A novel, green cloud point extraction and separation of phenols and flavonoids from pomegranate peel: An optimization study using RCCD

Journal of Environmental Chemical Engineering 7 (2019) 103306

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A novel, green cloud point extraction and separation of phenols and flavonoids from pomegranate peel: An optimization study using RCCD Pavankumar R. Morea, Shalini S. Aryaa,b, a b

T

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Food Engineering and Technology Department, Institute of Chemical Technology, Matunga, Mumbai, Maharashtra, 400019, India Department of Biotechnology, Engineering School of Lorena, University of São Paulo, CEP 12602-810, Brazil

A R T I C LE I N FO

A B S T R A C T

Keywords: Cloud point extraction Bioactives Pomegranate peel Green extraction RCCD Partition coefficient

Cloud point extraction (CPE) is one of the novel; environment-friendly; energy; time and cost-effective extraction technique. Therefore, in the present work preconcentration and separation of bioactive compounds from pomegranate peel, a major waste of the pomegranate processing industry was carried out using CPE. CPE works on the principle of entrapping the hydrophobic bioactive compound in the micelle. Surfactant concentration (5–11%), pH (4–8), temperature (30–60 °C), and salt concentration (4–12%) optimization was carried out to separate total phenols and flavonoids with maximum recovery (%R), partition coefficient (Ks/a), fraction concentration (fc), and minimum loss (%L) using the rotatable central composite design (RCCD). The optimum levels were 8.22% surfactant (Triton X-114), 4% salt (NaCl) at temperature of 36.80 °C and pH 4 which resulted in 95% recovery, 14.59 Ks/a, 0.95 fc and 5.27% loss of total phenols with the maximum desirability about 0.846. While in the case of total flavonoids at 8.27% Triton X-114 at pH 5.07/34.30 °C and 4.06% NaCl resulted, 98% recovery, 9.71 Ks/a, 1.01 fc, and 2% loss with 0.842 desirability. The total yield obtained for total phenols was 205.2 mg of GAE/g while for total flavonoids it was 60.05 mg of QE/g of pomegranate peel powder. Therefore, it can be concluded that this method can be successfully applied in the extraction of bioactive from any food systems for clean and green extractives label.

1. Introduction Food, agriculture, chemical, and pharma industries are participating in a comprehensive development for more energy saving, cost and time efficient, and eco-friendly (green) techniques for extraction and separation of bioactive compounds from food and its waste. Bioactive compounds are indispensable nutrients present in the plant, fruits, and its waste in very fewer amounts [1]. These compounds have all-embracing metabolic roles in living systems like antioxidation, chelation of metals, anti-dyspathetic, antibacterial, and clarification, etc. [2]. Fruits, vegetables and their waste are potential sources of bioactives like polyphenolics, flavonoids, antibiotics, alkaloids, natural colorants, and plant growing components [3] later is sensitive to temperature, light, and oxygen. Pomegranate (Punica granatum L.) plant from Punicaceae family have recently captured the huge attention by food, pharma, chemical processing industries due to its multifunctional, high nutritional and bioactive properties [4] and has been used anciently as folk and ayurvedic medicine [5]. Pomegranate is native to India and middle-east Asian countries. Among other fruits, pomegranate is highest in

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bioactive phytochemicals containing total polyphenols and flavonoids, which can be subcategorized as flavones, flavanones and anthocyanins [6]. It contains polyphenols like gallic acid, ellagic acid chlorogenic acids, catechins, etc. [7] and flavonoids such as quercetin, kaempferol, luteolin glycosides [8] and many more. Polyphenols and flavonoids mostly exist in peels, which contributes 50% of the total weight of pomegranate fruit, which is often discarded as a waste [9]. Peels have been reported as a source of good antioxidant, anti-inflammatory, antihepatotoxicity, anti-atherosclerotic capacity, and anti-microbial properties [10–12]. This waste could be explored as an emerging source of bioactive compounds that can be extracted and separated using cloud point extraction (CPE). This green extract then could be used efficiently in many green labeled food formulations. The conventional extraction techniques such as Soxhlet and orbital shaking which are not only time consuming but also environmentally hazardous as they emit volatile organic compounds. Other conventional extraction techniques that have been introduced are distillation, and organic solvent extraction [4,7], high-pressure water extraction [13], microwave assisted extraction (MAE), and solvent extraction assisted with ultrasonication (UAE) [8,14].

Corresponding author at: Food Engineering and Technology Department, Institute of Chemical Technology, Matunga, Mumbai, Maharashtra, 400019, India. E-mail addresses: [email protected] (P.R. More), [email protected], [email protected] (S.S. Arya).

https://doi.org/10.1016/j.jece.2019.103306 Received 23 June 2019; Received in revised form 17 July 2019; Accepted 20 July 2019 Available online 26 July 2019 2213-3437/ © 2019 Published by Elsevier Ltd.

Journal of Environmental Chemical Engineering 7 (2019) 103306

P.R. More and S.S. Arya

Fig. 1. Cloud point extraction (CPE) of polyphenols and flavonoids from pomegranate peel powder.

Novel technologies like supercritical fluid extraction [15], high voltage electrical discharge [9], liquid biphasic electric flotation system [16], and liquid-phase microextraction with some pre-treatments like salt and acid; alkali hydrolysis have been successfully reported to increase the recovery and extraction efficiency, reduce power, and solvent use. However, these techniques have limitations concerning environmental, consumers, and end product safety, nevertheless, these techniques also require costly and special instruments [2]. Cloud point extraction (CPE) is one of the most attractive novel; green; energy and cost-efficient extraction technique for bioactive compound preconcentration; [1,17] implies the cluster development of monomers of non-ionic surfactant that led to the formation of hydrophobic micelles followed by entrapment of hydrophobic bioactive into the micelles [1]. CPE uses a green solvent like water; which requires less operation with minimum disposal cost and works between the temperature range of 30–60 °C. The CPE of organic bioactives or inorganic molecules is carried out using nonionic surfactants as an extraction medium. Separation is done from cloud forming solution of surfactant and target sample by heating it above the cloud point temperature (CPT). In resultant, the surfactant forms micelles where the polar head faces towards the aqueous part and hydrophobic tail towards the lipophilic layer of target compound [1]. Furthermore, due to differences in densities of micelles and aqueous solutions; separation of two different phases takes place. This technique is also known as micelle-mediated extraction (MME) or liquid-concentration technique (LCT) [18]. As compared to other techniques; this has advantages over others like low cost, high speed, better efficiency, environmentally lower toxicity, and safety. Moreover, direct analysis of food constituents is complicated as it has complex structures. Hence, sample preparation is required. Loss of analytes is yet another challenge during sample preparation. Therefore, CPE is a suitable technique for isolation of analytes especially from food sources [1]. There have been demands for chemical, preservative and solventfree additives in food and nutraceuticals. Particularly they are demanding more natural, clean green labels. Therefore, extractives obtained from CPE (green technology) could be used as natural antioxidants, colorants, and preservatives in many food and nutraceutical formulations [19–21]. Considering all the above points, CPE assisted bioactive extraction has created a lot of interest among researchers. Use of CPE for the extraction of polyphenols from industrial waste is one of the major areas of study [21,22]. However, a scarce information available on the extraction of bioactives from pomegranate peel using CPE. Therefore, the present study is carried out to optimize CPE parameters like type and concentration of surfactant, temperature, pH, salt concentration by numeric optimization based on desirability.

