Two banana cultivars differ in composition of potentially immunomodulatory mannan and arabinogalactan

Banana (Musa acuminata and M. acuminata x M. balbisiana)fruit cell walls are rich inmannans, homogalacturonans and xylogalacturonan, rhamnogalacturonan-I, and arabinogalactans, certain forms of which is considered to have immunomodulatory activity.

Carbohydrate Polymers 164 (2017) 31–41 Contents lists available at ScienceDirect Carbohydrate Polymers journal homepage: www.elsevier.com/locate/carbpol Two banana cultivars differ in composition of potentially immunomodulatory mannan and arabinogalactan Tânia M Shiga a , Nicholas C Carpita c , Franco Maria Lajolo a,b , Beatriz Rosana Cordenunsi-Lysenko a,b,∗ a Department of Food Science and Experimental Nutrition, University of São Paulo, Av Prof Lineu Prestes, 580, Bloco 14, São Paulo, SP, 05508-000, Brazil Food Research Center (FoRC), São Paulo, SP, Brazil c Department of Botany & Plant Pathology, Purdue University, 915 West State Street, West Lafayette, IN 47907, USA b a r t i c l e i n f o Article history: Received 30 August 2016 Received in revised form 10 January 2017 Accepted 21 January 2017 Available online 22 January 2017 Keywords: Banana Polysaccharides Musa sp Mannan Arabinogalactan Cell wall Polysaccharide structure Bioactive polysaccharides a b s t r a c t Banana (Musa acuminata and M acuminata x M balbisiana) fruit cell walls are rich in mannans, homogalacturonans and xylogalacturonan, rhamnogalacturonan-I, and arabinogalactans, certain forms of which is considered to have immunomodulatory activity The cultivars Nanicão and Thap Maeo represent two widely variants with respect to compositional differences in the forms of these polysaccharides Nanicão has low amounts of mannan in the water-insoluble and water-soluble fraction Both cultivars have high amounts of water-soluble arabinogalactan These commelinoid monocots lack the (1 → 3),(1 → 4)␤-d-glucans of grasses, but Thap Maeo has higher amounts of non-starch glucans associated with wild species than does Nanicão High amount of callose was found in both cultivars As immunomodulatory activity is associated with the fine structure and interaction of these polysaccharides, breeding programs to introgress disease resistance from wild species must account for these special structural features in retaining fruit quality and beneficial properties © 2017 Elsevier Ltd All rights reserved Introduction Fruits are rich in soluble and insoluble fiber, comprising complex homo- and heteropolymers, such as pectins and hemicelluloses These polysaccharides are widely studied because their breakdown and solubilization during fruit ripening yield softness and juiciness to the pulp Moreover, there is a growing interest in the biological activity of its polysaccharides due to their potential beneficial effect to the human health Banana fruits contain various compounds, such as lectin, polysaccharides, flavonoids, alkaloids, steroids and glycosides that may produce physiological effects (Onyenekwe, Okereke, & Owolewa, 2013; Scarminio, Fruet, Witaicenis, Rall, & Di Stasi, 2012; Sansone, Sansone, Dias, Shiga, & Nascimento, 2016) The commercially available bananas are diploids or triploids of Musa acuminata (A genome) and Musa balbisiana (B genome), or ∗ Corresponding author at: Department of Food Science and Experimental Nutrition, University of São Paulo, Av Prof Lineu Prestes, 580, Bloco 14, São Paulo, SP, 05508-000, Brazil E-mail addresses: tatymish@usp.br (T.M Shiga), carpita@purdue.edu (N.C Carpita), fmlajolo@usp.br (F.M Lajolo), hojak@usp.br (B.R Cordenunsi-Lysenko) http://dx.doi.org/10.1016/j.carbpol.2017.01.079 0144-8617/© 2017 Elsevier Ltd All rights reserved are hybrids of the two species produced with their crossbreeding, resulting in classification groups AAA, AA, AB, AAB and ABB Cultivar Thap Maeo is a variant of cultivar Mysore and belongs to AAB genomic group, whilst Nanicão belongs to AAA genomic group Thap Maeo and Mysore cultivars are replacing traditional commercial cultivars because to their higher resistance to Black Sigatoka (black leaf-streak disease), a serious leaf spot disease of bananas that results in yield losses of 33–50% (Mobambo et al., 1993) Although resistance to Black Sigatoka is observed primarily in leaves, changes in the cell-wall composition related to disease resistance might be observed in the fruits, and these differences might impact fruit quality For instance, cultivar Mysore has anthocyanidin conjugates with