Modification of cell wall polysaccharides during retting of cassava roots

Food Chemistry 213 (2016) 402–409

Contents lists available at ScienceDirect

Food Chemistry journal homepage: www.elsevier.com/locate/foodchem

Modification of cell wall polysaccharides during retting of cassava roots Guillaume Legrand Ngolong Ngea a, Fabienne Guillon b, Jean Justin Essia Ngang a, Estelle Bonnin b, Brigitte Bouchet b, Luc Saulnier b,⇑ a b

University of Yaounde I, Cameroon INRA, UR 1268 Biopolymères, Interactions Assemblages, Nantes, France

a r t i c l e

i n f o

Article history: Received 25 March 2016 Received in revised form 24 June 2016 Accepted 29 June 2016 Available online 30 June 2016 Keywords: Softening Pectic polysaccharides Galactan Hemicelluloses Cell wall degrading enzymes Immunolabelling

a b s t r a c t Retting is an important step in traditional cassava processing that involves tissue softening of the roots to transform the cassava into flour and various food products. The tissue softening that occurs during retting was attributed to the degradation of cell wall pectins through the action of pectin-methylesterase and pectate-lyase that possibly originated from a microbial source or the cassava plant itself. Changes in cell wall composition were investigated during retting using chemical analysis, specific glycanase degradation and immuno-labelling of cell wall polysaccharides. Pectic 1,4-b-D-galactan was the main cell wall polysaccharide affected during the retting of cassava roots. This result suggested that better control of pectic galactan degradation and a better understanding of the degradation mechanism by endogenous endo-galactanase and/or exogenous microbial enzymes might contribute to improve the texture properties of cassava products. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction Cassava is one of the most important food crops in tropical countries and is used for human consumption in a large variety of food preparations. Retting of cassava roots is an integral part of cassava transformation for human consumption in subSaharan Africa. It is a conventional process that consists of soaking cassava roots in water for a 3–7 day period, during which the roots soften and other chemical modifications occur. Retting of cassava root is characterized by the degradation of endogenous cyanide compounds involved in the development of serious neurological disorders (Cardoso et al., 2005; Tshala-Katumbay et al., 2013). The retting process is also characterized by the production of organic acids and the softening of the root, which are both required to obtain products with acceptable organoleptic properties for consumers. The softening of the root is an important step for the production of high quality cassava products. Many investigations have focused on the role of starch in determining the cooking time and final texture of cassava roots, but no clear conclusions have been reached

Abbreviations: GalA, galacturonic acid; Ara, arabinose; Gal, galactose; Rha, rhamnose; HG, homogalacturonan; RGI, rhamnogalacturonan I; CWMs, cell wall materials. ⇑ Corresponding author. E-mail address: [email protected] (L. Saulnier). http://dx.doi.org/10.1016/j.foodchem.2016.06.107 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.

(Padonou, Mestres, & Nago, 2005; Safo-Kantanka & OwusuNipah, 1992). In the cases of many fruits and vegetables, the softening observed during cooking is influenced by the cell wall of the plant tissues (Waldron, Parker, & Smith, 2003). The final texture is likely to be dependent on the properties of cell wall components, their composition, proportions and interactions in relation to the different levels of structure (Waldron et al., 2003), including the molecular mechanism by which cells adhere to each other. In certain cassava cultivars, root tissues fail to soften even after a prolonged cooking time, which may be related to a higher level of chelator-insoluble pectic polysaccharides (Favaro et al., 2008). The detection of pectinolytic activities during spontaneous retting and the lack of cellulolytic activity supports the hypothesis that pectin might be involved in the root softening process (Ampe & Brauman, 1995). Pectinases detected during retting include three enzymes: pectin methylesterase, pectin/pectate lyase and polygalacturonase. It is unclear if these enzymes have plant and/or bacterial origins (Brauman, Kéléké, Malonga, Miambi, & Ampe, 1996). The use of fermentation starter exhibiting cell wall degrading enzymes activities have been reported to modify the texture of cassava products (Amoa-Awua & Jakobsen, 1995; Amoa-Awua, Frisvad, Sefa-Dedeh, & Jakobsen, 1997; Obilie, Tano-Debrah, & Amoa-Awua, 2003). In addition, different studies have suggested that the enzymatic hydrolysis of cell wall polysaccharides improves the detoxification of cassava roots (Mkpong, Yan,

