Physicochemical Properties and Surface Activities of Collagen

May 2, 2014 - ABSTRACT: A series of collagen hydrolysate-based surfactants (CHBS) were ... biological methods, collagen can be hydrolyzed into soluble...
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Physicochemical Properties and Surface Activities of Collagen Hydrolysate-Based Surfactants with Varied Oleoyl Group Grafting Degree Yuan-long Chi,†,‡ Qi-xian Zhang,† Xue-pin Liao,† Jian Zhou,† and Bi Shi*,† †

National Engineering Laboratory for Clean Technology of Leather Manufacture and ‡Department of Food Engineering, Sichuan University, Chengdu 610065, Sichuan, China ABSTRACT: A series of collagen hydrolysate-based surfactants (CHBS) were prepared by grafting different amount of oleoyl group onto collagen hydrolysate (CH). Their physicochemical properties and surface activities were investigated, and their emulsifying ability on rapeseed oil−water emulsion was evaluated. The results showed that, as increasing grafting degree of oleoyl group, the free amino content and isoelectric point of CHBSs and the size of CHBS particles formed in aqueous solution declined, while their molecular weight and surface hydrophobicity increased. The CHBSs with high grafting degree, such as CHBS-30, CHBS-40, and CHBS-50 (postfix datum represents mmol amount of oleic acid chloride to react with 10 g collagen hydrolysate), exhibited satisfactory wetting capacity and foaming capacity, and the CHBSs with low grafting degree, such as CHBS-10 and CHBS-20, presented good emulsifying capacity. The surface activity of CHBSs was closely correlated with the hydrophobicity of their molecules, as well as their capacity to reduce the surface tension of air−water interface. In addition, CHBS-20 showed good emulsifying ability on rapeseed oil−water emulsion in neutral and alkaline pH, even in the condition of low salt concentration. subsequently prepare collagen hydrolysate-based surfactant.9−11 In this study, CH was first extracted from waste pigskin shavings, and a series of CHBSs with varied grafting degrees of oleoyl group were prepared. Their physicochemical properties and surface adsorption characteristics were investigated, and then, the relationship between the grafting degree and surface activities, such as wetting property, foaming property, and emulsifying property, was studied. Furthermore, the emulsifying abilities of CHBSs in rapeseed oil−water emulsions under different pHs and NaCl concentrations were evaluated.

1. INTRODUCTION Collagen is a renewable biomass resource, widely existing in body tissues of mammals.1 Treated by physical, chemical, or biological methods, collagen can be hydrolyzed into soluble compounds with relatively low molecular weight, known as collagen hydrolysates (CH).2,3 The hydrolysates possess amphipathicity to some extent due to the fact that they contain both hydrophilic and hydrophobic amino acids. So, gelatin, a kind of collagen hydrolysate, can be used as a natural surfactant and commonly added into fruit juice, bread, cake, and candy.4 However, the surface activity of collagen hydrolysates is generally not as good as commercial surfactants, because they have excessively strong hydrophilicity caused by numerous hydrophilic groups, such as peptide bond, −OH, −COOH, and −NH2. Therefore, appropriate chemical modification in enhancing hydrophobicity of collagen hydrolysates would be needed in order to improve their surface activity and broaden their application fields. Our previous studies have confirmed that collagen hydrolysate-based surfactant (CHBS) could be prepared by grafting hydrophobic oleoyl onto the free amino group of collagen hydrolysate through amidation reaction.5,6 However, the effect of oleoyl group grafting degree on the physicochemical properties and the surface activity of CHBS was not investigated. Obviously, the understanding of these details is significant for the application of CHBS in different fields. Some industries inevitably produce a great amount of collagen wastes, such as skin shavings in tannery, bone residues in meat processing, and scales in aquatic product processing.7,8 Abandoning these collagen wastes would be a huge waste of biomass resource and might result in environmental problems if they are not properly disposed. Therefore, it is a meaningful work to extract collagen hydrolysates from these wastes and © 2014 American Chemical Society

