Obtainment and characterisation of pectin from sunflower heads purified by membrane separation techniques

Food Chemistry 318 (2020) 126476

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Obtainment and characterisation of pectin from sunflower heads purified by membrane separation techniques

T

Nerea Muñoz-Almagroa, Marin Prodanovb, Peter J. Wildec, Mar Villamiela, , Antonia Montillaa ⁎

a

Grupo de Química y Funcionalidad de Carbohidratos y Derivados, Instituto de Investigación en Ciencias de la Alimentación (CIAL) (CSIC-UAM) CEI (CSIC+UAM), Nicolás Cabrera, 9, Campus de la Universidad Autónoma de Madrid, 28049 Madrid, Spain b Grupo de Ingredientes Alimentarios Funcionales, Instituto de Investigación en Ciencias de la Alimentación (CIAL) (CSIC-UAM) CEI (CSIC+UAM), Nicolás Cabrera, 9, Campus de la Universidad Autónoma de Madrid, 28049 Madrid, Spain c Quadram Institute Bioscience, Norwich Research Park, Norwich, UK

ARTICLE INFO

ABSTRACT

Keywords: Pilot-scale Microfiltration Ultrafiltration Galacturonic acid Yield Functional properties

A pilot-scale extraction of sunflower pectin with 0.74% (w/v) sodium citrate (72 °C, 194 min) and different procedures of purification including alcohol precipitation, ultrafiltration (UFDF) and microfiltration (MFDF) with diafiltration were carried out. Considering the alcohol treatment, the yields were similar at laboratory and pilot-scale (~8.9%), demonstrating the efficiency of the scale-up. With respect to membrane processes, the best results were obtained with UFDF, showing the highest yield (13.3%) and pectin concentration higher than 90%. In all cases, pectins presented very low amount (~1%) of glucose and mannose, monosaccharides not included in the pectin structure. Detailed NMR analysis and functional properties (emulsifying and viscosity) that were also assessed corroborated the good quality of UFDF obtained pectin. These results point out that the obtainment of sunflower pectin of good quality can be achieved at pilot-scale by the extraction with sodium citrate and purification with membrane separation, eco-friendly alternatives to conventional procedures.

1. Introduction

This wide variety of applications has generated an industrial demand of pectin around 45.106 t which continues increasing since 2011 (MoralesContreras, Rosas-Flores, Contreras-Esquivel, Wicker, & Morales-Castro, 2018). Consequently, a continuous search to find different pectin sources from the commercial ones (citrus peel and apple pomace) that could provide not only competitive yields but also other structural features, is currently being carried out (Marić et al., 2018). In this sense, sunflower heads (SH) are considered a source of special interest due to their important content (15–25%) of low-methoxyl pectin (LMP) and high availability as an agricultural by-product (Iglesias & Lozano, 2004). In general, depending on extracting agents, the yield and characteristics of sunflower pectin can vary wildly (Iglesias & Lozano, 2004; Kang, Hua, Yang, Chen, & Yang, 2015; Sahari, Akbarian, & A., & Hamedi, M., 2003). In a previous study, we found

Pectins are complex heteropolysaccarides consisting of a linear chain of α-1,4-linked D-galacturonic acid (GalA) partly methylesterified, as well as neutral sugar side chains composed by arabinans, galactans and arabinogalactans (Broxterman & Schols, 2018; Marić et al., 2018). In the food industry, pectins highlight for their thickening, emulsifying and gelling properties in the formulation of products such as jams, sauces and low calories jellies (Marić et al., 2018; Naqash, Masoodi, Rather, Wani, & Gani, 2017). Moreover, these heteropolysaccarides also present health benefits, such as promotion of the growth of Bifidobacterium and Lactobacillus in the human gut and stabilising blood pressure (Ferreira-Lazarte, Kachrimanidou, Villamiel, Rastall, & Javier Moreno, 2018; Zhang, Zhang, Liu, Ding, & Ye, 2015).

Abbreviations: Ara, arabinose; DM, degree of methoxylation; ELSD, Evaporative Light Scattering Detector; FAO, Food and Agriculture Organisation of United Nations; FT-IR, Fourier-Transform Infrared Spectroscopy; Gal, Galactose; GalA, galacturonic acid; Glc, glucose; HPSEC, High Performance Size Exclusion Chromatography; Lab-Conv-1Ext, Lab-Conv-2Ext, Lab-Conv-3Ext and Lab-Conv-4Ext, sunflower pectin extracted and purified by conventional method for first, second, third and fourth times, respectively; LMP, low-methoxyl pectin; Man, mannose; Mw, molecular mass; NMR, Nuclear Magnetic Resonance; Pilot-Conv, sunflower pectin extracted and purified by conventional method at pilot scale; Pilot-MFDF, pectin purified by microfiltration; Pilot-NF-Conv, extract filtered by nanofitration and precipitated with ethanol; Pilot-NF, sample filtered by nanofitration; Pilot-UFDF, sunflower pectin recovered by ultrafiltration; Rha, rhamnose; SEM, Scanning Electron Microscope; SH, sunflower heads; TSS, Total Soluble Solids; Xyl, xylose ⁎ Corresponding author at: Instituto de Investigación en Ciencias de la Alimentación (CIAL) (CSIC-UAM), C/Nicolás Cabrera 9, Campus de la Universidad Autónoma de Madrid, E-28049 Madrid, Spain. E-mail address: [email protected] (M. Villamiel). https://doi.org/10.1016/j.foodchem.2020.126476 Received 17 May 2019; Received in revised form 17 January 2020; Accepted 23 February 2020 Available online 24 February 2020 0308-8146/ © 2020 Elsevier Ltd. All rights reserved.

