Structure-function relationships of pectic polysaccharides from broccoli by-products with in vitro B lymphocyte stimulatory activity

To study structure-function relationships of pectic polysaccharides with their immunostimulatory activity, broccoli by-products were used. Pectic polysaccharides composed by 64 mol% uronic acids, 18 mol% Ara, and 10 mol% Gal, obtained by hot water extraction, activated B lymphocytes in vitro (25–250 μg/mL).

Carbohydrate Polymers 303 (2023) 120432 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Structure-function relationships of pectic polysaccharides from broccoli by-products with in vitro B lymphocyte stimulatory activity ´nia S Ferreira a, b, *, Alexandra Correia c, d, e, Artur M.S Silva a, Dulcineia Ferreira Wessel a, f, g, So Susana M Cardoso a, Manuel Vilanova c, d, h, Manuel A Coimbra a, ** a LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal CICECO - Aveiro Institute of Materials, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal i3S, Instituto de Investigaỗ ao e Inovaỗ ao em Saúde, Universidade Porto, 4200-135 Porto, Portugal d IBMC, Instituto de Biologia Molecular e Celular, Universidade Porto, 4150-180 Porto, Portugal e DGAOT, Faculdade de Ciˆencias, Universidade Porto, Rua Campo Alegre, 4169-007 Porto, Portugal f School of Agriculture, Polytechnic Institute of Viseu, 3500-606 Viseu, Portugal g CITAB, University of Tr´ as-os-Montes e Alto Douro, 5001-801 Vila Real, Portugal h ICBAS, Instituto de Ciˆencias Biom´edicas de Abel Salazar, Universidade Porto, 4050-313 Porto, Portugal b c A R T I C L E I N F O A B S T R A C T Keywords: Brassica by-products Sulfated pectic polysaccharides Arabinogalactans NMR Methylation analysis Enzyme modified pectin Structure-function relationships To study structure-function relationships of pectic polysaccharides with their immunostimulatory activity, broccoli by-products were used Pectic polysaccharides composed by 64 mol% uronic acids, 18 mol% Ara, and 10 mol% Gal, obtained by hot water extraction, activated B lymphocytes in vitro (25–250 μg/mL) To disclose active structural features, combinations of ethanol and chromatographic fractionation and modification of the polysaccharides were performed Polysaccharides insoluble in 80 % ethanol (Et80) showed higher immunosti­ mulatory activity than the pristine mixture, which was independent of molecular weight range (12–400 kDa) and removal of terminal or short Ara side chains Chemical sulfation did not promote B lymphocyte activation However, the action of pectin methylesterase and endo-polygalacturonase on hot water extracted polysaccharides produced an acidic fraction with a high immunostimulatory activity The de-esterified homogalacturonan region seem to be an important core to confer pectic polysaccharides immunostimulatory activity Therefore, agri-food by-products are a source of pectic polysaccharide functional food ingredients Introduction Broccoli by-products are a source of pectic polysaccharides (Petkoư ăfer et al., 2017), which information about wicz & Williams, 2020; Scha their structure and potential bioactivities is scarce (Busato et al., 2020; Xu et al., 2015; Zhang et al., 2017) In general, pectic polysaccharides are complex heteropolysaccharides with a high proportion of GalpA (Waldron & Faulds, 2007) They include various fragments of linear and ramified regions covalently connected It is well established that the linear region consists of (α1→4)-D-GalpA residues (homogalacturonan region) carrying methyl ester groups and that can also be acetylated A backbone of alternating (α1→4)-D-GalpA and (α1→2)-L-rhamnopyr­ anosyl (L-Rhap) residues, ramified in the Rha by galactans, arabinoga­ lactans, and/or arabinans of varying structure is named type I rhamnogalacturonan (Vincken et al., 2003; Yapo, 2011) The diversity of pectic polysaccharide structures is dependent on plant source, stages of maturity, plant part, or processing Pectic polysaccharides found in several plants have been related to health effects, from anticancer to immunomodulatory activities (Jin et al., 2021; Ramberg et al., 2010; Yin et al., 2019) Immunomodulatory polysaccharides may strengthen innate and adaptive immunity by directly interacting with distinct cellular and humoral components of the immune system, or indirectly through complex reaction cascades between immune components (Ferreira et al., 2015) The immunosti­ mulatory activity of pectic polysaccharides has been associated to the branched regions (e.g arabinans (Dourado et al., 2004; Dourado et al., 