Effect of yeast mannan treatments on ripening progress and modification of cell wall polysaccharides in tomato fruit

Food Chemistry 218 (2017) 509–517

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Effect of yeast mannan treatments on ripening progress and modification of cell wall polysaccharides in tomato fruit Fang Xie, Shuzhi Yuan, Hanxu Pan, Rui Wang, Jiankang Cao ⇑, Weibo Jiang College of Food Science and Nutritional Engineering, China Agricultural University, 17 Qinghuadonglu Road, Beijing 100083, PR China

a r t i c l e

i n f o

Article history: Received 2 June 2016 Received in revised form 12 September 2016 Accepted 14 September 2016 Available online 15 September 2016 Keywords: Tomato Yeast mannan Ethylene Cell wall polysaccharides Neutral sugars

a b s t r a c t Yeast mannan treatments effectively delayed colour change and firmness decline and inhibited ethylene production in two cultivars of tomato fruit during storage. The yeast mannan treatment maintained the integrity of tomato pericarp cell wall architecture and suppressed the modification of water-soluble and insoluble pectic polysaccharides in the cell wall. A decrease in the neutral sugars, including D-galactose, L-arabinose and L-rhamnose, in water-insoluble pectin and an increase in these sugars in water-soluble pectin were inhibited by yeast mannan. The contents of D-xylose and D-mannose in the hemicellulose fraction were significantly higher in the yeast mannan-treated fruit after storage. The activities of several cell wall-modifying enzymes, including pectinmethylesterase, polygalacturonase and b-galactosidase, were suppressed in fruit treated with yeast mannan during storage. Overall, the yeast mannaninduced delay in the ripening progress of tomato fruit might occur via the strong suppression of ethylene synthesis, causing inhibition of solubilization and depolymerization of cell wall polysaccharides. Ó 2016 Elsevier Ltd. All rights reserved.

1. Introduction Tomato (Solanum lycopersicum L.) is cultivated worldwide and is an important fruit used for consumption as well as processing. However, the tomato is a climacteric fruit and is prone to rapid ripening and softening after harvest, which greatly limits storage, transportation, processing and marketing of the fruit. In past decades, numerous technologies have been developed for delaying ripening progress and extending postharvest life of tomato fruit, including refrigeration (Rugkong et al., 2010), controlled or modified atmosphere storage (Fagundes et al., 2015), application of an ethylene remover (Martínez-Romero et al., 2009), treatments with the ethylene inhibitor 1-methylcyclopropene (Wang, Cao, Lin, Sun, & Jiang, 2010), UV-C radiation (Barka, Kalantari, Makhlouf, & Arul, 2000; Bu, Yu, Aisikaer, & Ying, 2013) and exposure to ozone (Rodoni, Casadei, Concellón, Alicia, & Vicente, 2010). Nevertheless, novel strategies with natural sources for delaying fruit ripening progress need to be explored. Fruit ripening and tissue softening involves a wide range of biochemical and physiological changes in the cell wall architecture. The primary cell wall of fruit is a set of highly complex composites of structurally diverse polysaccharides, consisting of cellulose, hemicellulose and pectin (Mohnen, 2008; Ordaz-Ortiz, Marcus, & Knox, 2009). Cellulose molecules aggregate into microfibrils to ⇑ Corresponding author. E-mail address: [email protected] (J. Cao). http://dx.doi.org/10.1016/j.foodchem.2016.09.086 0308-8146/Ó 2016 Elsevier Ltd. All rights reserved.

provide the tough, load-bearing fibres of cell walls. Hemicellulose is a group of polysaccharides with a backbone made up of crosslinked b-D-(1 ? 4)-glycans, comprising xyloglucans, xylans and mannans, which are proposed to tether the microfibrils, providing a load-bearing molecular framework for both primary and secondary cell walls. Pectic substances constitute a highly complex and heterogeneous group of polysaccharides that possess backbones consisting of a linear chain of (1 ? 4)-a-D-GalA (galacturonic acid) in the primary cell wall and middle lamella and that participate in cell-to-cell adhesion. In general, cellulose microfibrils embedded in a gel matrix of pectic and hemicellulosic polysaccharides form the cell wall architecture, contributing to tissue firmness and flexibility. Pectic polysaccharides are abundant in the fruit cell wall. The structural domains and neutral sugar compositions of at least four components of pectic polysaccharides have been previously characterized (Mohnen, 2008; Round, Rigby, MacDougall, & Morris, 2010). The structural complexes include homogalacturonan (HG), xylogalacturonan (XGA), rhamnogalacturonan I (RG I) and rhamnogalacturonan II (RG II). HG, a linear homopolymer of a-1,4-linked D-GalA that comprises
65% of pectin, is often partially methylesterified at the O-6 carboxyl and sometime O-acetylated at O-2 or O-3. The branched XGA is an HG substituted at O-3 with a b-linked D-xylose (Xyl). RG I, represents 20–35% of pectin, contains a backbone of the repeating disaccharide unit of (1 ? 2)-a-L-Rha-(1 ? 4)-a-D-GalA that is predominantly substituted at the O-4 of rhamnose (Rha) residues by neutral sugar side