2. MATERIALS AND METHODS 2.1. Chemicals. Waste pigskin shavings were provided by a local tannery. Protease Alcalase 2.5L (550746 units/g) was provided by Novozymes Inc. (Shanghai, China). Oleic acid chloride (OAC), 1-anilinonaphthalene-8-sulfonic acid (ANS), alkylphenols polyoxyethylene (OP-10), sodium dodecyl benzenesulfonate (SDBS), and other chemicals were of analytical grade. 2.2. Preparation of Collagen Hydrolysate (CH) and Collagen Hydrolysate-Based Surfactant (CHBS). CH was prepared according to our previous work.9 Briefly, pigskin shavings was first cut into short strips (2 to 3 cm in length), and soaked in 9-fold of water (w/v) at 60 °C for 30 min. The pH of solution was adjusted to 9.0 by 0.5 M NaOH. A certain amount of Alcalase 2.5 L (2200 units/g pigskin) was added, and then hydrolysis reaction was conducted at 60 °C for 3 h under stirring. The reaction was terminated by inactivating the Received: Revised: Accepted: Published: 8501

February 18, 2014 May 1, 2014 May 2, 2014 May 2, 2014 dx.doi.org/10.1021/ie5007068 | Ind. Eng. Chem. Res. 2014, 53, 8501−8508

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enzyme at 100 °C for 5 min. Then, the CH solution (10%, w/ v) was obtained. As for the graft modification reaction, 100 mL of the obtained CH solution was transferred to a 250 mL four-necked flask equipped with a Hopkins’ condensing tube, and 10 mL of acetone was added as a diluent. The mixture was stirred in a water bath of 30 °C for 2 h, while a certain amount of OAC (5 mmol, 10 mmol, 20 mmol, 30 mmol, 40 mmol, and 50 mmol) and 7.5 M NaOH solution were added drop by drop at a molar ratio of 1:2 (OAC/NaOH). The mixture was then heated to 60 °C, and the acetone in it was recovered. After reacting for another 2 h at 60 °C, the mixture was cooled to room temperature and adjusted to pH 7.0 with 1 M HCl solution, and then, CHBS (CHBS-5, CHBS-10, CHBS-20, CHBS-30, CHBS-40 and CHBS-50) solutions were obtained. The CH solution and CHBS solutions were filtrated, precipitated under saturated (NH4)2SO4 solution, and dialyzed to remove salt, and the purified CH and CHBSs were obtained after freeze-drying. They were used for determinations of physicochemical properties and surface activities. The schematic diagram of collagen hydrolysate-based surfactant (CHBS) is shown in Figure 1.

and the consumed volume of 0.1 M NaOH solution was recorded as V2 (mL). Free amino group content (C, mM 100g−1) was calculated according to the equation: C = 100 × 0.1 × (V1 − V2)/0.5. 2.3.3. Molecular Weight and Distribution. Molecular weight of CH and CHBSs was determined by an Agilent 1200 high performance size exclusion chromatography (HPSEC, Agilent, U.S.A.) equipped with one Agilent G1311A pump, two connected HPSEC columns (PLgel10000 and PLgel Mixed-C, 300 mm × 7.5 mm, 5 μm, Agilent, U.S.A.), and one DAWN HELEOS II multiangle light scattering detector (Wyatt Technologies, U.S.A.). Sample solution (20 μL) (10 g L−1 CH or CHBSs in 100 mM nitrate buffer, pH 7.0, containing 0.02% (w/v) sodium azide) was injected and elution was conducted at 35 °C using 100 mM nitrate buffer (pH 7.0) at a flow rate of 1.0 mL min−1. Elution data was processed using Agilent Chemstation (version B.04.01), and molecular weight information including weightaverage molecular weight (Mw), number-average molecular weight (Mn), and polydispersity index (Mw/Mn, PDI) were calculated. 2.3.4. Surface Hydrophobicity. Surface hydrophobicity of CH and CHBSs was determined as described by AlizadehPasdar et al., where 1-anilinonaphthalene-8-sulfonic acid (ANS) was used as a fluorescence probe.13 Briefly, 10 g L−1 of CH and CHBS solutions was prepared and then diluted in 10 mM sodium phosphate buffer (pH 7.4). Concentrations of the diluted CH or CHBS solutions were 1 g L−1, 2 g L−1, 3 g L−1, and 5 g L−1. Four mL of the diluted sample solution was mixed with 40 μL of 8 mM ANS in 0.1 M sodium phosphate buffer (pH 7.4). Fluorescence intensity of the mixture was measured at an excitation wavelength of 374 nm and an emission wavelength of 485 nm using an F-4010 versafluor fluorometer (Hitachi, Japan). The initial slope (S0) of fluorescence intensity versus sample concentration plot was calculated by linear regression analysis, and used as an index of surface hydrophobicity. The bigger value of S0 characterized stronger surface hydrophobicity. 2.3.5. Particle Size. Particle size of CH and CHBSs dispersed in 10 mM sodium phosphate buffer (pH 7.0) with the concentration of 0.5 g L−1 was measured using dynamic light scattering technique. The measurements were performed in triplicate at 25 °C by a Zetasizer nano ZS (Malvern Instruments, U.K.). 2.3.6. ζ-Potential. ζ-Potential analysis of CH and CHBSs was performed by using laser Doppler electrophoresis, and the measurements of sample solutions were performed in triplicate at 25 °C by a Zetasizer nano ZS. For isoelectric point (IEP) calculation, samples were first dispersed in a series of sodium acetate buffers (pH 3.0 to 6.0), and their concentrations were all 0.5 g L−1. The pH value at zero ζ-potential was referred as IEP. 2.4. Determination of Surface Activities. 2.4.1. Surface Tension. Surface tension measurements of CH and CHBSs were performed in triplicate at 25 °C by an OCA-200 contact angle meter (Dataphysics, Germany) according to a sessile drop method described by Lucas et al. with some modifications.14 A series of CH or CHBS solutions with different concentrations were obtained by dilution of distilled water. The pH of the aqueous CH solution (0.5 g L−1) was 5.8, and the pHs of the aqueous CHBS solutions (0.5 g L−1) were all around 7.0. Prior to measurement, sample solution was left for 30 min to attain equilibrium. Critical micelle concentration