2006; Popov & Ovodov, 2013), arabinogalactans (Hamed et al., 2022; Zou et al., 2017), or their combination (Westereng et al., 2008; * Correspondence to: S.S Ferreira, LAQV-REQUIMTE, Department of Chemistry, University of Aveiro, 3810-193 Aveiro, Portugal ** Corresponding author E-mail addresses: soniasferreira@ua.pt (S.S Ferreira), mac@ua.pt (M.A Coimbra) https://doi.org/10.1016/j.carbpol.2022.120432 Received 19 July 2022; Received in revised form 18 November 2022; Accepted December 2022 Available online December 2022 0144-8617/© 2022 The Author(s) Published by Elsevier Ltd This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/) S.S Ferreira et al Carbohydrate Polymers 303 (2023) 120432 Westereng et al., 2009) In fact, the removal of the linear regions of pectic polysaccharides by endo-polygalacturonase has been shown to improve their complement fixing activity (Togola et al., 2008) whereas no effect or the opposite effect was observed by the removal of the branching regions with exo-α-L-arabinofuranosidase and galactosidases (Kiyohara et al., 2010; Nergard et al., 2005) Decrement of complement fixing activity or splenocytes proliferation was also observed by removal of arabinofuranosyl (Araf) residues with weak acid hydrolysis (Diallo, ăck, & Michaelsen, 2001, 2003; Wang, Liu, & Fang, 2005; Paulsen, Liljeba Duan et al., 2010; Inngjerdingen et al., 2007) However, the removal of Araf residues after enzymatic degradation of pectic polysaccharides from Glinus oppositifolius (L.) Aug DC (Aizoaceae) aerial parts did not affect their complement fixing activity or their ability to stimulate Peyer’s patches cells to produce secreted factors able to induce bone marrow cell proliferation (Inngjerdingen et al., 2007) On the other hand, the removal of homogalacturonan after endo-polygalacturonase treatment of pectic polysaccharides from Pterospartum tridentatum (L.) Willk inflorescences decreased the nitric oxide production by macro­ phages (Martins et al., 2017) These results show that different structural features of pectic polysaccharides can be involved in the triggering or modulation of immune responses In addition, chemical sulfation of polysaccharides has been shown to modulate their immunostimulatory activity (Ferreira et al., 2015) However, considering pectic poly­ saccharides, contradictory data have been reported with studies showing improvements (Du et al., 2010) or no effect on spleen cells proliferation (Wang et al., 2018) Therefore, the establishment of structure-function relationships of immunomodulatory pectic poly­ saccharides is far from being fully revealed Following the current state of knowledge about polysaccharides immunostimulatory activity, the pectic polysaccharides from broccoli by-products were extracted, fractionated and/or modified with 80 % ethanol solution, size exclusion chromatography, enzymatic treatments, chemical sulfation, and anion-exchange chromatography to study in vitro the potential immunostimulatory activity and to establish structure-function relationships separated from un-extracted residue by centrifugation (24,652g, ◦ C, for 30 min) followed by filtration in a fritted funnel (G-3) The soluble material was concentrated in a rotary evaporator and dialysed using membranes with 12 kDa cut-off to obtain HW This sample and the residue were freeze-dried for yield determination HW carbohydrates were analysed and further purified, fractionated, and derivatized (Fig 1) 2.4 Ethanol precipitation of HW Polysaccharides from HW were fractionated according to their sol­ ubility in ethanol solutions HW (100 mg) was hydrated by the addition of 20 mL of water, heated at 40 ◦ C, for min, and vortexed Some insoluble particles, 151 kDa 20 Et80-II 41–151 kDa 34 Et80-III 12–41 kDa 25 11 Et80-IV 10 hand, (1→4)-Galp residues are diagnostic of type I arabinogalactans, and t-Glcp, (1→4)-Glcp, and (1→4,6)-Glcp may be related with the occur­ rence of residual starch (Femenia et al., 2000), although t-Glcp and (1→4)-Glcp may be components of pectic polysaccharides, as cellobiose was reported in plum pectic polysaccharides (Nunes et al., 2012) Also, tXylp and (1→4)-Xylp may derive from pectic polysaccharides and/or polysaccharides tightly associated to pectic polysaccharides (Schols et al., 1990) The potential immunostimulatory activity of HW extract, was assessed in vitro in murine splenocyte cultures Cells viability was not affected by incubation with HW extract (100 mg/L) In addition, CD3+ cells (T cells) did not show significant up-regulated expression of the early