cell-wall polymers (regarded to possess strengthening and defense functions against pathogens) whilst, Nanicão lacks of anthocyanidins linked to the cell wall and has higher susceptibility to disease (Bennett et al., 2010) Chemical composition and structure of the cell wall components are the principal determinants of fruit texture, so characterization of newly introduced cultivars for disease resistance is essential to assure texture is not lost Polysaccharides for the human health are highly dependent on their structure (degree of branching, molecule size and monosaccharide composition) that confers water 32 T.M Shiga et al / Carbohydrate Polymers 164 (2017) 31–41 holding properties, solubility and availability for fermentation by colonic bacteria, and immunomodulatory effects (Blackwood, Salter, Dettmar, & Chaplin, 2000) In this work, we evaluated the composition and linkage structure of banana fruit wall polysaccharides to assess the impact of introgression of a diverse germplasm to increase disease resistance on the fine structure of wall polysaccharides that impart proper pulp softening and beneficial properties for human health Material and methods 2.1 Material Banana fruits Musa acuminata cv Nanicão (genomic group AAA) were purchased from CEAGESP (São Paulo State, Brazil) and M acuminata x M balbisiana cv Thap Maeo (genomic group AAB) were harvested in the plantation located in Itapetininga (São Paulo State, Brazil) Fruits were classified as ripe, based on ethylene and respiration levels, and amounts of starch and soluble sugars Ripe banana fruits were peeled, frozen in liquid N2 and freeze-dried The freeze-dried materials were ground in mortar and boiled at 70–80 ◦ C in methanol: chloroform (1:1, v/v) for 30 to remove fat, pigments and to inactivate enzymes The suspensions were filtered in sintered glass funnel, and the residues washed extensively with acetone to remove pigments The residues were dried at ambient temperature and kept in a desiccator until use The defatted material was used to cell wall polysaccharide extraction, enzymatic assay and ␤-glucan determination 2.2 Extraction of cell wall polysaccharides About g of fruit pulp was soaked in 50 mL of 0.08 M sodium phosphate, pH 6.0 Exactly 100 ␮L of heat-stable ␣-amylase from B licheniformis (Megazyme International Ireland, EC 3.2.1.1) was added, and the mixture was incubated at 40 ◦ C for h The pH was then adjusted to 4.5 with HCl, and 200 ␮L of amyloglucosidase from A niger (Megazyme International Ireland Limited, Ireland, EC 3.2.1.3) was added The mixture was incubated at 35 ◦ C for h, then adjusted to pH 7.5 with NaOH, and 100 ␮L of protease of B licheniformis (Megazyme International Ireland Limited, Ireland, EC 3.4.21.14) was added, and the mixture was incubated at 35 ◦ C for an additional hour The hydrolysate was centrifuged at 9000 g, and the pellet was washed extensively with vol of 50 mL of deionized water About 100 mL of DMSO (90% in water, v/v) was added to the residue, and the mixture was sonicated in ultra-sound bath for h (2x) and centrifuged at 10,000g for 10 min, and the supernatant was discarded The pellet was washed times with 90% DMSO and then washed exhaustively with water The residue was re-suspended in water, and freeze-dried and called water-insoluble polysaccharides (WIP) The aqueous supernatant (hydrolysis buffer) and the wash solutions were combined and brought to 80% EtOH (v/v) The ethanolic mixture was heated to 70 ◦ C for 15 and ice-cooled to precipitate the polysaccharides and other material, which were pelleted at 9000 g and washed extensively with chilled 80% EtOH The ethanolinsoluble material was re-suspended in water, frozen in liquid N2 and freeze-dried This fraction was named water-soluble polysaccharide (WSP) 2.3 Monosaccharide determination and linkage analysis 2.3.1 Carboxyl reduction of uronosyl residues Duplicate samples of water-soluble and insoluble banana cell wall materials were carboxyl-reduced with NaBD4 after activation with a water-soluble carbodiimide, as described by Kim and Carpita (1992) and modified by Carpita and McCann (1996) A colorimetric assay for uronic acids in the presence of neutral sugars (FilisettiCozzi & Carpita, 1991) was used to confirm that the reduction of the carboxyl groups was 95% or greater For each of sets of materials, two samples of each were used for monosaccharide and linkage analysis 2.3.2 Monosaccharide distribution Monosaccharides were obtained from uronosyl-reduced cell wall material (1–2 mg) by hydrolysis in mL of M trifluoroacetic acid (TFA) at 120 ◦ C for 90 One-half millilitre of t-butyl alcohol was added, and the TFA-alcohol mixtures were evaporated in a stream of nitrogen, reduced with NaBH4 , and alditol acetates were prepared according to Gibeaut and Carpita (1991) Derivatives were separated by gas-liquid chromatography (GLC) on a 0.25 mm × 30 m column of SP-2330 (Supelco, Bellefonte, PA) Temperature was held at 80 ◦ C during injection then rapidly ramped to 170 ◦ C at 25 ◦ C min−1 , and then to 240 ◦ C at ◦ C min−1 with a 10 hold at the upper temperature Helium flow was mL min−1 with split-less injection The electron impact mass spectrometry (EIMS) was performed with a Hewlett-Packard MSD at 70 eV and a source temperature of 250 ◦ C The proportion of 6,6-dideuteriogalactosyl was calculated using pairs of diagnostic fragments m/z 187/189, 217/219 and 289/291 according to the equation described in Kim and Carpita (1992) that accounts for spillover of 13 C 2.3.3 Linkage analyses For linkage analysis, about mg of polysaccharides dried in a vacuum desiccator over P2 O5 were suspended in mL of dry DMSO (Pierce silylation grade) were per-O-methylated with Li+ methylsulfinylmethanide, prepared by adding 1.25 mL of 2.5 M nbutyllithium in hexanes (Aldrich) to the polysaccharides purged by Argon Upon evaporation of the hexanes and formation of Li+ alkoxide ions, methyl iodide (Aldrich) was added according to Gibeaut and Carpita (1991) The per-O-methylated polymers were recovered after addition of water to the mixture and partitioning into chloroform The chloroform extracts were washed five times with a three-fold excess of water each, and the chloroform was evaporated in a stream of nitrogen gas The partly methylated polymers were hydrolyzed in M TFA for 90 at 120 ◦ C One-half millilitre of t-butyl alcohol was added, the TFA-alcohol mixture was evaporated in a stream of nitrogen gas, and the sugars were reduced with NaBD4 and acetylated The partly methylated alditol acetates were separated on the same column used to alditol acetates After a hold at 80 ◦ C for during injection, the derivatives were separated in a temperature program of 160 ◦ C to 210 ◦ C at ◦ C min−1 , then to 240 ◦ C at ◦ C min−1 , with a hold of at the upper temperature All derivative structures were confirmed by electron-impact mass spectrometry (Carpita & Shea, 1989) 2.4 Enzymatic digestion of cell wall polysaccharides 2.4.1 Cell wall polysaccharides composition Suspensions containing 10 mg mL−1 of water-soluble and water-insoluble polysaccharides in buffer were incubated with the following glycosidases at 40 ◦ C for h A blank without enzyme were also carried out in parallel 2.4.1.1 Xyloglucan determination Polysaccharide suspensions were hydrolyzed using 10 U mL−1 of xyloglucanase from Paenibacillus sp (E.C 3.2.1.151, E-XEGP, Megazyme International Ireland, Wicklow, Ireland) in 100 mM sodium acetate buffer, pH 5.5 2.4.1.2 Mannan determination Polysaccharide suspensions were hydrolyzed using 3.9 U mL−1 of endo-(1 → 4)-␤-d-mannanase from T.M Shiga et al / Carbohydrate Polymers 164 (2017) 31–41 Bacillus sp (E.C 3.2.1.78, E-BMABS, Megazyme) in 100 mM glycine buffer, pH 8.8 2.4.1.3 Arabinan determination Polysaccharide suspensions were hydrolyzed using 15 U mL−1 of endo-/exo-arabinanase of Cellvibrio japonicus (E.C 3.2.1.99, E-ARBACJ, Megazyme) in 100 mM potassium phosphate buffer, pH 7.0 2.4.1.4 Xylan determination Polysaccharide suspensions were hydrolyzed using U mL−1 of ␤-xylanase from Trichoderma viride (E.C 3.2.1.8, E-XYTR1, Megazyme) in 100 mM sodium acetate buffer, pH 4.5 2.4.1.5 Homogalacturonan determination Polysaccharide suspensions were hydrolyzed using U mL−1 of endopolygalacturonase from Aspergillus niger (E-PGALS, E C 3.2.1.15, Megazyme) in 100 mM sodium acetate buffer, pH 4.0 The reactions were terminated by boiling for 15 and the suspensions were centrifuged at 10,000g and the supernatants filtered through 45-␮m pore filter and analyzed in a PA1 pellicular anion-exchange analytical column (Dionex Corp., Sunnyvale, CA, USA, 250 × mm) with its respective guard column, using a DX500 HPAEC-PAD System (Dionex) equipped with a GP40 gradient pump an ED40 detector with a gold working electrode and Ag/AgCl reference electrode and a AS50 autosampler to detect cell wall polysaccharide oligomers The oligosaccharides were gradient eluted at 30 ◦ C at flow rate of mL min−1 , using 65 mM NaOH and NaOAc Isocratic elution was employed for min, using 65 