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that the yield of extraction of sunflower pectins with sodium citrate was 2-fold lower than the corresponding with nitric acid, however their structural and techno-functional properties were better than those obtained with the strong acid (Muñoz-Almagro, Rico-Rodriguez, Wilde, Montilla, & Villamiel, 2018b). To increase this lower yield, the consecutive extractions of pectin could be an interesting alternative. After extraction, the most commonly method for purification of pectin is the precipitation with ethanol (Adetunji, Adekunle, Orsat, & Raghavan, 2017; Yapo, Wathelet, & Paquot, 2007). Alcohol treatment is ready-to-operate, however, the lack of selectivity and its high alcohol consumption especially in scale-up production are their main drawbacks (Conidi, Cassano, Caiazzo, & Drioli, 2017; da Silva, Hamerski, & Scheer, 2012; Kang et al., 2015; Yapo et al., 2007). Hence, it is necessary the application of more selective techniques that replace the alcohol treatment. In this context, membrane separation processes, such as microfiltration (MF) and ultrafiltration (UF) combined with diafiltration (DF), represent environmental-friendly alternatives to conventional technologies as result not only of their easy control and mild conditions of pressure and temperature but also their low maintenance and operating costs (Conidi et al., 2017; Galanakis, 2015). In the last years, MFDF and UFDF have gained a considerable interest for the concentration of pectin from apple pomace (Qiu, Tian, Qiao, & Deng, 2009), sugar beet pulp (Hatziantoniou & Howell, 2002; Yapo et al., 2007), citrus pectin (da Silva et al., 2012) and sunflower heads (Kang et al., 2015). However, to the best of our knowledge, no studies are available on the comparison of structural characteristics and technological properties of pectin purified by both filtration membranes (MFDF, UFDF) and precipitation with ethanol at pilot scale. Considering the above exposed, the aim of this study has been the extraction of sunflower pectin in a pilot plant using sodium citrate solution with the purpose of studying the yield and structural characteristics of this polysaccharide purified by different membrane processes (UFDF and MFDF). Moreover, the impact of pectin structure on their emulsifying and rheological properties was also assessed. In addition, assays at laboratory scale were carried out to know the effect of consecutive extractions on the yield and composition of pectin.

the extraction was done in a bach reactor of 30 L and provided with a thermostatic system. The suspension was hold by a Nylon mesh strainer bag with a pore size of 30 µm. After extraction, the liquid phase was separated by racking for its further purification. In the case of the consecutive extractions, the wet solid was mixed again with sodium citrate and macerated at time and temperature, previously mentioned above. 2.3. Pectin purification Prior to the alcohol precipitation and membrane purification, extracted solution obtained at pilot scale was heated at 50 °C and clarified through a Büchner funnel with a cellulose-based filter plate No. 0, SA990 to remove suspended solids. Fig. 1 Supplementary shows a scheme of the assays carried out. 2.3.1. Conventional ethanol precipitation method For ethanol precipitation two volumes of 96% acidified ethanol (0.2% HCl v/v) were used and this solution was kept at 4 °C overnight. The mixture was centrifuged at 3700×g and the precipitate washed firstly with 96% acidified ethanol (0.04% HCl v/v) and later with ethanol (96%). The recovered pectin was lyophilised and stored at −20 °C. 2.3.2. Purification of sunflower pectin by filtration membranes In all membrane processes, a tubular spiral heat exchanger was immersed into the feed tank to keep the temperature at 50 °C during the filtration, reducing the viscosity and consequently, improving membrane permeability. All purifications were carried out in a concentration mode at constant transmembrane pressure (PTM, 0.8 bar). This parameter is the driving force behind the process. Matter transference (%) was calculated over time in each membrane process, following the equation:

Transference(%) =

Xp XR

× 100

where XR and XP are the values of Total Soluble Substances (TSS) related to the retentate and permeate, respectively.

2. Materials and methods

2.3.2.1. Ultrafiltration combined with diafiltration (UFDF). A volume of 4 L of the sunflower pectin extract was concentrated to 2.1 L in a preparative cross-flow pressure-driven UF unit (Fig. 2b Supplementary) with a spiral-wound cellulose membrane (model Prep-scale 6, 10 kDa) from Millipore (Merck, Darmstadt, Germany) (Table 1 Supplementary), after three DF cycles of 2 L each one (until reach the initial volume in the reactor). The diafiltered retentate was lyophilised, while the permeate was wasted.

2.1. Materials and chemicals Standard monosaccharides (arabinose, xylose, galactose, rhamnose, glucose and galacturonic acid (GalA)), β-phenylglucoside, hexamethyldisilazane, trifluoroacetic acid, citric acid monohydrate, sodium citrate tribasic dihydrate and Pullulan Standard (0.34–805 kDa), a glucan polymer based on α(1,6) linked maltotriose units were purchased from Sigma (St. Louis, MO, USA). Ethanol (96%, v/v) was acquired from VWR (Barcelona, Spain) while ammonium acetate was obtained from Panreac Applichem (Darmstadt, Germany). Sterilising cellulose-based filter plate (SA-990) with thickness of 3.3 mm and a pore size of 0.4–0.6 µm was obtained from Filtrox Southern Europe (Barcelona, Spain). Dried sunflower heads (SH) without seeds were supplied as a generous gift by Syngenta (Madrid, Spain). The SH samples were grinded and sieved (particle size ≤ 100 µm).

2.3.2.2. Microfiltration and diafiltration (MFDF) combined with nanofiltration (NF). Filtration of the extract was carried out by a selfdesigned multifunctional pilot pressure-driven cross-flow membrane unit (Fig. 2a Supplementary) (Morales, Smiderle, Piris, Soler-Rivas, & Prodanov, 2018). In the case of MF, 15 L of the extract was concentrated to 5 L by a multichannel ceramic membrane from CeraMen Corporation (Waltham, MA, USA) after three DF cycles of 7.5 L each one (until reach the initial volume in the reactor). The diafiltered retentate was lyophilised. Otherwise, 26 L of MF permeate were concentrated by NF up to 5.3 L with a spiral wound membrane Nanomax 50 from Millipore, nominal pore size, 0.5 μm (Bedford, MA, USA) (Table 1 Supplementary). Subsequently, the NF permeate was wasted while the retentate divided in two halves. One of them was precipitated with ethanol. The pectin was separated by centrifugation and lyophilised. The other half of the NF retentate was directly lyophilised for its characterisation. Afterward of these purification processes, the yields of extraction were calculated as follows:

2.2. Pectin extraction Before extraction, 0.02 or 1 kg (laboratory or pilot scale) SH were washed with MilliQ water (S/L ratio 1/30 w/v). The wet solid was extracted with 0.74% (w/v) sodium citrate (original S/L ratio 1:20 w/v) at 72 °C and pH 3.3 (adjusted with citric acid) for 194 min. These conditions were selected based on a previous study (Muñoz-Almagro et al., 2018b) whose parameters were optimised by a response surface methodology (RSM) taking into account the yield of extraction. Afterwards, the mixture was centrifuged at 3700×g for 10 min and recovered by conventional treatment at laboratory scale. At pilot scale, 2

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Table 1 Molecular parameters and sugar composition (% monosaccharides from the total quantified) of sunflower pectin extracted at laboratory and pilot scales using different purification methods. Lab scale

