Effects of pressurized hot water extraction on the yield and chemical characterization of pectins from Campomanesia xanthocarpa Berg fruits

Journal Pre-proof Effects of pressurized hot water extraction on the yield and chemical characterization of pectins from Campomanesia xanthocarpa Berg fruits

Isabela Pereira Dias, Shayla Fernanda Barbieri, Damian Estuardo López Fetzer, Marcos Lúcio Corazza, Joana Léa Meira Silveira PII:

S0141-8130(19)39142-1

DOI:

https://doi.org/10.1016/j.ijbiomac.2019.12.261

Reference:

BIOMAC 14289

To appear in:

International Journal of Biological Macromolecules

Received date:

8 November 2019

Revised date:

18 December 2019

Accepted date:

30 December 2019

Please cite this article as: I.P. Dias, S.F. Barbieri, D.E.L. Fetzer, et al., Effects of pressurized hot water extraction on the yield and chemical characterization of pectins from Campomanesia xanthocarpa Berg fruits, International Journal of Biological Macromolecules(2018), https://doi.org/10.1016/j.ijbiomac.2019.12.261

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© 2018 Published by Elsevier.

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Effects of pressurized hot water extraction on the yield and chemical characterization of pectins from Campomanesia xanthocarpa Berg fruits

Isabela Pereira Diasa*, Shayla Fernanda Barbieria*, Damian Estuardo López Fetzerc, Marcos Lúcio Corazzac, Joana Léa Meira Silveiraa,b**

a

Postgraduate Program in Biochemistry Sciences, Sector of Biological Sciences,

Department of Biochemistry and Molecular Biology, Federal University of Paraná,

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b

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Federal University of Paraná, Curitiba, PR, 81531-990, Brazil.

c

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CEP 81.531-980, Curitiba-PR, Brazil.

Department of Chemical Engineering, Federal University of Paraná, CEP 81531-990

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Curitiba, PR, Brazil.

*

These authors equally contributed to this work.

**

Abstract

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+55-41-3361-1665.

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Author to whom correspondence should be addressed: E-mail: [email protected]; Tel.:

Pressurized hot water extraction (PHWE), known as a ―green‖ extraction technique, was used to obtain polysaccharide from the pulp of gabiroba (Campomanesia xanthocarpa Berg) fruits. The effects of pressure, temperature, and flow rate on pectin yields were analyzed through a full factorial design experiment 23. The optimal extraction conditions to achieve maximum pectin yield (5.70 wt%) were pressure of 150 bar, temperature of 120 ºC, and flow rate of 1.5 mL min-1. The high pressure (100 bar) promoted an increase in galacturonic acid content (36.0%) compared to conventional

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hot water extraction (CEGP) with 25.7%. Differences in the proportion of homogalacturonan (HG) and rhamnogalacturonan (RG-I) domains ranging from 16.3 to 35.4% and 61.7 to 80.1%, respectively, were observed for each pectin sample according to the extraction conditions. The mono-dimensional (13C-NMR) and bi-dimensional (1H/13C HSQC-NMR) analyses confirmed the presence of HG and RG-I regions and indicated the presence of arabinogalactans type I (AG-I) and arabinogalactans type II (AG-II) in the PHWE pectin samples, which was not found for pectins from gabiroba

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method for extracting pectins from gabiroba fruits.

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pulp obtained by CEGP. The results showed that PHWE proved to be a promising

Keywords: Gabiroba; Pressurized hot water extraction (PHWE); Full factorial design

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1. Introduction

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experimental; Pectin; NMR analysis; HPSEC-MALLS-RI.

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Pectin is an important component of the diet, and it can be naturally present in fruits and vegetables or intentionally added (INS 440) during the processing and preparation of foods [1]. This polysaccharide is a health-promoting functional ingredient widely used in the food industry as an emulsifier, texturizer, thickener, and gelling agent [1,2]. In addition, it can be applied in biomedical [3], pharmaceutical [4], and cosmetics fields [5,6]. Application of pectins is related to the diversity in their structure and physicochemical properties such as monosaccharide composition, degree of methylesterification, molecular weight, and gelation properties, which are dependent on the pectin source and extraction method applied in its isolation from the plant material [1,5,7].

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Pectin is structurally and functionally the most complex polysaccharide in plant cell walls, having important functions in plant growth, morphology, development, and defense [8]. It is generally composed of the following: homogalacturonan (HG), a linear polymer formed by D-galacturonic acid residues α(1 → 4) linked, which may be partially methyl-esterified at C-6 and acetyl-esterified at O-2 and/or O-3 position [7,9]; rhamnogalacturonan I (RG-I), a repeating disaccharide [→2)-α-L-Rhap-(1→4)-α-DGalAp-(1→]n with neutral side chains mainly composed of arabinans, galactans, and

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arabinogalactans (AG) [9,10]; and rhamnogalacturonan II (RG-II), which comprises a

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homogalacturonic backbone branched by complex side chains containing the rare

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monosaccharides apiose, O-methyl-xylose, and O-methylfucose [5,8]. The industrial production of pectins is an important ally to agribusiness and can

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favor the ecosystem as an alternative for adding value to agrowaste and solid residues

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extracted from citrus peels (85.5%), apple pomace (14.0%), and beet pulp (0.5%) [7,11]. On a commercial scale, pectin production requires large amounts of raw material

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including acid solvents such as sulfuric, nitric, and hydrochloric associated with high-

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temperature (60–100 °C) processing. However, these extraction conditions can lead to degradation of the polymer structure such as demethoxylation [12], as well as corrosion in equipment and piping and environmental problems like water pollution by the acidic effluents produced [13,14].

In view of this, environmentally friendly technologies have been emerging as alternatives to the traditional acid extraction method. Cleaner extraction techniques such as enzyme-assisted [14], microwave-assisted [15,16], ultrasound-assisted [17,18], and pressurized hot water extraction [19,20] have been employed in the isolation of macromolecules from plants. In this context, pressurized hot water extraction (PHWE), also called subcritical water extraction (SWE), superheated water extraction, and pressurized liquid extraction

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(PLE) allow the fast and efficient extraction of macromolecules from plants using water as a solvent [21,22]. In PHWE, water is maintained at a temperature between normal boiling point (100 ºC) and critical point (374 ºC) under pressure that is high enough to keep the water in the liquid state [23,24]. In these conditions, the water exhibits physical advantages such as high diffusion, low viscosity, low surface tension, and increased vapor pressure. Such properties provide effective mass transfer and higher solubility of more hydrophobic compounds [13,21,22,25].

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PHWE has been mainly employed for extraction of phenolic compounds [26],

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essential oils [26], lipids [27], and antioxidant compounds [28]. Moreover, PHWE has

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been identified as a sustainable approach to polysaccharide extraction, e.g., glucans from mushrooms (Pleurotus ostreatus) [29] and hemicellulose from wood [30].

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Some studies have shown pectin extraction through PHWE in laboratory scale

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under different conditions—temperature 90 to 175 °C, pressure 3 to 100 bar, and processing time 5 to 100 minutes—using different raw materials: apple (Malus

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domestica) pomace, tangerine (Citrus reticulata) peel [19], mango (Mangifera indica)

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peel [31], pomelo (Citrus grandis Osbeck) peel [32,33], sugar beet (Beta vulgaris) pulp, [34] and cacao (Theobroma cacao L.) pod husk [20]. In those studies, the authors mainly evaluated the yield of pectins obtained and some chemical parameters such as degree of methyl esterification, monosaccharide composition, and molar mass. The use of a suitable extraction method alongside a good understanding of the individual and interactive effects of the process parameters temperature, pressure, and flow rate (in a semi-batch or continuous extraction processes) are essential to maximizing pectin extraction yield. However, not only the pectin yields should be considered, but also their structure and chemical composition because these are known to determine their applications [7,13,25].

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In our research group, Campomanesia xanthocarpa Berg (gabiroba), a Brazilian native species belongs to Myrtaceae family [35], was studied as a source of polysaccharides [36,37] with food [37,38] and bioactive properties [39]. Between the polysaccharides, a crude pectin from gabiroba pulp was obtained by hot water extraction using a batch process [37]. It was composed of 31.0% of HG with a degree of methyl-esterification of 60% and 65.3% RG-I. This structure presented different rheological properties, according to its concentration in solution, favorable to the

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application of gabiroba pectin in different systems [37]. Furthermore, the crude and

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purified gabiroba pectins presented antitumor potential, as demonstrated by their

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cytotoxic effect in human glioblastoma cell lines [39].

Therefore, considering the biotechnological and therapeutic potential already

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presented by the gabiroba pectins, the aim of this study was to extract these

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polysaccharides using a PHWE semi-batch technique, evaluate the influence of extraction parameters (pressure, temperature, and flow rate) by full factorial design

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using the pectin extraction yield as the response variable, in addition to determine and

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compare the chemical composition of gabiroba pectins obtained by PHWE at different conditions. Furthermore, to the best of our knowledge, there are no reported data presented concerning the application of PHWE extraction for polysaccharide recovery from Myrtaceae fruits.

2. Materials and methods

2.1. Plant material

Campomanesia xanthocarpa fruits were collected in Irati-Paraná, Brazil, located at coordinates 25° 25′ south latitude, 50° 36′ west longitude, and 25° 17′ south latitude,

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50° 30′ longitude west, in November 2018 during the ripening period. The fruits were selected and washed; the peels and seeds were removed in a Macanuda removing device (Model SPI-DMJI/2013), and the pulp sample was then stored in a freezer at –20 ºC. A moisture analyzer (Model-OHAUS MB25) was used to evaluate the total solids content of the pulp.

