Cascade Valorization of Apple Pomace into Polyphenols and Pectins by Green Extraction Processes

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IN

DEGREE PROJECT CHEMICAL SCIENCE AND ENGINEERING, SECOND CYCLE, 30 CREDITS

STOCKHOLM SWEDEN 2020,

Cascade Valorization of Apple Pomace into Polyphenols and Pectins by Green Extraction Processes

TEODÓRA GÁL

KTH ROYAL INSTITUTE OF TECHNOLOGY

SCHOOL OF ENGINEERING SCIENCES IN CHEMISTRY, BIOTECHNOLOGY AND HEALTH

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TRITA TRITA-CBH-GRU-2020:244

www.kth.se

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In this project, apple pomace from a Swedish cider making factory as a by-product was used as a raw material to extract valuable compounds. The extraction was focused on pectin and phenolic compounds with antioxidant activity. For the extraction procedures environmentally- friendly processes were chosen without using any harsh chemicals. Phenolic compounds were initially extracted from the pomace using 50% aqueous ethanol and then the composition, total phenolic content and antioxidant activity were studied in these extracts. The main focus was on pectins, which were extracted by subcritical water at three different pH conditions (pH 3, 5 and 7) and two different temperatures (120°C and 140°C) in 5-, 10- and 15 minute sequences. Then the pectins were characterized in terms of extraction yield, sugar composition, molecular weight and antioxidant activity and the results were compared in terms of the effect of pH, temperature and extraction time. The gelling properties of the different pectins were also studied as a proof of concept in an empirical experiment, where highly viscous liquids were obtained at 5% pectin and 60% sucrose content. In summary, the extracted phenolic compounds have potential to function as naturally derived antioxidants in cosmetics and the pectin may be used as a rheology modifier in water-based formulations of low pH without any additional chemical modifications.

SAMMANFATTNING

I det här projektet extraherades värdefulla komponenter från äpple-pomace som är en restprodukt från cidertillverkning. Restprodukten var tillhandahållen av en svensk cidertillverkare. Extraktionen fokuserades mot pektin och fenolföreningar som uppvisar antioxiderande aktivitet. Extraktionsprocessen designades ur ett miljövänligt perspektiv, inga skadliga kemikalier användes. Fenolära substanser extraherades initialt från pomacen med en vattenlösning innehållande 50% etanol. Därefter studerades kompositionen och den antioxiderande aktiviteten i dessa extrakt. Fokuseringen i projektet låg främst på pektin. Pektin extraherades med subkritiskt vatten vid tre olika pH (3,5 och 7) samt vid två skilda temperaturer (120°C och 140°C) i antingen 5, 10 och 15 minuters sekvenser. Pektinet karakteriserades med avseende på extraktionsutvinningsgrad, sockerkomposition, molekylvikt och antioxiderande aktivitet. Resultaten jämfördes för att undersöka effekten av pH, temperatur och extraktionstid.

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Gelningsegenskapen hos olika pektin studerades som ett bevis på koncept i ett empiriskt experiment där hög-viskösa vätskor bestående av 5% pektin och 60% sackaros framställdes.

Extraherade fenolföreningar har potential att fungera som naturligt utvunna antioxidanter i kosmetiska produkter och pektin kan tänkas användas som reologimodifierare i vattenbaserade formuleringar med låga pH-värden utan behov av kemiska modifieringar.

1. INTRODUCTION

1.1 Valorization of food waste in bioeconomy

Nowadays, creating a successful bioeconomy is crucial for the world due to climate change and overexploitation of natural resources. In order to achieve this, a smart and sustainable use of biomass for multiple uses (food, feed, chemicals, fibers, fuels) is necessary.¹ Significant amount of agricultural residues and by-products are generated from crop production and food processing e.g. straw, corncobs, corn stover or cutoffs from fruit and vegetable processing.² These materials are rich in value-added compounds that can be valorized by using physical, chemical and biotechnological treatments.²

1.2 Significance of apple pomace

There is a large amount of consumer products on the market that are made from apples, e.g.

apple juice, vinegar, cider and jam. In the production of these foodstuff, a by-product called apple pomace is generated, which is composed of skin and flesh (95%), seeds (2% to 4%) and stems (1%).³ This by-product has a high potential for further use due to its high antioxidant, dietary fiber and pectin content. Considering the fact that the worldwide production of apples is around 70 million tons⁴ and that the overall wet apple pomace production reached 3600 kilotons in 2010⁵ it would be advantageous to further utilize the pomace by extracting its components of high value. Currently, apple pomace is utilized for technological and nutritional purposes in pharmaceutical, cosmetic and nutrition applications (e.g. for feeding livestock)⁶. The rich content of bioactive molecules (e.g. polyphenols) and pectin in apple pomace puts it in the focus of scientific research.

The composition of apple pomace can vary depending on its origin and the type of apple. The main components of apple pomace are cellulose (12.8-17.6%), hemicelluloses (0.7-5%), starch (5.6-17.9%), pectin (3.5-14.3%), lignin (1.5-12.8%), proteins (2.9-5.1%) and lipids (1.2- 4.9%).^7–9 It may also contain trace amounts of mineral components such as calcium (0.06- 0.1%), iron (31.8- 38.3 mg/kg), magnesium (0.02-0.36%) and phosphate (0.07-0.076%).^3,7

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The monosaccharide composition of apple pomace may vary depending on the plant source (geographic location, single cultivar or industrial apples). The main sugars in apple pomace are glucose (16.8-22.7%), fructose (22.8-23.6%), sucrose (1.8-5.4%), arabinose (14-23%), galactose (6-15%) and xylose (0.06-1.1%).^7,8

Similarly, the content of phenolic compounds in apple pomace can vary depending on the apple source and their total amount accounts for 5.5-13.9 g gallic acid equivalent/kg apple (measured with Folin-Ciocalteau assay)⁶. The main types of phenolic compounds in apple pomace include phenolic acids (197.2-1543 mg/kg), flavanols (43.5-2414 mg/kg), dihydrochalcones (190.5- 2536 mg/kg) and flavonols (389.2-1195 mg/kg). Phenolic compounds are known to have antioxidant activity¹⁰ and thus, provide apple pomace with bioactive properties. For example, Diñeiro García et al. have reported that the antioxidant activity of apple pomace from single cultivar and industrial apples against DPPH was 11.1-15.9 g AA/kg and 4.5-10.3 g AA/kg, respectively⁶.

