stearic acids (saturated fatty acids), oleic acid (monounsaturated fatty acid), linoleic and α-linolenic acids (PUFAs) were identified in dark chocolate samples.Saturated fatty acids (palmitic and stearic acids) are the major fatty acids in all chocolate samples due to the incorporation of cocoa butter as fat medium in the chocolate formulation.The control sample contains the highest amount of saturated fatty acids (66.12%).Results showed that adding different SIOO significantly decreased (p<0.05) the content of saturated fatty acids (57.02-64.11%)compared to the control sample.At the same time, the content of PUFAs (linoleic and α-linolenic acids) in dark chocolate increased significantly (p<0.05) with the concentrations of SIOO (from 1.0-5.0%).These findings were reasonably expected due to the high concentration of PUFAs present in SIO (Rodzi et al. 2022).Li & Liu (2019) reported that the addition of different corn oil-based oleogels (monoglyceric stearate, β-sitosterol and lecithin and ethyl cellulose) in dark chocolate decreased significantly (p<0.05) the saturated fatty acids (21.17-21.84%)compared to the control sample (dark chocolate with 100% cocoa butter) (37.86%).The authors also proved that the PUFAs (57.24-57.88%) of dark chocolate increased significantly by adding different corn oil-based oleogels.Enrichment of PUFAs from SIO-based oleogel in dark chocolate might lower heart diseases, prevent cancer risk and cardiovascular diseases, and improve highdensity lipoprotein blood (Nguyen et al. 2020).Therefore, adding sacha inchi oil oleogels at different concentrations decreases the saturated fatty acids and enhances the content of PUFAs in dark chocolate.
Table 2 presents the Total Polyphenol Content (TPC) of dark chocolate added with different percentages of SIOO.The result showed that the TPC for dark chocolate added with 2.5 and 5.0% SIOO (27.35-28.69mg GAE/g) were significantly higher (p<0.05)than F1 and F2 samples (24.39-25.32mg GAE/g).However, the difference in TPC between F1 and F2 samples is insignificant (p>0.05).The lowest concentration that the high amount of cocoa liquor used in dark chocolate contributed to the great source of polyphenols, which provides numerous positive health benefits such as prevention of cancer, cardiovascular and inflammatory diseases as well as protection of low-density lipoprotein cholesterol against oxidative stress.It clearly showed that adding 2.5 and 5.0% sacha inchi oil oleogel significantly increased the total phenolic content of dark chocolate.
A higher amount of SIOO (2.5 and 5.0%) can definitely improve the antioxidant activity of dark chocolate.To our knowledge, there has yet to be published data on the effects of vegetable oil oleogel on the antioxidant activity of chocolate or all types of food products.Ramos-Escudero et al. (2019) reported that 27 commercial SIO samples contain several lipid-soluble antioxidant compounds, such as tocopherols (γ-& δ-) and sterols (campesterol and stigmasterol), which attributed to the antioxidant activity.Also, polyphenolic compounds play a synergistic role with other antioxidant compounds (tocopherols and sterols) in SIO (Cárdenas et al. 2021;Liu et al. 2014).Moreover, tocopherols and polyphenols act as antioxidants and provide health benefits in preventing hypertension, atherosclerosis and certain cancers (Liu et al. 2014).The health benefits associated with this high radical scavenging activity of dark chocolate added with sacha inchi oil oleogel are worth further investigation.
The mean scores for each attribute of dark chocolate added with different percentages of SIOO are presented in Table 3. Results showed that the mean scores for the overall acceptability of the dark chocolate samples were 4.22-5.20,which corresponded to "neither like nor dislike" and like" based on the seven-point hedonic scale.Dark chocolate samples (F1, F2 and F3) received similar score (5.01-5.20)"like" (p>0.05) for overall acceptability.Not only that, mean scores for texture, aroma, taste, bitter aftertaste and glossiness of dark chocolate added with SIOO (1.0 & 2.5%) were similar (p>0.05) with the control sample.However, panelists gave the lowest sensory score (4.22) (p<0.05) for the overall acceptability of the F4 sample.Besides that, mean scores for different sensory attributes

CONCLUSION
This research uses different percentages of sacha inchi oil oleogel as a partial cocoa butter replacer to produce dark chocolate with reduced saturated fat.Enriching sacha inchi oil oleogel in dark chocolate significantly increased the content of polyunsaturated fatty acids by lowering the saturated fatty acids compared to the standard dark chocolate.Sacha inchi oil oleogel significantly improved the dark chocolate's total phenolic content and antioxidant activity compared to the standard dark chocolate.When sacha inchi oil oleogels at concentrations of 1.0% and 2.5% are used together with cocoa butter in the formulation, dark chocolate has similar attributes to standard dark chocolate.However, the panelists rated the lowest score for all sensory attributes of dark chocolate added with 5% sacha inchi oil oleogel.These findings verify that healthier dark chocolate with higher polyunsaturated fat content, total phenolic content and antioxidant activity and lower saturated fat content, as well as optimal sensory acceptability can be obtained by adding sacha inchi oil oleogel in the production of dark chocolate.

Table 1 .
Fatty acid profiles of the dark chocolate added with different percentages of sacha inchi oil oleogels

Table 2 .
Total polyphenol content and free radical scavenging of the dark chocolate added with different percentages of sacha inchi oil oleogels

Table 3 .
Mean scores for each sensory attribute of dark chocolate added with different percentages of sacha inchi oil oleogel Values are mean±standard deviation (n=50) a-c Values in each column with different letters are significantly different (p<0.05).Dark chocolate formulations containing sacha inchi oil oleogel: F1: Dark chocolate without sacha inchi oil oleogel; F2: Dark chocolate with 1.0% sacha inchi oil oleogel; F3: Dark chocolate with 2.5% sacha inchi oil oleogel; F4: Dark chocolate with 5.0% sacha inchi oil oleogel