Pilot scale

Conventional

Conventional

Ultrafiltration

Microfiltration

Nanofiltration

Pilot-Conv

Pilot-UFDF

Pilot-MFDF

Pilot-NF-Conv

Lab-Conv-1Ext Yield (%) GalA Xyl Ara Rha Gal Man Glc GalA/Rha Ara + Gal/Rha DM (%)

ef

8.8 ± 0.3 80.9 ± 2.1abcd 3.7 ± 0.2c 5.9 ± 0.3 g 3.5 ± 0.2de 2.6 ± 0.4ab 1.0 ± 0.2c 2.4 ± 0.2c 23.1 ± 1.1b 2.4 ± 0.2b 20 ± 0.9a

Lab-Conv-2Ext d

4.1 ± 0.1 81.3 ± 1.4abcd 3.9 ± 0.3c 5.4 ± 0.2f 3.8 ± 0.1ef 2.5 ± 0.2ab 0.8 ± 0.1b 2.3 ± 0.2c 21.4 ± 0.9ab 2.1 ± 0.2b 20 ± 0.3a

Lab-Conv-3Ext d

4.3 ± 0.2 79.4 ± 2.8abc 3.9 ± 0.2c 5.6 ± 0.2 fg 4.0 ± 0.3f 2.9 ± 0.3b 1.2 ± 0.1d 2.6 ± 0.3c 19.9 ± 1.3a 2.1 ± 0.3b 20 ± 1.2a

Lab-Conv-4Ext c

f

2.0 ± 0.2 89.4 ± 1.8d 2.2 ± 0.0b 3.9 ± 0.2d 1.8 ± 0.1a 0.4 ± 0.1a 0.4 ± 0.1a 0.9 ± 0.2b 49.7 ± 2.1f 2.4 ± 0.2b 17 ± 0.5a

9.1 ± 0.3 83.0 ± 2.0abcd 3.8 ± 0.2c 4.6 ± 0.2e 3.1 ± 0.2 cd 2.6 ± 0.3ab 0.8 ± 0.1b 2.2 ± 0.2c 26.8 ± 0.5a 2.3 ± 0.1a 33 ± 1.0b

g

12.3 ± 0.4 90.2 ± 3.3bcd 0.3 ± 0.1a 3.0 ± 0.1c 3.0 ± 0.2c 2.9 ± 0.2b 0.2 ± 0.0a 0.6 ± 0.1ab 33.4 ± 0.3d 2.0 ± 0.1a 38 ± 1.8c

e

8.4 ± 0.1 87.9 ± 2.6 cd 4.2 ± 0.8c 2.2 ± 0.2b 2.5 ± 0.1b 2.2 ± 0.1ab 0.2 ± 0.0a 0.8 ± 0.1b 35.2 ± 0.3de 1.8 ± 0.2c 40 ± 1.2c

a

0.3 ± 0.0 77.2 ± 0.3a 0.7 ± 0.2a 1.8 ± 0.1ab 3.0 ± 0.2c 16.0 ± 2.2c 1.0 ± 0.1bc 0.3 ± 0.0c 25.7 ± 0.3b 5.9 ± 0.2d 49 ± 2.1d

Pilot-NF 1.2 ± 0.1b 77.6 ± 0.5ab 0.8 ± 0.1a 1.5 ± 0.2a 2.1 ± 0.2ab 17.1 ± 1.3c 0.3 ± 0.0a 0.6 ± 0.1ab 37.0 ± 0.2e 8.9 ± 0.5e 19 ± 1.0a

Note: values with different small case superscript letters (a-e) in the same line indicate significant differences estimated by Tukeýs test (P < 0.05); Xyl: xylose; Ara: arabinose; Rha: rhamnose; Gal: Galactose; Man: mannose; Glc: glucose; GalA: galacturonic acid (Codes see Abbreviations section or Fig. 1 Supplementary).

Fig. 1. Estimation of Mw (kDa) and main fragments found (%) in the sunflower pectin extracted and purified a) at laboratory and b) pilot scale. Note: values with different small case superscript letters (a-e) indicate significant differences in Mw, estimated by Tukeýs test (P < 0.05). (Codes see Abbreviations section or Fig. 1 Supplementary). *Mw upper to elution volume. **In this fragment modified pectins and salts overlap.

a digital refractometer 30PX/30GS (Mettler Toledo GmBH, Schwerzenbach, Switzerland). Turbidity was determined by a TN100 infrared turbidimeter (TermoFisher Scientific, Spain). The pH was measured using a pHmeter (Mettler Toledo GmBH, Schwerzenbach, Switzerland). Electrical conductivity was determined by direct measurement of samples using a conductivity meter (GLP 31EC, Crison Instruments, Barcelona, Spain).

Yield(%) = [weight of dried extracted pectin(g)]/[weight of dried powder(g)] × 100

2.4. Physicochemical analyses During the processes of UFDF, MFDF and NF, several aliquots of the retentates and permeates were analysed for their turbidity, content of TSS and electrical conductivity. TSS of the extracts were measured with

2.5. Structural characterisation The estimation of molecular mass (Mw) by HPSEC-ELSD of pectin 3

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4

(caption on next page)

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Fig. 2. The 1H NMR spectra of sunflower pectins. a) Lab-Conv-1Ext; b) Lab-Conv-4Ext; c) Pilot-UFDF; d) Pilot-MFDF; e) 1H 1H COSY spectrum of Pilot-UFDF; f) 1H 13C HSQC spectrum of Pilot-UFDF GA: 4-α-GalpA, GA′: 4-α-GalpA(OMe), GA″: →4)-3-O-Ac-α-GalpA-(1→; Araf: α-L-Araf; Rhap: α-L-Rhap. (Codes see Abbreviations section or Fig. 1 Supplementary).

rate changed from 0.1 to 1200 s−1. Rheological tests were performed in the range 0.05–10 Hz (0.5% strain).

