65
MICROWAVE-ASSISTED EXTRACTION OF PECTIN FROM BABY JAVA ORANGE (Citrus sinensis) PEEL AND ITS CHARACTERISATION
Susinggih Wijana*, Nur Lailatul Rahmah, Siti Insyirah Fadhilah
Department of Agroindustrial Technology – Faculty of Agricultural Technology –Universitas Brawijaya Jl. Veteran – Malang 65145
Corresponding Author, email: [email protected]
Submitted : 30 March 2023 Revised : 18 April 2023 Accepted : 29 April 2023 ABSTRACT
Baby java orange (Citrus sinensis) is the main ingredient for various products. Pectin as a baby java orange peel substance is extracted using various methods. Microwave-assisted extraction is a method that uses heat energy dissipated by the volume setting from the transmitting medium.
The objective of the study was to evaluate the effect of orange peel powder: acid solution ratio (1:10, 1:15, 1:20 b/v) and extraction time (5, 10, 20 min) on characteristics of extracted pectin by using MAE (Microwave-Assisted Extraction) at 2.450 MHz. The best result was a 15 minutes extraction time and 1:15 as the material: acid solution ratio. The treatment resulted in a 51.3%
yield, 10.8% water content, 5% ash content, 775.41 equivalent weight, and 6.49 methoxyl content. The intense FTIR spectrum in the ―CO―CH3and ―COOH groups showed the same result as the standardised pectin.
Keywords : Baby Java Orange; MAE; Orange Peel, Pectin
INTRODUCTION
Orange is one of the most abundant commodities in the world. By 2013/2014, FAO reported that orange production was approximately 68.93 million tons and 21.16 million tons for processing (FAO, 2017).
According to the traded in 2009, sweet oranges (Citrus sinensis) accounted for approximately 60% of orange production for fresh fruit and processed juice consumption (Xu et al., 2014). Its production undergoes an enormous amount of orange peel going to waste. Baby java orange is one of the sweet oranges for processed juice consumption. This orange has a thick peel, about 44% (Martati and Ciptadi, 2020), and the potential to waste more leather than other sweet oranges.
Orange peel contains significant pectin (Boukroufa et al., 2015; Methacanon et al., 2014). The pectin content of orange peel is generally 20-30% in dry weight (Zioga et al., 2022). Pectin is an additive widely used in the pharmaceutical and food industry, such as jelly, jam, and dessert, as well as a texture- smoothing substance because it acts as a gelling agent (Chandel et al., 2022; Mada et al.,
2022). The annual demand for pectin reached 30,000 tons in 2009 and exceeded 60,000 tons in 2015 (Ciriminna et al., 2016).
This fact shows that the world's pectin demand is relatively high because the sweet orange characteristic differs from others, so there is enormous potential to develop pectin extraction from baby java orange peel waste.
Pectin could be extracted by several kinds of methods, such as traditional heating (conventional extraction), microwave-assisted extraction (MAE), ultrasound-assisted extraction (UAE), ultra- high pressure assisted extraction (UHPE) (Grassino et al., 2016; Guo et al., 2012;
Maran et al., 2013). Conventional extraction methods in pectin extraction take much longer. A long extraction time may degrade the molecular chain of pectin and eventually result in a low yield (Maran et al., 2013). However, unlike the case with UAE, UHPE and MAE have in common regarding extraction time where extraction time is short. This research used MAE because it is the most suitable one compared to other extraction methods.
66 MAE takes less time, reduce solvent use, and less amount of money (Amarante et al., 2020;
Maran et al., 2013). MAE uses microwaves to accelerate extraction through quick and efficient solution heating (Alam et al., 2019;
Eskilsson and Björklund, 2000). A microwave (MW) is an electromagnetic wave of two oscillating perpendicular fields, i.e. electric and magnetic. Later applications commercially utilise frequency bands at 0.915 and 2.450 GHz, where most microwave ovens operate at 2.450 GHz (wavelength, 12.2 cm;
energy 0.94 J/mol) (Noor et al., 2021).