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chains. The side chains contain abundant individual, linear, or branched (1 ? 5)-a-L-Araf and (1 ? 4)-b-D-Galp residues. RG II, makes up
10% of pectin, has a backbone of (1 ? 4)-a-D-GalA like HG, but with complex side chains of several types of neutral sugars, including Rha, arabinose (Ara) and galactose (Gal). Depolymerization of these structural domains and solubilization of pectic polysaccharides causes the modification of cell wall polysaccharides and ultimately the disassembly of cell wall architecture, as a result of the combined action of several cell wall-modifying enzymes, such as pectinmethylesterase (PME), polygalacturonase (PG) and b-galactosidase (b-Gal) (Bennett & Labavitch, 2008; Gapper, Mcquinn, & Giovannoni, 2013; Mohnen, 2008). Recently, polysaccharides such as chitosan and oligochitosan isolated from shrimp or crab shells (Jongsri, Wangsomboondee, Rojsitthisak, & Seraypheap, 2016; Yan et al., 2011) have been extensively studied and were shown to have the ability to improve fruit quality and extend fruit postharvest life. On the contrary, oligosaccharides prepared from tomato cell walls were found to enhance ethylene synthesis and promote ripening progress of the fruit (Ma, Zhou, Wang, Chen, & Qu, 2016). The effect of polysaccharides on fruit ripening may be influenced by their physiochemical diversities, different sources, heterogeneous group of the compositions, and even the capacity of film-formation on the surface. Therefore, novel polysaccharides, in particular, derivatives or extracts from cell walls of microorganisms, need to be developed and tested for postharvest application. Yeast mannan (YM, the b-1,4-linked backbone containing D-mannose residues, mainly mannoproteins), an important component of polysaccharides of the yeast cell wall, comprises
40% of the yeast cell wall dry mass (Aguilar-Uscanga & François, 2003; Yamabhai, Sakubol, Srila, & Haltrich, 2016). YM is usually prepared from the waste beer yeast slurry as a side-product in brewing factories and is currently used as a stabilizing agent in the wine industry and as an additive in feeding. Additionally, it was well evidenced that YM has strong antioxidant capacities (Korcová, Machová, Filip, & Bystricky´, 2015; Machová & Bystricky´, 2013) and various health-promoting effects for humans (Yamabhai et al., 2016). These properties make YM a promising natural biodegradable additive in food processing or storage of fresh fruits. Despite this, little information concerning the use of YM in postharvest treatment of fruits is available to date. Accordingly, the present work probes the effect of YM on ripening progress of different cultivar tomato fruit after harvest. Microstructural changes in the cell wall architecture, alterations of different cell wall polysaccharide fractions, and the neutral sugar compositions, activities of cell wall-modifying enzymes of tomato fruit as influenced by the YM treatment were investigated. 2. Materials and methods 2.1. Fruit material and yeast mannan treatments Fruit from two tomato (Solanum lycopersicum L.) cultivars (‘Tuofu’ and ‘Hongmofen’) were harvested at the mature-green stage from a commercial garden in Tangshan city, Hebei province, China. The fruit were immediately transported to the laboratory within 5 h after harvest and were selected according to uniformity of size, colour and shape. Tomatoes with physical injuries, visual blemishes or infections were discarded. All fruit were dipped into 0.1% (v/v) sodium hypochlorite solution for 60 s for surface sterilization, then rinsed with tap water and air-dried. Yeast mannan (YM) was obtained from Angel Yeast Co., Ltd., China, and was extracted and prepared from the waste beer yeast slurry. YM solutions at concentrations of 0 (control), 1.0 and 5.0 g L1 were prepared with distilled water containing 0.01% (v/v) Tween-20 as a surfactant.

‘Tuofu’ tomatoes were immersed in the YM solutions and sealed in a stainless steel tank under vacuum condition of 0.01 MPa for 30 s to allow infiltration. Then, air was allowed slowly back into the tank and the fruit were continually immersed for an additional 10 min under air pressure conditions. After removal and air-drying, the fruit were stored at 23 ± 1 °C with 85–90% relative humidity (RH). ‘Hongmofen’ tomatoes were similarly immersed in the YM solutions under the vacuum condition of 0.02 MPa for 2 min and then under air pressure for an additional 10 min as described above. The ‘Hongmofen’ tomato were then stored at 16 ± 1 °C, 85–90% RH. Examinations and sampling were conducted at certain intervals during storage. ‘Tuofu’ tomato pericarp tissues were sampled and immediately dipped in liquid nitrogen and then frozen at 40 °C for further biochemical measurements. 2.2. Colour change index Tomatoes (180 fruit for each treatment representing three replicates) were used for estimating the extent of skin colour change from green to red, by visually measuring the percentage of the colour-changed area to the total surface of each fruit, using a rating scale, where 0 = no change (total green), 1 = less than 10%, 2 = 10–25%, 3 = 25–50%, 4 = 50–75%, 5 = 75–100% (pink), 6 = 100% (total red). The colour change index was calculated using the following formula: Colour change index ¼

P ðcolour scale  the fruit number of corresponding scaleÞ the highest scale  the number of total fruit  100