activation marker CD69 on their surface, indicating that the ex­ tracts did not stimulate T cells In contrast, higher proportions of CD19+ cells (B cells) expressed CD69 upon stimulation by HW extract, as compared to non-stimulated control (16 % vs 7.7 % of control, Fig 2) As no lipid A was detected in mg/mL of HW (limit of quantification of 100 ng/mL), it was excluded a possible lipopolysaccharide contamina­ tion (de Santana-Filho et al., 2012) These results show that pectic polysaccharides from broccoli have potential immunostimulatory ac­ tivity, as observed for pectic polysaccharides from other sources (Dourado et al., 2004; Ferreira et al., 2015; Martins et al., 2017; Popov & Ovodov, 2013; Togola et al., 2008; Westereng et al., 2008, 2009; Zou et al., 2017) B cells can be found along the intestinal tract in Peyer’s patches and can be reached by pectic polysaccharides through M cells or dendritic cells (Huang et al., 2017; Komban et al., 2019) This could constitute a route for direct B cell activation, despite the low absorption of pectic polysaccharides Nevertheless, indirect B cell activation may occur through cytokines produced by polysaccharide-stimulated enter­ ocytes or phagocytes Recognition of pectic polysaccharides may occur through pattern recognition receptors (Beukema et al., 2020), expressed on enterocytes and on dendritic cells and macrophages located in Pey­ er’s Patches or in the lamina propria (Farache et al., 2013) The detected B cell stimulatory effect suggests a potential effect of these poly­ saccharides in strengthening antibody-mediated immunity in the intes­ tinal tract and their suitability to be used in functional food ingredients 3.2 Purification and modification of broccoli hot water extractable pectic polysaccharides In order to evaluate the characteristics of pectic polysaccharides from HW extract that may be involved in their immunostimulatory ac­ tivity, two strategies were defined: Fractionation by ethanol precip­ α-Litation followed by size-exclusion chromatography, arabinofuranosidase treatment or chemical sulfation Ethanol precipi­ tation allows to obtain fractions with different proportions of neutral sugars to uronic acids and size-exclusion chromatography allows to obtain fractions with different molecular weight The α-L-arabinofur­ anosidase treatment and chemical sulfation allow to modify the poly­ saccharide side chains by removal of terminally-linked arabinose residues and incorporation of sulfate groups in the structure; Purifi­ cation by enzymatic treatment with pronase and α-amylase followed by pectin methylesterase and endo-polygalacturonase treatments This strategy allows to remove proteins, residual starch, and part of the homogalacturonan backbone, resulting in fractions richer in pectic polysaccharide side chains This enzymatic treated fraction was sub­ mitted to an anion-exchange chromatography to separate the different domains according to their charge density S.S Ferreira et al Carbohydrate Polymers 303 (2023) 120432 Fig Percentage of B cells activated (CD69+) by treatment with polysaccharides from broccoli by-products at the concentrations of 25, 100, and 250 mg/L Culture medium alone (RPMI) was used as negative control and lipopolysaccharides (LPS) were used as positive control Statistical differences between RPMI (negative control) and different stimulus are indicated above bars (* p < 0.01) Statistical differences among samples are also highlighted (* p < 0.01) 3.2.1 Ethanol precipitation of HW extract To find the immunostimulatory active structural motifs of HW polysaccharides, a fractionation according to the solubility of the poly­ saccharides in ethanol solutions was followed The HW extract was dissolved in cold water, centrifuged, and ethanol was added to the su­ pernatant The extract was soluble in 50 % ethanol but precipitated when the solution reached 80 % ethanol, yielding a fraction insoluble in 80 % ethanol (Et80) that was separated from the 80 % ethanol soluble material (SnEt) The Et80 fraction accounted for 85 % of HW and con­ tained 64 % of polysaccharides (Table 1) This fraction had UA (65 mol %) as major sugar, followed by Ara (17 mol%), Gal (10 mol%), and Rha (1 mol%), similar in carbohydrate content and composition with the pristine HW extract The SnEt fraction accounted for 14 % of HW mass and had only 34 % of carbohydrates, mainly Ara (43 mol%) and UA (36 mol%), allowing to infer the presence of highly branched pectic poly­ saccharides, rich in Ara, in agreement with their high solubility in