samples was determined following the method described by MuñozAlmagro, Rico-Rodriguez, Villamiel, & Montilla (2018a). A TSK-Gel guard column (6.0 mm × 400 mm) connected in series with two TSKGel columns G5000 PWXL (7.8 mm × 300 mm, 10 μm) and G2500 PWXL (7.8 mm × 300 mm, 6 μm) (Tosoh Bioscience, Stuttgart, Germany) were used in the samples separation (0.1% w/v, 50 µL). The pectin elution was carried out using 0.01 M NH4Ac as mobile phase at 0.5 mL/min for 50 min. The purified lyophilised pectins were analysed by GC-FID and FT-IR in order to determine the content of neutral sugars and GalA as well as degree of methoxylation (DM), respectively, according to the methods reported by Muñoz-Almagro et al. (2018b). Regarding the monosaccharide composition, the initial temperature, 150 °C, was held for 17 min, and then was rose to 165 °C at 1 °C/min, then increased at a rate of 10 °C/min to 200 °C and up to 380 °C at 50 °C/min. A DB-5HT capillary column (15 m × 0.32 mm × 0.10 µm (J&W Scientific, Folson, California, USA) was used to eluate the samples at 1 mL/min using nitrogen as carrier gas. For DM determination, the pectins were mixed with bromide potassium and analysed in a Bruker IFS66v (Bruker, US). The FT-IR spectra showed a frequency range of 400–4000 cm−1 at a resolution of 4 cm−1 (mid infrared region) with 250 co-added scans. The DM of pectin was determined as the relation between the peak area at 1734 cm−1 (COO-R) over the sum of the peak areas of 1734 cm−1 (COO-R) and 1612 cm−1 (COO−). For Nuclear Magnetic Resonance (NMR) spectroscopy analysis, mono-dimensional (13C- and 1H-) and bi-dimensional (COSY, HSQC) spectra were acquired at 30 °C. The samples were solubilised in D2O and the chemical shifts were expressed as δ (ppm), using this solvent as internal references at 4.64 ppm. Morphological observations of the sunflower pectins, obtained by different methods of purification, were performed using a DSM 950 scanning electron microscope (SEM, Zeiss Iberia, Madrid, Spain) at 10 mm of distance and 7 kV accelerating voltage with a magnification of 100x.

2.7. Statistical analysis Extraction and purification trials were carried out in duplicate whereas structural analysis, emulsifying properties and viscosity measurements were performed in triplicate. Data were expressed as mean values ± SD. Tukey’s test to evaluate the differences were performed using IBM® SPSS® Statistics 25 (IBM Corporation, Armonk, NY, USA). 3. Results and discussion 3.1. Laboratory scale. Structural characterisation of sunflower pectin extracted sequentially and purified by conventional method Sunflower pectins obtained by four consecutive extractions were evaluated in this study. Extraction yields, GalA content and neutral sugars contents are included in Table 1. As expected, the first extraction was the most efficient with yields significantly higher (8.8%, equivalent to 46% of total yield) than the subsequent extractions. The second and third extractions yielded around 4% of additional pectin, while in the last treatment only a 2% was extracted. Similarly, Chang, Dhurandhar, You, and Miyamoto (1994) carried out two consecutive extractions of sunflower pectin with sodium hexametaphosphate, obtaining yields of 8.8% in the first and 3.3% in the following treatment. The relatively low yield of the first extraction can be explained because most of the pectin is tightly bound to the hemicelluloses making more difficult its release (Kang et al., 2015). In our work, the global yield reached was 19.2%, demonstrating the potential of sunflower heads use as renewable pectin source. As it was expected, GalA was the most abundant monosaccharide present in all pectin samples, varying in a narrow range (79.4–89.4%). With respect to the content of main neutral sugars (Table 1), their sum was around 15% and was kept constant during the first three treatments. Taking into account the content of GalA and neutral sugars it is clear that the structure of pectin is composed mainly of homogalacturonan (HG) as well as a small part of arabinogalactan and arabinan branches to the rhamnogalacturonan-I (RG-I) chains (GalA/Rha, ~21). Nonetheless, in the last extraction, it is noticeable that pectin suffered an important degradation since the total content of neutral sugar sides decreased to 9.2%. This pectin was more enriched in HG than the others previously recovered. Regarding the susceptibility of the neutral sugar linkages in pectin to acid hydrolysis, is more labile than GalA-GalA (Kang et al., 2015). Other aspect that is evident in the subsequent extractions is that the content of the monosaccharides nonderived from pectin were considerably inferior in the fourth extraction, being the glucose the main interference with a proportion 2-fold higher than mannose. As illustrated in Fig. 3a Supplementary and Fig. 1a, this process demonstrated to be effective for the recovery of sunflower pectin (> 686 kDa), reaching values upper than 450 mg/g. With the subsequent extractions, a slight decrease in the concentration and Mw was observed, being in the latter significant, in the third and fourth extraction steps, probably due to the degradation of pectin. With respect to the changes in the functional groups, in general, hardly any modifications were found in the spectral profiles of pectin recovered in the three first treatments (Fig. 5a Supplementary). Nonetheless, it is noticeable a prominent reduction of the intensity of all bands in the last extraction. The band that appears on 2936 cm−1 assigned to the vibrations of C-H (–CH, –CH2, –CH3), showed the same

2.6. Technological properties The determination of these properties was carried out for sunflower pectins purified with membrane techniques at pilot scale. 2.6.1. Emulsifying properties Surface tension measurements were performed using the pendant drop technique with drop tensiometer FTA200 (First Ten Angstroms, Portsmouth, VA, USA). Sunflower oil was placed in a glass cuvette. The droplet of aqueous phase, formed at the tip of the needle, was immerged into the sunflower oil. Surface tension of pectin samples (0.01–1% w/v) was measured at 25 °C by triplicate, capturing an image every five seconds for 25 min. Deionised water was used as the control. Oil/water (O/W) emulsions consisted of extracted pectins (1% w/v) and sunflower oil (1:9 v/v). The mixture was emulsified with a highspeed homogeniser Ultra-Turrax T25 (IKA® England LTD, Oxford, UK) at a speed of 10000 rpm for 1 min. The droplet size distribution in the emulsions was measured using a Coulter LS13 320 Laser Diffraction Particle Sizer (Beckman Coulter Inc., California) and the surface mean diameter D3,2 was recorded. The emulsions were analysed in triplicate. 2.6.2. Rheological properties Rheological measurements were done using an AR 2000 rheometer (TA Instruments Ltd., Crawley, U.K.). Data were analysed with the computer software Rheology Advantage (TA Instruments, Waters Co., Ltd). A cone (40 mm diameter, 1°) was employed for viscosity (1% w/v) using a gap size of 85 µm at 25 °C. For steady flow studies, the shear 5

