DESC:INTEGRATED BIO-REFINERY PROCESS FOR SUSTAINABLE PRODUCTION OF LOW METHOXY PECTIN

FIELD OF INVENTION
[0001] The present disclosure described herein, in general, relates to sequential extraction of free sugars, pectin methyl esterase (PME) enzyme and low methoxy (LM) pectin from fruit/vegetable wastes generated by fruit/vegetable processing industries. In particular, the present disclosure relates to developing an integrated biorefinery process for sustainable production of low methoxy (LM) pectin directly from fruit/vegetable waste.

BACKGROUND
[0002] Pectin is a commonly used additive in the food industry as a gelling agent, stabilizing agent, emulsifier and in food packaging as edible coatings. Pectin is also currently explored in various other sectors such as pharmaceuticals as carriers for drug delivery, and in biomedicines.

[0003] Generally, the pectin extraction methods from fruit peels often lack an emphasis on the co-products. Conventionally, low methoxy (LM) pectin is produced by chemical de-esterification using acids and alkalis. The utilization of chemicals such as acids and alkalis in pectin extraction generates waste effluents that needs to be pre-treated before disposal.

[0004] In one of the prior arts, the fruit peels are blanched to remove the soluble molecules and to inactivate the enzymes. The peels are then dried and utilized as a raw material for pectin extraction. However, this process results in the generation of wastewater with high biological oxygen demand (BOD) and chemical oxygen demand (COD).

[0005] In yet another prior art, an evaporation unit process has been used to concentrate pectin to reduce the ethanol requirements in subsequent steps. However, the evaporation unit process by itself is an energy intensive process.

[0006] In yet another prior art, the low methoxy pectin is produced by de-esterification of high methoxy (HM) pectin using acids/alkalis in combination with ultrasound or microwave assisted approaches. As stated above, the utilization of chemicals such as acids and alkalis in pectin extraction creates waste effluents that need to be pre-treated before disposal.

[0007] Thus, there remains a need in the art to develop an integrated green, sustainable, and economical process for the sequential extraction of free sugars, pectin methyl esterase (PME) enzyme followed by low methoxy (LM) pectin production from fruit/vegetable waste generated by fruit/vegetable processing industries.

OBJECT OF THE INVENTION
[0008] The primary object of the present disclosure is to provide a pre-processing of fruit/vegetable waste to sequentially extract free sugars and pectin methyl esterase (PME) enzyme prior to the extraction of low methoxy (LM) pectin from fruit/vegetable waste generated by fruit/vegetable processing industries.

[0009] Yet another object of the present disclosure is to concentrate the pectin in the fruit/vegetable peels by removal of other water soluble molecules through the pre-processing stages.

[0010] Yet another object of the present disclosure is to utilize ultrafiltration in the integrated extraction process to reduce 80% ethanol requirements.

[0011] Yet another object of the present disclosure is to provide a green, sustainable, and economical biorefinery process to extract multiple valuable commercial biomolecules from fruit/vegetable waste generated by fruit/vegetable processing industries.

SUMMARY OF THE INVENTION

[0012] In an aspect of the present disclosure, an integrated process for the sequential extraction of free sugars, pectin methyl esterase (PME) enzyme and followed by low methoxy (LM) pectin from the waste orange peels has been disclosed. The process is aimed at providing a green, sustainable, and economical process for sequentially extracting free sugars, pectin methyl esterase (PME) enzyme and low methoxy (LM) pectin from the waste orange peels.

[0013] In an aspect of the present disclosure, fresh fruit waste samples from industry processing of Valencia orange (IO) and Pink Lady and Granny Apple (IA) were procured from a local fruit juicing industry in Dandenong, Melbourne, Australia. Valencia oranges (DO) were purchased from a local market in Dandenong, Melbourne and the oranges were peeled, and the peels were used for the experiments. Juice processing waste samples from a commercial juice extraction business (BM) were collected from Clayton, Melbourne as mixed fruit and vegetable waste samples. The moisture content was analyzed using a moisture analyzer (Mettler Toledo, HE53). The samples were stored at -20°C until further use.

[0014] In an aspect of the present disclosure, a process for the extraction/recovery of water-soluble free sugars from the fruit samples (IO, DO, IA and BM) has been disclosed. A pre-defined amount of fresh IO/DO/IA/BM peels are blended with a pre-defined volume of water in a solid to liquid ratio in the range of 1:1 to1:30 and blended the resulting mixture in batches to obtain a fine slurry. The pre-defined amount of fruit/vegetable peels samples is in the range of 400 grams and the pre-defined volume of water is in the range of 0.4 to 12 liters. The slurry mixture is then transferred to a Schott bottle and incubated at a temperature in the range of 4? to 40? in an incubator shaker at a speed in the range of 100-300 rpm for a time duration in the range of 5 to 90 minutes. After the incubation time, the slurry is filtered in a fruit filter press machine to separate the solid and liquid fractions. The solid fraction is stored at -20°C as water washed IO-WP/DO-WP/IA-WP/BM-WP peels for further utilization. The liquid fraction comprises the extracted water-soluble free sugars. The liquid fraction is further centrifuged, and the free sugars recovered are quantified using HPLC analysis.

