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Effect of molecular structure on emulsifying properties of sugar beet pulp pectin
Accepted Manuscript Effect of molecular structure on emulsifying properties of sugar beet pulp pectin Hai-ming Chen, Xiong Fu, Zhi-gang Luo PII:
S0268-005X(15)30096-5
DOI:
10.1016/j.foodhyd.2015.09.021
Reference:
FOOHYD 3144
To appear in:
Food Hydrocolloids
Received Date: 1 May 2015 Revised Date:
22 August 2015
Accepted Date: 18 September 2015
Please cite this article as: Chen, H.-m., Fu, X., Luo, Z.-g., Effect of molecular structure on emulsifying properties of sugar beet pulp pectin, Food Hydrocolloids (2015), doi: 10.1016/j.foodhyd.2015.09.021. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Ca2+ Ca2+
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Emulsion droplets Ca2+
Side chain
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Ferulic acid
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Effect of molecular structure on emulsifying properties of sugar beet
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pulp pectin
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Hai-ming Chen1,2, Xiong Fu1, Zhi-gang Luo1*
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1. College of Light Industry and Food Sciences, South China University of Technology,
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381 Wushan Road, Guangzhou 510640, PR China
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2. College of Food Sciences & Engineering, Hainan University, 58 People Road, Haikou,
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China
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* Corresponding author.
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Tel.: +86 20 8711 3845; Fax: +86 20 8711 3848
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Email: [email protected] (Z. G. Luo).
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Abstract: To investigate the impact of each functional group on the emulsifying
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properties of sugar beet pulp pectin (SBPP), seven enzymes were studied in a particular
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order. Compositions of SBPP and enzymatically modified SBPPs were determined, and
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the structures of SBPPs were characterized by FT-IR. In addition, the contribution of
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each functional group was evaluated based on the variation in emulsion characteristics.
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The results showed that protein, ferulic acid-araban/galactan-protein complexes and
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ferulic acid played important roles in improving the surface activity, emulsifying capacity
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and emulsifying stability of SBPP and the extent of the decrease in the emulsifying
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activity followed the order: ferulic acid > ferulic acid-arabinogalactan-protein complexes
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> protein. The decrease of methyl ester groups mainly affected the particle sizes of the
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emulsion. In addition to particle sizes, the cream index of the emulsion increased with the
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hydrolysis of acetyl groups. Arabinose and galactose less affected emulsifying properties
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than other functional groups.
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Keywords: Sugar beet pulp pectin, Enzyme, Structure, Emulsifying property.
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1. Introduction Pectin is widely used in the food industry for its gelling, thickening, and stabilizing
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properties (Voragen, Pilnik, Thibault, Axelos, & Renard, 1995; Funami, Zhang, Hiroe,
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Noda, Nakauma, Asai, Cowman, Al-Assaf, & Phillips, 2007). Commercial pectins are
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extracted from citrus peel and apple pomace in most instances (Funami, Nakauma,
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Ishihara, Tanaka, Inoue, & Phillips, 2011). A relatively new type of pectin, sugar beet
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pulp pectin (SBPP), has recently received much attention (Siew and Williams 2008;
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Fissore, Rojas, Gerschenson, & Williams, 2013).
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As shown in Fig. 1, SBPP is a heteropolysaccharide with a chain structure of
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(1→4)-linked α-D-galacturonic acid (GalA) units interrupted by the insertion of
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(1→2)-linked L-rhamnopyranosyl residues. Rhamnosyl residues (20 ~ 80%) can be
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substituted with side chains (‘hairy’ region) consisting of neutral sugars, such as
41
D-galactose, L-arabinose, D-xylose, D-glucose, D-mannose, L-fucose and D-glucuronic
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acid. In addition, lateral chains contain phenolic acids such as ferulic acid, which are
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linked to the arabinose and galactose residues via ester linkages (Fry, 1983). In addition,
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there is a higher concentration of the proteinaceous materials bound to the side chains
45
through covalent linkages (Williams, Sayers, Viebke, Senan, Mazoyer, & Boulenguer,
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2005). Some of the acid groups of the GalA in the linear chain structure (‘smooth’ region)
47
can be partially methyl-esterified and O-acetylated at the C-2 and/or C-3 positions (Siew
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& Williams, 2008). Compared to other conventional pectins, SBPP tends to exhibit a
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higher degree of acetylation (DA) and a greater number of neutral sugar side chains (rich
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in hairy regions) (Siew & Williams, 2008). In addition, SBPP has a greater number of
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feruloyl groups attached to the galactose and arabinose side chains (Colquhoun, Ralet,
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Thibault, Faulds, & Williamson, 1994; Guillon, Thibault, Rombouts, Voragen, & Pilnik,
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1989; Ralet, Thibault, Faulds, & Williamson, 1994; Rombouts & Thibault, 1986) and a
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greater amount of proteinaceous material bound to the lateral chains through covalent
55
linkages (Funami et al., 2007; Williams et al., 2005). Because of these differences in
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structural characteristics, SBPP does not have the capability to form gels like
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conventional pectins, but it possesses excellent emulsifying properties (Voragen et al.,
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1995; Funami et al., 2007; Williams et al., 2005; Li et al., 2012). According to Endreß &
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Rentschler (1999), the emulsifying ability of beet pectin can be explained by the high
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percentage of acetyl groups in its chemical structure. Nevertheless, Leroux, Langendorff,
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Schick, Vaishnav, & Mazoyer, (2003) studied the emulsifying ability of sugar beet pectin
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in relation to its chemical structure and concluded that there was no evidence that its
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emulsifying ability correlates with the number of acetyl groups in its structure but, rather,
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is related to its high concentration of proteinaceous components (Williams et al., 2005).
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Siew & Williams (2008) suggested that proteins and/or ferulic acid groups adsorb onto
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the surfaces of the oil droplets and stabilize the emulsions. Leroux et al., (2003)
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concluded that a highly methyl-esterified pectin is able to reduce the interfacial tension
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between the water and the oil phases. It is likely its hydrophobicity (due to its
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COOCH3-groups) that gives pectin its emulsifying properties. SBPP was fractionated
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using hydrophobic affinity chromatography, and three fractions with different proportions
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of protein, ferulic acid and weight-average molecular mass were obtained (Williams et al.,
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2005). They concluded that the emulsification properties of SBPP were influenced by the
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protein, ferulic acid groups, proportion of ester groups, and molecular mass distribution
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of the fractions. SBPPs were structurally modified using protease to degrade the
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proteinaceous moiety, polygalacturonase (PG) to cleave the carbohydrate backbone, and
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a arabinanase/galactanase (ABN/GAL) combination to cleave the lateral chains (Funami
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et al., 2011). Methyl and acetyl groups were removed with galacturonic acid (GalA) via
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the modification of PG. In addition, protein and ferulic acid exist in the side chain
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(mainly covalently bonding with arabinose and galactose); therefore, the combination of
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ABN/GAL can alter four factors (arabinose, galactose, protein and ferulic acid) at the
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same time. None of the previous papers provided a clear explanation for the relationship
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between each factor and the emulsification ability of pectins.
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The objective of the present study was to investigate the emulsifying properties of
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SBPP in relation to its structural characteristics. SBPPs were structurally modified using
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protease
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endo-α-1,4-polygalacturonase
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4-galactanase (GAL), feruloyl esterase (FAE), pectin methyl esterase (PME) and pectin
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acetyl esterase (PAE) to degrade the proteinaceous moiety, GalA unit, arabinose,
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galactose, ferulic acid, DM and DA subunits, respectively. The contribution of each of
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these structural units to the emulsification of SBPP was assessed through enzymatic
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modification in a particular order.
of
pepsin
food-grade
acid
endo-β-1,5-arabinanase
protease,
(ABN),
PE),
endo-β-1,
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(combination
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2. Materials and methods
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2.1. Materials and chemicals
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Sugar beet pulp pectin was purchased from CP Kelco (Lille Skensved, Denmark).
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Medium-chain triglyceride (MCT) was purchased from the Nisshin Oillio Group (Tokyo,
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Japan). GalA and bovine serum albumin were purchased from Sigma-Aldrich Chemical
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Co. (Milwaukee, WI, USA). PG (EC 3.2.1.15), ABN (EC 3.2.1.99), GAL (EC 3.2.1.89),
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and FAE (EC 3.1.1.73) were obtained from Megazyme International Ireland Ltd (Bray,
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Ireland). PME (E.C. 3.1.1.11) and PAE (E.C. 3.1.1.6) were purchased from
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Sigma-Aldrich Chemical Co. (Milwaukee, WI, USA). Pepsin (E.C. 3.4.23.1) and
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food-grade acid protease were purchased from Aladdin reagents Co., Ltd (Shanghai,
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China). The Viscozyme L9 enzyme was a commercial preparation obtained from Novo
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Nordisk (Copenhagen, Denmark), and because it was a multienzyme complex,
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purification was required before use (Garna, Mabon, Wathelet, Paquot, 2004).
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2-Deoxy-D-glucose, myo-inositol, L-rhamnose (Rha), L-arabinose (Ara), D-xylose (Xyl),
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D-galactose (Gal), D-glucose (Glc), D-galacturonic acid (GalA), ferulic acid (FA),
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succinic acid, glacial acetic acid and methanol were purchased from Sigma-Aldrich
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Chemical Co. (Milwaukee, WI, USA). All other chemicals were of analytical grade
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unless otherwise noted.
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2.2. Enzymatic modification by PME, PAE and PG In this section, SBPP was enzymatically modified by PME, PAE and PG in turn as
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shown in Fig. 2a. Firstly, SBPP was diluted to 1.0 w/v% in a 100 mM citrate buffer (pH
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7.0) with mechanical shearing at 2000 rpm for 15 min. Into these dispersions, PME (30
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unit per 1.0 g SBPP) was added and incubated in a sealed conical flask at 30 °C for 16 h
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with gentle stirring (Hotchkiss et al., 2002). The hydrolyzed mixture was heated at 100
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°C for 1 min to stop the enzymatic reaction and dialyzed against distilled de-ionized
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water at 20 °C through a dialysis membrane with a 10 kg/mol molecular weight cut off,
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followed by freeze drying. The freeze dried samples (SBPP-a1) were stored in a
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desiccator at 20 °C until use. Secondly, SBPP-a1 was diluted to 1.0 w/v% in a 100 mM
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citrate buffer (pH 6.0) and PAE (10,000 unit per 1.0 g SBPP) was added and incubated at
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30 °C for 30 min with gentle stirring. The reaction was stopped by treatment at 100 °C
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for 1 min, and the hydrolyzed mixture was dialyzed and freeze-dried. Thirdly, The freeze
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dried samples (SBPP-a2) was diluted to 1.0 w/v% in a 100 mM citrate buffer (pH 3.5)
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with mechanical shearing at 2000 rpm for 15 min. PG (10 unit per 1.0 g SBPP) was
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added and incubated at 40 °C for 20 h with gentle stirring (Funami et al., 2011). The
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reaction was stopped by heating at 100 °C for 1 min, and the hydrolyzed mixture was
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dialyzed and freeze-dried. The freeze dried samples (SBPP-a3) were stored in a desiccator
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at 20 °C until use.
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2.3. Enzymatic modification by PE, FAE and GAL or ABN.
SBPP was enzymatically modified by PE, FAE and GAL or ABN in turn as shown in
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Fig. 2b. First, protein degradation was accomplished following the method of Funami et
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al. (2011) using pepsin (700 unit per 1.0 g SBPP) and food-grade acid protease (70 unit
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per 1.0 g SBPP) at pH 3.0 and 30 °C. SBPP-b1 was obtained by enzyme deactivation,
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dialysis and freeze-drying as described above. Then, SBPP-b1 was catalyzed by FAE (0.3
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unit per 1.0 g SBPP) at pH 6.0 and 50 °C for 20 h. SBPP-b2 was obtained after enzyme
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deactivation, dialysis and freeze-drying. Finally, SBPP-b3 or SBPP-b4 was obtained from
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SBPP-b2, which was enzymatically modified by ABN (7 units per 1.0 g SBP) or GAL (5
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units per 1.0 g SBP) at pH 3.5 and 40 °C for 24 h.