Figure 1. Schematic diagram of collagen hydrolysate-based surfactant (CHBS).

2.3. Determination of Physicochemical Properties. 2.3.1. Fourier Transform-Infrared (FT-IR) Spectroscopy. FT-IR spectra of CH and CHBSs were measured using a Nicdet Is10 FT-IR spectrometer (Thermofisher, U.S.A.) with potassium bromide disc technique. 2.3.2. Free Amino Group Content. Free amino group content of CH and CHBSs was determined using a formaldehyde titration method as described by Gump et al. with modifications.12 Briefly, 0.5 g of CH or CHBSs was dissolved in 60 mL of degassed distilled water, and the pH was adjusted to 8.2 by 0.01 M NaOH solution under magnetic stirring. Formaldehyde solution (20 mL) (10−15%) was then added slowly. After mixing for 3 min, the pH of solution was titrated to 9.2 by 0.1 M NaOH solution, and the consumed volume of 0.1 M NaOH solution was recorded as V1 (mL). The samples without adding CH or CHBSs were used as control, 8502

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(cmc, g L−1) and the surface tension at cmc (γcmc, mN m−1) were acquired from the breakpoint of surface tension (γ) versus the logarithm of concentration (log C, g L−1) curve. 2.4.2. Surface Adsorption Characteristics. The surface adsorption characteristics of CH and CHBSs at water−air interface were calculated from the plot of surface tension versus the logarithm of concentration obtained in section 2.4.1. Γmax (mol m−2) is the maximal surface excess concentration of a surfactant at air−water interface. It was calculated according to the Gibbs equation: Γmax= −(dγ/d log C)/2.303nRT, where R is gas constant (8.314 J mol−1 K−1), T is absolute temperature (K), n is the number of molecular species in solution (n = 1 for nonionic compounds and n = 2 for ionic compounds),15 and dγ/d log C is the slope of linear portion of the plot before cmc. Then the minimal area occupied per molecule adsorbed at water−air interface (Amin, Å2) was calculated according to the equation: Amin = 1/NAΓmax, where NA is Avogadro’s number. Additionally, c20 that is the concentration required to lower the surface tension of water by 20 mN m−1 and pc20 (negative log c20) were calculated. 2.4.3. Wetting Activity. Static contact angle measurements of CH and CHBSs were performed in triplicate at room temperature by an OCA-200 contact angle meter using a sessile drop method described by Minko et al.16 Paraffin surface has low free energy and is difficult to be wetted by water, and thus, it is commonly used for static contact angle measurements in evaluating the wetting capacity of a surfactant. Advancing contact angle (θa) and receding contact angle (θr) of five drops placed on paraffin surface were determined, and the results were averaged to give the mean values of θa and θr for each sample. OP-10, a representative nonionic surfactant, is generally used as wetting agent, foaming agent, and emulsifying agent. SDBS, a representative anionic surfactant, is generally used as wetting agent and foaming agent. Both of them were used for comparison with CHBSs. 2.4.4. Foaming Activity. Foaming capacity (FC) and foam stability (FS) of CH and CHBSs were evaluated as described by Agyare et al. with modifications.17 Briefly, 5 g L−1 of CH or CHBS solution was prepared and kept at a water bath of 40 °C for 30 min. Sample solution (15 mL) was added into a 100 mL cylinder with stopper, and it was turned over 10 times at room temperature and left undisturbed. The foam volume was recorded as V0 (t = 0 min, mL) and V30 (t = 30 min, mL). FC and FS were then calculated using the equations: FC (%) = (V0/Vi) × 100 and FS (%) = (V30/V0) × 100, where Vi (mL) is the volume of initial liquid before shaking. OP-10 and SDBS were also used for comparison. 2.4.5. Emulsifying Activity. Emulsifying activity index (EAI) of CH and CHBSs was evaluated as described by Lin et al. with some modifications.18 Briefly, 15 mL of CH or CHBS solution (5 g L−1) and 15 mL of oleic acid were mixed in a 50 mL cylinder with stopper. The cylinder was left in a water bath of 40 °C for 75 min and turned over for ten times every 15 min during the period, and then it was left undisturbed at 40 °C for 24 h. The emulsification phase volume (VE, mL) and the total volume (V, mL) were recorded, and EAI was calculated by the equation: EAI (%) = (VE/V) × 100. OP-10 was used to compare with CHBSs. 2.5. Stability of Rapeseed Oil−Water Emulsion Stabilized by CHBSs. Oil-in-water emulsification tests were performed as described by Aewsiri et al. with minor modifications.19 Oil-in-water emulsions were prepared by homogenizing the mixture of rapeseed oil and CHBS solution