G.L. Ngolong Ngea et al. / Food Chemistry 213 (2016) 402–409

Chism, & Sayre, 1990; Santana, Vásquez, Matehus, & Aldao, 2002; Sornyotha, Kyu, & Ratanakhanokchai, 2010). Until now, most of the studies related to the softening of cassava roots have focused on understanding enzyme activities and microbial strains involved in the retting process, but these studies have not precisely linked softening with cell wall component modifications. Precise knowledge of cell wall polysaccharides and cell wall architecture of the roots is essential for understanding the texture modifications occurring during the retting process. In this study, chemical, enzymatic and microscopy methods were combined to analyse the nature of cell wall polysaccharides, identify key polysaccharides, determine their distribution in fresh cassava root, and characterize their modifications in retted root and retted root flour. 2. Material and methods 2.1. Plant material and enzymes Cassava (Manihot esculenta, Crantz) roots were harvested close to Yaounde (Cameroon) from healthy young plants (12 months old) of the bitter cultivar (Ntol) in August 2014. They were soaked in paraffin to preserve them from dehydration and oxidation. They were stored in a cold room at 4 °C. Cassava flour produced through traditional process of retting, grinding, drying and milling was purchased in Cameroon. The flour was stored in a cold room at 4 °C. Endo-1,4-b-D-glucanase (Cellulase) from Trichoderma longibrachiatum (E-CELTR), endo-1,4-b-D-galactanase from Aspergillus niger (E-GALN), heat-stable a-amylase from Bacillus licheniformis (E-BLAAM) and amyloglucosidase from Aspergillus niger (EAMGDF) were purchased from Megazyme (Bray, Ireland). 2.2. Retting of cassava roots Fresh cassava roots were peeled and then cut into cubes of 2– 3 cm3 that were immersed in tap water (150 mL/100 g of fresh root) and left until the roots softened at room temperature for 7 days. 2.2.1. Preparation of Alcohol Insoluble Residue (AIR) The top and bottom ends of fresh roots were removed, and the resulting middle portion was peeled. This material was then used for subsequent analyses or separated into central and peripheral areas, which were analysed separately. Pieces of retted roots were directly analysed. The samples of fresh or retted roots were treated with boiling alcohol to destroy any enzymatic activity and to eliminate alcohol soluble components, especially carbohydrates with a low degree of polymerization (such as free glucose, sucrose, among others). Pieces of fresh or retted roots were heated at 95 °C for 10 min in an appropriate volume of 96% EtOH to obtain a final EtOH concentration of 70%. The EtOH supernatant was eliminated by filtration, and successive washings with 70% EtOH were applied to the alcohol insoluble residue (AIR) until no remaining carbohydrate was detected in the supernatant using a phenol test (DuBois, Gilles, Hamilton, Rebers, & Smith, 1956). The residue was rinsed with 96% EtOH, then rinsed with acetone and dried overnight in an oven at 40 °C. AIR yields were calculated using the starting mass of the fresh and retted root samples. 2.2.2. Destarching of AIR AIR was de-starched using conditions derived from a starch assay (McCleary, Gibson, & Mugford, 1997). AIR (50 mg) was suspended in 1 mL of 50 mM sodium MOPS buffer (pH 7) heated at

403

100 °C, and then 0.2 mL of heat-stable alpha-amylase (60 U) was added and the mixture was incubated at 100 °C for 20 min. After cooling to 50 °C, the pH was adjusted to 4.5 with 0.8 mL of 200 mM acetate buffer (pH 4.5). Then, amyloglucosidase (40 U) was added to the mixture, which was incubated at 50 °C for 60 min. After cooling, 4 volumes of 96% EtOH were added and the resulting slurry was centrifuged (6000g, 10 min). The pellet was washed successively with 80% and 96% EtOH and then dried overnight in an oven at 40 °C. De-starched AIR is referred to as cell wall material (CWM). 2.2.3. Treatment of CWM with pure, specific glycosyl-hydrolases CWM obtained from fresh and retted cassava roots was degraded with different glycosyl-hydrolases to detect the presence of galactan and xyloglucan. CWM (5 mg) was suspended in 1 mL of distilled water containing endo-galactanase (20 U) or endoglucanase (20 U). The endo-glucanase could hydrolyse xyloglucan as well as cellulose. The suspension was gently agitated using a rotary wheel and digested overnight (16 h) at 40 °C. After centrifugation (20 °C, 20 min, 10,600g), the supernatant was placed in a boiling water bath for 10 min to inactivate enzymes and then was filtered through a 0.45-lm filter and frozen. Samples were diluted 5 times prior to the analysis of degradation products with high performance anion exchange chromatography (HPAEC). The samples (10 lL) were injected into a Carbopac PA-200 (5  250 mm) analytical column maintained at 25 °C (Dionex, http://www.dionex.com) and eluted at a flow rate of 0.4 mL min 1 using a gradient of ultrapure water (A), 1 M NaOAc (B) and 0.5 M NaOH (C) solutions. The gradient conditions were: 0 min (A, 60%; C, 40%), 30 min (A, 43%; B, 17%; C, 40%), 35 min (A, 35%; B, 25%; C, 40%), 36 min (A, 20%; B, 40% C, 40%), 38 min (A, 20%; B, 40%; C, 40%), and 39–65 min (A, 60%; C, 40%). A pulse amperometric detector (TSP EC2000, http://www.thermo.com) was used for the detection. Oligosaccharide identification was based on retention times compared to reference oligosaccharides obtained from purified xyloglucan that was available in the laboratory collection (Ray, Vigouroux, Quémener, Bonnin, & Lahaye, 2014). 2.3. Determination of the sugar composition of AIR and CWM samples CWM samples were ultra-ground in liquid nitrogen using a Spex 6700 cryogenic grinder (Spex. Industries, USA). The neutral sugar compositions were identified, after acid hydrolysis, using gas chromatography of alditol acetates (Englyst & Cummings, 1988). Acid hydrolysis of AIR or CWM was carried out as previously described (Saulnier, Marot, Chanliaud, & Thibault, 1995): the samples were pre-hydrolysed with 72% (w/ w; 26 M) sulphuric acid for 30 min at 25 °C and then hydrolysed into monomers at 100 °C for 2 h in 1 M sulphuric acid. Uronic acid content was determined in the acid hydrolysis supernatant using an automated m-phenylphenol method (Thibault, 1979) and galacturonic acid as a standard. Starch was analysed in AIR according to AOAC method 996.11 (McCleary et al., 1997) with a modification for quantifying glucose using HPAEC instead of an enzyme assay. 2.4. Sample preparation for microscopy Transverse sections (500 lm thick) were hand-cut from fresh and retted cassava roots. Small cubes of approximately 1 mm3 were then prepared from root transverse sections using a razor blade under a binocular dissecting microscope. Both the central and edge areas of the roots were sampled. Fragments were then fixed in a mixture of 3% paraformaldehyde and 0.5% glutaraldehyde in 0.1 M sodium phosphate buffer (pH 7.2) for one night at 4 °C. After washing, the samples were dehydrated in a graded aqueous

