Surfactant-Induced Competitive Displacement of Potato Pectin

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Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Surfactant-Induced Competitive Displacement of Potato Pectin− Protein Conjugate from the Air−Water Interface Daiki Murayama,† Daisuke Ando,‡ and Shinya Ikeda*,†,# †

Department of Food Science, University of Wisconsin−Madison, Madison, Wisconsin 53706, United States Department of Biochemistry, University of Wisconsin−Madison, Madison, Wisconsin 53706, United States

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‡

ABSTRACT: Potato pectin contains some proteinaceous components and exhibits emulsifying and emulsion stabilizing abilities. The objective of this study was to elucidate the effect of the pectin moiety of the pectin−protein conjugate present in pectic extracts from potato tubers on their interfacial properties. Potato pectin was extracted from highly purified cell wall materials to avoid the contamination of unconjugated proteins. The abilities of the potato pectin to adsorb to graphite surfaces and to resist surfactant-induced competitive displacement from the air−water interface were investigated using atomic force microscopy. The pectin moiety of the potato pectin was capable of adsorbing to graphite surfaces even after alkali treatment. Furthermore, the potato pectin exhibited enhanced resistance to surfactant-induced competitive displacement from the interface as a result of the formation of network structures of self-assembled pectin moieties at the interface. The present results suggest the importance of the pectin moiety with regard to the interfacial properties of potato pectin. KEYWORDS: Solanum tuberosum L., pectic polysaccharides, atomic force microscopy, competitive displacement, surface pressure

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INTRODUCTION Potato pectin has unique molecular characteristics that differentiate it from the readily available pectins that are derived commonly from citrus peels or apple pomace and are used widely as gelling and stabilizing agents in the food industry.1 Pectin is a complex heterogeneous polysaccharide in the plant cell wall and is composed mainly of homogalacturonan (HG) and rhamnogalacturonan I (RG-I) that typically account for approximately 65 and 20−35% (w/w) of the entire molecule, respectively.2 HG is an unbranched chain of 1,4linked galacturonic acid (GalA) residues that may be methyl esterified at the C-6 position and acetylated at the O-2 or O-3 position.3,4 RG-I consists of a backbone of alternating rhamnose and GalA residues and side chains of neutral sugar residues such as galactan, arabinan, and arabinogalactan branching from the rhamnose residues. 3,4 The main component of potato pectin is not HG but RG-I, which accounts for 75% (w/w) of the entire molecule.1 Furthermore, the side chains consist predominantly of long chains of β-1,4galactan with an average degree of polymerization of 50.5,6 As a result, the galactan side chains account for ca. 67% (w/w) of the entire RG-I of potato pectin.7 Potato pectin also has a relatively low degree of esterification of ca. 17−30% and a much higher degree of acetylation of ca. 9−15% than commercially available citrus or apple pectin.1,8 These structural characteristics may confer distinctive functional properties of potato pectin as a food ingredient. Certain pectic extracts, such as those extracted from sugar beet9−11 and soybean,12,13 have been shown to contain proteinaceous components that are covalently linked to polysaccharide chains. The presence of a surface active protein moiety enables such a pectin−protein conjugate to adsorb to air−water and oil−water interfaces and function as a foaming and emulsifying agent. Additionally, a bulky pectin moiety is expected to extend from the interface into the aqueous phase © XXXX American Chemical Society