[0015] In another aspect of the present disclosure, a process for the extraction/recovery of pectin methyl esterase (PME) enzyme from the water washed peel samples (IO-WP, DO-WP, IA-WP and BM-WP) has been disclosed.A pre-defined concentration of pH in the range of 6 to 10 potassium phosphate buffer is added to the water washed IO-WP/DO-WP/IA-WP/BM-WP peels (solid fraction stored at -20°C) in a solid to liquid ratio in the range of 1:1 to 1:30 and blended the resulting mixture in batches to obtain a fine slurry. The pre-defined amount of pre-processed peels is 200 grams and the pre-defined concentration of potassium phosphate buffer is in the range of 0.01 to 2M. The slurry mixture is then incubated in an incubator shaker at temperature in the range of 4? to 40? in an incubator shaker at a speed in the range of 100-300 rpm for time duration in the range of 5 to 90 minutes. After the incubation time, the slurry is filtered in a fruit/vegetable filter press machine thereby separating the solid and liquid fractions. The solid fraction is washed with 0.1 to 2 liter water thereby removing the remaining salts due to the utilization of buffers. The final pre-processed peels (IO-WBWP, DO-WBWP, IA-WBWP and BM-WBWP) is stored at -20°C for further utilization. The liquid fraction comprises the crude PME enzyme. The liquid fraction is further centrifuged, and the supernatant is utilized to determine the PME enzyme activity.

[0016] In yet another aspect of the present disclosure, the process for the extraction of pectin using hydrothermal processing from pre-processed peels (IO-WBWP, DO-WBWP, IA-WBWP and BM-WBWP) has been disclosed. The moisture content of IO/Do/IA/BM and IO-WBWP/DO-WBWP/IA-WBWP/BM-WBWP processed peels is determined using moisture analyzer. A pre-defined amount of the sample is blended with a pre-defined volume of Milli Q water for a time duration ranging from 5-60 seconds and to finely blend the peel and water mixture. The pre-defined amount of pre-processed peels is 10 grams and the pre-defined volume of water is in the range of 10 to 300 ml. The blended mixture is transferred to a reactor vessel using Milli Q water to make up the solid to liquid ratio to in the range of 1:5 to 1:30. The hydrothermal processing of the fresh ground samples is carried out using a 500 ml capacity hydrothermal batch reactor (Ollital Technology, China). The extraction of the pectin is carried out at a temperature in the range of 80 to 180°C for time duration in the range of 5 to 90 minutes. The hydrolysate is centrifuged in the range of 2000-10000 rpm for time duration in the range of 5 to 30minutes thereby separating the solid and liquid fractions. The liquid fraction is further filtered in Grade 2 sintered crucible to remove the suspended solids.

[0017] The filtered solution is centrifuged again until a clear supernatant is obtained. Ethanol is added to the clear supernatant in an ethanol to pectin solution ratio in the range of 1:1 to 1:4 for precipitating the pectin present in the clear supernatant. The resulting mixture is kept in an incubator shaker with continuous stirring with a speed in the range of 100-300 rpm at a temperature in the range of 4°C to 40°C for a time duration ranging from 0.1 to 24 hours. The precipitated pectin is centrifuged at speed in the range of 2000 to 10000 rpm for 5 to 30 mins and the precipitated pectin pellet is collected. The pectin pellet is further washed 1-3 times depending on the sample until a lighter supernatant is obtained. The washed pectin pellet is dried in a temperature in the range of 40-60°C until a constant weight is attained.

[0018] In yet another aspect of the present disclosure, the step of extracting low methoxy pectin from pre-processed peels is carried out using an alternate extraction method selected from microwave assisted extraction, ultrasound assisted extraction, and/or enzyme assisted extraction.

[0019] These and other objects, features, and advantages will become apparent to those of ordinary skill in the art from a reading of the following detailed description.

BRIEF DESCRIPTION OF DRAWINGS

[0020] The exemplary embodiments of the present invention have been described with reference to the accompanying drawings below:
[0021] Figure 1 illustrates flowchart of overall integrated process of sequential extraction of free sugars, pectin methyl esterase (PME) enzyme and low methoxy (LM) pectin from fruit/vegetable waste generated by fruit/vegetable processing industries in accordance with the present disclosure.
[0022] Figure 2 illustrates graphical analysis of LM-pectin yields in the processed peels at various stages of extraction process using hydrothermal treatment in accordance with the present disclosure.
[0023] Figure 3 illustrates mass balance of precipitated pectin yields and galacturonic acid yields at various stages of extraction process in accordance with the present disclosure.
[0024] Figure 4 illustrates graphical analysis of molecular weight and polydispersity index (PDI) analysis of extracted pectin samples in pre-processed peels in accordance with the present disclosure.
[0025] Figure 5 illustrates graphical analysis of galacturonic acid content of pectin in pre-processed peels in accordance with the present disclosure.
[0026] Figure 6 illustrates graphical analysis of degree of esterification (DE) of extracted pectin samples in pre-processed peels in accordance with the present disclosure.
[0027] Figure 7 illustrates characterization of the extracted pectin samples by using FTIR in accordance with the present disclosure.
[0028] Figure 8 illustrates apparent viscosity of extracted pectin samples from pre-processed peels in accordance with the present disclosure.
[0029] Figure 9 illustrates the analysis of protein content of pectin samples using carbon, hydrogen and nitrogen as detected in pectin co-products in accordance with the present disclosure.
[0030] Figure 10 illustrates the color analysis of commercial pectin and extracted pectin using hydrothermal process in accordance with the present disclosure.
[0031] Figure 11 illustrates experimental analysis of gelling test for extracted pectins in accordance with the present disclosure.
[0032] Figure 12 illustrates thermal gravimetric analysis (TGA) curves of commercial pectin and extracted pectin samples in accordance with the present disclosure.
[0033] Figure 13 illustrates 1H-NMR spectra of commercial pectin samples and extracted pectin samples in accordance with the present disclosure.
[0034] Figure 14(a) illustrates SEM images of commercial pectin and extracted pectin samples from pre-processing peels in accordance with the present disclosure.
[0035] Figure 14(b) illustrates optical microscopy images of commercial pectin and extracted pectin samples from pre-processing peels in accordance with the present disclosure.
[0036] Figure 15 illustrates the Mass balance of IO-WP membrane samples in accordance with the present disclosure.
[0037] Figure 16 illustrates the Mass balance of IO-WBWP membrane samples in accordance with the present disclosure.
[0038] Figure 17 illustrates graphical analysis of flux (J) of water and extracted pectin samples from pre-processing peel samples in accordance with the present disclosure.
[0039] Figure 18 illustrates the effect of membrane processing on IO-WP peel samples in accordance with the present disclosure.
[0040] Figure 19 illustrates the effect of membrane processing on IO-WBWP peel samples in accordance with the present disclosure.
[0041] Figure 20 illustrates graphical analysis of composition of free sugars extracted from pre-processing steps using HPLC in accordance with the present disclosure.
[0042] Figure 21 illustrates graphical analysis of the free sugars extracted using pre-processing extraction and hydrothermal extraction in accordance with the present disclosure.
[0043] Figure 22 illustrates graphical analysis of fractionation and mass balance of extracted pectin after hydrothermal treatment of peels in accordance with the present disclosure.
[0044] Figure 23 illustrates graphical analysis of PME enzyme activity of fresh pre-processing peel samples in accordance with the present disclosure.

