Effects of Process Parameters on the Color Quality of Anthocyanin-Based Colorants from Conventional and Microwave-Assisted Aqueous Extraction of Sweet Potato ( Ipomoea batatas L.) Leaf Varieties

: The study aimed to evaluate the effects of process parameters (time and raw material weight (RMW)) of conventional (boiling for 10–45 min) and microwave-assisted (2–8 min) aqueous extraction on the color quality (i.e., lightness ( L *), chroma ( C *), and hue ( H °) of anthocyanin –based colorants of red and Inubi sweet potato ( Ipomoea batatas L.) leaves. Using response surface methodology, it was found that RMW and boiling time (BT) and microwave time (MT) generally had a significant ( p < 0.05) effect on the color quality of the extract from both extraction methods. The effects were found to vary depending on the extraction method and variety of the leaves used. Both extraction methods produced a brown to brick-red extract from the Inubi variety that turned red-violet to pink when acidified. The red sweet potato leaves produced a deep violet colored extract that also turned red-violet when acidified. It is recommended that the anthocyanin content of the extracts be measured to validate the impact of the methods on the active agent. Nevertheless, the outcomes in this study may serve as baseline data for further studies on the potential of sweet potato leaf colorants as a colorant with functional properties.


Introduction
Color is a significant trait of sensory quality evaluation and a key attribute that affects the perception of an individual to foods [1][2][3][4][5]. Producers apply colorants to make food products more appealing and desirable to consumers [6,7]. Depending on the origin, colorants can be synthetic (dyes) or naturally derived (pigments) [8]. Natural colorants are more preferred because of the detrimental issues on safety and health by synthetic colorants [9,10]. Particularly, red colorants comprise the majority of commercially available colorants that are being added to foods [11]. Examples of natural red colorants include carotenoids, betacyanins, and anthocyanins [12,13].
Anthocyanins are the most important water-soluble natural pigments among the group of flavonoids [14][15][16]. Anthocyanins are known for their excellent antioxidant properties that can protect the human body from oxidative stress, thereby decreasing the risk of chronic disease such as aging, diabetes, cardiovascular diseases, and cancer [17][18][19].
Aside from the health-promoting benefits of these pigments, anthocyanins have a role as approved natural food colorants [20,21]. A wide range of color hues such as red, violet, purple, and blue in plants such as fruits, flowers, leaves, and tubers can be produced by anthocyanins at varying pH [22][23][24]. Natural food colorants containing anthocyanins are applied to foods and beverages such as yogurt, jellies, juices, and wine [25]. However, several problems arise when anthocyanins are exposed to various processing and storage conditions [26][27][28][29]. Factors such as pH, temperature, light, oxygen, and metal ions affect the stability of anthocyanins [14,[30][31][32]. In a study by Bakowska-Barczak [33], it was revealed that anthocyanins with acylated substituents increased stability to heat and light and low sensibility to pH changes. This indicates the suitability of anthocyanins as natural food colorants that can be used in the food industry [25,34,35]. Moreover, vegetable sources are more stable for food application because acylated anthocyanins are predominantly found in these commodities [23,28,36].
One of the potential sources of acylated anthocyanins is sweet potato (Ipomoea batatas L.), the seventh most important food crop and the second most grown root and tuber crop next to cassava [37][38][39][40]. Amongst the parts of sweet potato, the leaves contain significantly higher amounts of anthocyanins [37,[41][42][43][44][45]. The anthocyanins in sweet potato leaf (SPL) have the same potential as natural colorants in foods [46,47] with superior shelf life and equal stability in light and heat, such as those in red cabbage [48,49].
In the Philippines, sweet potato is widely produced, especially in the areas of the Bicol region, Central Luzon, Central Visayas, and Eastern Visayas [50,51]. Numerous sweet potato varieties (i.e., Inubi, red, SP native) are unique in terms of leaf color [50]. The colored pigments found in SPL can be used as natural colorants in foods [34,46,47]. There are current studies on the extraction of anthocyanins and the determination of phenolic content in SPL; however, there are no reports yet on the use of SPL from local varieties [52][53][54][55]. Furthermore, the potential of local SPL utilization as a source of natural colorant has been poorly noticed. The objective of the study is to evaluate the effects of process parameters (time and raw material weight (RMW)) of conventional and microwave-assisted aqueous extraction on the color quality (i.e., lightness (L*), chroma (C*), and hue (H°) of anthocyanin-based colorants of red and Inubi sweet potato (Ipomoea batatas L.) leaves.