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Fig. 2. (continued) 6

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area in all samples, indicating that HG did not suffer depolymerisation during the repeated process. The strong band around 1734 cm−1, related to the asymmetric tension of methylesters of the carbonyl groups increased its intensity, except in the four extraction, and the signal near 1612 cm−1, belonging to the symmetrical tension of the carboxylate ions, exhibited a reduction as a result of the variation of the degree of methyl esterification (DM). These qualitative modifications registered resulted in quantitative modifications in the DM, as illustrated in Table 1. All pectins analysed were of low-methoxyl exhibiting values of DM around 20%. Results indicated that it is possible to carry out extractions increasing pectin yield without modifying the techno-functional properties established by DM. Fig. 2a and b shows the 1H NMR spectra of the pectins recovered with alcohol precipitation at laboratory scale corresponding to the first and the last extractions. After four consecutive extractions, a slight decrease of the signals at 4.08, 4.14 and 4.31 ppm was detected. This could be due to a degradation of neutral sugar side-chains caused for long times and high temperature in acid medium (Kang et al., 2015), in agreement with the data of the monomeric composition of the first and the last extracted pectin. In fact, the intensity of the double of doublets ranging 2.54–2.77 ppm, corresponding to sodium citrate, was higher after several stages of extraction. On the other hand, both spectra depicted, two signals at 2.0 ppm (1.93 and 2.04 ppm) which were derived from acetyl groups binding at 3-O- and 2-O-GalA while a strong signal around 3.67 ppm was related to the methoxyl groups of GalA (Wang et al., 2016). Microstructure of pectin, analysed by SEM, extracted consecutively up to four times and purified by alcohol treatment are shown in Fig. 6(a–d) Supplementary. As illustrated, the pectin extracted in the first step presented two different microstructures. One of them showed a smooth surface with some long cracks and another one consisted of several irregular and rough surfaces. However, from second extraction up to the last one, the last type of microstructure that turned into more heterogeneous, compacted and flaky in shape is present. A slight and increasing compaction of the structure was observed in the successive extraction stages.

therefore there are still compounds in the retentate that could be eliminated. On the other hand, as expected in the successive DF cycles, a considerable concentration of the extract was observed (Table 2 and Fig. 4 Supplementary). Thus, proportionally the area of peak displayed at 24 min corresponding to pectin increased, whereas the fraction assigned to salts at 32 min decreased with the number of DF cycles producing a notable enrichment of the pectin initial extract, 22% vs. 96% and 78% pectin for UF and MF final extract, respectively. In the purification by UF, despite of that the maximum area of peak attributed to pectin was reached during the first DF cycle, it was necessary to carry out more cycles (2–4) to remove salts and achieve a greater purification of the pectin (Fig. 4 Supplementary). The efficiency of the pectin concentration was evaluated by electrical conductivity during the purification process (Table 2). This parameter is directly related to the content of electrolytes. Thereby, values around zero could indicate the removal of the salts from extracting agent (Morales et al., 2018). Thus, the conductivity decreased considerably with respect to the initial value in MF and UF processes. This parameter also highlights that only one DF step was necessary in UF, or perhaps two cycles. Contrary to this, the three cycles of DF, applied during the MF treatment, were somehow insufficient to achieve a good purification. 3.2.2. Structural analysis of sunflower recovered material As indicated in Table 1, the yield obtained with conventional method in the pilot plant (9.1%) was similar to that observed in the case of laboratory (8.8%), indicating the efficacy of the scale up. With respect to membrane processes, the UFDF gave rise to the highest pectin yield. These results are in accordance with several authors that also found a greater effectiveness in the recovery of sunflower and sugar beet pectin, when purified the extract by UF and afterwards lyophilised it (Hatziantoniou & Howell, 2002; Kang et al., 2015). On the contrary, Yapo et al. (2007) compared purification methods of pectin (alcohol treatment and UFDF) on sugar beet pulp, reporting better results with the former. At the same Mw cut-offs (10 kDa), these dissimilarities could be due to the bimodal distribution of the sugar beet pulp pectin with two peaks, one with Mw around 140 kDa and other of 35 kDa. In the case of MFDF coupled with NF, an important consideration was the low yields obtained in the NF step; an almost complete pectin recovery was obtained with MFDF (8.4%). The compositional and structural characteristics of the pectin obtained by the different methods are shown in Table 1. GalA was the most predominant carbohydrate in all samples showing values higher than 65%. This limit is established by the FAO to consider the pectin a food ingredient (E440). Purification of this extract by UF gave rise to pectins with the highest amount of this acid monosaccharide (90.2%). Yapo et al. (2007) found also more GalA content with UF purification than with conventional alcohol precipitation (72.1 vs 55.9%) in sugar beet pectin. On the contrary, Kang et al. (2015) exhibited a notable lower GalA content (49.4%) when the extract was recovered by UF, probably as consequence of the degradation suffered by pectin under the extraction conditions, stronger than the applied in this study (85 °C during 240 min). The distribution of domains, corresponding to ratio GalA/Rha and (Ara + Gal)/Rha suggested that all pectin samples were based on a backbone of GalA (HG domain) linked to several neutral sugar branches (RG-I). On the other hand, NF is a technique that could allow the recovery of residual pectin enriched in neutral sugar, especially galactose, with a significant role in biological activities (Kang et al., 2015). Regarding other monosaccharides (glucose and mannose) derived from polysaccharides such as cellulose and hemicellulose, their contents were similar in all samples purified with membrane techniques (0.8–1.4%) and lower than those found in pectin precipitated with ethanol at laboratory and pilot scale (≥3%). This fact could corroborate that membrane-based separation improve the quality of the

3.2. Pilot scale 3.2.1. Evaluation of the effectiveness of membrane filtration processes for pectin recovery Turbidity, permeate flow values, total soluble substances (TSS), electrical conductivity and % pectin concentration were measured over the time to assess the global purification process of the extracted pectin by UF, MF and NF (Table 2). As expected, TSS and turbidity values of retentate increased due to the concentration process within each cycle whereas the corresponding to permeate decreased. Furthermore, in all cases, the increase of the turbidity and TSS contents of the retentates were inversely proportional to their filtration fluxes (Table 2). According to Gimenes, Silva, Hamerski, and Scheer (2014), the linear and branched structure of the pectin molecules allow, for mobility and deformation of the chain under the action of an external force, their insertion into the membrane pores and their passage through them. However, the increase of the concentration favours to gel-like fouling of the pectin on the membrane surface. Due to that, the resistance of pectin to flow increases and it needs the application of greater force to pass through (Galanakis, 2015; Qiu et al., 2009). Therefore, a long operation results in a prominent accumulation of pectin by fouling and consequently, in more considerable losses (da Silva et al., 2012). As can be observed by transference data, UF was the most effective membrane technique for the purification of pectin, reducing the transference at 0% at the end of the third DF cycle, highlighting that pectin is almost pure (concentration 94%). On the contrary, applying the same PTM (0.8 bar), the percentages of transference of the purification by MF and NF slightly reached 29 and 33%, respectively; 7

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Table 2 Reduction of permeate flux, pectin concentration, total soluble solids (TSS), turbidity, and electrical conductivity of retentate (R) and permeate (P) during the membrane filtration.