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2.4. Combined enzymatic modification by GAL and ABN
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SBPP was enzymatically modified by GAL combined with ABN as shown in Fig. 2c.
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SBPP was diluted to 1.0 w/v% in a 100 mM citrate buffer (pH 3.5) with mechanical
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shearing at 2000 rpm for 15 min. Into these dispersions, ABN (7 units per 1.0 g SBP) and
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GAL (5 units per 1.0 g SBP) were added and incubated in a sealed conical flask at 40 °C
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for 24 h with gentle stirring (Funami et al., 2011). SBPP-c was obtained by enzyme
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deactivation, dialysis and freeze-drying.
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2.5. Enzymatic modification by FAE
SBPP was enzymatically modified by FAE directly as shown in Fig. 2d. SBPP was
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diluted to 1.0 w/v% in a 100 mM citrate buffer (pH 6.0) with mechanical shearing at
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2000 rpm for 15 min. Into these dispersions, FAE (0.3 units per 1.0 g SBP) was added
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and incubated in a sealed conical flask at 50 °C for 20 h with gentle stirring. SBPP-d was
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obtained by enzyme deactivation, dialysis and freeze-drying.
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2.6. Determination of concentrations of galacturonic acid and neutral sugar Pectin samples were hydrolyzed using a combination of chemical and enzymatic
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hydrolysis as described by Garna et al. (2004). Pectin (100 mg) was hydrolyzed with 0.2
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mol/L trifluoroacetic acid (TFA) (5 mL) at 80 °C for 72 h. The hydrolysate was adjusted
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to pH 5 with NH4OH (14 mol/L) and diluted to 25 mL. Ten milliliters of this solution was
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mixed with 10 mL of purified Viscozyme L9 (Novo Nordisk, Denmark) in a 20 mmol/L
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pH 5 sodium acetate buffer. The GalA concentration was measured using a Dionex
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DX-500 Bio-LC system (Dionex Corp., Sunnyvale, Calif., USA) equipped with a
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CarboPAc PA1 column (250 × 4 mm; Dionex Corp., Sunnyvale, Calif., USA) in
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combination with a CarboPac guard column (25 × 4 mm; Dionex Corp., Sunnyvale,
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Calif., USA). All measurements were carried out at a temperature of 30 °C and with a
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gradient reaching 170 mmol/L CH3COONa·3 H2O and 100 mmol/L NaOH for 13 min at
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a flow rate of 1 mL/min. The column was washed with 100 mmol/L NaOH for 10 min
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before the next injection. Twenty-five microliters of sample was injected. Detection was
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realized using a pulsed amperometric detector with a post-injection flow rate of 200
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µL/min of 900 mmol/L NaOH.
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2.7. Determination of protein concentration
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A Bradford protein assay kit (Pierce; Thermo Fisher Scientific Inc., USA) was used to
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determine the total protein concentration of the pectin samples based on their absorbance
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at 595 nm, which is the wavelength of the color produced by the reaction between the
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protein and Coomassie Brilliant Blue G-250. BSA was used as the standard for
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calibration within the working range of 0 to 750 mg/mL (Funami et al., 2011).
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2.8. Degree of esterification determination
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Methoxy and acetyl groups were released from pectin by saponification with 0.2 mol/L
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NaOH in 50% isopropanol at 4 °C for 2 h. Methanol and acetic acid levels were
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determined by HPLC on an Aminex HPX 87H column (300 × 7.8 mm; Bio-Rad
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Laboratories, Inc., Hercules, CA, USA) using succinic acid as the internal standard.16
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Degree of methylation (DM, mol of MeOH per 100 mol of galacturonic acid) and degree
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of acetylation (DA, mol of AcOH per 100 mol of galacturonic acid) were calculated from
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the amount of MeOH and AcOH, respectively, and from the amount of galacturonic acid
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(Voragen et al., 1986).
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2.9. Fourier-transform infrared spectroscopy (FT-IR)
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The FT-IR spectra of pectin samples were recorded using a Perkin Elmer Spectrum
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RXIFT-IR Spectrometer (Perkin Elmer Instruments, USA) at room temperature. The
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sample powder was blended with KBr powder and pressed into tablets before spectra
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were obtained over the range of 2000 to 800 cm-1 with an 8 cm-1 resolution.
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2.10. Evaluation of emulsifying properties
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2.10.1 Emulsion preparation
A SBPP or modified-SBPP solution was prepared by dissolving 1 g of pectin in 80 mL
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of ultrapure water with continuous stirring at room temperature for 24 h. Into this solution,
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1 mL of a 10% (w/v) benzoic acid solution was added as a preservative, and citric acid
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(10%, w/v) was used to adjust the final pH of the emulsions to 3.0. Then, distilled
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de-ionized water and 15 g of MCT were added to achieve a final mass of 100 g. The
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mixtures were then pre-homogenized using an Ultra-Turrax device at a speed of 24,000
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rpm for 3 min to form coarse emulsions, which were subsequently homogenized over
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three passes using an ultra-high-pressure homogenizer (Nano DeBEE, USA) operated at
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50 MPa.
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2.10.2. Determination of the surface and interfacial tension
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The surface tension of the SBPP and modified-SBPP solutions was measured at 25 °C
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using a DCAT21 surface tensiometer (Dataphysics Co., Germany). The measuring cup
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was placed in a stainless steel jacket on a perpendicularly movable platform. A platinum
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plate (24 mm × 10 mm × 0.1 mm) was gently placed at the bottom of a sensitive
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electronic balance and immersed in the measured solutions by slowly raising the platform.
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The force (mN/m) required to detach the plate from the surface was recorded as the
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dynamic surface tension. Finally, the surface tension was calculated based on
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balance-force measurements by applying the appropriate correction factors (Li et al.,
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2013). Interfacial tension measurements were carried out at the oil/water interface using
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1.0% (w/w) pectin solution prepared at room temperature by dissolving dried SBPP and
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modified-SBPP in fresh Milli-Q water containing 0.02% sodium azide as a bacteriocide.
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A constant volume of 1.0% (w/w) pectin solution was gently poured into a thermostated
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glass dish at 23 °C. Immediately afterwards, fish oil was gently layered on top of the
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pectin solution. The interfacial tension was measured after 1 h of equilibrium from the
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pull on the mica plate. Measurements were performed three times for each solution (Yapo,
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Robert, Etienne, Wathelet, & Paquot, 2007).
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2.10.3. Particle-size analysis of the emulsions
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A Malvern Mastersizer 2000 (Malvern, UK) was used to determine the droplet sizes of
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the emulsions using static multi-angle light-scattering analysis, which was performed in
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triplicate. A few drops of the emulsion were added to the water in the dispersion unit of
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the instrument with stirring at 2000 rpm until the degree of obscuration was
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approximately 15%. Values of 1.45 and 0.001 were used for the refractive index and
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absorption index of MCT, respectively, and values of 1.33 and 0 were used for the
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refractive index and absorption index of the water dispersant, respectively. The average
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size of the droplets in the emulsions was assessed in terms of the surface-weighted mean
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diameter d32 and volume-weighted mean diameter d43 (Li et al., 2012).
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2.10.4. Evaluation of emulsion storage stability
Freshly prepared emulsions were heated in a water bath at 80 °C for 30 min followed
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by centrifugation at 8000 g for 10 min. The extent of creaming was characterized by the
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creaming index (CI, %), which was determined according to the method developed by
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Sciarini, Maldonado, Ribotta, Perez, & Leon, (2009) as follows:
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CI = (height of the serum layer/total height of the emulsion) × 100%
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2.11. Statistical analysis
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Analyses of variance were performed, and the mean values ± standard deviations were
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evaluated by Duncan’s multiple-range test (p < 0.05) using SPSS version 13.0 statistical
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software (SPSS Inc., Chicago, IL, USA). Origin (Origin Lab Co., Pro.8.0, USA) software
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was used for data processing and to create charts.
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3. Results and discussion
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3.1. Influence of enzymes on the component of SBPP
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The components of enzymatically modified SBPPs are shown in Table 1. PME was
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used to hydrolyze methyl esters of pectin to pectate and methanol. PAE was used to
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hydrolyze acetyl residues, yielding polygalacturonic acid. After hydrolysis by the
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respective enzymes for 30 min, DM and DA values were reduced from 67 and 23.9 to 7.5
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and 5.1%, respectively. The concentrations of other components increased slightly. PG randomly hydrolyzes (1→4)-α-D-galactosiduronic linkages in pectate and other
260
galacturonans. In our research, the concentration of GalA decreased from 43.57 to
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21.67% when the enzyme digestion time was 24 h. Along with GalA, methyl and acetyl
262
groups were hydrolyzed. However, DM and DA are expressed as mol of methyl and
263
acetyl groups, respectively, per 100 mol of GalA (Fry, 1983). Therefore, the values of
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DM and DA were unchanged. The impact of PG on pectin mainly occurs in the backbone.
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Therefore, the concentrations of the neutral sugars, which mainly distribute in the side
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chains, increased sharply.
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Pepsin and food-grade acid protease were used to decompose the proteinaceous
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materials. As shown in Table 1, the concentration of protein decreased from 5.2 to 0.42%
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when the enzyme digestion time was 16 h, and these values were almost equivalent to
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those of a previous report (Funami et al., 2011). The concentrations of GalA and neutral
271
sugars increased with the concentration of protein decreasing. Methyl esterification and
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O-acetylation only reacted with the GalA of the backbone. Therefore, DM and DA did
273
not vary with the changing of protein concentration. The concentration of FA decreased
274
from 1.71 to 1.10% after treatment for 16 h. FAE catalyzes the hydrolysis of the
275
4-hydroxy-3-methoxycinnamoyl (feruloyl) group from an esterified sugar, which is
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usually arabinose in ‘natural’ substrates. The concentration of FA decreased from 1.10 to
277
0.22% when the enzyme digestion time was 20 h. The protein concentration reduced from
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0.42 to 0.36% in response to enzymatic hydrolysis for 20 h. The reduction in the
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concentration of protein may be due to the cross-linkage of protein and FA, which also
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occurs in the product of SBPP-c. The concentrations of all the neutral sugars increased
281
slightly, and the DM and DA values were nearly invariable. ABN hydrolyzes (1→5)-α-arabinofuranosidic linkages in (1→5)-arabinans. GAL
283
specifically hydrolyzes (1→4)-β-D-galactoside linkages in type I arabinogalatans. As
284
shown in Table 1, the concentration of Ara and Gal decreased from 13.11 to 3.81% and
285
12.63 to 2.33%, respectively. These values were almost identical to those in a previous
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report (Funami et al., 2011). Moreover, the concentrations of FA and protein were
287
somewhat reduced because Ara and Gal are mainly distributed in lateral chains and some
288
phenolic acids, such as ferulic acid, are linked to the arabinose and galactose residues via
289
ester linkages (Fry, 1983). In addition, the amounts of other neutral sugars increased
290
slightly and the DA and DM values were stable.
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3.2. FT-IR analysis
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The FT-IR spectra of SBPP and modified SBPP with different enzymes are shown in
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Fig. 3. In the spectrum of SBPP, the wavelength range of 950 to 1200 cm-1 is considered
295
the ‘fingerprint’ region of carbohydrates because it enables the identification of the major
296
chemical groups (Černá et al., 2003). The five bands at 1149, 1106, 1043, 1013 and 954
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cm-1 correspond to the skeletal C-O and C-C vibration bands of glycosidic bonds and the
298
pyranoid ring (Monfregola, Leone, Vittoria, Amodeo, & De Luca, 2011). There is no
299
distinct difference among the spectra of SBPP, SBPP-b1 and SBPP-c. FAE mainly
300
hydrolyzes the feruloyl groups that are esterified with arabinose or galactose. Therefore, a
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large number of ester bonds were broken and the infrared vibrations of SBPP-d in the
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ranges of 1745-1750 and 1616-1634 cm-1, which are attributed to the esterified and
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ionized carboxyl groups of pectin molecules (Pappas et al., 2004), varied considerably,
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especially esterified carboxyl groups. The spectrum of SBPP-a1 exhibited the same
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variation trend as that of SBPP-d because PME can cut carbomethoxy (COOCH3) and
306
remove methoxyl residues, yielding polygalacturonic acid. Compared with SBPP, the
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stretching intensities of COOH at 1616-1634 cm-1 of SBPP-a3 dropped appreciably due to
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the hydrolysis of galacturonic acid.