The pectin extraction time of various commodities has been widely studied and generally has a significant effect on yield and pectin levels (Chan and Choo, 2013; Faravash and Ashtiani, 2007; Kliemann et al., 2009;
Masmoudi et al., 2008). Pectin extraction needs fast extraction to avoid many impurities at alcohol precipitate and pectin hydrolysis (Garna et al., 2007). Therefore, the extraction time is the factor studied in this pectin extraction. Besides extraction time, other factors such as suitable pH, volume, and concentration of acid solution play a significant role in pectin extraction (Faravash and Ashtiani, 2007; Garna et al., 2007; Pinheiro et al., 2008), so it need a sufficient volume of the acid solution, in appropriate concentration, to soak the samples during the extraction process (Mandal et al., 2007).
Protopectin in the stem can be converted to water-soluble pectin in the hot acid environment (acid hydrolysis) and then extracted. Acid medium destroys the linkage between the carboxyl group and the bivalent cation of the molecular chain, which creates the water-soluble pectin molecular (Wang et al., 2014). This research used the weak organic acid solution (citric acid), which is more environmentally friendly, to avoid the utilises environmentally harmful solvents (Zanella and Taranto, 2015).
This study explored the effect of extraction time and baby java orange peel powder – acid solution ratio on extracted pectin by the MAE method using a citric acid solution.
METHOD
The main material used was baby java orange (Citrus sinensis) peel waste from
Selorejo, Malang, Indonesia. It was selected from baby java orange with a weight range of 165 to 187 g, green until yellowish green in colour, a thickness of peel 3.1-3.5 mm, maturity level being, and free from damage.
Baby Java Orange Peel Powder Preparation
After washing, baby java orange peel was cut into 1 x 1 cm and then steamed and boiled for 15 min to soften the stem of baby java orange peel and dried at 60°C for 24 h in a dryer cabinet (laboratory modified instrument) to reduce the water content.
The dried peel was ground by blender and then sieved 40-mesh. Peel powder was analysed for yield (Li et al., 2012) and moisture content (AOAC, 1995; Wilhelm et al., 2004).
Pectin Extraction from Baby Java Orange Peel Powder (modification of Huang et al.
(2010))
10 g of baby java orange peel powder was dissolved in a different volume of 1.38 M citric acid solution (pH 1.5). The differences volume was set as a ratio between baby java orange peel powder (solid) to extractant citric acid solution (liquid), known as the solid/liquid ratio.
Solid/liquid ratios of 1:10, 1:15, and 1:20 (w/v) were investigated in this research.
The mixture was stirred using a magnetic stirrer for 15 minutes and then extracted using the microwave for 5, 10, and 15 minutes at 2450 MHz. The extract was filtered, and the pulp-free filtrate was coagulated by 1:1 of 96% ethanol for two h.
Pectin was separated by filter cloth, then washed with 96% ethanol 1:1 and dried at 55 °C for 12 h in the oven. The dried pectin was stored in a dark container for some pectin analysis, such as moisture content (AOAC, 1995; Wilhelm et al., 2004), equivalent weight, and methyl content (Owens et al., 1952). All of the treatments were repeated three times. From the best combination of the ratio between baby java orange peel powder to extractant citric acid solution and extraction time, pectin was an analysis for Fourier Transform Infra-Red spectrophotometer (FTIR) (8400S/
Shimadzu IRPrestige 21).
67 Equivalent Weight (Owens et al., 1952)
The values of equivalent weights were used for calculating the anhydrobiotic acid (AUA) content and the degree of esterification. Equivalent weight was determined by weighing 0.5 g pectin in a 250 mL conical flask and moistening it with 5 mL of ethanol. 1 g of NaCl was added to sharpen the endpoint. 100 mL of free carbon dioxide distilled water and six drops of red phenol indicator were added. The mixture was then stirred rapidly to ensure that all the pectic substance had dissolved and no lumps were retained on the sides of the flask. Titration was done slowly (to avoid possible de- esterification) with 0.1 N standardised NaOH until the indicator's colour changed to pink (pH 7.5) and persisted for at least 30 seconds.