2.3. Fruit firmness measurement Pericarp firmness was measured on four separated but equidistant peeled sites on the equator of each fruit using a hand penetrometer (GY-1, Zhejiang, China) equipped with a flat tip (3.5 mm diameter). Firmness was expressed as the maximum force (N) attained during the penetration. 2.4. Ethylene production Three tomatoes representing a replicate (three replicates in a treatment) were placed in a 2-L glass container hermetically sealed with a rubber stopper for 60 min. One millilitre of headspace gas sample withdrawn from the container with a syringe was injected into a gas chromatograph (7890F, Tianmei Co., Shanghai, China), equipped with a flame ionization detector (FID) and a stainless steel column (inner diameter 2 mm  length 3 m) packed with activated alumina (80/100 mesh) to measure the concentration of ethylene according to Wang et al. (2010). Ethylene production was calculated according to the calibration curve and expressed as lL kg1 h1 of fruit weight (FW). 2.5. Microstructure observation of pericarp tissue Pieces of tomato pericarp tissue (2 mm  1 mm  1 mm) excised from the equatorial zone of the fruit were first fixed in 2.5% (v/v) glutaraldehyde solution and then successively postfixed, dehydrated and embedded according to the method described by Bu et al. (2013). The prepared pieces of samples were cut with a LEICA UC6 ultramicrotome (Leica Microsystems GmbH, Wetzlar, Germany) to obtain ultra-thin sections with a thickness of 70 nm. The ultra-thin sections were stained by alkaline lead citrate and uranyl acetate for 15 min, then observed using a transmission electron microscope (TEM) (Model JEM-1230, JEOL, Tokyo, Japan).

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2.6. Preparation, fractionation and analysis of cell wall materials 2.6.1. Cell wall materials preparation Fifty grams of tomato pericarp tissue was homogenized in 200 mL of 80% (v/v) ethanol, boiled for 30 min and then vacuum filtered after cooling. The residue was then boiled in fresh 80% ethanol three times to inactivate endogenous enzymes and remove free sugars. The insoluble residue was sequentially stirred in 100 mL of chloroform-methanol solution (1:1, v/v) for 30 min and vacuum filtered. The residue was further washed with 200 mL of acetone and partially dried by applying a mild vacuum. The residue was then dried to constant weight at 40 °C and stored in a desiccator at 23 °C. The resulting residue is referred to as alcohol-insoluble materials (AIM) as previously described by Huber and O’Donoghue (1993). 2.6.2. Fractionations of AIM A sequential extraction procedure for different fractions of the cell wall polysaccharides in AIM was employed according to Houben, Jolie, Fraeye, Loey, and Hendrickx (2011) and Rodoni et al. (2010). Briefly, aliquots of 500 mg of AIM were extracted thrice with deionized water for 4 h for water-soluble pectin (WSP). The residue was then extracted thrice with 50 mmol L1 t rans-1,2-diaminocyclohexane-N,N,N0 ,N0 -tetraacetic acid (CDTA) for 4 h for chelator-soluble pectin (CSP). Subsequently, the residue was extracted thrice with 50 mmol L1 Na2CO3, containing 20 mmol L1 NaBH4, for 24 h for sodium carbonate-soluble pectin (SSP). Finally, the AIM residue was extracted thrice with 1.0 mol L1 potassium hydroxide (KOH), containing 20 mmol L1 NaBH4, for 4 h to extract the hemicellulose fraction (HF). Each fraction was combined and neutralized to pH 6.5 and dialyzed against distilled water (molecular cut-off 3000 Da) thrice for 24 h. All of the polysaccharide fractions were concentrated with a rotary vacuum evaporator and then freeze-dried. 2.6.3. Galacturonic acid (GalA) content The GalA content in the AIM for each polysaccharide fraction was measured using the carbazole colorimetry method (Bitter & Muir, 1962) and a standard curve of GalA. The GalA content was expressed as mg g1 of AIM 2.6.4. Neutral sugar compositions The neutral sugar composition in each polysaccharide fraction was measured by the hydrolysis method with trifluoroacetic acid (TFA) according to Houben et al. (2011). A 10-mg sample was hydrolysed with 4.0 mL of 4 mol L1 TFA in an acid hydrolysis tube for 2 h at 120 °C. After cooling, the TFA was blown dry with N2 and then diluted with 10 mL of ultrapure water. The dilution was filtered through a 0.2 lm microporous membrane, and the filtrate containing the liberated neutral sugars was analysed using a high-performance anion exchange chromatography (HPAEC) system (Dionex ICS 3000, USA) with a pulsed amperometric detector and an ion exchange Carbopac PA-20 analytical column (3 mm  150 mm). The solvents used in the gradient elution system were water, 250 mmol L1 NaOH and 1 mol L1 NaAc. The flow rate was adjusted to 0.5 mL min1, and the column temperature was set to 35 °C. The injection volume was 10 lL. Calibration was performed with standard solutions of L-rhamnose (Rha), L-arabinose (Ara), D-glucose (Glc), D-galactose (Gal), D-mannose (Man) and D-xylose (Xyl). The neutral sugar content was expressed as mg g1 of AIM. 2.7. Activities of cell wall-modifying enzymes Tomato pericarp tissue (10.0 g) was homogenized using a cold pestle and mortar in 50 mL of ice-cold 95% (v/v) ethanol. The