404

G.L. Ngolong Ngea et al. / Food Chemistry 213 (2016) 402–409

ethanol series, progressively infiltrated with London Resin White (LRW) acrylic resin and then embedded in gelatine capsules. The resin was polymerized for 4 days at 55 °C without an accelerator. Semi-thin sections (1 lm) were cut on an ultramicrotome (UC7; Leica Microsystems, Germany) equipped with a diamond knife. 2.4.1. Light microscopy Semi-thin sections (1 lm) were stained for 10 min in a 0.02% acridine orange solution prepared in 0.01 M sodium phosphate buffer at pH 7.2 to make the cell walls and starch fluorescent. Sections were observed using an AZ100M fluorescence microscope (Nikon) equipped for epifluorescence and images were acquired with a Retiga EXI Aqua camera (RGB module). The samples were excited between 461 and 489 nm with the chromatic beam splitter set at 500 nm, and the long pass emission filter was above 515 nm.

polysaccharides and their interactions are generally involved in the softening of plant tissues (Johnston, Hewett, & Hertog, 2002). The recovery of alcohol insoluble residue (AIR) from fresh root (FR), retted root (RR) and cassava flour (CF) essentially reflected the moisture content of the starting product (Table 1). The AIR yields (expressed as % of fresh weight) of FR and RR were quite similar with the RR exhibiting a lower AIR yield due to a higher water content after the retting process, whereas the AIR yield of the flour was much higher because it was already a dry powder. Starch was the major component and represented nearly 90% (w/ w dry matter) of the AIR. It was therefore necessary to remove starch to track possible changes in cell-wall polysaccharide composition. 3.1. Composition of fresh cassava roots cell wall material (CWM)

2.4.2. Immunolabelling Antibodies were used to map the distribution of pectic epitopes. Immunolabelling was carried out as previously described (Guillemin et al., 2005; Herbette et al., 2015). For each immunolabelling, two blocks of embedded samples from fresh and retted roots were analysed. Semi-thin sections were floated on a drop of phosphate-buffered saline (PBS) supplemented with 3% bovine serum albumin to block non-specific labelling for 30 min. Sections were then incubated in buffer containing the primary Abs (1/10 dilution) supplemented with 1% bovine serum albumin and 0.05% Tween 20 for 1 h at room temperature. The sections were washed extensively in the buffer used for diluting the primary Abs and then incubated for 1 h at room temperature with secondary Abs (Goat anti-rat IgG; Life Technologies A11081) coupled to AlexaFluor 546 diluted 1:100 (v/v) in the buffer used for diluting primary Abs. The sections were washed in buffer as described above and rinsed twice in distilled water. The semi-thin embedded sections were observed on a LEICA DMRD fluorescence microscope equipped with epifluorescence. A band-pass filter (515–560 nm) was used for excitation; the chromatic beam splitter was set at 580 nm, and the long pass emission filter was above 590 nm. These antibodies were LM5 to target (1-4)-b-D-galactan (Jones, Seymour, & Knox, 1997), LM6 to target linear (1-5)-a-L-arabinan (Willats, Marcus, & Knox, 1998), LM19 to target unesterified homogalacturonan (Verhertbruggen, Marcus, Haeger, Ordaz-Ortiz, & Knox, 2009) and LM20 to target methyl-esterified homogalacturonan (Verhertbruggen et al., 2009).

Cell wall represented approximately 4.7% of the fresh cassava roots weight, which was consistent with the values between 2.63 and 4.92 g 100 g 1 reported for other cultivars (Favaro et al., 2008). Neutral sugars represented approximately 73% and galacturonic acid approximately 17% of total carbohydrate of the CWM, which agrees with the results of a previous study (Kajiwara & Maeda, 1983). CWM composition was close to the values previously reported in other studies for various cassava cultivars (Favaro et al., 2008; Kajiwara & Maeda, 1983; Salvador, Suganuma, Kitahara, Tanoue, & Ichiki, 2000). Glucose was the main constituent of CWM and probably originated primarily from cellulose. A high content of galactose and galacturonic acid, and lower amounts of arabinose and rhamnose were also measured. This suggests a strong presence of pectic polysaccharide as previously reported (Favaro et al., 2008; Kajiwara & Maeda, 1983). Pectic polysaccharides have two major domains: homogalacturonan made exclusively of galacturonic acid, and rhamnogalacturonan I (RGI) which is composed of a main chain of alternating galacturonic acid and rhamnose residues substituted by side chains of neutral sugar. The side chains are attached to the rhamnose residues in RGI and are mainly constituted of b-1,4 linked-D-galactose (galactan) and a-1,5 linked arabinose (arabinan) residues. Mannose, xylose and fucose were also observed in CWM, but we detected a higher proportion of mannose than the proportion reported by other researchers (Favaro et al., 2008). These neutral sugars might originate from different hemicellulosic polysaccharides, such as mannan, xyloglucan and xylan.

3. Results and discussion

3.2. Changes in CWM composition during retting

Cassava roots are widely consumed in sub-Saharan Africa. Retting leads to tissue softening that allows cassava roots to be processed into flour and other products. To explain how firm fresh root becomes soft and spongy after the retting process, we looked for modifications in cell wall composition because cell wall

CWM isolated from RR represented approximately 3.9% of the fresh root weight, which indicated a decrease in CWM yield compared to FR. The CWM of RR and CF had a similar composition that was different from FR (Table 1). CWM composition of RR and CF indicated a strong decrease in Gal and GalA content compared to

Table 1 Yield and starch content of alcohol insoluble residues (AIR) obtained from cassava fresh roots, retted roots and flour. The carbohydrate composition of cell wall material (CWM) was prepared from de-starched AIR.

