UTILIZATION OF RESPONSE SURFACE METHODOLOGY IN THE OPTIMIZATION OF ROSELLE ICE CREAM MAKING

This research was carried out to develop a functional ice cream product with natural colorant derived from an optimum set of roselle calyces extract and citric acid concentrations. Although citric acid can improve red color stability of rosella, its addition is l imited due to the acidic and bitter aftertaste it imparts. Response surface methodology (RSM) was employed to analyze the effect of roselle calyces extract and citric acid on physicochemical characteris tics and sensory acceptance of an ice cream. A central composite design consis ting of two independent variables (roselle calyces extract and citric acid cocentrations) at five levels (-1.41421, -1, 0, +1, and +1.41421) with 13 runs (formulations) was prepared to establish the optimum set of variables. Higher concentration of roselle calyces extract significantly increased the total anthocyanin content and color acceptance, while decreased the oHue and pH of the ice cream. Higher concentration of citric acid significantly increased the overrun and color acceptance, but decreased the v iscosity , oHue, pH, texture, taste acceptance, and overall acceptance of ice cream. The optimum scores of consumer sensory acceptance were met at 11.5% roselle calyces extract and 1.5% citric acid concentrations.


INTRODUCTION1
Color is an extremely impor tant quality parameter and is directly related to the food acceptability.Since ice cream should have a delicate, attractive color that readily suggests to the consumer what the flavor is, most flavors of ice cream require the addition of at least a small amount of color (Marshall and Arbuckle, 2000;Durge et al. 2013).Carmoisine, ponceau 4R, and car mine are food colors which are most often used to impart red color in ice cream (Deshpande, 2002).However, due to their toxicity,they are banned in some countries (Saddleback, 2009;Amin et al. 2010).Consequently, there is an increase concern in research of colorants as their substitution in ice cream.
Roselle (Hibiscus sabdariffa L.) calyces contain anthocyanins, Dp-3-sambubioside and Cy-3-sambubioside (70.9:29.1),which are responsible for the brilliant red color of roselle calyces.In addition, anthocyanins possess antioxidant properties which may account for some of beneficial effects against diseases (Ilori and Odukoya, 2005).Due to their unique color and flavor, long history of safe usage, and no repor ted toxicity; the calyces are commonly used to make jellies, jams, sauces, and beverages (Tsai et al. 2002;Mohd-Esa et al. 2010).Roselle calyces possess a potential to be utilized as natural colorant and antioxidant source in ice cream (Ilori and Odukoya, 2005;Cisse et al. 2011).However, the anthocyanins are most strongly colored at low pH (around 3) by exhibiting their wellknown purple-red color, while at alkaline-neutral pH values, they are inherently unstable and are converted to their corresponding carbinol bases, which are colorless (O'Connell and Fox, 2001).
The concentration of roselle calyces extract and citric acid which can lower the pH will affect the degree of coloration of the products and the concentration is limited due to the acidic and bitter after taste they impar ted (Mounigan and Badrie, 2007;D'Heureux-Calix and Badrie, 2004;Durge et al. 2013).Hence, this research is carried out to observe the effect of different concentrations of roselle calyces extract and citric acid on the physicochemical characteristics and consumer sensory acceptance of ice cream and deter mine the optimum set of roselle calyces ex tract and citric acid levels added to produce the most sensory acceptable ice cream.

Materials
The main materials used for the making of ice cream are sun-dried roselle (Hibiscus sabdariffa L.) calyces ( m.c. of 15.83% ) obtained from Bogor.

Determination of roselle calyces extract concentration
The deter mination of roselle calyces ex tract concentration used for ice cream making was based on the sensory evaluation using Consumer Rejection Threshold (CRT) method (Prescott et al. 2005) to determine the level limit of roselle calyces in ice cream which cannot be accepted anymore by the consumers (CRT value).Thirty panelists were involved.Six concentration of roselle calyces ex tract were used in the making of ice cream, which are 0.9, 1.8, 3.6, 7.2, 14.4, and 28.8% .CRT was measured using ser ies of six paired preference tests, one for each roselle calyces extract concentration level.

Response surface experimental design
The experimental design used in this research is Response Surface Methodology for optimization which was conducted according to Central Composite Design (CCD) with a quadratic model (Lee et al. 2006;Mendes et al. 2001) to study the combined effect of two independent variables, which were roselle calyces ex tract and citr ic acid levels.Each independent variable had five levels, which were 1.41, 1, 0, +1, and +1.41.The concentration level below the CRT value was deter mined as the highest level of independent variable of roselle calyces extract concentration for optimization (+1.41421), while the lowest level (-1.41421) was set to 5% .A total of 13 combinations (including five replicates at the center point with each value coded as 0) were carried out in random order according to a CCD configuration for the two chosen variables.RSM was done using Minitab 14 software.