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2.2. Experimental design

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Response surface methodology (RSM) was used in the design of the experiment

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and to determine an adequate model for pectin extraction using the PHWE technique. Three-factor, two-level (−1, 1) full factorial design was chosen to investigate the effect

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of process variables and optimize the pectin yield. In developing the regression

(1)

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equation, the tested factors were coded according to the equation:

The variables considered were pressure (X1) ranging between 50 and 150 bar, temperature (X2) between 80 and 120 °C, and flow rate (X3) between 1.5 and 4.5 mL min-1 (Table 1). A total of 11 test runs under these conditions, including three replicates for the central point, were performed for the statistical modeling. The experimental data were fitted to a first order polynomial equation to establish the relationship between independent variables and responses. The generalized form of the equation is:

(2)

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where Y represents yield as response variable and X1, X2, and X3 are the uncoded values of the independent variables of extraction: pressure (bar), temperature (°C), and flow rate (mL min-1), respectively. β0, β1, β2, β3, β12, β13, and β23 are constant coefficients where β0 is a constant; β1, β2, and β3 are coefficients for linear terms, and β12, β13, and β23 are the coefficients for interaction terms. The effects of process variables were analyzed statistically using an analysis of variance (ANOVA). The quality of the models was evaluated using the following statistical tests: p-value

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(p = probability), regression coefficient (R2), and lack of fit. The p-value, in this case,

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accounts for the probability of obtaining model values either greater than or equal to the

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experimental result [40].

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2.3. Equipment and experimental procedure (PHWE method)

A semi-batch extraction was used to extract pectin from gabiroba pulp with high

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pressure and hot water as solvent. A schematic diagram of the experimental setup used

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in this work is presented in Fig. 1.

Fig. 1. Schematic diagram of the dynamic pressure hot water extraction system (PHWE). V1 and V2: cylinder valves; V3: back pressure regulator valve.

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Around 25 g of thawed gabiroba pulp was placed inside the PHWE extractor vessel (20.44 cm3 inner volume, length L = 17.2 cm, diameter Φ = 1.24 cm). Both ends of the extractor were equipped with stainless steel filters with pore sizes of 0.5 mm. The temperature was controlled by a heating jacket equipped with electrical resistance and thermocouple sensors. Water was pumped into the extraction vessel using a highpressure liquid pump (Eldex, model 2SM, USA) and the constant volumetric flow mode. The pressure was adjusted and controlled using a back-pressure regulator (V3)

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(model KPB1SOA Swagelok, UK).

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First, the vessel was loaded with the raw material, and the pretreatment was

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applied: 550 mL of ethanol 99.9% was pumped at 80 °C and 50 bar using a constant flow rate around 4 mL min-1, resulting in the insoluble alcohol residues (AIR).

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Subsequently, ethanol was replaced by 18 MΩ.cm degassed ultrapure water, pH 6.9

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(Master system-MS2000, Gehaka, Brazil). After the adjusted extraction conditions (pressure, temperature, and flow rate) were performed according to the pre-established

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experimental design (Table 1), the system was kept in static equilibration for 15

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minutes. Afterward, in the static period, dynamic extraction was performed until there was 500 mL of aqueous extract.

The total volume of aqueous extract was collected at 20 °C from the PHWE system and then centrifuged (5,000 × g, 4 °C for 15 min). The supernatant was removed using a rotary evaporator vacuum (802, Fisatom) at 60 °C and dialyzed using 6-8 kDa membrane (Spectrum labs) against ultrapure water to conductivity similar to that of water (0.05 µS cm-1). After dialysis, the retained membrane fraction was precipitated with ethanol PA (3:1 v/v) (Dipalcool), followed by centrifugation (5000 × g, 4 °C for 25 min). Finally, the precipitate was washed three times with ethanol PA (Sigma), dried in a vacuum oven (EDGCON5P with FastVac DV-200N-250 vacuum pump) at room temperature (25 °C), and named as PEGP (Fig. 2).

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Fig. 2. Scheme of extraction of pectins from the pulp of gabiroba fruits by pressurized hot water

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extraction (PHWE).

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Pectin extraction yield was calculated as the ratio between the dry weight of

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pectins (PEGP) after ethanolic precipitation and the dry weight of gabiroba pulp as follows in Eq. 3:

(

)

( ) ( )

(3)

2.4. Conventional extraction of gabiroba pectin (CEGP)

Conventional aqueous extraction was carried out to extract pectins from gabiroba pulp in order to compare the yield and chemical structure of these polysaccharides with the samples obtained by PHWE extraction.

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The CEGP was performed according to Barbieri [37] with some modifications. Gabiroba pulp (25 g) was submitted to treatment with 99% ethanol under reflux for 30 min at 80 ºC, providing the alcohol insoluble residues (AIR). Afterwards, the pectins were extracted with ultrapure water at 100 ºC, 1:20 solid:liquid, for 2.77 h. These parameters correspond to similar conditions at the central point present in the factorial design (run PEGP9, PEGP10, and PEGP11 in Table 1), but under atmospheric pressure (1.01 bar). The aqueous extracts were obtained by centrifugation (12,000 × g; 25 min at

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4 °C), dialyzed (6-8 kDa), and the volume was reduced under vacuum followed by

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treatment with 99% ethanol (3:1, v/v) to precipitate the pectins. The precipitate was

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dried in a vacuum oven (EDGCON5P with FastVac DV-200N-250 vacuum pump) at

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room temperature (25 °C) and named as CEGP.

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2.5. Determination of galacturonic acid and neutral monosaccharides

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Uronic acid content was measured using the colorimetric m-hydroxybiphenyl

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method with galacturonic acid solution (0 - 250 g mL-1) as a standard according to Blumenkrantzand and Asboe-Hansen [41]. The sulfuric acid/tetraborate was added to tubes containing the sample, and the tubes were cooled in an ice-water bath. Then, the tubes were shaken in a vortex mixer, heated in a boiling water bath (6 min), and cooled in an ice-water bath. After adding reagent and shaking for 5 min, the absorbance of the samples was determined at 520 nm. The identity of the uronic acid was determined by anion exchange chromatography with pulse amperometric detection (HPAEC-PAD). Pectins were hydrolyzed with 2 mol L−1 TFA (8 h, 100 °C), then dried and washed three times with methanol until the acid was totally removed [37]. Samples (1 mg mL-1) were filtered through a membrane of 0.22 μm and injected into a Thermo Scientific Dionex ICS-

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5000/DC/SP/ASDV (USA) with CarboPac PA20 column (3×150 mm) according to Nagel, Sirisakulwat, Carle, and Neidhart [42]. Data were collected and analyzed using the ChromeleonTM 7.0 Chromatography Data System software. The neutral monosaccharides were determined by total acid hydrolysis with 2 mol L−1 TFA for 8 h at 100 °C, followed by conversion to alditol acetates by NaBH4 reduction (100 °C for 10 min) [43] and acetylation with acetic anhydride (Ac2O)pyridine (1:1 v/v, 1 mL) at 100 °C for 30 min [44]. The resulting alditol acetates were

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extracted with CHCl3, and the samples were analyzed in a Thermo Scientific Trace GC

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Ultra gas chromatograph with a mixture of He and N2 with compressed air as a carrier

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gas at 1 mL min−1 and using a DB-225-MS column (0.32 mm internal diameter x 30 m x film thickness 0.25 μm) programmed from 100 °C to 230 °C at a heating rate of 60 °C

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compared with standards.

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min−1. The alditol acetates were identified by their profiles, and retention times were

The proportions of homogalacturonans (HG) and ramnogalacturonans type I (RG-

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I) were calculated according to M'Sakni et al. [45] where:

–

( )

(4) ( )

(5)

2.6. High performance size exclusion chromatography

The elution profile of the soluble polysaccharides was analyzed by high performance size exclusion chromatography coupled with multi-angle laser light scattering (DSP-F, Wyatt Technology, USA) and refractive index detectors (Waters 2410, USA) (HPSEC-MALLS-RI). The chromatography was carried out on a Waters system containing four gel-permeation columns packed with Ultrahydrogel® 2000, 500,

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250, and 120 connected in series, with exclusion limits of 7×106, 4×105, 8×104, and 5×103 g mol−1, respectively. The flow rate was 0.6 mL min−1 with 0.1 mol L−1 NaNO2 in the mobile phase and 0.2 g L-1 NaN3 as a preservative at a temperature of 25 °C. The data were collected and processed using Wyatt Technology ASTRA software, version 4.70.07. The elution profile of pectin from citrus peels (PCP, Sigma Aldrich P9135) was used as a standard for comparing the data obtained.