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homogenate was incubated for 10 min at 4 °C and then centrifuged at 12,000g for 20 min at 4 °C. The precipitate was re-suspended with 10 mL of ice-cold 80% (v/v) ethanol, incubated and then centrifuged as described above. The precipitate was further extracted with 5 mL of 50 mmol L1 sodium acetate buffer (pH 5.5, containing 1.8 mol L1 NaCl) and centrifuged as above. The supernatant was collected as the crude enzyme extract. Polygalacturonase (PG) activity was determined according to Barka et al. (2000) and Bu et al. (2013). Enzyme extract (0.5 mL) was mixed with 1 mL of 50 mmol L1 sodium acetate buffer (pH 5.5) and 0.5 mL of 10 g L1 polygalacturonic acid. A blank was prepared by boiling the enzyme extract for 5 min before mixed. The mixture was incubated for 1 h at 37 °C. Next, 1.5 mL of 3,5-dinitrosalicylic acid (DNS) reagent was added, and the reaction was stopped by heating at 100 °C for 5 min. After cooling, the solution was diluted to 25 mL with distilled water, and the absorbance at 540 nm was measured. The PG activity was calculated against a standard calibration of GalA and expressed as lg h1 g1 FW. The activity of b-galactosidase (b-Gal) was measured according to Pressey (1983) and Rodoni et al. (2010) with slight modification. The reaction mixture consisted of 0.5 mL of enzyme extract and 0.5 mL of 50 mmol L1 p-nitrophenyl-b-D-galactopyranoside. After incubation for 30 min at 37 °C, the reaction was stopped by addition of 2.0 mL of 1.0 mol L1 Na2CO3. A blank was prepared by adding Na2CO3 before incubation. Free p-nitrophenol was measured spectrophotometrically at 400 nm, and the b-Gal activity was estimated in relation to a standard curve of p-nitrophenol and expressed as mmol h1 g1 FW. Pectinmethylesterase (PME) activity was measured using a continuous spectrophotometric assay (Hagerman & Austin, 1986; Hyodo et al., 2013). The pericarp tissue was homogenized in icecold 1.5 mol L1 NaCl solution and centrifuged at 12,000g for 30 min at 4 °C. The supernatant was collected and adjusted to pH 7.5 with NaOH. Then, 2.0 mL of 5g L1 pectin (Sigma, from citrus fruit) and 0.15 mL of 0.1 g L1 bromothymol blue, which was prepared in 3 mmol L1 potassium phosphate buffer (pH 7.5), and 0.85 mL of distilled water were mixed. The reaction was started by adding 100 lL of enzyme extract. The rate of decrease in the absorbance at 620 nm was recorded. The result was expressed as U g1 FW. A unit (U) of PME activity was defined as a 0.1 decrease in absorbance at 620 nm per min. 2.8. Statistical analysis All treatments and analyses, unless otherwise stated, were performed in triplicate, and the mean and standard error of experimental values were calculated. Differences among means of values were analysed by one-way analysis of variance (ANOVA) using SPSS 19.0 for Windows (SPSS Inc., Chicago, IL, USA). Mean separations were performed using Duncan’s multiple range test. Differences were considered statistically significant at P < 0.05. 3. Results 3.1. The effect of YM treatments on colour change of tomato fruit The skin colour of the tomato fruit of both cultivars changed gradually from green to pink and then to completely red during storage (Fig. 1A and B). However, the colour change was significantly slowed by YM treatments. The skin colour of ‘Tuofu’ tomato treated with 1.0 and 5.0 g L1 YM changed to light pink from green after 8 and 6 d of storage at 23 °C, respectively, compared to the control, which changed to pink after 4 d of storage (Fig. 1A). The skin colour of ‘Hongmofen’ tomato treated with 1.0 and 5.0 g L1 YM changed to light pink from green after 15 and 12 d of storage

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Fig. 1. Effect of YM treatments on ripening progress and colour change index of ‘Tuofu’ (A, C) and ‘Hongmofen’ (B, D) tomato fruit during storage. Green-mature ‘Tuofu’ and ‘Hongmofen’ tomato fruit were treated with 0 (control), 1.0 and 5.0 g L1 YM and stored at 23 ± 1 °C and 16 ± 1 °C, respectively. Each column is the mean for three replicates (60 fruit in each replicate), and those marked with different lower-case letters within the same time were significantly different (P < 0.05) according to Duncan’s multiple range test. The vertical bar indicates the standard error. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

at 16 °C, respectively, compared to the control, which changed to pink after 9 d of storage (Fig. 1B). Increases in the colour change index of both cultivar tomatoes were significantly (P < 0.05) inhibited by the YM treatments during storage, especially at the concentration of 1.0 g L1 (Fig. 1C and D). 3.2. The effect of YM treatments on firmness and ethylene production Both ‘Tuofu’ and ‘Hongmofen’ tomato fruit gradually softened during storage. However, the decrease in firmness was significantly inhibited by the treatment with 1.0 g L1 YM, though 5.0 g L1 YM resulted in relatively less inhibition on the decline in firmness of ‘Hongmofen’ (Fig. 2A and B). In particular, the firmness of ‘Tuofu’ tomato fruit treated with 1.0 g L1 YM was 18.9% and 35.1% higher (P < 0.05) than that of the control after 4 and 10 d of storage (Fig. 2A), respectively. The ethylene production of ‘Tuofu’ tomato fruit increased gradually, reaching the maximum after 8 d of storage, and then remained relatively high and stable (Fig. 2C). However, the increase in ethylene production was greatly inhibited by both YM treatments. The ethylene production of ‘Tuofu’ tomato fruit treated with 1.0 and 5.0 g L1 YM was detected after 4 d of storage and was 70.8% and 49.4% lower (P < 0.05) than that of the control after 8 d of storage, respectively. Unlike ‘Tuofu’, the ethylene production in ‘Hongmofen’ tomato fruit started to increase dramatically after 3 d of storage and reached the maximum after 6 d of storage. However, the increase in ethylene production was inhibited by the YM treatments (Fig. 2D). In particular, the ethylene production of ‘Hongmofen’ tomato fruit treated with 1.0 g L1 YM was 28.8% and 42.4% lower (P < 0.05) than that of the control after 6 and 15 d of storage, respectively.