at a ratio of 1:9 (v/v) at 18000 rpm for 3 min using a FA25 lab high-shear dispersing emulsifier (Fluko, Germany). Concentrations of CHBSs and rapeseed oil in the emulsion were 1% (w/v) and 10% (v/v), respectively. 0.2 mg L−1 of NaN3 was added as an antimicrobial agent. Prior to analysis, emulsions were 5-fold diluted with 10 mM sodium phosphate buffer (pH 7.0). The particle size and ζ-potential of oil droplet were measured to characterize the stability of the emulsion. To study the stability of the emulsion in storage process, the 5-fold diluted emulsion was left undisturbed overnight and then removed the large droplets and nonemulsified oil on the surface. Subsequently, it was stored at 20 °C for 15 d, and the particle size and ζ-potential were recorded. The emulsion stabilized by OP-10 was used for comparison. All measurements were performed in triplicate and the results were expressed as average and standard deviation. In view of practical application, the effects of pH and NaCl concentration on the stability of the emulsion were investigated. A series of emulsions with different pHs (3.0 to 9.0) or with different NaCl concentrations (0 to 500 mM) were prepared and stored at 20 °C for 24 h, and then their particle sizes and ζ-potentials were measured.

3. RESULTS AND DISCUSSION 3.1. Physicochemical Properties of CHBSs. In order to evaluate whether the oleoyl group of OAC has been successfully grafted onto CH, the FT-IR spectra of CH and CHBSs were measured and shown in Figure 2. CHBSs

Figure 2. FT-IR spectra of CH (a), CHBS-5 (b), CHBS-10 (c), CHBS-20 (d), CHBS-30 (e), CHBS-40 (f), and CHBS-50 (g).

displayed strong absorptions at 1655 and 1561 cm−1, due to the stretching vibrations of amide CO (amide I) and the bending vibrations of amide N−H (amide II), respectively.20 These two absorption bands of CHBSs were stronger in intensity than those of CH, mainly arose from the newly synthetic amide (NH−CO) through reaction between the amino groups of CH and the acyl chloride of OAC. Additionally, CHBSs presented three stronger absorption peaks at 2922 cm−1, 2857 and 1446 cm−1 compared with CH. These three peaks are attributed to the asymmetric stretching vibrations, the symmetric stretching vibrations and the bending vibrations of methylene, respectively,21 which proves the introduction of aliphatic chain of OAC in the products. All these observations suggest that CHBSs were successfully prepared by grafting the oleoyl group of OAC onto 8503