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2.7. Nuclear magnetic resonance (NMR) spectroscopy

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Mono-dimensional (13C- and 1H-) and bi-dimensional (1H/13C HSQC) NMR spectra were acquired at 70 °C on a Bruker AVANCE III 400 NMR (Bruker, Germany)

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spectrometer, operating at 9.5 T, observing 1H at 400.13 MHz and 13C at 100.61 MHz,

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and equipped with a 5-mm multinuclear inverse detection probe with z-gradient. The samples were solubilized in D2O, and the chemical shifts were expressed as δ (ppm)

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using the resonances of −CH3 groups of acetones (1H at δ 2.22; 13C at δ 30.20) as

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internal references. All pulse programs were supplied by Bruker. TopSpin software, version 3.1, was used for the evaluation of data. The values of degree of methyl-esterification (DM) were determined by 1H-NMR spectroscopy integrating the hydrogen areas corresponding to H-1 and H-5 of unesterified α-D-GalAp units and H-1 and H-5 of esterified α-D-GalAp units [46,47]. Briefly, the samples were deuterium-exchanged three times by freeze drying with a D2O solution and finally dissolved in D2O and transferred to a 5 mm NMR tube. The 1HNMR spectra were acquired at 70 °C on a Bruker AVANCE III 400 NMR spectrometer, observing 1H at 400.13 MHz. Chemical shifts were expressed as δ (ppm).

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2.8. Statistical analysis

Results were statistically evaluated by analysis of variance (ANOVA) at 95% level of confidence using the Statistica 10 software (Statsoft Inc., USA) in order to identify significant differences between the responses analyzed in the full factorial design (23). The same software was used to construct the response surfaces.

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3. Results and discussion

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3.1. Effect of the variables on pectin yield using pressurized hot water extraction

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(PHWE)

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In this study, the full factorial design experimental 23 was applied to investigate the effect of independent variables (pressure, temperature, and flow rate) on extraction

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yield of PEGP samples (Table 1), which was calculated on the basis of the dry pulp

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weight (16.45% total solids). Eleven runs for eight factorial points and three replicates at the central point (100 bar, 100 ºC, and 3.0 mL min-1) were carried out. The design of the experiments, the pectin extraction yield, and predicted responses (YP) are given in Table 1.

Table 1 Full factorial design of experiments with independent variables and their levels, experimental and predicted data (yield) of PEGP samples. Variables

Unit

(X1) Pressure (X2) Temperature

Actual levels -1

0

+1

bar

50

100

150

ºC

80

100

120

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(X3) Flow rate

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1.5

3.0

4.5

X1

X2

X3

Yield (wt%)

YPb (wt%)

PEGP1

50

80

1.5

5.46

5.38

PEGP2

150

80

1.5

3.52

3.44

PEGP3

50

120

1.5

5.18

5.42

PEGP4

150

120

1.5

5.70

5.88

PEGP5

50

80

4.5

4.33

4.56

PEGP6

150

80

4.5

2.39

2.62

PEGP7

50

120

4.5

4.72

4.60

PEGP8

150

120

4.5

5.12

5.06

PEGP9

100

100

3.0

4.87

4.62

PEGP10

100

100

3.0

PEGP11

100

100

3.0

CEGP

1.01

100

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4.63

4.62

4.92

4.62

5.05

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Validation X1

X2

PEGP4a

PEGP4c

120

1.5

Yield (wt%)

Mean (wt%)

SDc (wt%)

5.66

0.06

5.70 5.71 5.59

PHWE pectin samples; bYield predicted by the statistical model; cSD: standard deviation; CEGP:

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a

150

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PEGP4b

X3

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Triplicates

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conventional extraction gabiroba pectin.

The yield of the PEGP samples ranged from 2.39 wt% to 5.70 wt% according to the PHWE conditions applied (Table 1). The optimal extraction conditions for achieving maximum yield were pressure of 150 bar, temperature of 120 ºC, and flow rate of 1.5 mL min-1, with which it was possible to obtain a pectin yield of up to 5.70 wt% in the PEGP4 sample. Under these conditions, the analyses were performed in triplicate (PEGP4 a; b; c) showing a mean value of 5.66 ± 0.06 wt% (Table 1). The experimental yield was close to the predicted yield (5.88 wt%), which demonstrates the validation of the optimized condition. In addition, to confirm the reproducibility of the experimental design, the central point (PEGP 9-11) was analyzed in triplicate. The results obtained in these analyses

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showed a similar yield for each sample, with mean value 4.80 wt% ± 0.15 wt% (Table 1). In order to compare the efficiency of the PHWE extraction, a semi-batch process with hot aqueous extraction commonly used at laboratory scale (batch process) for pectin extraction from gabiroba pulp (CEGP) was performed under conditions of 1.01 bar, 100 ºC, and 2.77 h, similar to conditions used for the PEGP9, PEGP10, and PEGP11 samples at central point (100 bar, 100 ºC, and 3.0 mL min-1, related to 2.77 h)

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in the experimental design. The yield obtained by conventional extraction was 5.05

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wt%, close to that obtained at the central point (4.80 wt% average). However, it can be

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observed that under higher pressure and temperature conditions (150 bar and 120 °C), there was an increase of up to 5.70 wt% in the pectin yield.

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Table 2 presents the analysis of variance (ANOVA) used to evaluate the

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significance of each variable on extraction yield of PEGP samples present in the full

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factorial design experimental 23.

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Table 2

ANOVA analysis for the regression model of the PEGP sample yields. SS

DF

MS

F-value

p-value

1.095

1

1.095

45.6

0.021

3.150

1

3.150

131.1

0.008

X3

1.361

1

1.361

56.6

0.017

X1 X2

2.880

1

2.880

119.8

0.008

X1 X3

0.002

1

0.002

0.1

0.810

X2X3

0.186

1

0.186

7.7

0.109

Lack of fit

0.143

2

0.071

3.0

0.252

Pure error

0.048

2

0.024

Total SS

8.865

10

X1 X2

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0.96

Adjusted R²

0.93

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X1: pressure; X2: temperature; X3: flow rate; SS: sum of squares; DF: degrees of freedom; MS: square mean; R2: regression coefficient; F-value: Fisher value; p-value: probability value.

As can be seen, the p-values of linear coefficients (X1: pressure; X2: temperature; X3: flow rate, Table 2) and interaction term coefficients X1X2 were lower than 0.05 (p < 0.05), indicating the significant effects of these parameters on PHWE pectin yield. The lack of fit

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tests were not significant (p = 0.252), indicating that the models had adequate accuracy for

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predicting pectin yield using any combination of independent factors within the range of

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this study. The large values of the coefficients of determination (R2) at 0.96 and adjusted

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R2 at 0.93 showed that the model can be used with a strong confidence level to predict the

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extraction process (Table 2).

The linear equation was obtained by removing non-significant terms (p > 0.05)

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such as the interaction between pressure (X1) and flow rate (X3), with p = 0.810, and interaction between temperature (X2) and flow rate (X3), with p = 0.109. The

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relationship between the response (yield) and independent variables (pressure, temperature, and flow rate) is shown below in Eq. 6:

(

)

(

)

(

)

(

)

(6)

where X1 (pressure), X2 (temperature), and X3 (flow rate) are the coded variables.

Equation 6 and the data from the variance analysis (ANOVA) presented in Table 2 demonstrate that the yield of the PEGP samples was affected by the three variables (pressure, temperature, and flow rate) through a linear effect.

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The individual and interactive effects that the process variables (pressure, temperature, and flow rate) had on the PEGP yield are also shown in Fig. 3 by threedimensional (3D) surface plots: when the slope of the response surface is relatively steep, it means that the response value has greater effect with the change of extraction conditions. In this work, the 3D response surfaces were obtained by keeping one of the

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variables constant at zero level while varying the other two variables (Fig. 3).

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Fig. 3. Response surface plot showing the effect of the interaction between (a) temperature and pressure, (b) pressure and flow rate, and (c) flow rate and temperature on the yield of the PEGP samples.

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In Fig. 3a the response surface shows the correlation between pressure and temperature variables. The pectin yield increased with the increase in temperature and pressure to 120 ºC and 150 bar, respectively. According to the literature, a high temperature associated with pressure (that maintains water in the liquid state) affects the mass transfer rate to favor extraction, enhancing the solubility of the solute and the diffusion coefficient [13,22,29]. These conditions may cause cell deformation and cell membrane damage that facilitates the solvent’s permeability in the plant tissues,

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promoting the extraction of pectins that are more tightly bound to the cell wall [48].

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Some authors such as Chen, Fu, and Luo [34] also describe the influence of pressure on

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pectin yield, showing that a pressure around 100 bar combined with a temperature of

pulp pectin (24.63%) by PHWE.

re

120 ºC for 30 min constituted an ideal condition for obtaining a high yield of sugar beet

lP

Flow rate was also a significant variable to be considered in pectin extraction by PHWE. Fig. 3b and 3c show that the highest yield was achieved when a lower flow rate

na

(1.5 mL min-1) was used. The flow rate is directly related to the total extraction time,

Jo ur

which in this work ranged from 1.85 hours (4.5 mL min-1) to 5.55 hours (1.5 mL min-1), to obtain a total 500 mL of aqueous extract for each run. Different authors have described time as an important variable in pectin extraction [49,50,51] because extended contact times between extracting solvent and plant material provide greater mass transfer of solid particles in the solution [52]. However, some studies show that excessive time exposure under high temperatures leads to degradation of the pectin chain molecules [32,53]. As seen in Fig. 3c, the highest yield was achieved at the highest temperature of 120 ºC and at the lower flow rate of 1.5 mL min-1. This may have been due to changes in the physical properties of water at different temperatures. According to the NIST database [54], higher temperatures reduce the viscosity of liquid solvents; the water’s

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20

viscosity decreased from 3.5x104 Pa s to 2.3x104 Pa s when the temperature was increased from 80 °C to 120 °C, allowing a better penetration of matrix particles and improving the efficiency of the extraction process [55]. In addition, increases in vapor pressure and rapid thermal desorption of target compounds from matrices could enhance extraction efficiency [25]. Other studies using PHWE to obtain pectins also observed temperature as a significant variable [19,32,34,48,56]. Liew et al. [32] showed that pectin yields obtained

of

from pomelo (Citrus grandis Osbeck) peels at 120 °C were at least three times (4.3 to

ro

20.4%) higher than the yields at 90 °C.