DETAILED DESCRIPTION OF THE INVENTION

[0045] The invention will be described in detail below with reference to the drawings and specific embodiments. This embodiment is implemented on the premise of the technical solution of the present invention, and detailed implementation manners and specific operation procedures are given, but the scope of protection of the present invention is not limited to the following embodiments.

[0046] In the present invention, a process for an integrated process for the sequential extraction of free sugars and pectin methyl esterase (PME) enzyme from fresh peels prior to the extraction of low methoxy (LM) pectin directly from fruit/vegetable waste generated by fruit/vegetable processing industries has been disclosed.

[0047] In an embodiment of the present invention, figure 1 illustrates a flowchart for integrated extraction process of free sugars, pectin methyl esterase (PME) enzyme and low methoxy (LM) pectin from fruit/vegetable waste generated by fruit/vegetable processing industries. In order to perform the study, fresh fruit waste samples from industry processing of Valencia orange (IO) and Pink Lady and Granny Apple (IA) were procured from a local fruit juicing industry in Dandenong, Melbourne, Australia. Valencia oranges (DO) were purchased from a local market in Dandenong, Melbourne and the oranges were peeled, and the peels were used for the experiments. Juice processing waste samples from a commercial juice extraction business (BM) were collected from Clayton, Melbourne as mixed fruit and vegetable waste samples. The moisture content was analyzed using a moisture analyzer (Mettler Toledo, HE53). The samples were stored at -20°C until further use.

[0048] In an embodiment of the present invention, a process for the extraction/recovery of water-soluble free sugars from the fruit samples (IO, DO, IA and BM) has been disclosed. A pre-defined amount of fresh IO/DO/IA/BM peels are blended with a pre-defined volume of water in a solid to liquid ratio in the range of 1:1 to 1:30 and blended the resulting mixture in batches to obtain a fine slurry. The pre-defined amount of fruit/vegetable peels samples is in the range of 400 grams and the pre-defined volume of water is in the range of 0.4 to 12 liters. The slurry mixture is then transferred to a Schott bottle and incubated at a temperature in the range of 4? to 40? in an incubator shaker at a speed in the range of 100-300 rpm for a time duration in the range of 5 to 90 minutes. After the incubation time, the slurry is filtered in a fruit filter press machine to separate the solid and liquid fractions. The solid fraction is stored at -20°C as water washed IO-WP/DO-WP/IA-WP/BM-WP peels for further utilization. The liquid fraction comprises the extracted water-soluble free sugars.

[0049] In an embodiment of the present invention, a process for the extraction/recovery of pectin methyl esterase (PME) enzyme from the water washed peel samples (IO-WP, DO-WP, IA-WP and BM-WP) has been disclosed. A pre-defined concentration of pH in the range of 6 to 10 potassium phosphate buffer is added to the water washed IO-WP/DO-WP/IA-WP/BM-WP peels (solid fraction stored at -20°C) in a solid to liquid ratio in the range of 1:1 to 1:30 and blended the resulting mixture in batches to obtain a fine slurry. The pre-defined amount of pre-processed peels is 200 grams and the pre-defined concentration of potassium phosphate buffer is in the range of 0.01 to 2M. The slurry mixture is then incubated in an incubator shaker at temperature in the range of 4? to 40? in an incubator shaker at a speed in the range of 100-300 rpm for time duration in the range of 5 to 60 minutes. After the incubation time, the slurry is filtered in a fruit/vegetable filter press machine thereby separating the solid and liquid fractions. The solid fraction is washed with 0.1 to 2 liter water thereby removing the remaining salts due to the utilization of buffers. The final pre-processed peels (IO-WBWP, DO-WBWP, IA-WBWP and BM-WBWP) is stored at -20°C for further utilization. The liquid fraction comprises the crude PME enzyme.