1.3 Pectin

Pectin is a high molecular weight (Mw) heteropolysaccharide, which is abundant in the cell wall and middle lamella of higher plants. It is mainly used in the food, cosmetic, pharmaceutical and textile industry as a gelling agent, thickener or stabilizer.¹¹ Generally, the main feedstocks for commercial pectin production are apple and citrus peel.¹² The structure of pectin is quite complex due to the substitutions of neutral and uronic sugars. Structurally, pectin is composed of a α-1,4-linked D-galacturonic acid (GalA) backbone that is partly methyl-esterified.

Four fundamental constitutents of the pectin structure are homogalacturonan (HG), xylogalacturonan, rhamnogalacturonan-I (RG-I) and rhamnogalacturonan-II (RG-II). HG is linear and consists of 72-100 GalA residues and it represents about 60% of pectin. In xylogalacturonan the GalA residues are partially substituted by β-(1,3)-linked D-xylose residues at C2 or C3 positions. RG-I accounts for around 7-14% of pectin and consists of alternating units of α-(1,4)-galacturonosyl and α-(1,2)-rhamnosyl.¹³ About 20-80% of the rhamnosyl residues in the RG-I region have side chains consisting of L-arabinose and D- galactose residues in different proportions and various levels of branching. These side chains can even include β-1,4-linked galactans with degrees of polymerization up to 47.¹⁴ Substitution of rhamnose units by xylose or glucose have also been reported to exist¹³. RG-II is a rather short constituent of pectin, with 7-9 of GalA units and contains four heteropolymeric side chains.

These side chains are uncommon monosaccharides such as apiose, aceric acid or fucose^13,15.

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The RG-II region contains 12 different types of sugars, which are connected by over 20 types of linkages.¹⁴ The complex structure of pectin and its components are shown in Figure 1.

Figure 1. Structural organization of pectin¹⁶

The heterogeneous and branched structure of pectin allows it to form gels through different mechanisms, depending on the degree of methoxylation (DM). High methoxylated pectins (HM pectins, 50-80% DM) can form a gel in acidic medium at a pH lower than 3.5 in the presence of a co-solute, which is typically sucrose. Sucrose is used at concentrations around 55% to facilitate hydrophobic interactions between methoxyl groups.

The low pH is required as it reduces the dissociation of carboxyl groups, allowing them to form hydrogen bonds. Low methoxylated pectins (LM pectins, 25-50% DM) can form gels by the aid of divalent cations. When a divalent cation such as Ca²⁺ is added to LM pectin, the calcium ions form bridges between the dissociated carboxyl groups and a physical hydrogel is formed, where the cross-links are held together by ionic interactions. It has been suggested that these gels are not formed merely due to ionic interactions but the mechanism might involve the formation of egg-box complexes, similar to the formation of alginate gels¹⁵.

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7 1.4 Extraction of pectin

Due to the heterogeneous structure of pectin it is a difficult task to extract it and optimize the extraction procedures to obtain the desired pectin structure for specific purposes. According to Adetunji et. al.(2017) the extraction involves two main steps, which are the hydrolysis of pectin from the cell wall and then the solubilization of pectin.¹⁷ A conventional method for extracting pectin from apple pomace is using the aqueous solution of a mineral acid, e.g. HCl (pH 1.5- 3.0), and high temperature (70-100°C).^3,18 On the other hand, other methods offer more environmentally-friendly extraction options , such as enzymatic, ultrasound-enhanced and subcritical water extractions³. Subcritical water extraction (SWE) has a high potential as it has already been proven to be effective for the hydrolysis of lignocellulosic materials and enhancement of pectin extraction rates and yields from citrus peel^3,11. Subcritical water extraction is achieved by a high pressure (10-11 MPa) applied to liquid water, which is able to reach temperatures higher than its boiling point without changing its phase. Adetunji et.al.

(2017) have stated that the high efficiency of SWE is due to the several physical changes that the water undergoes, i.e. a higher diffusion coefficient, lower viscosity and surface tension, increased vapor pressure and decreased dielectric constant.¹⁷ Moreover, it is also beneficial to use because here the solvent is only water that can be recycled easily and it preserves the high molecular weight of polysaccharides and functional groups¹⁹. Thus, SWE is a green method that should gain more focus in the future when it comes to valorization of by-products from the food industry. For example, Wang et.al. (2014) have performed SWE on apple pomace and citrus peel without the addition of any acid or alkaline compound and obtained pectins in 5 minutes with a yield of 16.7%.¹¹ On the contrary, Marcon et.al. (2005) have reached 16.8%

pectin yield by using the conventional acidic extraction (5% citric acid) for 80 minutes²⁰. This suggests that subcritical water can be just as effective as traditional acidic extraction in shorter time.

1.5 Applications of pectin

Pectin is a biocompatible natural polymer that is being studied to make different gels for advanced purposes, from cosmetics to drug delivery systems.^21,22 For example, Ro et.al. (2015) have shown that pectin possesses a strong ability to stabilize retinyl palmitate, which is an antioxidant used in cosmetics known to be unstable in most formulations when stored for a longer period of time, even at 4°C²¹. Lupi et.al. (2014) have demonstrated that pectin is also able to form oil in water (O/W) emulsion gels for cosmetic purposes.²³

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Besides being biocompatible, pectin has other beneficial properties such as muco-adhesiveness, ease of dissolution in basic environments and the ability to form gels in acidic conditions, which make it suitable for drug delivery systems in different parts of the body (gastric and nasal system, colon)²⁴. Pectin also appears to be able to inhibit cancer metastasis and primary tumor growth in multiple types of cancer in animals²⁵, which offers a possibility for a very advanced application.

2. AIM OF PROJECT

The initial aim of the project was to study the effect of pH, temperature and time on subcritical water extraction of pectin from apple pomace by analyzing the yields, sugar composition, molecular weight, antioxidant activity and gelling properties. The second aim of the project was to extract valuable phenolic compounds of apple pomace and analyze them in terms of chemical composition and antioxidant activity.

3. MATERIALS AND METHODS 3.1 Raw material

Apple pomace was obtained from a cider factory in Väddö Musteri (Sweden) as a pressed fruit cake. The pomace was freeze-dried and then milled into fine powder, which was then stored at -18°C until extraction.

3.2 Extraction procedures

3.2.1 Extraction of phenolics

The milled apple pomace was mixed with 50 % ethanol at a pomace : ethanol ratio of 1:10. The suspension was stirred for 1 h and the container was covered with aluminum foil. Then the suspensions were filtered under vacuum and the filtrate was put into a dark container and was stored at +4°C until analysis. The pellet was freeze-dried and used for the extraction of pectins.