The neutralised solution was used for the methoxyl determination. Equation (1) was used to calculate the equivalent weight (EW).
EW= sample weight (g) × 1000
volume NaOH (ml) × N NaOH………(1)
Methoxyl Content (Owens et al., 1952)
25 ml of 0.25 N NaOH of methoxyl (MeO) was determined by adding to the titrated solution, which was then shaken thoroughly and allowed to stand for 30 min at room temperature in a stoppered flask.
Twenty-five mL of 0.25 N HCl was added and titrated to the same endpoint (pink). Equation (2) was used to calculate the methoxyl content (MC).
MC=ml NaOH × N NaOH × 31
mg sample x 100%……(2)
Structural Analysis
A solid sample is mixed with a Potassium bromide (KBr) ratio of 1:10. The mixture is crushed and then pressed into KBr pellets. The KBr pellet is placed on a tablet holder and then measured with an FTIR. FTIR spectra were used to obtain information on chemical structure with wavelengths ranging from 4,000-400 cm-1. FTIR spectra result compared to standard pure pectin from literature to know the proximity of the pectin extract functional group to the standard.
Statistical Analysis
The experiment was arranged based on a completely randomised design in three replicates. The results were analysed by analysis of variance (ANOVA) using Statistical Program for Social Science (SPSS) 17.0. Differences were considered statistically at P≤0.05. Duncan Multiple Range Tests were conducted when there was a significant difference between the treatments. The Least Significant Difference (LSD) test was conducted when there was no interaction between the factor. To determine the most suitable treatment, the researcher compared the result of the data analysis and the parameters. Multiple Attribute was used to determine the most suitable treatment (Zeleny, 1982). The control group is determined by comparing the result with other research.
RESULTS AND DISCUSSION Pectin Yield
The pectin yield of baby java orange peel using the MAE method significantly differed with advancing extraction time and baby java orange peel powder to acid solution ratio (Figure 1), between 22.33%
and 51.3%. The length of extraction time for pectin affects the pectin characteristics. The longer extraction time, more pectin yield is produced. During 10 to 15 min experienced, a significant increase in yield.
The extended contact between material and solvent will provide a more significant opportunity to hydrolyse the protopectin in materials to increase the yield of pectin (Mada et al., 2022; Sayed et al., 2022).
Figure 1. The average pectin yield of baby java orange peel where material: acid solution ratio and extraction time became the factor chart
68 The 1:10 ratio produces a constant yield at any given time, while the ratio of 1:15 has the most yield results because of the energy and time spent long enough to extract material.
The yield generated on the variation of 1:20 with a wide range of long extraction times does not change significantly. In addition, variations in the ratio of 1:20 produce a yield of at least compared to the variation ratio of 1:10 and 1:15. According to Vayupharp and Laksanalamai (2015) and Zhang et al. (2018), at the highest ratio variation, excessive solvent volume requires a longer time in the extraction process.
Adetunji et al. (2017) reported that solid /liquid ratio and time extraction were necessary for pectin yield. The results of the present study by Adetunji et al. (2017) and de Oliveira et al. (2015) were similar to our findings. Increasing the solid/liquid ratio between 1:10 and 1:15 caused an increasing volume of citric acid solution. The citric acid solution volume is directly proportional to the medium disperses heat. This leads to acid penetration in hydrolysing protopectin. Baby java orange peel powder becomes easier pectin so that the yield increases. However, a solid/liquid ratio increase of up to 1:20 causes a decrease in pectin yield. Citric acid, a weak acid, is not fully ionised in water. It is suspected that the higher volume of the acid solution, the more H+ and free acid ions, allowing the resulting pectin to undergo further hydrolysis to pectic acid.