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Table 1. Free Amino Group Content, Molecular Weight, Surface Hydrophobicity Index, Particle Size, and Isoelectric Point of CH and CHBSs sample CH CHBS-5 CHBS-10 CHBS-20 CHBS-30 CHBS-40 CHBS-50 a

free amino content (mmol 100 g−1)a

Mw (PDI) (g mol−1)

± ± ± ± ± ± ±

4396 (1.308) 7382 (1.290) 10260 (1.384) 14820 (1.218) 18000 (2.046) 21470 (2.553) 22430 (2.496)

49.17 47.44 36.38 23.00 16.41 14.86 14.68

3.33 0.97 1.16 0.87 0.54 0.51 0.36

surface hydrophobicity index (S0)a 5.37 27.13 28.58 33.40 43.16 68.99 58.76

± ± ± ± ± ± ±

1.91 2.62 6.28 9.58 10.44 1.81 7.34

particle size (μm)a

isoelectric point (IEP)

± ± ± ± ± ± ±

4.48 4.13 4.02 3.90 3.81 3.62 3.58

0.32 0.203 0.188 0.172 0.157 0.156 0.149

0.033 0.023 0.022 0.017 0.024 0.021 0.012

Data is expressed as the average and standard deviation of three independent replicates.

3.2. Surface Activities of CHBSs. Collagen hydrolysate (CH) is the water-soluble mixture of polypeptides, and exhibits relatively low surface activity owing to lack of hydrophobic groups. After grafting hydrophobic oleoyl groups, CHBSs may show better surface activity at air−water interface. Surface tension (γ) of solution against the logarithm of concentration (log C) of CH and CHBSs were plotted in Figure 3. The γ of

CH. As the amount of OAC increased, CHBSs exhibited an increasing trend in intensity of amide group and methylene absorption bands, which was presumably due to the increasing amount of oleoyl chains grafted onto CH. However, CHBS-50 did not exhibit the strongest absorption in these characteristic bands as expected, which implies that the oleoyl group grafting degree of CHBS-40 has approached to the maximum. The oleoyl group grafting degree of CHBSs had significant effects on the free amino group content, molecular weight (Mw), polydispersity index (PDI), surface hydrophobicity index (S0), and isoelectric point (IEP) of CHBS molecules, as well as the particle size of CHBS particles formed in aqueous solution, as shown in Table 1. As the grafting degree of CHBSs increased, the free amino group content decreased gradually, while the Mw and S0 exhibited an increasing trend overall, which are the situations as expected. CHBS-50 presented the minimal free amino group content (14.68 mmol 100 g−1) and the maximal Mw (22430 g mol−1). The free amino content, Mw and S0 changed quickly as the offer of OAC increased from 0 to 30 mmol, and then slowed down as further increase of OAC offer. When OAC amount reached 40 mmol (CHBS-40), the maximal PDI and S0 were detected, suggesting that the reactivity between CHBS and OAC was declining and other side reactions might become the dominant when more than 40 mmol OAC was used. These observations indicated that the grafting degree of CHBS-40 almost reached the maximum, which is consistent with the result obtained in FT-IR spectra (Figure 2). When CHBSs disperse in aqueous solution, they tend to aggregate and form colloidal particles, owing to the intermolecular forces between CHBS molecules, such as hydrogen bond and hydrophobic interaction. The particle size of CHBS particles exhibited a decreasing trend as the increase of grafting degree, which might be due to the reason that the grafted hydrophobic groups weakened the polar affinity among CHBS molecules, and thus, their aggregation action declined correspondingly. Small particle size favors the formation of compact molecule arrangement at air−water interface,22 and therefore, it is inferred that the CHBSs with higher grafting degree might possess stronger capacity of reducing interfacial tension. The IEP of CHBSs decreased from 4.48 (CH) to 3.58 (CHBS-50), which suggests that CHBSs would have poor solubility in acidic condition and might possess low surface activity. CHBSs are anionic surfactants with carboxyl groups with a pKa of around 4.8. In acidic condition, CHBSs are weaker in forming micelles due to the polyelectrolyte effect, and the protonated CHBSs will decrease the cmc (critical micelle concentration) substantially. These physicochemical properties of CHBSs are closely related to their surface adsorption properties, and thus would have a great influence on their surface activities.

Figure 3. γ-log C plots of CH (■), CHBS-5 (●), CHBS-10 (▲), CHBS-20 (∇), CHBS-30 (★), CHBS-40 (□), and CHBS-50 (⧫).