ethanol (Fernandes et al., 2019), together with non-carbohydrate com­ pounds that are usually adsorbed to the polysaccharides and are solu­ bilized with ethanol (Fernandes et al., 2020; Gonỗalves et al., 2018) When incubated with splenocytes, fraction Et80 stimulated 11 % to 37 % of B cells in a dose-response relationship from 25 to 250 mg/L, respectively (Fig 2), showing a higher immunostimulatory activity than 100 mg/L of initial HW As the sugars and the glycosidic linkage composition of fraction Et80 were comparable to those of the initial HW, the higher immunostimulatory activity of fraction Et80 seems to be due to the removal of non-carbohydrate compounds in the EtSn Even in small amounts, the EtSn compounds may prevent the expression of polysaccharide immunostimulatory activity, similarly to the effect of chlorogenic acids mixed with coffee arabinogalactans (Passos et al., 2021) 3.2.1.1 Size-exclusion chromatography of Et80 fraction Polysaccharides from Et80 fraction were submitted to size-exclusion chromatography on a Sephacryl 400-HR to characterize their molecular weight distribution (Fig 3.a) and evaluate the effect of this parameter in their immunosti­ mulatory activity Although it was not possible to resolve poly­ saccharides by size, eluted compounds were separated in four fractions: Et80-I consisted on the material with molecular weight higher than 151 S.S Ferreira et al Carbohydrate Polymers 303 (2023) 120432 Fig Chromatograms of a) size exclusion chromatography of Et80, with indication of recovered fractions (FI, FII, FIII, and FIV), blue dextran (void volume), dextrans (12 kDa, 80 kDa, and 270 kDa), and Glc (inner volume of the column) elution volumes and b) anion-exchange chromatography of Enz_HW with indication of recovered fractions (A, B, and C) kDa, accounting for 20 % of the eluted material, and had 37 % of polysaccharides; Et80-II consisted on the material with molecular weight between 41 and 151 kDa, accounting for 34 % of the eluted material, and was the richest fraction in polysaccharides (73 %); Et80-III consisted on the material with molecular weight between 12 and 41 kDa, accounting for 25 % of the eluted material, which contained 59 % of sugars and represented the peak of material that absorbs at 280 nm, ie, diagnostic of the presence of phenolic compounds; Et80-IV consisted on the material with molecular weight lower than 12 kDa, yielded 11 %, and had vestigial amounts of carbohydrates, only % Carbohydrate composition and linkage analysis of fractions Et80-I, Et80-II, and Et80-III (Tables and 2) revealed their resemblance with initial Et80 The higher molecular weight polysaccharides (Et80-I) were richer in Ara (26 mol%) whereas the lower molecular weight poly­ saccharides (Et80-III) had higher amount of UA (60 mol%) Linkage analysis showed that the branching degree of arabinans, estimated by the ratio between the branching units (sum of (1→2,5)-, (1→3,5)-, and twice (1→2,3,5)-Araf linkages) and total Ara, of fraction Et80-II (0.29) was similar to the initial Et80 (0.27), lower in Et80-I and Et80-III (0.21 and 0.13, respectively) The branching degree of galactans, estimated by the ratio between the branching units ((1→3,6)-Galp linkages) and total Gal, was also lower for fractions Et80-I and Et80-III (0.37 and 0.24, respectively) than for fraction Et80-II and initial Et80 (0.45 and 0.42, respectively), indicating that the branching degree of arabinans and galactans was correlated To evaluate the contribution of molecular weight to fraction Et80 immunostimulatory activity, fractions Et80 I, Et80 II, and Et80 III were incubated with spleen cells All fractions presented immunostimulatory activity comparable to Et80 (Fig 2), showing a lack of molecular weightimmunostimulatory activity relationship in Et80, despite the variation of arabinans and galactans branching degree increase of the relative content of terminally- and 6-linked Gal residues These results show that the Ara residues were removed from positions 2, 3, and of arabinans and of galactans, in accordance with the data reported for other arabinans and arabinogalactans (Ferreira, Passos, Cepeda, et al., 2018; Peng et al., 2016; Taylor et al., 2006) The α-Larabinofuranosidase treatment resulted in arabinans and galactans with a branching degree of 0.21 and 0.22, respectively, lower than the 0.27 and 0.42 of the initial Et80 fraction The remaining 58 % Ara residues in Et80_Ara should be part of longer arabinan chains, as α-L-arabinofur­ anosidase preferably acts on single and short chain Ara