Contrary to Kulkarni and Vijayanand (2010), a strong acid obtained the highest yield at a solid/liquid ratio of 1:30. The pectin yield also increased with increased extraction time. The same results were obtained by Bagherian et al. (2011) and de Oliveira et al.
(2015). Theoretically, microwave radiation loosens the cell wall matrix. It leads to the severing of the parenchymal cells (Kratchanova et al., 2004). The longer extraction time, more skin tissues are rapidly and extensively opened up by the microwave (Bagherian et al., 2011). However, a higher solvent would decrease the microwave adsorption of material because the solvent absorbed more energy. Therefore, the break of the material cell wall and mass transmission might be negatively influenced (Belkheiri et al., 2021; Mahmoud et al., 2022) and decrease the pectin yield.
Moisture Content
The moisture content of baby java orange peel pectin using the MAE method was between 5.73% and 9.83% wet basic (Figure 2). The moisture content is less than 12%, meeting the International Pectin Procedures Association (IPPA) requirements. The 1:10 ratio showed no increase in moisture content either at 5, 10, or 15 min of extraction time. At a 1:15 ratio showed an increase in moisture content from 5 to 10 min, then decreased at 15 min.
Meanwhile, at a 1:20 ratio, the moisture content increased from 10 to 15 min. This shows that extraction time influenced the increase in moisture content of pectin.
Longer extraction times can increase pectin hydrolysis, resulting in more degradable and shorter pectin chains (Maneerat et al., 2017).
Figure 2. Average water content (%) of baby java orange peel pectin where material: acid solution ratio and extraction time became the factor chart
Interactions between the old premises and materials extraction time ratio show a significant influence (p-value <0.05). The variations of materials to acid solutions ratio at 1:10 and 1:15 unchanged the moisture content significantly. However, moisture content increase at a 1:20 ratio.
The moisture content of pectin from baby java orange peel powder was significantly affected by the solid/liquid ratio and extraction time. Commonly, the results showed an increasing solid/liquid ratio affected by increasing moisture content, except for a ratio of 1:10. More volume of solvent contributed to the water- pectin bond (it might be monolayer or
69 capillary water) so that some of the pectins extracted still contain the water. The more volume of water, the possibility of more impurities that bind to the water that dissolves in the extraction process so that the extract still contains water. Increasing time extraction also affected increasing moisture content except for 15 min.
Equivalent Weight
Pectin equivalent weight measures free galacturonic acid content or unesterified in the pectin molecular chain (Wathoni et al., 2019). The average equivalent weight of baby java orange peel pectin extracted using MAE with the treatment of extraction time and the ratio between materials and acid solution was between 542.56 and 775.41. ANOVA with a 95% significance level (α = 0.05) showed that the material and solution ratios and extraction time (Figure 3) had significantly influenced the equivalent weight.
Figure 3. The average equivalent weight of baby java orange peel where material: acid solution ratio and extraction time became the factor chart
However, the results showed that the interaction between the two factors was not significantly different toward equivalent weight because the p-value was more than 0.05. The extraction time of 5 min and 10 min did not have a significant difference in equivalent weight, but at 15 min, the equivalent weight of pectin increased. The same effect was on the ratio where the 1:10 and 1:15 ratios did not differ significantly, but equivalent weight increased at a 1:20 ratio.
The increase in equivalent weight is thought because of the acid hydrolysis process.
According to Lekhuleni et al. (2021), the
increase and decrease in equivalent weight depend on the amount of free (non- esterified) galacturonic acid. A high equivalent weight will produce a high gel.
Otherwise, low will have low gel formation because pectin will be highly degraded.
The equivalent weight of pectin was the total content of free galacturonic acid (not esterified) in the molecular chains of pectin (Wignyanto et al., 2014). The equivalent weight measured anhydrobiotic acid (AUA) content and esterification rate (Ismail et al., 2012). The longer extraction time and higher solution volume during the extraction process, the more heat it produced, so increasing the non-esterified carboxyl content happened. The esterification process is determined by the precipitation process using ethanol. The replacement of the H atom of galacturonic acid, a pectin unit by a methyl group, can be prevented by water bound to the galacturonic acid polymer. The water- bound increase in the galacturonic acid may cause much free galacturonic acid.