Fresh Root (FR) Retted Root (RR) Cassava flour (CF)

AIR

Carbohydrate composition of CWM g/100 g of CWM

Yielda

Starchb

Rhac

Fucc

Arac

Xylc

Manc

Galc

Glcc

Gal. A.c

Totalc

36.4 32.9 84.8

86.1 (0.6) 88.0 (1.6) 89.4 (0.9)

1.0 (0.1) 0.0 (0.0) 0.7 (0.7)

0.8 (0.1) 0.3 (0.3) 0.5 (0.5)

3.2 (0.2) 1.0 (0.1) 1.9 (0.1)

3.8 (0.3) 4.7 (0.2) 4.4 (0.1)

8.0 (0.1) 8.9 (0.2) 8.9 (0.3)

15.4 (0.5) 4.6 (0.3) 5.3 (0.3)

28.4 (2.2) 30.1 (1.0) 30.7 (0.4)

12.2 (0.2) 6.0 (0.2) 6.9 (0.2)

72.7 (3.6) 55.7 (1.5) 59.2 (1.1)

Data are the mean of duplicate measurements, and the values in bracket are the average difference. a g/100 g of fresh root, retted root or flour. b g/100 g in AIR. c Carbohydrate components: Rha: rhamnose; Fuc: fucose; Ara: arabinose; Xyl: xylose; Man: mannose; Gal: galactose; Glc: glucose; Gal A.: galacturonic acid; Total: sum of components.

405

G.L. Ngolong Ngea et al. / Food Chemistry 213 (2016) 402–409

FR. Arabinose was also severely reduced by the retting process. Conversely, glucose, mannose and xylose content slightly increased in the CWM of RR and CF. Similar effects were observed for RR and CF, although the samples were obtained from different sources of cassava. The major changes in CWM composition suggested that the retting process primarily affected pectic polymers, such as galactan, homogalacturonan and arabinan. The measurement of pectin-methylesterase and polygalacturonate lyase activities during retting already indirectly supported the degradation of the homogalacturonan part of the pectin and the role of these enzymes in the softening of cassava root tissue during retting (Brauman et al., 1996). However, pectin is a very complex family of polysaccharides that encompasses various types of neutral and acidic polymers (Bonnin, Garnier, & Ralet, 2014). 1,4 linked galactan and 1,5 linked arabinan are the main constituents of side chains attached to RGI, but type II arabinogalactan (1,31,6 linked galactan backbone) might also occur in Cassava roots either as part of arabinogalactan-protein or as a minor side chain component of RGI. To confirm the origin of changes occurring in cassava cell walls, especially in the galactose content, we used specific glycosyl-hydrolases to identify the cell wall polysaccharides present in cassava root. 3.3. Identification of cell wall polysaccharides using specific glycanase degradation To determine the type of pectic galactan and the possible presence of xyloglucan in CWMs of FR and RR, we used endo-1,4-b-Dgalactanase and endo-1,4-b-D-glucanase, respectively. HPAEC chromatograms of CWMs from FR and RR digested with the enzymes are shown in Fig. 1. The action of endo-1,4-b-D-galactanase on FR CWM mainly released galactose and 1,4 linked galactobiose as well as minor amounts of higher DP oligosaccharides, which were not identified. Endo-1,4-b-D-galactanase thus confirmed the presence of linear 1,4 linked linear galactans that are possibly linked as side-chains

150

to the rhamnogalacturonan backbone of pectic polysaccharides. Conversely, endo-1,4-b-D-galactanase released neither galactose nor galactobiose from RF CWM, which suggests that the linear 1,4 linked galactans present in FR CWM were completely degraded during retting. However, retted root CWM still contained galactose (1/3 of initial quantity) that most likely originated from other wall components, such as xyloglucan. As reported in potato tubers (Oxenboll Sørensen et al., 2000), this polymer may contain b-1,2-linked galactosyl residues that are not hydrolysed by endo-galactanase. Additionally, xyloglucans were revealed by the action of endo1,4-b-D-glucanase on FR and RR CWMs. The enzyme released glucose and cellobiose from cellulose, and a series of peaks eluting between 13.6 and 15 min were identified as xyloglucanoligosaccharides (XyGOs: XXXG, XXFG, XLXG and XLFG). Some of these XyGOs contained galactose (XXFG, XLXG and XLFG) (Ray et al., 2014). The abundance of glucose and cellobiose was slightly higher in the hydrolysate from RR than from FR CWM, which agrees with the slight enrichment in glucose already observed in RR CWM. The level of XyGOs released from CWMs indicated that the retting process did not affect xyloglucan polymers. 3.4. Characterization of cell wall organization in cassava roots and changes after retting The results from composition and enzymatic profiling showed that pectin was prominently affected during retting process. Consequently, the distribution of pectic polysaccharides in CWM and changes after retting were more specifically investigated by immunocytochemistry using LM19, LM20, LM5 and LM6 that recognize un-esterified homogalacturonan, methyl-esterified homogalacturonans, linear galactan and arabinan chains linked to RGI, respectively. Semi-thin sections of fresh roots sampled from the median zone of the root were first examined using acridine orange, which stained cell walls rich in pectins orange and granular starch green (Figs. 2 and 3). Three regions were considered: the periderm and

A-FR

125

A-RR

Gal

100 Gal2

Detector response (nC)

75 50

Gal

25 0 250

Gal2

B-FR

Glc2

B-RR

200 Glc2

150 100

Glc Glc

50 0

XyGOs 0

5

10

15

XyGOs 20 0

5

10

15

20

Elution time (min.) Fig. 1. HPAEC profile of degradation products obtained after endo-galactanase (A) and endo-glucanase (B) treatment of CWMs isolated from fresh root (FR) and retted root (RR). Gal: galactose; Glc: Glucose; Gal2: (1,4)b-D-Galactobiose; Glc2: (1,4)b-D-Glucobiose; XyGOs: Xyloglucan-oligosaccharides.