3.3. The effect of YM treatment on microstructural change in tomato pericarp tissue Changes in the microstructure of the cell wall region of ‘Tuofu’ tomato pericarp tissue were observed using TEM (Fig. 3A–E). The tomato cell wall at harvest (0 d) maintained its integrity, and plasma membrane remained close to the cell wall with a clear intercellular layer between cell walls (Fig. 3A). Then, the cell wall began to swell, and the plasma membrane separated from the cell wall after 6 d of storage. In addition, the intercellular layer disappeared (Fig. 3B). After 12 d of storage, the cell wall became more swollen and loosened with scattered arrangements of filaments (Fig. 3C). However, the cell wall and middle lamella were still arranged in a dense formation in the YM-treated fruit after 6 d of storage (Fig. 3D). The demarcation line between the plasma membrane and cell wall was still obviously visible in the YM-treated fruit even after 12 d of storage. Although the middle lamella partially dissolved, the cell wall was still relatively intact (Fig. 3E). 3.4. The effect of YM treatment on cell wall polysaccharide fractions The GalA content is usually used as a measure of pectin levels in different cell wall polysaccharide fractions. The GalA content in WSP increased gradually in both the control and the YM-treated tomato after a transient decrease. The GalA content in the YM-treated fruit was significantly (P < 0.05) lower than that of the control after 8 d of storage (Fig. 4A). CSP was the most abundant pectic polysaccharide fraction in AIM of tomato pericarp cell wall, and the GalA content in CSP also increased gradually, but the increase was greatly enhanced by the YM treatment during storage (Fig. 4B). The GalA content in SSP in the control fruit decreased to a relatively stable level similar to that in the

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Fig. 2. Effect of YM treatments on firmness and ethylene production of ‘Tuofu’ (A, C) and ‘Hongmofen’ (B, D) tomato fruit during storage. Green-mature ‘Tuofu’ and ‘Hongmofen’ tomato fruit were treated with 0 (control), 1.0 and 5.0 g L1 YM and stored at 23 ± 1 °C and 16 ± 1 °C, respectively. Each column or value is the mean for three replicates (10 fruit for firmness and three fruit for ethylene in each replicate), and those marked with different lower-case letters within the same time were significantly different (P < 0.05) according to Duncan’s multiple range test. The vertical bar indicates the standard error.

YM-treated tomato after a transient increase (Fig. 4C). CSP and SSP are usually designated water-insoluble pectin (WIP). The GalA content in WIP in the control tomato increased after 4 d of storage, but it was significantly enhanced and reached a much higher level in the YM-treated fruit (Fig. 4D). The GalA content in WIP in the YM-treated tomato was 20.7% higher (P < 0.05) than that in the control fruit after 4 d of storage.

In the HF, Xyl was the absolute predominant neutral sugar but its content decreased during storage. However, the Xyl content in the YM-treated fruit was much higher than that in the control fruit after 10 d of storage. Meanwhile, the Man content in the YM-treated tomato increased up to 2-fold relative to that in the control (Fig. 5D). 3.6. The effect of YM on activities of cell wall-modifying enzymes

3.5. The effect of YM on neutral sugar compositions in cell wall polysaccharide fractions Different sugar profiles were observed in the cell wall polysaccharide fractions of the tomato pericarp tissue after 10 d of storage. Gal was the predominant neutral sugar in the WSP fraction, followed by Ara, Rha, Glc and, to a lesser extent, Man (Fig. 5A). The contents of these neutral sugars remarkably increased in WSP in the control fruit, but the increase was significantly inhibited in the YM-treated tomato. In the CSP fraction, Gal, Ara, Rha, Glc and, to a lesser extent, Xyl were also the main neutral sugars (Fig. 5B). The contents of these neutral sugars increased in the control fruit and the increase was greatly enhanced in the tomato treated with YM. In the SSP fraction, considerable abundant Gal, Rha and Ara were detected in the tomato at harvest and their levels were much higher than those in WSP and CSP at any storage stage (Fig. 5C). The contents of Gal, Ara, Rha, Glc and Xyl sharply decreased in SSP in the control fruit, but the decrease in contents of these neutral sugars was significantly inhibited in the YM-treated tomato, except Rha.

The PME activity in the control tomato increased and reached a high level after 4 d of storage, but the PME activity in the YM-treated tomato was significantly lower than that in the control during the whole storage (Fig. 6A). The PG activity in the control tomato increased slowly and gradually before 6 d of storage but then increased rapidly and reached a peak value at 10 d of storage (Fig. 6B). The PG activity in the YM-treated fruit changed in a similar trend with the control but was significantly (P < 0.05) inhibited after 6 d of storage. The b-Gal activity in the control fruit increased gradually during storage, whereas the increase in b-Gal activity was effectively inhibited by the YM treatment after 4 d of storage (Fig. 6C). 4. Discussion Ethylene has been shown to be essential for the initiation and completion of ripening and softening of tomatoes (Gapper et al., 2013; Martínez-Romero et al., 2009) and other climacteric fruits (Bennett & Labavitch, 2008). Thus, suppression of ethylene