According to the pectin standard, the equivalent weight of pectin was in the range of 600-800 (International Pectin Producers Association), between 542.56 and 775.41.
Methoxyl Content
Results of ANOVA with a 95%
confidence level (α = 0.05) showed that the material and acid solution ratio does not have a significant impact (p-value >0.05).
The average methoxyl content of baby java orange peel pectin can be seen in Figure 4.
There is no interaction between the material and acid solution ratio with the extraction time, where the p-value is more than 0.05.
The material and solution ratio does not affect the results of methoxyl content.
However, long extraction times affect the results of methoxyl content. Methoxyl content increases with increasing extraction time because more free carboxyl groups are esterified to methyl esters which can increase methoxyl levels in pectin (Parasu et al., 2021).
In microwave, the transfer of energy follows two mechanisms: dipole rotation, involving the reversal of dipoles in polar molecules, and ionic conduction, involving
70 the displacement of charged ions present in the solvent (Routray and Orsat, 2012). One advantage of this heating type is the medium's homogeneous temperature distribution. Venkatesh and Raghavan (2004) noted that conventional thermal processing has no temperature gradient.
Figure 4. Average methoxyl content (%) of baby java orange peel pectin where material:
acid solution ratio and extraction time became the factor chart
Methoxyl content is determined by the degree of esterification (DE). DE is the percentage of carboxyl groups esterified with methanol (Santos et al., 2013; Seixas et al., 2014).
This research used ethanol to esterify the galacturonic acid as a pectin unit to give an alternative alcohol kind in the esterification process. Chain length and degree of methylation were critical in determining the properties of pectins, particularly gel formation. Ultimately esterified pectins would have 16% methoxyl content but do not occur naturally. The usual range is 9 to 12% ester methoxyl, although some pectin may have a shallow methoxyl content (deMan, 1999).
International Pectin Producers Association divided the methoxyl content of pectins into two parts. Low Methoxyl Pectin (LMP) contained 2.5-7.2%, while High Methoxyl Pectin (HMP) contained more than 7.2%. The longer extraction process, more pectin dissolves in the solvent. The amount of methoxyl pectin extracted from the acid hydrolysis process will accumulate in the esterification process using ethanol, thereby increasing the amount of methoxyl in pectin.
The results showed that the pectin was included in the LMP type, which was between
4.17-6.49%, with a degree of esterification lower than 50%.
Functional Group of Extracted Pectin In this study, we choose an extraction time of 15 min and a ratio of 1:15 as the best treatment based on the Zeleny method (Masud and Ravindran, 2008). The combination resulted in 51.3% yield, 10.80%
water content, 5.00% ash content, 775.4 grams equivalent weight, and 6.49%
methoxyl content. It resulted in the highest yield, equivalent weight, and methoxyl content compared to other factors.
Galacturonate acid was identified after selecting the most suitable treatment; the galacturonate acid content was 59.54%. The substance played an essential role in determining the functional characteristics of the pectin solution. The level of the galacturonante acid may influence the structure and texture of pectin gel (Constenla and Lozano, 2003).
The FTIR spectrum of pure pectin is shown in Figure 5. The spectrum peaked at 3,402 cm-1 due to the stretching of ―OH groups. The peak at 2,939 cm-1 indicated C―H stretching vibration. The peaks at 1,460 and 1,332 cm-1 could be assigned to ―CH2
scissoring and ―OH bending vibration peaks, respectively. The peak at 1,010 and 1,647 cm-1 suggested ―CH―O―CH―
stretching and C＝O stretching vibration peaks. The peak at 1,157 cm-1 suggested the presence of ―CH―OH in aliphatic cyclic secondary alcohol (Coimbra et al., 1998;
Synytsya et al., 2004).
