Introduction
The large amount of food waste worldwide has a massive impact on the raw materials  in food production, creating a global environmental and social imbalance that affect low-income countries the most[1]. In the UK alone, 9.5 million tonnes of food waste is produced, not only releasing 36 million tonnes of CO2 emission equivalents, but also costing the government £19 billion[2]. It does not help that 70% of this waste could have been eaten, in a world where up to 783 million people have limited access to food[3]. This also does not fall in line with the Sustainable Development Goal (SDG) target 12.3 (‘By 2030 halve per capita global food waste… and reduce food losses along production and supply chains, including post-harvest losses’) where it has only reduced by 13.2% globally[4], and with less than 7 years left of the SDGs implementation, drastic changes must happen in the near future in order to reach this target.
Approximately 14% of food produced is lost between harvest and retail[5], and in the case of fruits this is due to either processing before being put on retail, purposely thrown away due to the products aesthetics or spoiling before reaching retail[6]. Processed fruit generates 2 types of waste: liquid waste, as a result of juicing and water used to wash the fruit, and solid waste, for example the peel that equates to around 15 to 60% of the solid waste[7]. The solid fruit waste can be valorised by extracting compounds that can be used in multiple industries, e.g. semisolids, i.e. creams or ointments, with oils extracted from the seed[8].
Mangoes (Mangifera indica) are an interesting fruit with high solid waste valorising potential; there are many cultivars of mangoes harvested around the world, such as Alphonso from India and Tommy Atkins  from the United States of America. They are a fruit growing in popularity, with 43,000 tonnes being imported into the UK in 2022[9]. The by-products of mango processing totals 35-60% of the weight of the fruit (peel, kernel, seed)[10], and contains several extractable compounds, including antioxidants such as xanthone C-glycosides[11] and dietary fibres[12]. With each variety of mango having different amounts of extractable compounds, specific species can be exploited for valorisation in different industries. One extractable compound, with potential use in multiple industries, that all mangoes contain a large amount is pectin.
Pectin is a polysaccharide used by plants for cell wall adhesion[13] and has influences in other parts of  plants morphogenesis such as root elongation[14]. It is a family of galacturonic acids derived from the monomer D-galacturonic acid, a uronic acid; with the cell walls of mango peel containing approximately 52% uronic acid[15], there is large prospects for valorising the peel for galacturonic acids, with potential to compete with the main sources of commercially available pectin, which are citrus peel and apple pomace[16]. Homogalacturonan (HG) is the main monomer of the polysaccharide chain, and they can be methylated on C6 or acetylated on O2 or O3 on the carbohydrate ring to form esters[17]; some of the C6 esters can be unmethylated, leaving a negative charge, which can interact with calcium ions that helps support the adhesion of pectin chains and gelatinisation, dubbed the ‘egg-box’ model[18], as shown in Figure 2. This gelling effect caused by the HG-calcium ion interactions means that when extracted, pectin has applications in the food industry as a thickening agent in jams, jellies, and milk products[19], making it a non-meat alternative to gelatine for the diets of people cannot consume specific meat products, which includes Muslims, Hindus, vegetarians and vegans. It can also be used in medicine, for example to coat drugs and formulate capsules that are pH dependent for delivery to specific parts of the body[20]. 
Pectin extraction has been done industrially since the late 20th century via acid stewing, but it is not very efficient;. the vat required to heat up the water and maintain the temperature for several hours requires a lot of energy[22], in addition to the acid that can potentially produce unwanted byproducts, thus decreasing the yield and quality of the pectin that can be extracted. Microwave technology can breakdown the mango peel without the need for acid, as well as requiring less energy to heat the mango[22], making it a viable method replacement of acid extraction. Microwave-assisted extraction has been researched on and trialled since the 2000’s[23, 24], with one of the more noteworthy papers looking at a Life Cycle Assessment (LCA) of extracted pectin from citrus peel at pilot scale[22]. 
In 2009, the FAO published a pectin characterisation tests and purity parameters in the JEFCA Monographs[24], with purity tests including loss on drying, galacturonic acid contents and nitrogen contents, setting requirements to ensure safety for human consumption. While at a laboratory-scale study, the parameters cannot be tested in the required method due to the amount of pectin needed being unable to be made in a small timescale , approximations of some can be found through other methods that require much less yield. 