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3.3. Enzymatic modification by PME, PAE and PG
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Food hydrocolloids perform two essential functions that determine emulsification
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effectiveness: providing good emulsifying activity and good emulsion stability (Funami
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et al., 2011). Emulsifying activity and emulsion stability were assessed by measuring the
314
droplet size of the fresh emulsion and cream index (CI), respectively. The efficacy of a
315
surface-active molecule is generally evaluated by its ability to decrease the surface
316
tension of a solution and the interfacial tension of an emulsion. According to Fig. 2a,
317
SBPP was sequentially hydrolyzed by PME, PAE and PG, yielding SBPP-a1, SBPP-a2
318
and SBPP-a3, respectively. The surface and interfacial tension of SBPP-a1 (45.2 mN/m
319
and 24.3 dyne cm-1) was significant higher than that of SBPP (44.3 mN/m and 17.5 dyne
320
cm-1) (Fig. 4) because the character of pectin is predominantly hydrophilic and the loss of
321
methyl ester groups, which are hydrophobic, results in the decrease of surface activity.
322
Yapo et al. (2007) investigated the relationship between SBPP and its surface properties
323
and the results showed that the surface and interfacial tension of the extracted pectin
324
which has higher DM were significant higher than that of the pectin with lower DM. The
325
particle size of SBPP-a1 increased from 0.409 (d3,2) and 0.621 µm (d4,3) to 0.649 and
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0.873 µm after PEM treatment. This reason may be that PME hydrolyze methyl esters of
327
pectin to carboxyl groups or pectate and methanol. The amount of carboxyl group
328
increased after the treatment of PME and High-methoxyl SBPP (DM > 50%) converted
329
to low-methoxyl SBPP-a1 (DM < 50%). The low-methoxyl pectin gels under the
330
condition of a high concentration of calcium ions. And the variation of DM has a
331
significant impact on the interaction between SBPP and calcium ions. When the DM of
332
pectin decreases to a certain degree, the increase of carboxyl groups improve the
333
sensitivity of pectin molecules toward Ca2+ (Hotchkiss et al., 2002), which will make the
334
oil droplets move closer together. Therefore, the particle size of the emulsion stabilized
335
by SBPP-a1 increased. No significant difference was found in the CI of pectins before and
336
after PME modification (Table 2).
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SBPP-a2 was obtained using PAE with SBPP-a1 as material. Compared with SBPP-a1,
338
the DA of SBPP-a2 was reduced from 23.9 to 5.1% (Table 1), whereas the surface tension
339
of the SBPP-a2 solution increased from 45.2 to 48.0 mN/m. And the interfacial tension of
340
the SBPP-a2 stabilized emulsion increased from 24.3 to 30.1 dyne cm-1 when compared
341
with that of the emulsion prepared by SBPP-a1 (Fig. 4). These results suggest that the
342
contribution of acetyl groups to the surface activity of pectin was small but not negligible.
343
Dea & Madden (1986) considered that highly acetylated SBPP is more surface-active
344
than commercial, high-methoxy or low-methoxy pectins. This results were also consistent
345
with the conclusion obtained by Zouambia, Moulai-Mostefa, & Krea (2009). As shown in
346
Table 2, the particle sizes of SBPP-a2 increased from 0.649 and 0.873 µm to 0.881 and
347
1.148 µm after PAE modification. In addition, the CI of the emulsion prepared by
348
SBPP-a2 increased significantly from 24 to 41%, indicating that the acetyl groups have an
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impact on both emulsifying stability and emulsifying capacity. This effect occurs because
350
the aggregation of oil droplets can be prevented by acetyl groups though steric and
351
mechanical stabilization effects. Moreover, acetyl groups can also decrease the sensitivity
352
of pectin molecules toward calcium ions. As a result, SBPP cannot gel at high
353
concentrations of calcium ions (Pippen, McCready, & Owens, 1950).
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SBPP-a3 was obtained by hydrolyzing SBPP-a2 with PG. Compared with SBPP-a2, the
355
GalA concentration of SBPP-a3 reduced from 46.55 to 21.67% (Table 1), and the surface
356
tension of the SBPP-a3 solution increased from 48.0 to 49.1 mN/m. And the interfacial
357
tension of the SBPP-a2 stabilized emulsion increased from 30.1 to 37.3 dyne cm-1 when
358
compared with that of the emulsion prepared by SBPP-a1 (Fig. 4). As shown in Table 2,
359
the particle size of SBPP-a3 increased from 0.881 and 1.148 µm to 0.932 and 1.161 µm
360
after PG modification. In addition, the CI of the emulsion prepared by SBPP-a3 increased
361
significantly from 41 to 45%. These results suggest that the emulsifying properties of
362
pectin declined with the decrease in GalA concentration.
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3.4. Enzymatic modification by PE, FAE and GAL or ABN
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To investigate the contribution of each functional group on the side chain to the
366
emulsifying activity and emulsifying stability of SBPP, PE, FAE, GAL and ABN were
367
applied to yield SBPP-b1, SBPP-b2, SBPP-b3 and SBPP-b4, respectively (Fig. 2b). As
368
shown in Fig. 4, the surface and interfacial tension of SBPP changed greatly after the
369
synergistic effect of pepsin and a food-grade acid protease. With the protein
370
concentration decreasing from 5.2 to 0.42%, the surface tension of the SBPP-b1 solution
371
increased from 44.4 to 54.4 mN/m at 25 °C. And the interfacial tension of the SBPP-b1
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stabilized emulsion increased from 17.5 to 29.4 dyne cm-1 when compared with that of
373
the emulsion prepared by SBPP-b (Fig. 4). These results demonstrated that the surface
374
activity of SBPP reduced as the protein concentration decreased. This is due to the
375
protein itself has high surface activity. This observation was consistent with those of
376
Leroux et al. (2003) and Funami et al. (2007) who both indicated that reducing the level
377
of protein in the SBPP significantly lowered its surface active properties. Compared with
378
SBPP, the particle size of the emulsion stabilized by SBPP-b1 increased from 0.409 (d3,2)
379
and 0.621 µm (d4,3) to 2.636 and 3.360 µm, which indicated that protein concentration
380
had a positive effect on the emulsifying capacity of pectin. This result can be explained
381
by the hydrophobic behavior of protein, which acts as ‘bridge’ between the
382
polysaccharide and the oil phase. Therefore, pectin molecules can adsorb onto the
383
oil–water interface as an anchor, whereas the carbohydrate moiety forms a hydrated layer,
384
preventing the aggregation or coalescence of the emulsion droplets (Nakamura, Yoshida,
385
Maeda, Furuta, & Corredig, 2004). The CI of the emulsion stabilized by SBPP-b1
386
increased from 23 to 70%, which showed that protein levels also has a strong impact on
387
the stability of emulsion. This result was consistent with the results from Leroux et al.
388
(2003) who showed that protein associated with the pectin played a key role in the
389
stabilization of the rapeseed oil or orange oil emulsions.
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The surface tension of the SBPP-b2 solution and the properties of the emulsion, which
391
was prepared using SBPP-b2 as the surfactant, were changed greatly when compared with
392
those of SBPP-b1. As shown in Fig. 4, the surface tension of the SBPP-b2 solution
393
increased sharply from 54.4 to 58.0 mN/m and the interfacial tension of the SBPP-b2
394
stabilized emulsion increased from 29.4 to 40.0 dyne cm-1 when compared with that of
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the emulsion prepared by SBPP-b1, which suggested that ferulic acid had an important
396
effect on the surface activity of pectin. This may be the reason that ferulic acid itself is
397
hydrophobic and, like protein, acts as a ‘bridge’ connection between the oil droplet and
398
the polysaccharide. Siew and Williams (2008) also revealed that the adsorbed fractions of
399
pectin sample were rich in protein and ferulic acid and speculated that the reduction in
400
surface tension of sugar beet pectin may be due to its higher protein and/or ferulic acid
401
concentrations. The particle size of the emulsion stabilized by SBPP-b2 increased from
402
2.636 (d3,2) and 3.360 µm (d4,3) to 6.032 (d3,2) and 8.110 µm (d4,3), which indicated that
403
ferulic acid, in the absence of protein, plays a key role in the emulsifying capacity of
404
pectin. Furthermore, the impact of ferulic acid concentration on the emulsifying stability
405
of pectin is significant according to the CI data shown in Table 2.
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Then, based on SBPP-b2, SBPP-b3 and SBPP-b4 were obtained through the
407
modification of GAL and ABN, respectively. No significant difference in the surface and
408
interfacial activity or emulsifying capacity was found between SBPP-b2 and SBPP-b3 or
409
SBPP-b4 by comparing the data for surface tension (Fig. 4) and particle sizes (Table 2).
410
However, the CI values of both emulsions, stabilized by SBPP-b3 (78%) or SBPP-b4
411
(79%), increased slightly. This finding suggested that the carbohydrate moiety in pectin
412
molecules exhibited a stabilizing capability, though the effect was much lower than that
413
of the proteinaceous and ferulic acid materials. This finding was in line with the paper
414
reported by Dickinson (2003) who suggested that the presence of non-adsorbing moieties
415
could affect creaming stability by providing the protective layer that confers effective
416
steric stabilization and inducing depletion flocculation during extended storage. What’s
417
more, the changes of the viscosity in continuous phase, caused by enzyme modification,
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418
may also have an effect.
419 420
3.5. Enzymatic modification by GAL/ABN and FAE Oligosaccharide-protein complexes always exhibit good emulsifying properties
422
(Akhtar & Dickinson, 2003). In series b of the enzymatic modification (Fig. 2b), protein
423
and ferulic acid were almost completely hydrolyzed, and we could not determine the
424
impact of the oligosaccharide-protein complexes on the emulsifying properties of pectin.
425
Hence, in this section, ferulic acid-oligosaccharide-protein complexes were removed by
426
catalysis with GAL and ABN. In this way, we were able to determine the effects of
427
ferulic acid-oligosaccharide-protein complexes on the emulsion properties. Another
428
phenomenon observed in series b was that the particle size of the emulsion stabilized by
429
FAE modified pectin (SBPP-b2) increased rapidly. Based on this result, we can conclude
430
that the ferulic acid concentration plays an important role in the emulsifying properties of
431
pectin, but only under low protein concentration conditions. To investigate the influence
432
of ferulic acid concentration on the emulsifying properties of SBPP, especially under high
433
protein levels, SBPP was directly modified by FAE in series d.
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SBPP-c and SBPP-d were hydrolyzed with GAL/ABN and FAE from SBPP,
435
respectively. The surface tension of the SBPP-c solution was 54.6 mN/m, which was
436
significant (p < 0.05) higher than that of SBPP (44.4 mN/m) and slightly higher with that
437
of SBPP-b1 (54.4 mN/m) but lower than that of SBPP-d (55.8 mN/m). These results
438
suggested that the covalently linked ferulic acid-arabinogalactan-protein conjugate plays
439
a more important role in the surface activity of pectin than protein alone but a less
440
important role than that of ferulic acid. The interfacial tension of the emulsions stabilized
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by SBPP-c (38.3 dyne cm-1) and SBPP-d (38.6 dyne cm-1) had little difference but
442
significant (p < 0.05) higher than that of SBPP (17.5 dyne cm-1). As shown in Table 2,
443
the particle sizes of the emulsion prepared by SBPP-c were 1.920 (d3,2) and 2.760 µm
444
(d4,3), which are much higher than those of SBPP (0.409 and 0.621 µm for d3,2 and d4,3,
445
respectively) and SBPP-b1 (2.636 and 3.360 µm for d3,2 and d4,3, respectively) but lower
446
than those of SBPP-d (3.050 and 4.142 µm for d3,2 and d4,3, respectively). These results
447
indicated that the effect of functional groups on the emulsifying activity decreased in the
448
following order: ferulic acid > ferulic acid-arabinogalactan-protein complexes > protein.
