Effect of functionalization of raw materials on the quality of instant chocolate drink with sensory, physicochemical and anti-diabetic parameters

The cocoa bean is a component of the cocoa pod, which is frequently used as an ingredient in foods and drinks. Numerous healthy substances, such as polyphenols, which act as antioxidants, antiaging, anti-inflammatory, antidiabetic, and antihypertensive agents, are found in cocoa beans. The goal of this study is to create a useful instant beverage that acts as an antidiabetic. This study involved the creation of a combined instant chocolate powder and cocoa liquid. According to the findings, the Forastero variety processed cocoa powder had the highest total polyphenol content, namely 425.179 pm 0.01 mg GAE/g and the lowest polyphenol content was owned by Kalla cocoa, namely 172.768 pm 2.93 mg GAE/g. The formulation’s overall polyphenol content ranged from 3.125 pm 1.89 to 150.446 pm 17.04 mg GAE/g. The highest inhibition percentage with the lowest IC50 value was found in the formulation A31 sample and the lowest inhibition percentage with the highest IC50 value was in the formulation B32 sample. The higher the value of the total polyphenol content of the cocoa powder sample, the higher the percentage of inhibition of alpha-amylase activity. The physicochemical analysis is carried out by measuring the color. The color change in cocoa powder is due to changes in the structure of the cocoa anthocyanin-type polyphenols during the fermentation, drying and roasting processes. The sensory analysis of the ranking method showed the best formulations of B23 and B33, according to the panelists.


Introduction
Cocoa is one of every of Indonesia's main plantation commodities which could be developed.In latest years, the hobby with inside the biologically lively additives contained in cocoa beans has multiplied hastily because of their useful consequences on human fitness.The blessings of cocoa (Theobroma cacao L) that's wealthy in phenolic compounds as a chocolate meals or drink for the fitness of the frame are broadly known, which include stopping and decreasing the chance of coronary heart disorder and cancer [1,2], boom insulin sensitivity, lower insulin resistance, and reduce systolic blood pressure [3].
Cocoa beans produced by farmers are generally unfermented cocoa beans, so their quality as a raw material for processed chocolate food products is in the low category.This is due to the weak characteristic taste of cocoa due to the absence of aroma precursor compounds.Meanwhile, the quality of cocoa beans for cortthe chocolate industry is largely determined by the content of polyphenolic 1230 (2023) 012159 IOP Publishing doi:10.1088/1755-1315/1230/1/012159 2 compounds, aroma, and taste.The content of chocolate aroma and flavor compounds can be obtained through the complete fermentation process of wet cocoa beans before drying.
The forastero variety of cocoa beans has a high polyphenol content.Meanwhile, the aroma and taste of the fermented and roasted Sulawesi clone varieties of cocoa beans are urgently needed for the process of developing cocoa beans as a raw material for chocolate products, especially functional chocolate.Therefore, this research was conducted to determine the total content of polyphenolic compounds in cocoa powder from different seed varieties and cocoa powder processed by the instant chocolate beverage product industry, to determine the effect of cocoa powder on sensory parameters, color, and antidiabetic properties of polyphenol extracts from various types of instant chocolate drinks tested in vitro.

Manufacturing of cocoa powder
The cocoa beans of the Sulawesi clone variety are sorted, fermented for five days, then dried and roasted at 105ºC for 10 minutes to form a distinctive chocolate aroma and taste.For the forastero variety cocoa beans, after sorting and then drying.The dried cocoa beans are removed from the outer skin so that the nibs are obtained.Cocoa nibs are put into a stone mill to become a coarse cocoa paste (cocoa liquor).The cocoa paste is pressed using a press machine at 50ºC with a pressure of 58 MPa for 60 minutes until all the fat separates from the cake.The cake was crushed using a hammer machine using a 0.8 mm sieve until fine cocoa powder was obtained.

Cocoa powder polyphenol extraction
As much as 2 grams of fat-free cocoa powder was put into a 250 mL Erlenmeyer, then added with 20 mL of acetone: water solvent with a ratio of 7:3 (v/v).Then put it in the incubator shaker for 24 hours at room temperature in a closed state and protected from direct light.After that, it was centrifuged at 3500 rpm for 20 minutes, and the supernatant was separated.The total extract was then put in a rotary evaporator at 40ºC to evaporate the acetone to obtain a viscous polyphenolic extract.The polyphenol extract was then dried in a room with a temperature of 20ºC to obtain polyphenol extract powder [4].

Determination of total polyphenol levels
Up to 100 mg polyphenol extract powder was dissolved with Aquabidest up to 10 ml.Pipette 1 ml and dilute to 10 ml with Aquabidest.Pipette 2 ml and dilute to 10 ml with Aquabidest.Pipette 1 mL of extract directly into a 25 mL volumetric flask. 1 ml of 1N Folin-Ciocalteu reagent was added, then shaken and left for 5 minutes.5 mL 10 % Na2CO3 was added, then pressed out with Aquabidest in a 25 mL volumetric flask.Shake and leave at room temperature for 1 hour.The absorbance was measured at a wavelength of 745 nm.The same procedure was carried out to prepare gallic acid mother liquor with concentrations of 0 ppm, 20 ppm, 40 ppm, 60 ppm, 80 ppm, and 100 ppm as the basis for making a standard curve to calculate the total polyphenol content.