The first aim of this study is to show a comparison between acid and microwave-assisted extraction, showing that microwave technology is a feasible alternative to the traditional method. A second aim of this study is to obtain approximate purity values of parameters that can be tested on a larger batch of pectin, and to propose if a move to a larger scale of research is needed. This batch will be extracted via the most optimal conditions determined by the initial comparison. The final aim of this study is to compare the results obtained from this research to the results from this paper[22] and suggest whether mango peel is viable competitor of citrus peel for pectin extraction.

 
Methods
Materials                                                                                                                                                    Keitt mangoes, from Brazil, were sourced from a local supermarket and processed to acquire the peel via a sharp knife, with the flesh removed with a  stainless steel spoon. From 10 mangoes, 817g of peel was obtained.
All chemicals used, i.e. ethanol, HCl, H2SO4 and phenolphthalein, were purchased from Fischer Scientific and used without further purification. 
Pectin extraction and purification                                                                                                                                                                        Pectin extraction was attempted using the traditional method (acid stewing) and microwave-assisted extraction. The traditional method consisted of  a 1hr reflux on 10g of peel in aqueous HCl. 4 separate reactions took place , with HCL concentrations of 0.01M and 0.03M, at peel to solvent ratios of 1:30 and 1:10 respectively, tested at temperatures of 85⁰c and 100⁰c. The microwave-assisted extraction uses a CEM MARS 6 extraction system, in which 5g of peel and 50ml of water was put into each easy prep plus vessel with a stirrer flea. 4 different temperatures were tested (100⁰c, 120⁰c, 140⁰c, and 160⁰c), with the power set to 600W. 
Both methods resulted with aqueous solutions containing pectin. Residual mango is filtered out with a sinter funnel before isolating the pectin from the solution with addition of excess ethanol (c.a. 2.5:1 ratio of ethanol to water) under rapid stirring for 20 minutes. The solution was left overnight for maximum precipitation, before centrifugation for further isolation, which was done with a Thermo Megafuge Centrifuge at 3900rpm for 20 minutes (acceleration value at 9 and deceleration value at 3), with falcon tubes containing 40ml of a 5:3 ratio of solution to ethanol. The resultant pectin pellet was extracted and purified by boiling in excess ethanol for 15 minutes before hot filtering with a sinter funnel to obtain an off-white solid. The solid was put into the freezer for 4 hours before undergoing freeze-drying with a Labylo Freeze Drier to obtain the final product[10].                              
Characterisation of Pectin
Degrees of Esterification (DE) using infrared spectroscopy (IR)                                                                  Attenuated total reflectance IR (ATR-IR) was performed using a Perkin Elmer Spectrum 400 IR. The spectrum was taken at a range of 4000cm-1 to 650cm-1 with a blank background reading. After the spectrum is produced, it is converted to absorbance, from transmittance, and with the areas of the ester and acid peaks calculated (c.a. 1750cm-1 and 1600cm-1 respectively), the DE was determined with the following equation (Eqn 1):                                                                                                       DE (%) = (Ester area)/(Ester area+Acid area)×100
Degrees of Esterification via titration (DEt)                                                                                            500mg was moistened with 2ml of ethanol before being dissolved in 100ml of deionised water. 5 drops of phenolphthalein was added before subjecting the solution to a titration with 0.5M NaOH (result denoted as initial titre). Thereafter 10ml of 0.5M NaOH was added and mixed vigorously, with the solution allowed to stand for 15 minutes. 10ml of 0.5 HCl was added after, and the solution was shaken until the pink colour disappeared. 5 drops of phenolphthalein was added to the solution and it was titrated again with 0.5M NaOH to a faint pink colour persisted after shaking (end point). This was recorded as the final titre. The DEt was calculated with the following equation (Eqn 2):                                                                                               DEt (%) (Equation 2) = (final titre)/(initial titre+final titre)×100[25]. 
Galacturonic acid contents                                                                                                                     100mg of pectin (denoted as W) is mixed with 1ml of 72% H2SO4 and subjected to shaking in a water bath (40⁰c), being stirred every 5 to 10 minutes for 2 hours with a glass stirring rod. Thereafter, 28 ml of deionised water is added to the mixture, before autoclaved for 1 hour at 121⁰c. The resulting hydrolysed sample is filtered through a sinter funnel, and the liquid is further filtered through a RC 0.22µm filter dish before subjected for galacturonic acid analysis via HPLC[26].