449
The CI of the emulsion stabilized by SBPP-c (69%) was much higher than that of SBPP
450
(23%) but similar to that of SBPP-b1 (70%) and SBPP-d (72%), which suggested that
451
carbohydrate covalently bound to a small amount of protein and ferulic acid exhibited
452
excellent emulsifying stability.
454
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3.6. Emulsifying mechanism of sugar beet pectin A deduced mechanism was drawn schematically (Fig. 5) to explain the emulsifying
456
properties of sugar beet pectin. The ferulic acid and proteinaceous materials (hydrophobic
457
groups) adsorb onto the surface of emulsion droplets, decreasing the interfacial tensions
458
between the water and oil phases. It is evident that the emulsification properties of sugar
459
beet pectin are influenced by the accessibility of the protein and ferulic acid groups to the
460
surface of the oil droplets (Williams et al., 2005). Protein and ferulic acid make a main
461
contribution to the emulsifying capacity of SBPP. The lateral chains of SBPP, especially
462
arabinose and galactose, connect the main chain with protein and ferulic acid. The
463
presence of the non-adsorbing lateral chains mainly affect creaming stability by steric
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effects (Dickinson, 2003). Polygalacturonic acid (main chain), together with neutral
465
sugars in the side chain, forms a layer of film, protecting oil droplets from aggregation
466
and fusion. The thickness of the film is affected greatly by the concentration of GalA
467
(Funami et al., 2011). In addition, methyl and acetyl groups in the main chain play a role
468
in preventing carboxyl groups of GalA from moving toward calcium ions. GalA, methyl
469
and acetyl groups in the main chain make a main contribution to the emulsifying stability
470
of the emulsion prepared by SBPP.
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471
4. Conclusion
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The impact of each functional group on the emulsifying properties of SBPP was
474
investigated by enzymatic modification using seven enzymes in a particular order. The
475
structures of SBPPs were characterized based on FT-IR. In addition, the contribution of
476
each functional group was evaluated based on the variation in emulsion characteristics.
477
The results showed that the composition of SBPP or enzymatically modified SBPPs
478
varied in a broad range after enzymatic hydrolysis. FTIR spectroscopy showed that the
479
characteristic absorption intensities of SBPP had no distinct difference from the spectra
480
of SBPP-b1 and SBPP-c, but had an obvious variation, especially in the ranges of
481
1745-1750 and 1616-1634 cm-1, which are attributed to the esterified and ionized
482
carboxyl groups, when compared with SBPP-a1, SBPP-a2, SBPP-a3, and SBPP-d. Protein,
483
ferulic acid-araban/galactan-protein complexes and ferulic acid played an important role
484
in improving the surface activity, emulsifying capacity and emulsifying stability of SBPP.
485
The decrease of methyl ester groups mainly affected the particle sizes of the emulsion. In
486
addition to particle sizes, the cream index of the emulsion was significantly affected by
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487
acetyl groups. Arabinose and galactose less affected emulsifying properties than other
488
functional groups.
489
Abbreviations
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SBPP, sugar beet pulp pectin; FT-IR, Fourier Transform Infrared Spectroscopy; GalA,
492
galacturonic acid units; PG, polygalacturonase; ABN, arabinanase; GAL, galactanase; PE,
493
protease (combination of pepsin and food-grade acid protease) FAE, feruloyl esterase;
494
PME, pectin methyl esterase; PAE, pectin acetyl esterase; MCT, medium-chain
495
triglyceride; Rha, rhamnose; Ara, arabinose; Xyl, xylose; Gal, galactose; Glc, glucose;
496
GalA, galacturonic acid; FA, ferulic acid; DM, degree of methylation; DA, degree of
497
acetylation; CI, cream index.
498
Acknowledgements
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This research was supported by the Guangzhou Science and Technology Program
501
(2013J4500036), the National Natural Science Foundation of China (21376097), the
502
program for New Century Excellent Talents in University (NCET-13-0212), the
503
Guangdong Natural Science Foundation (S2013010012318), and the Fundamental
504
Research Funds for the Central Universities, SCUT (2013ZZ0070).
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Table 1 Monosaccharide composition, galacturonic acid, protein, ferulic acid and degree of esterification of different enzymatically modified sugar beet pectinsA
586
Protein
Ferulic acid
(%)
(%)
DM
DA
1.21±0.01a
67±0.4b
23.9±0.2a
5.48±0.03b
1.17±0.01b
7.5±0.1e
23.6±0.3a
46.55±0.45d
5.75±0.02a
1.20±0.01a
8.0±0.1d
5.1±0.6c
3.26±0.02a
21.67±0.31i
5.14±0.04c
1.11±0.01c
8.1±0.3d
5.3±0.3c
2.29±0.02f
1.22±0.01g
45.22±0.37e
0.42±0.00f
1.10±0.01c
69±0.5a
23.5±0.6a
12.63±0.05b
2.35±0.01e
1.28±0.01f
45.37±0.44e
0.36±0.01g
0.22±0.00e
67±0.7b
23.8±0.3a
15.36±0.05b
2.33±0.04h
2.76±0.01d
1.56±0.01c
47.06±0.58c
0.26±0.01h
0.16±0.00g
68±0.6ab
24.3±0.5a
13.96±0.03b
3.81±0.01i
2.89±0.01g
2.88±0.01c
2.01±0.03b
48.59±0.21b
0.21±0.00i
0.12±0.00h
67±0.7b
23.9±0.2a
SBPP-c
14.46±0.02a
3.11±0.03j
2.05±0.01i
3.88±0.01b
2.01±0.01b
58.26±0.39a
2.83±0.01e
0.66±0.01d
68±0.8ab
24.0±0.3a
SBPP-d
7.56±0.02e
9.61±003g
EP
Constitution sugars (%) Sample
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585
10.59±0.03d
1.32±0.01e
44.26±0.13g
4.13±0.02d
0.17±0.00f
65±0.5c
22.3±0.3b
Ara
Gal
Glc
Xyl
GalA
SBPP
6.72±0.03g
9.03±0.04h
9.86±0.07f
0.66±0.00j
1.00±0.01h
43.57±0.13h
5.20±0.02c
SBPP-a1
7.23±0.05f
9.70±0.02f
10.22±0.04e
0.74±0.01i
1.23±0.02g
44.82±0.20f
SBPP-a2
7.55±0.06e
9.91±0.06e
10.62±0.07d
0.88±0.01h
1.40±0.01d
SBPP-a3
14.02±0.04b
27.19±0.09a
25.92±0.04a
5.27±0.03a
SBPP-b1
10.81±0.07d
12.94±0.04d
12.06±0.02c
SBPP-b2
10.96±0.09d
13.11±0.03c
SBPP-b3
13.57±0.03c
SBPP-b4
588
A
589
(a-j) are significantly different (p < 0.05).
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587
0.98±0.01g
SC
Rha
The data are averages and standard deviations of triplicate measurements. Values in each column with different superscript letters
ACCEPTED MANUSCRIPT
590
Table 2. The oil droplet sizes and creaming index (CI) of emulsions stabilized by SBPP
591
and modified SBPP Samples
Particle size A d4,3 (µm)
SBPP
0.409 ± 0.003i
0.621 ± 0.004i
SBPP-a1
0.649 ± 0.003h
0.873 ± 0.005h
SBPP-a2
0.881 ± 0.005g
1.148 ± 0.007g
SBPP-a3
0.932 ± 0.006f
1.161 ± 0.010g
SBPP-b1
2.636 ± 0.021d
3.360 ± 0.028e
70 ± 2cd
SBPP-b2
6.032 ± 0.034b
8.110 ± 0.041c
76 ± 2b
SBPP-b3
6.150 ± 0.036a
8.340 ± 0.036b
78 ± 2ab
SBPP-b4
6.155 ± 0.038a
8.492 ± 0.047a
79 ± 2a
SBPP-c
1.920 ± 0.018e
2.760 ± 0.022f
69 ± 1d
SBPP-d
3.050 ± 0.027c
4.142 ± 0.031d
72 ± 2c
SC
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24 ± 1g 41 ± 1f
45 ± 1e
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each column with different superscript letters (a-i) are significantly different (p < 0.05).
EP
595
23 ± 1h
The data are averages and standard deviations of triplicate measurements. Values in
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A
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d3,2 (µm)
592 593
CI (%)
29
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596
Figure captions
597
Fig. 1. Schematic figure of SBPP and enzymes used in the experiments. GalA:
599
Galacturonic acid; Rha: Rhamnose; Gal: Galactose; Ara: Arabinose; FA: Ferulic acid.
600
Fig. 2. Flow chart for the enzymatic modification of SBPP.
601
Fig. 3. FT-IR spectra of SBPP and modified SBPPs.
602
Fig. 4. The surface tension of SBPP and modified SBPP solutions.
603
Fig. 5. Schematic drawing of the emulsion stabilized by sugar beet pectin.
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Fig. 1. Schematic figure of SBPP and enzymes used in the experiments. GalA: Galacturonic acid; Rha: Rhamnose; Gal: Galactose; Ara: Arabinose; FA: Ferulic acid.
31
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Fig. 2. Flow chart for the enzymatic modification of SBPP.
32
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SBPP-a1 SBPP-a2
SBPP-a3
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SBPP-b1 SBPP-c
COOCH3 2000
1800
COOH 1600
1400
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SBPP-d SBPP
1200
1000
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Wavenumber （ cm-1）
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Fig. 3. FT-IR spectra of SBPP and modified SBPPs.
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800
40
30
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50
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55
34
at er
45
SC
60
SB SB PP P SB P-c PP w -d
S SB BP P P SB P-b P 1 SB P-b P 2 SB P-b PP 3 -b 4
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S SB BPP P SB P-a P 1 SB P-a PP 2 -a 3
surface tension (mN/m) 50
S SB BP P P SB P-c PP 1 W d at er
EP 40
S SB BP P SB PPb P 1 SB P-b 2 SB PPPP b3 -b 4
S SB BP P P SB Pa P 1 SB P-a PP 2 -a 3
-1
Interfacial tension (dyne cm )
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70
65
20
10
0
Fig. 4. The surface and interfacial tension of SBPP and modified SBPP solutions.
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Ca2+
Ca2+
Ca2+
Ca2+
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Ca2+
:C
Main chain
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Emulsion droplets
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Ca2+
Ca2+
Ferulic acid
Side chain
Protein
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Fig. 5. Schematic drawing of the emulsion stabilized by sugar beet pectin
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ACCEPTED MANUSCRIPT Seven enzymes were studied in a particular order. Compositions of SBPP and enzymatically modified SBPPs were determined. The structures of SBPPs were characterized
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Ferulic acid was an important factor on emulsifying ability of SBPP.
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The contribution of each functional group to emulsifying ability was evaluated.
S0268-005X(15)30096-5
DOI:
10.1016/j.foodhyd.2015.09.021
Reference:
FOOHYD 3144
To appear in:
Food Hydrocolloids
Received Date: 1 May 2015 Revised Date:
22 August 2015
Accepted Date: 18 September 2015
Please cite this article as: Chen, H.-m., Fu, X., Luo, Z.-g., Effect of molecular structure on emulsifying properties of sugar beet pulp pectin, Food Hydrocolloids (2015), doi: 10.1016/j.foodhyd.2015.09.021. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Ca2+ Ca2+
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Emulsion droplets Ca2+
Side chain
Ca2+
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Ferulic acid
Ca2+
Protein Main chain
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1
Effect of molecular structure on emulsifying properties of sugar beet
2
pulp pectin
4
Hai-ming Chen1,2, Xiong Fu1, Zhi-gang Luo1*
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1. College of Light Industry and Food Sciences, South China University of Technology,
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381 Wushan Road, Guangzhou 510640, PR China
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2. College of Food Sciences & Engineering, Hainan University, 58 People Road, Haikou,
9
China
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* Corresponding author.