and provide additional colloidal stability arising from steric hindrance against the coalescence of gas bubbles and oil droplets.10,14 However, a small-molecule surfactant added afterward is capable of adsorbing to void spaces in an existing interfacial film of the preadsorbed protein, increasing the surface pressure of interfacial domains occupied by the surfactant and competitively displacing the proteins from the interface.15,16 Sugar beet pectin was reported to exhibit enhanced resistance to surfactant-induced competitive displacement from the air−water interface because its pectin moiety formed cross-linked two-dimensional networks at the interface and strengthened the interfacial film mechanically.11 Alkali treatment of the pectin removed the pectin networks from the interface so that the interfacial film behaved in a manner similar to that of pure protein.11 Pectic extracts from soybean were also suggested to exhibit enhanced resistance to surfactant-induced competitive displacement from oil droplet surfaces.12 The diameter of oil-in-water emulsion droplets stabilized using the soybean extract did not change for at least 1 h after the addition of a small-molecule surfactant to the aqueous phase, while in the presence of β-galactosidase, the diameter decreased first, as a result of hydrolysis of the pectin moieties, and then increased as a result of the coalescence of oil droplets, which was considered to be induced by competitive displacement of the protein moieties from the droplet surfaces.12 Potato pectin was recently reported to contain proteinaceous components in either free or conjugated forms and has a greater ability to stabilize oil-in-water emulsions than commercial citrus and apple pectins.8 Five different potato Received: Revised: Accepted: Published: A

March 20, 2019 June 6, 2019 July 9, 2019 July 9, 2019 DOI: 10.1021/acs.jafc.9b01773 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry pectin samples were prepared using five different acids for extraction, while there were no obvious correlations between the emulsion stabilities and the protein contents of these pectin samples.8 It is therefore critical to understand how different forms of proteinaceous components in potato pectin affect its functionality. The objective of this study was to elucidate the effect of the pectin moiety of the pectin−protein conjugate present in pectic extracts from potato tubers on its interfacial properties. The cell wall material (CWM) isolated from potato tubers was treated with phenol to ensure that unconjugated protein components were removed prior to the extraction of pectin. Part of the extracted potato pectin was subjected to alkali treatment to cleave the covalent linkage between the pectin and protein moieties and comparatively investigate the abilities of the alkali-treated and untreated potato pectins to adsorb to hydrophobic graphite surfaces and to resist surfactant-induced competitive displacement from the air−water interface.

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Compositional Analysis. Colorimetric Analysis. Total sugar contents were determined using the phenol-sulfuric acid method.17 Total uronic acid contents were measured using an m-hydroxydiphenyl method18 and reported as GalA contents because only trace amounts of glucuronic acid are found in potato cell walls.19 Total protein contents were measured using a bicinchoninic acid assay kit (Thermo Scientific, Rockford, IL, U.S.A.). All measurements were triplicated. All reported contents are expressed on a dry weight basis. Nuclear Magnetic Resonance (NMR) Spectroscopy. Pectin samples (5%, w/w) were dissolved in D2O with continuous stirring at 60 °C for at least 30 min. Chemical shifts (δ) were expressed in ppm relative to acetone-d6 (δC/δH 30.89/2.22 ppm) as an internal reference.20 NMR spectra were acquired on an AVANCE III 700 MHz spectrometer (Bruker BioSpin, Bruker, Billerica, MA, U.S.A.) equipped with a cryogenically cooled 5 mm QCI 1H/31P/13C/15N gradient probe with inverse geometry. NMR experiments were performed at 25 °C. 1H and 13C NMR spectra, 1H−13C heteronuclear single-quantum coherence (HSQC), and 1H−13C heteronuclear multiple-bond correlation (HMBC) spectra were recorded using the standard Bruker procedures. Bruker’s Topspin software (ver. 3.1) was used to process spectra. Surface Pressure (Π)−Area (A) Isotherm. The stock solutions of the potato pectin and the alkali-treated potato pectin were spread to give 3 or 0.5 mg of the proteinaceous components in the potato pectin or the alkali-treated potato pectin, respectively, on a clean water surface of area 230 cm2 formed on a polytetrafluoroethylene (PTFE) trough (KSV Instruments, Helsinki, Finland). Interfacial films were incubated at room temperature for at least 12 h prior to compression at a constant rate of 2 mm/min to 30 cm2 while surface pressure was being monitored by using a balance equipped with a Wilhelmy plate (KSV Instruments, Helsinki, Finland), followed by expansion at the same rate to 230 cm2. This rate of change in area was adopted to avoid kinetic effects on the determination of isotherms.21 Surfactant-Induced Competitive Displacement. Interfacial films were prepared by spreading 3 or 0.5 mg of the proteinaceous components in the potato pectin or the alkali-treated potato pectin, respectively, onto the air−water interface formed on the PTFE trough and incubating at room temperature for at least 12 h until a constant surface pressure was observed. The interfacial film was then compressed at a constant speed of 2 mm/min until the surface pressure reached 10 mN/m. In some selected experiments, 1 mL of 5 M CaCl2 was injected into the subphase under the film. To induce the competitive displacement, 500 μL of 1 mM Tween 20 was injected into the subphase at 2 h intervals. Interfacial films were transferred onto freshly cleaved mica surfaces at the end of each of the 2 h intervals using the Langmuir−Blodgett method.22 A freshly cleaved piece of mica sheets was lowered down through an interface and then pulled back out at a constant rate of 0.14 mm/s. Transferred films were allowed to air-dry and were imaged in n-butanol using atomic force microscopy (AFM). AFM. Stock solutions of the potato pectin and the alkali-treated potato pectin were diluted to 1 μg/mL using Milli-Q water. An aliquot (10 μL) of a diluted pectin solution was deposited onto a freshly cleaved highly oriented pyrolytic graphite surface (Bruker, Santa Barbara, CA, U.S.A.) and incubated at room temperature for 10 min. The graphite surface was then rinsed with 10 mL each of Milli-Q water, ethanol, and n-butanol, and imaged in n-butanol using AFM. Potato pectins deposited onto graphite surfaces and their interfacial films transferred onto mica surfaces were imaged using a microscope (BioScope Catalyst, Bruker, Santa Barbara, CA, U.S.A.) operated in peak force tapping mode and a silicon nitride cantilever (ScanAsystFluid, Bruker Nano Surfaces, Camarillo, CA, U.S.A.) having a nominal spring constant of 0.7 N/m and a resonant frequency of approximately 150 kHz. The obtained topographical images were processed and analyzed using NanoScope Analysis software ver. 1.40 (Bruker, Santa Barbara, CA, U.S.A.). To evaluate the interfacial area coverage by pectin, three images were analyzed at a given surfactant concentration. Five cross-sections in each of these images were arbitrarily selected and analyzed to evaluate the thickness of an interfacial film.