In order to obtain a concentrated mixture of phenolic extracts, the solvent was evaporated using rotary evaporator at 36°C in the dark. The concentrated extract was then transferred into falcon tubes and was freeze-dried for further analysis.

3.2.2 Subcritical water extraction

Subcritical water extraction was employed to extract the pectins from apple pomace using an accelerated solvent extractor system (Dionex, ASE^TM 350, Thermo Scientific^TM Sunnyvale, CA, USA).

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The extraction was carried out at two different temperatures (120°C and 140°C) at three different pH conditions using citrate buffer (pH 3 and 5) and water (pH 7). Approximately 2 mg of dried pomace was mixed with the inert material and sequentially extracted for 5, 10 and 15 minutes with a pressure of 1500 psi for each pH and temperature condition. The extractions were performed in duplicates in order to ensure the reproducibility of the extraction and the sufficient amount of material to work with for the characterization studies. After extraction the pH of the extracts was measured, then dialysis was performed for 2-3 days using 3.5 kDa MWCO membranes and finally the samples were freeze-dried.

3.3 Methods of analysis

3.3.1 Protein content

Soluble protein content of apple pomace was determined using the dye-binding Bradford assay²⁶. 10 mg/ml solutions of apple pomace were prepared in Eppendorf tubes. The samples were solubilized by stirring at room temperature for 1 h at 500 rpm, then centrifuged (23°C, 10 min, 10 rpm) and the supernatant was used for the analysis. 7.7 ml dye was diluted in 27.3 ml of MilliQ water. 900 µl of the diluted dye reagent and 100 µl of sample solution were mixed and then incubated at room temperature for 15 minutes. The absorbance of the samples was measured at 595 nm against deionized water as blank. The measurement was performed in triplicates and BSA solutions at concentrations of 0.025, 0.05, 0.075, 0.15 and 0.2 mg/ml were used for calibration.

3.3.2 Total phenolic content

Total phenolic content of the phenolic extracts was determined using Folin-Ciocalteau assay. 5 ml suspensions of samples were prepared in 50% and 80% ethanol at concentrations of 100 mg/ml. The samples were covered with aluminum foil to protect them from light and stirred for 1 h. The samples were then filtered on filter paper and flushed with additional solvent (1 ml).

The samples were then diluted tenfold with deionized water and aliquots of 100 µl were mixed with 100 µl of Folin-Ciocalteau reagent in dark Eppendorf tubes. After 2 minutes, 800 µl of sodium carbonate (Na2CO3) solution was added and the samples were incubated at 40°C for 20 minutes in the dark. The absorbance was measured spectrophotometrically at 765 nm using a UV-vis spectrophotometer (Varian Cary 50 UV-VIS, Agilent) against a water blank.

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Gallic acid solution was used as a standard (at concentrations of 0.01, 0.02, 0.04, 0.06 and 0.08 mg/ml) to prepare the calibration curve and the results were expressed in g gallic acid (GAE)/100 g apple pomace.

3.3.3 Extraction yields

The yields of SWE were determined gravimetrically and calculated according to the following equation:

𝑌𝑖𝑒𝑙𝑑(%) =(𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑑𝑟𝑦 𝑒𝑥𝑡𝑟𝑎𝑐𝑡 + 𝑓𝑎𝑙𝑐𝑜𝑛 𝑡𝑢𝑏𝑒) − 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑓𝑎𝑙𝑐𝑜𝑛 𝑡𝑢𝑏𝑒 𝑤𝑒𝑖𝑔ℎ𝑡 𝑜𝑓 𝑎𝑝𝑝𝑙𝑒 𝑝𝑜𝑚𝑎𝑐𝑒 𝑢𝑠𝑒𝑑 𝑓𝑜𝑟 𝑒𝑥𝑡𝑟𝑎𝑐𝑡𝑖𝑜𝑛 ∙ 100

3.3.4 Monosaccharide composition Sulfuric acid hydrolysis

Sulfuric acid hydrolysis was performed on the apple pomace and on the solid residues left after SWE. Apple pomace was weighed to 3 mg (in triplicates) and the residues were weighed to 4 mg (in duplicates, for every pH and temperature) and then mixed with 125 µl of 72% sulfuric acid (H2SO4) solution at room temperature for 1 h. 1375 µl of deionized water was then added to the vials, vortexed and then hydrolyzed at 100°C for 3 h. The samples were then filtered through Chromacol 0.45 µm filters and, diluted with MilliQ water (1:10 v/v) and injected into high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) system.

Methanolysis

Two-step methanolysis was performed on the apple pomace and the pectin extracts according to the method described by Martínez-Abad et al. (2018)²⁸. The apple pomace was weighed between 2-3 mg in triplicates and the pectin extracts (all pH-s, temperatures and times) were weighed between 1-1.5 mg in glass vials. 1000 µl of 2M HCl in MeOH was added to the vials, which were flushed with argon afterwards.

The vials were closed and left in a heating block at 100°C for 5 hours. The vials were then cooled down and the solutions were neutralized with 200 µl pyridine and they were left to dry under air at 35°C overnight. In the next step, 1 µl of 2M TFA was added to the vials and then they were kept at 120°C for 1 hour. After cooling, the samples were filtered through Chromacol 0.45 µm filters.

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100 µl of the filtrates was transferred to glass tubes and then they were dried under air at 35°C.

They were then redissolved in 1 ml of MilliQ water and transferred to HPAEC-PAD vials.

A CarboPac PA1 column was used in the HPAEC-PAD system. The column was maintained at 30°C at a flow rate of 1 ml/min. The eluent program was employed as follows: deionized water, 300 mM sodium hydroxide, 200 mM sodium hydroxide + 170 mM sodium acetate, 1 M sodium acetate. Standard solutions of fucose, arabinose, rhamnose, galactose, mannose, xylose, fructose, galacturonic acid, glucuronic acid and 4-O-methyl glucuronic acid of 0.005, 0.01, 0.02, 0.05 and 0.1 mg/ml were used for calibration.²⁷

3.3.5 Starch content

The starch content of apple pomace and phenolic extracts was determined using the Total Starch Assay Kit (Megazyme, Wicklow, Ireland). 1 mg of apple pomace was weighed in Eppendorf tubes (in triplicates) and wetted with 20 µl of 80 % (v/v) ethanol, then 300 µl of thermostable α-amylase solution was added. The samples were incubated in a boiling water bath for 12 minutes and vortexed in every 4 minutes to ensure the solubilization of starch. Afterwards, the gelatinized samples were cooled down and treated with 10 µl of amyloglucosidase. at 50°C for 30 minutes. The samples were then added up to 1 ml by MilliQ water and centrifuged at 3000 rpm for 10 minutes. Then 40 µl was taken out from the supernatant and mixed with 960 µl of GOPOD reagent. The mixtures were then incubated at 40°C for 20 minutes and transferred into spectrophotometer cuvettes. The absorbance was measured at 510 nm, where the GOPOD reagent was used as blank. A solution of maize starch was used as control and glucose solutions of different concentrations (0.005, 0.01, 0.05, 0.1, 0.5 and 1.0 mg/ml) were used for standard calibration.