406

G.L. Ngolong Ngea et al. / Food Chemistry 213 (2016) 402–409

pe

A1

A2

100 m

B sx

xv

100 m

Fig. 2. Transverse section of fresh cassava root stained with acridine orange (A1) Outer region of the root, including the periderm and cortex; (A2) Starchy flesh of the root with part of the cortex at the top of the figure; (B) Central region of the root, including the starchy flesh and central vascular fibres on the right side of the figure. pe: periderm; xv: xylem vessels; sx: secondary xylem. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

A2-FR

the cortex region (Fig. 2A1), the outer part of the starchy flesh that contains a portion of the cortex and mostly storage parenchyma cells filled with starch granules (Fig. 2A2), and the central part of the root that comprises starchy flesh and central vascular fibre with xylem vessels surrounded by secondary xylem cells (Fig. 2B). Images of the A2 and B regions are shown at a higher magnification in Fig. 3 for both fresh and retted roots. In fresh root tissues, the cells were tightly attached together. Storage parenchyma cells had a polyhedral shape with a conserved turgescent appearance and were highly cohesive both in the different parts of the starchy flesh of the root (Fig. 3A2-B-FR). The cell walls were stained orange with acridine orange. Tissues in retted roots lost cell-cell adhesion, which was clearly visible in the central part of the root (Fig 3B-RR), and the cells exhibited a more rounded shape. The green staining of cell walls with acridine orange indicated that their composition had changed (Fig. 3A2-B-RR). Starch granules inside the cells were still present with no change in their shape. Immunolabelling of fresh and retted roots is displayed on Figs. 4 and 5 for the outer part of the starchy flesh (region A2; Fig. 4) and the central part of the root (region B; Fig. 5), respectively. Strong labelling of cell walls was observed with anti-galactan LM5 Ab in the storage parenchyma and medullar storage parenchyma, but no labelling was found in the bundle of fresh root (Fig. 5B-FR), which indicates that galactan chains are not homogeneously distributed in fresh root tissues. Labelling of cell walls completely disappeared after retting (Figs. 4 and 5), which confirmed the loss of galactan indicated by endo-galactanase profiling. Using anti-arabinan LM6 Ab, faint labelling of cell walls in storage parenchyma and strong labelling of the cell wall of cells surrounding xylem vessels were observed in fresh root. Apart from the strong labelling around the vessels, the antibody labelled the cell walls of bundle region similarly to the walls of parenchymal cells. Nearly no labelling was observed in retted root, which indicated that the amount of arabinan diminished considerably after retting. This outcome matched the results indicated by the CWM composition analysis. LM19 Ab was specific for un-esterified homogalacturonan bound strongly to storage parenchyma cells of fresh roots. The

B-FR

sg sg

A2-RR

B-RR

sg

sg

Fig. 3. Transverse sections of fresh (FR) and retted (RR) in starchy flesh cassava roots stained with acridine orange A2-FR) outer region of a fresh root; B-FR) central region of a fresh root; A2-RR) outer region of a retted root; B-RR) central region of a retted root. sg: starch granules. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

407

G.L. Ngolong Ngea et al. / Food Chemistry 213 (2016) 402–409

LM5

A2-FR

LM5

A2-RR

LM5

B-FR

LM5

B-RR

LM6

A2-FR

LM6

A2-RR

LM6

B-FR

LM6

B-RR

LM19

B-RR

LM20

B-RR

xv

LM19

A2-FR

LM19

A2-RR

LM19

B-FR sx

LM20

A2-FR

LM20

A2-RR

50 m

LM20

B-FR

50 m

Fig. 4. Transverse sections of fresh (FR) and retted (RR) root from the outer part (A2) of starchy flesh cassava roots. Immunofluorescence labelling with LM5 antigalactan Ab; LM6 anti-arabinan Ab; LM19 anti-non-esterified-homogalacturonan Ab; and LM20 anti-methyl-esterified-homogalacturonan Ab.

Fig. 5. Transverse sections of fresh (FR) and retted (RR) in the central region (B) of starchy flesh cassava roots. Immunofluorescence labelling with LM5 anti-galactan Ab; LM6 anti-arabinan Ab; LM19 anti-non-esterified-homogalacturonan Ab; and LM20 anti-methyl-esterified-homogalacturonan Ab. xv: xylem vessels.

labelling was less intense in the cells close to the secondary xylem. LM20 Ab was specific for methyl-esterified homogalacturonan labelled cell corners in storage parenchyma cells and the entire cell wall of secondary xylem cells in FR roots. LM19 and LM20 labelling patterns suggested that low methyl-esterified and methylesterified homogalacturonans were not distributed equally in the starchy parenchyma and secondary xylem cells. Cell wall labelling with LM19 Ab in retted root tissue was reduced in the storage parenchyma and was very scarce in the secondary xylem region. Labelling with LM20 was almost abolished. All together, these results suggested that homogalacturonans were partly degraded during retting.