2. Experimental 2.1. Material and chemical The fresh Pomegranate (Punica granatum L.) fruits of Bhagwa verity were procured from Sahakari Bhandar, Matunga, Mumbai, Maharashtra, India. Fruits were sorted and washed. After removing the arils from the fruits, pomegranate peels were dried at 40 °C in hot air oven until constant weight. The dried peels were then powdered using a laboratory grinder. This powdered sample was then passed through 40micron size sieve. This uniform sized pomegranate peel powder was stored in a dry and airtight plastic bottle in a freezer at −20 °C until final use. Methanol, Na2CO3, AlCl3, potassium acetate, NaCl, HCl, and NaOH were purchased from SD Fine Chemicals Mumbai, India. Triton X-100 ((C2H4O)n C14H22O, n = 9–10), Triton-114((C2H4O)n C14H22O, n = 7–8), Tergitol NP-12 (Nonylphenol Ethoxylate), gallic acid, quercetin, Folin–Ciocalteu’s reagent, DPPH, and ABTS were procured from Sigma–Aldrich Chemicals Co. (St. Louis, MO, USA). Distilled water was prepared using a Sartorius arium® advance series water purification system (Mumbai, India). All other chemicals used for assay were of analytical grade. 2.2. Cloud point extraction (CPE) of total phenols and flavonoids from pomegranate peel powder The CPE of polyphenols and flavonoids was performed as per the method of Katsoyannos et al. [21] with slight modifications (Fig. 1). In brief, the mixture of pomegranate peel powder (0.5 g), distilled water, and surfactant (Triton X-114%, v/v) was taken in a centrifuge tube and was vortexed for 1 min, followed by adjustment of a pH. Tubes were kept on the shaker for 30 min at 37 ± 2 °C followed by centrifugation at 10,000 RPM for 10 min. The supernatant was transferred to other tubes. Salt (NaCl) was added to the sample solution for smoothing of phase separation by reducing the cloud point temperature (CPT) due to salting out effect with an increase in the density of aqueous water phase and kept in a thermostatic water bath for 30 min. The sample was then centrifuged for 10 min at 8000 rpm followed by measurement of volumes of the aqueous and surfactant phases and used for the calculation of responses. The aqueous bottom phases were separated by syringe. Remaining surfactant-phase was allowed to settle at the bottom of the tube as it was highly viscous. All the experimental trials were taken in triplicates and each sample was analyzed six times and response values reported are mean of resulting values from each assay. 2.3. Experimental design for optimization of the extraction process Extraction process parameters such as surfactant concentration (%, v/v), pH, temperature (°C), and salt concentration (%, w/v) were varied 2

Journal of Environmental Chemical Engineering 7 (2019) 103306

P.R. More and S.S. Arya

2.5. Calculations of responses

as per the RCCD. The lower (−1) and upper (+1) limit in coded form were set each of four parameters according to the resultant observation obtained from CPT. As per the experimental design, 30 experimental runs were carried out which consisted of sixteen-factor points (at +1 and −1 level), eight axis points (at ± α = +2 and −2 level), and six repetitive runs at 0 level or middle point. Four responses, i.e., recovery (Y1, %R), partition coefficient (Y2, Ks/a) and loss during extraction (Y3, %L), and concentration fraction (Y4, fc) of the total phenolic and total flavonoid content were measured for each experimental run. A quadratic polynomial equation was modeled (Eq. (1)) for all four responses (Yi) as a function of x1, x2, x3, and x4.

All the responses for total phenolic content (TPC) and total flavonoid content (TFC) ware calculated after CPE. The total phenol and total flavonoid recovery by CPE from the sample were determined as per Eq. (6).

Recovery (%R) =

+ β9 x2 x 4 + β10 x3 x 4 + β11 x12+ β12 x 22 + β13 x 32 + β14 x42

Ks / a =

(1) Where, βi (i = 1–14) are the regression coefficients whereas; x1, x2, x3 and x4 are coded forms (ranging from −1 to +1) of surfactant concentration (x1, %; v/v), pH (x2), temperature (x3, °C) and salt concentration (x4, %; w/v), respectively. All factor values were converted from real to coded using Eq. (2)–(5).

(Surfactant conc. ;X1 − value of center point; 0 level) (difference between levels )

(2)

x2 =

(pH ; X2 − value of center point; 0 level) (difference between levels )

(3)

x3 =

(Temperature ; X3 − value of center point ; 0 level) (difference between levels )

x4 =

(Salt conc. ;X 4 − value of center point; 0 level) (difference between levels )

(Cim × Vim)

× 100

(6)

Cim representing the bioactives concentration in the initial sample mixture of volume Vim, and Csf representing the bioactives concentration of in the surfactant phase of volume Vsf. Partition coefficient (Ks/a) of bioactives in surfactant/aqueous was calculated according to Eq. (7).

Yi = β0 + β1 x1 + β2 x2 + β3 x3 + β4 x 4 + β5 x1 x2 + β6 x1 x3 + β7 x1 x 4 + β8 x2 x3

x1 =

(Csf × Vsf )

Csf Caf

(7)

Where Csf and Caf are bioactives concentration in the top surfactant phase and bottom aqueous phase at equilibrium, respectively. The fraction concentration (fc) of bioactives in the surfactant phase (Csf) to the initial sample mixture (Cim) was determined according to Eq. (8).

fc =

Csf (8)

Cim

2.6. Optimization by a numerical optimization method The numerical optimization technique [25] was used to detect the optimum values of variables (X1, X2, X3, and X4). It was done to maximize the value of the overall desirability (D), according to Eq. (9).

(4)

1

Overall desirability(D) = (d1r1 × d 2r2 × d3r3 × d4r4 ) r 1+ r 2+ r3+ r4 (5)

(9)

th

Where di desirability value of each (i ) response and factors individually and ri indicate the relative importance (on the scale of 1–5) of respective responses and factors. D and di values are ranging from 0.0 to 1.0; nearest values to 1 represent the most desirability. The di value was determined using Eqs. (10) and (11).

2.4. Analytical methods 2.4.1. Determination of total phenolics content Total phenolics content of extract of pomegranate peel obtained after CPE was determined by Folin-Ciocalteu assay with slight modifications [23]. Gallic acid solution was used as standard (10 mg mL−1) in 80% methanol to give suitable concentrations (0.1–1 mg mL−1) for a standard curve. For the analysis, 0.1 mL of extract or standard gallic acid solution, 0.1 mL of 80% methanol, 0.1 mL of Folin–Ciocalteu reagent and 0.7 mL of Na2CO3 were added into 2 mL Eppendorf’s. The samples were vortexed immediately and incubated in the dark for 20 min at room temperature. After incubation, all samples were centrifuged at 8000 RPM at 4 °C for 8 min. The absorbance of the supernatant was then measured at 735 nm in 1 mL quartz cuvettes using a UV–vis spectrophotometer (Jasco, V-730). The results were reported as milligram of gallic acid equivalent per gram (mg GAE/g pomegranate peel powder).

When maximization ofYidi =

Yi − Li Ui − Li

(10)

When minimization ofYidi =

Ui − Yi Ui − Li

(11)

Where Li is the lower limit of response (Yi) and Ui is the upper limits of the response (Yi). From suggested values for x1, x2, x3 and x4, the true values for surfactant concentration (%), pH, temperature (°C) and salt concentration (%) were calculated by using Eqs. (2)–(5). The optimized condition was then compared with the predicted and experimental values of the responses. 2.7. Statistical analysis The significant variance between the average values (n = 3) was analyzed using a one-way ANOVA. Both the least significant difference (LSD) and Tukey's HSD tests were applied using SPSS software (IBM SPSS Statistics software version 16.0). Design-Expert version 7.0.0 (Stat-Ease Inc., Minneapolis, MN) was used for the application of response surface methodology and numeric optimization.

2.4.2. Determination of total flavonoids content Total flavonoids content was determined by aluminum chloride spectrophotometric assay, according to the modified method of Zhishen et al. [24]. Aqueous sample extract (0.5 mL) was poured in a 5 mL glass test tube, followed by addition of 2.5 mL of 60% methanol. After about 5 min, 0.1 mL of 10% AlCl3 and 1 M potassium acetate solutions were added, and total reaction volume was made up to 5 mL with distilled water. The resultant mixture solution was vortexed in and was kept for 15 min in dark and absorbance was measured at 510 nm using a UV–vis spectrophotometer (Jasco, V-730). Total flavonoids content was expressed as quercetin equivalent per gram (mg QE/g pomegranate peel powder).