mM NaOH, followed by NaOAc gradient mM) ending at 40 A cleaning step of 10 of NaOH 200 mM containing 200 mM NaOAc was added 2.4.1.6 Monosaccharide determination The hydrolysates were brought to 80% EtOH by adding vol of absolute EtOH and centrifuged at 10,000g The supernatant was transferred to a new vial, dried under N2 stream, re-suspended in M trifluoroacetic acid (TFA) and hydrolyzed at 120 ◦ C for 90 (Kim & Carpita, 1992) The hydrolysates were dried with mL of t-butyl alcohol, and suspended in water, and the monosaccharides were analyzed on a CarboPac PA10 pellicular anion-exchange analytical column (250 × mm, with the corresponding guard column) using a DX500 HPAEC-PAD System (Dionex), according to Cordenunsi et al (2008), to detect xylose (Xyl), glucose (Glc), arabinose (Ara), mannose (Man), rhamnose (Rha), fucose (Fuc), galactose (Gal), galacturonic acid (GalA) and glucuronic acid (GlcA) released from cell wall polysaccharides hydrolysis 2.5 Determinations of ˇ-glucan 2.5.1 Pretreatment of water-insoluble polysaccharides Water-insoluble polysaccharides were assayed with and without pre-treatment with protease from Bacillus licheniformis (EC 3.4.21.14, E-BSPRT from Megazyme) Water-insoluble polysaccharides were submitted to protease pre-treatment to remove resident enzymes About g of waterinsoluble polysaccharide was incubated with 35 U of protease for h at 60 ◦ C with 50 mL of 0.08 M phosphate buffer, pH 7.5 The reaction was terminated by boiling for 15 to inactivate the enzyme and centrifuged at 10,000g The pellet was recovered and exhaustively washed with 80% EtOH and freeze-dried 2.5.1.1 Banana pulp preparation For banana pulp ␤-glucan assays, the pulp was defatted as described above and washed exhaustively with 80% EtOH in order to remove all free-sugar The pulp samples ® were washed with acetone and dried in a SpeedVac concentrator 33 2.5.1.2 Sample preparation Mixtures containing mg mL−1 of water-insoluble or water-soluble-polysaccharides and a suspension containing 30–50 mg mL−1 of defatted banana pulp were prepared in 150 mM sodium phosphate buffer, pH 6.5 2.5.1.3 Laminarinase assay Banana cell wall and defatted banana pulp suspensions were incubated for h at 60 ◦ C with U mL−1 of laminarinase (EC 3.2.1.6, endo-1,3(4)-␤-glucanase from Clostridium thermocellum, E-LICACT, Megazyme) 2.5.1.4 Lichenase treatment Banana cell wall and defatted banana pulp suspensions were incubated for h at 40 ◦ C with U mL−1 of lichenase (EC 3.2.1.73, endo-(1 → 3),(1 → 4)-␤-d-glucanase from Bacillus subtilis, Megazyme) Banana isolated cell walls material, the incubation times were up to 24 h The supernatants of laminarinase and lichenase and hydrolysis were recovered, filtered through 45 ␮m pore filter and analyzed using a DX500 HPAEC-PAD System (Dionex) as mentioned in the item 2.4.1 in order to detect ␤-glucan oligomers Samples of the supernatant obtained from laminarinase and lichenase hydrolysis ® were dried in a CentriVap , suspended in M TFA and hydrolyzed at 100 ◦ C for h, mL of t-butyl alcohol was added and the mixture ® was dried in a CentriVap The monosaccharides were suspended in water and analyzed by HPAEC-PAD (Dionex) as earlier Controls without laminarinase was also carried out as described earlier The oligomers and glucose released after cell wall polysaccharide hydrolysis were separated on a CarboPac PA10 pellicular anionexchange analytical column in the same way mentioned above Reaction blanks without enzyme were carried out in parallel Results 3.1 Composition of cell wall polysaccharides Owing to an enrichment of water-soluble polysaccharides, the fruit pulps of Thap Maeo banana were denser than those of Nanicão, with polysaccharide mass almost 2.5-fold higher (Fig S1) 3.1.1 Monosaccharide composition The compositions of water-soluble and insoluble polysaccharides in banana fruit pulp were vastly different, at least one-half of the monosaccharide composition was Gal, with the remainder in Man (Fig 1A and C) Ara and Xyl were also present, but Glc was less than mol% of the water-soluble fraction The uronosyl residues in water-soluble and water-insoluble polysaccharides were carboxylreduced with NaBD4 to convert them to their respective neutral sugars, distinguishing them as their 6,6-dideutero neutral sugars by MS GalA and GlcA were detected as smaller constituents of the water-soluble fractions In stark contrast, glucans were the major polysaccharides of water-insoluble fraction (Fig 1A and C), when calculated in the absence