[0050] In another embodiment of the present invention, the process for the extraction of pectin using hydrothermal processing from pre-processed peels (IO-WBWP, DO-WBWP, IA-WBWP and BM-WBWP) has been disclosed. The moisture content of IO/Do/IA/BM and IO-WBWP/DO-WBWP/IA-WBWP/BM-WBWP processed peels is determined using moisture analyzer. A pre-defined amount of the sample is blended with a pre-defined volume of Milli Q water for a time duration ranging from 5-60 seconds and to finely blend the peel and water mixture. The pre-defined amount of pre-processed peels is 10 grams and the pre-defined volume of water is in the range of 10 to 300 ml. The blended mixture is transferred to a reactor vessel using Milli Q water to make up the solid to liquid ratio to in the range of 1:5 to 1:30 The hydrothermal processing of the fresh ground samples is carried out using a 500 ml capacity hydrothermal batch reactor (Ollital Technology, China). The extraction of the pectin is carried out at a temperature in the range of 80 to 180°C for time duration in the range of 10-40 mins. The hydrolysate is centrifuged in the range of 2000-10000 rpm for time duration in the range of 5 to 30minutes thereby separating the solid and liquid fractions. The liquid fraction is further filtered in Grade 2 sintered crucible to remove the suspended solids.

[0051] The filtered solution is centrifuged again until a clear supernatant is obtained. Ethanol is added to the clear supernatant in an ethanol to pectin solution ratio in the range of 1:1 to 1:4 for precipitating the pectin present in the clear supernatant. The resulting mixture is kept in an incubator shaker with continuous stirring with a speed in the range of 100-300 rpm at a temperature in the range of 4°C to 40°C for a time duration ranging from 0.1 to 24 hours. The precipitated pectin is centrifuged at speed in the range of 2000 to 10000 rpm for 5 to 30 mins and the precipitated pectin pellet is collected. The pectin pellet is further washed 1-3 times depending on the sample until a lighter supernatant is obtained. The washed pectin pellet is dried in a temperature in the range of 40-60°C until a constant weight is attained.

[0052] In an embodiment of the present invention, a process for membrane assisted concentration of the hydrothermal treated crude pectin solution to reduce the ethanol requirements of the process has been disclosed. The crude pectin solution is concentrated up to 10 times using ultrafiltration thereby reducing the ethanol requirements. The retentate is further precipitated and washed with ethanol.

[0053] The hydrothermal treated crude pectin solution is concentrated using membranes with molecular weight cut off (MWCO) of 30 kDa with a diameter of 47mm the ultrafiltration is carried out with an HP 4750 Stirred cell (Sterlitech corporation, USA). The membrane is pre-conditioned with Milli Q water at the same operating conditions as that for the sample.

[0054] For membrane assisted concentration of crude pectin solution samples, 50 ml of hydrolysate is added to stirred vessel and the Teflon coated magnetic stir bar is fitted in place to provide continuous stirring to assist filtration process by preventing concentration build up. The pressure is set at 50 psi using nitrogen gas until 90% of feed volume is collected as permeate. The mass of permeate collected at equal intervals of 30 secs is recorded using SPDS data collection software to estimate the flux. The concentration level of the retentate is calculated using volume concentration ratio (VCR) which is determined by the following formula equation (1):
VCR=VF/VR

[0055] Where, VF stands for initial feed volume and VR stands for final retentate volume.

[0056] An aliquot of the feed and permeate is compared for composition of extracted free sugars by HPLC analysis. The composition of feed and permeate is used to calculate the rejection (R) by using the following formula equation (2):

[0057] Where CF is the concentration of the sugars in the feed, CP is the concentration of the sugars in the permeate.

[0058] In another embodiment of the present disclosure, the step of extracting low methoxy pectin from pre-processed peels is carried out using an alternate extraction method selected from microwave assisted extraction, ultrasound assisted extraction, and/or enzyme assisted extraction.

[0059] Figure 2 illustrates graphical analysis of LM-pectin yields in the processed peels at various stages of extraction process using hydrothermal treatment in accordance with the present disclosure. The extracted pectin in the IO peels increased in concentration due to removal of soluble molecules in the pre-processing stages and the simultaneous concentration of pectin as illustrated in figure 2. In terms of pectin yields from the IO peels as starting material, figure 2 illustrates that the pectin yields reduced due to minor pectin losses during the pre-processing stages. When calculated in terms of galacturonic acid % yields, figure 2 illustrates that there were negligible pectin losses. This is due to the entrapment of other soluble molecules like sugars and polyphenols during pectin precipitation that gives higher yields but comparatively lesser purity of precipitated pectin. Whereas in the case of precipitated pectin from pre-processed peels, it is observed that the purity of pectin is better due to the removal of soluble molecules in the initial pre-processing peel washing stages.

[0060] Figure 3 illustrates mass balance of precipitated pectin yields and galacturonic acid yields at various stages of extraction process in accordance with the present disclosure. The mass balance of precipitated pectin yields and corresponding galacturonic yields is represented for IO, IO-WBWP and IO-WBWP peels. Figure 3 illustrates that even though the pectin yields reduced, the galacturonic acid yields increased with the pre-processing stages. Further, figure 2 illustrates that the concentration of pectin in the IO peels increased from 16.9 % to 34.5 % during the pre-processing stages due to removal of soluble solids.

Working Example
[0061] The present invention is now further described by the following non-limiting example.