3. Results and discussion 3.1. Screening of surfactant type and extraction time A screening of aqueous solutions of three surfactants (Triton X-100, Triton-114, and Tergitol NP-12) at varied extraction time (20, 30, and 3

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P.R. More and S.S. Arya

Table 1 % Recovery of total phenolic and flavonoid compounds using CPE at 45 °C; 7% (v/v) surfactant, and pH 4. Time (min)

Triton X-100 a

20 30 40

b

Triton X-114 a

c

a

Tergitol NP-12

b

a

c

%R (TPC )

%R (TFC )

%R (TPC )

%R (TFC )

%Ra (TPCb)

%Ra (TFCc)

68.69 ± 0.69aA 87.67 ± 0.92cA 83.42 ± 0.83bB

62.26 ± 0.62aA 86.88 ± 0.84cB 80.10 ± 0.73bB

83.05 ± 0.69aC 98.09 ± 1.19bC 96.93 ± 1.75bC

85.88 ± 0.68aC 96.96 ± 1.13cC 92.96 ± 0.55bC

73.37 ± 2.86aB 79.09 ± 0.97bB 76.08 ± 0.52abA

74.12 ± 0.51aB 82.03 ± 0.57cA 78.31 ± 0.35bA

The different alphabets (small letters and capital letters) in superscript signifies that the average values belong to distinct subsets across the columns and rows at 95% confidence interval. Values are showed as average ± SD (n = 3). a % Recovery. b Total phenolic compounds. c Total flavonoid compounds.

observation from Fig. 2(B) indicates that an increase in pH from 2 to 9; adversely decreased CPT. This might be because of the solubilization of surfactant in the water at lower pH, which avoids the micelle formation. On the other hand, the pH value above 8 also adversely decreased the %R, which might be due to the dissociation of bioactives in alkaline conditions [30]. Hence, form above observations limits of CPE parameters- set for further optimization in the range of; 5–11% (v/v) Triton X-114 concentration; pH; 4– 8, 4–12% (w/v) salt (NaCl), and 30–60 °C temperature which was slightly higher than minimum observed CPTs from Fig. 2(A and B).

40 min.) at 45 °C, 7% (v/v) surfactant, and pH 4 were performed. From Table 1 it can be seen that using all three surfactants (Triton X-100, Triton X-114, and Tergitol NP-12) %R of polyphenols and flavonoids increased with the increase in time from 20 min to 30 min. This might be due to the fact that surfactants take 30 min for entrapment of hydrophobic bioactives (TPC and TFC) into hydrophobic micelles that separate into two phases. With a similar instance, He et al. [26] also performed CPE for 30 min. who also recorded with the increase in time from 30 to 40 min, a slight decrease in the values of %R of both TPC and TFC in all three surfactants were recorded. This reflects that CPE up to 40 min at 45 °C can be the onset point of degradation of bioactives. This type of behavior also signified by observing variability within these three different time intervals (Table 1). Further %R of TPC and TFC were significantly different within Triton X-100, Triton X-114, and Tergitol NP-12 (Table 1). Among all three of its, Triton X-114 showed the maximum extraction recovery about 98.09% of TPC and 96.96% TFC followed by Triton X-100 (87.67% TPC and 86.88% TFC) and Tergitol NP-12 (79.09% TPC and 82.03% TFC) at 30 °C (Table 1). This might be because Triton X-114 has HLB (hydrophilic-lipophilic balance) value of 12.4, which might be more compatible for bioactives than Triton X-114 (HLB-13.4) and Tergitol NP-12 (HLB-13.8). Our results are on a similar line with Leite and coresearcher [27] who observed better extraction of chlorophyll at 41 °C and 30 min extraction time using surfactant Triton X-114 having HLB of 12.4. Based on these experimental results, surfactant-Triton X114 and extraction time of 30 min was selected for further optimization of factors.

3.3. Experimental design Once the selection of limits for factors were varied as per the rotatable central composite design (RCCD); all the points obtained from RCCD were converted into coded values using equation numbers (2)–(5). (Table 2) The lower limit (−1 in coded form) and upper limit (+1 in coded form) set for surfactant concentration, pH, temperature and salt concentration were 5; 11 (%), 4; 8, 30; 60 (°C), and 4; 12 (%) respectively. They were selected according to the observation from CPT records (Fig. 2A and B). All the response values were obtained after 30 experimental runs conducted according to RCCD and are illustrated in Table 2. A quadratic polynomial model (Eq. (1)) was developed for every response (Yi) as a function of x1, x2, x3, and x4. The interaction effect of all factors on %R, %L, and Ks/a were discussed using response surface plots (Figs. 3–8). Fraction concentration (fc) values in all interactions were completely analogous to recovery; hence, corresponding response surfaces were not plotted below and but discussed from Tables 3 and 4.

3.2. Selection of limits of CPE process variables

3.4. Response surface models

After screening of surfactant (Triton X-114) and extraction time, the maximum and minimum limit of each process variable (surfactant concentration (%, v/v), pH, temperature (°C), and salt concentration (%, w/v)) was selected by finding the highest CPT by visual observation. From Fig. 2(A) it was observed that with an increase in Triton X114 concentration (1–15%, v/v); an increase in CPT was noted, i.e. 24.3–37.5 °C. This could be due to the higher surfactant concentration that facilitates the development of more micelles, which resultant needs more temperature to separate the surfactant phase. On the other hand, a minimum 5% surfactant has been reported as the adequate cloud forming and surfactant phase volume for effective decantation of the aqueous phase after CPE. However, the surfactant can be used in the range of 5–15% (v/v), but most advisable was below 11% [28]. From Fig. 2(A) it can be observed that with the increase in salt (NaCl) concentration (1–15%, w/v); a decrease in CPT from 38.2 to 17 °C was recorded. This behavior observed might be due to the salting out effect, which causes the dehydration of the surfactant phase that leads to effective phase separation [29]. After 12% (w/v) salt concentration, there was no more change in CPT (Fig. 2(A)) was noted. Similarly, pH is also a crucial factor in effective cloud formation. The

The effect of varying CPE parameters on responses (% surfactant concentration, pH, temperature ºC, and % salt concentration) as function of recovery (%R), partition coefficient (Ks/a), loss (%L), and fraction concentration (fc) were successfully modeled using a quadratic polynomial model (Eq. (1)). The coded form of coefficients for all the terms (linear, square, and interaction) in the model has been outlined in Table 3. 3.4.1. Linear terms The linear terms in the quadratic equation (Eq. (1)) were significantly affected on the changes in recovery (%R), partition coefficient (Ks/a), loss (%L), and concentration fraction (fc) of CPE of TPC and TFC (Table 3). However, few exceptions have been observed in the case of salt conc. (on %R and %L) and surfactant conc. (on Ks/a), for which the respective linear terms were insignificant at p > 0.05 (Table 4). The relative impact of the linear term of temperature (°C) was most influencing for change in three responses (negative to %R; concentration fraction in surfactant phase; Csf, and positive to %L) except Ks/a 4

Journal of Environmental Chemical Engineering 7 (2019) 103306

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Fig. 2. Cloud point temperature (CPT) at varying surfactant and salt concentration (A) and pH values (B).

%L, and Ks/a were discussed using response surface plots (Figs. 3–8). fc values in all interactions were completely analogous to %R. Therefore, corresponding response surfaces were not plotted whereas have been discussed in Tables 3 and 4.

followed by pH, surfactant concentration and salt concentration for CPE of TPC and TFC (Table 3). The highest influencing factor on Ks/a was pH followed by salt conc., temperature, and surfactant concentration for TPC while for TFC; pH was the only affecting factor on Ks/a. Among all significant factors at p < 0.05 surfactant concentration showed the least negative influence on %R and fc which suggested that initially, it affects positively however later it observed to be negative (Table 3) for both CPE cases.