of Glc, GalA was more easily observed as a principal constituent, with modest amounts of Xyl, Ara, Gal and Man (Fig 1A and C) The great excess of GalA compared to Rha, indicated that HG and its derivatives are the most abundant pectin Nanicão and Thap Maeo cultivars differ markedly in the monosaccharide composition of their water-soluble polysaccharides (Fig 1B and D) Thap Maeo had significantly more Man (24 mol%) and slightly more GalA (2 mol%) than Nanicão, and consequently, significantly less Gal, Xyl, Ara and GalA (11, 6, and mol%, respectively) (Fig 1B) Although glucans were the dominant polysaccharide of the water-insoluble fractions, Nanicão had 30 mol% larger proportions (Fig 1D) Because of this difference, the 34 T.M Shiga et al / Carbohydrate Polymers 164 (2017) 31–41 Fig Thap Maeo (Genomic group AAB – grey bars) and Nanicão (Genomic group AAA – black bars) water-soluble and water-insoluble polysaccharide composition (A and C) The values of Thap Maeo minus Nanicão values (B and D) Monosaccharide composition considering and excluding glucose content (at right) Rha, rhamnose; Fuc, fucose; Ara, arabinose; Xyl, xylose; Man, mannose; Gal, galactose; Glc, glucose; GalA, galacturonic acid; GlcA, glucuronic acid N = insoluble fractions of Thap Maeo had more GalA, Ara, Xyl and Gal (14, 6, 6, and mol%, respectively) (Fig 1D) 3.1.2 Linkage analysis of banana fruit pulp cell walls Banana polysaccharides were characterized by high amounts of water-soluble mannan and glucomannan, especially in Thap Maeo that had 5-fold more 4-Man and 8-fold more 4,6-Man (Fig 2A and B) Nanicão and Thap Maeo water-insoluble polysaccharides were composed of medium amounts of mannans and glucomannans, confirmed by 4-Man and 4,6-Man residues (both mol%, 0.1 and 0.2 mol%, respectively) and 4-Glc (22 and 14 mol%) (Fig 2C and D) Both banana cultivars showed high amounts of water-insoluble homogalacturonan and rhamnogalacturonan, especially Thap Maeo Thap Maeo had three-fold more 4-GalA, seven-fold more t-Rha and 2,4-Rha and fold more 2-Rha (Fig 2C and D) In contrast, Nanicão had more water-soluble homogalacturonan and glucurono(arabino)xylan, containing about 2-times more 4-GalA, 2,4-Xyl, and 3,4-Xyl (Fig 2A and B, Table 1) T.M Shiga et al / Carbohydrate Polymers 164 (2017) 31–41 35 Fig Thap Maeo (Genomic group AAB – grey bars) and Nanicão (Genomic group AAA – black bars) water-soluble and water-insoluble polysaccharides linkage analysis (A and C) The values of Thap Maeo minus Nanicão values (B and D) Monosaccharide composition considering glucose content Rha, rhamnose; Fuc, fucose; Ara, arabinose; Xyl, xylose; Man, mannose; Gal, galactose; Glc, glucose; GalA, galacturonic acid; GlcA, glucuronic acid; t, terminal; p, pyranose; f, furanose N = Xyloglucan was the major hemicellulose, composed of 4-Glc, 4,6-Glc, t-Xyl, and 2-Gal, along with xylan (4-Xyl, 2,4-Xyl and 3,4Xyl)(Fig 2A and C) Fucose was found in trace amounts In Nanicão, xyloglucan and xylan were more insoluble Nanicão contained 2fold more 4-Glc and 4,6-Glc and 18 times more t-Xylp (Fig 2C and D) Both cultivars showed high amounts of water-soluble arabinogalactans and galactans However, in Thap Maeo, t-Araf and 4-Gal were 3-fold higher in the water-insoluble polysaccharides, whilst in water-soluble polysaccharides was 3-fold and 2-fold lower (Fig 2C, D, A and B, respectively) 3.2 Digestion of cell wall polysaccharides with hydrolytic enzymes Enzymatic hydrolysis confirmed linkage analysis and revealed that the major constituent of Nanicão and Thap Maeo banana pulp cell wall were mannan, (Figs 3, and S2 ) The oligomers obtained from mannanase, arabinanase and xyloglucanase enzymatic digestion were hydrolyzed using TFA M to obtain the monosaccharide composition (Figs S2 and ) According to linkage analysis, both banana cultivar have high amounts of homogalacturonans and small amounts of rhamnogalacturonan I, mainly in water-insoluble polysaccharides However, endopolygalacturonase hydrolysis produced a small peak containing high 36 T.M Shiga et al / Carbohydrate Polymers 164 (2017) 31–41 Table Thap Maeo and Nanicão bananas cell wall polysaccharide composition (For interpretation of the references to colour in this table, the reader is referred to the web version of this article.) molecular weight fragments (Figs and 4) and small amounts of GalA (Figs S2 and 5) The (1 → 4)-␤-d-mannanase-digestible mannans were found mainly in the water-soluble polysaccharides of Thap Maeo banana (Figs 3, and and S2) The mannans