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Fig. 3. Effect of the YM treatment on microstructural changes of tomato pericarp cell walls. Green-mature ‘Tuofu’ tomato fruit were treated with 0 (control) and 1.0 g L1 YM and stored at 23 ± 1 °C. A: tomato fruit at harvest (0 d). B and C: the control fruit at 6 and 12 d of the storage. D and E: the YM-treated fruit at 6 and 12 d of the storage. The observation was carried out using transmission electron microscopy, and micrographs were taken at 6,000 magnification (bar = 5 lm). CW: cell wall; ML: middle lamella; PM: plasma membrane.

synthesis might delay the fruit ripening progress. In this study, strong suppression of ethylene production was found in both cultivar tomatoes (‘Tuofu’ and ‘Hongmofen’) with YM treatments. The YM treatments also effectively inhibited colour change and firmness decline, and therefore delayed the ripening and softening progress of tomato fruit during storage. However, no report on the postharvest application of YM is available yet. Fruit ripening is typically accompanied by modification of cell wall polysaccharides, notably pectin and hemicellulose, which are strongly promoted by ethylene (Bennett & Labavitch, 2008; Gapper et al., 2013). Pectin can be fractionated to WSP and water-insoluble pectin (WIP): CSP and SSP. The WSP is thought to represent cell wall pecticpolymers solubilized in vivo while remaining in the apoplast. CSP is generally considered to represent ionically bound pectin in the cell wall by calcium bridges, whereas SSP is considered to be the pectin highly esterified to other cell wall polysaccharides by covalent bonds (Houben et al., 2011; Redgwell, Fischer, Kendal, & Macrae, 1997). In this study, the levels of WSP and CSP greatly increased in tomato fruit during storage while SSP levels decreased, indicating the gradual solubilization of WIP into WSP. Such changes could lead to the loss of cohesion of pectin gel matrix, cell wall dissolution and cell separation, as supported by the previous reports (Ali, Chin, & Lazan, 2004; Rugkong et al., 2010; Van der linden, Sila, Duvetter,

Baerdemaeker, & Hendrickx, 2008; Xin et al., 2010). However, the solubilization of WIP was effectively inhibited by the YM treatment, contributing to strengthening the cell wall structure and maintaining fruit firmness during storage. The two pectin backbone polymers, RG I and RG II, contain abundant Rha, Ara and Gal in side chains (Mohnen, 2008; Round et al., 2010). Rha, Ara and Gal were found at the highest levels in SSP in comparison to other pectin fractions of tomato fruit at harvest. However, these neutral sugars decreased greatly in SSP but increased significantly in WSP and CSP during storage, indicating that RG I and RG II were depolymerized from the pectin backbone in insoluble SSP and decomposed into soluble WSP, in concordance with previous reports (Inari, Yamauchi, Kato, & Takeuchi, 2002; Redgwell et al., 1997; Round et al., 2010; Xin et al., 2010). Nevertheless, the YM treatment elevated the levels of these neutral sugars in SSP and CSP and decreased their levels in WSP of the fruit after storage, compared to the control. These results suggest that the depolymerization of RG I and RG II in insoluble pectin was suppressed by the YM treatment, which is beneficial for maintaining cell wall integrity and slowing textural changes leading to fruit softening. Hemicellulose, which is rich in Xyl and Man, was also reported to be modified during fruit ripening (Houben et al., 2011; OrdazOrtiz et al., 2009). In this study, HF were also depolymerized, as

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Fig. 4. Effect of the YM treatment on GalA content in WSP (A), CSP (B), SSP (C) and water-insoluble pectin (D) of tomato fruit during storage. Green-mature ‘Tuofu’ tomato fruit were treated with 0 (control) and 1.0 g L1 YM and stored at 23 ± 1 °C. Each value is the mean for three replicates (10 fruit in each replicate), and those marked with different lower-case letters within the same time were significantly different (P < 0.05) according to the T-test. The vertical bar indicates the standard error.

Fig. 5. Effect of the YM treatment on neutral sugar contents in WSP (A), CSP (B), SSP (C) and HF (D) of tomato fruit during storage. Green-mature ‘Tuofu’ tomato fruit were harvested (0 d) and treated with 0 (control) and 1.0 g L1 YM and then stored at 23 ± 1 °C for 10 d. Each column is the mean for three replicates (10 fruit in each replicate), and those marked with different lower-case letters within the same component were significantly different (P < 0.05) according to Duncan’s multiple range test. The vertical bar indicates the standard error.