[0062] Free Sugar Extraction
[0063] For the extraction of water-soluble free sugars, 400 grams of fresh IO/DO/BM peels are blended with 1.2 liters of water to obtain a solid to liquid ratio of 1:3 (w/v) and 1:5 for IA peels and blended the resulting mixture in batches to obtain a fine slurry. The slurry mixture is then transferred to a Schott bottle and incubated at 10? in an incubator shaker at 150 rpm for 30 minutes. After the incubation time, the slurry is filtered in a fruit filter press machine to separate the solid and liquid fractions. The solid fraction is stored at -20°C as water washed IO-WP/DO-WP/IA-WP/BM-WP peels for further utilization. The liquid fraction comprises the extracted water-soluble free sugars. The liquid fraction is further centrifuged, and the free sugars recovered are quantified using HPLC analysis.

[0064] Extraction of pectin methyl esterase (PME) enzyme
[0065] For the extraction of pectin methyl esterase enzyme, 0.1M of pH 8 potassium phosphate buffer is added to the water washed IO-WP/DO-WP/ /BM-WP peels (solid fraction stored at -20°C) in a solid to liquid ratio of 1:3 (w/v) and 1:5 for IA-WP peels and blended the resulting mixture in batches to obtain a fine slurry. The slurry mixture is then incubated in an incubator shaker for a time duration of 30 minutes at 10°C. After the incubation time, the slurry is filtered in a fruit filter press machine thereby separating the solid and liquid fractions. The solid fraction is washed with 0.25 liter water thereby removing the remaining salts due to the utilization of buffers. The final pre-processed peels (IO-WBWP, DO-WBWP, IA-WBWP and BM-WBWP) is stored at -20°C for further utilization. The liquid fraction comprises the crude PME enzyme. The liquid fraction is further centrifuged, and the supernatant is utilized to determine the PME enzyme activity.

[0066] Extraction of pectin
[0067] For the extraction of pectin, 10g of the pre-processed peels (IO-WBWP, DO-WBWP, IA-WBWP and BM-WBWP) is blended with 50 ml of Milli Q water for 20 seconds to reduce the particle size by fine blending. The blended mixture is transferred to a reactor vessel using Milli Q water to make up the solid to liquid ratio to 1:10. The hydrothermal processing of the fresh ground samples is carried out using a 500 ml capacity hydrothermal batch reactor (Ollital Technology, China). The extraction of the pectin is carried out at 121°C for 20 minutes. The hydrolysate is centrifuged at 4500 rpm for 10 minutes thereby separating the solid and liquid fractions. The liquid fraction is further filtered in Grade 2 sintered crucible to remove the suspended solids.

[0068] The filtered solution is centrifuged again until a clear supernatant is obtained. Ethanol is added to the clear supernatant in an ethanol to pectin solution ratio of 2:1 for precipitating the pectin present in the clear supernatant. The resulting mixture is kept in an incubator shaker with continuous stirring at 150 rpm at 10°C for 1 hour. The precipitated pectin is centrifuged at 4500 rpm for 10 minutes and the precipitated pectin pellet is collected. The pectin pellet is further washed 1-3 times depending on the sample until a lighter supernatant is obtained. The washed pectin pellet is dried in a temperature in the range of 55°C until a constant weight is attained.

[0069] The hydrothermal extracted pectin is compared with conventional acid extraction of the pectin. The conventional acid is carried out by blending the fresh peels as mentioned previously. After adjusting the Solid to Liquid ratio to 1:10, the pH is adjusted to 1.6 using HCl and heated to 90°C for 90 mins. The qualitative analysis of the hydrothermal extracted pectin is done through its comparison with the conventional acid extracted pectin yields.

[0070] Characterization of extracted pectin
[0071] In another embodiment of the present invention, the extracted pectin is characterized to study its molecular weight, galacturonic acid, degree of esterification and rheological properties.

[0072] Molecular Weight
[0073] Figure 4 illustrates graphical analysis of molecular weight (MW) and polydispersity index (PDI) analysis of extracted pectins in pre-processed peels in accordance with the present disclosure. Figure 4 illustrates that the molecular weight of non-processed and processed IO samples ranged between 48.1-52 kDa. The IO-WBWP samples have the highest molecular weight of 52 kDa. The PDI value for IO-WBWP samples is 3.7 as compared to IO and IO-WP samples with PDI of 2.9. This is due to the presence of low molecular weight compounds like buffer salts.

[0074] Comparatively, the conventional IO pectin samples has better MW than hydrothermal samples. This is due to the presence of proteins in the precipitated pectin molecules. Due to harsh acidic conditions in the conventional process, proteins have co-extracted and precipitated along with pectin during the alcohol precipitation process. The proteins extracted from orange peels typically have a molecular weight range of 24-36 kDa. Presence of proteins is the reason for a comparatively higher PDI of 4.4 observed in the conventional acid extracted pectin samples.

[0075] Galacturonic acid
[0076] Figure 5 illustrates graphical analysis of galacturonic acid content of pectin in pre-processed peels in accordance with the present disclosure. The pectin extracted from orange peels has a galacturonic acid content of 65% (higher purity of pectin). The pre-processed samples contain a relatively higher amount of galacturonic acid content, and the values is much closer to the highly purified commercial sigma pectin samples. This is due to the initial washing of peels to remove the soluble molecules, and this results in increasing the purity of pectin. Further figure 5 illustrates that the galacturonic acid content of conventional acid extracted pectin is better than hydrothermal extracted IO samples. This is due to the removal of neutral sugar side chains due to acidic conditions that ultimately increases the overall galacturonic acid percentage in the conventional pectin samples.