MATERIALS AND METHODS

Preparation of Potato Pectin. Isolation of CWM. Potato (S. tuberosum L. cv. Russet Burbank) tubers cultivated at the Hancock Agricultural Research Station (Hancock, WI, U.S.A.) under standard conditions were washed and peeled using a commercial peeler. Piths of the parenchyma region of tubers were cut from the peeled potatoes and then diced into 1 cm cubes. The cubes (250 g) were homogenized in 500 mL of chilled sodium acetate buffer (0.2 M, pH 5.5) using a Waring blender for 30 s. After the homogenate was filtered through a 100-μm nylon mesh, the residue was rinsed with 1 L of deionized water, homogenized in 250 mL of the chilled buffer using the Waring blender for 30 s, and filtered through a nylon mesh. The residue was rinsed with 1 L of deionized water and homogenized in 250 mL of the chilled buffer at 10000 rpm for 5 min using a disperser (T25 digital ULTRA-TURRAX, IKA, Staufen, Germany). The slurry was filtered through a nylon mesh and rinsed with 1 L of deionized water. After excess water was removed, the residue was suspended in 200 mL of Tris buffer-saturated phenol (0.5 M Tris−HCl, pH 8), stirred at 500 rpm for 30 min at room temperature, filtered through a doubled muslin cloth, and rinsed with 2 L of deionized water. This step was repeated once again. The recovered CWM was then dispersed in the same weight of Milli-Q water, dialyzed using a 3.5 kDa cutoff tubing (Pierce Biotechnology, Rockford, IL, U.S.A.), and lyophilized. Extraction of Potato Pectin. Potato pectin was extracted from the CWM according to the method of Yang et al.8 with slight modifications. The CWM (2 g) was dispersed in 150 mL of MilliQ water and adjusted to pH 2.00 using 6 M HCl. The slurry was heated at 90 °C for 60 min with continuous stirring using a magnetic stirrer. After being cooled to room temperature, the slurry was centrifuged at 15000 g for 30 min. The supernatant was collected, mixed with three volumes of absolute ethanol, and incubated at 4 °C overnight. Precipitated pectin was collected by centrifugation at 15000 g for 15 min. Obtained pellets were rinsed with 200 mL of 70, 80 and 90% (v/v) ethanol twice in this sequence and dispersed in 75 mL of Milli-Q water. The dispersion was stirred overnight at room temperature, dialyzed against deionized water, and centrifuged at 15000 g for 15 min. The supernatant containing pectin was collected and stored as an aqueous solution. Sodium azide (0.02%, w/w) was added to prevent bacterial growth. Alkali Treatment of Potato Pectin. A stock solution containing NaOH and NaBH4 was mixed with pectin solutions to give a final concentration of 0.25 M NaOH and 0.25 M NaBH4. These solutions were incubated at 50 °C for 5 h and dialyzed using a 3.5 kDa cutoff membrane.13 The dialysate containing alkali-treated pectin was collected and stored as an aqueous solution, to which 0.02% (w/w) sodium azide was added. B

