DESC:AN INTEGRATED PROCESS FOR RECOVERY OF PECTIN AND A SUGAR-POLYPHENOL MIXTURE FROM HORTICULTURAL WASTE
TECHNICAL FIELD
[001] The present disclosure described herein, in general, relates to recovery of pectin and a sugar-polyphenol mixture from mango processing waste.
BACKGROUND
[002] Generally, the fruit peel pectin extraction methods used often lacks an emphasis on the by-products. Conventionally, pectin is extracted in an acid hydrolysis process. The peel slurry is heated at (90–120) °C for a minimum of 2.5 h. Among acids, particularly mineral acids, such as nitric acid, hydrochloric acid and sulphuric acid are used. Microwave and sonication assisted extraction are found to be good alternatives to conventional extraction. In the reported studies, it is seen that, microwave power of 413 W exposure to fruit peel slurry in presence of acid (pH 2.7) for 134 seconds helps in recovery of 28.8% pectin. A variety of fruit peel was used in a study, in which heating the slurry for 90 min at 90°C in presence of acidified water (pH 2.5) recovered around 16.6% of pectin. Ultrasonication (180W) under the same experimental condition resulted in a reduced duration of 20 min, which helped in extraction of 15.8 percent of pectin. The degree of esterification (DE) >65% was obtained in both conventional and ultrasonication methods. Apart from high DE pectins, low DE pectins are also obtained from fruit peels with the help of lemon juice assisted sonication.
[003] In the acid based conventional processes, often the pectin is contaminated with traces of acid. Apart from that, the by-product of this process generates large volumes of acidified water, which requires additional treatment before disposal. The pectin wastewater contained 10,000-12,000 mg/L COD (chemical oxygen demand) and 5,000-6,000 mg/L BOD (biological oxygen demand). As per the regulatory requirement, the defined range for chemical parameters prior to discharge are; BOD < 15 mg/L, nitrogen < 8 mg/L, suspended solids < 20 mg/L and pH– 6.5-7.5. It may be observed in the above study that industrial food by-product effluent regulations are strict and thus, considerable efforts are required to treat such waste streams.
OBJECT OF THE INVENTION
[004] It is prime object of present disclosure to provide an integrated method to extract pectin from a horticultural waste.
[005] It is another object of present disclosure to provide an integrated method to extract polyphenol from a horticultural waste.
[006] It is yet another object of present disclosure to provide multiple coproducts during extraction process.
SUMMARY
[007] In one aspect of the present disclosure, an environment-friendly hydrothermal process to extract pectin from a horticultural waste, namely mango peels, at a reasonable temperature and pressure is disclosed. The mango peels are blanched in hot water at 90°C -100°C for 5 min. Further, the blanched peels are dried at 50°C for 7 h in a hot air food drier until the constant weight is achieved. The dried peels are ground in a mixer grinder and the powder is passed through a 500µ size sieve. The dried powder is packed in an air tight container and stored at 4 ± 1°C until further use. A slurry of peel is prepared by mixing water and the dried powder. The ratio in the present invention may vary from 1:10 to 1:20 parts of solid to solvent, wherein a pressure range of 12 to 18 Psi at a temperature range of 115°C to 125°C is maintained. More preferably the slurry (8g of dry peel/160 ml distilled water) is treated under pressure (16.2 ± 2.1 Psi) and 121.0 ± 1.5 °C. As a control for the pectin yield, conventional process is performed on peel slurry where it is heated under reflux at 90°C for 2.5 h. The treated slurry is filtered through a nylon cloth and the solid residue is separated. The residual solids are dried at 105°C until constant weight is achieved and stored in air tight bags until further use. The viscous liquid is further centrifuged at 4500 rpm for 20 min to remove any suspended solids. The clear supernatant liquid is decanted and absolute ethyl alcohol is added in 1:1 ratio. Pectin is allowed to precipitate overnight, wherein the supernatant is precipitated overnight to filter the pectin mass from supernatant.
Further, distillation is conducted to recover alcohol from the mixture to obtain the resultant by-product liquid by distillation under vacuum at 45°C to separate/recover the alcohol from the filtrate using a rotary evaporator such that the concentrated filtrate obtained is rich in sugars and polyphenols. High average yields of pectin (15.7%, 27.2%) are obtained from the mango peels.