[0077] Degree of esterification (DE)
[0078] Figure 6 illustrates graphical analysis of degree of esterification (DE) of extracted pectins in pre-processed peels in accordance with the present disclosure. Figure 6 illustrates that the DE of the commercial citrus sigma pectin is closer to the DE of the extracted orange pectin. Noticeably, IO-WBWP samples has a very low DE pectin. This is due to enzymatic de-esterification that have simultaneously occurred during the PME extraction step resulting in de-esterification of pectin from 51.9 % DE to 19.7 % DE.

[0079] Figure 7 illustrates characterization of the extracted pectins by using FTIR in accordance with the present disclosure. The characteristic peaks representing pectin present in the commercial pectin samples is found in all the extracted pectin molecules. The predominant peaks corresponding to O-H stretch which is present in the hydrogen bonds of galacturonic acid (3200-3600 cm-1), CH and CH2 present in the methyl groups of pectin (2800-3000 cm-1), the COOCH3 group of methyl ester peak (1740 cm-1) and COOH group of carboxylic acid (1630 cm-1), the CH3 bending of methyl ester (1350-1450 cm-1), and C-O stretch of glycosidic bond (1000-1200 cm-1) were all present in the extracted pectin molecules confirming the structure of pectin.

[0080] Rheological properties
[0081] Figure 8 illustrates the apparent viscosity of extracted pectins from pre-processed peels in accordance with the present disclosure. Figure 8 illustrates that all three samples show clear shear thinning properties that classify the sample as pseudoplastic that are the characteristic of pectin polysaccharide. The better shear thinning properties of IO-WBWP sample is because of comparatively a greater number of non-esterified groups that readily form hydrogen bonds with neighbouring pectin chains. This resulted in increased viscosity of the IO-WBWP pectin sample.

[0082] Protein content of pectin samples using CHN analysis
[0083] The protein content present in the precipitated pectin is characterized using CHN analysis. Figure 9 illustrates the analysis of protein content of pectin samples using carbon, hydrogen and nitrogen as detected in pectin co-products in accordance with the present disclosure. Figure 9 illustrates that conventional acid assisted process has a comparatively higher amount of protein compared to pectin extracted using hydrothermal and commercial sigma citrus pectin. This is due to the extraction of protein due to the harsh acidic conditions in conventional process that simultaneously co-precipitated along with pectin molecules. The IO-WBWP pectin samples have the least protein content similar to the highly purified commercial sigma pectin samples. This is due to the removal of enzymes in the pre-processing step that have produced a low protein content pectin.

[0084] Color of pectin
[0085] The color of pectin plays an important factor in its application since it affects the appearance of the final product. Figure 10 illustrates the color analysis of commercial pectin and extracted pectin using hydrothermal process in accordance with the present disclosure. The lightness value L* for CS pectin is highest with a 78.76 lightness value. The IO pectin sample has a comparatively lower lightness value of 55.82. The lower lightness value of the IO sample is due to coloured compounds captured during extraction and precipitation. The improvement of lightness value due to pre-processing stages and the lightness value increased from 55 in IO peels to 62 in IO-WP peels to 71 in IO-WBWP peels as illustrated in figure 10.

[0086] Gelling properties of pectin
[0087] Gelling properties of pectin is as result of dehydration of pectin chains in presence of sugars. This results in an intermediate stage between the solution and precipitation and thereby forms a gel. The gelling ability of the extracted pectin is tested for the IO and IO-WP samples. Figure 11 illustrates experimental analysis of gelling test for extracted pectins in accordance with the present disclosure. The gels formed by the extracted pectin is stable during the inversion physical test. The appearance and colour of the pectin gels prepared from the hydrothermal process is similar to commercial pectin gel as illustrated in figure 7.

[0088] Thermal gravimetric analysis (TGA)
[0089] Figure 12 illustrates thermal gravimetric analysis (TGA) curves of commercial pectin and extracted pectin samples in accordance with the present disclosure. The extracted pectin samples show three stages of mass loss and the pattern is similar to commercial pectin as illustrated in Figure 8 that indicates similar thermal stability. The first stage is the 50-200°C phase where the volatile molecules are evaporated. The second phase is the 200-400°C phase where the galacturonic acid chains undergo thermal degradation along with pectin decarboxylation that results in solid char. The last phase is the 400-650°C that is due to slow char decomposition.

[0090] 1H-NMR
[0091] The 1H-NMR spectra identify the characteristics peaks present in the pectin structure. Figure 13 illustrates 1H-NMR spectra of commercial pectins and extracted pectins in accordance with the present disclosure. The D2O solvent peak is identified at 4.5 ppm. The protons present in the methoxy group of the esterified unit of galacturonic acid gives a high-intensity peak at 3.8 ppm. The protons present in the C2, C3, C4, and C5 is identified at 3.7 ppm, 3.9 ppm, 4.4 ppm, and 4.9 ppm respectively. The same peaks are also identified in commercial pectin samples. The 1H-NMR data further confirms the structure of extracted pectin.

[0092] Optical microscopy and scanning electron microscopy (SEM) images of pectin samples.
[0093] The micrographs of the film surfaces are captured to study the structural morphology and homogeneity of the film. Figure 14(a) illustrates SEM images of commercial pectin and extracted pectins from pre-processing peels in accordance with the present disclosure. Figure 14(b) illustrates optical microscopy images of commercial pectin and extracted pectins from pre-processing peels in accordance with the present disclosure. Figures 14 (a) and 14 (b) illustrated uniform morphology of the pectin film without any pores. The pectin film has homogenous film-forming abilities that is confirmed by optical microscopy and scanning electron microscopy (SEM) images as illustrated in figure 14 (a) and 14(b).