3.5. Evaluation of optimum condition in different interaction terms The surfactant concentration, pH, temperature, and salt concentration play a key role in the CPE system. This is because non-ionic surfactant act as the main extraction medium, pH effects on the micellesolute interaction [28], temperature helps surfactant to rich critical micelle concentration (CMC), and salt reduces the CPT of surfactant.

3.4.2. Interaction terms The interaction effects among all four factors (x1×x2, x1×x3, x1×x4, x2×x3, x2×x4, and x3×x4) on %R, %L, and fc of TPC were significant at p < 0.05. Whereas among all, only three factors (x1×x3, x1×x4, and x3×x4) significantly affected Ks/a (Table 3). In the case of CPE of TFC, almost all interaction terms were found significantly affecting on %R, % L and fc at p < 0 except pH and salt concentration (x2×x4). There was only one interaction term (x2×x3) effect on Ks/a found to be significant (Table 3).

3.5.1. Surfactant concentration-pH (x1x2) The relative effect of the positive interaction term (x1×x2; surfactant concentration and pH) on %R of TFC (66–86%) and TFC (66–90%) was higher than the corresponding linear impact of variables. This reflects that by keeping the temperature and salt concentration constant at middle point (45 °C and 8%, v/v); increase in surfactant concentration along with pH; the %R and fc of TPC and TFC also increases (Fig. 3a and d) till optimum maximum value. Furthermore, increased surfactant concentration should improve the %R; however, pH limited this. This interaction might be because of the dissociation of phenolics at alkaline conditions. Therefore, there was a strong interaction with water molecule rather than micelle [30] was noted. This decides the state of analytes exist. This phenomenon might be related to the highest extraction %R is achieved at pH values less than pKa value of analytes, where the uncharged form of the target analytes gets entrapped into micelles [31]. For this instance, similar results also reported by Padilha et al. and Kiai et al. [28,31]. On the other hand, Zhou et al. [32] observed a maximum %R of flavonoids from Apocynum venetum leaf samples at pH 8 and 1.2% w/v surfactant. Chatzilazarou et al. [22] reported the lesser recovery; perhaps, these results were due to other types of surfactants with different concentrations or no other interaction factors along with pH in the system. From Fig. 3(b and e) and Table 3 negative interaction terms (x1×x2) of %L recognize the increment in surfactant from 5 to 11% v/v within the pH range of 4–8. The %L was found to be very less (below 14%) till 7 to 8%; v/v surfactant and pH 5. Afterward, it was found to be increased above 35 and 40% of TPC and TFC at the highest level of variables. This behavior attributed due to degradation or dissociation of bioactives in alkaline solution [30]. Similar results were reported by Tang et al. [33]. For CPE, the influence on Ks/a due to extraction parameters are not discussed much, on that note, it has been taken into consideration in this study. The x1x2 interaction was found non-significant on Ks/a at p < 0.05 (Table 4) for both TPC and TFC, although surfactant is an

3.4.3. Square terms The square terms in the equation (Eq. (1)) of all CPE parameters of both TPC and TFC significantly contributed to the changes in the case of almost all the responses of TPC and TFC. The only exception had been realized in the case of Ks/a, for which square term of the temperature was not contributing significantly at p < 0.05 (Table 3). 3.4.4. Anova From Table 4 it was observed that, while fitting to Eq. (1), the R squared values (R2) for recovery (%R), partition coefficient (Ks/a), loss (%), and fraction concentration (fc) were 0.9840; 0.9265; 0.9840; 0.9840 (for total phenols) and 0.9798; 0.9476; 0.9798;0.9802 (for total flavonoids) respectively and adjusted R2 values for all four responses were 0.9691; 0.8580; 0.9691; 0.9692 (for total phenols) and 0.9610; 0.8988; 0.9610; 0.9617 respectively. No significant differences between R2 and adj. R2 values were observed. This indicated the desirability of the best fitting of the model. A low p-value (< 0.0001) along with high f-value (> 13) for each model also demonstrated that they were significant for CPE. The lack of fit values for all four responses (for total phenolics: 0.0556; 0.1281; 0.0556; 0.0549 and for total flavonoids: 0.2454; 0.1783; 0.2454; 0.2998) for the given model were recorded as insignificant; showing that the variance within the data is not due to noise within the system. Preferably, it is due to the factors or varying CPE parameters (Table 4). In the present investigation, the experimental design (RCCD) has been formulated and interactions of all respective factors affecting on CPE of bioactives had been examined by using an empirical quadratic polynomial model (Eq. (1)) and interaction effect of all factors on %R, 5

6

5 (−1) 5 (-1) 5 (-1) 5 (-1) 5 (-1) 5 (-1) 5 (-1) 5 (-1) 11 (+1) 11 (+1) 11 (+1) 11 (+1) 11 (+1) 11 (+1) 11 (+1) 11 (+1) 2 (-2) 8 (0) 8 (0) 8 (0) 14 (+2) 8 (0) 8 (0) 8 (0) 8 (0) 8 (0) 8 (0) 8 (0) 8 (0) 8 (0)

4 (−1) 4 (-1) 4 (-1) 4 (-1) 8 (+1) 8 (+1) 8 (+1) 8 (+1) 4 (-1) 4 (-1) 4 (-1) 4 (-1) 8 (+1) 8 (+1) 8 (+1) 8 (+1) 6 (0) 2 (-2) 6 (0) 6 (0) 6 (0) 10 (+2) 6 (0) 6 (0) 6 (0) 6 (0) 6 (0) 6 (0) 6 (0) 6 (0)

30 30 60 60 30 30 60 60 30 30 60 60 30 30 60 60 45 45 15 45 45 45 75 45 45 45 45 45 45 45

(−1) (-1) (+1) (+1) (-1) (-1) (+1) (+1) (-1) (-1) (+1) (+1) (-1) (-1) (+1) (+1) (0) (0) (-2) (0) (0) (0) (+2) (0) (0) (0) (0) (0) (0) (0)

4 (−1) 12 (+1) 4 (-1) 12 (+1) 4 (-1) 12 (+1) 4 (-1) 12 (+1) 4 (-1) 12 (+1) 4 (-1) 12 (+1) 4 (-1) 12 (+1) 4 (-1) 12 (+1) 8 (0) 8 (0) 8 (0) 0 (-2) 8 (0) 8 (0) 8 (0) 16 (+2) 8 (0) 8 (0) 8 (0) 8 (0) 8 (0) 8 (0)

86.88 75.48 78.54 83.84 73.24 72.24 54.15 70.40 92.65 76.37 62.18 54.74 84.84 73.70 51.02 51.97 71.16 81.42 67.25 94.65 50.61 65.93 36.67 93.98 80.26 79.38 81.36 80.88 80.22 83.36

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

2.65mno 0.86fghijk 1.88fghijklm 2.07klm 0.58efghij 2.10efghi 1.92bc 2.19def 1.84no 2.83fghijkl 0.62cd 4.24bc 3.69lmn 3.65efghij 0.66b 2.38b 1.13efg 1.79jklm 0.59de 4.52no 2.16b 2.91de 2.37a 3.06no 8.21ijklm 1.96ghijklm 0.74jklm 1.16ijklm 1.46hijklm 0.73klm

5.54 ± 0.22g 4.15 ± 0.08f 3.72 ± 0.14def 10.19 ± 0.10j 2.81 ± 0.01cd 2.59 ± 0.05bc 1.28 ± 0.06a 6.54 ± 18.4h 19.39 ± 1.00l 3.11 ± 0.07cde 3.92 ± 0.06ef 1.37 ± 0.12a 9.40 ± 0.43gh 2.65 ± 0.22c 1.15 ± 0.04a 1.20 ± 0.05a 1.65 ± 0.05ab 5.57 ± 0.23gh 2.64 ± 0.12c 25.34 ± 0.34m 1.52 ± 0.05a 1.39 ± 0.19a 1.01 ± 0.04a 17.16 ± 0.76k 8.29 ± 0.70i 5.46 ± 0.17g 5.52 ± 0.30g 5.48 ± 0.20g 8.31 ± 0.09i 5.54 ± 0.05g