Table 1. Chocolate instant drink formulation
Of the 18 instant chocolate drinks that have been made, the ranking is tested for taste and aroma attributes, with a scale of 1-5 (criteria 1: really dislike, 2: don't like, 3: neutral, 4: like, 5: really like) to find out the preference level of the panelists.

Determination of physicochemical properties and antidiabetic activity test of instant chocolate drink
The physicochemical tests in this study was the color test.The color test was carried out using a Minolta CR-400 Chromameter.The chromameter tool is turned on and then calibrated between the values Y, x, y and the calibration plate.Press color space until it appears on the screen L*, a*, b*.Aim the chromameter head at the instant drink sample then press measure enter until it beeps or the light turns on so that sample L*, a*, b* values appear on the screen.
The determination of antidiabetic activity begins with preparing a 20 mM phosphate buffer solution pH 6.9, amylase enzyme solution, 1% starch solution and DNS solution.Mother liquor samples were made with concentrations of 1, 2, 5, 10, and 15 mg/mL.Then 100 µL of the test sample solution was pipetted into a test tube and 100 µL of the amylase enzyme was added.Samples were incubated for 20 minutes at 37 o C. Added 100 µL of starch then incubated for 10 minutes at 37 o C. The sample was added with 200 µL of DNS solution and heated with boiling water for 5 minutes then allowed to stand at room temperature for 5 minutes and diluted with 3.5 mL of aquabidest.Absorption was measured at ʎmax = 540 nm with a UV-VIS spectrophotometer [5].The antidiabetic value of the sample will be compared with the value of acarbose and the control solution.Acarbose solutions were made with various concentrations, namely 0.006 mg/mL, 0.02 mg/mL, 0.04 mg/mL, and 0.06 mg/mL.Likewise for the control solution and the blank were carried out as in testing the antidiabetic value of the sample.

Total polyphenols
The total polyphenols of the various cocoa samples tested is shown in Table 2.The total polyphenol content in cocoa powder was calculated using the standard gallic acid curve.Based on the measurement of the standard curve, a linear line equation is obtained y = 0.007x + 0.025.Forastero variety cocoa powder without fermentation had the highest total polyphenol content, namely 425.179±0.01mg GAE/g, then mixed cocoa powder samples (forastero: Kalla 75:25) amounted to 389.732±4.95mg GAE/g.The lowest polyphenol content was found in the control cocoa powder (Kalla cocoa) of 172.768±2.93mg GAE/g.The total polyphenol content of the tested cocoa powder instant drink formulation samples is shown in Table 3.
Based on the data above, the total polyphenol content of the formulation ranged from 3.125±1.89-150.446±17.04mg GAE/g.Formulation A31 (forastero 100% 8 grams) contained the highest total polyphenols of 150.446±17.04mg GAE/g, while formulation B33 (Kalla 100% 4 grams) contained the lowest total polyphenols of 3.125±1.89mg GAE/g.The presence of a high content of polyphenolic compounds in cocoa powder and product formulations greatly contributes to nourish the body, especially as an antidiabetic.The processing of cocoa beans affects the reduction of the content of polyphenolic compounds in cocoa beans and processed cocoa products.The processing processes that can reduce the reduction in polyphenol content are fermentation, drying, roasting and other processing processes such as the production process to produce chocolate products, although the effect is not as great as that of the previous processing process.