Sweet Potato Leaves (SPL)
Two varieties of SPL were used in the study-Inubi and a red variety. Red and violet pigments can be observed throughout the leaf blade and veins of red SPL, whereas those of Inubi SPL can only be seen on the veins (Figure 1). Inubi SPL were obtained from Sapang Multipurpose Cooperative, Moncada, Tarlac, Philippines. Red SPL was procured from Fresh-Q Industries, Brgy. Sulucan, Angat, Bulacan, Philippines. The leaves were procured at the age of 3 months and transported to the laboratory, where they were stored inside the chiller (~4 °C) prior to analysis.

Conventional Extraction Method
Conventional extraction was based on the modified method of Liao et al. [56]. Preweighed SPL were washed repeatedly with tap water until no visible dirt could be seen. Extraction was performed by boiling SPL with 500 mL distilled water (Table 1) with occasional stirring every 5 min. After boiling, the mixture was allowed to cool at room temperature (~28 °C) for 10 min followed by initial filtration using a sieve (W.S. Tyler, Mentor, OH, USA) with mesh no. 120 to separate the leaves from the extract. The SPL extract (SPLE) was then filtered further using a Buchner filtration setup with Whatmann No. 1 filter paper. Stabilization of the color was employed by adjusting the pH of the extract to 3 using 1% (w/v) food-grade citric acid (Neco Philippines, Inc., Sta Cruz, Manila, Philippines) solution since it was reported that acid prevents the degradation of non-acylated anthocyanin pigments in the sample [57]. The obtained SPLE was stored in chilling temperature until analysis.

Microwave-Assisted Extraction (MAE)
The microwave-assisted extraction (MAE) was based on Song et al., and Bhuyan et al. [55,58]. An ordinary countertop microwave oven (Whirlpool x2-20ES, Whirlpool, Benton Harbor, MI, USA) with a fixed microwave power output of 800 W was chosen for the extraction of colorants from SPL. Pre-weighed and pre-washed SPL was placed in a 1000 mL beaker and mixed with 500 mL distilled water. The beaker was placed in the middle of the oven over a rotating dish and was exposed to microwave radiation at different times (Table 1). After heating, the beaker was left for 10 min at room temperature to cool. The extract was then filtered and acidified similar to the treatment done in the conventional extraction method.

Identifying the Limits of Process Parameters
The upper and lower limits of the process parameters are shown in Table 2. The minimum weight of 50 g SPL was based on a leaf-to-water ratio of 1:10 parts used by [59], whereas the maximum weight of 125 g (1:4) was based on a preliminary experiment (data not shown). The minimum and maximum time (min) of boiling (BT) was also based on another preliminary run where it was observed that shorter boiling time extracted less color from the leaves and longer boiling time resulted in the extract to dry out. The upper and lower limits of microwave time (MT) were defined by also taking into account the results obtained in preliminary tests (data not shown) as well as the significant parameters in typical MAE process for leaves [60].

Modeling of Responses
Different RMW-BT and RMW-MT combinations were generated using Design-Expert ® Application V.7.0.0 (DX7, Stat-Ease Inc., Minneapolis, MN, USA). A central composite rotatable response surface methodology (RSM) design ( Table 2) was used. Central composite RSM designs are known to be widely used for fitting quadratic or second-order response surfaces, making them applicable for process optimizations [61]. Runs were separated into two blocks, with each block corresponding to experimental days. Each experimental run was subjected to color measurements (L*, C*, H°). The responses were then subjected to multiple linear regression analysis to approximate empirical models, the coefficient of variation (%C.V.), the R-squared, and the adjusted R-squared. Empirical models developed were based on the general equation for second-order responses as shown in Equation (1), where Y, β, and X represent the predicted response, regression coefficients, and independent variable, respectively [61,62].

  
The transformation of the models was based on the recommendation of the software based on a Box-Cox test. The most accurate model was then obtained with several statistical tests performed by DX7, including the F-test, lack-of-fit test, externally and internally studentized residuals, DFFITS, and Cook's distance.

Color Measurement
The color of the sample runs (LAB) was measured using a bench spectrophotometer (ColorFlexEZ, Hunter Associates Laboratory Inc., Reston, VA, USA). Approximately 35 mL of the sample SPLE was placed in the sample cup holder and the L*, a*, and b* coordinates were recorded. The chroma (C*) and hue (H°) values were then calculated using the obtained a* and b* coordinates using Equations (2) and (3), respectively.
The color coordinates a* and b* do not directly describe the quality of the color and should not be interpreted separately, hence the reason behind using C* and H° values instead [63]. An angle of 180° was added to the hue value in cases where a negative a* value was recorded whereas an angle of 360° was added when a positive a* and a negative b* value were obtained [64]. A total of three replicates were done per experimental run and the data were expressed as mean ± standard deviation (SD). Color charts were created based on the obtained LAB coordinates using Adobe Color CC (Adobe Systems, San Jose, CA, USA).

Verification of Models
Significant models were verified by being subjected to validation experiments. Two random points were chosen from the design space and were run in triplicate, after which the samples were again subjected to color measurements. Models were considered valid and accurate when the actual response obtained from analysis fell on the confidence and/or predicted interval of the predicted response.