The FTIR spectrum of the best baby java orange pectin is shown in Table 1. In Figure 5a, the spectrum peaked at 3,444.63 cm-1 due to the stretching of ―OH groups.
The peak at 3,010.67 cm-1 was due to the
―CO―CH3 groups and C-H vibrational modes, including CH, CH2, and CH3
stretching and bending vibrations (Chen et al., 2014; Williams and Fleming, 1995). The group specifically showed methoxyl content.
The peak at 2,634.58 cm-1 is due to the
―COOH groups. The peak at 1,394.44 cm-1 was due to the ―COO― (carboxylate ions) and ―OH bending. The peak at 1,141.78 cm-1 suggested the presence of ―CH―OH in aliphatic cyclic secondary alcohol.
71
Figure 1 The Comparison of FTIR spectrum of pure pectin (Mishra et al., 2009) (a); baby java lemon best pectin (simplo) (b); and the best Pectin FTIR spectrum (simplo) and best pectin (duplo) (c)
Table 1. Result of FTIR spectrum of best treatment pectin (functional group of extracted pectin) Wavenumber (cm-1)
Interpretation Type of Vibration No. Node a Node b
Standardized Pectin (Mishra et al., 2009)
Data Reference (Williams and Fleming, 2007)
1. 3444.63 3388.70 3402 3200-3650 –OH (alcohol, phenol) Stretch
2. 2634.58 2632.65 2939 2850-3000 C–H aliphatic Stretch
3. 1394.44 1394.44 1460 1370-1465 –CH2 aliphatic
4. 1217.00 1215.07 1332 1260-1410 –OH Bending
5. 1078.13 1078.13 1010 1000-1300 –CH–O–CH– (alcohol, eter, ester, and carboxylic acid)
Stretch
6. 1647.10 1645.17 1647 1615-1650 C＝O Stretch
7. 1141.78 1141.78 1157 1000-1300 –CH–OH
72
Table 2. Comparison between the most suitable treatment and the control Testing Parameter Standardized Quality of
Pectin (IPPA)
Most Suitable Treatment (MAE)
Maceration (Meilina and Sailah, 2005)
Yield - 51.3% 23.12%
Moisture content Max. 12% 10.8% 11.08%
Ash content Max. 10% 5% 1.27%
Equivalent weight 600–800 775.41 2429.95
Metoxyl content Low 2.5–7.12%
High >7.12%
6.49% 6.05%
Galacturonate content Min. 35% 59.54% 48.61%
Similar to Figure 5a, Figure 5b also showed a similar result of absorption. The spectrum peaked at 3,388.70 cm-1 due to the stretching of ―OH groups. The peak at 3,002.96 cm-1 was due to the ―CO―CH3
groups and C-H vibrational modes, including CH, CH2, and CH3 stretching and bending vibrations (Chen et al., 2014; Williams and Fleming, 1995). The group specifically showed methoxyl content. The peak at 2,632.65 cm-1 was due to the ―COOH groups. The peak at 1,394.44 cm-1 was due to the ―COO―
(carboxylate ions) and ―OH bending. The peak at 1,141.78 cm-1 suggested the presence of ―CH―OH in aliphatic cyclic secondary alcohol. In this study, the results of the comparison between the most suitable treatment and the control can be seen in Table 2.
CONCLUSION
The material and acid solution ratio and extraction time of pectin from baby java orange peel significantly influenced the yield, water content, and ash content. Still, there was no significant influence on the equivalent weight and methoxyl content. The most suitable baby java orange peel pectin was obtained when the ratio of material: the acid solution was 1:15, the extraction time was 15 min, and the treatment resulted in a 51.3%
yield. The chemical parameter result involved 10.8% water content, 5% ash content, 775.41 grams of equivalent weight, 6.49% methoxyl (low methoxyl), and 59.54% galacturonate content. IR Spectra showed that the functional group supports the pectin.
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