The production of ice cream
The extraction of roselle calyces was perfor med according to Tsai et al. (2002) with modification.The dried calyces were extracted with water using vacuum in the ratio of 1:10 (w/v) at 60ºC for 2 hours, then were rapidly filtered through filter cloth to obtain the roselle calyces extract.The making of ice cream was perfor med according to Handayani et al. (2009) with modification, the procedure of which can be seen in Figure 1.The ice cream for mulation is according to the low-fat ice cream for mulation by Marshall and Arbuckle (2000) with modification.Formulation of ice cream per 1 kg mix used in this research is shown in Table 1.

Physico-chemical characteristics and sensory analyses of ice cream
The physical analyses per formed were deter mination of viscosity using Brookfield viscometer (Hwang et al. 2009), overrun (Marshall et al. 2003), meltdown test (Hwang et al. 2009), and color measurement using chromameter (Madeira et al. 2003;Duangmal et al. 2008).The chemical analyses perfor med were determination of pH using pH meter (AOAC,  et al. 2009).Sensory analysis was perfor med using scor ing test on texture and melting characteristic in mouth; and hedonic test on color, aroma, taste, texture, melting characteristic in mouth, and overall.

Best formulation ice cream
The best for mulation of ice cream was deter mined from the result of hedonic test, which was the one with optimum set of roselle calyces extract and citric acid levels.Its physical and chemical characteristics, as well as its consumer sensory acceptance were analyzed.The analyses per for med were the same with the ones per for med on ice cream in each design, in addition to the deter mination of total phenolic content using Folin-Ciocalteu method (Slinkard and Singleton, 1977;Hwang et al. 2009), antioxidant activity analysis using DPPH method (Hwang et al. 2009), chemical composition analysis using proximate analyses (AOAC, 2005).

Characteristics of analyses of roselle calyces extract
Some analyses were per formed on roselle calyces ex tract to observe its general characteristics, in which the result is shown in Table 2.According to Cisse et al. (2011), the pH, total soluble solid, anthocyanin content, and phenolic content of roselle calyces extract are 2.30±0.05,45±2 g/kg, 7.1±0.5 g/kg TSS, and 29.1±1.0g/kg TSS, respectively.There is no significant difference between the result obtained and result from Cisseet et al. (2011) in terms of pH and TSS.However, the anthocyanin content and phenolic content are significantly lower, which may be caused by the different condition of plantation, drying, and storage of roselle.The pH of the roselle calyces ex tract is quite low due to the presence of various acid compounds, dominated by citric acid and hibiscus acid, which impart acidic taste (Mounigan and Badrie, 2007;D'Heureux-Calix and Badrie, 2004).The anthocyanins pigments, Dp-3-sambubioside and Cy-3-sambubioside, concentrated in the roselle calyces are responsible for the red color of roselle calyces ex tract (Ilori and Odukoya, 2005;Cisse et al. 2011).Hence, it can be utilized as natural colorant.The IC50 value shows that it possesses antioxidant properties and hence it can be considered as antioxidant source.

Concentration of roselle calyces extract
By counting the number of panelists preferring control sample in each pair of samples, the proportion can be obtained which then is plotted.By using 5% significance criterion (0.7) as a function of roselle calyces extract concentrations based on binomial distr ibution tables for paired preference tests (N = 30), the CRT value can be obtained (Prescott et al. 2005), as shown in Figure 2.

Figure 2. Proportion of panelists preferring control sample
The graph in Figure 2 shows that the CRT value of roselle calyces extract concentration in ice cream is 18.28% .Hence, for the making of ice cream, the roselle calyces ex tract concentration as the highest level of independent variable of roselle calyces extract concentration for optimization (+1.41421) is lower than 18.28% , which is 18% .The panelists or the consumer s were not able to accept ice cream with the addition of roselle calyces extract more than that limit because roselle calyces has acidic and bitter after taste due to the presence of various acid compounds, which are dominated by citr ic acid and hibiscus acid (Mounigan and Badrie, 2007;D'Heureux -Calix and Badrie, 2004).