3.3.6 DPPH scavenging activity

The antioxidant activity of phenolic extracts freshly extracted from apple pomace, concentrated phenolic extracts (after freeze-drying) and 10-minute pectin extracts from all pH and temperatures was determined according to the method of Brand-Williams et al. 1995 with modifications²⁹. The solutions of fresh phenolic extracts were used at 8 mg/ml apple pomace suspensions in duplicates (in 50% EtOH and 80% EtOH), and the concentrated phenolic extracts (triplicates) and the 10-minute extracts (duplicates) were prepared at 10 mg/ml (in MeOH) and . 50 ml of 0.6 mM 1,1-diphenyl-2-picrylhydrazyl (DPPH) stock solution was prepared and diluted to 0.12 mM, 0.3 mM and 0.2 mM to be used for the fresh phenolics, concentrated phenolics and pectin extracts, respectively.

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A 96-well plate was used to analyze the antioxidant activity. The samples were added in different concentrations into the wells along with methanol. The fresh phenolics and the concentrated phenolics were added to the wells in 100 µl, 80 µl, 60 µl, 40 µl, 20 µl, 10 µl and the pectin samples were added in 100 µl, 80 µl, 60 µl, 40 µl, 20 µl, 10 µl, 6 µl and 1µl volumes.

100 µl of the DPPH solution was added last to the wells and then the UV absorbance was measured at 517 nm at the beginning (t0), after incubation at room temperature in the dark for 30 minutes (t30) and 60 minutes (t60) against methanol as blank. The radical scavenging activity of samples was expressed as EC50 representing the 50% reduction in the initial concentration of DPPH.

3.3.7 Phenolic acid profiling by HPLC

The phenolic acid content of samples was determined according to Menzel et.al. (2019)³⁰ using the same extracts as in the DPPH assay (freshly extracted from apple pomace) by diluting hundredfold from 83,3 mg/ml suspensions. The diluted samples were transferred to HPLC vials and the analysis was performed using a Waters HPLC system (Waters 2695 separation module, Waters 2996 photodiode array detector; USA) coupled to a UV/Vis detector, equipped with a C18 guard column and an SB-C18 separation column (Zorbax SB-C18 5 µm particle size, 4.6x250 mm, Agilent, Santa Clara, CA, USA). The separation column was kept at 25°C and the flow rate was 1 ml/min. The components of the mobile phase were 2% (v/v) acetic acid in water (eluent A) and 100% methanol (eluent B). The gradient was as follows: 100-75% A (11 minutes), 75.25% A (4 minutes), 64% A (10 minutes), 55% A (10 minutes), 35% A (3 minutes), 100% A (3 minutes) and 100% A (4 minutes). Between injections, the column was washed with 100% B for 10 minutes and equilibrated to the starting conditions for 5 minutes. The overall run time was 60 minutes and the injection volume was 20 µl for all samples and standards. A standard calibration was recorded using chlorogenic acid, caffeic acid, cinnamic acid, ferulic acid and gallic acid (5 µg/ml, 10 µg/ml, 25 µg/ml, 50 µg/ml, 100 µg/ml). UV-VIS spectra between 210 and 400 nm were recorded at a rate of 1.25 scans/s. The chromatograms recorded at 270 nm and 325 nm were used to detect the unknown phenolic acids.

3.3.8 Molecular weight distribution

The molecular weight distribution of the extracts was determined using size exclusion chromatography (SEC). First, the samples were dissolved in 0.01 M sodium hydroxide (NaOH) at 2 mg/ml concentration at 30°C overnight and then filtered through 0.2 µm nylon filters.

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The filtered samples were analyzed on an Ultimate-3000 HPLC system (Dionex, Sunnyvale, CA, USA) equipped with both RI (Waters, Milford, MA, USA) and UV/Vis (Dionex, Sunnyvale, CA, USA) detectors. The separation was performed using a column set-up consisting of a Suprema guard column (50 × 8 mm, 10 µm particle size), a Suprema 30 Å column and two Suprema 1000 Å columns (300 × 8 mm, 10 µm particle size) connected in series (Polymer Standard Services, Mainz, Germany). Standard calibration with 10 pullulan standards (Polymer Standard Services, Mainz, Germany) ranging between 342 and 708,000 Da was used for determining the molecular weight. The mobile phase was 10 mM NaOH and the oven was set at 40 °C and the cooler at 30 °C, the flow rate was 1 ml/min.³¹

3.3.9 Fourier transform infrared spectrometry (FTIR)

FTIR spectra of the selected extracts (pH3 120°C 10-min, pH5 120°C 10-min, pH7 120°C 10- min, pH5 140°C 5-min, 10-min and 15-min) were recorded using an FTIR instrument (Perkin Elmer Spectrum 100, Norwalk, CT, USA) equipped with a single reflection accessory unit (Golden Gate, Graseby Speac Ltd, Kent, England) for Attenuated Total Reflection (ATR). The spectral range was 600-4000 cm^-1 at a resolution of 4 cm^-1.16 scans were recorded for each sample, and the spectra were automatically baseline corrected and normalized using Spectrum software (Perkin Elmer).

3.4 Gelling properties of pectin extracts

The study of gelling properties was based on the work of Chandra R. Vinthanage et.al. (2010)³² and the concentration of pectin, sucrose and Ca²⁺(in 0.2 % calcium-chloride (CaCl2) solution) was varied. The pH was also varied along with the abovementioned factors in the second part of the experiment. The pectin and the sucrose (when required) were dissolved together in a buffer solution (pH 3.3, 3.0 or 2.5 depending on the recipe) by mixing at 100°C. The mixture was vortexed a few times to further enhance dissolution. The CaCl2 solution was added when the mixture cooled down and then it was vortexed and then kept in the fridge. The study of gelling properties was carried out in two rounds. In the first round, 1% pectin was used for making gels (1ml in volume), from the extracts of pH5-140°C-5-min and pH5-140°C-10-min (8 gels were made in this round). Four types of recipes were made according to Table 1.