residues (Taylor et al., 2006) The sample Et80_Ara was incubated with spleen cells, resulting in stimulation of B cells to the same extension of that observed for Et80 fraction These results show that the removal of terminal Ara or small Ara chains did not interfere with immunostimulatory activity of broccoli pectic polysaccharides These results were in accordance with the lower contribution of Ara residues of pectic polysaccharides with immuno­ modulatory activity (Inngjerdingen et al., 2007), showing the impor­ tance of the core (1→3,6)- and (1→6)-β-D-galactan for the expression of their activity, in accordance with the reported arabinogalactans anti­ complement activity (Peng et al., 2016; Yamada & Kiyohara, 2007; Zou et al., 2019) 3.2.1.3 Chemical sulfation of Et80 fraction To study the effect of chemical modification with sulfate groups, Et80 fraction was incubated with chlorosulfonic acid and pyridine, giving origin to Et80_Sulf sub­ fraction Et80_Sulf had 52 % of total sugars and a sugar composition resembling Et80 fraction The sulfation was observed in the FTIR spec­ – O absorption band at 1260 cm− trum by the presence of a strong S– (Fig 4, Castro et al., 2006) In addition, linkage analysis showed the increase of 2,5-, 3,5-, and 2,3,5-linked Ara, indicating the presence of sulfate groups in 2, and positions Considering Gal residues, it was possible to observe a slight decrease in 3- and 4-Gal with an increase of all other residues, allowing to infer some extent of sulfation of the gal­ actan moiety Similarly, the glucan moiety seems to have been also sulfated at position 6, inferred by the decrease of 4-Glc and the increase of 4,6-Glc When incubated with spleen cells, Et80_Sulf showed the same immunostimulatory activity of Et80 fraction Similar results were observed for other sulfated pectic polysaccharides that retained the same spleen cell proliferation activity (Wang et al., 2018) It is possible that the sulfated polysaccharides contribute positively to the 3.2.1.2 α-L-Arabinofuranosidase treatment To evaluate the influence of terminally-linked Ara of arabinans and arabinogalactans on the immu­ nostimulatory activity of Et80 fraction, it was treated with α-L-arabi­ nofuranosidase This enzymatic treatment allowed to recover 79 % of the mass of Et80 fraction and 58 % of Ara in Et80_Ara subfraction Et80_Ara had 61 % of total sugars and was mainly composed by UA (64 mol%), Gal (14 mol%) and Ara (10 mol%) Linkage analysis revealed that the α-L-arabinofuranosidase treatment promoted the decrease of the relative content of terminally-, 2,5-, and the 3,5-linked Ara residues, as well as 3,6-linked Gal residues This treatment allowed to observe an S.S Ferreira et al Carbohydrate Polymers 303 (2023) 120432 Fig FTIR spectra of samples Et80 and Et80_Sulf acquired by ATR immunostimulatory activity of the sample, but the modification of the galactan moiety may hidden the structural galactans features also important for the bioactivity of these polysaccharides, with a net zero balance motives from hot water extracted broccoli pectic polysaccharides are related with lateral chains rich in branched Ara with UA residues that can confer acidity to the polymer These structural features can be ob­ tained by enzymatic modification of the hot water extract by pectin methylesterase and endo-polygalacturonase, followed by anionexchange chromatography Moreover, it seems that the poly­ saccharides recovered in Enz_HWA had a combination of polysaccharide structures that may mask the activity of Enz_HWB, as the pristine Enz_HW, which contained the same structural features, did not present the same extent of activity 3.2.2 Pectin methylesterase and endo-polygalacturonase treatment of purified HW extract To isolate the branched regions of pectic polysaccharides, the hot water extract from broccoli was treated with pronase and α-amylase, to remove proteins and starch-like polysaccharides, respectively This was followed by treatment with pectin methylesterase and endopolygalacturonase to methyl de-esterify and hydrolyse homogalactur­ onan domains from pectic polysaccharides After these enzymatic treatments, 32 % of HW material was recovered as high-molecularweight material in Enz_HW fraction, which was composed by 60 % of sugars (Table 1) This enzymatic treatment removed a large extend of HW Glc (78 %), in accordance with the presence of starch-like poly­ saccharides Enz_HW was composed mainly by UA (37 mol%), Ara (33 mol%), and Gal (19 mol%), showing a 84 % UA removal, in accordance with the degradation