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of polygalacturonic acid (Barka et al., 2000). It has been demonstrated that activities of PME and PG increased during tomato ripening (Ali et al., 2004; Hyodo et al., 2013; Inari et al., 2002; Rugkong et al., 2010; Van der linden et al., 2008). In this study, similarly, an early increase of PME activity and a delayed but pronounced increase in PG activity were observed in tomato fruit during storage, which were all suppressed by the YM treatment, concomitant with the inhibitory effect of YM on the modification of cell wall polysaccharides. b-Gal might act as a pectin-debranching enzyme and was reported to have the ability to simultaneously modify pectin and hemicellulose. Galactose loss from pectin side chains by b-Gal has been proposed as one reason for the increase of soluble pectin (Redgwell et al., 1997). Rugkong et al. (2010) found that b-Gal in tomato slightly increased during storage, but Ali et al. (2004) reported that no significant change of b-Gal activity in unripe and ripe tomatoes. Pressey (1983) found three classes of b-Gal in tomato had various activity changes during ripening. In the present study, the b-Gal activity increased gradually in tomato but the increase was greatly slowed by the YM treatment, which was consistent with the changes in neutral sugar compositions in different polysaccharide fractions as affected by YM. As a result, the microstructure of fruit tissue might become loose and even destroyed due to the disassembly of the cellulose-hemicellulose network of cell wall architecture resulted from the degradation of pectic and hemicellulosic polysaccharides matrix (Bu et al., 2013; Mohnen, 2008). In this study, the YM treatment effectively delayed the disassembly of cell wall architecture and therefore maintained the integrity of microstructure of tomato pericarp tissue during storage. Mannans, belonging to the hemicellulosic heteroglycans, have been observed in the cell wall of tomato and may be involved in ripening of the fruit (Ordaz-Ortiz et al., 2009) or not (Prakash et al., 2012). Although whether YM treatment directly alters the metabolism of mannans in tomato cell walls is still unknown, YM treatment on delaying the tomato ripening progress may be closely related to its strong inhibitory effect on ethylene synthesis of the fruit. 5. Conclusion

Fig. 6. Effect of the YM treatment on activities of PME (A), PG (B) and b-Gal (C) of tomato fruit during storage. Green-mature ‘Tuofu’ tomato fruit were treated with 0 (control) and 1.0 g L1 YM and stored at 23 ± 1 °C. Each value is the mean for three replicates (10 fruit in each replicate), and those marked with different lower-case letters within the same component were significantly different (P < 0.05) according to the T-test. The vertical bar indicates the standard error.

the levels of Xyl and Man decreased in HF during tomato ripening, which agreed with the previous findings in cherry tomatoes (Inari et al., 2002). However, the depolymerization of HF was suppressed by the YM treatment. Solubilization and depolymerization of cell wall polysaccharides are a consequence of the coordinated action of several cell wall-modifying enzymes, such as PME, PG and b-Gal, which are strongly stimulated by ethylene during ripening (Bennett & Labavitch, 2008; Gapper et al., 2013). PME catalyses the demethylesterification of cell wall polygalacturonan to generate demethylated pectin that can be more easily hydrolysed by PG (Bennett & Labavitch, 2008; Hyodo et al., 2013). PG catalyses the hydrolytic cleavage of galacturonide linkages of the main chain

Collectively, the YM treatment strongly suppressed ethylene synthesis of tomato fruit during storage, causing the inhibition of activities of several cell wall-modifying enzymes. As a consequence, the solubilization and depolymerization of cell wall polysaccharides were inhibited, and therefore the integrity of cell wall architecture was maintained by the YM treatment. Ultimately, the YM treatment effectively suppressed the progress of ripening and softening of tomato fruit during storage. These results indicated that the application of YM could provide a novel and promising approach for extending fruit postharvest life. Acknowledgements This work was supported by the National Basic Research Program of China (‘973’ program, No. 2013CB127104) and the Special Fund for Agro-scientific Research in the Public Interest of China (No. 201303075). References Aguilar-Uscanga, B., & François, J. M. (2003). A study of the yeast cell wall composition and structure in response to growth conditions and mode of cultivation. Letters in Applied Microbiology, 37(3), 268–274. Ali, Z. M., Chin, L. H., & Lazan, H. (2004). Comparative study on wall degrading enzymes, pectin modifications and softening during ripening of selected tropical fruits. Plant Science, 167(2), 317–327.

F. Xie et al. / Food Chemistry 218 (2017) 509–517 Barka, E. A., Kalantari, S., Makhlouf, J., & Arul, J. (2000). Impact of UV-C irradiation on the cell wall-degrading enzymes during ripening of tomato (Lycopersicon esculentum L.) fruit. Journal of Agricultural & Food Chemistry, 48(3), 667–671. Bennett, A. B., & Labavitch, J. M. (2008). Ethylene and ripening-regulated expression and function of fruit cell wall modifying proteins. Plant Science, 175(1), 130–136. Bitter, T., & Muir, H. M. (1962). A modified uronic acid carbazole reaction. Analytical Biochemistry, 4(4), 330–334. Bu, J., Yu, Y., Aisikaer, G., & Ying, T. (2013). Postharvest UV-C irradiation inhibits the production of ethylene and the activity of cell wall-degrading enzymes during softening of tomato (Lycopersicon esculentum L.) fruit. Postharvest Biology & Technology, 86(3), 337–345. Fagundes, C., Moraes, K., Pérez-Gago, M. B., Palou, L., Maraschin, M., & Monteiro, A. R. (2015). Effect of active modified atmosphere and cold storage on the postharvest quality of cherry tomatoes. Postharvest Biology & Technology, 109, 73–81. Gapper, N. E., Mcquinn, R. P., & Giovannoni, J. J. (2013). Molecular and genetic regulation of fruit ripening. Plant Molecular Biology, 82(6), 575–591. Hagerman, A. E., & Austin, P. J. (1986). Continuous spectrophotometric assay for plant pectin methyl esterase. Journal of Agricultural & Food Chemistry, 34(3), 440–444. Houben, K., Jolie, R. P., Fraeye, I., Loey, A. M. V., & Hendrickx, M. E. (2011). Comparative study of the cell wall composition of broccoli, carrot, and tomato: Structural characterization of the extractable pectins and hemicelluloses. Carbohydrate Research, 346(9), 1105–1111. Huber, D. J., & O’Donoghue, E. M. (1993). Polyuronides in avocado (Persea americana) and tomato (Lycopersicon esculentum) fruits exhibit markedly different patterns of molecular weight downshifts during ripening. Plant Physiology, 102(2), 473–480. Hyodo, H., Terao, A., Furukawa, J., Sakamoto, N., Yurimoto, H., Satoh, S., & Iwai, H. (2013). Tissue specific localization of pectin-Ca2+ cross-linkages and pectin methyl-esterification during fruit ripening in tomato (Solanum lycopersicum). PLoS ONE, 8(11). e78949. Inari, T., Yamauchi, R., Kato, K., & Takeuchi, T. (2002). Changes in pectic polysaccharides during the ripening of cherry tomato fruits. Food Science & Technology Research, 8(1), 55–58. Jongsri, P., Wangsomboondee, T., Rojsitthisak, P., & Seraypheap, K. (2016). Effect of molecular weights of chitosan coating on postharvest quality and physicochemical characteristics of mango fruit. LWT – Food Science and Technology, 73, 28–36. Korcová, J., Machová, E., Filip, J., & Bystricky´, S. (2015). Biophysical properties of carboxymethyl derivatives of mannan and dextran. Carbohydrate Polymers, 134, 6–11. Ma, Y., Zhou, L., Wang, Z., Chen, J., & Qu, G. (2016). Oligogalacturonic acids promote tomato fruit ripening through the regulation of 1-aminocyclopropane-1carboxylic acid synthesis at the transcriptional and post-translational levels. BMC Plant Biology, 16(1), 1–11.