Loss on drying                                                                                                                                                             25mg (denoted as M0) of the sample in a sample vial is placed in a biomass drying oven at 105⁰c for 2 hours. After leaving to cool until the sample reaches room temperature, the mass is recorded after (denoted as M1) and the difference is calculated with the subsequent equation (Eqn 3):                   Loss on drying (%) = (M0-M1)/M0×100 
Nitrogen contents                                                                                                                                            The percentage of nitrogen was obtained using CHN analysis with a Thermo Flash CHNS-O, and is given as a percentage.
Total insolubles                                                                                                                                    After filtration of the sample for galacturonic acid analysis, the mass of the sinter funnel with the hydrolysed solid is measured (denoted as W0). The solid was washed with deionised water before the sinter funnel is placed in a biomass drying oven for 1 hour at 105⁰c. The mass of the sinter funnel with the dried solid is measured (denoted as W1). The calculation can be performed with the equation (Eqn 4):  Insoluble pectin (%) = (W0 - W1)/W×100[26].    




















Results and Discussions
Entry	Conditions	DE (%)	Yield (%)
Acid reflux
1	85⁰c 	0.03M HCl	37.05	0.44
2	100⁰c	0.03M HCl	37.04	0.80
3	85⁰c	0.01M HCl	34.54	2.80
4	100⁰c	0.01M HCl	30.82	0.99
Microwave
5	100⁰c	32.09	0.02
6	120⁰c	43.16	0.33
7	140⁰c	42.73	2.07
8	160⁰c	33.24	0.36
Determination of the best method and condition
The degrees of esterification obtained from acid reflux conditions ranged from 30.82% to 37.05%, in comparison to microwave conditions which range from 32.09% to 43.16%, as shown in Table 1.      DE is defined as a percentage of the preservation of the esterified carboxyl bonds in comparison to the total amount of carboxyl bonds in pectin[28]. It is a measurement of pectin quality that is easily influenced by the extraction conditions, and as it can be determined quickly through IR, it makes for an ideal parameter to compare the two methods, and the different conditions for each of them. Pectin can be classed as either high methoxy pectin (HMP), where the DE is greater than 50%, or low methoxy pectin (LMP), in which DE is less than 50%. LMP’s are seen as more valuable due to gelation able to be achieved with calcium ions at a wider range of pH in comparison to HMP, which require an environment with high sugar content[28]; however a pectin with a low DE tends to be more brittle due to the dependency on calcium ions to maintain the gelation[29]. From Table 1, it can be concluded that entry 7 performed the best of any of the conditions tested as it shows a balance between DE and yield; the DE is low enough to meet the LMP threshold, but not too low that if left to form a gel, it is not too brittle, and the yield obtained is much larger than most of the other tested conditions. This was enough justification that the batch of pectin to be further characterised was extracted with this method and condition. It was interesting to observe that the highest yield was entry 3 was a 35% increase to what was recorded with the microwave at 140⁰c; an unexcepted result as it has been recorded that at more optimal conditions, microwave-assisted extraction should have a greater yield recorded in comparison to acid stewing[10, 22]. This led to a side experiment, in which 2 more microwave reactions at 140⁰c took place with the same amount of mango peel and 50ml of 0.01M HCl and H2SO4 solution instead of water, with the aim is only to see whether the replacement of water for weak acid solution would have an effect on the yield or DE.. Sulphuric acid is tested as it is another acid that can be used in acid extraction[30]. 
Microwave extraction with acid solution     
Acid	DE (%)	Yield (g)
HCl	50.6012	0.0521
H2SO4	54.7417	0.0738
The data in Table 2 shows that the addition of acid had increased DE to 54.7417% and 50.6012%, but decreased yield by approximately 64% and 75% for H2SO4 and HCl respectively.
The increase in DE indicates that the use of acid solution in the microwave vessels compared to deionised water aids in the preservation of the esterified carboxyl groups; this may have occurred as under acidic conditions, the ester bonds are less likely to hydrolyse compared to the glycosidic bonds that are located in the main pectic polysaccharide chain, and the microwave more likely enhances this effect with more even energy distribution in comparison to acid stewing. The decrease in yield could also have happened for the same reason, in which the combination of both is favoured for glycosidic bond hydrolysis; this leads to higher amounts of premature pectin chain termination, which is more solubilised in the acidic conditions and are not so easily recovered in the purification process. A higher DE for H2SO4 comes from the way the sulphate ions interact with the pectin environment, affecting the solubility and stability of the extractable polysaccharide in a way that aids the release from the peel. The reason that H2SO4 also recorded a higher yield than HCl is that it is a diprotic acid[31] instead of a monoprotic acid; the donation of 1 more proton per molecule, and the accumulation of these extra protons lead to more effective protonation of the ester linkages, which promotes hydrolysis of pectin to more extractable fragments. While these results are noteworthy, this experiment was to only see what effect the combination of acid solution and microwave technology would do for pectin extraction, and has no bearing on the overall conclusion of this report. 