BRIEF DESCRIPTION OF THE DRAWINGS
[008] Figure 1 illustrates flowcharts for pectin extraction process and yield of pectin in accordance with the present disclosure.
[009] Figure 2, illustrates SEM images of original peel and residue after hydrothermal treatment in accordance with the present disclosure.
[0010] Figure 3, illustrates graphical analysis of degree of esterification (DE) of extracted pectins in accordance with the present disclosure.
[0011] Figure 4, illustrates molecular weight analysis of pectins in accordance with the present disclosure.
[0012] Figure 5, illustrates characterization of the pectins by using FTIR in accordance with the present disclosure
[0013] Figure 6, illustrates experimental analysis of gelling test for pectins in accordance with the present disclosure.
[0014] Figure 7, illustrates the analysis of carbon, hydrogen and nitrogen as detected in pectin co-products in accordance with the present disclosure.
[0015] Figure 8, illustrates concentration of polyphenols detected in pectin co-products in accordance with the present disclosure.

DETAILED DESCRIPTION
[0016] In one embodiment of the present disclosure, figure 1 illustrates flowchart for pectin extraction process as disclosed in the present invention. In order to perform the study, variety of mango peels are used. First, the mango peels are blanched in hot water at 90°C -100°C for 5 min. Further, the blanched peels are dried at 50°C for 7 h in a hot air food drier until the constant weight is achieved. The dried peels are ground in a mixer grinder and the powder is passed through a 500µ size sieve. The dried powder is packed in an air tight container and stored at 4 ± 1°C until further use. A slurry of peel is prepared from dried power of mango peels in water as 1:20 w/v (1g of solid to 20 ml of water). The solid to liquid ratio is optimized in a separate single factor study (effect of dilution on pectin yields) and solid to liquid ratio of 1:20 is chosen for this study. The ratio in the present invention may vary from 1:10 to 1:20 parts of solid to solvent, wherein a pressure range of 12 to 18 Psi at a temperature range of 115°C to 125°C is maintained. The natural pH of the prepared slurry are maintained to be in the range of 4-4.3 at 1:20 solid to liquid ratio. The slurry (8g of dry peel/160 ml distilled water) is treated under pressure (16.2 ± 2.1Psi) which is chosen on the basis of best yield for pectin when compared at different pressure levels) and 121.0 ± 1.5 °C. As a control for the pectin yield, conventional process is performed on peel slurry where it is heated under reflux at 90°C for 2.5 h. The treated slurry is filtered through a nylon cloth and the solid residue is separated. The solids are dried at 105°C until constant weight is achieved and stored in air tight bags until further use. The viscous liquid filtrate is further centrifuged at 4500 rpm for 20 min to remove any suspended solids. The clear supernatant liquid is decanted and absolute ethyl alcohol is added in 1:1 ratio. Pectin is allowed to precipitate overnight, wherein the supernatant is precipitated overnight to filter the pectin mass from liquid. Further, distillation is conducted to recover alcohol from the mixture to obtain the resultant by-product liquid by distillation under vacuum at 45°C to separate/recover the alcohol from the filtrate using a rotary evaporator; wherein the concentrated filtrate obtained is rich in sugars and polyphenols. The suspended gelatinous mass is filtered through cheese cloth, the filtered liquid is concentrated under vacuum and stored to quantify polyphenols and sugars. The filtered gel is dried under vacuum at 45°C until constant weight is achieved. The yield is calculated as the percentage of dry peel weight.
[0017] Figure 2 illustrates SEM images of original peel and residue after hydrothermal process. Scanning electron microscopy (SEM) is carried out on the peel samples before and after the treatment to assess the effect of hydrothermal treatment. The samples are applied to double-sided adhesive tape and coated with platinum (using a low accelerating potential (20kV) for 300s) before imaging. The micrographs of the treated peels are compared. Since the hydrothermal treatment (without acid) resulted in a significant yield of pectin from varieties of mangoes, further characterisation is performed on the pectin that is extracted through this processing pathway. The intact mango peels are observed to have a fibrous appearance as seen in the micrographs (Figures 2a and 2b). As compared to the intact peel, structural disruption is observed in case of the hydrothermally treated peels. The porous structure as observed in the SEM images maybe treated as an indication of the pectin removal. Further characterization of the residue may be required to determine its porosity and the application potential.