-p

The results obtained through the experimental design (Table 1), Equation 6, and response surface (Fig. 3) showed that the extraction conditions pressure, temperature,

re

and flow rate affected the pectin yield obtained from gabiroba pulp by PHWE.

lP

According to these data, it was observed that high pressure (150 bar) and high temperature (120 ºC) combined with a lower flow rate (1.5 mL min-1/ 5.55 h) that

na

provides a longer contact time between solvent and raw material were shown to be the

Jo ur

most favorable conditions for obtaining the highest yield of gabiroba pectin (5.70 wt%) observed in the PEGP4 sample (Table 1). However, in addition to its high yield, the chemical composition of the polysaccharide of interest must be taken into account as it directly reflects its application.

Thus, the PEGP4 sample obtained as an optimal condition by experimental design, the PEGP2 sample (150 bar, 80 ºC, and 1.5 mL min-1/ 5.55 h) that presented the lowest content of galacturonic acid (GalA, Table 3), and the PEGP10 sample (100 bar, 100 ºC, and 3.0 mL min-1/2.77 h; at central point) that presented a higher GalA content (Table 3) in relation to the tested conditions were selected to study their chemical composition. The PWHE samples was also compared with the CEGP—obtained by

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21

conventional extraction with hot water (1.01 bar, 100 ºC, and 2.77 h)—to evaluate the influence of extraction processes on the chemical composition of these pectins.

3.2. Chemical characterization of gabiroba pectins

The influence of extraction conditions on the chemical structure of pectins extracted from gabiroba pulp through PHWE was evaluated by colorimetric,

of

spectrometric, and spectroscopic techniques.

ro

The monosaccharide analyses (Table 3) revealed that all samples of pectic

-p

polysaccharides obtained from the pulp of gabiroba fruits presented arabinose (Ara: 44.3 to 59.7%), followed by galacturonic acid (GalA: 17.2 to 36.1%) and galactose

re

(Gal: 8.9 to 18.7%) as the major monosaccharides, varying their proportions according

lP

to the extraction conditions. In addition, minor amounts of rhamnose (Rha: 0.6 to 1.5%), xylose (Xyl: 0.3 to 1.3%), mannose (Man: 0.5 to 2.8%), glucose (Glc: 0.5 to

na

1.6%), and fucose (Fuc: 0.1 to 0.3%) were also present in these pectic structures. The

Jo ur

small amounts of Xyl, Man, and Glc possibly came from hemicelluloses detected in the gabiroba pulp [36].

These results are in agreement with the monosaccharide composition of gabiroba pectins recently described by Barbieri et al. [37]. These authors used hot water aqueous extraction in a batch processes for 28 h, giving a crude fraction pectin mainly composed by 54.5% Ara, 33.5% GalA, and 7.6% Gal. According to the literature, similar monosaccharide composition was observed in water extraction pectins from other fruits belonging to the Myrtaceae family such as araçá (Psidium catteleianum), which presented Ara (50.3%), GalA (30%), and Gal (10.4%) [57], and in a mixture of two species of guavira (Campomanesia pubescens and C. adamantium) composed of Ara (46.7%), GalA (44.6%), and Gal (5.5%) [58].

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Table 3 Monosaccharide composition of pectins obtained from the gabiroba pulp.

X1b

X2b

X3b

GalAc

Rha

Fuc

Ara

Xyl

Man

Gal

Glc

HGd

RG-Id

CEGP

1.01

100

-

25.7

1.5

0.3

54.4

0.3

1.0

15.8

1.0

24.2

73.2

PEGP1

50

80

1.5

34.0

1.2

0.1

44.4

0.9

2.8

14.9

1.6

32.8

61.7

PEGP2

150

80

1.5

17.2

0.9

0.2

59.7

0.4

1.7

18.7

1.3

16.3

80.1

PEGP3

50

120

1.5

24.5

0.6

0.3

57.7

1.0

1.5

13.5

1.1

23.9

72.3

PEGP4

150

120

1.5

34.8

1.2

0.2

44.4

1.2

1.7

15.2

1.3

33.6

62.0

PEGP5

50

80

4.5

35.2

1.0

0.2

44.3

0.9

0.7

16.9

0.8

34.2

63.4

PEGP6

150

80

4.5

29.6

1.1

0.1

54.1

0.8

0.6

13.0

0.6

28.5

69.4

PEGP7

50

120

4.5

30.8

1.3

0.2

46.2

1.0

1.4

17.9

1.1

29.5

66.7

PEGP8

150

120

4.5

29.6

0.8

0.2

49.3

1.3

1.0

16.4

1.2

28.8

67.4

PEGP9

100

100

3.0

36.1

1.0

0.1

49.5

0.6

0.6

11.4

0.5

35.0

63.0

PEGP10

100

100

3.0

36.0

0.7

0.1

52.4

1.0

0.5

8.9

0.5

35.4

62.6

PEGP11

100

100

3.0

35.0

0.6

0.1

46.6

0.7

0.5

16.0

0.5

34.4

63.8

re

-p

of

Run

ro

Monosaccharide composition (%) a

CEGP: conventional extraction gabiroba pectin. PEGP1-11: PHWE pectin samples. % of peak area of monosaccharide composition relative to the total peak area, determined by GLC.

b

X1: pressure (bar); X2: temperature (ºC); X3: flow rate (mL min-1).

PAD. d

na

Uronic acids, determined using the m-hydroxybiphenyl method [41]. GalA was identified by HPAEC-

HG / RG-I according to M’sakni et al. [45].

Jo ur

c

lP

a

Some studies have shown that the content of GalA may vary according to raw material and the extraction conditions [19,33,51]. In this work, the GalA content of pectins obtained by PHWE was affected by pressure and temperature varying between 17.2% and 36.1% (Table 3). It was observed that a lower temperature (80 ºC) promoted the extraction of pectins with lower GalA content (17.2%) in the PEGP2 sample compared to the other tested conditions. Already, when the gabiroba pectin was extracted at a higher temperature (100ºC) and 100 bar, an increase in GalA content to 36.0% in the PEGP10

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23

sample was observed (Table 3). However, when the temperature was increased to 120 °C in the PEGP4 sample, a slight decrease in the GalA content to 34.7% was observed (Table 3). This reduction is probably associated with elevated extraction temperature that may also lead to degradation or depolymerization of the pectin. Wang et al. (2014) also showed a decrease in GalA content from 44.4% to 40.1% in apple pomace pectin when the temperature of subcritical water extraction (PHWE) increased from 130 °C to 150 °C, respectively.

of

Under atmospheric pressure (1.01 bar) for 2.77 h, the CEGP sample obtained by

ro

conventional aqueous extraction at 100 ºC presented a minor GalA content of 25.7%

-p

(Table 3) as compared to the PEGP10 sample extracted by PHWE in the same conditions (100 ºC, 2.77 h), but under the higher pressure of 100 bar, which showed a

differences

in

the

proportions

lP

The

re

10.3% increase in GalA content (36.0%).

of

homogalacturonan

(HG)

and

rhamnogalacturonan (RG-I) domains in the pectin samples were evaluated by applying

na

Equations 4 and 5 [45] and using the monosaccharide composition data (Table 3).

Jo ur

All the gabiroba pectin samples obtained from PHWE (PEGP 1-11) and CEGP presented a main chain consisting mainly of the RG-I domain, which ranged from 61.7% to 80.1% depending on the extraction conditions (Table 3). According to the literature [59], a higher proportion of RG-I-rich pectin can be obtained by using water as a solvent when compared to extraction using mineral acids at pH 1–3 and 80–90 °C (the process commonly used to obtain commercial HG-rich pectins) because the hot acid results in the hydrolysis of the neutral side chains of the RG-I region. The PEGP2 sample presented a high RG-I (80.1%) and low HG (16.3%) content compared to the other pectin samples obtained by PHWE. However, when the pectin was extracted at a high temperature (PEGP4 sample), an 18.1% decrease in the RG-I composing its structure, compared to the PEGP2 sample, was observed.

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Differences of pressure applied at conventional extraction (CEGP) and PHWE (PEGP10) at 1.01 bar and 100 bar, respectively, suggest that under atmospheric pressure the extraction of pectins formed mainly by the RG-I domain is favored, as observed in the CEGP sample with RG-I (73.6%) and HG (24.2%) proportions, while the use of higher pressure leads to extraction of pectins composed of a lower proportion of RG-I (62.8%) and a higher proportion of HG (35.4%) compared to CEGP. According to the literature [8, 9, 10], the RG-I proportion reflects the rhamnose content present in the

of

main pectin chain along with arabinose and galactose that compose the neutral side

ro

chains. The differences in Ara and Gal content could indicate changes in the side chains.