[0094] Membrane assisted concentration.
[0095] The amount of ethanol required for the precipitation process is reduced in the present disclosure with the membrane assisted concentration with dead end filtration set up. Figure 15 illustrates the Mass balance of IO-WP membrane samples in accordance with the present disclosure. Figure 16 illustrates the Mass balance of IO-WBWP membrane samples in accordance with the present disclosure. Figure 15 and Figure 16 illustrate that the retentate is concentrated to 10 times of its feed volume to achieve a volume concentration ratio (VCR) of 10. This means the volume of ethanol required for the precipitation process is reduced by 90 % due to membrane assisted concentration. During the ultrafiltration more than 87 % of pectin for IO-WP sample and more than 75 % of pectin for IO-WBWP sample from the feed is collected as retentate with hardly any loss from the permeate stream as illustrated in figure 15 and figure 16.

[0096] Figure 17 illustrates graphical analysis of flux (J) of water and extracted pectins from pre-processing peel samples in accordance with the present disclosure. The flux for water is in the range of 150 to 130 and the flux for the IO pre-processed samples is in the range of 25 to 15, better than IO sample flux. The comparison of hydrothermal extracted pectin with and without membrane processing is studied as illustrated in figure 15 and figure 16. The comparison is intended to ensure that the quality of pectin is not compromised in the pursuit of ethanol reduction. It is observed from the membrane-processed pectin is superior in quality compared to without membrane processing for both IO-WP and IO-WBWP samples. The membrane processed samples have an added advantage of reduced protein content as illustrated in Figure 18 and Figure 19.

[0097] HPLC of free sugars.
[0098] The free sugars extracted during the pre-processing step is analyzed for the monosaccharides content. Figure 20 illustrates graphical analysis of composition of free sugars extracted from pre-processing steps using HPLC in accordance with the present disclosure. Fructose, glucose and sucrose are identified in the water fraction samples. The total sugar yields on dry basis is calculated as 21 % for IO samples, out of which 10 % is glucose, 9 % is fructose and 2 % is sucrose. These values are closer to the reported values in literature of 23 % total sugars (8 % glucose, 12 % fructose, and 3 % sucrose). Similarly, the total free sugars are 30.6 % for IA samples, 14 % for DO samples and 31.9 % for BM sample. This is a promising by-product that is further used as a starting material to produce valuable biochemicals.

[0099] Figure 21 illustrates graphical analysis of the free sugars extracted using pre-processing extraction and hydrothermal extraction in accordance with the present disclosure. The hydrothermal extraction recovered 29.4 % of total free sugars. The water extraction pre-processing step recovered 21 % of total free sugars. The simultaneous hydrothermal extraction of IO-WP peels recovered 6.8 % of remaining sugars and the hydrothermal extraction of IO-WBWP peels recovered 0.5 % of remaining sugars. A total of 28.3 % sugars are recovered from the pre-processing method in a sequential approach.

[0100] Figure 22 illustrates graphical analysis of fractionation and mass balance of extracted pectin after hydrothermal treatment of peels in accordance with the present disclosure. With the pre-processing of the samples, the pectin content increases and the total dissolved solids content decreases. The separation of free sugars as a by-product in the initial pre-processing stages helps to concentrate the pectin and to decrease the per unit impacts from the pectin production process both from life cycle and techno economic perspective.

[0101] Figure 23 illustrates graphical analysis of PME enzyme activity of fresh pre-processing peel samples in accordance with the present disclosure. The PME enzyme activity from DO samples has the highest as expected since orange peels has abundant PME enzymes. The DO samples consist of only the orange peels, where the enzymes are abundantly present. Even though IO sample is of the same variety of orange, it consists of mixture of peels and pomace fraction. The total enzyme activity is comparatively lesser than that of DO samples. The citrus sample, apple pomace sample and the boost mix sample that is majorly carrot waste are good sources of PME enzyme.

[0102] The quantity of PME enzyme required to de-esterify pectin produced from 1 kg of fresh peels is 2000 units of PME. To de-esterify 1 kg of HM pectin produced from 25 kg fresh peels, 50000 units of PME is required. This means 1 kg of fresh peels produce more than enough PME enzyme to de-esterify pectin produced from 25 kg fresh peels. The PME enzymes are produced in surplus and is a high value by-product in the extraction process.

[0103] The extracted pectin and the by-products have various industrial applications. The pectin is widely used in food industries as a gelling agent, stabilizer, and emulsifier and in food packaging as edible coatings. Pectin is also currently explored in various other sectors such as pharmaceuticals as carriers for drug delivery, in nutraceuticals as a prebiotic, in cosmetics as a texturizer for creams, stabilizer for shampoos, and in biomedicine as wound-healing patches.

[0104] The LM pectin forms gel at low sugar levels in the presence of calcium and has higher industrial applications as compared to HM pectin that forms gels at higher sugar concentration. LM pectin is industrially applied in low calorie food products. The by-product free sugars are industrially applicable in fermentation to obtain biobased products like vinegar. The by-product PME enzyme solution is further concentrated and purified as a commercial product. The PME enzyme is industrially applicable as aids for fruit/vegetable juice extraction, fruit/vegetable juice clarification and pectin de-esterification.

[0105] While the present invention has been described above in terms of specific embodiments, it is to be understood that the invention is not intended to be confined or limited to the embodiment disclosed herein.
,CLAIMS:We Claim:

1. An integrated process for sequentially extracting free sugars, pectin methyl esterase (PME) enzyme and low methoxy (LM) pectin from fruit/vegetable wastes, the process comprising the steps of:
a) extracting water-soluble free sugars with water washed peel samples from the fruit waste samples;
b) extracting pectin methyl esterase enzyme with pre-processed peels from the water washed peel samples obtained in step (a);
c) extracting low methoxy pectin through hydrothermal processing from the pre-processed peels obtained in step (b).