Ks/a (Y2)

13.12 ± 2.65bc 24.52 ± 0.86efghi 21.46 ± 1.88defgh 16.16 ± 2.07cd 26.76 ± 0.58ghij 27.76 ± 2.10hijk 45.85 ± 1.92m 29.60 ± 2.19ijk 7.35 ± 1.84ab 23.63 ± 2.83efghi 37.82 ± 0.62l 45.26 ± 4.24m 15.16 ± 3.69cd 26.30 ± 3.65fghij 48.98 ± 0.66m 48.03 ± 2.38m 28.84 ± 1.13ijk 18.58 ± 1.79cde 32.75 ± 0.59jkl 5.35 ± 1.49a 49.39 ± 2.16m 34.07 ± 2.9kl 63.33 ± 2.37n 6.02 ± 3.06a 19.74 ± 1.28cdef 20.62 ± 1.96defg 18.64 ± 0.74cde 19.12 ± 1.16cde 19.78 ± 1.46cdef 16.64 ± 0.73cd

%L (Y3)

0.87 0.75 0.79 0.84 0.73 0.72 0.54 0.70 0.93 0.76 0.62 0.55 0.85 0.74 0.51 0.52 0.71 0.81 0.67 0.95 0.51 0.66 0.37 0.94 0.80 0.79 0.81 0.81 0.80 0.83

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

fc (Y4)

0.03mno 0.01fghijk 0.02ghijklm 0.02klm 0.01efghij 0.02efghi 0.02bc 0.02defg 0.02no 0.03ghijkl 0.01 cd 0.04bc 0.04lmn 0.04efghij 0.01b 0.02b 0.01efgh 0.02jklm 0.01def 0.05° 0.02b 0.03de 0.02a 0.03° 0.08ijklm 0.02hijklm 0.01jklm 0.01ijklm 0.01ijklm 0.01klm

84.64 ± 3.44ghi 84.39 ± 0.48ghi 93.45 ± 2.73j 90.91 ± 1.48ij 74.14 ± 1.07ef 66.19 ± 2.82de 51.39 ± 0.87b 63.48 ± 0.40cd 88.20 ± 3.27ghij 74.90 ± 2.33f 59.80 ± 0.98bcd 58.90 ± 1.62bcd 102.85 ± 1.20k 81.71 ± 0.28fgh 59.11 ± 2.24bcd 58.25 ± 2.06bcd 54.63 ± 0.81b 75.31 ± 2.29f 79.69 ± 1.91fg 120.10 ± 9.45l 41.48 ± 1.27a 58.04 ± 0.99bcd 57.47 ± 2.15bc 109.39 ± 2.21k 85.50 ± 4.53ghij 86.44 ± 0.46ghij 89.69 ± 1.48hij 93.53 ± 3.22j 88.26 ± 0.89hij 90.14 ± 2.06hij

%R (Y1)

Salt conc. (%, w/v)

%R (Y1)

Temperature (°C)

Surfactant conc. (%, v/v)

pH

Responses for TFC

Responses for TPC

CPE conditions (coded value)

6.25 ± 0.12fghij 7.80 ± 0.53ghijkl 8.37 ± 0.60ijkl 7.63 ± 0.02ghijkl 2.30 ± 0.06abcd 3.42 ± 0.21abcdef 0.72 ± 0.03a 2.23 ± 0.02abcd 9.58 ± 0.23jkl 4.18 ± 0.15bcdef 8.41 ± 0.45ijkl 8.49 ± 1.97ijkl 5.84 ± 0.10efghi 4.84 ± 0.22defgh 1.29 ± 0.05ab 2.68 ± 0.09abcde 1.43 ± 0.02abc 10.10 ± 0.20kl 4.70 ± 1.26cdefg 10.91 ± 0.86l 3.13 ± 0.17abcdef 1.66 ± 0.03abcd 2.71 ± 0.15abcde 9.04 ± 0.12ijkl 7.91 ± 1.53ghijkl 8.10 ± 3.54hijkl 7.55 ± 1.82ghijk 8.16 ± 1.86hijkl 8.50 ± 0.11ijkl 9.63 ± 1.85hijkl

Ks/a (Y2)

15.36 ± 3.44def 15.61 ± 0.48def 6.55 ± 2.73c 9.09 ± 1.48cd 25.86 ± 1.07gh 33.81 ± 2.82hi 48.61 ± 0.87k 36.52 ± 0.40ij 11.80 ± 3.27cdef 25.10 ± 2.33g 40.20 ± 0.98ijk 41.10 ± 1.62ijk −2.85 ± 1.20b 18.29 ± 0.28efg 40.89 ± 2.24ijk 41.75 ± 2.06ijk 45.37 ± 0.81k 24.69 ± 2.29g 20.31 ± 1.91fg −20.10 ± 9.45a 58.52 ± 1.27l 41.96 ± 0.99ijk 42.53 ± 2.15jk −9.39 ± 2.21b 14.50 ± 4.53cdef 13.56 ± 0.46cdef 10.31 ± 1.48cde 6.47 ± .3.22c 11.74 ± 0.89cde 9.86 ± 2.06cde

%L (Y3)

Values are showed as average ± SD (n = 3). The different small alphabets in superscript signifies that the average values belong to distinct subsets across the columns at 95% confidence interval. TPC: total phenolic compounds, TFC: total flavonoid compounds, %R: % recovery, Ks/a: partition coefficient, %L: % loss fc: fraction concentration.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

Run k

Table 2 Matrix summary of the factors and responses calculated in cloud point extraction (CPE) of total phenolic compounds and total flavonoid compounds according to RCCD.

0.85 0.84 0.93 0.91 0.74 0.66 0.51 0.63 0.88 0.75 0.60 0.59 1.03 0.82 0.59 0.58 0.55 0.75 0.80 1.20 0.41 0.58 0.57 1.09 0.86 0.86 0.90 0.94 0.88 0.90

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

fc (Y4)

0.03hij 0.00hij 0.03k 0.01jk 0.0fg 0.03ef 0.0b 0.00de 0.03hijk 0.02g 0.01cde 0.00bcde 0.01l 0.00ghi 0.02bcde 0.02bcde 0.0ab 0.02g 0.02gh 0.09m 0.0a 0.01bcde 0.02bcd 0.02l 0.05hijk 0.00hijk 0.01ijk 0.03k 0.01hijk 0.02ijk

P.R. More and S.S. Arya

Journal of Environmental Chemical Engineering 7 (2019) 103306

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Fig. 3. Response surfaces describing the interaction between surfactant concentration (%, v/v) and pH on the different %R (a; d), %L (b; e), and Ks/a (c; f) of TPC and TFC respectively.

From Fig. 3c and f, it has been observed that Ks/a was maximum at the middle range of surfactant and near to lower range of pH with parabolic interactions within factors. The negative coefficients (Table 3) of the linear term of pH are given in Table 3. It reflects that a rise in pH lowers the Ks/a, probably this behavior observed is due to the dissociation and breaking of hydrogen bonds which lead to

important factor for partitioning which leads to rising in Ks/a when the concentration of surfactant increased [34]. The hydrophobic surfactant phase which is a strong hydrogen bond acceptor may have enhanced the extraction rate of most hydrophobic organic components like phenols and some flavonoids in the surfactant phase from pomegranate peel [34].