DOI: 10.1021/acs.jafc.9b01773 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry Statistical Analysis. All data were presented as mean ± standard deviation. The t-test and one-way analysis of variance followed by Turkey’s HSD test were performed using the SPSS package (SPSS, Version 23.0, SPSS Inc., Chicago, IL, U.S.A.) at a 5% significant level.

HMBC (not shown) spectra of the potato pectin and are listed in Table 1. The HSQC spectrum of the alkali-treated potato pectin (Figure 1b) shows two major anomeric signals at the same chemical shifts as those of the potato pectin as well as all of the signals of D-GalA and D-galactose listed in Table 1. These results are aligned with the results of the colorimetric determination of the GalA content in that the alkali treatment had little effect on the GalA content of potato pectin. The signal at δC/δH 54.23/3.83 ppm in the HSQC spectrum of the potato pectin (Figure 1a) was assigned to the carboxymethyl group in GalA, while those at δC/δH 21.58/2.12 and δC/δH 21.91/2.21 ppm were assigned to the acetyl group substituted at the O-2 and O-3 positions of GalA, respectively.28 None of these signals were detected in the HSQC spectrum of the alkali-treated potato pectin (Figure 1b), suggesting that not only proteinaceous components but also methyl and acetyl groups of the potato pectin were cleaved effectively as a result of the alkali treatment. It is therefore likely that the hydrophobicity−hydrophilicity balance of the alkali-treated potato pectin shifted to the hydrophilic side to a certain extent. It is also worth noting that no signal of α-glucose was detected in any NMR spectra, confirming that starch in the cell wall materials for pectin extraction was appropriately removed by the repeated homogenization and rinsing. Adsorption to Graphite Surfaces. Visualizing the molecular structure of a polysaccharide−protein conjugate can be challenging because the molecule tends to form clumps, particularly if it is deposited onto a substrate from a solution and air-dried.29 Thus, in this study, surface active components dissolved in aqueous sample solutions were allowed to adsorb to graphite surfaces spontaneously and were imaged without being air-dried. The topographical AFM image of the potato pectin adsorbed on to the graphite surface (Figure 2a) shows filamentous structures representing the molecular chains of pectin and globular structures representing proteinaceous components. The pectin chains appear to have self-assembled to certain degrees and formed networks, while the globular structures appear to be attached to the pectin network. These structural features resemble those of sugar beet pectin visualized using AFM.9,30 The heights of the network structures in Figure 2a varied from ca. 0.8 to 1.9 nm of which the lowest value is comparable to the height of the single molecular chain of pectin.31,32 Therefore, the variability in the height of the pectin network is most likely to have resulted from the side-by-side aggregation of molecular chains to various degrees. The heights of the globular structures also varied from 2.3 to 12.4 nm of which the lowest value is comparable to that of the protein moiety of sugar beet pectin.9 A possibility is that proteinaceous components in the potato pectin had similar sizes and aggregated to various degrees, while another possibility that the sizes of the proteinaceous components were not uniform cannot be excluded. The topographical AFM image of the alkali-treated potato pectin adsorbed to the graphite surface shows far fewer and