of homogalacturonans The remaining UA in Enz_HW is expected to belong to small homogalacturonan regions or regions with acetyl groups (Searle-van Leeuwen et al., 1996), as well as rhamnogalacturonans and type II arabinogalactans containing GlcA (Vincken et al., 2003; Yapo, 2011) Ara and Gal were also lost after the enzymatic treatment (49 % and 47 %, respectively), indicating that these residues were constituents of small side chains, which were removed upon dialysis This inference takes into consideration the absence of side enzyme effects on other polysaccharides beyond the homogalacturonan moieties To obtain pectic polysaccharides with different charge properties, the Enz_HW fraction was submitted to an anion-exchange chromatog­ raphy (Table and Fig 3.b) As the compounds eluted mainly with 0.125 M of NaCl in the buffer, it was inferred that they constituted an acidic fraction (Enz_HWB, 50 %) The fraction that eluted with buffer without NaCl (Enz_HWA) was not retained in the column, accounting for 36 % of Enz_HW This non retained fraction had lower amount of UA (34 mol%) than the retained fraction (40 mol%), allowing to infer that Enz_HWB had more exposed UA residues than Enz_HWA In addition, although the amount of Ara and Gal was similar, Enz_HWB had an Ara: Gal ratio of whereas Enz_HWA had a ratio of Linkage analysis showed that Enz_HWB had also a slightly higher proportion of 3,5- and 2,3,5-linked Ara than Enz_HWA, allowing to infer the presence of ara­ binans with higher branching degree (0.33) than Enz_HWA (0.24) (Table 2) Accordingly, 4- and 3,6-linked Gal were recovered mainly in the non-retained fraction Noncarbohydrate compounds from Enz_HW (14 %) were eluted with 0.250 M of NaCl (Enz_HWC) The incubation of Enz_HWB with spleen cells induced a stimulation of 46 % of B lymphocytes (Fig 2), higher than the activation observed for the hot water extract (16 %) and even Et80 fraction (26 %), considering the same amount of stimulus (100 μg/mL) However, Enz_HW and Enz_HWA stimulated less B lymphocytes (13 % and 10 %, respectively) These results allow to infer that the active structural 3.3 Detailing of structural features from broccoli pectic polysaccharides by NMR spectroscopy To detail the structural features of immunostimulatory poly­ saccharides from broccoli, fraction Et80, the one with higher immu­ nostimulatory activity when strategy (ethanol precipitation) was used to obtain the polysaccharides, and Enz_HWB, the fraction with higher immunostimulatory activity derived from strategy (enzymatic treat­ ment and anion exchange chromatography separation), were analysed by NMR spectroscopy The 13C NMR, HSQC, and HMBC spectra are represented in Figs 5, 6, and According to these 1D and 2D NMR spectra, methylation analysis (Table 2), and literature about pectic polysaccharides rich in UA, arabinans, and arabinogalactans (Cardoso et al., 2002; Dourado et al., 2006; Fernandes et al., 2019; Hamed et al., 2022; Makarova et al., 2013; Makarova et al., 2016; Schols et al., 1990; Shakhmatov et al., 2014; Shakhmatov et al., 2017; Shakhmatov et al., 2019), intense signals of the anomeric carbon in the 13C NMR spectrum were attributed to C-1 of α-Araf (106.3–109.2 ppm), α-GalpA (99.2–100.2 ppm), and β-Galp (103.1–104.1 ppm) for both Et80 and Enz_HWB Based on literature about α-glucans (Chen et al., 2017; Guo et al., 2015), the signal at 99.5 ppm of Et80 was attributed to C-1 of α-Glcp Low intense signals of C-1 from β-Galp (102.9 ppm), α-Rhap (99.2 and 100.1 ppm), and β-GlcpA (102.9 ppm) were also observed for both Et80 and Enz_HWB Prominent signals in the region of carboxyl groups were observed in the 13C NMR at δC range of 170–171 ppm, indicating the existence of UA with different vicinities Methyl and acetyl esterification were suggested by both one-bond 13C–1H correlation of NMR resonances, in the HSQC spectrum (Fig 6), and by three-bond 13C–1H correlation of NMR res­ onances, in the HMBC spectrum (Fig 7) The signal of CH3 of methyl esterified UA at 52.6/3.69 ppm and the signal of CH3 of acetyl esterified UA at 20.3/2.04 ppm were observed in the HSQC spectrum The signals of the carboxyl carbon correlation with methyl groups of CH3CO- at 173.6/2.06 ppm and 172.9/2.05 ppm (176.0/2.09) ppm were observed in the HMBC spectrum (Makarova et al., 2013; Shakhmatov et al., 2017, 2019) The methyl esterification was residual in Enz_HWB in contrast with Et80 intense signal of CH3O-group, which justify different chemical shifts for GalA anomeric carbon and C-5 (Fig 6) Some assignments were