CH and CHBSs first declined rapidly as their log C increased, and then decreased slowly until a defined concentration (cmc) was reached. It was also discovered that, as the grafting degree increased from CH to CHBS-50, the ability of CHBS in reducing surface tension of solution was improved. CHBS-50 was used as an example to obtain the cmc and the γcmc (γ at cmc) of CHBSs, as presented in Figure 3, and these values were discussed as follows. A surfactant in aqueous solution will tend to be adsorbed at the air−water interface, thus resulting in a sharp decline of surface tension of solution. So, the surface adsorption characteristics of a surfactant are generally used as an index for assessing its surface activity.23,24 Table 2 listed the cmc, γcmc, Γmax (maximal surface excess concentration), Amin (minimal area occupied per molecule), and pc20 (negative of log c20, representing the concentration required to lower the surface tension of water by 20 mN m−1). Obviously, both the cmc and γcmc declined gradually as the grafting degree increased, indicating that the CHBSs with higher grafting degree possess stronger capability in reducing surface tension of air−water interface. These results suggest that the CHBSs with higher grafting degree have better surface adsorption performance, which coincides with the conclusion of Faustino et al. in the research of the surfactants derived from amino acids.25 The area 8504

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Table 2. Surface Adsorption Characteristics of CH and CHBSs at Air−Water Interface at 25 °C

a

Table 3. Contact Angles of CH and CHBS Solutions (5 g/L) on Paraffin Surface

sample

cmc (g L−1)

γcmca (mN m−1)

106 Γmax (mol m−2)

Amin (Å2)

pc20

CH CHBS-5 CHBS-10 CHBS-20 CHBS-30 CHBS-40 CHBS-50

17.84 2.97 1.27 0.97 0.52 0.45 0.40

42.38 34.50 33.98 32.88 30.78 29.45 29.09

2.32 2.67 2.75 3.23 4.17 4.44 4.50

71.60 62.21 60.40 51.43 39.84 37.41 36.91

−0.88 0.13 0.51 0.60 0.74 0.80 0.85

contact angle sample water CH CHBS-5 CHBS-10 CHBS-20 CHBS-30 CHBS-40 CHBS-50 OP-10 SDBS

γwater = 72.82 mN m−1.

occupied per molecule (A) provides the information regarding the arrangement of molecules adsorbed at air−water interface. The Amin decreased gradually from CH to CHBS-50, indicating that the molecules of CHBSs with higher grafting degree were packed more closely at the interface, and thus effectively reduced the surface tension of solution. The A depends principally on the size of hydrophilic head.26 The smaller hydrophilic head is, the smaller the A is. The hydrophilic head of CHBSs, particularly for −NH2, was consumed by the grafting reaction, and thus considerably diminished. Reasonably, the CHBS-50 that had highest grafting degree presented the minimal Amin at air−water interface. Γmax is negatively related with Amin, and therefore, CHBS-50 also achieved the maximal value of Γmax. This should be due to the fact CHBS-50 has the highest content of hydrophobic oleoyl group, and the hydrophobic effect of surfactants is a main driving force for adsorption at the interface.23 The pc20, a characteristic index of surfactants’ efficiency, increased as the increase of grafting degree. In general, the CHBSs with higher grafting degree, such as CHBS-30, CHBS-40, and CHBS-50, possess better surface adsorption effectiveness and efficiency and could be expected to having better wetting and foaming activities. In comparison with the amino acid−based surfactants which possess similar structural feature to CHBSs, CHBSs showed comparative capacity in reducing surface tension and presented similar surface adsorption characteristics with monomeric surfactants but could not achieve a surface activity as good as gemini surfactants.22,27 It could be due to the reason that CHBSs are the mixture of oleoyl group grafting polypeptides, which negatively affects their interfacial properties. On the other hand, a part of hydrophobic groups of CHBSs would be wrapped in their micromolecular structure, which leads to the decline of surface activity to some extent.28 The advancing contact angle (θa) and receding contact angle (θr) of CH and CHBS solutions on paraffin surface were measured to characterize the wetting property. As shown in Table 3, the θa and θr of water on paraffin surface were all around 134°, indicating that the surface was barely wetted. The contact angles of CH solution were smaller than water, but the paraffin surface still cannot be wetted (θ > 90°). As the grafting degree increased from CHBS-5 to CHBS-30, the contact angles decreased quickly. The contact angles of CHBS-30 were about 78°, indicating that the paraffin surface was partly wetted (θ < 90°). When the grafting degree further increased, the contact angles declined correspondingly, and the smallest contact angles were obtained when CHBS-50 was used. These results demonstrated that the CHBSs with higher oleoyl group grafting degree has a stronger wetting capacity, which coincided with the viewpoint that the surfactants with lower γcmc possess