-p

Therefore, as evidenced by monosaccharide composition (Table 3), the PHWE samples presented a decrease in Ara and Gal content that suggests a break in the side chains of

re

gabiroba pectins.

lP

The pectin samples (Table 1) from gabiroba pulp were also analyzed by high performance size exclusion chromatography (HPSEC-MALLS-RI) (Fig. 4) to verify the

na

macromolecular distribution.

Jo ur

All the PHWE pectin samples resulted in heterogeneous elution profiles (Supplementary Fig. S1). The chromatograms for CEGP (Fig. 4a), PEGP2 (Fig. 4b), PEGP4 (Fig. 4c), PEGP10 (Fig. 4d), and PCP (Fig. 4e) are presented to show qualitative differences between elution profile peaks. The chromatograms show four peaks detected by RI at around 40 (I), 45 (II), 50 (III), and 54 min (VI), indicating the presence of high and low mass populations that make up the structure of these pectins. The light-scattering detector at 90° (LS) shows a major peak around 40 min for all samples analyzed, corresponding to the largest molar mass and the first peak (I) detected by refractive index (RI), which confirms the ability to obtain high molar mass pectin populations through PHWE and CEGP.

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According to our previous study, which isolated a homogalacturonan from gabiroba fruits after a fractionation process [37], it is possible that this pectin population eluted in peak (I), corresponding to the HG region. This hypothesis can be confirmed by comparing the gabiroba pectin samples with the elution profile of a pectin sample obtained from citrus peel (called PCP) used as a commercial standard (Sigma Aldrich). A homogeneous elution profile of the PCP—mainly formed by GalA (81.9%), followed by Gal (11.5%), Ara (3.7%), and Rha (1.6%)—corresponding to 80.3% of HG can be

of

observed by RI and LS detectors in Fig. 4e, with a major peak detected at 42 min (I),

ro

corroborating the data obtained in this study. The major peaks present in the elution

-p

profile, ranging between 45 and 54 min as detected by refractive index (RI) for CEGP, PEGP2, PEGP4, and PEGP10 samples, probably correspond to the RG-I region being in

Jo ur

na

lP

re

agreement with the monosaccharide composition (Table 3).

26

Jo ur

na

lP

re

-p

ro

of

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Fig. 4. HPSEC elution profile of gabiroba pectins obtained from the pulp of gabiroba fruits according to the experimental design. Refractive index (RI). Light scattering (LS 90°). (a) CEGP, (b) PEGP2, (c) PEGP4, (d) PEGP10, and (e) PCP.

The chemical structures of CEGP, PEGP2, PEGP4, and PEGP10 were investigated by 13C-NMR (Fig. 5) and 1H/13C HSQC-NMR (Fig. 6, Table 4), analyzing the influence of extraction conditions on the chemical structure of pectins extracted from gabiroba pulp through PHWE. The main peaks and correlations observed in each spectrum were compared with the NMR data available in the literature [37,39,60,61,62].

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27

For the CEGP (Fig. 5a), PEGP4 (Fig. 5c), and PEGP10 (Fig. 5d), the signals of the C-6 of unesterified α-D-GalAp units were assigned at around δ 174.0 ppm. Signals of methyl-esterified α-D-GalAp units could not be observed in the 13C-NMR spectrum for all samples; however, signals of methyl and acetyl groups linked to the α-D-GalAp units appear close to δ 52.9 and δ 20.0 ppm, respectively. For PEGP2 (Fig. 5b), the signals attributed to the presence of α-D-GalAp units are not observed, probably due to a low amount of GalA (17.2%, Table 3).

of

The 13C-NMR also indicated the presence of arabinogalactans (AG) in the

ro

CEGP and PEGP samples. Signals around δ 109.0, δ 107.0, and 106.0 ppm were

-p

attributed to α-L-Araf (C-1), while signals for anomeric carbon (C-1) of (1→4)-linkedβ-D-Galp units were found at δ 104.3 ppm. The signal around δ 16.0 ppm was assigned

re

to α-L-Rhap(1→ 2)-linked units (Fig. 5), showing the presence of the RG-I region in

Jo ur

na

lP

agreement with the monosaccharide composition (Table 3).

28

Jo ur

na

lP

re

-p

ro

of

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Fig. 5. 13C-NMR spectrum of (a) CEGP, (b) PEGP2, (c) PEGP4, and (d) PEGP10. Samples were dissolved in deuterium oxide (D2O) and data collected at probe temperature of 70 ºC. Acetone (δ30.2) was used as internal standard. Chemical shifts are expressed in δ, ppm.

The 1H/13C HSQC-NMR spectrum shows the correlations between carbon and hydrogen with signals at different intensities for each monosaccharide unit that makes up the structure of gabiroba pectins extracted by PHWE under different pressure,

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29

temperature, and flow rate conditions. The spectrums of CEGP, PEGP2, PEGP4, and PEGP10 (Fig. 6a, b, c, and d, respectively; Table 4) revealed C-1/H-1 correlations from HG assigned to →4)-α-D-6MeGalAp-(1→ (unit A) at δ 100.1/4.96 and of →4)-α-DGalAp-(1→ (unit B) at δ 99.5/5.14 units. The remaining methyl-esterified units of α-DGalAp ring correlations were assigned at δ 68.5/3.77 (C-2/H-2), δ 68.9/3.91 (C-3/H-3), δ 78.2/ 4.45 (C-4/H-4), and δ 70.5/5.06 (C-5/H-5) units, while the signals at δ 68.4/3.75 (C-2/H-2), δ 68.4/3.98 (C-3/H-3), δ 78.2/4.45 (O-substituted C-4/H-4), and δ 71.6/4.70

of

(C-5/H-5) were attributed to α-D-GalAp unesterified units. The signal at δ 52.8/3.82

ro

corresponding to CH3-C6 of →4)-α-D-6MeGalAp-(1→ was observed for all samples

-p

[61,63].

The methyl esterification degree (DM) of pectin samples obtained by PHWE

re

extraction and CEGP were determined by 1H-NMR spectroscopy. All samples presented

lP

a high degree of methyl-esterification (DM> 50%), which showed 79.6%, 68.3%,

Jo ur

na

52.0%, 61.8% for the CEGP, PEGP2, PEGP4, PEGP10, respectively.

Jo ur

na

lP

re

-p

ro

of

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30

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31

Fig. 6. 1H/13C HSQC-NMR correlation map of (a) CEGP, (b) PEGP2, (c) PEGP4, and (d) PEGP10. Samples were dissolved in deuterium oxide (D2O) and data collected at probe temperature of 70 ºC. Acetone (δ30.2/2.22) was used as internal standard. Chemical shifts are expressed in δ, ppm.

In addition to the typical signals of homogalacturonan, signals attributed to RG-I backbone were assigned at δ 98.7/4.93 (C-1/H-1), δ 16.5/1.25 (C-6/H-6) to unsubstituted →2)-α-L-Rhap-(1→ (unit C) and at 16.5/1.28 ppm to substituted →2,4)-

of

α-L-Rhap-(1→ (unit D) (Fig. 6; Table 4) [37,64].

Glycosil units

C

1

H

→4)-α-D-GalAp-(1→

C

1

na

H

13

C

C

→2)-α-L-Rhap-(1→

1

Jo ur

H

1

2

3

4

5

100.1

68.5

68.9

78.2

70.5

4.96

3.77

3.91

4.45

5.06

99.5

68.4

68.4

78.2

71.6

5.14

3.75

3.98

4.45

4.70

nd

nd

nd

nd

lP

→4)-α-D-6MeGalAp-(1→

13

B

Chemical shifts, δ (ppm)

Nucleus 13

A

-p

H and 13C NMR chemical shifts of gabiroba pectins.

re

1

ro

Table 4

98.7 4.93

nd

nd

nd

nd

H

C

E

t-β-L-Araf-(1→

1

H

13

F

82.2

63.2

5.09

4.04

3.90

3.79

-

79.7

76.5

84.1

61.4

H

5.15

4.28

3.98

3.98

3.75

C

107.6

81.1

76.7

82.2

66.4

H

5.08

4.13

4.01

4.19

3.81

C

107.0

79.9

83.9

82.5

61.3

H

5.17

4.36

3.99

4.17

3.81

107.1

80.9

nd

82.2

66.7

→5)-α-L-Araf-(1→

13

I

74.4 nd

107.0

1

H

101.6

C

13

G

1.28

t-α-L-Araf-(1→ 1

→3)-α-L-Araf-(1→ →3,5)-α-L-Araf-(1→

1

13

C

-

16.5 nd

1

13

nd

1.25

C

→2,4)-α-L-Rhap-(1→

nd

-OCH3 52.8 3.81

16.5

13

D

6

-

-

-

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H

5.20

4.22

C

104.3

71.8

1

H

4.63

13

C

1

H

13

M

→4)-α-D-Galp

→4)-β-D-Galp-(1→

60.9

3.53

3.78

3.90

3.66

3.75

96.5

72.4

71.9

77.4

74.5

60.9

4.58

3.58

3.76

4.12

3.82

3.71

nd

nd

nd

92.4

1

H

5.30

13

C

104.3

71.9

73.4

77.6

74.6

61.0

1

H

4.61

3.68

3.75

4.14

3.70

3.81

103.0

70.4

81.5

74.5

69.0

3.81

3.91

73.4

62.7

3.75

3.82

73.6

69.0

3.88

3.91

nd

→3,6)-β-D-Galp-(1→ H

13

C

4.48

4.23

103.0

→3)-β-D-Galp-(1→ 1

H

4.53

13

C

102.6

70.8

1

4.53

81.9 3.79

71.1

72.2

4.47

nd

nd 3.68

lP

H

3.85

4.30

re

→6)-β-D-Galp-(1→ nd: not determined.