Analysis of in vitro antidiabetic properties of instant chocolate drink formulation
The content of polyphenols in cocoa beans is a group of natural antioxidants that are beneficial to the health of the body.The chocolate instant drink formula made from cocoa beans exhibits antioxidant and antidiabetic activity.In this study, the antidiabetic properties were tested for the activity of inhibiting the amylase enzyme.The results of in vitro testing for antidiabetic properties can be seen in Table 4.The parameter used to show antidiabetic activity is the Inhibition Concentration (IC50) value, namely the concentration of polyphenolic extracts that can inhibit 50% of the activity of the amylase enzyme.Concentration variations are made so that they can be used to create a regression equation to calculate IC50.The results of the analysis of α-amylase (IC50) inhibitory activity show that the smaller the IC50 value of a compound, the greater the inhibitory effect on α-amylase activity [6].This is based on the analysis data in Table 4 that the highest percentage of inhibition was 60.81% in the cocoa powder formulation A31 sample (forastero 100% 8 grams) with the lowest IC50 value of 11.56 µg/mL and the lowest inhibition percentage of 29.01% in the sample of cocoa powder formulation B32 (100% Kalla 6 grams) with the highest IC50 of 34.19 µg/mL.While the control (acarbose) used had an IC50 value of 14.284 µg/mL.From the results of the analysis, it is also known that the inhibitory activity of α-amylase is correlated with the content of total polyphenol levels.The higher the value of the total polyphenol content of the cocoa powder sample, the greater the inhibition percentage of α-amylase activity so that it is effective for use as an antidiabetic.
The inhibitory effect of cocoa powder extract is due to the predominant presence of procyanidin compounds in cocoa.The procyanidin compound consists of 3.6% procyanidin B, 0.75% procyanidin B5, and 2.28% procyanidin C1.The high molecular weight cocoa procyanidin fraction (High-DP>3) inhibits the glucosidase enzyme (small intestine) with an IC50 value of 50 µg/mL.Procyanidin compounds slow down glucose absorption through the inhibition of the glucosidase enzyme in the small intestine.Polyphenol compounds can act as digestive enzyme inhibitors (α-amylase and α-glucosidase).
Polyphenolic compounds are also capable of acting as natural inhibitors of enzymes that play a role in hydrolyzing carbohydrates so that they can help inhibit increased blood glucose levels [7].As a comparative anti diabetic, acarbose is used, a diabetes drug that competitively inhibits the action of enzymes (maltase, isomaltase, sucrose, and glucoamylase) which can delay the breakdown of sucrose and carbohydrates in the small intestine.From the data in Table 4 the inhibitory effect of the formulation samples on the amylase enzyme was greater than that of acarbose which served as a control.

Sensory evaluation of chocolate instant drink formulation products
The ranking test in this study was carried out on product quality for all sensory quality parameters, where the first order always stated the highest level of sensory preference and the next order showed a lower level.The number of panelists in this test was 31 untrained panelists.The test attributes are in the form of aroma and taste (sweet, sour, astringent, and bitter).The ranking test sample is 18 samples.The sample ranking test uses 5 rating scales, namely scale 1 really dislike, 2 don't like, 3 neutral, 4 like, and 5 really like.The results of the ranking test were processed using the SPPS Friedman Test.

Figure 1. Ranking test results
Based on all the assessment attributes in the ranking test, according to the panelists the best sample is sample B33 (Kalla 100% 4 grams) and the less good is sample A31 (Forastero 100% 8 grams).The aroma of cocoa is due to the components tetramethylpyrazine and trimethyl pyrazine [8].While the sour taste in cocoa is due to the presence of acetic acid, lactic acid, and citric acid in cocoa beans.The emergence of a bitter taste in the sample is due to the distinctive taste of chocolate which has alkaloid components such as theobromine and caffeine, phenolic components, pyrazine, several peptides and free amino acids.The tannins contained in cocoa also give the sample an astringent impression.

Analysis of color properties of chocolate instant drink formulation (L*, a*, b*)
One of the most important sensory quality elements for food is color.Color is a material property that is thought to originate from the spread of the light spectrum.Food color can be measured using a colorimeter instrument with a hunter color system [9]mill.In color measurement, the higher the L value, the higher the brightness level.The quality of the cocoa powder is largely determined by the intensity of the brown color.The alkalization process affects the intensity of the color.Unrealized cocoa powder generally has a light brown color, while alkaline cocoa powder has a color intensity ranging from reddish brown to very dark red-brown [10].Chromatic color results for these different types of products show a purple color.This is also reflected in the o Hue value calculation, for products A11, A12, A13, A21, A22, A23, A31, A32, A33, B11, B12, B13 it varies from 41.049-48.121.Chromatic colors in products B21, B22, B23, B31, B32 and B33 show a red-yellow color in the range of 54.146 to 55.738.The decrease in shine is due to the addition of powder to the product.The more cocoa powder is in the product, the darker the color intensity of the product, because cocoa powder has a dark color appearance.

Conclusion
Unfermented forastero variety cocoa powder had the highest total polyphenol content, namely 425.179±0.01mg GAE/g.While the Sulawesi clone variety cocoa powder had a polyphenol content of 209.821±7.67mg GAE/g and the lowest polyphenol content was owned by Kalla cacao, namely 172.768±2.93mg GAE/g.The more cocoa powder added to the product, the darker the color intensity of the product because cocoa powder has a dark color appearance.From the results of the organoleptic test ranking method with aroma and taste parameters, the B23 and B33 formulations were the most preferred.The formulated cocoa powder polyphenolic extract has an IC50 value ranging from 11.56 to 34.19 mg/mL.The results of the analysis of α-amylase (IC50) inhibitory activity show that the smaller the IC50 value of a compound, the greater the inhibitory effect on α-amylase activity so that it is effectively used as an antidiabetic.

Table 2 .
Total polyphenols of cocoa powder after press stage.

Table 3 .
The total polyphenols content of formulated powder.

Table 4 .
Inhibitory effect of powder formulation on amylase enzyme

Table 5 .
Color analysis data.