cell to cell separation and tissue softening. A similar decrease in the galactose and arabinose content of pectins were also observed during the ripening of fruits, whereas pectin structure was drastically altered concomitantly with tissue softening (Brummell, 2006). Several studies support the involvement of homogalacturonan in cell wall stiffening and in cell to cell adhesion (Bouton et al., 2002; Jarvis, Briggs, & Knox, 2003; Leboeuf, Guillon, Thoiron, & Lahaye, 2005; Liners, Gaspar, & Van Cutsem, 1994; Mouille et al., 2007), and it has long been thought that degradation of pectin by polygalacturonase was the main cause of fruit softening. However, attempts to control fruit softening using genetic engineering of pectin methyl esterase and polygalacturonase had limited success (Goulao & Oliveira, 2008; Roberts, Elliott, & Gonzalez-Carranza, 2002). Much less is known about the function of galactan and arabinan pectic side chains in cell walls and their role in tissue softening. Several studies indicated that galactan and arabinan side chains of rhamnogalacturonan contributed to cell wall mechanical properties and the texture of tissue changes of various plant

3.5. Role of galactan and arabinan in tissue softening During retting, intense changes occurred for homogalacturonan, galactan and arabinan. These modifications were accompanied by

408

G.L. Ngolong Ngea et al. / Food Chemistry 213 (2016) 402–409

organs. Galactan deposition in developing pea cotyledons was correlated with an increase in wall firmness (McCartney, Ormerod, Gidley, & Knox, 2000), and transgenic potato tubers in which the amount of galactan was reduced exhibited more brittle tissues compared to wild type (Ulvskov et al., 2005). Alteration of rhamnogalacturonan arabinan side chains was also associated with loosely adhering cells (Iwai, Ishii, & Satoh, 2001; Leboeuf et al., 2005; Orfila et al., 2002). In apples, loss of branched arabinan occurred before loss of texture (Peña & Carpita, 2004). Adsorption of arabinan and galactan side chains onto primary cell wall cellulose has been demonstrated in vitro, which suggests that cellulose microfibrils may be tethered by neutral side chains belonging to the same pectic macromolecule (Zykwinska, Thibault, & Ralet, 2007), but the role of these polymers in cell adhesion was not clearly established. 4. Conclusions Cassava root cell walls were deeply altered during retting with the loss of pectin related constituents, including galactose, galacturonic acid, arabinose and rhamnose. Meanwhile, the cellulose and hemicellulosic network remained unchanged, which supported the hypothesis that modification of pectins, and more specifically galactan attached to rhamnogalacturonan, were involved in the softening and the loss of the intercellular cohesion of cassava root tissues. Interestingly, the cell wall compositions of cassava flour and retted root were very similar and presented both low amounts of galactose and galacturonic acid. This observation might be very important for quality control to ensure that any flour present in the market went through a proper retting process. This study complements earlier work that suggested there were pectinolytic activities and a lack of cellulolytic activities during the retting process. In this respect, further studies are needed to investigate the plant or microbial origins of the depolymerizing enzymes that act during retting. A better control of pectic galactan degradation, and understanding the mechanism of their degradation by endogenous cassava and/or exogenous microbial enzymes, could help to improve texture properties of cassava end products and would drive the development of an enzyme-assisted retting process. Acknowledgements We are indebted to Jacqueline Vigouroux and Laurent Helary for their technical advice and assistance with the polysaccharide and microscopy analyses. This work was supported by a grant from the Department of Cooperation and Cultural Action at the embassy of France in Cameroon. References Amoa-Awua, W. K., Frisvad, J. C., Sefa-Dedeh, S., & Jakobsen, M. (1997). The contribution of moulds and yeasts to the fermentation of ‘‘agbelima” cassava dough. Journal of Applied Microbiology, 83(3), 288–296. http://dx.doi.org/ 10.1046/j.1365-2672.1997.00227.x. Amoa-Awua, W. K. A., & Jakobsen, M. (1995). The role of Bacillus species in the fermentation of cassava. Journal of Applied Bacteriology, 79, 250–256. http://dx. doi.org/10.1111/j.1365-2672.1995.tb03134.x. Ampe, F., & Brauman, A. (1995). Origin of enzymes involved in detoxification and root softening during cassava retting. World Journal of Microbiology & Biotechnology, 11, 178–182. http://dx.doi.org/10.1007/BF00704644. Bonnin, E., Garnier, C., & Ralet, M.-C. (2014). Pectin-modifying enzymes and pectinderived materials: Applications and impacts. Applied Microbiology and Biotechnology, 98(2), 519–532. http://dx.doi.org/10.1007/s00253-013-5388-6. Bouton, S., Leboeuf, E., Mouille, G., Leydecker, M.-T., Talbotec, J., Granier, F., & Truong, H.-N. (2002). QUASIMODO1 encodes a putative membrane-bound glycosyltransferase required for normal pectin synthesis and cell adhesion in Arabidopsis. The Plant Cell, 14(10), 2577–2590. http://dx.doi.org/10.1105/ tpc.004259. Brauman, A., Kéléké, S., Malonga, M., Miambi, E., & Ampe, F. (1996). Microbiological and biochemical characterization of cassava retting, a traditional lactic acid