nd

nd

1

na

The 1D and 2D NMR spectra also provide information about the arabinose components of CEGP, PEGP2, PEGP4, and PEGP10 samples. Unusual signals at δ

Jo ur

p

74.8

C

C

O

69.0

13

13

N

73.5

of

L

→4)-β-D-Galp

3.92

ro

K

t-β-D-Galp-(1→

-

3.90

-p

J

32

101.6/5.09 (C-1/H-1), δ 74.4/4.04 (C-3/H-3), δ 82.2/3.90 (C-4/H-4), and δ 63.2/3.79 (C5/H-5) were assigned to terminal β-L-Araf-(1→ (unit E). Despite few reports of the presence of the t-β-L-Araf-(1→ units, this signal was also recently observed for pectins obtained from guavira pomace [56], a fruit that belongs to the Myrtaceae family as does the gabiroba fruit. The terminal of Ara in the α configuration (unit F) was observed at δ 107.0/5.15 (C-1/H-1). Furthermore, the other anomeric signals—(C-1/H-1) at δ 107.6/5.08, δ 107.0/5.17, and δ 107.1/5.20 ppm—were attributed to →5)-α-L-Araf-(1→ (G), →3)-α-L-Araf-(1→ (H) and →3,5)-α-L-Araf-(1→ (I) units, respectively (Fig. 6, Table 4) [58,60].

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The 1H/13C HSQC-NMR spectrum (Fig. 6) also shows the correlation at δ 104.3/4.63 ppm assigned to nonreducing terminal β-D-galactose units (J), whereas the anomeric signals at δ 96.5/4.58 and δ 92.4/5.30 ppm indicated the presence of α and β, reducing end galactose units (units K and L). The intense correlations in the type-I arabinogalactan (AG-I) backbone were observed at δ 104.3/4.61, δ 71.9/3.68, δ 73.4/3.75, δ 77.6/ 4.14, δ 74.6, 3.70, and δ 61.0/3.81 ppm, which was attributed to C1/H-1, C-2/H-2, C-3/H-3, C-4/H-4, C-5/H-5, and C-6/H-6 from →4)-β-D-Galp-(1→

of

(unit M) [37,61,63]. AG-I has been obtained by aqueous extraction from fruits such as

ro

starfruit (Averrhoa carambola L.) [64], cubiu (Solanum sessiliflorum D.) [63], and

-p

tamarillo (Solanum betaceum) [65].

For the PEGP2, PEGP4, and PEGP10 samples, other correlations were

re

observed in the HSQC spectrum (Fig. 6b, c, d; Table 4). The signals assigned at δ

lP

103.0/4.48, δ 103.0/4.53, and δ 102.6/4.53 ppm, corresponding to →3,6)-β-D-Galp-(1→ (N), →3)-β-D-Galp-(1→ (O), and →6)-β-D-Galp-(1→ (P) units, respectively, are

na

characteristic components of type-II arabinogalactans [61,62,66]. These polysaccharides

Jo ur

consist of a (1→3)-linked-β-D-Galp backbone containing short side chains of α-L-Araf(1→6)-[β-D-Galp-(1→6)]n. The galactosyl residues of these side chains can be substituted with α-L-Araf-(1→3) units [10]. The signals attributed to AG-II were not observed in the sample extracted with water by the conventional method without the use of high pressure, as shown in the CEGP spectrum (Fig. 5a). The investigation of the chemical structure of pectins obtained through monosaccharide composition as well as HPSEC-MALLS-RI, 13C-NMR, and 1H/13C HSQC analyses confirmed that it is possible to extract pectins from the gabiroba pulp using the PHWE technique. Although all samples obtained by PHWE presented a main

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34

chain composed mainly by RG-I regions with a minor proportion of HG, the HG/RG-I ratio ranged in relation to the extraction condition. In evaluating the differences in PHWE samples in low temperature conditions (80ºC), it was possible to obtain a low yield (3.52 wt%) and low GalA content (17.2%) as observed in the PEGP2 sample. In addition, the GalA signals in the HSQC spectrum showed low intensity for the HG compared to the other samples. When the temperature increased to 120 ºC, an increase in the yield and GalA content of the samples was

of

observed. An optimal condition, demonstrated by experimental design, can be reached

ro

to obtain a higher pectin yield (5.70 wt%) from the PEGP4 sample. However, according

-p

to results obtained in yield that were in agreement with the chemical structure, it is possible to suggest that the most favorable condition for pectin extraction from

re

gabiroba pulp is at 100 bar of pressure and 100 ºC of temperature as tested at PEGP10

lP

(central point) (Table 1, Table 3). In these milder conditions, a high GalA content (36.0%) and yield of 4.63% of PEGP10 sample was obtained without changes of

na

chemical composition, with the presence of RG-I region with a minor proportion of HG,

Jo ur

in addition to AG-I and AG-II, as shown by NMR analyses.

4. Conclusions

Pectin was extracted from gabiroba pulp by pressurized hot water (PHWE), and the

yield and chemical structure were affected by pressure, temperature, and flow rate conditions. According to the experimental design, the linear equation, and the surface response, the use of 150 bar pressure and temperature of 120 ºC led to a higher pectin yield (5.70 wt%). Furthermore, this high pressure also promoted an increase of 10.3% in galacturonic acid content compared to conventional hot water extraction. Gabiroba pectin samples extracted through PHWE presented a high degree of methyl-

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esterification with a main chain composed mainly by RG-I regions with a minor proportion of homogalacturonans with in different HG / RG-I ratios for each pectin sample. 13C-NMR and 1H/13C HSQC data also suggest the presence of AG-I and AG-II in the gabiroba pulp extracted by PHWE. AG-II structures were not observed in the CEGP sample extracted with water without high pressure. Overall, considering the quantity and quality of the extracted pectins, it can be suggested that PHWE is a promising, environmentally friendly technique for the extraction of these

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polysaccharides.

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Acknowledgements

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The authors gratefully acknowledge the following Brazilian agencies for financial

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support: the National Council for Scientific and Technological Development - CNPq, the Coordination for the Improvement of Higher Education Personnel - CAPES, the

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Araucaria Foundation, the Nanoglicobiotec and Ministry of Science and

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Technology/CNPq, and the Federal University of Parana - Brazil. J.L.M.S. is a research member of the CNPq Foundation (nº 476950/2013-9; 308296/2015-0; 309225/2018-3); M.L.C is a research member of the CNPq Foundation (Grant num. 305393/2016-2); I.P.D is the beneficiary of a post-graduation scholarship (nº 1798682) provided by CAPES, and S.F.B. is the beneficiary of a post-doctoral scholarship from Coordination of Superior Level Staff Improvement - CAPES, nº 88887.335103/2019-00. The authors would like to thank the NMR Center of UFPR for recording the NMR spectra, the Brazilian Agricultural Research Corporation/Embrapa Forestry, Rossana Catie Bueno de Godoy, Cristiane Helm, and Maria Cristina Medeiros Mazza for providing the gabiroba pulp.

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References

[1] S.Y. Chan, W.S. Choo, D.J. Young, X.J. Loh, Pectin as a rheology modifier: Origin, structure, commercial production and rheology, Carbohydr. Polym. 161 (2017) 118-139. https://doi.org/10.1016/j.carbpol.2016.12.033 [2] Z.J. Kermani, A. Shpigelman, H.T.T. Pham, M. Ann, A.M.V. Loey, M. E. Hendrickx, Functional properties of citric acid extracted mango peel pectin as related to its chemical structure, Food Hydrocoll. 44 (2015) 424-434. https://doi.org/10.1016/j.foodhyd.2014.10.018 [3] F. Munarin, M.C. Tanzi, P. Petrini, Advances in biomedical applications of pectin gels, Int. J. Biol. Macromol. 51 (4) (2012) 681-689. https://doi.org/10.1016/j.ijbiomac.2012.07.002

of

[4] G. Smistad, S. Bøyum, S.J. Alund, A.B.C Samuelsen, M. Hiorth, The potential of pectin as a stabilizer for liposomal drug delivery systems, Carbohydr. Polym. 90 (3) (2012) 1337–1344. https://doi.org/10.1016/j.carbpol.2012.07.002

ro

[5] F. Naqash, F.A. Masoodi, S.A. Rather, S.M. Wani, A. Gani, Emerging concepts in the nutraceutical and functional properties of pectin-A Review, Carbohydr. Polym. 168 (2017) 227239. https://doi.org/10.1016/j.carbpol.2017.03.058

re

-p

[6] A. Noreen, Z-I-U. Nazlic, J. Akrama, I. Rasulb, N.Y. Manshaa, R. Iqbald, S. Tabasuma, M. Zubera, K.M. Zia, Pectins functionalized biomaterials; a new viable approach for biomedical applications: A review, Int. J. Biol. Macromol. 101 (2017) 254-272. https://doi.org/10.1016/j.ijbiomac.2017.03.029

lP

[7] F. Dranca, M. Oroian, Extraction, purification and characterization of pectin from alternative sources with potential technological applications, Food Res. Int. 113 (2018) 327–350. https://doi.org/10.1016/j.foodres.2018.06.065

na

[8] D. Mohnen, Pectin structure and biosynthesis, Curr. Opin. Plant Biol. 11 (3) (2008) 266–277. https://doi.org/10.1016/j.pbi.2008.03.006