2. The process as claimed in claim 1, wherein the step of extracting water-soluble free sugars with water washed peel samples from the fruit/vegetable waste samples comprises the steps of:
a) mixing a pre-defined amount of fruit/vegetable peel samples with a pre-defined volume of water in a solid to liquid ratio in the range of 1:1 to 1:30;
b) blending the resulting mixture obtained in step (a) in batches to obtain a fine slurry;
c) transferring the slurry mixture to a Schott bottle and incubating the slurry mixture at a temperature in the range of 4°C to 40°C in an incubator shaker at a speed in the range of 100 to 300 rpm for a time duration in the range of 5 to 90 minutes;
d) filtering the incubated slurry mixture in a fruit filter press machine to separate the solid and liquid fractions, wherein the liquid fractions comprises the extracted water-soluble fruit sugars;
e) storing the solid fraction at -20°C as water washed peel samples for the extraction of pectin methyl esterase enzyme;
wherein the pre-defined amount of fruit/vegetable peels samples is 400 grams and the pre-defined volume of water is in the range of 0.4 to 12 liters.

3. The process as claimed in claim 1, wherein the step of extracting pectin methyl esterase enzyme with pre-processed peels from the water washed peel samples comprises the steps of:
a) adding a pre-defined concentration of potassium phosphate buffer to the water washed peel samples in a solid to liquid ratio in the range of 1:1 to 1:30, wherein the pH of the potassium phosphate buffer is in the range of 6 to 10;
b) blending the resulting mixture obtained in step (a) in batches to obtain a fine slurry;
c) incubating the slurry mixture in an incubator shaker at a temperature in the range of 4°C to 40°C in an incubator shaker at a speed in the range of 100 to 300 rpm for a time duration in the range of 5 to 90 minutes;
d) filtering the incubated slurry mixture in a fruit/vegetable filter press machine to separate the solid and liquid fractions, wherein the liquid fraction comprises the crude pectin methyl esterase enzyme;
e) washing the solid fractions with 0.1 to 2 liter water for removing the salts present in the solid fractions;
f) storing the washed solid fractions at -20°C as pre-processed peels for the extraction of pectin through hydrothermal processing;
wherein the pre-defined amount of water washed peel samples is 200 grams and the pre-defined concentration of potassium phosphate buffer is in the range of 0.01 to 2M.

4. The process as claimed in claim 1, wherein the step of extracting low methoxy pectin through hydrothermal processing from the pre-processed peels comprises the steps of:
a) blending a pre-defined amount of the pre-processed peel samples with a pre-defined volume of Milli Q water for a time duration in the range of 5 to 60 seconds;
b) transferring the blended mixture to a reactor vessel using Milli Q water to make up the solid to liquid ratio in the range of 1:5 to 1:30;
c) hydrothermal processing of the blended mixture in a hydrothermal batch reactor for the extraction of the pectin at a temperature in the range of 80 to 180°C for a time duration in the range of 5 to 90 minutes;
d) centrifuging the resulting hydrolysate in a range of 200 to 10,000 rpm for a time duration in the range of 5 to 30 minutes to separate the solid
e) filtering the liquid fraction in a grade 2 sintered crucible to remove the suspended solids;
f) centrifuging the filtered solution until a clear supernatant is obtained;
g) precipitating the pectin present in the clear supernatant with the addition of ethanol in an ethanol to pectin solution ratio in the range of 1:1 to 1:4;
h) incubating the resultant mixture in an incubator shaker with continuous stirring with a speed in the range of 100 to 300 rpm at a temperature in the range of 4 to 40°C for a time duration in the range of 0.1to 24 hours;
i) centrifuging the precipitated pectin at a speed in the range of 2000 to 10,000 rpm for 5 to 30 minutes;
j) collecting the precipitated pectin pellet;
k) washing the pectin pellet for obtaining a lighter supernatant;
l) drying the washed pectin pellet in a temperature in the range of 40 to 60°C until a constant weight is attained;
wherein the pre-defined amount of pre-processed peels is 10 grams and the pre-defined volume of water is in the range of 10 to 300ml.

5. The process as claimed in claim 4, wherein the process comprises the step of concentrating the supernatant obtained in step (f) upto 10 times through a process of membrane assisted ultrafiltration simultaneously reducing the ethanol requirements.

6. The process as claimed in claim 5, wherein the process of membrane assisted ultrafiltration comprises the steps of:
a) adding 50 ml of dried pectin pellet to stirred vessel and fitting the Teflon coated magnetic stir bar to provide continuous stirring for assisting in filtration process;
b) collecting 90% of feed volume as permeate, wherein the pressure is maintained in a range of 10 to 100 psi with nitrogen gas;
c) processing the retentate as claimed in claim 4 from steps (f) to step (l).

7. The process as claimed in claim 5, wherein the membrane has a molecular weight cut off in the range of 5 to 100kDa with a diameter of 47mm.

8. The process as claimed in claim 5, wherein the membrane is pre-conditioned with Milli Q water at the same operating conditions as that for the dried pectin pellet samples.

9. The process as claimed in claim 1, wherein the step of extracting of low methoxy pectin from pre-processed peels obtained in step (b) is carried out using an alternate extraction method selected from microwave assisted extraction, ultrasound assisted extraction, and/or enzyme assisted extraction.