not possible for GalA due to overlapping of signals and to its lower de­ gree of freedom when compared to other sugar residues (Dourado et al., 2006; Schols et al., 1990) Overall, the data indicate the presence of a S.S Ferreira et al Carbohydrate Polymers 303 (2023) 120432 Legend: Ara GlcA Gal GalA Rha Fuc Glc 13 Fig Representation of the C NMR spectra from a Et80 and b Et80_Ara Illustration of sugar residues using the nomenclature proposed by Neelamegham et al (2019) *Identification of negative signals of DEPT-135 NMR spectrum from Et80 methyl and acetyl esterified galacturonan in Et80 fraction and an acetyl esterified galacturonan in Enz_HWB (Table 3) Methyl esterification and acetylation, specifically at O-2 and/or O-3 positions, were previously observed in pectins from Brassica (Westereng et al., 2006) In 13C and 1H NMR spectra (Fig 5), C-6/H-6 of deoxy-sugar residues Rha were observed at 16.1–16.8/1.11–1.18 ppm (Shakhmatov et al., 2017) These results indicated the presence of t-Rhap, usually found in the non-reducing terminal of arabinogalactans, and the presence of (α1→2)-Rhap and (α1→2,4)-Rhap, typical of in RG-I regions, according with what was observed in methylation analysis The negative carbon signals in the 13C DEPT-135 (Fig 5) found at δC 69.4, 66.3–66.8, 60.9, 60.7, and 60.5 ppm can be attributed to the -CH2of sugar residues, namely the C-5 of Ara and the C-6 of Gal and Glc Other assignments of the proton and carbon signals of Ara, Gal, and Glc were reported in Table The identification of signals belonging to (α1→5)-Araf, (α1→3,5)-Araf, (α1→3)-Araf, (α1→2,5)-Araf, (α1→2,3,5)Araf, and t-α-Araf indicated the presence of arabinans (Dourado et al., 2006; Fernandes et al., 2019; Shakhmatov et al., 2014, 2017, 2019) Downfield chemical shifts of Ara residues were also found in HSQC spectrum, indicating linkages between Ara-Gal residues, as usually found in arabinogalactans (Makarova et al., 2016; Shakhmatov et al., 2014, 2017), and confirmed by the decreasing of these signals when Et80 fraction was treated with α-L-arabinofuranosidase (Fig 5.b), where terminally linked Ara residues were mainly removed from galactans (Leboeuf et al., 2004) The identification of signals belonging to t-β-Galp, (β1→4)-Galp, (β1→3)-Galp, (β1→6)-Galp, and (β1→3,6)-Galp agreed with the pres­ ence of 4-linked galactans, like the ones found in type I arabinoga­ lactans, and 6-linked galactans ramified at like the ones found in type II arabinogalactans Galactans are usually found in ramified regions of pectic polysaccharides and type II arabinogalactans can be found both as free polysaccharides and as side chains of rhamnogalacturonans (Makarova et al., 2016; Shakhmatov et al., 2017, 2019) In this work, no correlation was observed between these two polysaccharides and RG-I, which can be due to low intensity of these possible linkages A methyl group signal not related with carboxyl group of UA was S.S Ferreira et al Carbohydrate Polymers 303 (2023) 120432 Fig Representation of HSQC spectra of fraction Et80 and Enz_HWB The notations used are given in Table 10 S.S Ferreira et al Carbohydrate Polymers 303 (2023) 120432 Fig Representation of HMBC spectrum of fraction Et80 Table H and 13C chemical shifts (δ) of main residues found in Et80 and/or Enz_HWB Residue C-1 →4)-α-GalpA-(1→ GalA →4)-α-GalpA-(1→ GalA Me α-Rhap Rha →5)-α-Araf-(1→ A5 →3,5)-α-Araf-(1→ A35 →3)-α-Araf-(1→ A3 →2,5)-α-Araf-(1→ A25 α-Araf-(1→ At →2,3,5)-α-Araf-(1→ A235 α-Araf-(1→ At* β-Galp-(1→ Gt →6)-β-Galp-(1→ G6 →3,6)-β-Galp-(1→ G36 →3)-β-Galp-(1→ G3 →4)-β-Galp-(1→ G4 4-OMe-α-GlcpA-(1→ GA α-Glcp Glc C-2 C-3 C-4 C-5 C-6 H-1 H-2 H-3 H-4 H-5;5’ H-6;6’ 100.2 4.84 100.2 4.84 100.1 4.63 107.3 4.96 107.1 5.00 107.1 5.04 107.0 5.07 107.0 5.02 106.7 5.12 109.2 5.12 103.1 4.36 103.1 4.36 103.1 4.36 104.1 4.51 104.1 4.51 102.9 4.36 99.7 5.28 – – – – – – 81.0 4.01 79.1 4.16 81.0 4.01 86.7 4.04 81.7 3.99 84.5 4.18 – – 70.5 3.4 70.5 3.42 69.5 3.53 70.0 3.56 71.7 3.54 72.9 3.22 – 3.53 – 3.97 – – – – 76.6 3.88 83.9 3.95 83.7 3.83 79.4 4.09 76.4 3.83 80.9 4.22 – – 72.5 3.53 72.5 3.53 79.6 3.55 80.0 3.61 73.2 3.56 73.9 – – – 79.0 4.33 – – – – 82.3 4.08 81.1 3.89 81.3 3.99 81.5 4.17 83.9 3.92 81.9 3.99 – – – 3.84 – 3.80 – 3.97 – 4.04 77.5 4.05 82.1 3.23 – – 72.5 4.5 70.5 4.98 – – 66.5 3.67;3.75 66.8 3.67;3.75 60.9 3.60;3.69 66.8 3.67;3.75 60.9 3.60;3.69 66.5 3.67;3.75 – – – 3.53 – 3.84 – 3.84 – 3.53 – 3.54 – 4.17 – – 170.3 found in the HSQC spectrum at 60.0/3.35 ppm and the chemical shift of C-4/H-4 at 82.1/3.23 ppm confirmed the existence of 4-O-Me-GlcA