Regression analysis for determination of antioxidant activity of coconut sap under various heating temperature and concentration of lysine addition

F U L L P A P E R Regression analysis for determination of antioxidant activity of coconut sap under various heating temperature and concentration of lysine addition *Sulistyo, S.B. and Haryanti, P. Agricultural Engineering Study Program, Faculty of Agriculture, Jenderal Soedirman University, Purwokerto, Indonesia Food Technology Study Program, Faculty of Agriculture, Jenderal Soedirman University, Purwokerto, Indonesia


Introduction
Coconut sap is sugary exudates, sweet, oyster-white color and translucent, with nearly neutral pH that is obtained from the young inflorescence of a coconut tree (Cocos nucifera L.) (Borse et al., 2007). Coconut sap can be processed to beverage or as a raw material of coconut sugar. Because of its nutritious, coconut sap will suffer spontaneous fermentation and become alcoholic and acidic due to microbial activity (Hariharan et al., 2014). Granulated coconut sugar is produced from coconut sap through a lengthy heating process. The typical attribute of coconut sugar quality is the brown color that occurs during the heating process of the coconut sap. The brown color occurred during the heating of the sap is caused by Maillard reaction. This reaction is important in the production of sugar i.e. its impact to the flavor, color, and aroma (Asikin et al., 2014).
Maillard reaction intensity is commonly influenced by the composition of reactants, i.e. reducing sugar and amino acids contained in sap (Nagai et al., 2018), as well as the temperature of the heating process (Carciochi et al., 2016). According to Ho et al. (2008), reactant such as free amino acids are important as source of amino groups, free ammonia or nitrogen atoms through both deamination and retro-aldol reactions, while monosaccharide such as glucose and fructose play a role in the initial Maillard reaction by forming an abundant pool of high reactive C2, C2 and C4 dicarbonyl compounds.
The addition of lysine provided more basic amino groups, so the Maillard reaction occurred at the alkaline pH created the products via the 2.3-enolization track via 1-deoxy-2,3-dicarbonyl. This route will produce reductone compound that has antioxidant activity. Therefore, it is important to know the change of chemical and antioxidant properties of coconut sap induced with lysine which generated Maillard reaction products and melanoidin on the granulated coconut sugar. The Maillard reaction products and melanoidin were contributed to the antioxidant activity of granulated coconut sugar. Wijewickreme et al. (1999) reported that the amino eISSN: 2550-2166 © 2020 The Authors. Published by Rynnye Lyan Resources acid and reducing sugar in Maillard reaction were contributed to food color, flavor and antioxidant. In vitro studies, showed that MRPs may have antioxidant activity as they can role as metal chelators, radical scavengers (Kim, 2013). Yan et al. (2018) reported that MRPs derived from chitooligosaccharide and glycine model system exhibited strong antioxidant activity. The addition of this MRPs to fruit juices also increased the antioxidant capacity of these beverages. Karseno et al. (2018) stated that there was a significant correlation between the browning intensity and DPPH radical scavenging activity with a correlation coefficient (r) of 0.93.
The objectives of this research were to analyze the antioxidant activity of coconut sap added with various concentration of lysine during the heating process by means of regression analysis.

Materials
The coconut sap was tapped from spathes of tall variety of coconut trees (Cocos nucifera L.) cultivated in Sikapat Village, Sumbang District, Banyumas Regency, Central Java, Indonesia with the altitude of 500-1000 m above sea level. The tapping process was conducted during the daytime for 9 hours (0600 -1500) in fine weather with a temperature of 24-27°C. The relative humidity ranged about 91-92%.

Collection of coconut sap
The coconut sap was obtained from the tapped inflorescence of 15 coconut trees. The sap was collected into plastic containers which have been washed using hot water to minimize microbial contamination. The preservatives added were 1.7 g/L lime with the addition of 0.56 g/L of mangosteen peel powder. The procedure of the tapping process was as follows. The tip of the coconut inflorescence was cut by a sterilized stainless steel knife. A plastic container with the preservatives substance inside was placed covered up the cut inflorescence to collect the coconut sap. The coconut sap collected was measured the pH value and total soluble solids by a portable refractometer (Atago, Japan) immediately. The sap samples which their chemical properties meet the requirement for granulated coconut sugar production were then processed.