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RESULTS AND DISCUSSION Chemical Composition. The total sugar content of the potato pectin or the alkali-treated potato pectin was determined to be 93.3 ± 0.5% or 97.8 ± 0.3% (w/w), respectively. There was no significant difference (p > 0.05) in the GalA content between the potato pectin (16.9 ± 1.4%, w/ w) and the alkali-treated potato pectin (20.3 ± 2.3%, w/w), indicating that the β-eliminative degradation of HG during the alkali treatment was inhibited satisfactorily because of the presence of NaBH4.23 The protein content was significantly decreased (p < 0.05) from 1.4 ± 0.1% to 0.3 ± 0.0% (w/w) as a result of the alkali treatment, suggesting that the covalent linkage between the pectin and protein moieties in the potato pectin was broken effectively.24 It should also be noted that the protein content in the present potato pectin is considerably lower than a reported value of 2.8% (w/w) for a potato pectin extracted using almost identical conditions, including the type of acid, pH, temperature, and time,8 except that the CWM used to extract pectin in this study was treated with Tris buffersaturated phenol. The considerably lower protein content suggests that unconjugated proteins were effectively removed from the potato pectin prepared in the present study. Figure 1a shows the HSQC NMR spectrum of the potato pectin. Two major anomeric signals were identified at δC/δH

Figure 1. HSQC spectra of (a) the potato pectin and (b) the alkalitreated potato pectin in D2O at 25 °C.

101.01/5.43 and δC/δH 105.67/4.67 ppm and assigned to DGalA and D-galactose, respectively, on the basis of previously reported data in the literature.25−27 Other chemical shifts of DGalA and D-galactose were assigned using the HSQC and

Table 1. Chemical shifts (13C and 1H) of the main residues of the potato pectin in D2O at 25 °Ca chemical shift (ppm) residue

C1/H1

C2/H2

C3/H3

C4/H4

C5/H5

C6/H6

GalA galactose

101.01/5.43 105.67/4.67

73.01/3.67 73.23/3.71

72.61/3.87 74.73/3.81

74.85/3.99 79.10/4.20

78.29/3.69 75.93/3.75

n.d. 62.13/3.84

a

n.d.: not detected. C

DOI: 10.1021/acs.jafc.9b01773 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 3. Surface pressure−area isotherms of (solid line) the potato pectin and (dotted line) the alkali-treated potato pectin at the air− water interface during a compression−expansion cycle. Figure 2. Topographical AFM images of (a) the potato pectin and (b) the alkali-treated potato pectin adsorbed to graphite surfaces. The image size is 1.5 μm × 1.5 μm. The curves beneath the images represent the height profiles of the cross-sections highlighted and numbered in each image.

moiety should shift the hydrophobicity−hydrophilicity balance of the entire molecule to the hydrophilic side. Similar observation was reported for sugar beet pectin, where an alkali-treated pectin exhibited much lower surface tensions than the untreated pectin.11 Additionally, an okra pectin with a lower molecular weight was reported to exhibit faster decreases in the interfacial tension of n-hexadecane−water interfaces.33 It was also reported that a smaller pectin moiety of the citrus pectin−protein conjugate exhibited a more effective emulsifying ability.14 In Figure 3, both the potato pectin and the alkali-treated potato pectin showed a hysteresis between compression and subsequent expansion with a much smaller hysteresis width for the potato pectin. Compression of a pure protein film to a large degree tends to enhance the intermolecular association/ aggregation of protein molecules at the interface.34 As a result, the dissociation of the protein aggregates upon expansion may be delayed or inhibited, leading to a lower Π than that at the same A during compression.34 The smaller hysteresis observed for the potato pectin therefore indicates that the presence of the pectin moiety may physically interfere with protein− protein interactions at the interface. The elastic shear modulus of the interfacial film of alkali-treated sugar beet pectin formed at the air−water interface was reported to be approximately four times that of untreated sugar beet pectin,11 which can also be explained if the pectin moiety of the pectin−protein conjugate in sugar beet pectin weakened protein−protein interactions at the interface. Competitive Displacement. Figure 4a−h shows topographical AFM images of the interfacial films sampled during the competitive displacement of the potato pectin and the alkali-treated potato pectin from the air−water interface. Prior to the addition of the surfactant Tween 20 to the subphase, the interfacial areas were covered entirely by the pectins (Figure 4a,e). The interfacial film of the alkali-treated potato pectin revealed some globular structures similar in size to those observed in Figure 2b for the same pectin adsorbed to the graphite surface. The interfacial film of the potato pectin before the addition of the surfactant appears to have a smoother surface (Figure 4a) as compared to that of the alkali-treated