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Table 1. Pectin gel compositions (first round) Pectin

[%] pH Sucrose

[%]

CaCl2

[µl]

1 3.0 - 20

1 3.0 30 20

1 3.0 - -

1 3.0 30 -

In the second round, only the pH5-140°C-5' extracts were used for the gelling according to the following table:

Table 2. Pectin gel compositions (second round) Pectin

[%] pH Sucrose

[%]

CaCl2

[µl]

5 3.0 60 -

5 3.0 60 20

5 2.5 60 -

5 3.3 60 -

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15 4. RESULTS AND DISCUSSION

4.1 Properties of the raw material

The raw apple pomace, which was used as a feedstock for the pectin extraction, was analyzed in terms of monosaccharide composition; protein, starch and phenolic acid content; and antioxidant activity. The results of the chemical composition analysis are presented in Table 3 and the monosaccharide composition is presented in Table 4.

Table 3. Chemical composition of apple pomace and antioxidant activity of phenolic extracts Moisture content (%)^a 80.87

Total carbohydrate content (mg/g) from sulfuric acid

hydrolysis^b

879.7

Total carbohydrate content

(mg/g) from methanolysis^c 443.5 Starch content (%)^d 7.84 Protein content (%)^e 1.18 Total phenolic content (mg

GAE/100 g) ^f 6045

Chlorogenic acid (mg/kg) 36.00 Other phenolic acids (mg/kg) 26.11

EC50 (mg/mg DPPH)

[Fresh extract] 42.97

EC50 (mg/mg DPPH)

[Freeze-dried extract] 19.38

a Moisture content was determined by gravimetric analysis after freeze-drying;

b Carbohydrate content was determined by HPAEC-PAD after H2SO4 hydrolysis;

c Carbohydrate content was determined by HPAEC-PAD after methanolysis;

d Starch content was determined enzymatically using the total starch assay kit (Megazyme);

e Protein content was determined using the Bradford dye-binding assay;

f Total phenolic acid content was determined by the Folin-Ciocalteau assay.

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Table 4. Monosaccharide composition of apple pomace determined by HPAEC-PAD after sulfuric acid hydrolysis and methanolysis

Fuc Ara Rha Gal Glc Xyl Man GalA GlcA Fru

Sulfuric acid

hydrolysis 5% 3% n.d. 2% 27% 2% n.d. 1% n.d. 59%

Methanolysis 2% 13% 2% 6% 53% 5% 1% 17% 1% n.d.

n.d. Not detected

Two different methods of hydrolysis were used to analyze the sugar composition of apple pomace and the results showed a significant difference in terms of glucose, galacturonic acid and fructose content (Table 4). This can be explained by the different mechanisms of these hydrolysis methods. Sulfuric acid hydrolysis is harsh and is able to hydrolyze the glycosidic bonds in both the amorphous and crystalline regions of cellulose, thus it gives a better estimate for cellulose content. Uronic acids can be degraded during sulfuric acid hydrolysis, thus methanolysis is the suitable method for the quantification of uronic sugar content³³. In the present study, the biggest difference was seen in the fructose content of apple pomace, where it was 0% and 59% when analysed after methanolysis and sulfuric acid hydrolysis, respectively.

A possible explanation for this is related to the melting point of fructose (103°C), which might have degraded while the temperature is kept at 120°C during methanolysis. The glucose content was also high in both the sulfuric acid and the methanolysis samples. Glucose forms the backbone of cellulose, which is the main component in all plant cell walls³⁴, thus this was to be expected. In the methanolysis samples GalA was the second most abundant monosaccharide (17%), which indicates the presence of pectin as it is the main component of pectin backbone.

Fucose, rhamnose and glucuronic acid are also present in the apple pomace, which come from the branched regions of pectin (RG-I and RG-II). The methanolysis results show a high amount of arabinose (13%) that most probably comes from the side chains of pectin along with galactose and xylose. However, these two monosaccharides can be found in hemicelluloses as well, not only in pectin. There is a low amount of mannose in the apple pomace that comes from hemicelluloses, as it cannot be found in pectin.^13,35

The total phenolic content (TPC) of the apple pomace was found to be high (Table 3) compared to the values found in the literature.⁶ This may suggest that the raw material does not only contain high amount of phenolics but also other non-phenolic compounds (e.g reducing sugars and amino acids) that can interfere with the analysis.³⁰

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The fresh extracts did not show particularly high antioxidant activity however, after the rotary evaporation of ethanol and freeze-drying, this value increased to more than double. This was due to the more concentrated state of samples and indicated promising results with regards to the stability of antioxidant compounds. Accordingly, antioxidant compounds were not degraded during the rotary evaporation and conserved after storage at +4°C for several weeks. This suggested that a significant amount of antioxidants can be recovered from apple pomace with a rather simple extraction method. It is important to note that TPC and antioxidant activity did not show a correlation as other non-phenolic compounds (e.g. alkylresorcinols, carotenoids and sterols) can also act as antioxidants³⁶.

The phenolic acid profiling of apple pomace was performed using HPLC and the HPLC chromatogram is presented in Figure 2. The main phenolic acid in the apple pomace was found as chlorogenic acid (15.569 min) as in agreement with previous studies⁶. HPLC analysis displayed the presence of other phenolic acids (eluting after 30 min) however, these were not identified due to the lack of standards. This result suggests that apple pomace is a rich source of phenolic compounds as consistent with other studies^6,10.

Figure 2. HPLC chromatogram of phenolic acids in apple pomace

4.2 Subcritical water extraction of pectin

4.2.1 Total extraction yields

The total yields of the extractions, as shown in Table 5, showed general differences based on the pH of the SWE. Accordingly, SWE at pH 5 resulted in the lowest yields, whereas SWE at pH 7 gave the highest yields at both extraction temperatures. This could be explained by the pH7 extraction being milder compared to pH3 and pH5 extractions, which prevented the polysaccharides from acid hydrolysis and hence resulted in higher yields.