Effects of Process Parameters on the Color Quality of Anthocyanin-Based Colorants from Conventional (Boiling) Aqueous Extraction of Sweet Potato (Ipomoea batatas L.) Leaf Varieties
Statistical analyses on precision and accuracy indicated that all models can be used to predict the effect of RMW and BT on the color quality of sweet potato leaf colorants (SPLC) ( Table 3). A relatively high % coefficient of variation (CV) was obtained from the model showing the effect of process parameters on the hue value of red SPLC, indicating that the model may not be as accurate as compared to the other models. However, the models can still be used since its adequate precision of greater than 4 means that it can be used in navigating the design space.   I1  I2  I3  I4  I5  I6  I7  I8  I9  I10  I11  I12  I13  I14   I1  I2  I3  I4  I5  I6  I7  I8  I9  I10  I11  I12  I13  I14   R1  R2  R3  R4  R5  R6  R7  R8  R9  R10  R11  R12  R13  R14   R1  R2  R3  R4  R5  R6  R7  R8  R9  R10  R11  R12 R13 R14

Effects of Process Parameters on the Color Quality of Anthocyanin-Based Colorants from Microwave-Assisted Aqueous Extraction of Sweet Potato (Ipomoea batatas L.) Leaf Varieties
The effects of RMW and MT on the color quality of SPLC are summarized in Table 4. Statistical analyses on precision and accuracy indicated that all models for the color responses can be used to predict the color quality of SPLC except for the H° of red SPLC.
The models were used since their adequate precision of greater than 4 means that they can be used in navigating the design space. Moreover, a relatively high %CV was obtained from the model showing the effect of process parameters on the hue value of Inubi SPLC, indicating that the model may not be as accurate as compared to the other models. Table 4. Effects of microwave-assisted extraction process parameters on the color quality of SPLC.

Validation of Models
Response models were validated through verification studies using random points from the design space. The results (Table 5) showed that the models can be generally used to predict the effects of RMW, BT, and MT on the color quality of Inubi and red SPLC. All of the actual responses fell within the range of the prediction interval, indicating that the models can accurately predict the effect of RMW and BT on color quality (i.e., lightness (L*), chroma (C*), and hue (H°)) of Inubi and red SPLC. 15.09 ± 0.57 8.20 ± 1.36 b 5.60 ± 1.47 b 18.60 ± 1.64 1 Conditions: 75 g of SPL per 500 mL distilled water boiled for 20 min. 2 Conditions: 125 g of SPL per 500 mL distilled water boiled for 10 min. 3 Conditions: 50 g of SPL per 500 mL distilled water boiled for 30 min. 4 Conditions: 34.5 g of SPL per 500 mL distilled water extracted through MAE for 5 min. 5 Conditions: 50 g of SPL per 500 mL distilled water extracted through MAE for 2 min. 6 Conditions: 125 g of SPL per 500 mL distilled water extracted through MAE for 8 min. a Significantly different from the predicted value based on prediction interval. b Significantly different from the predicted value based on confidence interval. c Expressed as mean ± standard deviation.