[0018] Characterization of extracted pectin
[0019] Degree of esterification (DE)
[0020] Figure 3 illustrates graphical analysis on degree of esterification of extracted pectin. The extracted pectin are found to have a high DE, in an order of >50%. The degree of esterification values for commercial pectin sample, Calypso and Totapuri peel pectins are found to be 81.4 ± 1.2%, 87 ± 1.9%, 77.6 ± 4.2%, respectively. The DE values are believed to be dependent on sources and methods of processing for pectin extraction. Many of the qualitative parameters such as viscosity and gelation mechanism is dependent on DE value. The hydrogen bonding and hydrophobic interactions are found to play a major role in the formation of entangled networks between solute and solvent (water). The hydrophobic interactions are mainly imparted by the presence of esterified groups in the network when sucrose is present in the matrix. The effect of the ester groups in the hydrophobic interactions is previously determined by analytical techniques such as X-ray diffraction and thermodynamic studies. The increase in DE value is found to reduce the free energy of junction zone formation thus, favouring the stable junction zone formation, which in turn is required to form stable gels. Therefore, all of the extracted mango peel pectins, which are highly esterified are expected to form gels at an acidic pH, in the presence of sugars.
[0021] Figure 4 illustrates Molecular weight analysis of pectins. The molecular weight of extracted pectins are found to be lower than the commercial pectin as shown in the table. The molecular weights are, however, larger as compared to the mango peel pectins, obtained by Matharu et al. (2016), in a microwave assisted extraction study for mango peels as reference. The Mw/Mn ratio, also defined as “polydispersity index”, is a measure of relative molecular mass distribution. The indices as obtained in the extracted pectins are monodisperse and similar to the previous reports (lies in the range of 1.7-1.9) for different peel samples (Matharu et al., 2016). One of the reasons for a lower PDI in extracted samples as compared to commercial citrus pectin is lower range of molecular mass distribution. The molecular weight may influence the gel formation of pectin, when incorporated along with sugars. The gelling tests are further explained in the section “gelling test for pectins”.
[0022] ATR/FT-IR analysis
[0023] Figure 5 illustrates the ATR/FT-IR results for the mango peel pectins. The characteristic bands around 3300-3500 cm-1 denote O-H stretching. The broad absorption at this region may occur due to the hydrogen bonding of hydroxyl groups and carboxylic acid dimers of polysaccharide. The band at around 2950 cm-1 may be assigned to the C-H stretching of carbohydrates that make up the pectin. The detection of esterified vs non esterified (methylated) functional groups may be found in the fingerprint region 1750-1350 cm-1. Both the commercial sample and the extracted pectins produced an intense band around 1740 cm-1. This can be assigned to the carbonyl group of the methyl esters in the pectin. The patterns are found to be well correlated to that of commercial pectin. The shape of two peaks at around 1740 cm-1 are found to be slightly different in case of commercial citrus pectin vs. extracted mango peel pectins. The Totapuri peel pectin is found to be similar to commercial grade in terms of the peak shape. A slight change is observed in case of Calypso variety. This may be due to the different degree of esterification of the extracted pectins. The Totapuri peel pectin degree of esterification value is found to be closer to the commercial grade pectin while the Calypso peel pectins are found to be slightly higher than the DE value of commercial grade. Thus, the esterified peak shape intensity is more in the former. The band around 1600 cm-1 is due to the carboxylate group. Small bands at around 1200 cm-1 and 1100 cm-1 may be due to the glycoside ring, C-O stretching and O-H bending. Bands around 650 cm-1 may indicate the pyranoid ring structure of pectin. Similar observations are reported for tomato, citrus and pumpkin pectin, where a high intensity band at 1749 cm-1 is correlated with stretching of a methylated carboxyl (C=O).