Heating of coconut sap
A 10 L of coconut sap was filtered with a filter cloth, then was divided into four parts. The first part was sap sample without addition of amino acid, the second, third and fourth part was added with 0.25, 0.5 and 0.75 mM lysine, respectively. Each sap sample was poured into an aluminum pan and was subsequently heated by a gas stove. During the heating process, the sap sample was continuously agitated until the temperature of the sap reached 118°C (for about 50 mins). A 50 g of sap sample was collected at heating temperature of 40, 60, 80, 100 and 118⁰C for analyzing the antioxidant properties.

Chemical analysis of coconut sap
After the heating process of coconut sap, the next step was chemical analysis. Four antioxidant properties, i.e. total phenolic content (TPC), browning intensity, DPPH radical scavenging activity and chelating activity, were subsequently analyzed.

Total phenolic
The estimation of the total phenolic content of the coconut sap was performed according to the Folin-Ciocalteu method with slight modifications (Payet et al., 2005). A total of 30 µL of the sample were added with 150 µL of 10% Folin-Ciocalteu reagent in a test tube. After incubated for 8 mins, an amount of 120 µL of 7.5% Na2CO3 dissolved in distilled water must be added. The sample was then incubated for 1 hr at 30°C, and the absorbance at 765 nm was measured. For the blank measurement, the sample was replaced by an appropriate solvent which was subtracted from the absorbance at 765 nm. The measurement result was then obtained by reporting the absorbance in the standard curve of gallic acid used as the standard phenolic compound. The results were expressed in milligrams of gallic acid equivalent per 100 gram of sample (mgGAE/100 g of sample).

Browning intensity
The browning color of coconut sap samples during the heating process was measured according to the method of Ajandouz et al. (2001) with slight modification. The sap samples were adjusted with distilled water (1:25 w/v), then centrifuged at 1006 x g for 15 mins. The absorbance of browning was measured FULL PAPER using a Genesys 10S UV-Vis spectrophotometer (Thermo Scientific; Carlsbad, CA, USA) at 420 nm, as an index of the brown polymers formed in more advanced stages.

Radical scavenging activity
DPPH radical scavenging activity of coconut sap was measured using the method of Payet et al. (2005). A volume of 280 µL of 0.1 mM DPPH• methanolic solution was pipetted into each tube test followed by 20 µL of sample, or solvent for the blank. The mixture was subsequently incubated at room temperature for 30 minutes, and the absorbance at 515 nm was then measured with a spectrophotometer. The antioxidant activity can be evaluated as a percentage of the radical scavenging activity (RSA) using the following equation:

RSA (%) = (Ao-As)/Ao x 100
Where Ao is the absorbance of the blank and As is the absorbance of the sample at 515 nm after 30 mins.

Chelating activity
The ability of sap samples to chelate the metal ions Fe 2+ was investigated according to Kim (2013). One gram of sap samples with 5-fold dilution was prepared, then filtered with filter paper to obtain sap solution. One hundred microliters of coconut sap solution were added with 600 μL of distilled water and 100 μL of 0.2 mM FeCl 2 • 4H 2 O. The mixture was allowed to rest at room temperature for 30 s. The reaction mixture, which contained 100 μL of distilled water instead of sample, was served as the control. The reaction mixture was then added with 200 μL of 1 mM ferrozine and its changes in color were monitored at 562 nm with a Genesys 10S UV -Vis spectrophotometer (Thermo Scientific; Carlsbad, CA, USA) after 10 mins of resting time at room temperature. The chelating activities were calculated using the following equation:

Chelation activity (%)= (Ao-As)/Ao x 100
Where Ao is the absorbance of the control and As is the absorbance of the sample after 10 mins incubation.