fermentation for foo-foo (cassava flour) production. Applied and Environmental Microbiology, 62(8), 2854–2858. Brummell, D. A. (2006). Cell wall disassembly in ripening fruit. Functional Plant Biology. http://dx.doi.org/10.1071/FP05234. Cardoso, A. P., Mirione, E., Ernesto, M., Massaza, F., Cliff, J., Haque, M. R., & Bradbury, J. H. (2005). Processing of cassava roots to remove cyanogens. Journal of Food Composition and Analysis, 18(5), 451–460. http://dx.doi.org/10.1016/j. jfca.2004.04.002. DuBois, M., Gilles, K. a., Hamilton, J. K., Rebers, P. a., & Smith, F. (1956). Colorimetric method for determination of sugars and related substances. Analytical Chemistry, 28(3), 350–356. http://dx.doi.org/10.1021/ac60111a017. Englyst, H. N., & Cummings, J. H. (1988). Improved method for measurement of dietary fiber as non-starch polysaccharides in plant foods. Journal Association of Official Analytical Chemists, 71(4), 808–814. Favaro, S. P., Beléia, A., da Silva Fonseca Junior, N., & Waldron, K. W. (2008). The roles of cell wall polymers and intracellular components in the thermal softening of cassava roots. Food Chemistry, 108(1), 220–227. http://dx.doi.org/ 10.1016/j.foodchem.2007.10.070. Goulao, L. F., & Oliveira, C. M. (2008). Cell wall modifications during fruit ripening: When a fruit is not the fruit. Trends in Food Science and Technology, 19(1), 4–25. http://dx.doi.org/10.1016/j.tifs.2007.07.002. Guillemin, F., Guillon, F., Bonnin, E., Devaux, M.-F., Chevalier, T., Knox, J. P., & Thibault, J.-F. (2005). Distribution of pectic epitopes in cell walls of the sugar beet root. Planta, 222(2), 355–371. http://dx.doi.org/10.2307/23389051. Herbette, S., Bouchet, B., Brunel, N., Bonnin, E., Cochard, H., & Guillon, F. (2015). Immunolabelling of intervessel pits for polysaccharides and lignin helps in understanding their hydraulic properties in Populus tremula  alba. Annals of Botany, 115(2), 187–199. http://dx.doi.org/10.1093/aob/mcu232. Iwai, H., Ishii, T., & Satoh, S. (2001). Absence of arabinan in the side chains of the pectic polysaccharides strongly associated with cell walls of Nicotiana plumbaginifolia non-organogenic callus with loosely attached constituent cells. Planta, 213(6), 907–915. http://dx.doi.org/10.1007/s004250100559. Jarvis, M. C., Briggs, S. P. H., & Knox, J. P. (2003). Intercellular adhesion and cell separation in plants. Plant, Cell & Environment, 26(7), 977–989. Retrieved from Disque Dru:Documents:BiblioLuc:2003:Jarvis+2003.pdf. Johnston, J. W., Hewett, E. W., & Hertog, M. L. A. T. M. (2002). Postharvest softening of apple (Malus domestica) fruit: A review. New Zealand Journal of Crop and Horticultural Science, 30(3), 145–160. http://dx.doi.org/10.1080/ 01140671.2002.9514210. Jones, L., Seymour, G. B., & Knox, J. P. (1997). Localization of pectic galactan in tomato cell walls using a monoclonal antibody specific to (1[-]4)-[beta]-DGalactan. Plant Physiology, 113(4), 1405–1412. http://doi.org/113/4/1405 [pii]. Kajiwara, S., & Maeda, H. (1983). The monosaccharide composition of cell wall material in cassava tuber (Manihot utilissima). Agricultural and Biological Chemistry, 47(10), 2335–2340. Leboeuf, E., Guillon, F., Thoiron, S., & Lahaye, M. (2005). Biochemical and immunohistochemical analysis of pectic polysaccharides in the cell walls of Arabidopsis mutant QUASIMODO 1 suspension-cultured cells: Implications for cell adhesion. Journal of Experimental Botany, 56(422), 3171–3182. http://dx.doi. org/10.1093/jxb/eri314. Liners, F., Gaspar, T., & Van Cutsem, P. (1994). Acetyl- and methyl-esterification of pectins of friable and compact sugar-beet calli: consequences for intercellular adhesion. Planta, 192(4), 545–556. http://dx.doi.org/10.1007/BF00203593. McCartney, L., Ormerod, A. P., Gidley, M. J., & Knox, J. P. (2000). Temporal and spatial regulation of pectic (1?4)-beta-D-galactan in cell walls of developing pea cotyledons: Implications for mechanical properties. Plant Journal, 22(2), 105–113. http://dx.doi.org/10.1046/j.1365-313X.2000.00719.x. McCleary, B., Gibson, T., & Mugford, D. (1997). Measurement of total starch in cereal products by amyloglucosidase-alpha-amylase method: Collaborative study. Journal Association of Official Analytical Chemists, 80(3), 571–579. Mkpong, O. E., Yan, H., Chism, G., & Sayre, R. T. (1990). Purification, characterization, and localization of linamarase in cassava. Plant Physiology, 93, 176–181. http:// dx.doi.org/10.1104/pp.93.1.176. Mouille, G., Ralet, M. C., Cavelier, C., Eland, C., Effroy, D., Hématy, K., & Höfte, H. (2007). Homogalacturonan synthesis in Arabidopsis thaliana requires a Golgilocalized protein with a putative methyltransferase domain. Plant Journal, 50(4), 605–614. http://dx.doi.org/10.1111/j.1365-313X.2007.03086.x. Obilie, E. M., Tano-Debrah, K., & Amoa-Awua, W. K. (2003). Microbial modification of the texture of grated cassava during fermentation into akyeke. International Journal of Food Microbiology, 89(2–3), 275–280. http://dx.doi.org/10.1016/ S0168-1605(03)00294-0. Orfila, C., Huisman, M. M. H., Willats, W. G. T., Van Alebeek, G. J. W. M., Schols, H. A., Seymour, G. B., & Knox, J. P. (2002). Altered cell wall disassembly during ripening of Cnr tomato fruit: Implications for cell adhesion and fruit softening. Planta, 215(3), 440–447. http://dx.doi.org/10.1007/s00425-002-0753-1. Oxenboll Sørensen, S., Pauly, M., Bush, M., Skjøt, M., McCann, M. C., Borkhardt, B., & Ulvskov, P. (2000). Pectin engineering: modification of potato pectin by in vivo expression of an endo-1,4-beta-D-galactanase. Proceedings of the National Academy of Sciences of the United States of America, 97(13), 7639–7644. http:// dx.doi.org/10.1073/pnas.130568297. Padonou, W., Mestres, C., & Nago, M. C. (2005). The quality of boiled cassava roots: Instrumental characterization and relationship with physicochemical properties and sensorial properties. Food Chemistry, 89(2), 261–270. http://dx. doi.org/10.1016/j.foodchem.2004.02.033. Peña, M. J., & Carpita, N. C. (2004). Loss of highly branched arabinans and debranching of rhamnogalacturonan I accompany loss of firm texture and cell