Jo ur

[9] B.M. Yapo, Pectic substances: from simple pectic polysaccharides to complex pectins - A new hypothetical model, Carbohydr. Polym. 86 (2) (2011) 373–385. https://doi.org/10.1016/j.carbpol.2011.05.065 [10] A.J. Voragen, G.J. Coenen, R. Verhoef, H. Schols, Pectin, a versatile polysaccharide present in plant cell walls, Struct. Chem. 20 (2009) 263–275. https://doi.org/10.1007/s11224-009-9442 [11] R. Ciriminna, A. Fidalgo, R. Delisi, L.M. Ilharco, M. Pagliaro, Pectin production and global market, Agro Food Ind. Hi Tec. 27 (5) (2016) 17-20. [12] U. Einhorn-Stoll, H. Kunzek, Thermoanalytical characterisation of processing-dependent structural changes and state transitions of citrus pectin, Food Hydrocoll. 23 (1) (2009) 40–52. https://doi.org/10.1016/j.foodhyd.2007.11.009 [13] L.R. Adetunji, A. Adekunle, V. Orsat, V. Raghavan, Advances in the pectin production process using novel extraction techniques: A review, Food Hydrocoll. 62 (2017) 239-250. https://doi.org/10.1016/j.foodhyd.2016.08.015 [14] M. Marić, A.N. Grassino, Z. Zhuc, F.J. Barba, M. Brnčić, S.R. Brnčić, An overview of the traditional and innovative approaches for pectin extraction from plant food wastes and byproducts: Ultrasound-, microwaves-, and enzyme-assisted extraction, Trends Food Sci. Tech. 76 (2018) 28–37. https://doi.org/10.1016/j.tifs.2018.03.022

Journal Pre-proof

37

[15] S. Rahmati, A. Abdullah, O.L. Kang, Effects of different microwave intensity on the extraction yield and physicochemical properties of pectin from dragon fruit (Hylocereus polyrhizus) peels, Bioact. Carbohydr. Diet. Fibre 18 (2019) 1-8. https://doi.org/10.1016/j.bcdf.2019.100186 [16] P. Rodsamran, R. Sothornvit, Microwave heating extraction of pectin from lime peel: Characterization and properties compared with the conventional heating method, Food Chem. 278 (2019) 364–372. https://doi.org/10.1016/j.foodchem.2018.11.067 [17] B.B.V. Guandalini, N.P. Rodrigues, L.D.F. Marczak, Sequential extraction of phenolics and pectin from mango peel assisted by ultrasound, Food Res. Int. 119 (2019) 455–461. https://doi.org/10.1016/j.foodres.2018.12.011 [18] M. Kazemi, F. Khodaiyan, S.S. Hosseini, Eggplant peel as a high potential source of high methylated pectin: Ultrasonic extraction optimization and characterization, LWT - Food Sci. Tech. 105 (2019) 182–189. https://doi.org/10.1016/j.lwt.2019.01.060

ro

of

[19] X. Wang, Q. Chen, X. Lü. Pectin extracted from apple pomace and citrus peel by subcritical water, Food Hydrocoll. 38 (2014) 129-137. https://doi.org/10.1016/j.foodhyd.2013.12.003

-p

[20] N. Muñoz-Almagro, L. Valadez-Carmona, J.A. Mendiola, E. Ibáñez, M. Villamiel, Structural characterisation of pectin obtained from cacao pod husk. Comparison of conventional and subcritical water extraction, Carbohydr. Polym. 217 (2019) 69–78. https://doi.org/10.1016/j.carbpol.2019.04.040

lP

re

[21] M. Plaza, C. Turner, Pressurized hot water extraction of bioactives, TrAC-Trend Anal. Chem. 71 (2015) 39–54. https://doi.org/10.1016/j.trac.2015.02.022 [22] M. Plaza, M.L. Marina, Pressurized hot water extraction of bioactives, TrAC-Trend Anal. Chem. 116 (2019) 236-247. https://doi.org/10.1016/j.trac.2019.03.024

Jo ur

na

[23] C.C. Teo, S.N. Tan, J.W.H. Yong, C.S. Hew, E.S. Ong, Pressurized hot water extraction (PHWE), J. Chromatogr. A, 1217 (16) (2010) 2484–2494. https://doi.org/10.1016/j.chroma.2009.12.050 [24] A. Mustafa, C. Turner, Pressurized liquid extraction as a green approach in food and herbal plants extraction: A review, Anal. Chim. Acta 703 (1) (2011) 8-18. https://doi.org/10.1016/j.aca.2011.07.018 [25] S.M. Zakaria, S. M. M. Kamal, Subcritical Water Extraction of Bioactive Compounds from Plants and Algae: Applications in Pharmaceutical and Food Ingredients, Food Eng. Rev. 8 (1) (2016) 23–34. https://doi.org/10.1007/s12393-015-9119-x [26] M. Çam, E. Yuksel, H. Alasalvar, B. Basyigit, H. Sen, M. Yilmaztekin, A. Ahhmed, O. Sagdıc, Simultaneous extraction of phenolics and essential oil from peppermint by pressurized hot water extraction, J. Food Sci. Technol. 56 (1) (2019) 200–207. https://doi.org/10.1007/s13197-0183475-5 [27] H.K. Reddy, T. Muppaneni, Y. Sun, Y. LI, S. Ponnusamy, P.D. Patil, P. Dailey, T. Schaub, O.F. Holguin, B. Dungan, P. Cooke, P. Lammers, W. Voorhies, X. Lu, S. Deng, Subcritical water extraction of lipids from wet algae for biodiesel production, Fuel 13 (2014) 73–81. https://doi.org/10.1016/j.fuel.2014.04.081 [28] M. Plaza, V. Abrahamsson, C. Turner, Extraction and Neoformation of Antioxidant Compounds by Pressurized Hot Water Extraction from Apple Byproducts, J. Agric. Food Chem. 61 (2013) 5500-5510. https://doi.org/10.1016/j.fuel.2014.04.081

Journal Pre-proof

38

[29] F.R. Smiderle et al., D. Morales, A. Gil-Ramírez, L.I. Jesus, B. Gilbert-López, M. Iacomini, C. Soler-Rivas, Evaluation of microwave-assisted and pressurized liquid extractions to obtain dglucans from mushrooms, Carbohydrate Polymers 156 (2017) 165–174. https://doi.org/10.1016/j.carbpol.2016.09.029 [30] G. Gallina, Á. Cabeza, H. Grénman, P. Biasi, J. García-Serna, T. Salmi, Hemicellulose extraction by hot pressurized water pretreatment at 160 ºC for 10 different woods: Yield and molecular weight, The J. Supercrit. Fluid. 133 (2017) 716–725. https://doi.org/10.1016/j.supflu.2017.10.001 [31] H. Xia, A.S. Matharu, Unavoidable food supply chain waste: acid-free pectin extraction from mango peel via subcritical water, Faraday Discuss. 202 (2017) 31–42. https://doi.org/10.1039/C7FD00035A

of

[32] S.Q. Liew, W.H. Teoh, C.K. Tan, R.Y.G.C. Ngoh, Subcritical water extraction of low methoxyl pectin from pomelo (Citrus grandis (L.) Osbeck) peels, Int. J. Biol. Macromol. 116 (2018) 128– 135. https://doi.org/10.1016/j.ijbiomac.2018.05.013

-p

ro

[33] S.Q. Liew, W.H. Teoh, R. Yusoff, G.C. Ngoh, Comparisons of process intensifying methods in the extraction of pectin from pomelo peel, Chem. Eng. Process. 143 (2019). https://doi.org/10.1016/j.cep.2019.107586

re

[34] H.M Chen, X. Fu, Z.G. Luo, Properties and extraction of pectin-enriched materials from sugar beet pulp by ultrasonic-assisted treatment combined with subcritical water, Food Chem. 168 (2015) 302–310. https://doi.org/10.1016/j.foodchem.2014.07.078

lP

[35] G. M. Barroso, Sistemática das magnoliophytas, in G. M. Barroso (Ed.). Sistemática de angiospermas do Brasil-parte II, Editora Universidade de São Paulo, São Paulo, 1978, pp. 114– 126.