[0024] Figure 6 illustrate experimental analysis of gelling test for pectins. Under the experimental conditions, in presence of 65% and 75% w/w of sucrose, the gels are found to set within an hour. Thus, the pectins may also be classified under rapid set pectins. The solutions containing 55% of sucrose are not found to gel. Thus, the sucrose quantity that is greater than 55% w/w may be considered as optimum for jams and jellies in presence of the extracted pectins. The commercial pectin gelled in the presence of 65% and 75% w/w of sucrose but compared to mango peels, it took longer time for the gel setting. Figure 6a and 6b show the appearance of gels in different positions. As clearly observed from the images, the gels are stable on inversion of the beaker. The commercial pectin is found not to be stable at 1 h as shown in the Figure 6a. The visual appearance of the gels are also appealing due to the translucency. The gelling nature of mango peel pectins are found to be comparable to the commercial grade.
[0025] Co-products characterization
[0026] Structural carbohydrate composition of solid residue
[0027] The major component of the residual solid is determined as cellulose followed by lignin and hemicellulose. A major quantity of hexane and alcohol soluble extractives are also detected in the peels as well as the residue. In general, 18 ± 1.2% of total extractives (hexane followed by ethanol) are present in the dried peels while 28.0 ± 0.7% of extractives are present in the solid residue after pectin extraction. The increase in value is due to the removal of pectin from the total mass, which concentrates the other components present in the peel. The removal of extractives is important to prevent the interference of extractives in lignin determination as described in the reference protocol. The peels are found to contain cellulose (19-23%), hemicellulose (2.9-3.3%) and lignin (acid insoluble) (10.0-16%) prior to pectin extraction. The values obtained after pectin extraction are as follows; cellulose (37-39%), hemicellulose (7-9%) and acid soluble lignin (17-19%) in the residue. It may be observed that cellulose, hemicellulose and lignin get concentrated in the solids, which may again be due to the removal of pectin. It may also be inferred that the primary effect of pectin extraction is mainly observed for cellulose and hemicellulose quantities. Therefore, one of the applications for the residual solids may be ruminant feed. Alternatively, lignin separation may help in the extraction of a purified cellulose which can further be used as feedstock for biofuels and platform chemicals. The CHN analysis as shown in the Figure 7, Indicates the presence of a significant nitrogen percentage in the solid residues. The solids, therefore, can alternatively be added back to the soil as a nitrogen source.
[0028] Polyphenols in the pectin co-product
[0029] The retention time of the standards in a mixture are found to be 4.2 min for gallic acid, 7.2 min for mangiferin, 13.1 min for ellagic acid and 21.2 min for quercetin. The calibration curves are plotted in the range of 2.5 µg/ml–160 µg/ml (mangiferin) and 5–160 µg/ml (gallic acid, ellagic acid and quercetin). The known volume of the concentrated liquid is diluted with the mobile phase. The yield of polyphenols in the hydrolysed extract are shown in the Figure 8. A higher quantity of mangiferin is detected in the Indian cultivar, while gallic acid is higher in the Australian cultivar. The difference in values of the different extracts is beyond the control of the experimental protocol due to the variation that is induced by the geographical location, harvesting conditions and other environmental conditions. Among the polyphenols in the hydrolysed extract (mg/litre), gallic acid is obtained in the highest concentration (525-1405 mg/l) followed by mangiferin (7–162 mg/l), quercetin (40–49 mg/l) and ellagic acid (38–69 mg/l). The previously reported quantities of these polyphenols in peel-solvent extracts are gallic acid (14.7-16.6 g/kg), mangiferin (1.6-4.9 g/kg of peel), quercetin (65.3 mg/kg of peel) and ellagic acid (36.6 mg/g of peel) respectively. On a dry basis (data not shown), the highest yield of polyphenols obtained in the pectin co-products are; mangiferin (0.2g/kg of Totapuri peel), quercetin (70mg/ kg of Totapuri peel), ellagic acid (0.1mg/g of Totapuri peel) and gallic acid (5g/kg of Totapuri peel). The significant quantity of polyphenols in the pectin co-product indicates that even the co-product may be a good source of bioactives and, that the extract without hydrolysis may be added to various products as a natural preservative and antioxidant nutraceutical. In many pharmacological studies, the identified polyphenols are indicated in disease mitigation and cure. Mangiferin is the major component of a Cuba-based natural formulation that is used to cure elevated stress in patients. The formulation is prepared as a decoction of mango stem bark and consists of a mixture of polyphenols such as gallic acid, benzoic acid and mangiferin. Similarly, quercetin, gallic acid and ellagic acid are found to be useful as natural antioxidants in many studies.