A cross-peak at 82.1/3.35 ppm in the HMBC spectrum confirmed an Omethyl substituent at C-4 of β-D-GlcpA According to literature, these residues can occur as terminals of side chains of (β1→6)-Galp chains from type II arabinogalactans (Shakhmatov et al., 2014; Shakhmatov – 16.1–16.8 1.04–1.11 60.4 3.60;3.69 69.2 3.89;3.80 69.2 3.89;3.80 60.4 3.60;3.69 60.4 3.60;3.69 – – – – CH3O- 52.6 3.67 CH3COO 20.3 2.04 60 3.35 et al., 2017) NMR spectra interpretation corroborated glycosidic linkage data obtained by methylation analysis Furthermore, data revealed that Et80 and Enz_HWB had acetyl-esterified pectic polysaccharides and type II arabinogalactans with 4-O-Me-GlcpA residues NMR spectra also allowed to identify the main differences between fractions Et80 and 11 S.S Ferreira et al Carbohydrate Polymers 303 (2023) 120432 Enz_HWB as α-glucans and highly methyl-esterified pectic poly­ saccharides in Et80 fraction These polysaccharides seem to hinder the immunostimulatory activity of slightly charged pectic polysaccharides composed with galactans motifs and with branched Ara residues 2020, UIDP/50011/2020 & LA/P/0006/2020), and CITAB research Unit (FCT UID/AGR/04033/2019), for the financial support through national funds and, where applicable, co-financed by the FEDER, within the PT2020 Partnership Agreement SSF thank FCT for the individual doctoral grant (SFRH/BD/103003/2014) The authors also thank Monliz SA, Alpiarỗa, Portugal, for providing the broccoli by-products Concluding remarks References The results herein collected showed that broccoli by-products are a source of pectic polysaccharides with in vitro stimulatory activity on B lymphocytes Immunostimulatory pectic polysaccharides can be ob­ tained by preparation of an AIR, which can be stabilized by solvent drying, and extraction with hot water The highest immunostimulatory activity was observed for the acidic fraction of pectic polysaccharides purified with pronase and α-amylase, methyl de-esterified with pectin methylesterase, and partially degraded with endo-polygalacturonase (Enz_HWB) After these treatments to remove protein, starch, and partially degrade the polygalacturonan backbone, Enz_HWB was a fraction composed by galacturonans (40 mol%) residually methylesterified but acetyl-esterified, with ramified regions of 4-linked gal­ actans (0.5 mol%), arabinans (35 mol%, with a branching degree of 0.33), and type II arabinogalactans (15 mol%, with a branching degree of 0.32) with 4-O-Me-GlcA residues Pectic polysaccharides with immunostimulatory activity, although not as high as that observed for Enz_HWB, can also be obtained by a simple 80 % ethanol precipitation (Et80) of the hot water extract The lower immunostimulatory activity of Et80 can be due to the presence of starch derived α-glucans (7.6 mol%) and highly methyl esterified uronic acids The activity of the compounds in these fractions seems not to be dependent of their molecular weight (from 12 to 400 kDa), neither of the lower content of terminally linked single or small chain Ara residues (14 mol% of arabinans, with a branching degree of 0.27) Et80 also con­ tained ramified regions of 4-linked galactans (1.0 mol%) and highly ramified type II arabinogalactans (5.2 mol%, with a branching degree of 0.82) The presence of pectic polysaccharides with potential immunosti­ mulatory activity in broccoli by-products showed that these by-products 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contribution statement ´ nia S Ferreira: Conceptualization, Investigation, Methodology, So Data curation, Writing – original draft Alexandra Correia: Investiga­ tion, Methodology, Data curation, Writing – review & editing Artur M S Silva: Methodology, Data curation, Writing – review & editing Dulcineia Ferreira Wessel: Supervision, Writing – review & editing Susana M Cardoso: Supervision, Writing – review & editing Manuel Vilanova: Supervision, Writing – review & editing Manuel A Coim­ bra: Conceptualization, Data curation, Supervision, Writing – review & editing 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 Data availability Data will be made available on request Acknowledgements Thanks are due to University of Aveiro, Polytechnic Institute of Viseu, FCT/MCT, LAQV-REQUIMTE (UIDB/50006/2020 & UIDP/ 50006/2020), and CICECO-Aveiro 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(Table 2) confirmed the presence of pectic polysaccharides The high amount of (1→5)-Araf indicates the existence of arabinans in the ramified domain of pectic polysaccharides, as found in broccoli