θa (deg)a 134.6 120.6 106.8 97.5 91.4 78.7 77.4 71.0 83.3 80.7

± ± ± ± ± ± ± ± ± ±

1.0 2.1 5.4 3.1 2.7 0.9 1.4 1.6 2.2 2.4

θr (deg)a 134.2 120.2 107.3 96.3 90.6 77.1 75.9 70.9 81.9 79.9

± ± ± ± ± ± ± ± ± ±

1.1 2.4 4.4 3.1 2.2 0.8 0.9 1.4 4.3 2.8

Data is expressed as the average and standard deviation of five independent replicates. a

stronger wetting activity.29 In contrast experiments, SDBS presented better wetting capacity than OP-10, but they both exhibited weaker wetting capacity in comparison with CHBS30, CHBS-40, and CHBS-50. This could be due to the general phenomenon that the wetting capacity of anionic surfactants (CHBS and SDBS) is commonly better than nonionic surfactants (OP-10), and the anionic surfactants with branched alkyl structures (CHBS) always possess stronger wetting activity than those with linear alkyl structures (SDBS).30 It is well-known that many crops and injurious insects are difficult to be wetted by water-soluble pesticides, owing to the hydrophobic wax coat on their surface. Our previous study has proved that CHBS has good biocompatibility and biodegradability.6 So, CHBSs with high grafting degree might be a promising wetting agent to be used as assistant of pesticides. Effect of the grafting degree of CHBSs on the foaming properties is shown in Figure 4A. It was found that the foaming capacities of CHBSs were obviously better than CH, and the foaming capacity and foam stability of CHBSs exhibited an increasing trend overall as the grafting degree increased, presumably because the surfactant with stronger capacity to reduce surface tension generally possesses better foaming activities.30 The foaming properties of CHBS-20, CHBS-30, CHBS-40, and CHBS-50 were close to or even better than SDBS but worse than OP-10. Additionally, the foaming capacity and foam stability of CHBS-50 were slightly lower than CHBS-40, which might be associated with the relatively lower surface hydrophobicity of CHBS-50, as shown in Table 1. The emulsifying properties of CH and CHBSs are shown in Figure 4B. The emulsifying activity index (EAI) of CH was very low, implying that CH has a poor emulsifying capacity. After grafting oleoyl groups, the emulsifying ability of CHBSs was obviously enhanced, and CHBS-20 exhibited the maximal EAI (54.83%). As the grafting degree further increased, the EAI declined gradually, which might be due to the fact that the hydrophile−lipophile balance (HLB) of CHBSs with higher grafting degree was not suitable for stabilizing the emulsions containing oleic acid.31 Additionally, the EAI of CHBSs was higher than OP-10, probably because CHBS has similar structure to the molecule of oil phase (oleic acid) in the emulsions.32 3.3. Stability of Rapeseed Oil−Water Emulsions Stabilized by CHBSs. Particle size and ζ-potential are generally considered as criteria for assessing the stability of 8505

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Figure 4. Foaming properties (A) and emulsifying property (B) of CH and CHBSs. Bars represent standard deviation (n = 3).

Table 4. Particle Size and ζ-Potential of Rapeseed Oil−Water Emulsions Stabilized by CH and CHBSs at 20 °C ζ-potential (mV)a

particle size (μm)a sample CH CHBS-5 CHBS-10 CHBS-20 CHBS-30 CHBS-40 CHBS-50 OP-10 a

0-day 4.157 3.587 2.45 1.93 1.993 2.027 2.263 1.167

± ± ± ± ± ± ± ±

0.271 0.049 0.137 0.096 0.104 0.08 0.076 0.064

15-day 4.529 3.662 2.671 2.015 2.248 2.313 2.681 1.305

± ± ± ± ± ± ± ±

0-day

0.538 0.257 0.231 0.146 0.169 0.209 0.184 0.122

−16.97 −15.97 −18.27 −19.17 −19.05 −19.00 −18.73 −24.83

± ± ± ± ± ± ± ±

15-day 0.57 0.67 0.25 0.70 0.41 0.53 0.43 0.39

−6.05 −10.51 −12.48 −14.04 −13.75 −13.82 −12.95 −20.91

± ± ± ± ± ± ± ±

2.48 1.20 0.87 1.88 1.57 1.74 1.13 1.42

Data is expressed as the average and standard deviation of three independent replicates.