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Tel.: +86 20 8711 3845; Fax: +86 20 8711 3848
13
Email: [email protected] (Z. G. Luo).
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Abstract: To investigate the impact of each functional group on the emulsifying
16
properties of sugar beet pulp pectin (SBPP), seven enzymes were studied in a particular
17
order. Compositions of SBPP and enzymatically modified SBPPs were determined, and
18
the structures of SBPPs were characterized by FT-IR. In addition, the contribution of
19
each functional group was evaluated based on the variation in emulsion characteristics.
20
The results showed that protein, ferulic acid-araban/galactan-protein complexes and
21
ferulic acid played important roles in improving the surface activity, emulsifying capacity
22
and emulsifying stability of SBPP and the extent of the decrease in the emulsifying
23
activity followed the order: ferulic acid > ferulic acid-arabinogalactan-protein complexes
24
> protein. The decrease of methyl ester groups mainly affected the particle sizes of the
25
emulsion. In addition to particle sizes, the cream index of the emulsion increased with the
26
hydrolysis of acetyl groups. Arabinose and galactose less affected emulsifying properties
27
than other functional groups.
28
Keywords: Sugar beet pulp pectin, Enzyme, Structure, Emulsifying property.
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1. Introduction Pectin is widely used in the food industry for its gelling, thickening, and stabilizing
31
properties (Voragen, Pilnik, Thibault, Axelos, & Renard, 1995; Funami, Zhang, Hiroe,
32
Noda, Nakauma, Asai, Cowman, Al-Assaf, & Phillips, 2007). Commercial pectins are
33
extracted from citrus peel and apple pomace in most instances (Funami, Nakauma,
34
Ishihara, Tanaka, Inoue, & Phillips, 2011). A relatively new type of pectin, sugar beet
35
pulp pectin (SBPP), has recently received much attention (Siew and Williams 2008;
36
Fissore, Rojas, Gerschenson, & Williams, 2013).
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As shown in Fig. 1, SBPP is a heteropolysaccharide with a chain structure of
38
(1→4)-linked α-D-galacturonic acid (GalA) units interrupted by the insertion of
39
(1→2)-linked L-rhamnopyranosyl residues. Rhamnosyl residues (20 ~ 80%) can be
40
substituted with side chains (‘hairy’ region) consisting of neutral sugars, such as
41
D-galactose, L-arabinose, D-xylose, D-glucose, D-mannose, L-fucose and D-glucuronic
42
acid. In addition, lateral chains contain phenolic acids such as ferulic acid, which are
43
linked to the arabinose and galactose residues via ester linkages (Fry, 1983). In addition,
44
there is a higher concentration of the proteinaceous materials bound to the side chains
45
through covalent linkages (Williams, Sayers, Viebke, Senan, Mazoyer, & Boulenguer,
46
2005). Some of the acid groups of the GalA in the linear chain structure (‘smooth’ region)
47
can be partially methyl-esterified and O-acetylated at the C-2 and/or C-3 positions (Siew
48
& Williams, 2008). Compared to other conventional pectins, SBPP tends to exhibit a
49
higher degree of acetylation (DA) and a greater number of neutral sugar side chains (rich
50
in hairy regions) (Siew & Williams, 2008). In addition, SBPP has a greater number of
51
feruloyl groups attached to the galactose and arabinose side chains (Colquhoun, Ralet,
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Thibault, Faulds, & Williamson, 1994; Guillon, Thibault, Rombouts, Voragen, & Pilnik,
53
1989; Ralet, Thibault, Faulds, & Williamson, 1994; Rombouts & Thibault, 1986) and a
54
greater amount of proteinaceous material bound to the lateral chains through covalent
55
linkages (Funami et al., 2007; Williams et al., 2005). Because of these differences in
56
structural characteristics, SBPP does not have the capability to form gels like
57
conventional pectins, but it possesses excellent emulsifying properties (Voragen et al.,
58
1995; Funami et al., 2007; Williams et al., 2005; Li et al., 2012). According to Endreß &
59
Rentschler (1999), the emulsifying ability of beet pectin can be explained by the high
60
percentage of acetyl groups in its chemical structure. Nevertheless, Leroux, Langendorff,
61
Schick, Vaishnav, & Mazoyer, (2003) studied the emulsifying ability of sugar beet pectin
62
in relation to its chemical structure and concluded that there was no evidence that its
63
emulsifying ability correlates with the number of acetyl groups in its structure but, rather,
64
is related to its high concentration of proteinaceous components (Williams et al., 2005).
65
Siew & Williams (2008) suggested that proteins and/or ferulic acid groups adsorb onto
66
the surfaces of the oil droplets and stabilize the emulsions. Leroux et al., (2003)
67
concluded that a highly methyl-esterified pectin is able to reduce the interfacial tension
68
between the water and the oil phases. It is likely its hydrophobicity (due to its
69
COOCH3-groups) that gives pectin its emulsifying properties. SBPP was fractionated
70
using hydrophobic affinity chromatography, and three fractions with different proportions
71
of protein, ferulic acid and weight-average molecular mass were obtained (Williams et al.,
72
2005). They concluded that the emulsification properties of SBPP were influenced by the
73
protein, ferulic acid groups, proportion of ester groups, and molecular mass distribution
74
of the fractions. SBPPs were structurally modified using protease to degrade the
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proteinaceous moiety, polygalacturonase (PG) to cleave the carbohydrate backbone, and
76
a arabinanase/galactanase (ABN/GAL) combination to cleave the lateral chains (Funami
77
et al., 2011). Methyl and acetyl groups were removed with galacturonic acid (GalA) via
78
the modification of PG. In addition, protein and ferulic acid exist in the side chain
79
(mainly covalently bonding with arabinose and galactose); therefore, the combination of
80
ABN/GAL can alter four factors (arabinose, galactose, protein and ferulic acid) at the
81
same time. None of the previous papers provided a clear explanation for the relationship
82
between each factor and the emulsification ability of pectins.
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The objective of the present study was to investigate the emulsifying properties of
84
SBPP in relation to its structural characteristics. SBPPs were structurally modified using
85
protease
86
endo-α-1,4-polygalacturonase
87
4-galactanase (GAL), feruloyl esterase (FAE), pectin methyl esterase (PME) and pectin
88
acetyl esterase (PAE) to degrade the proteinaceous moiety, GalA unit, arabinose,
89
galactose, ferulic acid, DM and DA subunits, respectively. The contribution of each of
90
these structural units to the emulsification of SBPP was assessed through enzymatic
91
modification in a particular order.
of
pepsin
food-grade
acid
endo-β-1,5-arabinanase
protease,
(ABN),
PE),
endo-β-1,
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(PG),
and
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(combination
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93
2. Materials and methods
94
2.1. Materials and chemicals
95
Sugar beet pulp pectin was purchased from CP Kelco (Lille Skensved, Denmark).
96
Medium-chain triglyceride (MCT) was purchased from the Nisshin Oillio Group (Tokyo,
97
Japan). GalA and bovine serum albumin were purchased from Sigma-Aldrich Chemical
ACCEPTED MANUSCRIPT
Co. (Milwaukee, WI, USA). PG (EC 3.2.1.15), ABN (EC 3.2.1.99), GAL (EC 3.2.1.89),
99
and FAE (EC 3.1.1.73) were obtained from Megazyme International Ireland Ltd (Bray,
100
Ireland). PME (E.C. 3.1.1.11) and PAE (E.C. 3.1.1.6) were purchased from
101
Sigma-Aldrich Chemical Co. (Milwaukee, WI, USA). Pepsin (E.C. 3.4.23.1) and
102
food-grade acid protease were purchased from Aladdin reagents Co., Ltd (Shanghai,
103
China). The Viscozyme L9 enzyme was a commercial preparation obtained from Novo
104
Nordisk (Copenhagen, Denmark), and because it was a multienzyme complex,
105
purification was required before use (Garna, Mabon, Wathelet, Paquot, 2004).
106
2-Deoxy-D-glucose, myo-inositol, L-rhamnose (Rha), L-arabinose (Ara), D-xylose (Xyl),
107
D-galactose (Gal), D-glucose (Glc), D-galacturonic acid (GalA), ferulic acid (FA),
108
succinic acid, glacial acetic acid and methanol were purchased from Sigma-Aldrich
109
Chemical Co. (Milwaukee, WI, USA). All other chemicals were of analytical grade
110
unless otherwise noted.
112
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2.2. Enzymatic modification by PME, PAE and PG In this section, SBPP was enzymatically modified by PME, PAE and PG in turn as
114
shown in Fig. 2a. Firstly, SBPP was diluted to 1.0 w/v% in a 100 mM citrate buffer (pH
115
7.0) with mechanical shearing at 2000 rpm for 15 min. Into these dispersions, PME (30
116
unit per 1.0 g SBPP) was added and incubated in a sealed conical flask at 30 °C for 16 h
117
with gentle stirring (Hotchkiss et al., 2002). The hydrolyzed mixture was heated at 100
118
°C for 1 min to stop the enzymatic reaction and dialyzed against distilled de-ionized
119
water at 20 °C through a dialysis membrane with a 10 kg/mol molecular weight cut off,
120
followed by freeze drying. The freeze dried samples (SBPP-a1) were stored in a
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desiccator at 20 °C until use. Secondly, SBPP-a1 was diluted to 1.0 w/v% in a 100 mM
122
citrate buffer (pH 6.0) and PAE (10,000 unit per 1.0 g SBPP) was added and incubated at
123
30 °C for 30 min with gentle stirring. The reaction was stopped by treatment at 100 °C
124
for 1 min, and the hydrolyzed mixture was dialyzed and freeze-dried. Thirdly, The freeze
125
dried samples (SBPP-a2) was diluted to 1.0 w/v% in a 100 mM citrate buffer (pH 3.5)
126
with mechanical shearing at 2000 rpm for 15 min. PG (10 unit per 1.0 g SBPP) was
127
added and incubated at 40 °C for 20 h with gentle stirring (Funami et al., 2011). The
128
reaction was stopped by heating at 100 °C for 1 min, and the hydrolyzed mixture was
129
dialyzed and freeze-dried. The freeze dried samples (SBPP-a3) were stored in a desiccator
130
at 20 °C until use.
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131 132
2.3. Enzymatic modification by PE, FAE and GAL or ABN.
SBPP was enzymatically modified by PE, FAE and GAL or ABN in turn as shown in
134
Fig. 2b. First, protein degradation was accomplished following the method of Funami et
135
al. (2011) using pepsin (700 unit per 1.0 g SBPP) and food-grade acid protease (70 unit
136
per 1.0 g SBPP) at pH 3.0 and 30 °C. SBPP-b1 was obtained by enzyme deactivation,
137
dialysis and freeze-drying as described above. Then, SBPP-b1 was catalyzed by FAE (0.3
138
unit per 1.0 g SBPP) at pH 6.0 and 50 °C for 20 h. SBPP-b2 was obtained after enzyme
139
deactivation, dialysis and freeze-drying. Finally, SBPP-b3 or SBPP-b4 was obtained from
140
SBPP-b2, which was enzymatically modified by ABN (7 units per 1.0 g SBP) or GAL (5
141
units per 1.0 g SBP) at pH 3.5 and 40 °C for 24 h.
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142 143
2.4. Combined enzymatic modification by GAL and ABN
ACCEPTED MANUSCRIPT
SBPP was enzymatically modified by GAL combined with ABN as shown in Fig. 2c.