Characterisation of pectin extracted from microwave at 140⁰c
Purity 	Criteria (%)	Results (%)
DE	-	41.7780
DEt	-	46.1034
Loss on drying	<12	11.0336
Total insoluble	<3	4.0527
Nitrogen content	<2.5	0.58
Galacturonic acid content	>65	37.2361
 The results of all of the JEFCA purity parameters plus DE is recorded in Table 3.
Degree of Esterification                                                                                                                        The DE, determined via IR, was recorded at 41.7780%, which is similar to what was observed with the result from Table 1, showing consistency with analytical precision. While IR is a quick method to obtain a DE value, there are a few limitations. an example is baseline drift, in which the line either side of the peak does not return to absolute 0, but will instead be shown as an elevated baseline. This can come across due to sample characteristics or atmospheric absorption, and affect the DE as a true value cannot be determined. Titrimetry is another method of DE determination, and while it is a more versatile method with less sensitivity to sample conditions than IR, it still has limitations, such as endpoint determination. The combination of both methods can be used for cross verification of results,  and as similar values are recorded, then there is an increase in the confidence that the true DE value is within the range of both values (41.7780 – 46.1034%). Comparing this to what was attained from citrus peel in Garcia-Garcia et al’s paper[22] (72.8%), both have a vast difference in esterification, and are clearly meant for different applications, with the citrus restricted to high-sugar jams and jellies, as well as less potential uses in medicine[29].
Loss on drying                                                                                                                                       Loss on drying was found to be at 11.0336%, which meets the threshold of 12% maximum loss of sample mass. This primarily shows that the pectin has a desirable quality and integrity that is crucial for applications in the food industry, due to excessive moisture in pectin that could lead to microbial growth or spoilage, and decreasing shelf life. The adherence of the pectin batch to the JEFCA requirements is indicative of the efficacy of the purification process, as it shows that the use of the freeze-dryer in the drying stage is removing the desired amount of moisture without overly dehydrating the pectin, which could potentially compromise quality and functionality. Again comparing this to the results in the LCA paper (8.98%), the difference shows that process that they undertook was more effective in drying out excess moisture out of the pectin that what was done here, which would result in a longer shelf life than the mango pectin.
Total insolubles                                                                                                                                      The recorded value (4.0527%) is above the 3% limit. This could be due to the formation of calcium pectinate (CaP) salts; With the egg-box model of pectin and calcium interactions, this facilitates the cross-linking that is formed between the ions and the HG chains, forming a three-dimensional structure. This forms a CaP gel[32], and with changes to the conditions, in time this will crystalise to form an insoluble salt. The presence of this salt will contribute to the total weight of the insoluble fraction. The contrast between this result and what was obtained in Garcia-Garcia et al’s paper[22] (0.29%), despite it only being approximately 3.7%, is quite significant, as one is far below the threshold and the other is far above it, showing a vast difference in purity. While the presence of CaP is not ideal for applications in the food industry, there has been research for use of these salts in medicine, for example in colonic drug delivery with silica-coated beads[33], in which the aim would be to exploit the conditions in the gastrointestinal tract to allow for conditioned release of drugs, showing that there is still potential use for this sample for a different application despite not meeting the JEFCA requirement.
Nitrogen content                                                                                                                                       The nitrogen content recorded in Table 3 falls well within the 2.5% maximum threshold from the JEFCA requirements. This shows that the pectin chains have a high level of purity, with little nitrogen-containing compounds present. Nitrogen contents mainly test for two types of compounds, the majority being protein insolubles; while most proteins would be removed in the extraction and purification process, it is feasible that not all would be removed. These are considered impurities, and can affect the gelling properties of the pectin. The other compound that could be present at higher nitrogen content are alkaloids. These are naturally occurring toxic amines that are produced by plants as a defence mechanism, and consumption of these amines can lead to disruption in multiple parts of the body, such as the central nervous system[34]. It is important to test for nitrogen contents as  a result over the threshold means a possibility of the presence of alkaloids, making it unsafe for food and medicinal applications. 
