Effect of pH, Temperature and Time Combinations on Yield and Degree of Esterification of Mango Peel Pectin: A Box-Behnken Design Based Statistical Modelling

A 3-factor-3-level Box-Behnken design was employed to determine the effect of conditions: pH, temperature and time on yield and degree of esterification (DE) of mango peel pectin obtained using acid extraction method. Fifteen experimental runs with different combinations of pH (1.3, 2.5 and 3.7), temperature (60, 75 and 90 C) and time (45, 90 and 135 min) were performed on mango peel collected from fruit processing industry. Acid extraction method was used to extract pectin. Yield and the DE of mango peel pectin varied from 6.1 to 16.3% (dry weight basis) and 45.5 to 87.5%, respectively. Interactive effects of pH, temperature and time on the DE were significant at P<0.050. The empirical quadratic second degree polynomial model developed for DE in the study was significant (P=0.000) and well fitted to all experimental data with R of 99.48. The study highlighted that mango peel from fruit processing industry can be used to produce high methoxyl (DE > 50 %) or low methoxyl pectin (DE < 50 %) by controlling conditions during extraction and the model could be used to predict the DE of mango peel pectin for given conditions.


INTRODUCTION
Pectin is a polysaccharide of galacturonic acids with branches of neutral sugars such as Lrhamnose, L-arabinose and D-galactose (Wai et al., 2010). Galacturonic acids in pectin are methyl esterified to various extents (Wang et al., 2017). The ratio of esterified galacturonic acid units to total galacturonic acids determines the degree of esterification (DE) (Flutto, 2003). Based on the DE, pectin is classified as "High methoxyl (HM) pectin" (DE > 50 %) and "Low methoxyl (LM) pectin" (DE < 50 %) (Ranganna, 1986;Thakur et al., 1997). The DE determines gelling properties, solubility, emulsion activities, emulsion stability and release effects of pectin in complex food matrices, thus plays a unique role in food manufacturing (Srivastava and Malviya, 2011;Müller-Maatsch et al., 2016). However, as pectin is a constituent in plant cell wall, its yield and the DE depend on the chemical composition of plant cells and structure of pectin. Furthermore, nature of raw material, method and conditions such as pH, temperature, time, ionic strength of solution, solid: solution ratio of extraction affect yield and the DE of pectin (Yeoh et al., 2008;Begum et al., 2017;Sandarani, 2017).
Mango fruit is composed of approximately 11 -18% peel, 14 -22% seed and the rest being flesh (Mitra et al., 2013). In Sri Lankan context, mango peel and seed account for 51% of total fruit waste discarded by the fruit processing industry (Wathsala et al., 2017). As mango peel contains nearly 12 -18.5% pectin (Koubala et al., 2008;Girma and Worku, 2016), investigation of mango peel for pectin extraction is useful. Different extraction conditions are used to obtain pectin from fruit peels (Begum et al., 2017;Sandarani, 2017). Therefore, it is important to identify appropriate extraction conditions to obtain maximum possible yield of pectin. Hence, this study was conducted to investigate the effect of extraction conditions namely pH, temperature and time on yield and the DE of mango peel pectin and to optimize these conditions to extract maximum possible pectin by employing Response Surface Methodology (RSM).

Collection and preparation of materials
Fresh mango peel was obtained from fruit processing plants at CBL Natural Foods (Pvt) Limited, Minuwangoda, Sri Lanka and Kist Processing Plant, Kilinochchi, Sri Lanka. Peels were transported to the laboratories within 2 -3 h, cleaned, sorted, washed twice in potable running water and left for 10 min for moisture draining. The peels were disintegrated into pieces of approximately 1 cm 2 and dehydrated at 55 -60 o C for 7 -8 h in a dehydrator (TSM Products, D10 -32609, United States). The dehydrated pieces were ground in a tabletop laboratory grinder (Jaipan, India) and sieved through a laboratory sieve set (Ailmill, India) to make powder with particle size of 0.425 -0.850 mm. Mango peel powder was packaged in metalized polyester bags and stored in air tight polypropylene containers at ambient conditions until further use.
All chemicals used in the study were analytical grade purchased from Merck Chemicals, India, VWR chemicals, USA and Sigma Aldrich, Germany. Ethanol (96%, v/v) was procured from Lanka Sugar Company (Pvt) Limited for extraction of pectin.

Experimental design
A 3-factor-3-level Box-Behnken design of response surface methodology (RSM) was employed to design the experimental runs and investigate the effect of extraction conditions (pH, temperature and time) on yield and the DE of pectin. Fifteen experimental runs were carried out in triplicate represented by center points (Table 1). The conditions used were; pH of 1.3, 2.5 and 3.7, temperature of 60, 75 and 90 o C and time of 45, 90 and 135 min which were selected based on previous studies on various fruit peels (Kratchanova et al., 2004;Kanmani et al., 2014;Müller-Maatsch et al., 2016). The experimental runs were conducted in a randomized order.