145
SBPP was diluted to 1.0 w/v% in a 100 mM citrate buffer (pH 3.5) with mechanical
146
shearing at 2000 rpm for 15 min. Into these dispersions, ABN (7 units per 1.0 g SBP) and
147
GAL (5 units per 1.0 g SBP) were added and incubated in a sealed conical flask at 40 °C
148
for 24 h with gentle stirring (Funami et al., 2011). SBPP-c was obtained by enzyme
149
deactivation, dialysis and freeze-drying.
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151
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2.5. Enzymatic modification by FAE
SBPP was enzymatically modified by FAE directly as shown in Fig. 2d. SBPP was
153
diluted to 1.0 w/v% in a 100 mM citrate buffer (pH 6.0) with mechanical shearing at
154
2000 rpm for 15 min. Into these dispersions, FAE (0.3 units per 1.0 g SBP) was added
155
and incubated in a sealed conical flask at 50 °C for 20 h with gentle stirring. SBPP-d was
156
obtained by enzyme deactivation, dialysis and freeze-drying.
158
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2.6. Determination of concentrations of galacturonic acid and neutral sugar Pectin samples were hydrolyzed using a combination of chemical and enzymatic
160
hydrolysis as described by Garna et al. (2004). Pectin (100 mg) was hydrolyzed with 0.2
161
mol/L trifluoroacetic acid (TFA) (5 mL) at 80 °C for 72 h. The hydrolysate was adjusted
162
to pH 5 with NH4OH (14 mol/L) and diluted to 25 mL. Ten milliliters of this solution was
163
mixed with 10 mL of purified Viscozyme L9 (Novo Nordisk, Denmark) in a 20 mmol/L
164
pH 5 sodium acetate buffer. The GalA concentration was measured using a Dionex
165
DX-500 Bio-LC system (Dionex Corp., Sunnyvale, Calif., USA) equipped with a
166
CarboPAc PA1 column (250 × 4 mm; Dionex Corp., Sunnyvale, Calif., USA) in
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combination with a CarboPac guard column (25 × 4 mm; Dionex Corp., Sunnyvale,
168
Calif., USA). All measurements were carried out at a temperature of 30 °C and with a
169
gradient reaching 170 mmol/L CH3COONa·3 H2O and 100 mmol/L NaOH for 13 min at
170
a flow rate of 1 mL/min. The column was washed with 100 mmol/L NaOH for 10 min
171
before the next injection. Twenty-five microliters of sample was injected. Detection was
172
realized using a pulsed amperometric detector with a post-injection flow rate of 200
173
µL/min of 900 mmol/L NaOH.
174
2.7. Determination of protein concentration
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A Bradford protein assay kit (Pierce; Thermo Fisher Scientific Inc., USA) was used to
177
determine the total protein concentration of the pectin samples based on their absorbance
178
at 595 nm, which is the wavelength of the color produced by the reaction between the
179
protein and Coomassie Brilliant Blue G-250. BSA was used as the standard for
180
calibration within the working range of 0 to 750 mg/mL (Funami et al., 2011).
182
2.8. Degree of esterification determination
EP
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Methoxy and acetyl groups were released from pectin by saponification with 0.2 mol/L
184
NaOH in 50% isopropanol at 4 °C for 2 h. Methanol and acetic acid levels were
185
determined by HPLC on an Aminex HPX 87H column (300 × 7.8 mm; Bio-Rad
186
Laboratories, Inc., Hercules, CA, USA) using succinic acid as the internal standard.16
187
Degree of methylation (DM, mol of MeOH per 100 mol of galacturonic acid) and degree
188
of acetylation (DA, mol of AcOH per 100 mol of galacturonic acid) were calculated from
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189
the amount of MeOH and AcOH, respectively, and from the amount of galacturonic acid
190
(Voragen et al., 1986).
191
2.9. Fourier-transform infrared spectroscopy (FT-IR)
193
The FT-IR spectra of pectin samples were recorded using a Perkin Elmer Spectrum
194
RXIFT-IR Spectrometer (Perkin Elmer Instruments, USA) at room temperature. The
195
sample powder was blended with KBr powder and pressed into tablets before spectra
196
were obtained over the range of 2000 to 800 cm-1 with an 8 cm-1 resolution.
197
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2.10. Evaluation of emulsifying properties
199
2.10.1 Emulsion preparation
A SBPP or modified-SBPP solution was prepared by dissolving 1 g of pectin in 80 mL
201
of ultrapure water with continuous stirring at room temperature for 24 h. Into this solution,
202
1 mL of a 10% (w/v) benzoic acid solution was added as a preservative, and citric acid
203
(10%, w/v) was used to adjust the final pH of the emulsions to 3.0. Then, distilled
204
de-ionized water and 15 g of MCT were added to achieve a final mass of 100 g. The
205
mixtures were then pre-homogenized using an Ultra-Turrax device at a speed of 24,000
206
rpm for 3 min to form coarse emulsions, which were subsequently homogenized over
207
three passes using an ultra-high-pressure homogenizer (Nano DeBEE, USA) operated at
208
50 MPa.
AC C
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209 210
2.10.2. Determination of the surface and interfacial tension
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The surface tension of the SBPP and modified-SBPP solutions was measured at 25 °C
212
using a DCAT21 surface tensiometer (Dataphysics Co., Germany). The measuring cup
213
was placed in a stainless steel jacket on a perpendicularly movable platform. A platinum
214
plate (24 mm × 10 mm × 0.1 mm) was gently placed at the bottom of a sensitive
215
electronic balance and immersed in the measured solutions by slowly raising the platform.
216
The force (mN/m) required to detach the plate from the surface was recorded as the
217
dynamic surface tension. Finally, the surface tension was calculated based on
218
balance-force measurements by applying the appropriate correction factors (Li et al.,
219
2013). Interfacial tension measurements were carried out at the oil/water interface using
220
1.0% (w/w) pectin solution prepared at room temperature by dissolving dried SBPP and
221
modified-SBPP in fresh Milli-Q water containing 0.02% sodium azide as a bacteriocide.
222
A constant volume of 1.0% (w/w) pectin solution was gently poured into a thermostated
223
glass dish at 23 °C. Immediately afterwards, fish oil was gently layered on top of the
224
pectin solution. The interfacial tension was measured after 1 h of equilibrium from the
225
pull on the mica plate. Measurements were performed three times for each solution (Yapo,
226
Robert, Etienne, Wathelet, & Paquot, 2007).
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2.10.3. Particle-size analysis of the emulsions
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229
A Malvern Mastersizer 2000 (Malvern, UK) was used to determine the droplet sizes of
230
the emulsions using static multi-angle light-scattering analysis, which was performed in
231
triplicate. A few drops of the emulsion were added to the water in the dispersion unit of
232
the instrument with stirring at 2000 rpm until the degree of obscuration was
233
approximately 15%. Values of 1.45 and 0.001 were used for the refractive index and
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absorption index of MCT, respectively, and values of 1.33 and 0 were used for the
235
refractive index and absorption index of the water dispersant, respectively. The average
236
size of the droplets in the emulsions was assessed in terms of the surface-weighted mean
237
diameter d32 and volume-weighted mean diameter d43 (Li et al., 2012).
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238 239
2.10.4. Evaluation of emulsion storage stability
Freshly prepared emulsions were heated in a water bath at 80 °C for 30 min followed
241
by centrifugation at 8000 g for 10 min. The extent of creaming was characterized by the
242
creaming index (CI, %), which was determined according to the method developed by
243
Sciarini, Maldonado, Ribotta, Perez, & Leon, (2009) as follows:
244
CI = (height of the serum layer/total height of the emulsion) × 100%
245
2.11. Statistical analysis
(1)
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Analyses of variance were performed, and the mean values ± standard deviations were
248
evaluated by Duncan’s multiple-range test (p < 0.05) using SPSS version 13.0 statistical
249
software (SPSS Inc., Chicago, IL, USA). Origin (Origin Lab Co., Pro.8.0, USA) software
250
was used for data processing and to create charts.
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3. Results and discussion
253
3.1. Influence of enzymes on the component of SBPP
254
The components of enzymatically modified SBPPs are shown in Table 1. PME was
255
used to hydrolyze methyl esters of pectin to pectate and methanol. PAE was used to
256
hydrolyze acetyl residues, yielding polygalacturonic acid. After hydrolysis by the
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257
respective enzymes for 30 min, DM and DA values were reduced from 67 and 23.9 to 7.5
258
and 5.1%, respectively. The concentrations of other components increased slightly. PG randomly hydrolyzes (1→4)-α-D-galactosiduronic linkages in pectate and other
260
galacturonans. In our research, the concentration of GalA decreased from 43.57 to
261
21.67% when the enzyme digestion time was 24 h. Along with GalA, methyl and acetyl
262
groups were hydrolyzed. However, DM and DA are expressed as mol of methyl and
263
acetyl groups, respectively, per 100 mol of GalA (Fry, 1983). Therefore, the values of
264
DM and DA were unchanged. The impact of PG on pectin mainly occurs in the backbone.
265
Therefore, the concentrations of the neutral sugars, which mainly distribute in the side
266
chains, increased sharply.
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Pepsin and food-grade acid protease were used to decompose the proteinaceous
268
materials. As shown in Table 1, the concentration of protein decreased from 5.2 to 0.42%
269
when the enzyme digestion time was 16 h, and these values were almost equivalent to
270
those of a previous report (Funami et al., 2011). The concentrations of GalA and neutral
271
sugars increased with the concentration of protein decreasing. Methyl esterification and
272
O-acetylation only reacted with the GalA of the backbone. Therefore, DM and DA did
273
not vary with the changing of protein concentration. The concentration of FA decreased
274
from 1.71 to 1.10% after treatment for 16 h. FAE catalyzes the hydrolysis of the
275
4-hydroxy-3-methoxycinnamoyl (feruloyl) group from an esterified sugar, which is
276
usually arabinose in ‘natural’ substrates. The concentration of FA decreased from 1.10 to
277
0.22% when the enzyme digestion time was 20 h. The protein concentration reduced from
278
0.42 to 0.36% in response to enzymatic hydrolysis for 20 h. The reduction in the
279
concentration of protein may be due to the cross-linkage of protein and FA, which also
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occurs in the product of SBPP-c. The concentrations of all the neutral sugars increased
281
slightly, and the DM and DA values were nearly invariable. ABN hydrolyzes (1→5)-α-arabinofuranosidic linkages in (1→5)-arabinans. GAL
283
specifically hydrolyzes (1→4)-β-D-galactoside linkages in type I arabinogalatans. As
284
shown in Table 1, the concentration of Ara and Gal decreased from 13.11 to 3.81% and
285
12.63 to 2.33%, respectively. These values were almost identical to those in a previous
286
report (Funami et al., 2011). Moreover, the concentrations of FA and protein were
287
somewhat reduced because Ara and Gal are mainly distributed in lateral chains and some
288
phenolic acids, such as ferulic acid, are linked to the arabinose and galactose residues via
289
ester linkages (Fry, 1983). In addition, the amounts of other neutral sugars increased
290
slightly and the DA and DM values were stable.
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3.2. FT-IR analysis
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The FT-IR spectra of SBPP and modified SBPP with different enzymes are shown in
294
Fig. 3. In the spectrum of SBPP, the wavelength range of 950 to 1200 cm-1 is considered
295
the ‘fingerprint’ region of carbohydrates because it enables the identification of the major
296
chemical groups (Černá et al., 2003). The five bands at 1149, 1106, 1043, 1013 and 954
297
cm-1 correspond to the skeletal C-O and C-C vibration bands of glycosidic bonds and the
298
pyranoid ring (Monfregola, Leone, Vittoria, Amodeo, & De Luca, 2011). There is no
299
distinct difference among the spectra of SBPP, SBPP-b1 and SBPP-c. FAE mainly
300
hydrolyzes the feruloyl groups that are esterified with arabinose or galactose. Therefore, a
301
large number of ester bonds were broken and the infrared vibrations of SBPP-d in the
302
ranges of 1745-1750 and 1616-1634 cm-1, which are attributed to the esterified and
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ionized carboxyl groups of pectin molecules (Pappas et al., 2004), varied considerably,
304
especially esterified carboxyl groups. The spectrum of SBPP-a1 exhibited the same
305
variation trend as that of SBPP-d because PME can cut carbomethoxy (COOCH3) and
306
remove methoxyl residues, yielding polygalacturonic acid. Compared with SBPP, the
307
stretching intensities of COOH at 1616-1634 cm-1 of SBPP-a3 dropped appreciably due to
308
the hydrolysis of galacturonic acid.