Regression analysis of total phenolic content (TPC)
As seen in Figure 1, the mean total phenolic content for all concentration of lysine addition follows a logarithmic regression. According to our experiment, mean total phenolic content of coconut sap during heating tended to decrease and had a strong relationship with heating temperature (T) from room temperature (28 o C) until 100 o C with coefficient of determination (R 2 ) value of 0.9764 and the mathematical expression between these parameters was TPC = -0.104×ln(T) + 0.7986. However, when the coconut sap was continuously heated until the temperature of 118 o C, the value of mean total phenolic content rose sharply. This increase might be caused by the breakdown of cellular constituents which release bound phenolic compounds from the ester bonds due to the thermal process (Ng et al., 2020). The relationships between TPC and heating temperature for different concentration of lysine addition also followed logarithmic regression and tended to decrease, indicated by the negative coefficient of ln(T), for a certain range of heating time, as seen in Table 1. The experiment results revealed that the addition of 0.75 mM lysine contributed to the highest increase of final TPC to the initial TPC. The addition of 0.75 mM lysine escalated the TPC up to 167%. On the other hand, based on the multiple linear regression results the combination of lysine concentration addition and the heating temperature had less significant effect on the changes of TPC during heating. This can be seen from the small adjusted R 2 , i.e. 0.177.

Regression analysis of browning intensity
The relationship between the average of browning intensity (BI) of sap during heating with various concentration of lysine addition and the heating temperature was relatively significant (Figure 2). The changes in browning intensity during heating followed an exponential regression function. According to our experiment, the changes of browning intensity can be expressed as BI = 0.013×e 0.0372×T with R² = 0.7676. Moreover, the changes of BI during heating in each concentration of lysine addition had the same pattern, i.e. exponential regression, as seen in Figure 3. In the heating process of coconut sap from room temperature to 80 o C the browning intensity slightly increased, however, it escalated significantly when the heating temperature mM lysine presented the highest final browning intensity, i.e. around 42 times compared to the initial browning intensity, which was indicated by the absorbance of 420 nm. The combination of lysine addition and heating temperature resulted in a fair correlation to the TPC with adjusted R 2 of 0.56. Figure 4 shows the relationship between mean DPPH scavenging activity and heating temperature of coconut sap with various lysine concentration added to the sap. The changes of DPPH scavenging activity followed an exponential regression with the mathematical expression of DPPH = 0.5884×e 0.0296(T) and R² of 0.5619. As the same as browning intensity, DPPH scavenging activity of coconut sap slightly increased during heating from room temperature until the temperature of 100 o C and subsequently escalated greatly at heating temperature of 118 o C. For each concentration of lysine addition, the changes of DPPH scavenging activity also followed the exponential regression function ( Table 2). The addition of lysine to coconut sap gave a significant difference of final and initial DPPH (Δ DPPH ). The higher the lysine concentration added, the smaller the DPPH difference. As seen in Table 3, the treatment of K1 (no lysine addition) provided the highest DPPH difference, i.e. 35 times of initial DPPH, which was calculated using the following formula:  Furthermore, the results of multiple linear regression show that the combination of lysine concentration added to sap and heating temperature revealed less significant correlation to DPPH scavenging activity. The multiple R (coefficient of correlation) was 0.7335 while the adjusted R 2 was 0.494.

Regression analysis of chelating activity
The changes of mean chelating activity (CA) of coconut sap during heating process followed a quadratic regression function with R 2 value of 0.94 and in the range of 28-100 o C heating temperature. The regression formula of this correlation was CA = -0.0019×T 2 + 0.2069×T + 2.9159, as seen in Figure 5. The coefficient value of T 2 is negative which indicates that the chelating activity increased at the beginning step of heating until reached a maximum value and then decreased until the end of the heating process. The effect of concentration of lysine addition to the chelating activity of coconut sap during heating also produced changes of chelating activity which followed a quadratic regression curve (Table 4).

Conclusion
The changes in the total phenolic content of coconut sap against temperature during heating process followed a logarithmic regression function. The correlation between total phenolic content and heating temperature was quite strong until the temperature reached 100 o C. Moreover, the changes in both browning intensity and DPPH scavenging activity of coconut sap against heating temperature followed an exponential regression. A quadratic regression function can represent the relationship between the chelating activity of coconut sap and heating temperature since the correlation of FULL PAPER Figure 4. The relationship between heating temperature and DPPH scavenging activity with various concentration of lysine addition.

Concentration of lysine addition (mM)
Regression equation   Table 2. Regression equation of the relationship between DPPH and heating temperature with various concentration of lysine addition Figure 5. The relationship between heating temperature and chelating activity with various concentration of lysine addition.