in Nanicão were oligomeric, soluble in 80% EtOH, and found even in samples that were not treated with mannanase (Figs S2 and 5) Only small amounts mannan from Thap Maeo pulps were solubilized by EtOH (Figs and S2) While mannans were the predominate form of the polymer of water-soluble polysaccharides, glucomannans predominated in water-insoluble polysaccharides (Figs S2 and 5) Enzymatic treatment with arabinanase released almost no oligomers (Figs and 4), as evidenced by the low amounts of Ara released (Figs S2 and 5) Enzymatic treatment confirmed the results obtained by linkage analysis Only low amounts of 5-Araf residues were found in the water-insoluble polysaccharides of either banana cultivars (Figs and S3), most probably released from short-chains of arabinans On the other hand, high amounts of 3-, 6- and 3,6-Gal were found, confirming the presence of righ amounts of arabinans Probably, the majority of t-Araf found belongs to arabinogalactans (Fig 2) The high amounts of t-Araf and 3,6-Gal in the water-soluble polysaccharide, suggests that arabinogalactans are water-soluble and has a highly branched structure (Fig 2A) Xyloglucanase treatment released oligomers predominantly in the water-insoluble fractions (Figs and 4) In both banana cultivars, water-soluble polysaccharides had small amounts of xyloglucan On other hand, water-insoluble polysaccharides showed higher amounts of xyloglucans, as can be seen by the presence of Glc, Gal and Xyl, (Figs S2 and 5), as well as the 2-Xyl and 4,6-Glc residues (Fig 2) Xylanase did not produce significant amounts of oligomers despite the presence of 4- and 3,4-Xyl (Figs and 4) and of t-Araf residues (Fig 2) 3.2.1 Digestion of glucans with ˇ-glucanases To confirm the chemical nature of glucose-rich polymer, the water-insoluble polysaccharide fraction was assayed using laminarinase The water-soluble polysaccharides not have significant amounts of glucose, however, almost all glucose are composed of 3,4-Glc and 4-Glc We also tested the defatted banana pulp with laminarinase Banana water-insoluble polysaccharide without pre-treatment with protease were hydrolyzed and assayed for ␤-glucan Banana pulp treated with laminarinase produced high amounts of glucose in control samples (without laminarinase) and in samples containing enzyme (Fig S4) Both samples produced high amounts of glucose, showing ␤-glucanase activity (Fig S4) Laminarinase hydrolyzes (1 → 3)-␤-d- linkages of ␤-glucans or polysaccharide connected by ␤-1,3 linkages containing a mixture of (1 → 3)-␤-d- linkages and either (1 → 4)-␤-d- linkages or (1 → 6)␤-d- linkages That means that laminarinase could be hydrolyzing callose or there was endogenous ␤-glucanase acting in banana samples To remove interference of endogenous enzymes, samples were pre-treated with protease, and boiled for 15 to inactivate the protease added Lichenase digests (1 → 4)-␤-d-glucosyl units in polymers that contain (1 → 3)-␤-d-glucosyl at the non-reducing end (Anderson & Stone, 1975), and not ␤-d-glucans containing only (1 → 3)-␤-d- or (1 → 4)-␤-d- bonds Thus, the enzyme digests the mixed-linkage (1 → 3)-␤-d-, (1 → 4)-␤-d-glucan but not cellulose or callose Samples treated with protease were hydrolyzed with laminarinase and lichenase and resulted in high amounts of glucose, whilst control produced only small amounts of glucose (Fig S5) Both cultivars showed similar profiles (Fig S5) Water-soluble polysaccharides treated with laminarinase produced oligomers in a short period of time (15 − h) (Fig 6) Lichenase, in turn, required a very long period of hydrolysis to T.M Shiga et al / Carbohydrate Polymers 164 (2017) 31–41 37 Fig Oligomer profile of Nanicão banana cell wall polysaccharides The water-soluble and water-insoluble polysaccharides were hydrolyzed using mannanase, arabinanase, xyloglucanase, xylanase and endopolygalacturonase Red line, blank without enzyme; black line, samples containing enzyme (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) release oligomers (24 h) (Fig 6) Hence, the digestion of both banana cultivars using laminarinase and lichenase revealed no (1 → 4)-␤-d-glucosyl units containing (1 → 3)-␤-d-glucosyl at the non-reducing end (Fig 6) Oat, barley and laminarin were also hydrolyzed using laminarinase and lichenase to show the effectiveness of the enzymes used in the ␤-glucan assays in a well-known samples containing ␤-glucans (Fig S6) Discussion 4.1 Bananas polysaccharide composition Banana non-starch polysaccharide is composed of (gluco)mannan and some mannan, made up of (1 → 4)-␤-dlinked glucose, (1 → 4)-␤-d-linked mannose and small amounts of galacto(gluco)mannan Homogalacturonans and rhamnogalacturonan I were the main acidic pectins in banana Homogalacturonans were not totally digested by endopolygalacturonase and produced only small amounts of oligomers, the majority with high molecular weight The lack of digestion suggests that the enzyme could not hydrolyze the galacturonans, possibly because galacturonans in banana are acetylated or methylated The neutral and acidic pectin and hemicellulose ratios were different between cultivars revealing structural differences in the cell wall composition Differences in the genomic group may be correlated to those differences The differences in the polysaccharide composition and solubility may affect banana texture loss during ripening, as shown in our previous work (Shiga et al., 2011) Just small amounts of arabinan was detected in both banana pulps The majority of t-Araf were more likely released from arabinogalactan rather than arabinan The large amounts of t-Araf residues along with low amount of 5-Araf revealed that banana pectin has short branches of arabinan Corroborating the results of linkage analysis, the enzymatic hydrolysis with arabinanase did not produced high amounts of oligomers neither arabinose monomers after TFA hydrolysis 38 T.M Shiga et al / Carbohydrate Polymers 164 (2017) 31–41 Fig Oligomer profile of Thap Maeo banana cell wall polysaccharides The water-soluble and water-insoluble polysaccharides were hydrolyzed using mannanase, arabinanase, xyloglucanase, xylanase and endopolygalacturonase Red line, blank without enzyme; Black line, samples containing enzyme (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) There are two different types of arabinogalactan in banana pulp Type I arabinogalactan, composed of (1 → 4)-␤-d-galactose with t-Araf sidechain attached at the O-3 of the galactose units Type II arabinogalactan that has a short (1 → 3)-␤-d- and (1 → 6)-␤-dgalactan chains connected to each other by (1 → 3, → 6)-linked branch point residues Type II arabinogalactan constitute the major arabinogalactan in banana cell wall pulp, whilst type I arabinogalactan in average amounts Cultivars Nanicão and Thap Maeo showed similarities in their polysaccharide compositions when water-soluble and waterinsoluble polysaccharides were combined (Figs and Fig S3) Basically, the main difference between both cultivars was correlated to the polysaccharides solubility and mannan content (Table 1) In Thap Maeo, galacturonans, xyloglucan and xylans showed to be more insoluble in water, while mannans were far more soluble In terms of its nutritional characteristics, banana cell wall has an interesting polysaccharide composition, since it is rich in mannans, galactans and galacturonans Studies conducted with various plant materials showed that neutral polysaccharides such as arabinogalactans, arabinoxylans and mannans have a potential immunomodulatory activity (Classen, Thude, Blaschek, Wack & Bodinet, 2006; Im et al., 2010; Ramberg, Nelson, & Sinnott, 2010; Liu, Willför, & Xu, 2015) According to our previous works results, banana has high amounts of mannose-rich polysaccharides and both, Thap Maeo and Nanicão non-starch polysaccharides, are able to activate macrophages (Cordenunsi et al., 2008; Shiga et al., 2011; Sansone, Miranda Brito Sansone, Shiga, & Nascimento, 2016) However, the effects depended of concentration and cultivar origin Polysaccharide immunomodulatory activity is associated with the highly branched structure of arabinogalactans (Paulsen & Barsett, 2005) According to ours results, banana has arabinogalactans containing high amounts of t-Araf side-chains Banana showed high amounts of 3-, 6- and 3,6- and 3,4-Gal, characteristic of arabinogalactans Hence, the majority of t-Araf residues seems to belong to arabinogalactans rather than to arabinans and are attached to galactose at O-3 position T.M Shiga et al / Carbohydrate Polymers 164 (2017) 31–41 39 Fig Hydrolysis profile of Thap Maeo banana cell wall oligomers using M TFA The water-soluble and water-insoluble polysaccharides were hydrolyzed using mannanase, arabinanase, xyloglucanase, xylanase and endopolygalacturonase and the oligomers obtained hydrolyzed with M TFA Red line, standard; Black line, samples containing monosaccharides hydrolyzed by endopolygalacturonase (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 40 T.M Shiga et al / Carbohydrate