Jo ur

na

[36] S.F. Barbieri, A.C. Ruthes, C. L. O. Petkowicz, R.C.B. Godoy, G.L. Sassaki, A. Santana-Filho, Extraction, purification and structural characterization of a galactoglucomannan from the gabiroba fruit (Campomanesia xanthocarpa Berg), Myrtaceae family, Carbohydr. Polym. 174 (2017) 887–895. https://doi.org/10.1016/j.carbpol.2017.07.015 [37] S.F. Barbieri, S.C. Amaral, A.C. Ruthes, C.L.O. Petkowicz, N.C. Kerkhoven, Silva, E.R.A., J.L.M. Silveira, Pectins from the pulp of gabiroba (Campomanesia xanthocarpa Berg): Structural characterization and rheological behavior, Carbohydr. Polym. 214 (2019) 250-258. https://doi.org/10.1016/j.carbpol.2019.03.045 [38] S.F. Barbieri, C.L.O. Petkowicz, R.C.B. Godoy, H.C.M. Azeredo, C.R.C. Franco, J.L.M. Silveira, Pulp and jam of gabiroba (Campomanesia xanthocarpa Berg): Characterization and rheological properties. Food Chem. 263 (2018), 292–299. https://doi.org/10.1016/j.foodchem.2018.05.004 [39] S.C. Amaral, S.F .Barbiere, A.C. Ruthes, J.M. Bark, S.M.B, Winnischofer, J.L.M. Silveira, Cytotoxic effect of crude and purified pectins from Campomanesia xanthocarpa Berg on human glioblastoma cells, Carbohydr. Polym. 224 (2019). https://doi.org/10.1016/j.carbpol.2019.115140 [40] G.E.P. Box, W.G. Hunter, J.S. Hunter, Statistics for Experimenters: An Introduction to Design, Data Analysis and Model Building, John Wiley and Sons Inc., New York, 1978. [41] N. Blumenkrantz, G. Asboe-Hansen, New method for quantitative determination of uronic acids, Anal. Biochem. 54 (2) (1973) 484–489. https://doi.org/10.1016/0003-2697(73)90377-1

Journal Pre-proof

39

[42] A. Nagel, S. Sirisakulwat, R. Carle, S. Neidhart, An acetate-hydroxide gradient for the quantitation of the neutral sugar and uronic acid profile of pectins by HPAECPAD without postcolumn pH adjustment, J. Agr. Food Chem. 62 (2014) 2037–2048. https://doi.org/10.1021/jf404626d [43] M.L Wolfrom, A. Thompson, Reduction with sodium borohydride, in R.L. Whistler, M.L. Wolfrom, J.N. BeMiller (Eds.), Methods in carbohydrate chemistry, New York and London: Academic Press Inc., 1963, pp. 65–68. [44] M.L Wolfrom, A. Thompson, Acetylation, in R.L. Whistler, M.L. Wolfrom, J.N. BeMiller (Eds.), Methods in carbohydrate chemistry, New York and London: Academic Press Inc., 1963, pp. 211–215.

of

[45] N.H. M’sakni, H. Majdoub, S. Roudesli, L. Picton, D.L. Cerf, C. Rihouey, C. Morvan, Composition, structure and solution properties of polysaccharides extracted from leaves of Mesembryanthenum crystallinum, Eur. Polym. J., 42 (4) (2006) 786-795. https://doi.org/10.1016/j.eurpolymj.2005.09.014

-p

ro

[46] H. Grasdalen, O.E. Bakøy, B. Larsen, Determination of the degree of esterification and the distribution of methylated and free carboxyl groups in pectins by 1H NMR spectroscopy, Carbohydr. Res. 184 (1988) 183–191. https://doi.org/10.1016/0008-6215(88)80016-8

re

[47] C. Rosenbohm, I. Lundt, T.M.I.E. Christensen, N.W.G. Young, Chemically methylated and reduced pectins: preparation, characterisation by 1H NMR spectroscopy, enzymatic degradation, and gelling properties, Carbohydr. Res. 338 (7) (2003) 637-649. https://doi.org/10.1016/S00086215(02)00440-8

lP

[48] X. Guo, D. Han H. Xi, L. Rao, X. Liao, X. Hu, J. Wu, Extraction of pectin from navel orange peel assisted by ultra-high pressure, microwave or traditional heating: A comparison, Carbohydr. Polym. 88 (2) (2012) 441– 448. https://doi.org/10.1016/j.carbpol.2011.12.026

Jo ur

na

[49] S.S. Hosseini, F. Khodaiyan, M.S. Yarmand, Optimization of microwave assisted extraction of pectin from sour orange peel and its physicochemical properties, Carbohydr. Polym. 140 (2016) 59–65. https://doi.org/10.1016/j.carbpol.2015.12.051 [50] E. Chaharbaghi, F. Khodaiyan, S.S. Hossein, Optimization of pectin extraction from pistachio green hull as a new source, Carbohydr. Polym. 173 (2017) 107–113. https://doi.org/10.1016/j.carbpol.2017.05.047 [51] C. Colodel, L.C. Vriesmann, R.F. Teófilo, C.L.O. Petkowicz, Extraction of pectin from ponkan (Citrus reticulata Blanco cv. Ponkan) peel: Optimization and structural characterization, Int. J. Biol. Macromol. 117 (2018) 385–391. https://doi.org/10.1016/j.ijbiomac.2018.05.048 [52] B. Pasandide, F. Khodaiyan, Z.E. Mousavi, S.S. Hosseini, Optimization of aqueous pectin extraction from Citrus medica peel, Carbohydr. Polym. 178 (2017) 27–33. https://doi.org/10.1016/j.carbpol.2017.08.098 [53] J.P. Maran, Statistical optimization of aqueous extraction of pectin from waste durian rinds, Int. J. Biol. Macromol. 73 (2015) 92–98. https://doi.org/10.1016/j.ijbiomac.2014.10.050 [54] NIST, National Institute of Standards and Technology. https://webbook.nist.gov/chemistry/fluid/, 2017 (accessed 14 October 2019). [55] N. Nastić, J. Švarc-Gajić, C. Delerue-Matos, S. Morais, M.F. Barroso, M.M. Moreira, Subcritical water extraction of antioxidants from mountain germander (Teucrium montanum L.), J. Supercrit. Fluids, 138 (2018) 200–206. https://doi.org/10.1016/j.supflu.2018.04.019

Journal Pre-proof

40

[56] K. Klinchongkon, N. Chanthong, K. Ruchain, P. Khuwijitjaru, S. Adachi, Effect of ethanol addition on subcritical water extraction of pectic polysaccharides from passion fruit peel, J. Food Process. Preserv. 41 (2017) 1-9. https://doi.org/10.1111/jfpp.13138 [57] L.C Vriesmann, C.L.O Petkowicz, P.I.B. Carneiro, M.E. Costa, E. Beleski-Carneiro. Acidic polysaccharides from Psidium cattleianum (Araçá), Braz. Arch. Biol. Techn. 52 (2) (2009) 259– 264. http://dx.doi.org/10.1590/S1516-89132009000200001 [58] V.S. Schneider, M. Iacomini, L.M.C. Cordeiro, β-L-Araf-containing arabinan and glucuronoxylan from guavira fruit pomace, Carbohydr. Res. 481 (2019) 16–22. https://doi.org/10.1016/j.carres.2019.06.005

of

[59] Y. Mao, R. Lei, J. Ryan, F.A. Rodriguez, B. Rastall, A. Chatzifragkou, C. Winkworth-Smith, S. E. Harding, R. Ibbett, E. Binner, Understanding the influence of processing conditions on the extraction of rhamnogalacturonan-I ―hairy‖ pectin from sugar beet pulp, Food Chem: X 2 (2019). https://doi.org/10.1016/j.fochx.2019.100026

-p

ro

[60] R.R. Klosterhoff, J.M. Bark, N.M. Glänzel, M. Iacomini, G.R. Martinez, S.M.B. Winnischofer, L.M.C. Cordeiro, Structure and intracellular antioxidant activity of pectic polysaccharide from acerola (Malpighia emarginata), Int. J. Biol. Macromol. 106 (2018) 473–480. https://doi.org/10.1016/j.ijbiomac.2017.08.032

lP

re

[61] G.E. Nascimento, M. Iacomini, L.M.C. Cordeiro, New findings on green sweet pepper (Capsicum annum) pectins: Rhamnogalacturonan and type I and II arabinogalactans, Carbohydr. Polym. 171 (2017) 292–299. https://doi.org/10.1016/j.carbpol.2017.05.029

na

[62] J.M. Rodrigues, M.E.R. Duarte, M.D. Noseda, Modified soybean meal polysaccharide with high adhesion capacity to Salmonella, Int. J. Biol. Macromol. 139 (2019) 1074–1084. https://doi.org/10.1016/j.ijbiomac.2019.08.038

Jo ur

[63] C. Colodel, R.M.G. Bagatin, T.M. Tavares, C.L.O. Petkowicz, Cell wall polysaccharides from pulp and peel of cubiu: A pectin-richfruit, Carbohydr. Polym. 174 (2017) 226–234. https://doi.org/10.1016/j.carbpol.2017.06.052 [64] C.L. Leivas, M. Iacomini, L.M.C. Cordeiro, Structural characterization of a rhamnogalacturonan I-arabinan-type I arabinogalactan macromolecule from starfruit (Averrhoa carambola L.), Carbohydr. Polym. 121 (2015) 224–230. https://doi.org/10.1016/j.carbpol.2014.12.034 [65] G.E. Nascimento, C.R. Corso, M.F.P. Werner, C. H. Baggio, M. Iacomini, L.M.C. Cordeiro, Structure of an arabinogalactan from the edible tropical fruit tamarillo (Solanum betaceum) and its antinociceptive activity, Carbohydr. Polym. 116 (2015) 300–306. https://doi.org/10.1016/j.carbpol.2014.03.032 [66] C.S. Tamiello-Rosa, T.M. Cantu-Jungles, M. Iacomini, L.M.C. Cordeiro, Pectins from cashew apple fruit (Anacardium occidentale): Extraction and chemical characterization, Carbohydrate Research 483 (2019). https://doi.org/10.1016/j.carres.2019.107752