15,569 32,205 33,817 35,050 36,378 37,061

AU

0,000 0,005 0,010 0,015 0,020 0,025 0,030 0,035

Minutes

0,00 5,00 10,00 15,00 20,00 25,00 30,00 35,00 40,00 45,00 50,00 55,00 60,00

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Table 5. Extraction yields at different pH and temperatures

120°C 140°C

SWE pH3 pH5 pH7 pH3 pH5 pH7

Yield (%) 20.8 18.7 21.2 25.4 23.2 42.8

The higher extraction temperature (140°C) resulted in distinctively higher yields at all pH conditions. This was in agreement with Wen-Jia Li et.al. (2019), where a peak point in the yield at 140°C was observed and explained by higher molecular mobility³⁵. The effect of pH was not very significant at 120°C, while at 140°C the pH7 extraction resulted in a much higher yield than at pH3 and pH5. Marcon et.al. (2005) have performed acidic pectin extraction in 5% (w/v) citric acid at different temperatures (50, 75 and 100°C), resulting in yields between 5.7-16.8%²⁰, while the yields obtained here are higher than that (18.7-42.8%). However, both acidic extraction and SWE show an increase in yield with increasing temperature²⁰. It is also important to note that the values shown in Table 5 represent extracts with different sugar compositions, thus these values do not necessarily represent the pectin yields.

4.2.2 Monosaccharide composition of extracts

The time evolution of the monosaccharide composition of all extracts at pH3, pH5 and pH7 is presented in Figure 3, 4 and 5, respectively. (A detailed monosaccharide composition of the extracts is shown in Table A.1 and A.2 in Appendix A).

Earlier studies reported the structure of pectin in detail^13,14. Accordingly, galacturonic acid (GalA) composes the main backbone of pectin and its content is significantly higher compared to other monosaccharides it indicates a linear pectin structure. The branching of pectin regions (RG-I, RG-II and xylogalacturonan) is determined by the abundance of neutral (Ara, Gal, and Rha) and uronic sugars (GlcA) (Figure 1). Relatively higher presence of Ara and Gal usually indicates the arabinan and/or arabinogalactan side chains of RG-I region. A higher abundance of Fuc, on the other hand, is considered to be the indication of RG-II side chains. Finally, the presence of xylose may show the xylogalacturonan region. The following discussion about the monosaccharide composition of the extracts was built on this branching pattern of pectin.

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Figure 3. Monosaccharide composition at pH3 at 120°C and 140°C

At pH 3, the monosaccharide composition of the extracts at both 120°C and 140°C showed a predominant GalA, Gal and Ara content, suggesting high abundance of pectin (Figure 3). The time evolution of SWE at 120°C demonstrated that glucose was mostly extracted during the first 5 min and gradually diminished with extraction time. This glucose can be attributed to starch. The 10-min extract exhibited the highest monosaccharide content, composing of GalA with a high amount of Ara and Gal. This indicated that 10-min extraction produced the most branched pectins, where the branches appeared to arise from RG-I pectin. In contrast, the least branched pectin was produced in the 15-minute extract, with a minor content of mannose that possibly originated from hemicelluloses³⁴. The low presence of Fuc and GlcA in the extracts may suggest RG-II pectin however, it is uncertain how long the branches are, as some other rare sugars (e.g. apiose) found only in RG-II were not detected due to lack of standards. As for SWE at 140°C, pH 3, a similar time evolution was observed in the total carbohydrate content of the extracts. On the other hand, the GalA and Ara content showed a clear decreasing trend whereas Gal content increased with time. The abundance of Glc, and Man and Xyl (ascribed to hemicelluloses) generally increased with time. These trends in the monosaccharide composition at 140°C indicated that more branched pectins along with higher amounts of starch and hemicelluloses were isolated by the combined effect of low pH and high temperature.

0 100 200 300 400 500 600 700 800

pH3 120 5 pH3 120 10 pH3 120 15 pH3 140 5 pH3 140 10 pH3 140 15

Monosaccharide composition [mg/g DW]

Fucose Arabinose Rhamnose GlcA Galactose Glucose Xylose Mannose GalA

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At pH 5, the monosaccharide composition of the extracts at both 120°C and 140°C increased as SWE prolonged (Figure 4). On the other hand, this increase was faster at 140°C, which could be explained by the capability to access the carbohydrate fractions easier than a lower temperature. As expected, the GalA content of the extracts decreased with time at both temperatures, showing that less pectin remained in the apple pomace to be extracted as SWE progressed. This was also evident in the monosaccharide composition of the residues after SWE, as discussed later (Figure 8). Moreover, the content of Ara, Gal and Rha clearly increased with time at both temperatures. This decrease in the GalA content and the increase of the Ara, Gal and Rha sugars revealed that the extracted pectins were less branched in the beginning of SWE and more branched fractions were isolated with longer extraction time.

0 100 200 300 400 500 600 700 800

pH5 120 5 pH5 120 10 pH5 120 15 pH5 140 5 pH5 140 10 pH5 140 15

Monosaccharide composition [mg/g DW]

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At pH 7, a general increasing trend was observed in the total monosaccharide content of the extracts at both 120°C and 140°C (Figure 5), similar to the extraction at pH 5. On the other hand, Total carbohydrate content of all time extracts at 120°C extracts was higher than the respective time extracts at 140°C. This may suggest that at a neutral pH and high temperature other cell wall components (e.g. proteins or lignin) were possibly co-extracted. The 5-min extracts at pH 7 at both extraction temperatures contained the lowest and the 10-min extracts contained the highest GalA unlike the general decreasing trend at pH 3 and pH 5 extracts.

Besides, Ara and Gal content also increased with time. This demonstrated that a mixture of xylogalacturonan, RG-I and RG-II pectins were isolated using mild pH conditions during SWE.

Taken together, it could be postulated that the branching pattern of pectins may be modulated by tuning the pH and temperature conditions of SWE.

4.2.3 Monosaccharide composition of residues after SWE

The monosaccharide content (%) of the residues remained after SWE was analyzed after sulfuric acid hydrolysis and presented in Figure 6. This comparison will not be suitable to compare the pectin content due to the fact that uronic acids degrade during sulfuric acid hydrolysis³³ however, it will show a good estimate for cellulose and hemicellulose content.

0 100 200 300 400 500 600 700 800

pH7 120 5 pH7 120 10 pH7 120 15 pH7 140 5 pH7 140 10 pH7 140 15

Monosaccharide composotion [mg/g DW]

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Figure 6. Monosaccharide content of the residues after sulfuric acid hydrolysis

In the residues, glucose was measured as the main component, which likely originated from cellulose and hemicellulose fractions that were resistant to be extracted by subcritical water.