shorter filamentous structures representing the molecular chains of pectin (Figure 2b). These pectin chains were most likely to be covalently linked to the proteinaceous components because functional groups such as methoxyl and acetyl groups that would otherwise provide the molecule with hydrophobicity had been removed by the alkali treatment as has been suggested on the basis of the NMR spectra (Figure 1). The presence of such pectin moieties covalently linked to protein moieties in the alkali-treated potato pectin in turn infers that the untreated potato pectin contained a larger quantity of the naturally occurring pectin−protein conjugate. The heights of the globular structures representing protein moieties in Figure 2b varied from 5.0 to 14.1 nm, while those in Figure 2a varied from 2.1 to 12.7 nm. The larger sizes of the protein moieties in the alkali-treated potato pectin indicate that the unconjugated protein moieties of the pectin were more prone to aggregate with each other in an aqueous solution. Π−A Isotherm. Π−A isotherms of the alkali-treated and untreated potato pectins were obtained to shed light on the effect of the pectin moiety on interfacial properties of the potato pectin−protein conjugate. To an air−water interface, of which A was 230 cm2, an aqueous solution of the potato pectin or the alkali-treated potato pectin containing 3 or 0.5 mg of protein moieties, respectively, was spread. The Π of the potato pectin was already lower than that of the alkali-treated pectin at this point prior to compression as shown in Figure 3. During compression, the Π of the potato pectin was fairly constant around 0.7 mN/m until A decreased to ca. 160 cm2, increased steeply with decreasing A, and reached 23.5 mN/m at the maximum compression to 30 cm2. The Π of the alkali-treated potato pectin started to increase at an earlier stage of compression to ca. 215 cm2 and reached 24.8 mN/m at the same maximum compression. It thus appears that the pectin moiety has an inhibitory effect on the surface activity of the pectin−protein conjugate presumably because the pectin D

DOI: 10.1021/acs.jafc.9b01773 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 4. Topographical AFM images of interfacial films of (top) the potato pectin, (middle) the alkali-treated potato pectin, and (bottom) the potato pectin with CaCl2 added to the subphase. The concentrations of Tween 20 in the subphases were (a, e, and i) 0 μM; (b, f, and j) 1.5 μM,; (c, g, and k) 2.5 μM; or (d, h, and l) 3.5 μM.

pectin (Figure 4e). At a surfactant concentration of 1.5 μM, the interfacial film of the potato pectin showed a number of irregularly shaped dark domains occupied by the surfactant and brighter pectin domains containing both filamentous structures representing pectin moieties and globular structures representing protein moieties (Figure 4b). The irregular shapes of the boundaries of the surfactant domains are an indication that the mechanical strength of the interfacial film was not uniform so that the failure of the film due to elevated surface pressures of the surfactant domains occurred preferentially in mechanically weaker regions.22,35,36 At the same surfactant concentration, the alkali-treated potato pectin showed larger and slightly more round-shaped surfactant domains, while it was difficult to detect the presence of filamentous pectin moieties (Figure 4f). As the surfactant concentration increased further, the surfactant domains continued to enlarge regardless of the pectin type. However, the potato pectin appears to cover a larger interfacial area than the alkali-treated potato pectin consistently at all examined surfactant concentrations up to 3.5 μM. Figure 4i−l reveals the effect of the addition of Ca2+ to the subphase on the competitive displacement of the potato pectin. The calcium ion is capable of cross-linking adjacent antiparallel nonesterified HG blocks through bridging of negatively charged carboxyl groups of GalA residues.37 Before adding the surfactant (Figure 4i), the interfacial film appeared to be much rougher than that of the same pectin without added Ca2+ (Figure 4a), implying that the addition of Ca2+ induced the