Extraction of pectin
Pectin from mango peel powder was extracted according to the method described by Kratchanova et al. (2004). Approximately 10 g of mango peel powder was mixed with 300 ml of distilled water and pH was adjusted to required levels indicated in Table 1 using 1 M hydrochloric acid. The suspension was left for 20 -30 min with occasional stirring for equilibrium. The pH was re-adjusted as required. Sample containers were partially covered with watch glasses and heated at predetermined temperatures for respective times (Table 1) in a water bath (Labtech, LWB-306DS). Subsequently, the hot suspension was filtered through a muslin cloth and cooled to 4 o C in an ice bath. An equal volume of 96% (v/v) ethanol was gradually added to the sample and slowly stirred for nearly 5 min. The solution was then allowed to rest for 1 h and the coagulated pectin was separated by filtering through a muslin cloth. Another equal volume of ethanol was added to the filtrate and coagulated pectin was separated as above. The isolated pectin was washed 5 times with 96% (v/v) ethanol, dried at 35 o C in an air convection incubator (Pol-Eko Aparatura -CLW 15, Poland) for overnight and weighed after cooled to ambient temperature. The dried sample was ground into fine powder and stored in airtight amber glass containers under ambient conditions until determination of the DE.

Determination of yield
The yield of pectin from mango peel was calculated by using the Equation 1.
where, W0 is the weight of dried pectin before grinding and packaging (g) and W1 is the weight of dried mango peel powder used for extraction (g).

Determination of the DE
According to Pasandide et al. (2015), the powdered sample (0.2 g) was added into a conical flask containing 20 ml of distilled water and 3 ml of 96% ethanol and thoroughly dissolved using a vortex mixer at 3000 rpm (Velp Scientifica -ZX3, USA

Yield of mango peel pectin
The conditions namely pH, temperature and time (combinations of 15 experimental runs) used for extracting pectin from mango peel affected the yield varied from 6.1 to 16.3% (dry weight basis). Combination of pH 2.5, 90 o C and 135 min resulted in the highest yield whereas pH 3.7, 60 o C and 90 min resulted in the lowest yield (Table 2).  (Yapo et al., 2007;Liew et al., 2018;Wang et al., 2014). Thus, the present study revealed that fresh mango peel from processing industry could be successfully used for commercial scale pectin extraction in Sri Lanka.

The DE of mango peel pectin
The presence of both HM pectin (DE > 50 %) and LM pectin (DE < 50 %) in the extracted mango peel pectin was evident (Table 3). Previous studies reported the presence of only HM pectin with DE ranging between 53 and 79% in mango peel pectin and the need for further de-esterification to obtain LM pectin (Berardini et al., 2005;Sirisakulwat et al., 2010;Geerkins et al., 2015;Patel, 2017;Oleivera et al., 2018). Such variations in the DE of pectin may probably be attributable to the differences in mango variety, ripening stage and extraction conditions. Nonetheless, the present study clearly showed that LM pectin can also be produced from mango peel by proper adjustment of extraction conditions (pH, temperature and time) without de-esterifying HM pectin, which is an addition in cost. were close to 1, which further ensured the fitness of the model with experimental data (Table  4). The value of R 2 (99.48) indicated that 99.48 % total variation by pH, temperature and time can be explained by the model to predict DE with only 0.52% of total variation that could not be revealed. These findings confirmed that the model could be used to predict DE of mango peel pectin for given extraction condition.  (Koubala et al., 2008;Faruque et al., 2016), durian peel (Wai et al., 2010) and banana peel (Emanga et al., 2008). The curvature in all 3D surface graphs (Figures 1, 2  and 3), demonstrated that the model carries quadratic effects of pH, temperature and time on DE that were statistically significant at P<0.050. Increase in DE was evident at higher level of pH and the highest was observed at upper left corner of the graph corresponds with the lowest time duration and the highest pH ( Figure 1). The similar feature could be observed in the graph of temperature and pH ( Figure 2). DE decreased with temperature and time and the highest DE could be seen in the upper left corner of graph that relates to lowest temperature and the highest time ( Figure 3).   (Figure 4). When the pH increases, inadequacy of H + ions to deesterify pectin during extraction was indicated by steep ascent in DE up to pH 2.5 and moderate upsurge thereafter up to pH 3.7. With the rise in temperature, as more thermal energy is available to hydrolyze the ester bonds, further de-esterification of pectin during extraction was evident in this study. Similar results were reported in previous studies for peels of citrus (Kanmani et al., 2014), mango (Sayah et al., 2014) and banana (Emanga et al., 2008). With increase in duration (time) of extraction, as the amount of thermal energy available to hydrolyze the ester bonds increases, de-esterification enhances, thereby reducing the DE of pectin (Adetunji et al., 2017). However, reduction in the DE was evident during extraction up to 90 min and an increase thereafter up to 135 min. This may probably be due to the presence of other constituents such as carbohydrates, proteins and bioactive compounds in mango peels, which may possibly interfere with the extraction process.

CONCLUSIONS
The main and interactive effects of pH, temperature and time significantly contribute to DE and yield of mango peel pectin. Mango peel could be a good commercial source for pectin production since it yielded 17.2% pectin that is comparable to other commercial sources such as apple pomace and sugar beet. The pH and time duration should be employed more carefully than temperature to obtain pectin with required DE since the effect of the former on DE is higher than that of the latter. The developed empirical quadratic second degree polynomial model can be used to predict the DE of mango peel pectin at commercial scale processing since the model was in good agreement with experimental data. Mango peel pectin with less than or more than 50% DE could be obtained when pH, temperature and time are manipulated according to the model. Hence, mango peel from fruit processing industries can be used as a single source to extract both HM and LM pectins, depending on their role in manufacturing of different foods whereas the commercial pectin resources used at the present give only high methoxyl pectin and require an additional de-esterification process step to produce low methoxyl pectin.