No fructose was detected in the residues and this could be due to the degradation at higher temperatures as discussed earlier. The xylose content of the residues could be ascribed to either pectin or hemicelluloses (e.g. xylans, xyloglucans). Comparing the different pH conditions, it was observed that the effect of pH on the monosaccharide composition of the residues was not significant with the exception of the residues at pH 3, 140°C. This residue contained higher glucose and lower arabinose and galactose those of other residues. This could be explained by the combined effect of low pH and high temperature that facilitated the isolation of pectin and hemicelluloses. With regards to the different extraction temperatures, higher glucose and lower hemicellulose content was detected in the residues from SWE at 140°C than those in the residues from SWE at 120°C at all pH conditions. This suggested that the higher extraction temperature is more selective towards pectin.

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

pH 3 120 pH5 120 pH7 120 pH3 140 pH5 140 pH7 140

Monosaccharide composition (% DW)

Fuc Ara Rha Gal Glc Xyl Man Fru GalA

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23 4.2.4 Molecular weight distribution

The molecular weight (Mw) distributions of the extracts were analyzed using size exclusion chromatography (SEC) (Figure 7, Figure 8 and Figure 9). The number- (Mn) and weight- average (Mw) molecular weight and dispersity index (D) of all samples are presented in Table 6.

Table 6. Number average- and weight-average molecular weight and dispersity index of the extracts

Mn (kDa)

Mw

(kDa) D

120°C pH3

5-min 211 255 1.21

10-min 121 216 1.78

15-min 204 244 1.20

pH5

5-min 172 237 1.37

10-min 182 228 1.25

15-min 165 216 1.31

pH7

5-min 224 258 1.15

10-min 150 202 1.35

15-min 133 185 1.39

140°C pH3

5-min 177 236 1.33

10-min 121 182 1.51

15-min 114 189 1.65

pH5

5-min 174 245 1.41

10-min 157 232 1.48

15-min 138 215 1.56

pH7

5-min 179 246 1.37

10-min 130 211 1.62

15-min 111 196 1.76

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Figure 7. Molecular weight (Mw) distributions at pH 3

-0,5 0 0,5 1 1,5 2 2,5 3

14 16 18 20 22

Signal [mV]

Time [min]

pH3 120 5 pH3 120 10 pH3 120 15 pH3 140 5 pH3 140 10 pH3 140 15

-0,5 0 0,5 1 1,5 2 2,5 3

14 16 18 20 22

Signal [mV]

Time [min]

pH5 120 5 pH5 120 10 pH5 120 15 pH5 140 5 pH5 140 10 pH5 140 15

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The extracts obtained from SWE at pH 3 showed bimodal Mw distributions (Figure 7), indicating the presence of low Mw and high-Mw populations of polysaccharides. The weight- average molecular weights (Mw) of pH 3 extracts at both temperatures were between 200-255 kDa, confirming their polymeric structure. The size distributions of the extracts obtained from SWE at 120°C were generally higher than those obtained at 140°C. This suggested that pectins were hydrolyzed at higher extraction temperature as in agreement with previous reports on polysaccharides¹⁹. At 120°C, the 5-min and 15-min extracts both showed high Mw and low dispersity (around 1.2) however, the 10-min extract had a lower Mw and the highest dispersity out of all the samples. The 120°C extracts showed a clearer tendency when it comes to Mw, where the weight-average molecular weight slowly decreased with time. The differences in dispersity between the 140°C extracts were not as big as in case of SWE at 120°C, implying the extraction of similar polysaccharide populations.

As for the Mw distribution of the extracts at pH 5, both the 120°C and 140°C extracts showed a decrease in molecular weight throughout the extraction time. The Mw of the extracts was between 215-250 kDa, which indicated that the pectins were again obtained in a polymeric form. At 120°C the 5-min extract showed monomodal Mw distribution (Figure 8) however it became bimodal over time (10- and 15-min extracts). This change in the Mw suggested that the pectins slowly hydrolyzed with time. The hydrolysis was stronger at 140°C as the 5-min extract already showed a bimodal distribution, which became trimodal in the 15-min extract.

-0,5 0 0,5 1 1,5 2 2,5 3

14 16 18 20 22

Signal [mV]

Time [min]

pH7 120 5 pH7 120 10 pH7 120 15 pH7 140 5 pH7 140 10 pH7 140 15

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The 140°C extracts were also a lot more polydisperse than the 120°C extracts, which confirmed that the temperature had a strong effect at pH 5 and the hydrolysis was faster at 140°C.

Comparing the Mw distribution of the extracts at pH 7 (Figure 9) the molecular weight of the 5-min extracts were almost the same at both temperatures and then the values started to decrease as SWE prolonged. Both at 120°C and 140°C the Mw distribution evolved from monomodal to trimodal throughout the extraction. On the other hand, the 140°C extracts exhibited a very sharp increase towards higher dispersity, demonstrating that the hydrolysis was stronger at this temperature, similarly to pH 3 and pH 5 extractions.

4.2.5 Antioxidant activity

The antioxidant activity of the 10-min extracts was measured using the DPPH assay and the results are presented as EC50 (mg extract/mg DPPH) in Table 7. The DPPH assay was affected by the presence of smaller particles that were precipitated in the pH 3 and pH 5 extract solutions.

Even though all mixtures were centrifuged, only the pH 7 extracts were clear enough to give a result that could be evaluated.

Table 7. Antioxidant activity of pH 7 extracts EC50

[mg/mg DPPH]

pH7 120°C 10-min 39.053

pH7 140°C 10-min 36.345

The extracts showed similarEC50 values, indicating that the extraction temperature did not have an effect on the antioxidant activity. The EC50 values were lower than that of the apple pomace, implying that the antioxidant activity was higher in the pectin extracts than in the raw material.

This suggests that some of the phenolic compounds were preserved as attached to the pectin chains. Different kinds of interactions between pectic polysaccharides and polyphenols have been evidenced by previous studies. For example, procyanidins form complexes with pectin through hydrogen bonding. Moreover, due to the structural flexibility of pectin, it can encapsulate certain phenolic compounds in its hydrophobic cavities. Arabinogalactan side chains can also form covalent bonds with ferulic acid derivatives as a result of biosynthetic processes³⁷. Therefore, it can be suggested that the pectin extracted from our raw material may show antioxidant activity due to its ability to form these types of interactions.

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27 4.2.6 Fourier transform infrared spectrometry (FTIR)

The chemical structure of the selected extracts was studied by FTIR and presented in Figure 10. Due to the large number of samples, only the pH3 120°C 10-min, pH5 120°C 10-min, pH7 120°C 10-min and pH5 140°C 5-, 10-, 15-min extracts were selected to be studied by FTIR and for gelling properties.