Polymers 164 (2017) 31–41 Fig Water-soluble polysaccharides from Nanicao and Thap Maeo bananas, digested with lichenase for h, 16 h and 24 h and with laminarinase for h Red line, blank without enzyme; Black line, samples containing enzyme (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) In our previous work, it was also observed high amounts of glucose in banana polysaccharides Hence, in the present work we decided to study the structure of banana glucans, since ␤-glucans is known to activate the immune system Samples hydrolyzed with laminarinase and control samples (without laminarinase) released large amounts of glucose Glucose could be originated from hydrolysis of ␤-glucan by laminarinase or by endogenous ␤-glucanase When samples were pre-treated with protease, and hydrolyzed by laminarinase, the amount of glucose released by control (without laminarinase) did not reduce significantly Samples pre-treated with protease were then hydrolyzed with lichenase and released high amounts of glucose Control samples (pre-treated with protease, without lichenase) released low amounts of glucose, showing that banana has ␤-glucanase activity that was inactivated by protease treatment Presence of mixed-linkage glucans was not found, since lichenase produced oligomers only after 24 h of hydrolysis Lichenase, hydrolyzes (1 → 4)-␤-d-glucosidic linkages in polymers containing (1 → 3) and (1 → 4) bonds, however, not hydrolyzes ␤-d-glucans containing only (1 → 3) or (1 → 4) bonds Hence, lichenase cannot hydrolyze callose Laminarinase is an endoglucanase that hydrolyzes (1–3) or (1–4) linkages only when the glucose residue whose reducing group is involved in the linkage to be hydrolyzed is itself substituted at C-3 The oligomers produced by laminarinase probably were derived from callose, which was found in large amounts in banana cell wall Conclusions Banana fruit cell wall is characterized by high amounts of highly branched arabinans and mannans and small amounts of short-chains of arabinans and galacto(gluco)mannan Homogalacturonans and rhamnogalacturonan I were the main acidic pectins in banana and were resistant to hydrolysis by endopolygalacturonase Thap Maeo and Nanicão differ with respect to its cell wall polysaccharide composition ratio and solubility of polysaccharides and, in proportion of each polysaccharide in the water-soluble and water-insoluble fractions Both cultivars also showed that banana fruits may have a potential biological activity, mainly due to its high arabinogalactan and mannan contents Thap Maeo had more watersoluble galacturonans, galactans, arabinogalactans and ␤-glucan as callose, hence more ␤-glucanase activity However, in both cultivars studied mixed-linkage glucans were not present This is an important characteristic, since it makes Thap Maeo to have higher probability to producing immunomodulatory activity T.M Shiga et al / Carbohydrate Polymers 164 (2017) 31–41 Acknowledgements The authors acknowledge financial support of Núcleo de Apoio Pesquisa em Alimentos e Nutric¸ão – NAPAN and Coordenac¸ão para a Pesquisa de Nível Superior – CAPES, for financial support (Process BEX-10734/13-9) We also thank the valuable help of Anna T Olek, Matheus Romanos Benatti and John F Klimek from Purdue University for their 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Witaicenis, A., Rall, V L., & Di Stasi, L C (2012) Dietary intervention with green dwarf banana flour (Musa sp AAA) prevents intestinal inflammation in a trinitrobenzenesulfonic acid model of rat colitis Nutrition Research, 32(3), 202–209 Shiga, T M., Soares, C A., Nascimento, J R O., Purgatto, E., Lajolo, F M., & Cordenunsi, B R (2011) Ripening-associated changes in the amounts of starch and non-starch polysaccharides and their contributions to fruit softening in three banana cultivars Journal of the Science of Food and Agriculture, 91, 1511–1516  ... results, banana has arabinogalactans containing high amounts of t-Araf side-chains Banana showed high amounts of 3-, 6- and 3,6- and 3,4-Gal, characteristic of arabinogalactans Hence, the majority of. .. branched arabinans and mannans and small amounts of short-chains of arabinans and galacto(gluco )mannan Homogalacturonans and rhamnogalacturonan I were the main acidic pectins in banana and were resistant... are two different types of arabinogalactan in banana pulp Type I arabinogalactan, composed of (1 → 4)-␤-d-galactose with t-Araf sidechain attached at the O-3 of the galactose units Type II arabinogalactan