Fig. 4. Response surfaces describing the interaction between surfactant concentration (%, v/v) and temperature (°C) on the different %R (a; d), %L (b; e), and Ks/a (c; f) of TPC and TFC respectively. 7

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Fig. 5. Response surfaces describing the interaction between surfactant concentration (%, v/v) and salt concentration (%, w/v) on the different %R (a; d), %L (b; e), and Ks/a (c; f) of TPC and TFC respectively.

was significant on almost all the responses of CPE of TPC, except for Ks/ a of TFC which was observed as non-significant at p < 0.05 (Table 4). Table 3 shows the impact of the negative coefficient of interaction term (x1x3) %R and fc using response surfaces (Fig. 4(a and d)) obtained from the quadratic model (Eq. (1)). This signifies the %R of both TPC and TFC. As the concentration was increased with an increase in surfactant and temperature till the maximum stationary ridge point, this was well described by maximum stationary ellipses of Fig. 4(a and d). Probably

solubilization of TPC and TFC to aqueous phase [35]. With that instance, quite similar reports have been mentioned by Vicente et al. [36]. According to them; lower Ks/a was achieved at higher pH. Hence, the rise in pH value of the solution to alkaline conditions more than pKa values will reduce TPC and TFC extraction in the surfactant phase.

3.5.2. Surfactant concentration-temperature (x1x3) The interaction of surfactant concentration and temperature (x1x2)

Fig. 6. Response surfaces describing the interaction between pH and temperature (°C) on the different %R (a; d), %L (b; e), and Ks/a (c; f) of TPC and TFC respectively. 8

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Fig. 7. Response surfaces describing the interaction between pH and salt concentration (%, w/v) on the different %R (a; d), %L (b; e), and Ks/a (c; f) of TPC and TFC respectively.

increase in temperature from 30 to 60 °C of CPE system led to %L of TPC and TFC up to 45 to 50%. Positive coefficients of interaction term also reflect the same. This type of relationship might be due to a reduction in the surfactant phase volume at higher temperature [37]. Benkhedja et al. [38] also noted a 90% efficiency at 10% (w/v) surfactants and 42 °C; unlike wang et al. [39] observed the 30 °C was the optimum temperature for successful CPE. Some other studies also reflect the degradation of phenolic and flavonoid compounds at higher

this behavior observed due to the use of salt as another factor that minimizes the CPT of the system led to achieving maximum %R and fc at the resulted temperature (about 35 °C) near to CPT. Tang et al. [33] reported the maximum %R of flavonoids from Crotalaria sessiliflora at 90 °C perhaps type of surfactant, and thermostable compounds were different. The minimum stationary ellipses (Fig. 4(b and e)) showed that the increasing or decreasing surfactant from the center point with an

Fig. 8. Response surfaces describing the interaction between temperature (°C) and salt concentration (%, w/v) on the different %R (a; d), %L (b; e), and Ks/a (c; f) of TPC and TFC respectively. 9

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Table 3 The coefficients of the terms (in coded form) signifies at p < 0.05 in the quadratic model showing the effect of process parameters on all responses in cloud point extraction (CPE) of TPC and TFC. Responses for TPC

Intercept x1-Surfactant conc. (%, v/v) x2- pH x3-Equilibrium temp. (°C) x4-salt conc. (%, w/v) x1×x2 x1×x3 x1×x4 x2×x3 x2×x4 x3×x4 x1×x1 (x12) x2×x2 (x22) x3×x3 (x32) x4×x4 (x42)

Responses for TFC

%R (Y1)

Ks/a (Y2)

%L (Y3)

fc (Y4)

%R (Y1)

Ks/a (Y2)

%L (Y3)

fc (Y4)

80.91 −3.683333 −4.5875 −7.905 −1.0875* 1.89375 −5.42125 −2.69125 −1.525 2.18 3.43 −4.809375 −1.611875 −7.040625 3.548125

6.43 0.21** −1.34 −0.98 −1.32 −0.19** −2.10 −2.23 0.36** 0.76** 2.12 −1.56 −1.09 −1.50 3.35

19.09 3.68 4.59 7.91 1.09* −1.89 5.42 2.69 1.53 −2.18 −3.43 4.81 1.61 7.04 −3.55

0.81 −0.036 −0.046 −0.078 −0.012 0.020 −0.055 −0.026 −0.016 0.023 0.035 −0.047 −0.016 −0.070 0.036

88.93 −2.13 −4.69 −6.92 −2.34 7.39 −6.34 −2.35 −3.97 −0.054** 3.15 −10.22 −5.56 −5.09 6.46

8.31 0.42* −2.26 −0.35** −0.22** 0.34** −0.17** −0.52* −0.91 0.47* 0.37** −1.54 −0.64 −1.19 0.38*

11.07 2.13 4.69 6.92 2.34 −7.39 6.34 2.35 3.97 0.054** −3.15 10.22 5.56 5.09 −6.46

0.89 −0.021 −0.047 −0.070 −0.024 0.074 −0.063 −0.023 −0.041 −0.00625 0.032 −0.10 −0.056 −0.051 0.064

TPC: total phenolic compounds, TFC: total flavonoid compounds, %R: % recovery Ks/a: partition coefficient, %L: % loss, fc: fraction concentration, x1, x2, x3 and x4 represent the dimensionless coded values of surfactant concentration (%), pH, temperature (°C) and salt concentration (%), not significant. * 0.05 < p < 0.1. ** p > 0.10.

up to 50 °C. For concern comparable reports available [41].

temperature [22]. Quadratic model (Eq. (1)) suggest the most appropriate surfactant concentration and temperature of CPE system was near 8.5% (v/v) at 40 °C and 8.5% (v/v) at 35 °C for responses (%R, %L, and fc) of TPC and TFC. The relative effect of interaction between surfactant and temperature on Ks/a has been studied. The negative interaction coefficient (-2.10) of Ks/a shows significant influence in case TPC while for TFC, it has an insignificant relationship at p < 0.05 (Table 4). From quadratic Eq. (1) and rising ridges of Fig. 4(c), it was observed that the antagonistic relation of Ks/a of TPC towards the temperature. The increase in surfactant concentration (5–8%) led to increasing the Ks/a of TPC, while the rise in temperature (30–60 °C), the Ks/a values were found to be decreased (Fig. 4(c)). Assumption of this behavior due to a rise in the surfactant concentration increases the hydrophobicity of the surfactant phase (micelle phase), by this instance El-Abbassi et al. [34] showed analogues towards the surfactant, but on the other hand, he also reported the increment in Ks/a at 90 °C. Probably this happens due to a different type of surfactant having quite higher CPT. Besides this, some reports available of which shows the separation of some biomolecule comparable at the temperature range (23–45 °C) [26,36,40]. Table 4 showed that the coefficient of the interaction term (x1x3) for Ks/a of TFC was insignificant. The simple maximum pattern of Fig. 4(f) reflects the truthiness of this relationship. In this interaction, Ks/a was highest near at center of response plot (Fig. 4(f)) from which an increase in the magnitude of surfactant conc. and temperature led to a decrease in Ks/a although surfactant and temperature are important factors for phase separation that might be a possible role of thermostability of flavonoids

3.5.3. Surfactant concentration-salt concentration (x1x4) The interaction of surfactant concentration-salt concentration, at pH 6 and 45 °C, the %R of TPC and TFC ranges from 70 to 86% (Fig. 5(a and d)) and 75 to 95%. Analogously fc of same responses ranges from 0.70 to 0.86 and 0.75 to 0.95, from Table 3 it was observed that the coefficients for %R and fc were negative for both TPC and TFC, conflicting coefficients observed for the square term of salt (x42). The saddle pattern of Fig. 5(a and d) indicates that the increment or decrement of surfactant concentration from the center point decreases the %R. Whereas it shows the antagonistic relation towards the salt concentration. Generally, electrolytes like NaCl addition decreases the solubility of bioactives in water by the salting-out effect. Hence, hydrogen bonds between a water molecule and polar head of surfactant become weaker [42] which reflects in the phase separation and concentration of solute in the surfactant phase and improve %R [43]. This relationship showed the %R and fc initially rises till stationary point. Such behavior might be observed due to other constant factors viz. pH and temperature limit it. As salt decreases the CPT (Fig. 2(a)) up to 17 °C and current interaction was at temperature 45 °C for 30 min that probably led to degradation of boactives [22]. Surfactant and salt also significantly influence the %L of TPC and TFC by saddle effect (Fig. 2(b and e)) from Table 3 this behavior was signified by observation of coefficient of positive interaction and negative square term of salt (−3.55). The minimum loss was observed at surfactant 8% while an increase or decrease of it led to an increment in %L of TPC and TFC was noted. While