G.L. Ngolong Ngea et al. / Food Chemistry 213 (2016) 402–409 separation during prolonged storage of apple. Plant Physiology, 135(3), 1305–1313. http://dx.doi.org/10.1104/pp.104.043679. Ray, S., Vigouroux, J., Quémener, B., Bonnin, E., & Lahaye, M. (2014). Novel and diverse fine structures in LiCl–DMSO extracted apple hemicelluloses. Carbohydrate Polymers, 108, 46–57. http://dx.doi.org/10.1016/ j.carbpol.2014.03.017. Roberts, J. A., Elliott, K. A., & Gonzalez-Carranza, Z. H. (2002). Abscission, dehiscence, and other cell separation processes. Annual Review of Plant Biology, 53, 131–158. http://dx.doi.org/10.1146/annurev.arplant.53.092701.180236. Safo-Kantanka, O., & Owusu-Nipah, J. (1992). Cassava varietal screening for cooking quality: Relationship between dry matter, starch content, mealiness and certain microscopic observations of the raw and cooked tuber. Journal of the Science of Food and Agriculture, 60(1), 99–104. http://dx.doi.org/10.1002/jsfa.2740600116. Salvador, L. D., Suganuma, T., Kitahara, K., Tanoue, H., & Ichiki, M. (2000). Monosaccharide composition of sweetpotato fiber and cell wall polysaccharides from sweetpotato, cassava, and potato analyzed by the highperformance anion exchange chromatography with pulsed amperometric detection method. Journal of Agricultural and Food Chemistry, 48(8), 3448–3454. http://dx.doi.org/10.1021/jf991089z. Santana, M. A., Vásquez, V., Matehus, J., & Aldao, R. R. (2002). Linamarase expression in cassava cultivars with roots of low- and high-cyanide content. Plant Physiology, 129(4), 1686–1694. http://dx.doi.org/10.1104/pp.000927. Saulnier, L., Marot, C., Chanliaud, E., & Thibault, J.-F. (1995). Cell wall polysaccharide interactions in maize bran. Carbohydrate Polymers, 26(4), 279–287. http://dx. doi.org/10.1016/0144-8617(95)00020-8. Sornyotha, S., Kyu, K. L., & Ratanakhanokchai, K. (2010). An efficient treatment for detoxification process of cassava starch by plant cell wall-degrading enzymes.

409

Journal of Bioscience and Bioengineering, 109(1), 9–14. http://dx.doi.org/10.1016/ j.jbiosc.2009.06.021. Thibault, J.-F. (1979). Automated-method for the determination of pectic substances. Lebensmittel-Wissenschaft Und-Technologie, 12, 247–251. Tshala-Katumbay, D., Mumba, N., Okitundu, L., Kazadi, K., Banea, M., Tylleskar, T., & Muyembe-Tamfum, J. J. (2013). Cassava food toxins, konzo disease, and neurodegeneration in sub-Sahara Africans. Neurology, 80(10), 949–951. http:// dx.doi.org/10.1212/WNL.0b013e3182840b81. Ulvskov, P., Wium, H., Bruce, D., Jørgensen, B., Qvist, K. B., Skjøt, M., & Sørensen, S. O. (2005). Biophysical consequences of remodeling the neutral side chains of rhamnogalacturonan I in tubers of transgenic potatoes. Planta, 220(4), 609–620. http://dx.doi.org/10.1007/s00425-004-1373-8. Verhertbruggen, Y., Marcus, S. E., Haeger, A., Ordaz-Ortiz, J. J., & Knox, J. P. (2009). An extended set of monoclonal antibodies to pectic homogalacturonan. Carbohydrate Research, 344(14), 1858–1862. http://dx.doi.org/10.1016/ j.carres.2008.11.010. Waldron, K. W., Parker, M. L., & Smith, A. C. (2003). Plant cell walls and food quality. Comprehensive Reviews in Food Science and Food Safety, 2(4), 128–146. Willats, W. G. T., Marcus, S. E., & Knox, J. P. (1998). Generation of a monoclonal antibody specific to (1?5)-a-l-arabinan. Carbohydrate Research, 308(1–2), 149–152. http://dx.doi.org/10.1016/S0008-6215(98)00070-6. Zykwinska, A., Thibault, J.-F., & Ralet, M.-C. (2007). Organization of pectic arabinan and galactan side chains in association with cellulose microfibrils in primary cell walls and related models envisaged. Journal of Experimental Botany, 58(7), 1795–1802. http://dx.doi.org/10.1093/jxb/erm037.