[0030] Sugars in the pectin co-product
[0031] Figure 8, illustrates concentration of polyphenols detected in pectin co-products. As discussed above, on acid hydrolysis of the extract with 2M TFA, the yield of sugars also increases significantly along with polyphenols. Prior to acid hydrolysis the reducing sugar content of liquid is 32.4-39.9 g/l while after acid hydrolysis it is found to be 126.6-139.2 g/l. This may be due to the hydrolysis of the glycosidic linkage, post which the polyphenols such as quercetin and mangiferin become free in the mixture. The released sugars from such chemical hydrolysis thus, improved the overall yields of sugars. In a separate study done on bioethanol production from mango peels, approximately 300 g/l (or 30% w/v) of sugars are obtained when the peel slurry is digested with pectinase. The sugar yield in pectin co-product is approximately half of the reported quantity. Therefore, as an alternative, if the polyphenols could be separated from the mixture, the sugars may be used to produce biofuels in an integrated system which will further add value to the overall process.
[0032] The acid hydrolysis part used in this study is mainly required for the quantification of polyphenols. In general, the mixture of polyphenols are found to be more effective compared to single molecule in many antioxidant studies and thus, the need of hydrolysis may be overlooked. Therefore, the concentrated coproduct obtained may directly be incorporated in food products as a “sugar and antioxidant mixture”
[0033] Although the present disclosure has been described in the context of certain aspects and embodiments, it will be understood by those skilled in the art that the present disclosure extends beyond the specific embodiments to alternative embodiments and/or uses of the disclosure and obvious implementations and equivalents thereof. Thus, it is intended that the scope of the present disclosure described herein should not be limited by the disclosed aspects and embodiments above.

,CLAIMS:1. A hydrothermal process to extract pectin and sugar-poylphenol mixture from mango processing waste, the process comprising:
preparing a slurry of mango peel powder by mixing at least 1g of dried powder of mango peels in approximate 20 ml of water at solid to liquid ration;
maintaining the slurry at a pH range between 4 - 4.3;
treating the slurry under pressure of 16.2 ± 2.1 Psi and at a temperature of 121.0 ± 1.5 °C;
filtering the slurry, treated under pressure, through a nylon cloth, wherein the solid residue is separated;
drying the solid residue obtained at 105°C until constant weight is achieved;
centrifuging the viscous liquid filtrate at 4500 rpm for 20 min to remove any suspended gelatinous mass, wherein the said suspended gelatinous mass is filtered through cheese cloth to obtain a clear supernatant liquid;
decanting the clear supernatant liquid;
adding absolute ethyl alcohol in 1:1 ratio to the clear supernatant liquid;
precipitating the supernatant overnight to obtain pectin and
wherein the by product liquid having alcohol and water is distilled under vacuum at 45°C to separate the alcohol from the filtrate using a rotary evaporator; wherein the concentrated filtrate obtained is rich in sugars and polyphenols.

2. The process as claimed in claim 1, to obtain dried powder of mango peel comprises the steps of:
- blanching the mango peels in hot water at 90°C for 5 mins;
- drying the blanched mango peels at 50°C for 7 hours in a hot air food drier until a constant weight is achieved;
- grinding the dried peels obtained in a mixer grinder;
- passing the ground powder obtained through a 500µ size sieve;
- packing the dried power in an air tight container and
- storing the dried powder at 4 ± 1°C.

3. The process as claimed in claim 1, wherein the alcohol used can be recovered and be recycled in the hydrothermal process.

4. The method as claimed in claim 1, wherein the filtered gel is dried under vacuum at 45°C until constant weight is achieved, wherein the yield is calculated as the percentage of dry peel weight.

5. The method as claimed in claim 1, wherein scanning electron microscopy (SEM) image analysis carried out on original peel and hydrothermally treated residual peel indicated higher pectin removal due to porous structure observed in the SEM images on treated residual peel.

6. The method as claimed in claim 1, wherein increase in degree of esterification was found to reduce the free energy of junction zone formation, thereby favoring the stable junction zone formation to form stable gels.