309 310
3.3. Enzymatic modification by PME, PAE and PG
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Food hydrocolloids perform two essential functions that determine emulsification
312
effectiveness: providing good emulsifying activity and good emulsion stability (Funami
313
et al., 2011). Emulsifying activity and emulsion stability were assessed by measuring the
314
droplet size of the fresh emulsion and cream index (CI), respectively. The efficacy of a
315
surface-active molecule is generally evaluated by its ability to decrease the surface
316
tension of a solution and the interfacial tension of an emulsion. According to Fig. 2a,
317
SBPP was sequentially hydrolyzed by PME, PAE and PG, yielding SBPP-a1, SBPP-a2
318
and SBPP-a3, respectively. The surface and interfacial tension of SBPP-a1 (45.2 mN/m
319
and 24.3 dyne cm-1) was significant higher than that of SBPP (44.3 mN/m and 17.5 dyne
320
cm-1) (Fig. 4) because the character of pectin is predominantly hydrophilic and the loss of
321
methyl ester groups, which are hydrophobic, results in the decrease of surface activity.
322
Yapo et al. (2007) investigated the relationship between SBPP and its surface properties
323
and the results showed that the surface and interfacial tension of the extracted pectin
324
which has higher DM were significant higher than that of the pectin with lower DM. The
325
particle size of SBPP-a1 increased from 0.409 (d3,2) and 0.621 µm (d4,3) to 0.649 and
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0.873 µm after PEM treatment. This reason may be that PME hydrolyze methyl esters of
327
pectin to carboxyl groups or pectate and methanol. The amount of carboxyl group
328
increased after the treatment of PME and High-methoxyl SBPP (DM > 50%) converted
329
to low-methoxyl SBPP-a1 (DM < 50%). The low-methoxyl pectin gels under the
330
condition of a high concentration of calcium ions. And the variation of DM has a
331
significant impact on the interaction between SBPP and calcium ions. When the DM of
332
pectin decreases to a certain degree, the increase of carboxyl groups improve the
333
sensitivity of pectin molecules toward Ca2+ (Hotchkiss et al., 2002), which will make the
334
oil droplets move closer together. Therefore, the particle size of the emulsion stabilized
335
by SBPP-a1 increased. No significant difference was found in the CI of pectins before and
336
after PME modification (Table 2).
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SBPP-a2 was obtained using PAE with SBPP-a1 as material. Compared with SBPP-a1,
338
the DA of SBPP-a2 was reduced from 23.9 to 5.1% (Table 1), whereas the surface tension
339
of the SBPP-a2 solution increased from 45.2 to 48.0 mN/m. And the interfacial tension of
340
the SBPP-a2 stabilized emulsion increased from 24.3 to 30.1 dyne cm-1 when compared
341
with that of the emulsion prepared by SBPP-a1 (Fig. 4). These results suggest that the
342
contribution of acetyl groups to the surface activity of pectin was small but not negligible.
343
Dea & Madden (1986) considered that highly acetylated SBPP is more surface-active
344
than commercial, high-methoxy or low-methoxy pectins. This results were also consistent
345
with the conclusion obtained by Zouambia, Moulai-Mostefa, & Krea (2009). As shown in
346
Table 2, the particle sizes of SBPP-a2 increased from 0.649 and 0.873 µm to 0.881 and
347
1.148 µm after PAE modification. In addition, the CI of the emulsion prepared by
348
SBPP-a2 increased significantly from 24 to 41%, indicating that the acetyl groups have an
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impact on both emulsifying stability and emulsifying capacity. This effect occurs because
350
the aggregation of oil droplets can be prevented by acetyl groups though steric and
351
mechanical stabilization effects. Moreover, acetyl groups can also decrease the sensitivity
352
of pectin molecules toward calcium ions. As a result, SBPP cannot gel at high
353
concentrations of calcium ions (Pippen, McCready, & Owens, 1950).
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SBPP-a3 was obtained by hydrolyzing SBPP-a2 with PG. Compared with SBPP-a2, the
355
GalA concentration of SBPP-a3 reduced from 46.55 to 21.67% (Table 1), and the surface
356
tension of the SBPP-a3 solution increased from 48.0 to 49.1 mN/m. And the interfacial
357
tension of the SBPP-a2 stabilized emulsion increased from 30.1 to 37.3 dyne cm-1 when
358
compared with that of the emulsion prepared by SBPP-a1 (Fig. 4). As shown in Table 2,
359
the particle size of SBPP-a3 increased from 0.881 and 1.148 µm to 0.932 and 1.161 µm
360
after PG modification. In addition, the CI of the emulsion prepared by SBPP-a3 increased
361
significantly from 41 to 45%. These results suggest that the emulsifying properties of
362
pectin declined with the decrease in GalA concentration.
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3.4. Enzymatic modification by PE, FAE and GAL or ABN
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To investigate the contribution of each functional group on the side chain to the
366
emulsifying activity and emulsifying stability of SBPP, PE, FAE, GAL and ABN were
367
applied to yield SBPP-b1, SBPP-b2, SBPP-b3 and SBPP-b4, respectively (Fig. 2b). As
368
shown in Fig. 4, the surface and interfacial tension of SBPP changed greatly after the
369
synergistic effect of pepsin and a food-grade acid protease. With the protein
370
concentration decreasing from 5.2 to 0.42%, the surface tension of the SBPP-b1 solution
371
increased from 44.4 to 54.4 mN/m at 25 °C. And the interfacial tension of the SBPP-b1
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stabilized emulsion increased from 17.5 to 29.4 dyne cm-1 when compared with that of
373
the emulsion prepared by SBPP-b (Fig. 4). These results demonstrated that the surface
374
activity of SBPP reduced as the protein concentration decreased. This is due to the
375
protein itself has high surface activity. This observation was consistent with those of
376
Leroux et al. (2003) and Funami et al. (2007) who both indicated that reducing the level
377
of protein in the SBPP significantly lowered its surface active properties. Compared with
378
SBPP, the particle size of the emulsion stabilized by SBPP-b1 increased from 0.409 (d3,2)
379
and 0.621 µm (d4,3) to 2.636 and 3.360 µm, which indicated that protein concentration
380
had a positive effect on the emulsifying capacity of pectin. This result can be explained
381
by the hydrophobic behavior of protein, which acts as ‘bridge’ between the
382
polysaccharide and the oil phase. Therefore, pectin molecules can adsorb onto the
383
oil–water interface as an anchor, whereas the carbohydrate moiety forms a hydrated layer,
384
preventing the aggregation or coalescence of the emulsion droplets (Nakamura, Yoshida,
385
Maeda, Furuta, & Corredig, 2004). The CI of the emulsion stabilized by SBPP-b1
386
increased from 23 to 70%, which showed that protein levels also has a strong impact on
387
the stability of emulsion. This result was consistent with the results from Leroux et al.
388
(2003) who showed that protein associated with the pectin played a key role in the
389
stabilization of the rapeseed oil or orange oil emulsions.
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The surface tension of the SBPP-b2 solution and the properties of the emulsion, which
391
was prepared using SBPP-b2 as the surfactant, were changed greatly when compared with
392
those of SBPP-b1. As shown in Fig. 4, the surface tension of the SBPP-b2 solution
393
increased sharply from 54.4 to 58.0 mN/m and the interfacial tension of the SBPP-b2
394
stabilized emulsion increased from 29.4 to 40.0 dyne cm-1 when compared with that of
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the emulsion prepared by SBPP-b1, which suggested that ferulic acid had an important
396
effect on the surface activity of pectin. This may be the reason that ferulic acid itself is
397
hydrophobic and, like protein, acts as a ‘bridge’ connection between the oil droplet and
398
the polysaccharide. Siew and Williams (2008) also revealed that the adsorbed fractions of
399
pectin sample were rich in protein and ferulic acid and speculated that the reduction in
400
surface tension of sugar beet pectin may be due to its higher protein and/or ferulic acid
401
concentrations. The particle size of the emulsion stabilized by SBPP-b2 increased from
402
2.636 (d3,2) and 3.360 µm (d4,3) to 6.032 (d3,2) and 8.110 µm (d4,3), which indicated that
403
ferulic acid, in the absence of protein, plays a key role in the emulsifying capacity of
404
pectin. Furthermore, the impact of ferulic acid concentration on the emulsifying stability
405
of pectin is significant according to the CI data shown in Table 2.
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Then, based on SBPP-b2, SBPP-b3 and SBPP-b4 were obtained through the
407
modification of GAL and ABN, respectively. No significant difference in the surface and
408
interfacial activity or emulsifying capacity was found between SBPP-b2 and SBPP-b3 or
409
SBPP-b4 by comparing the data for surface tension (Fig. 4) and particle sizes (Table 2).
410
However, the CI values of both emulsions, stabilized by SBPP-b3 (78%) or SBPP-b4
411
(79%), increased slightly. This finding suggested that the carbohydrate moiety in pectin
412
molecules exhibited a stabilizing capability, though the effect was much lower than that
413
of the proteinaceous and ferulic acid materials. This finding was in line with the paper
414
reported by Dickinson (2003) who suggested that the presence of non-adsorbing moieties
415
could affect creaming stability by providing the protective layer that confers effective
416
steric stabilization and inducing depletion flocculation during extended storage. What’s
417
more, the changes of the viscosity in continuous phase, caused by enzyme modification,
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418
may also have an effect.
419 420
3.5. Enzymatic modification by GAL/ABN and FAE Oligosaccharide-protein complexes always exhibit good emulsifying properties
422
(Akhtar & Dickinson, 2003). In series b of the enzymatic modification (Fig. 2b), protein
423
and ferulic acid were almost completely hydrolyzed, and we could not determine the
424
impact of the oligosaccharide-protein complexes on the emulsifying properties of pectin.
425
Hence, in this section, ferulic acid-oligosaccharide-protein complexes were removed by
426
catalysis with GAL and ABN. In this way, we were able to determine the effects of
427
ferulic acid-oligosaccharide-protein complexes on the emulsion properties. Another
428
phenomenon observed in series b was that the particle size of the emulsion stabilized by
429
FAE modified pectin (SBPP-b2) increased rapidly. Based on this result, we can conclude
430
that the ferulic acid concentration plays an important role in the emulsifying properties of
431
pectin, but only under low protein concentration conditions. To investigate the influence
432
of ferulic acid concentration on the emulsifying properties of SBPP, especially under high
433
protein levels, SBPP was directly modified by FAE in series d.
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SBPP-c and SBPP-d were hydrolyzed with GAL/ABN and FAE from SBPP,
435
respectively. The surface tension of the SBPP-c solution was 54.6 mN/m, which was
436
significant (p < 0.05) higher than that of SBPP (44.4 mN/m) and slightly higher with that
437
of SBPP-b1 (54.4 mN/m) but lower than that of SBPP-d (55.8 mN/m). These results
438
suggested that the covalently linked ferulic acid-arabinogalactan-protein conjugate plays
439
a more important role in the surface activity of pectin than protein alone but a less
440
important role than that of ferulic acid. The interfacial tension of the emulsions stabilized
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by SBPP-c (38.3 dyne cm-1) and SBPP-d (38.6 dyne cm-1) had little difference but
442
significant (p < 0.05) higher than that of SBPP (17.5 dyne cm-1). As shown in Table 2,
443
the particle sizes of the emulsion prepared by SBPP-c were 1.920 (d3,2) and 2.760 µm
444
(d4,3), which are much higher than those of SBPP (0.409 and 0.621 µm for d3,2 and d4,3,
445
respectively) and SBPP-b1 (2.636 and 3.360 µm for d3,2 and d4,3, respectively) but lower
446
than those of SBPP-d (3.050 and 4.142 µm for d3,2 and d4,3, respectively). These results
447
indicated that the effect of functional groups on the emulsifying activity decreased in the
448
following order: ferulic acid > ferulic acid-arabinogalactan-protein complexes > protein.