Ultrafiltration (UFDF)

Concentration 1ª Diafiltration cycle 2ª Diafiltration cycle 3ª Diafiltration cycle

Microfiltration (MFDF)

Concentration 1ª Diafiltration cycle 2ª Diafiltration cycle 3ª Diafiltration cycle

Nanofiltration (NF)

Concentration

Time (min)

Permeate flux (L/h.m2)

Reduction Permeate Flux (%)

0 24 36 46

351.9 203.7 111.1 425.9

0 42 68 0

50 61 63

388.9 314.8 370.4

14 30 0

74 83 98

351.9 333.3 340.2

103 108

Pectin Concentration (%)

TSS (°Brix)

Turbidity (NTU)

Conductivity (mS/cm)

R

P

Transference (%)

R

P

R

P

1 1.3 1.4 0.2

0.4 0.4 0.3 0.2

40.0 30.8 21.4 100

47.5 145 171 24.3

4.16 2.68 1.06 1.88

1.33 1.40 2.23 0.42

2.45 2.22 2.46 0.06

74

0.3 0.4 0.2

0.1 0.1 0.2

33.3 25.0 100

34.2 96.1 17.8

0.44 0.45 0.79

0.49 0.88 0.15

0.31 0.33 0.16

5 10 0

91

0.3 0.4 0.4

0.1 0.1 0.4

33.3 25.0 100

43.4 65.4 16.0

0.47 0.32 0.41

0.32 0.22 0.22

0.13 0.11 0.02

296.3 277.8

6 7

94

0.4 0.5

0.1 0

25.0 0

18.6 20.1

0.34 0.27

0.12 0.10

0.03 0.02

0 42 86 97

76.9 59.2 53.8 92.3

0 23 30 0

1.3 1.4 1.5 0.5

1 0.9 0.9 0.5

76.9 64.3 60.0 100

124 168 243 115

0.45 0.39 0.32 0.50

1.28 2.54 2.95 0.68

2.48 1.31 0.98 1.51

120 165 182

78.5 57.7 95.4

15 37 0

38

0.8 0.9 0.5

0.5 0.5 0.5

62.5 55.6 100

146 211 111

0.40 0.35 0.23

0.88 1.58 0.98

0.89 0.86 0.83

204 230 261

84.6 73.1 92.3

11 23 0

53

0.5 0.6 0.4

0.4 0.3 0.4

80.0 50.0 100

155 231 139

0.19 0.14 0.28

0.95 0.98 0.46

0.79 0.82 0.47

281 290

80.8 75.4

12 18

78

0.4 0.7

0.3 0.2

75.0 29.0

211 325

0.20 0.20

0.56 0.63

0.44 0.23

0 12 23

175.7 148.6 135.1

0 15 23

0.4 0.4 0.6

0.4 0.3 0.2

100 75.0 33.3

0.82 1.07 2.06

0.71 0.51 0.12

0.64 1.10 2.75

0.23 0.35 0.70

22

22

2

obtained pectin in comparison with traditional method. The HPSEC-ELSD chromatographic profiles of the sunflower pectin purified by conventional method and filtration membranes, UFDF and MFDF (Fig. 4 Supplementary), displayed at tr 24 min a peak that was the most abundant fraction identified as pectin. Furthermore, a minor peak around 18 min, which was upper than elution volume and could be attributed to the presence of hemicelluloses from cell wall that could partially remain glued to the pectin after extraction. Qiu, Yadav, Chau, and Yin (2020) found that this fragment corresponded to the nonfragmented high molar mass arabinoxylans associated with protein. Besides, in NF samples, there was a narrow peak in the range of 30–34 min, corresponding to sodium citrate and modified pectins, since the presence of GalA in this fraction was confirmed (Table 1). With respect to the quantitation (Fig. 1b), the UFDF pectin had a lower Mw (452 kDa) than Pilot-Conv (793 kDa) and MFDF (620 kDa) pectins. This could be due to fragments with smaller Mw that are recovered with UF and this could cause a decrease in the average Mw. Kang et al. (2015) obtained a greater difference when UF or precipitation with alcohol for the purification of pectin were used. As for the purification of the pectin, MF and UF demonstrated to be the most effective techniques reaching concentration values upper than 700 mg/ g, without presence of salts. Conventional method was not as selective as indicated by the minor presence of pectin (500 mg/g). Quantitative results confirmed that pectin was not found in samples purified by NF, only there were modified pectins with Mw around 9 kDa. According to the effect of membrane purification on the DM (Fig. 5b Supplementary), no notable differences were observed in the infrared spectral patterns of the pectins thus purified, with the exception of the samples related to NF. These results suggested in most of the cases, that

the HG backbone did not suffer important modifications after purification. In general, the marked band around 1741 cm−1, related to the tension of esters of the carbonyl groups, increased its intensity, whereas the signal at 1632 cm−1, belonging to the tension of the carboxylate ions, reduced its area as consequence of the variation of the DM. Notable differences in the bands 1424, 1240, 1146 and 1050 cm−1were attributed to –C-O-C glycoside ring bond as well as C-O stretching in COOH. From the analysis of the DM (Table 1), it is noted that, although all samples were low methoxylated pectin, DM varied from 30 to 41%, higher values than those found for laboratory pectin (17–20%); special cases were again NF samples that shown the extreme data (19–49%). Kang et al. (2015) found values in sunflower pectin purified with alcohol and by UF in the range 22.6–27.3%. Fig. 5 Supplementary, Fig. 2b shows a more detailed structural characterisation (NMR 1H, 1H COSY and 1H, 13C HSQC) of pectins recovered by conventional method and by UF and MF. Signals displayed in COSY spectra at 1.19 and 1.22 corresponded to the methoxyl groups of O-2 and O-2,4 linked L-rhamnose only were observed in the sunflower pectins purified by filtration membranes. Regardless purification method used, all samples exhibited two signals around δH 2.00 ppm (2.02 and 2.13 ppm) corresponded to methyl protons from acetyl groups binding at 2-O- and 3-O-GalA which correlated with signals at δC 20.07 and 20.39 ppm (Popov et al., 2014; Tamaki, Konishi, & Tako, 2008). Despite of that all pectins showed a rhamnose H-4 signal at 3.18 ppm, Pilot-MFDF presented the most intense signal in comparison to the others. In the HSQC spectrum (Fig. 2f), the resonance of the methyl group carbon at δ 52.90 ppm correlated with signals of methyl ester protons at δ 3.75 ppm. As illustrated in COSY analysis (Fig. 2e), the presence of 8