Table 4 ANOVA for the model representing the influence of process parameters on all four responses in cloud point extraction (CPE) of total phenolic compounds and total flavonoid compounds. Responses for TPC

P-value Plof -value F-value Flof -value R2 Adjusted R2

Responses for TFC

%R (Y1)

Ks/a (Y2)

%L (Y3)

fc (Y4)

%R (Y1)

Ks/a (Y2)

%L (Y3)

fc (Y4)

< 0.0001 0.0556 65.93 4.49 0.9840 0.9691

< 0.0001 0.1281 13.51 2.87 0.9265 0.8580

< 0.0001 0.0556 65.93 4.49 0.9840 0.9691

< 0.0001 0.0549 66.10 4.52 0.9840 0.9692

< 0.0001 0.2454 52.07 1.91 0.9798 0.9610

< 0.0001 0.1783 19.39 2.36 0.9476 0.8988

< 0.0001 0.2454 52.07 1.91 0.9798 0.9610

< 0.0001 0.2998 52.97 1.66 0.9802 0.9617

TPC: total phenolic compounds, TFC: total flavonoid compounds, %R: % recovery, Ks/a: partition coefficient, %L: % loss, fc: fraction concentration and lof: Lack of fit. 10

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Fig. 7(b and e) rise in temperature and salt led to an increase in loss from 10 to 26% of TPC. While insignificant interaction was observed for %L of TFC as it showed the loss in negative value (Table 2). The partition coefficient of TPC showed a significant interaction effect while it is insignificant for TFC. For TPC coefficient value of both interaction and square term (Table 3) shows the maximum Ks/a (about 12) at pH 5.7 and 8% salt concentration; further, increase in pH, Ks/a drops down to 3 (Fig. 7(c)) with falling saddle pattern. The Ks/a of TFC in the sample are affected by linear (x2 and x4) and square terms (x22 and x42) (Table 3) only, but interaction term changing the Ks/a is not statistically significant (P > 0.1) (Table 4). Padilha et al. [28] and Vicente et al. [45] reports comparable results with the contest of this interaction. Thus, for 8% salt concentration, the contours from the response (Fig. 7(f)) is almost parallel or slight concave after the pH 6.

as salt increases; %L was also increased. The effect of the same interaction term (x1x2) on partition coefficient ranges for TPC and TFC were 3–12 and 6.5–9.5 respectively (Fig. 5(c and f)). As the surfactant increases; the Ks/a [34] initially it rises but an increase in salt concentration, decrease in Ks/a of TPC was recorded (Fig. 5(c)). In the case of TFC mound shape behavior was observed (Fig. 5(f)) which describes that surfactant positively influences the Ks/a but increase in salt effect negatively (Table 3). This behavior might be attributed to the salting-out effect of surfactant which facilitates the phase separation and decreases the CPT. At lower CPT interaction causes at 45 °C led to a reduction in the surfactant phase which also entraps fewer bioactives attributed to a decrease in Ks/a. Padilha et al. [28] showed the same reports within the domain of 0–10 wt% NaCl with 6% is optimum salt concentration. He et al. [26] also observed analogous findings. On the other hand, the current study was in the range of 4–12 % (w/v) salt which was very high as compared to other researches [30,44], this is the probable reason of current contrary results.

3.5.6. Temperature-salt concentration (x3x4) The temperature-salt concentration interaction, at 8% surfactant and pH 6, the %R and fc of TPC ranges 66 to 92% and 0.66 to 0.92, respectively (Fig. 8(a)) and for same responses of TFC ranges from 76 to 101.2% and 0.76 to 1.02, respectively (Fig. 8(d)). The positive coefficients of %R and fc for both TPC and TFC are significantly contributing for change due to interaction, conflicting to these both linear (x3 and x4) and square (x32) coefficients are negative (Table 3). The saddle pattern of relationship in interaction indicates the increase and decrease in salt from the center point with a simultaneous increase in temperature; decreases the %R of polyphenols and flavonoids from pomegranate peels (Fig. 8(a and d)). Meanwhile, the opposite saddle pattern was observed which shows the increase in loss percentage about −7 to 21% of TPC and −4.7 to 21.3% of TFC (Fig. 8(b and e)). While analyzing the relationship, although electrolytes are important for phase separation. This study works at a very high range of salt, along with temperature above 40 °C may degrade the bioactives on a higher temperature [46]. Contrary to this, Tang et al. [33] finds better extraction recovery of flavonoids at more than 80 °C probably this is because of a surfactant having higher CPT. Partition coefficient (Ks/a) for TPC shows the significant influence; rather it was statistically insignificant for TFC as p > 0.10. (Table 4). With the reference of the interaction of pH and salt (x2x4), this (x3x4) it shows ups and downs in responses values of Ks/a (Fig. 8(f)) by showing the saddle effect on TPC. The Ks/a was initially very high at temperature 30 to 40 °C along with the salt up to 8% (v/v), (Fig. 8(c)) but as the temperature rises it gets decreased even salt is at center point, the negative coefficients of linear and square term of temperature might be responsible for such behavior [37,38]. In the case of flavonoids, response contour showed the saddle effect with rising and fall of temperature from 40 °C followed by rising ridges towards the temperature from 40 to 60 °C and salt concentrations. Hence from this interaction optimum value of observed with about 8% salt at minimum temperature (< 40 °C).

3.5.4. pH-temperature (x2x3) The influence in the pH and temperature represented as interaction term x2x3 (Table 3) was also analyzed by keeping other factors constant at center points (surfactant-8%, v/v, and salt-20%, w/v). The %R and parallelly fc of TPC and TFC ranges from 62 to 86%; 0.62 to 0.86 and 70 to 90%; 0.7 to 0.9 Fig. 6(a and d) and Table 2. In the rising ridge effect of interaction on %R of TPC, the rise or drops of temperature from 40 °C and simultaneous increment of pH from 4 to 8; decrease in %R was observed (Fig. 6(a)). The concentric elliptical relationship of factors on %R of TFC shows the increment in pH after 5 with the corneous rise in temperature from 30 to 60 °C resulted in the reduction of recovery (Fig. 6(d)). Parallelly exact opposite to %R, adverse behaviors of interaction term on %L of TPC and TFC was observed from falling ridge effect and concentric ellipse effect on %L of TPC and TFC respectively. On a similar note, Kiai et al. [31] and El-Abbassi et al. [34], reports that pH of a solution is increased phenols dissociation from surfactant phase led to the more bioactive loss. On the other hand, rise in temperature also might cause degradation of phenols and flavonoids [22]. Change in Ks/a by the influence of the same interaction term (x2x3) at constant other two variables at 8% Triton X-114 and 8% NaCl. Ks/a of TPC and TFC is ranging from 1.5 to 7.5 and 4.5 to 10 respectively (Fig. 6(c and f)). In the case of TPC, Ks/a was insignificantly affecting at p < 0.05 (Table 4). While for TFC, antagonistically rising ridge relationship of Ks/a can be observed from the concave contour line on response surface (Fig. 6(c and f)). It was also observed that for TPC rise or drops in temperature from 40 °C and for TFC increase and decrease in temperature from center point (45 °C) with continuous rise in pH from 4 to 8 led to decrease on partitioning, reference to this, Vicente et al. [45] also observed analogous reports. 3.5.5. pH-salt concentration (x2x4) For CPE of TPC, the pH and salt concentration interaction terms are significant for %R, fc, and %L whereas for TFC all the responses are insignificant at p < 0.05 (Table 4) when surfactant and temperature fixed to 8% (v/v) at 45 °C. The TPC the positive interaction coefficients and square term of salt (x42) indicates the increment of both pH and salt up to stationary point reduces the %R (72%) an increasing the pH and salt from 4 to 8 and 4 to 12; there was a minimum %R obtained (Fig. 7(a)) and fc (0.72). On the other hand, it was noted that coefficients of all responses for TFC are insignificantly affecting by interaction (Table 3) as it shows the saddle effect on %R. It can report that both terms were showing a negative effect on %R of TFC (Fig. 7(d)). Fig. 7(d) also showed the %R exceeds the limits (100%), which reflects the inadequacy of the interaction term. Probably this might be due to more interaction of bioactives with water molecules at higher pH [30]. Padilha et al. [28] also report possible reasons because of higher salt concentration. Meanwhile, the interaction effect on %L reported from