Response surface experimental design
The roselle calyces ex tract concentration obtained from CRT method, which is 18% , was set as the highest level of independent variable of roselle calyces extract concentration for optimization (+1.41421) in the response sur face design, while the lowest level (-1.41421) is set to 5% .The variables and their levels are shown in Table 3, while the experimental design is shown in Table 4. Optimized physical characteristics Some physical characteristics optimized includes: viscosity, overrun, meltdown proper ties and color.Figure 3 (a) and (b) show the contour and sur face plot of viscosity, respectively.Citric acid per centage significantly (p≤0.05)affects the viscosity.On the other hand, roselle percentage does not significantly (p>0.05)influence the viscosity.
Figure 4 (a) and (b) show the contour and sur face plot of overrun, respectively.Citric acid percentage significantly (p≤0.05)increases the overrun, which is coherent w ith the result of viscosity determination in which there is decreasing of viscosity along with the increasing of citric acid concentration used.According to Marshall and Arbuckle (2000), as the viscosity of the mix decreases, the rate of whipping increases.It is due to the decrease of mix surface tension which causes the air to be easier to go through the surface of the mix to be incorporated into the mix during aeration process, and therefore the overrun is higher.On the other hand, roselle percentage does not significantly (p>0.05)influence the overrun.
Figure 5 (a) and (b) show the con tour and sur face plot of melting rate, respectively.Roselle percentage significantly (p≤0.05)affects the melting rate.On the other hand, citric acid percentage does not significantly (p>0.05)influence the melting rate.
The ºHue of ice cream in various designs ranged from 13.02 to 51.40º.The ºHue in the range of 342-18º represents the red purple color, 18-54º represents red color, and 54-90º represents yellow red color (Hutchings, 1999).Hence, the color of ice cream samples is ranging from red purple to red, in which the lower ºHue represents darker red color which shows higher intensity of red color.Figure 6 (a) and (b) show the contour and surface plot of ºHue, respectively.
Roselle percentage significantly (p≤0.05)decreases the ºHue, which is coherent with the result of total anthocyanin content and pH deter mination in which there is increasing of total anthocyanin content and decreasing of pH of ice cream along with the increasing of roselle calyces ex tract concentration used.Anthocyanins pigments which are concentrated in roselle calyces, are responsible for the brilliant red color of roselle calyces (Ilori and Odukoya, 2005).Hence, higher concentration of roselle calyces ex tract added to the ice cream gives higher pigment concentration, and therefore the intensity of red color in the ice cream increases as the increasing of roselle calyces extract concentration, shown by the lower ºHue value.Higher concentration of roselle calyces extract added to the ice cream impar ts more acid compounds, and therefore the acidity of the ice cream increases (decreasing of pH) as the increasing of roselle calyces ex tract concentration.Acidic pH ( low pH) favours the appearance of the colored forms, the flavyliumcation AH + which is red (Selim et al. 2008;D'Heureux-Calix and Badrie, 2004).Hence, higher concentration of roselle calyces extract added to the ice cream increased the red color exhibition of roselle anthocyanins, and therefore the intensity of red color in the ice cream increases as the increasing of roselle calyces extract concentration, shown by the lower ºHue value.
Citric acid percentage significantly (p≤0.05)decreases the ºHue, which is coherent with the result of pH determination in which there is decreasing of pH of the ice cream along with the increasing of citric acid concentration used.Citric acid has the ability to lower the pH, and hence the pH of ice cream decreases as the increasing of citric acid concentra tion added (Smith and Hong-Shun, 2003;Durge et al. 2013).Acidic pH (low pH) favors the appearance of the colored for ms, the flavyliumcation AH + which is red (Selim et al. 2008;D'Heureux-Calix and Badrie, 2004).Hence, higher concentration of citric acid added to the ice cream increased the red color exhibition of roselle anthocyanins, and therefore the intensity of red color in the ice cream increases as the increasing of citric acid concentration, shown by the lower ºHue value.Figure 8 (a) and (b) show the contour and sur face plot of total anthocyanin content, respectively.Roselle percentage significantly (p≤0.05)affects the total anthocyanin content.According to Duangmal et al. (2008), per 100 grams dry weight of roselle calyces ex tract, there is about 1.5 g total anthocyanins, which consists of Dp-3-sambubioside and Cy-3-sambubioside (70.9:29.1),and also small amount of Dp-3-glucoside and Cy-3glucoside.Hence, higher concentration of roselle calyces extract added to the ice cream impar ts more anthocyanins, shown by the higher total anthocyanin content of ice cream.Citric acid percentage does not significantly (p>0.05)influences the total anthocyanin content.

Optimized sensory properties
Texture and melting characteristics in mouth are analyzed using panel to obtain the optimal ice cream.Figure 9 (a) and (b) show the contour and surface plot of texture (1 (extremely coarse) -5 (extremely smooth)), respectively.Citric acid percentage significantly (p≤0.05)decreases the tex ture of ice cream.On the other hand, roselle percentage does not significantly (p>0.05)influence the texture.