449
The CI of the emulsion stabilized by SBPP-c (69%) was much higher than that of SBPP
450
(23%) but similar to that of SBPP-b1 (70%) and SBPP-d (72%), which suggested that
451
carbohydrate covalently bound to a small amount of protein and ferulic acid exhibited
452
excellent emulsifying stability.
454
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3.6. Emulsifying mechanism of sugar beet pectin A deduced mechanism was drawn schematically (Fig. 5) to explain the emulsifying
456
properties of sugar beet pectin. The ferulic acid and proteinaceous materials (hydrophobic
457
groups) adsorb onto the surface of emulsion droplets, decreasing the interfacial tensions
458
between the water and oil phases. It is evident that the emulsification properties of sugar
459
beet pectin are influenced by the accessibility of the protein and ferulic acid groups to the
460
surface of the oil droplets (Williams et al., 2005). Protein and ferulic acid make a main
461
contribution to the emulsifying capacity of SBPP. The lateral chains of SBPP, especially
462
arabinose and galactose, connect the main chain with protein and ferulic acid. The
463
presence of the non-adsorbing lateral chains mainly affect creaming stability by steric
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effects (Dickinson, 2003). Polygalacturonic acid (main chain), together with neutral
465
sugars in the side chain, forms a layer of film, protecting oil droplets from aggregation
466
and fusion. The thickness of the film is affected greatly by the concentration of GalA
467
(Funami et al., 2011). In addition, methyl and acetyl groups in the main chain play a role
468
in preventing carboxyl groups of GalA from moving toward calcium ions. GalA, methyl
469
and acetyl groups in the main chain make a main contribution to the emulsifying stability
470
of the emulsion prepared by SBPP.
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471
4. Conclusion
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The impact of each functional group on the emulsifying properties of SBPP was
474
investigated by enzymatic modification using seven enzymes in a particular order. The
475
structures of SBPPs were characterized based on FT-IR. In addition, the contribution of
476
each functional group was evaluated based on the variation in emulsion characteristics.
477
The results showed that the composition of SBPP or enzymatically modified SBPPs
478
varied in a broad range after enzymatic hydrolysis. FTIR spectroscopy showed that the
479
characteristic absorption intensities of SBPP had no distinct difference from the spectra
480
of SBPP-b1 and SBPP-c, but had an obvious variation, especially in the ranges of
481
1745-1750 and 1616-1634 cm-1, which are attributed to the esterified and ionized
482
carboxyl groups, when compared with SBPP-a1, SBPP-a2, SBPP-a3, and SBPP-d. Protein,
483
ferulic acid-araban/galactan-protein complexes and ferulic acid played an important role
484
in improving the surface activity, emulsifying capacity and emulsifying stability of SBPP.
485
The decrease of methyl ester groups mainly affected the particle sizes of the emulsion. In
486
addition to particle sizes, the cream index of the emulsion was significantly affected by
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487
acetyl groups. Arabinose and galactose less affected emulsifying properties than other
488
functional groups.
489
Abbreviations
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490
SBPP, sugar beet pulp pectin; FT-IR, Fourier Transform Infrared Spectroscopy; GalA,
492
galacturonic acid units; PG, polygalacturonase; ABN, arabinanase; GAL, galactanase; PE,
493
protease (combination of pepsin and food-grade acid protease) FAE, feruloyl esterase;
494
PME, pectin methyl esterase; PAE, pectin acetyl esterase; MCT, medium-chain
495
triglyceride; Rha, rhamnose; Ara, arabinose; Xyl, xylose; Gal, galactose; Glc, glucose;
496
GalA, galacturonic acid; FA, ferulic acid; DM, degree of methylation; DA, degree of
497
acetylation; CI, cream index.
498
Acknowledgements
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This research was supported by the Guangzhou Science and Technology Program
501
(2013J4500036), the National Natural Science Foundation of China (21376097), the
502
program for New Century Excellent Talents in University (NCET-13-0212), the
503
Guangdong Natural Science Foundation (S2013010012318), and the Fundamental
504
Research Funds for the Central Universities, SCUT (2013ZZ0070).
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chemistry, 56(11), 4164-4171.
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Voragen, A. G. J., Pilnik, W., Thibault, J. F., Axelos, M. A. V., & Renard, C. M. G. C.
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(1995). Chapter 10: Pectins. In A. M. Stephen (Ed.), Food polysaccharides and their
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applications (pp. 287-339). New York: Marcel Dekker, Inc. Williams, P. A., Sayers, C., Viebke, C., Senan, C., Mazoyer, J., & Boulenguer, P. (2005).
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Elucidation of the emulsification properties of sugar beet pectin. Journal of
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Agricultural and Food chemistry, 53(9), 3592-3597.
Yapo, B. M., Robert, C., Etienne, I., Wathelet, B., & Paquot, M. (2007). Effect of
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extraction conditions on the yield, purity and surface properties of sugar beet pulp
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pectin extracts. Food Chemistry, 100(4), 1356-1364.
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Zouambia, Y., Moulai-Mostefa, N., & Krea, M. (2009). Structural characterization and
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surface activity of hydrophobically functionalized extracted pectins. Carbohydrate
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polymers, 78(4), 841-846.
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Table 1 Monosaccharide composition, galacturonic acid, protein, ferulic acid and degree of esterification of different enzymatically modified sugar beet pectinsA
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Protein
Ferulic acid
(%)
(%)
DM
DA
1.21±0.01a
67±0.4b
23.9±0.2a
5.48±0.03b
1.17±0.01b
7.5±0.1e
23.6±0.3a
46.55±0.45d
5.75±0.02a
1.20±0.01a
8.0±0.1d
5.1±0.6c
3.26±0.02a
21.67±0.31i
5.14±0.04c
1.11±0.01c
8.1±0.3d
5.3±0.3c
2.29±0.02f
1.22±0.01g
45.22±0.37e
0.42±0.00f
1.10±0.01c
69±0.5a
23.5±0.6a
12.63±0.05b
2.35±0.01e
1.28±0.01f
45.37±0.44e
0.36±0.01g
0.22±0.00e
67±0.7b
23.8±0.3a
15.36±0.05b
2.33±0.04h
2.76±0.01d
1.56±0.01c
47.06±0.58c
0.26±0.01h
0.16±0.00g
68±0.6ab
24.3±0.5a
13.96±0.03b
3.81±0.01i
2.89±0.01g
2.88±0.01c
2.01±0.03b
48.59±0.21b
0.21±0.00i
0.12±0.00h
67±0.7b
23.9±0.2a
SBPP-c
14.46±0.02a
3.11±0.03j
2.05±0.01i
3.88±0.01b
2.01±0.01b
58.26±0.39a
2.83±0.01e
0.66±0.01d
68±0.8ab
24.0±0.3a
SBPP-d
7.56±0.02e
9.61±003g
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Constitution sugars (%) Sample
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10.59±0.03d
1.32±0.01e
44.26±0.13g
4.13±0.02d
0.17±0.00f
65±0.5c
22.3±0.3b
Ara
Gal
Glc
Xyl
GalA
SBPP
6.72±0.03g
9.03±0.04h
9.86±0.07f
0.66±0.00j
1.00±0.01h
43.57±0.13h
5.20±0.02c
SBPP-a1
7.23±0.05f
9.70±0.02f
10.22±0.04e
0.74±0.01i
1.23±0.02g
44.82±0.20f
SBPP-a2
7.55±0.06e
9.91±0.06e
10.62±0.07d
0.88±0.01h
1.40±0.01d
SBPP-a3
14.02±0.04b
27.19±0.09a
25.92±0.04a
5.27±0.03a
SBPP-b1
10.81±0.07d
12.94±0.04d
12.06±0.02c
SBPP-b2
10.96±0.09d
13.11±0.03c
SBPP-b3
13.57±0.03c
SBPP-b4
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A
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(a-j) are significantly different (p < 0.05).
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0.98±0.01g
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The data are averages and standard deviations of triplicate measurements. Values in each column with different superscript letters
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Table 2. The oil droplet sizes and creaming index (CI) of emulsions stabilized by SBPP
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and modified SBPP Samples
Particle size A d4,3 (µm)
SBPP
0.409 ± 0.003i
0.621 ± 0.004i
SBPP-a1
0.649 ± 0.003h
0.873 ± 0.005h
SBPP-a2
0.881 ± 0.005g
1.148 ± 0.007g
SBPP-a3
0.932 ± 0.006f
1.161 ± 0.010g
SBPP-b1
2.636 ± 0.021d
3.360 ± 0.028e
70 ± 2cd
SBPP-b2
6.032 ± 0.034b
8.110 ± 0.041c
76 ± 2b
SBPP-b3
6.150 ± 0.036a
8.340 ± 0.036b
78 ± 2ab
SBPP-b4
6.155 ± 0.038a
8.492 ± 0.047a
79 ± 2a
SBPP-c
1.920 ± 0.018e
2.760 ± 0.022f
69 ± 1d
SBPP-d
3.050 ± 0.027c
4.142 ± 0.031d
72 ± 2c
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24 ± 1g 41 ± 1f
45 ± 1e
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each column with different superscript letters (a-i) are significantly different (p < 0.05).
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23 ± 1h
The data are averages and standard deviations of triplicate measurements. Values in
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A
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d3,2 (µm)
592 593
CI (%)
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Figure captions
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Fig. 1. Schematic figure of SBPP and enzymes used in the experiments. GalA:
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Galacturonic acid; Rha: Rhamnose; Gal: Galactose; Ara: Arabinose; FA: Ferulic acid.
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Fig. 2. Flow chart for the enzymatic modification of SBPP.
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Fig. 3. FT-IR spectra of SBPP and modified SBPPs.
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Fig. 4. The surface tension of SBPP and modified SBPP solutions.
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Fig. 5. Schematic drawing of the emulsion stabilized by sugar beet pectin.
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Fig. 1. Schematic figure of SBPP and enzymes used in the experiments. GalA: Galacturonic acid; Rha: Rhamnose; Gal: Galactose; Ara: Arabinose; FA: Ferulic acid.
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Fig. 2. Flow chart for the enzymatic modification of SBPP.
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SBPP-a1 SBPP-a2
SBPP-a3
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COOCH3 2000
1800
COOH 1600
1400
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SBPP-d SBPP
1200
1000
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Wavenumber （ cm-1）
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Fig. 3. FT-IR spectra of SBPP and modified SBPPs.
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800
40
30
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34
at er
45
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SB SB PP P SB P-c PP w -d
S SB BP P P SB P-b P 1 SB P-b P 2 SB P-b PP 3 -b 4
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S SB BPP P SB P-a P 1 SB P-a PP 2 -a 3
surface tension (mN/m) 50
S SB BP P P SB P-c PP 1 W d at er
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S SB BP P SB PPb P 1 SB P-b 2 SB PPPP b3 -b 4
S SB BP P P SB Pa P 1 SB P-a PP 2 -a 3
-1
Interfacial tension (dyne cm )
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65
20
10
0
Fig. 4. The surface and interfacial tension of SBPP and modified SBPP solutions.
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Ca2+
Ca2+
Ca2+
Ca2+
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:C
Main chain
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Emulsion droplets
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Ca2+
Ca2+
Ferulic acid
Side chain
Protein
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Fig. 5. Schematic drawing of the emulsion stabilized by sugar beet pectin
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ACCEPTED MANUSCRIPT Seven enzymes were studied in a particular order. Compositions of SBPP and enzymatically modified SBPPs were determined. The structures of SBPPs were characterized
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Ferulic acid was an important factor on emulsifying ability of SBPP.
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The contribution of each functional group to emulsifying ability was evaluated.