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methyl ester in the 6-position was evident from weak correlations between H1 and methyl ester protons (Petersen, Meier, Duus, & Clausen, 2008) at δ 5.25/3.76 ppm. The positions of carbonyl carbons in the 13C NMR spectrum (173.27 and 172.54 ppm) could be related with methyl protons at δ 3.70 ppm what could suggest that carboxyl groups of GalpA residues were present as methyl esters (spectra not shown) (Petersen et al., 2008). These results were also corroborated with the presence of signals δH1 and δH3 corresponding to → 4)-3-O-Ac-α-GalpA-(1→. Moreover, as it is shown in HSQC spectrum, Galp gave the matching signal at 3.73/67.79 and 3.78/67.95 ppm, showing UFDF, the highest intensity in 1H NMR spectra. With the exception of Pilot-MFDF, several proton signals attributed to arabinose such as H-2 (4.18 ppm), H-3 (4.00 ppm), H-4 (4.08 ppm) and H-5 (3.84 ppm) were observed in other samples, indicating the presence of terminal α-arabinoses especially in Pilot-UFDF (Ovodova et al., 2009). Non-esterified GalA residues were found at 5.0 ppm (H-1) and 4.90 ppm (H-5) (Wang et al., 2016). On the basis of these results, the lack of signals of terminal β-galactose and α-arabinose of Pilot-MFDF and the prominent intensity of signal 4Rhap which, suggested the presence of short chains of RGI, in agreement with the tendencies observed in emulsifying properties section. Regarding morphology of the samples purified by the conventional method and membrane processes at pilot plant (Fig. 6 Supplementary), SEM pictures corroborating the notable differences that exit among them. Pilot-Conv presented smooth and rough surfaces (Fig. 6e Supplementary). Pilot-UFDF and Pilot-NF (Fig. 6h and f Supplementary) were similar, showing larger, irregular, smooth and flaky in shape surfaces. Meanwhile, Pilot-MFDF consisted of small and rough surfaces with some long cracks strongly compacted (Fig. 6 Supplementary). 3.2.3. Technological characteristics of sunflower pectins obtained at pilot scale 3.2.3.1. Emulsifying properties. In order to better understand the emulsifying mechanism of pectin, the surface tension of pectin at the oil–water interface (O/W), the droplets size and surface tension of emulsion were investigated. As shown in Table 3, the surface tension was reduced up to 7 times in the case of sunflower pectin purified by microfiltration, whereas in the other pectins only decreased 3-fold this valour with respect to the control. Although low surface tension facilitated droplets formation, the protein associated to the pectin could affect also the emulsifying activity of this polysaccharide. Several studies have demonstrated that deproteinised polysaccharides, such as Acacia and fenugreek gums, exhibited higher surface tension (Brummer, Cui, & Wang, 2003; Dickinson, 2003). This was in line with the higher content of protein (10.5%) found in Pilot-MFDF samples in comparison with sunflower pectins precipitated with ethanol or purified by ultrafiltration (8.1–8.4%). These results were considerably lower than those of pomegranate and pumpkin pectins (≥52.5 mN/m) (Cui & Chang, 2014; Yang et al., 2018) but similar to that of sunflower pectin (19 mN/m) (Muñoz-Almagro et al., 2018b). Droplet size also defines the emulsifying activity and can be indicative of the stability of the emulsion (Kermani, Shpigelman, Pham, Van Loey, & Hendrickx, 2015). As illustrated Table 3, the D3,2 of

Fig. 3. Viscosity-shear rate profiles of sunflower pectins at different concentration (T = 25 °C, 0.05 – 2% (w/v)).

emulsions were significantly smaller than the control group. These results could indicate that the aggregation of sunflower pectins did not occur and the emulsions were more stable against oil droplet coalescence as compared to the control group. It is important to highlight that the emulsion prepared by Pilot-MFDF exhibited also the smallest droplet size (D3,2 = 16.86 µm), followed by Pilot-UFDF and Pilot-Conv. A plausible explanation for this behaviour could be related to lower percentage of monosaccharides belonging to RG-I such as rhamnose, arabinose and galactose that contained the pectin purified by microfiltration (6.9%) in comparison to the other samples (> 9%). Zhang et al. (2020) found that a high proportion of RG-I could be responsible for the bigger droplet size in emulsions, while the small droplet size of emulsions might be attributed to the surface tension lowering ability.

Table 3 The surface tension and droplets size of emulsions prepared with Pilot-Conv, Pilot-MFDF and Pilot-UFDF at concentration (1% (w/v)). Groups

D3,2 (µm)

Control Pilot-Conv Pilot-MFDF Pilot-UFDF

110.1 27.91 16.86 23.39

± ± ± ±

Surface tension (mN/m) 3.68d 2.31c 0.10a 0.52b

71.52 ± 4.3c 20.75 ± 1.7b 9.50 ± 0.22a 18.75 ± 0.67b

3.2.3.2. Steady-shear measurements. As depicted Fig. 3, the apparent viscosity of the three sunflower pectins obtained by different purification procedures depended on the shear rate at different concentrations. In general terms, all pectins solutions showed nearly Newtonian flow behavior at low concentrations (< 1.0% w/v). On the

a

Mean volume-weighted diameter. a-d: Results were expressed as mean value ± standard deviation. Values were compared with significant difference within the same columns (p < 0.05). 9

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contrary, a pseudoplastic flow became dominant when the concentration increased. The viscosity of pectin decreased rapidly with increasing the shear rate (from 1 to 100 s−1) but decreased less rapidly at higher shear rates (100–1000 s−1) at higher concentrations (> 1.0% w/v). With the exception of Pilot UFDF samples, very small viscosities were observed in all sunflower pectin at concentrations lower than 1.0% (w/v), which can be interpreted by the higher Mw of Pilot-Conv and Pilot-MFDF (452 vs ≥ 620 kDa). According to Hua, Wang, Yang, Kang, and Zhang (2015), the high Mw is related to a small number of molecules, which were too far apart to interact with another one. Besides, the fact that sunflower pectins purified by filtration membranes showed a higher viscosity than those precipitated with ethanol also could be attributed to their higher DM values (≥38%, Table 1). Low DM are associated to strong repulsions between pectin chains, which increase distance of molecules. When pectin concentration was above 1.0% (w/w), the initial viscosity was significantly increased, being the most viscous sample, Pilot-UFDF followed by Pilot-MFDF and Pilot-Conv. This change indicated that the intermolecular distance had been shortened due to increase of solid concentration. Besides, the intermolecular interactions, especially hydrogen bonding with hydroxyl groups became stronger (Hua et al., 2015).

Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.foodchem.2020.126476. References Adetunji, L. R., Adekunle, A., Orsat, V., & Raghavan, V. (2017). Advances in the pectin production process using novel extraction techniques: A review. Food Hydrocolloids, 62, 239–250. Broxterman, S. E., & Schols, H. A. (2018). Interactions between pectin and cellulose in primary plant cell walls. Carbohydrate Polymers, 192, 263–272. Brummer, Y., Cui, W., & Wang, Q. (2003). Extraction, purification and physicochemical characterization of fenugreek gum. Food Hydrocolloids, 17, 229–236. Chang, K. C., Dhurandhar, N., You, X., & Miyamoto, A. (1994). Cultivar/location and processing methods affect yield and quality of sunflower pectin. Journal of Food Science, 59, 602–605. Conidi, C., Cassano, A., Caiazzo, F., & Drioli, E. (2017). Separation and purification of phenolic compounds from pomegranate juice by ultrafiltration and nanofiltration membranes. Journal of Food Engineering, 195, 1–13. Cui, S. W., & Chang, Y. H. (2014). Emulsifying and structural properties of pectin enzymatically extracted from pumpkin. LWT - Food Science and Technology, 58, 396–403. da Silva, V. R., Hamerski, F., & Scheer, A. P. (2012). Pretreatment of aqueous pectin solution by cross-flow microfiltration: Analysis of operational parameters, degree of concentration and pectin losses. International Journal of Food Science and Technology, 47, 1246–1252. Dickinson, E. (2003). Hydrocolloids at interfaces and the influence on the properties of dispersed systems. Food Hydrocolloids, 17, 25–39. Ferreira-Lazarte, A., Kachrimanidou, V., Villamiel, M., Rastall, R. A., & Javier Moreno, F. (2018). In vitro fermentation properties of pectins and enzymatic-modified pectins obtained from different renewable bioresources. Carbohydrate Polymers, 199, 482–491. Galanakis, C. M. (2015). Separation of functional macromolecules and micromolecules: From ultrafiltration to the border of nanofiltration. Trends in Food Science and Technology, 42, 44–63. Gimenes, M. L., Silva, V. R., Hamerski, F., & Scheer, A. P. (2014). Pretreatment of aqueous pectin solution by cross-flow microfiltration: study on fouling mechanism. International Journal of Chemical Engineering and Applications, 5, 281–286. Hatziantoniou, D., & Howell, J. A. (2002). Influence of the properties and characteristics of sugar-beet pulp extract on its fouling and rejection behaviour during membrane filtration. Desalination, 148, 67–72. Hua, X., Wang, K., Yang, R., Kang, J., & Zhang, J. (2015). Rheological properties of natural low-methoxyl pectin extracted from sunflower head. Food Hydrocolloids, 44, 122–128. Iglesias, M. T., & Lozano, J. E. (2004). Extraction and characterization of sunflower pectin. Journal of Food Engineering, 62, 215–223. Kang, J. Q., Hua, X., Yang, R. J., Chen, Y., & Yang, H. (2015). Characterization of natural low-methoxyl pectin from sunflower head extracted by sodium citrate and purified by ultrafiltration. Food Chemistry, 180, 98–105. Kermani, Z. J., Shpigelman, A., Pham, H. T. T., Van Loey, A. M., & Hendrickx, M. E. (2015). Functional properties of citric acid extracted mango peel pectin as related to its chemical structure. Food Hydrocolloids, 44, 424–434. Marić, M., Grassino, A. N., Zhu, Z., Barba, F. J., Brnčić, M., & Brnčić, S. R. (2018). An overview of the traditional and innovative approaches for pectin extraction from plant food wastes and by-products: Ultrasound-, microwaves-, and enzyme-assisted extraction. Trends in Food Science & Technology, 76, 28–37. Morales-Contreras, B. E., Rosas-Flores, W., Contreras-Esquivel, J. C., Wicker, L., & Morales-Castro, J. (2018). Pectin from Husk Tomato (Physalis ixocarpa Brot.): Rheological behavior at different extraction conditions. 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4. Conclusions In this study we have first found that after three sequential extractions of sunflower pectin with sodium citrate, a 17% of pectin with adequate structural characteristics was obtained. However, the most striking results were those derived from the scale-up of the extraction and purification of pectin. The analyses of momomeric composition, degree of methyl esterification and Mw corroborated that the processes used led to an interesting variety of pectin with different structural characteristics, which could show diverse techno-functional properties. The recovery and purification of sunflower pectin by membrane techniques proved to be more efficient than the precipitation with ethanol in terms of concentration, galacturonic acid content and yield, especially in the case of ultrafiltration combined with diafiltration, moreover, the former is highly solvent consuming. With respect to the emulsifying properties, sunflower pectin purified by microfiltration exhibited not only the smallest droplet size but also the lowest surface tension, demonstrating to be the most efficient sunflower pectin. On the contrary, sunflower pectins purified by ultrafiltration highlighted for being the most viscous sample. The results here obtained demonstrate the usefulness of sunflower heads as renewable pectin source and the feasibility of sodium citrate and ultrafiltration as eco-friendly alternatives to the conventional obtainment of pectin. CRediT authorship contribution statement Nerea Muñoz-Almagro: Investigation, Methodology, Writing original draft. Marín Prodanov: Resources, Supervision. Peter J. Wilde: Resources, Supervision. Mar Villamiel: Funding acquisition, Supervision. Antonia Montilla: Funding acquisition, Supervision. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments This work has been funded by MINECO of Spain, Project AGL201453445- R. N. Muñoz-Almagro thanks to Ministry of Economy of Spain for providing her FPI predoctoral fellowship. 10

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