Optimized sensory acceptance
The acceptance of panelists was optimized in terms of color, aroma, taste, texture, and overall parameters.Figure 11 (a) and  (b) show the contour and sur face plot of color acceptance, respectively.Both roselle and citric acid percentage significantly (p≤0.05)increases the color acceptance, coherent with the result of color measurement in which there is decreasing of ºHue (increasing of red color intensity) of the ice cream along with the increasing of concentration of roselle calyces ex tract and citr ic acid used.Hence, the consumers prefer the ice cream to be in redder color.It is supported by the research done by Duangmal et al. (2008) which revealed that the observers preferred a roselle-colored drink to be in a shade of vivid deep red to pale dull red.Food colors are added to improve the acceptance of food products (Mudambi et al. 2006).In this research, the addition of roselle calyces extract is proven to improve the color acceptance of ice cream.with the result of pH deter mination in which there is decreasing of ice cream pH along with the increasing of citric acid concentration.Hence, the consumers prefer the ice cream to be in less acid taste.Roselle percentage does not significantly (p>0.05)influence the ice cream taste acceptance.Roselle percentage significantly (p≤0.05)affects the melting characteristic in mouth acceptance in the trend that is coherent with the result of scoring test on melting characteristic in mouth in which it increases (slower melting) up to cer tain point, then decreases (quicker melting), along with the increasing of roselle calyces extract level.Hence, the consumers prefer the ice cream to be melted slower in mouth.Citric acid percentage does not significantly (p>0.05)influence the melting characteristic in mouth acceptance.Figure 16 (a) and (b) show the contour and surface plot of overall acceptance, respectively.Citric acid percentage significantly (p≤0.05)affects the overall acceptance.On the other hand, roselle percentage does not significantly (p>0.05)influence the ice cream overall acceptance.

Best formulation ice cream
The best for mulation ice cream is deter mined by finding the best combination of independent variables (percentage of roselle and citric acid) to maximize the response functions of ice cream consumer sensory acceptance (color, taste, melting characteristic in mouth, and overall acceptance).These criteria are selected since the acceptability infor mation is extremely useful in deter mining the optimal design of food products since consumer is the ultimate judge, therefore these characteristics need to be assessed in relation to the acceptability of the product to the consumer (Lawless and Heymann, 2010).The response of aroma acceptance and texture acceptance are not included in the deter mination of best combination because different concentrations of roselle calyces extract and citric acid have no significant effect on these two parameters.

Figure 17. Response optimization
From the optimization plot in Figure 17, 11.5% roselle and 1.5% citric acid are obtained as the optimal solution for the variable combination in which the composite desirability is 0.86353.So, the ice cream which is made with combinations of 11.5% roselle calyces extract and 1.5% citric acid will have the highest consumer sensory acceptance among ice cream with other formulation.Table 5 shows the result of analyses perfor med on the best for mulation ice cream.The viscosity and overrun of the best for mulation ice cream is in the range for typical ice cream.The melting rate is a little bit too small and undesirable, but this melting characteristic is acceptable to the consumers.The pH is lower than normal ice cream due to the presence of acid compounds impar ted by the addition of roselle calyces ex tract and citric acid.In this low pH, the roselle anthocyanin is more stable and expressed red color in the ice cream which is acceptable to the consumers.Although the low pH impar ts acid taste to the ice cream, this taste is in the level of sensory acceptable to the consumers.The best for mulation ice cream is also acceptable to the con sumers in ter ms of aroma, texture, and overall.The best for mulation ice cream contains phenolic compounds and possesses antioxidant proper ties, shown by the scavenging effect.The fat content is a bit higher than the for mulation which may be due to the fat content imparted by the roselle calyces.However, it is still in the range of fat content for low-fat ice cream, hence the ice cream is still categorized as low-fat ice cream.The moisture content is also a bit higher than the for mulation.The ash content is impar ted by the mineral content in skim milk powder and roselle calyces.The protein content is a bit lower than the average values of protein content in low-fat ice cream.The carbohydrate content is higher than the average values of carbohydrate in low-fat ice cream which may be due to the high carbohydrate content in roselle calyces.

CONCLUSIONS
Higher concentration of roselle calyces ex tract significantly increased the total anthocyanin content and color acceptance of ice cream.In contrast, it significantly decreased the ºHue and pH of ice cream.Different concentrations of roselle calyces extract significantly affected the melting rate, melting characteristic in mouth, and melting characteristic in mouth acceptance of ice cream, but the trends were unclear.
Higher concentration of citric acid significantly increased the overrun and color acceptance of ice cream.However, it significantly decreased the viscosity, ºHue, pH, tex ture, taste acceptance, and overall acceptance of ice cream.The optimum set of roselle calyces extract and citric acid levels for making of ice cream were 11.5% roselle and 1.5% citric acid.
The color stability of roselle anthocyanins in ice cream during storage is needed to be observed, hence fur ther research can be done by conducting the stability test of the pigments on ice cream.Moreover, the characteristics of the ice cream can be compared with the commercial ones.The antioxidant activity of roselle calyces in ice cream and factors affecting it can also be studied further.

Table 2 .
Analyses result of roselle calyces extract

Table 3 .
Variables for optimization

Table 4 .
Central composite experimental design for optimization

Table 5 .
Result of analyses of best formulation ice cream