Effects of Process Parameters on the Color Quality of Anthocyanin-Based Colorants from Conventional (Boiling) Aqueous Extraction of Sweet Potato (Ipomoea batatas L.) Leaf Varieties
The RMW had significant (p < 0.05) negative linear effect on the lightness L* of both red and Inubi SPLC, implying that the sample becomes darker at increasing RMW ( Figure  2A,D). This may be due to increasing concentrations of colorant extracted at increasing amounts of leaves used. On the other hand, BT had a quadratic effect on the L* of both varieties of SPLC, with those of Inubi SPLC being significant (p < 0.05). This indicates that the color of SPLC continuously darkens with longer BT, which then starts to increase or become lighter upon reaching the minimum L* value. The decreasing L* may be a result of the increasing pigment concentration as it is extracted from the leaves, whereas the increasing L* value might be due to the formation of translucent extracts due to the color fading of extracted anthocyanin [65]. Moreover, increasing L* values are known to be the result of prolonged heating of anthocyanin, causing it to transition from the flavylium cation to the colorless and/or yellowish carbinol [66,67]. RMW and BT also had a significant (p < 0.05) effect on the chroma, C* of Inubi and red SPLC ( Table 3). The color of the Inubi SPLC ( Figure 2B,E) became more saturated, as observed by the increasing C* at a longer BT and greater RMW. An opposite effect was observed on the C* value of red SPLC ( Figure 2E), where the C* value decreased with increasing RMW and BT, which may be due to the differences in the variety and anthocyanin composition between the SPL varieties. The type of anthocyanin in the leaves may dictate the overall color stability of the colorant, as shown in the study by Loypimai et al. (2016), wherein cyanidin-3-O-glucoside anthocyanins in black rice bran colorants were found to be more thermally stable and degraded slower compared to pelargonidin and delphinidin and malvidin [68]. The opposite effect on the C* value of red SPLC could possibly mean that the type of anthocyanin found in this SPL variety degraded much faster compared to the anthocyanin present in Inubi SPLC. At a prolonged BT, both varieties of SPLC eventually became less saturated, which could be explained by the possible degradation of monomeric anthocyanin and the conversion of flavylium cations to their colorless carbinol/yellowish form [65][66][67]. Figure 2C,F show the effect of BT and RMW on the hue (H°) value of SPLC. BT had a significant (p < 0.05) negative linear effect on the H° of both Inubi and red SPLC, indicating that the color of the sample moved towards the red-violet region of the color space, which is towards 0° (Lawless & Heymann, 2010). This could be a result of the increase in concentration of anthocyanin extracted due to prolonged heating and possible self-association of the pigment (Kammerer, 2016;Boulton, 2001). It could also indicate that the heating condition on the extraction of the colorant was not too severe, leading to a yellow chalcone formation, as demonstrated by the anthocyanin extracts of Reyes and Cisneros-Zevallos (2007). However, it was observed that RMW had different effects on the color of the two SPLC. At increasing RMW, the H° value of Inubi SPLC ( Figure 2E) also increased, indicating that the color moved towards the yellow region (90°), whereas those of red SPLC had a decreasing H° (towards red). This may be due to the differences in variety of the leaves, since the leaf blades of Inubi are predominantly green compared to the red SPL, which is predominantly purple. The green color may indicate the presence of pigments such as chlorophyll, which is known to turn yellow when exposed to heat and acid (Von Elbe and Schwartz, 1996). Increasing the RMW of the Inubi might have resulted in the increase of chlorophyll concentration in the extract, which interfered with the red color of the anthocyanin. The predominant violet to red color of the red SPL might indicate a higher concentration of anthocyanin, which could have oppressed the possible color interference of its chlorophyll-a possible scenario that needs to be studied in future experiments.
Colors of all samples using the Inubi extract colors were observed to be similar, with a pink, red, brown, and maroon color (Figure 3). For the red SPLC, the colors were more similar and harder to distinguish from each other, with a red to red-violet coloration. Comparing the lightness, chroma and hue (LCH) values of the two varieties, it was observed that red SPLC had a darker, more saturated, and more red color than the Inubi SPL, which may be due to the differences in the variety of SPL used.

Effects of Process Parameters on the Color Quality of Anthocyanin-Based Colorants from Microwave-Assisted Aqueous Extraction of Sweet Potato (Ipomoea batatas L.) Leaf Varieties
Contour plots show that MT had a quadratic effect (p < 0.05) on L*, which implies that the color of SPLC continuously darkens with longer MT up to a certain time, and beyond this period L* values increase or become lighter ( Figure 4A,D). Contour plots (Figure 4B,E) also show that MT had a quadratic effect on C*, which implies that the color of SPLC becomes more saturated with longer MT and then starts to become less saturated upon reaching the maximum C* value. At prolonged MT, SPLC from both varieties eventually becomes less saturated, similar to the effect of BT in the conventional method of extraction. The increasing C° value may have been due to prolonged heating of anthocyanin, causing a shift in flavylium ions [28,69]. This causes self-association of the anthocyanin in the aqueous environment, thereby displacing the equilibrium towards the chalcone forms, which makes the samples' color more saturated [70]. A similar trend on the effect of BT and RMW on the hue value was observed in the effect of MT and RMW during MAE ( Figure 4C). There was no generated model for the H° value of the red SPLC.
The Inubi SPLC using MAE had a lighter pink to orange and less saturated color whereas the red SPLC had a darker red to maroon color ( Figure 5). Some runs yielded very light-colored samples, which can be attributed to the lesser RMW and shorter MT. Saturated and more red-colored samples were observed at a longer MT and greater RMW, similar to the conventional method.

Conclusions
The different process parameters showed that boiling time/microwave time and raw material weight affected the color quality of Inubi and red SPLC. RMW, BT, and MT generally had a significant (p < 0.05) effect on the color (L*, C*, H°) of both SPLC varieties. The extraction process parameters produced SPLC with red, red-violet, and pink colors. Verification experiments showed that the statistical models can accurately predict the effect of the process parameters on the quality of SPLC. The findings from the study revealed a more in-depth analysis on the possible effects of process parameters on the extraction of anthocyanin-based colorants from sweet potato leaves. The information obtained may be useful for possible optimization as well as exploring more the nature of colorants found in SPLC. It is recommended that the effect of process parameters of other extraction methods on the quality of SPLC and other possible sources of anthocyanin-based colorants be explored.