Figure 10. FTIR spectra of the chosen extracts

Between 3600 and 3000 cm^-1 a typical broad band was observed corresponding to the stretching of O-H groups. The band from 3000 to 2800 cm^-1 corresponded to the stretching of C-H bonds³⁸. The C-H stretching vibrations were observed to superimpose upon the broader O-H band as in agreement with Gnanasambandam et.al. (1999)³⁹. The spectral peaks between 2950 and 2750 cm^-1 can be attributed to O-CH3 band stretching as the pectin could be methyl-esterified.

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The bands between 1300 and 700 cm^-1 corresponded to the fingerprint region, representing glycosidic linkages, C-O-C vibration, C-C and C-O stretching³⁸.The peak seen at 1720 cm^-1 in all samples was ascribed to the ester carbonyl (C=O), and the band between 1640 and 1620 cm^-

1 indicated carboxylate ion stretching (COO-). The former may indicate that the extracted pectins were indeed methyl-esterified³⁹. Accordingly, all the selected extracts showed methyl esterification with similar band intensity at 1620 cm^-1. The COO- bands showed varying band intensity and it can be assumed that the more intense the band is, the lower the DM. In summary, all samples were methyl-esterified but to an unknown degree.

4.2.7 Gelling properties

The gelling properties of the extracted pectins were studied in an empirical way. As the degree of methylation was not determined, we investigated the effect of sucrose, Ca²⁺ and different pH conditions on the gel formation. In case of the recipes that were prepared with 1% pectin in a citrate buffer of pH 3, none of the samples formed particularly viscous structure however, the formulations that contained sucrose were more viscous than the others. The addition of Ca²⁺

did not have a significant effect on the viscosity, even when its concentration was increased.

When the pectin concentration was raised to 5%, more viscous were obtained compared to the 1% pectin concentration. Adding 60% (w/w) sucrose largely contributed to the rise in viscosity.

However, the subsequent addition of Ca²⁺ had the opposite effect and made the samples less viscous, along with the appearance of blurriness. This trial was performed in buffers of different pH (pH 3.0, pH 2.5 and pH 3.3) however the change in the pH did not result in significant differences between the samples. Smaller differences in the viscosity of samples, which could not be observed visually, by the change of pH could be further studied by rheology measurements.

The gelling behavior of the samples can be explained by the different structural features of the extracted pectins. Although the DM was not measured, SWE can be assumed to result in a high DM according to the work of Wang et.al. (2014)¹¹. Due to the fact that high methoxylated pectins gel at pH values lower than 3.5¹⁵, the pH of the buffer most probably facilitated the formation of gels. The two other structural elements that are known to affect the gelling properties are molecular weight and branching. The extracts that were used for the preparation of gels had a high molecular weight and a significant amount of side chains mostly composed of arabinan and galactan. The 5-min extract used for the gelling study contained about 50%

GalA and the 10-min extract only contained about 35% GalA, while both had high Mw.

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Hence, it could be suggested that the combination of high molecular weight and high level of branching prevented the formation of strong gels. According to Fraeye et.al. (2010), gel strength increases with molecular weight however, a high level of branching can cause steric hindrance and hamper pectin-pectin interactions¹⁵.

5. CONCLUSION

In this study, the valorization of apple pomace into pectins was evaluated using subcritical water extraction. The first step was to isolate the phenolic compounds with antioxidant activity to be evaluated in further applications. In the second step, the effect of temperature and different pH conditions on the extraction of pectins was studied. Due to the special circumstances (COVID- 19 pandemic), the scope of this thesis is only focused on the validation of subcritical water extraction from apple pomace and the proof of concept of the gelation of the extracted pectins.

Valuable antioxidant compounds, which exhibited remarkable radical scavenging activity, were extracted from apple pomace using a fast and easy method. Subcritical water extraction was successfully implemented to produce higher pectin yields from apple pomace compared to traditional acidic extraction techniques. The highest pectin yield was obtained from the extraction at 140°C, pH 7 with an average molecular weight of 218 kDa. On the other hand, the highest molecular weight was obtained from the extractions at 120°C, pH 3. These results confirmed that the features of extracted pectins could be modulated by adjusting the extraction temperature and pH conditions. The extracted pectins seemed to form viscous liquids, which hold a potential to be used as thickeners in acidic hydrogels in the presence of sucrose. The lack of visible gelling was likely due to the combination of high molecular weight and long branching of pectins. In addition, the pH7 extracts showed significant antioxidant activity despite the prior extraction of phenolic compounds, which can be potentially valorized in various future formulations.

In light of the global pandemic, it is crucial to recognize the need for a radical change in terms of sustainability. The United Nations has set 17 sustainable development goals to make the world a better place, covering different areas e.g. education, human rights and sustainability.

The goal of my project is to extract valuable compounds from apple pomace, a by-product of apple processing. This by-product would otherwise be thrown away and all the value in it would be lost. Thus, I believe that this project contributes to UN Goal 12, Responsible Consumption and Production as it highlights the efficient use of natural resources and reduces waste generation in the food industry.

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For future work, a quantitative analysis of degree of methylation could be conducted in order to better understand the gelling behavior of the subcritical extracted pectins. As the extracted pectins are anticipated to be relatively branched, the degree of branching could be further investigated by size exclusion chromatography. These two properties are fundamental for fine- tuning the properties pectin gels for different applications. In addition, the combination of the prior extracted antioxidant compounds and pectins in various formulations could offer a possibility for innovative products. Finally, chemical modification routes (e.g. amidation) could be investigated to produce pectins for advanced uses.

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31 Acknowledgements

I am incredibly grateful to have worked under the supervision of Francisco Javier Vilaplana Domingo, the Head of Division of Glycoscience. He has always managed to help me take the project into the right direction when I was uncertain and has always taken the time to listen to my progress. Nevertheless, he is not only a great professor but also a kind person who truly cares about his students and colleagues.

I would also like to acknowledge my co-supervisors Secil Yilmaz Turan and Carolin Menzel, who have treated me as an equal partner and have trusted my experiments. Caro has taught me that in science there are always some unexpected results, but that should not discourage me.

Secil has taught me that sometimes when certain things do not work it is not necessarily my fault. She has also given me lots of constructive criticism on writing my thesis, contributing a great deal to my finished work. Furthermore, both Caro and Secil have been very understanding when my schedule was busy due to my part-time job and I needed some extra help.

Lastly, I am grateful to all the other members of the Division of Glycoscience who have helped me during my project in these difficult times.

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Cascade Valorization of Apple Pomace into Polyphenols and Pectins by Green Extraction Processes

Cascade Valorization of Apple Pomace into Polyphenols and Pectins by Green Extraction Processes

TEODÓRA GÁL

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