3.6. Correlation of model predicted and actual experimental responses The principal aim of the present work was to improve the %R of total phenolics and total flavonoids, along with analysis of influence in Ks/a of both the responses by CPE. For validation of the obtained results after application of RCCD, the model predicted values were compared with the actual experimental values of %R and Ks/a from Fig. 9(a and b) and Fig. 10(a and b) it was observed that the actual values of both TPC and TFC were scattered close to the predicted straight line which signifies the satisfactory correlation within predicted and actual values. Whereas, the actual results of Ks/a of both TPC and TFC shows the little distance of scattered points from the prediction line but R2-values of both shows they were valid according to the model. Very high determination coefficient values of %R of both TPC (R2984) and TFC (R2-9798) represented that the significant fitting of model prediction with actual experimental values. Actual Ks/a value also 11

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Fig. 9. Correlation of model predicted against experimental values of recovery (%R) and partition coefficient (Ks/a) of total phenols by CPE.

98.21% recovery, 1.79% loss, Ks/a −9.71 and fc -1.01. For the validation of prediction vs. actual experiment was done at conditions analogous to suggested. With that instance observed results of the experiment were close to suggested which are illustrated in Table 5.

showed the good adjustment correlating to predicted data, it can observe from the determination coefficients of TPC (R2-9265) (Fig. (b)) and TFC (R2-9476) (Fig. 10(b)).

3.7. Numeric optimization

3.8. The yield of CPE of total phenolic content and total flavonoid content

Numerical optimization using Eq. (9) was carried out by targeting to get a maximum CPE recovery (%) with minimum loss (%) of bioactive compounds (TPC and TFC) which is summarized in Table 5. The relative importance (ri) has been given to each response and factors range from 1 (least important) to 5 (most important). While considering the maximum %R with minimum %L highest importance (ri = 5) has been given to %R and %L followed by partition coefficient; Ks/a considered second most important (ri = 4) as a high value of Ks/a also signifies the efficiency of CPE. fc shows analogous values to %R, so it was set to the center value (ri = 3). On the other hand, variables such as temperature and pH may be responsible for degradation at their higher values, so those factors also consider for the optimization by giving relative importance 3 and 4 to pH and temperature respectively. The suggested optimum conditions were 8.22%, v/v; surfactant concentration, at pH 4; 36.80 °C, and 4.00%, w/v; salt concentration for efficient (D = 0.846) CPE of TPC with parallel suggested 93.94% recovery, 13.83% loss, Ks/a-6.06 and fc-0.94. similarly, 8.27%; v/v, surfactant concentration at pH 5.07, 34.30 °C, and 4.06%, w/v; salt concentration for most desirable (D = 0.842) CPE of TFC with parallel suggested

Subsequently, the extract of dried pomegranate peel extracted using optimized CPE was used in the estimation of retained total phenols and total flavonoid contain respectively (Fig. 11) using well-known methods viz. Folin-Ciocalteu and aluminum chloride method. In this case, the total phenolic contain obtained was 205.2 ± 6.76 mg GAE/g of pomegranate peel powder and total flavonoid contain obtained was 60.05 ± 3.12 mg QE/g of pomegranate peel powder. These results reflect the most promising extraction efficiency of CPE as compare to other reports [4,9,15]. Hence, it was suggested that the optimum conditions suggested for both TPC and TFC in present work is efficient for cloud point extraction. 4. Conclusion Cloud point extraction is one of the upcoming novel, green, and energy efficient extraction and separation techniques that can be used for food bioctives. Rotatable central composite design (RCCD) along with response surface (RSM) interaction analysis is an obligatory

Fig. 10. Correlation of the model predicted against actual experimental values of recovery (%R) and partition coefficient (Ks/a) of total flavonoids by CPE. 12

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P.R. More and S.S. Arya

Table 5 The set of constraints for different parameters for optimizing the CPE conditions. CPE parameters

Target

Lower limit (Li)

Upper Limit (Ui)

Relative importance (ri)

Optimized conditions at D

Actual experimental conditions*

Surfactant conc. (%, v/ v) pH Temperature (°C) Salt conc. (%, w/v) %R Ks/a %L fc

In range

5

11

3

D = 0.846 8.22

9.00

Minimize Minimize In range Maximize Maximize Minimize In range

4 30 4 36.67 1.01 5.35 0.37

8 60 12 94.65 25.34 63.33 0.95

3 4 3 5 4 5 3

Surfactant conc. (%, v/ v) pH Temperature (ºC) Salt conc. (%, w/v) %R Ks/a %L fc

In range

5

11

Minimize Minimize In range Maximize Maximize Minimize In range

4 30 4 41.48 0.72 −20.1 0.41

8 60 12 120.1 10.91 58.52 1.2

Total phenol content

4.00 37.00 4.00 94.73 ± 0.96 14.59 ± 0.71 5.27 ± 0.22 0.95 ± 0.05

3

4.00 36.80 4.00 93.94 13.83 6.06 0.94 D = 0.842 8.27

3 4 3 5 4 5 3

5.07 34.30 4.06 98.21 9.71 1.79 1.01

5.00 34.00 4.00 95.87 ± 0.88 9.55 ± 0.64 4.13 ± 0.18 0.96 ± 0.06

Total flavonoid content

8.00

% R: % recovery, Ks/a: partition coefficient, %L: % loss, fc: fraction concentration, D: overall desirability value, *values are reported as mean ± SD (N = 6).

interest to investigate further the best efficiency using this method.

technique for the optimization of CPE conditions. Numerically optimized parameters of CPE of bioactives from pomegranate peel suggested by RSM were 8.22%; v/v surfactant (Triton X-114), 4%; w/v salt (NaCl) at 36.80 °C and pH 4 which gave maximum recovery, partitioning and lesser loss about 95%, 14.59, and 5.27% respectively of total phenols with the yield of 205 mg GAE/ g of pomegranate peel. While in case of total flavonoids, 8.27%; v/v Triton X-114, 4.06%; w/v NaCl at 34.30 °C and pH 5.07, yielded about 60 mg QE/g of pomegranate peel along with 98% recovery, partition coefficient 9.71, and 2% loss. Validation of the CPE parameters, from 30 RCCD experimental results were well acceptable for analyzing the trends and the relative importance of each CPE parameter. After the investigation of phase separation behavior using Triton X-114, this study can also be explored for the extraction of other bioactives and biomolecules. Furthermore, investigation of the most compatible surfactant for better separation and recovery from other bioactive and vital nutrients can be then accessed and an optimized process can be designed. CPE assisted with ultrasound, microwave, enzyme, and some other techniques are of

Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments This work was supported by TEQIP-III, Center of excellence in process intensification with focus on green technology, Institute of Chemical Technology, Mumbai, India. (Ref: ICT/REG/SSL/2306 Dated: 30th August 2018). We thank TEQIP-III, Institute of Chemical Technology, Mumbai, India who supported finance that greatly assisted the research.

Fig. 11. Total phenolic contain (TPC) and total flavonoid contain (TFC) from the optimized CPE. Values. are mean ± SD of six readings. 13

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