deposition of pectin molecules dissolved in the subphase onto the interfacial film of the preadsorbed pectin−protein conjugate through Ca2+-mediated cross-linking between pectin moieties. At higher surfactant concentrations, dark surfactant domains emerged but they appeared to be much smaller than those in the absence of Ca2+ added to the subphase at the same surfactant concentration. Therefore, the Ca2+ added to the subphase is considered to have enhanced the formation of cross-links between pectin moieties at the interface and strengthened the interfacial film. Additionally, the surfactant domains shown in Figure 4j−l appear to be more roundshaped than those shown in Figure 4b−d, indicating that the mechanical strength of the interfacial film was more evenly distributed across the entire film as a result of the enhanced cross-linking of pectin moieties residing at the interface. As shown in Figure 5, the interfacial area coverages of the alkali-treated potato pectin, the potato pectin, and the potato pectin with Ca2+ added to the subphase started to decrease at surfactant concentrations of 0.5, 1.0, and 1.5 μM, respectively. The potato pectin showed significantly larger interfacial area coverages than the alkali-treated pectin at surfactant concentrations of 0.5−2.5 μM (p < 0.05). Additionally, the rate of decrease in the interfacial area coverage of the potato pectin with increasing surfactant concentration up to 1.5 μM appeared to be lower than that of the alkali-treated potato pectin. These results reinforce the qualitative observation in the AFM images that the alkali-treated potato pectin was more susceptible to surfactant-induced displacement from the E

DOI: 10.1021/acs.jafc.9b01773 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Figure 5. Changes in the interfacial area coverage by (Δ) the potato pectin, (○) the alkali-treated potato pectin, and (□) the potato pectin with CaCl2 added to the subphase during competitive displacement by Tween 20. Error bars represent standard deviations.

Figure 6. Changes in the interfacial film thickness of (Δ) the potato pectin, (○) the alkali-treated potato pectin, and (□) the potato pectin with CaCl2 added to the subphase during competitive displacement by Tween 20. Error bars represent standard deviations.

interface than the potato pectin as a result of the absence of pectin moieties in interfacial films (Figure 4b,f). In addition to the Ca2+-mediated cross-linking, the presence of abundant long side chains of β-1,4-galactan in the potato pectin may potentially provide an alternative means of cross-linking, thereby strengthening its interfacial film, because the galactan side chains of RG-I have been shown to have an ability to selfassemble to form gel networks.38,39 At relatively high surfactant concentrations of 3.0−3.5 μM, the potato pectin and the alkalitreated potato pectin showed similar interfacial area coverages with no significant differences (p > 0.05). Therefore, the surface pressure of protein domains in these interfacial films is considered to have been in balance with that of surfactant domains. As expected, the interfacial area coverage of the potato pectin with Ca2+ added to the subphase was consistently and significantly larger than those of the potato pectin without added Ca2+ at surfactant concentrations of 1.0− 3.5 μM (p < 0.05). In Figure 6, the interfacial film thicknesses of the alkalitreated potato pectin and the potato pectin were shown to increase gradually with increasing surfactant concentrations up to 2.0 and 3.0 μM, respectively, and then reach a constant value of ca. 2.7 nm. These trends appear to be in parallel with those of the interfacial area coverages shown in Figure 5. Concurrent decreases in the area coverage and increases in the film thickness have been considered to result from protein molecules desorbed from the interface but not fully dissociated from the molecules residing at the interface.22 Interfacial films of the alkali-treated potato pectin were significantly thicker than those of the potato pectin at surfactant concentrations of 1.0−2.0 μM (p < 0.05), suggesting once again that the potato pectin was more resistant to surfactant-induced displacement from the interface. In contrast, the interfacial film thickness of the potato pectin with Ca2+ added to the subphase continuously increased with increasing surfactant concentration up to 3.5 μM without reaching a plateau and was significantly lower than that of the potato pectin without added Ca2+ at surfactant concentrations of 2.0 and 2.5 μM (p < 0.05). The addition of Ca2+ was most likely to enhance cross-linking