Galacturonic Acid Content                                                                                                                    As shown in Table 4, HPLC analysis determined galacturonic acid content to be 37.24%.                 
Contents	%
Galacturonic acid 	37.24
Cellulose	23.55
Xylose/Fructose/Mannose/Rhamnose	11.19
Arabinose	16.79
Levoglucosan	7.08
Others	4.19
 This is significantly lower than the minimum requirement set by JEFCA (>65%), and what was obtained in Garcia-Garcia et al paper on citrus peel (72.3%). A reason for this could be the raw material that was used, in which Keitt mangoes may have inherently low levels of galacturonic acid content; the results suggests this as a high presence of other sugars can be seen; 23.55% of the sample is the cellulose, which can be extracted and used as a dietary fibre[35]. 27.98% of the contents are sugars that are present in hemicellulose (Fructose & Mannose) and ‘hairy’ pectin (Xylose, Rhamnose & Arabinose). Hemicellulose are matrix polysaccharides that form hydrogen bonds to cellulose microfibrils[36] to help strengthen the cell wall. They can be formed by different monosaccharides, and can also be used as a dietary fibre[37]. ‘Hairy’ pectin is a region of the chain that has branching of other monosaccharides, as shown in Figure 3. This can disrupt the gelling behaviour of the pectin as they can interfere with the ‘egg-box’ interactions of calcium ions and the ‘smooth’ galacturonic acid pectin region. Levoglucosan is a product of cellulose degradation[38], whereas the results denoted as ‘Others’ in Table 4 are not, they are degradation products of galacturonic acids; these show that the microwave produces an unwanted side effect of breaking down polysaccharide chains, which can reduce purity of the sample and the yield obtained. The presence of all of these products at such a high proportion, in comparison to how little galacturonic acid there is in the sample shows a high level of impurity that no other JEFCA parameter has been able to measure, and presents the case that it is not ready to move to higher scale research.
Yield                                                                                                                                                     0.977g of pectin was extracted from 60g of Keitt mango peel, equating to a yield of 1.62%. Comparing to the literature, in 2016 paper by Avtar et al [10], a dry weight yield of 11.63% from 20g of dried mango peel, and in the LCA paper, 150g of pectin was extracted from 3kg of citrus peel, which reflects an extraction efficiency of 5%. What must be noted is that the pectin from this research was obtained through undried mango peel, which impacts how much can be extracted due to the high moisture content of fresh peel, which vary between ~59 to ~69% in different mango cultivars[39]. Based on these moisture numbers, a theoretical yield from dry peel can be obtained using the mean moisture content (64%) by finding the dry mass of the fresh peel and obtaining a yield percentage based on that; using this, the dry peel mass would be 21.6g (60g×0.36=21.6g), meaning yield would have been 4.61%. This is quite comparable to the citrus peel, but upscaled extraction to match the amount of peel could lead to a greater difference of yield. What this does is bring up the question of whether it is worth it to valorise Keitt mango peel for pectin as the primary source, or to obtain it as a side product, for applications in the food industry. Looking at the HPLC results, 23.55% of the 0.977g of pectin was actually cellulose as an unintended side product, and with knowledge that optimal cellulose extraction efficiency of mango peel has been recorded at 44.6%[40], it seems that it would be better to have cellulose as the main extraction product of Keitt mango peel, and to see whether pectin can be extracted as a side product.

Conclusion
The use of microwaves as a means to extract pectin from mango peel is a viable alternative and potential replacement of the traditional acid extraction method, with more optimal conditions providing the best compromise between DE and yield (42.7322% and 0.2071g respectively), showing the potential of microwave technology for valorisation of food wastes for biobased chemicals and materials. The data also shows that the source is not be a viable competitor of some of the main sources of pectin, such as citrus peel, but there is potential to compete with other LMP sources; this would need more research to optimise as the data obtained from the research also shows that using Keitt mango peel as a source of pectin for applications in the food industry is not a viable source, as some of the purity parameters  that JEFCA have set to regulate commercial pectin production (galacturonic acid contents and total insolubles) have not been met. There might be potential use for the pectin for medicinal uses instead, with some of the total insolubles potentially being CaP salts. However further research would need to occur to confirm the presence of the salts. 
In the comparison of the acid reflux reactions against the microwave reactions in this research paper, an unexpected result of acid reflux having a higher yield was observed, why would this have happened?