517

Machová, E., & Bystricky´, S. (2013). Antioxidant capacities of mannans and glucans are related to their susceptibility of free radical degradation. International Journal of Biological Macromolecules, 61(10), 308–311. Martínez-Romero, D., Guillén, F., Castillo, S., Zapata, P. J., Serrano, M., & Valero, D. (2009). Development of a carbon-heat hybrid ethylene scrubber for fresh horticultural produce storage purposes. Postharvest Biology & Technology, 51(2), 200–205. Mohnen, D. (2008). Pectin structure and biosynthesis. Current Opinion in Plant Biology, 11(3), 266–277. Ordaz-Ortiz, J. J., Marcus, S. E., & Knox, J. P. (2009). Cell wall microstructure analysis implicates hemicellulose polysaccharides in cell adhesion in tomato fruit pericarp parenchyma. Molecular Plant, 2(5), 910–921. Prakash, R., Johnston, S. L., Boldingh, H. L., Redgwell, R. J., Atkinson, R. G., Melton, L. D., ... Schröder, R. (2012). Mannans in tomato fruit are not depolymerized during ripening despite the presence of endo-b-mannanase. Journal of Plant Physiology, 169(12), 1125–1133. Pressey, R. (1983). B-Galactosidases in ripening tomatoes. Plant Physiology, 71(1), 132–135. Redgwell, R. J., Fischer, M., Kendal, E., & Macrae, E. A. (1997). Galactose loss and fruit ripening: high-molecular-weight arabinogalactans in the pectic polysaccharides of fruit cell walls. Planta, 203(2), 174–181. Rodoni, L., Casadei, N., Concellón, A., Alicia, A. R. C., & Vicente, A. R. (2010). Effect of short-term ozone treatments on tomato (Solanum lycopersicum L.) fruit quality and cell wall degradation. Journal of Agricultural & Food Chemistry, 58(1), 594–599. Round, A. N., Rigby, N. M., MacDougall, A. J., & Morris, V. J. (2010). A new view of pectin structure revealed by acid hydrolysis and atomic force microscopy. Carbohydrate Research, 345(4), 487–497. Rugkong, A., Rose, J. K. C., Sang, J. L., Giovannoni, J. J., O’Neill, M. A., & Watkins, C. B. (2010). Cell wall metabolism in cold-stored tomato fruit. Postharvest Biology & Technology, 57(2), 106–113. Van der linden, V., Sila, D. N., Duvetter, T., Baerdemaeker, J. D., & Hendrickx, M. (2008). Effect of mechanical impact-bruising on polygalacturonase and pectinmethylesterase activity and pectic cell wall components in tomato fruit. Postharvest Biology & Technology, 47(1), 98–106. Wang, M., Cao, J., Lin, L., Sun, J., & Jiang, W. (2010). Effect of 1-methylcyclopropene on nutritional quality and antioxidant activity of tomato fruit (Solanum lycopersicon L.) during storage. Journal of Food Quality, 33(2), 150–164. Xin, Y., Chen, F., Yang, H., Zhang, P., Deng, Y., & Yang, B. (2010). Morphology, profile and role of chelate-soluble pectin on tomato properties during ripening. Food Chemistry, 121(2), 372–380. Yamabhai, M., Sakubol, S., Srila, W., & Haltrich, D. (2016). Mannan biotechnology: from biofuels to health. Critical Reviews in Biotechnology, 36(1), 32–42. Yan, J., Li, J., Zhao, H., Chen, N., Cao, J., & Jiang, W. (2011). Effects of oligochitosan on postharvest Alternaria rot, storage quality, and defense responses in Chinese jujube (Zizyphus jujuba Mill. cv. Dongzao) fruit. Journal of Food Protection, 74(5), 783–788.