of pectin moieties at the interface and thereby the ability of the potato pectin to resist displacement from the interface. It is worth noting that the lowest measurable film thickness of the potato pectin in the presence of Ca2+ added to the subphase was significantly greater than those of both the potato pectin and the alkali-treated potato pectin (p < 0.05), which presumably resulted from the Ca2+-mediated cross-linking of pectin molecules dissolved in the subphase and those preadsorbed to the interface. In summary, the presence of the pectin moiety was demonstrated to be an important determining factor for properties of the naturally occurring pectin−protein conjugate in pectic extracts from potato tubers at the air−water interface. The potato pectin extracted from the CWM, from which free proteinaceous components were thoroughly removed, was treated with alkali to eliminate the surface activity of the pectin moiety, by removing methoxyl and acetyl groups, and to cleave the linkage between the pectin and protein moieties. Nevertheless, the presence of a small fraction of pectin− protein conjugate in the alkali-treated potato pectin was probed using AFM as a minor component that adsorbed to hydrophobic graphite surfaces spontaneously. Upon the compression of the interfacial film of the untreated potato pectin to a large degree, the pectin moiety appeared to have an inhibitory effect on protein−protein interactions at the interface presumably because of the bulkiness of the moiety. The pectin moiety also exhibited an inhibitory effect on surfactant-induced competitive displacement of the pectin− protein conjugate from the air−water interface. AFM images of interfacial films revealed that pectin moieties self-assembled and formed cross-linked two-dimensional networks at the interface. Such pectin networks are expected to reinforce the mechanical strength of the interfacial film and provide a more pronounced resistance to elevated surface pressures exerted by surfactant domains. The present results imply that potato F

DOI: 10.1021/acs.jafc.9b01773 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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Journal of Agricultural and Food Chemistry

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pectin is a promising material for developing novel strategies to create highly stable foams and emulsions.

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AUTHOR INFORMATION

Corresponding Author

*Phone: +81-155-49-5565. E-mail: [email protected]. ORCID

Shinya Ikeda: 0000-0002-5407-7615 Present Address #

Department of Life and Food Sciences, Obihiro University of Agriculture and Veterinary Medicine, Obihiro, Hokkaido 080− 8555, Japan (S.I.). Funding

This work was supported by the United States Department of Agriculture (U.S.D.A.) National Institute of Food and Agriculture, Hatch Project 1012819. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We thank Professor Jiwan P. Palta of the Department of Horticulture, the University of Wisconsin−Madison, for kindly providing raw potato tubers and Professor John Ralph of the Department of Biochemistry and the Great Lakes Bioenergy Research Center, the University of Wisconsin−Madison, for letting us use the NMR spectrometer.

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ABBREVIATIONS USED HG, homogalacturonan; RG-I, rhamnogalacturonan I; GalA, galacturonic acid; CWM, cell wall material; NMR, nuclear magnetic resonance; HSQC, heteronuclear single-quantum coherence; HMBC, heteronuclear multiple-bond correlation; PTFE, polytetrafluoroethylene; AFM, atomic force microscopy

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REFERENCES

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DOI: 10.1021/acs.jafc.9b01773 J. Agric. Food Chem. XXXX, XXX, XXX−XXX

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DOI: 10.1021/acs.jafc.9b01773 J. Agric. Food Chem. XXXX, XXX, XXX−XXX