Figure 5. ζ-Potential (A) and particle size (B) of the rapeseed oil−water emulsions stabilized by CH (▲) and CHBS-20 (□) under different pH conditions. Bars represent standard deviation (n = 3).

emulsions, which is supported by the DLVO theory.33,34 The small particle size of oil droplets or big absolute value of ζpotential promises less aggregation probability of the oil droplets in emulsion, and consequently achieve a better stability of the emulsion. The rapeseed oil−water emulsions were stabilized by CH and CHBSs for 15 days, and then, their particle size and ζ-potential were measured, as shown in Table 4. At 0-day, the particle size of the emulsion stabilized by CH exceeded 4 μm, suggesting that the emulsion was unstable. As the grafting degree of CHBSs increased, the particle size first decreased sharply and then increased slightly, and the minimal value of particle size was obtained when CHBS-20 was used, demonstrating that CHBS-20 possesses the best emulsifying capacity on rapeseed oil−water emulsions. After 15-day storage, the particle size of all the emulsions increased. However, the particle size by using CHBS-20 was also the smallest (only about 2 μm). As for ζ-potential, the absolute value of the

emulsion by using CHBS-20 was the highest, which was consistent with the measurement of the particle size. However, CHBS-20 still did not achieve as good emulsifying activity as commercial emulsifier OP-10. From the view of practical application, the emulsifying capacity of a surfactant at different pHs or in different salt concentrations are important criteria to evaluate its availability being used as an emulsifier. This is due to the facts that the ionization state of an anionic emulsifier (CHBS) may change with pH, and the presence of salt may disrupt the electric double layer of emulsion, which result in the change of emulsion stability. CHBS-20 presented the optimum emulsifying performance on rapeseed oil−water emulsion, so it was used in the following emulsifying stability tests. The ζ-potential and particle size of the emulsions stabilized by CH and CHBS20 in the pH range 3.0−9.0 are shown in Figure 5. The emulsion stability by using CHBS-20 at different pHs was much 8506

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Figure 6. ζ-Potential (A) and particle size (B) of the rapeseed oil−water emulsions stabilized by CH (▲) and CHBS-20 (□) under different NaCl concentrations. Bars represent standard deviation (n = 3).

amino content, isoelectric point and aggregated particle size in aqueous solution decreased. The CHBSs with high grafting degree possessed satisfactory foaming capacity and wetting capacity, and the CHBSs with low grafting degree had good emulsifying capacity. Therefore, the foaming capacity, wetting capacity and emulsifying capacity of CHBS can be well controlled by changing the grafting degree. As a kind of biomass-based surfactant, CHBSs might be promising to be used in different fields according to their grafting degree. Meanwhile, the present work may have displayed a potential alternative of reusing collagen waste.

better than that using CH as expected. As the pH increased from 3.0 to 9.0, the ζ-potential switched from positive charge to negative charge, and the absolute value of the ζ-potential in the pH range 7.0−9.0 was obviously higher than that of pH 3.0− 6.0, indicating that the emulsion stabilized by CHBS-20 has a better stability in neutral and weak alkaline conditions. The zero ζ-potential in the two emulsions was all near pH 4.0, which is consistent with the result of the isoelectric point (IEP) measurement in Table 1, and shows that the emulsions are unstable around pH 4.0. Most molecules of CHBS-20 are protonated around pH 4, and the protonated molecules will decrease the solubility and cmc of CHBS-20 substantially, which result in a significant decline of the emulsion stability.30 In addition, the particle size of oil droplets by using CHBS-20 was always smaller than that by using CH and kept around 2.7 μm in neutral and weak alkaline conditions. However, the particle sizes by using CHBS-20 and CH were all increased rapidly to about 3.8 μm at pH 4.0 owing to the aggregation of oil droplets around zero ζ-potential. These results indicated that CHBS-20 could exhibit good emulsifying capacity in neutral and weak alkaline conditions. Figure 6 displayed the ζ-potential and particle size of the emulsions stabilized by CH and CHBS-20 under different NaCl concentrations. Obviously, the CHBS-20 containing emulsions were also more stable than the emulsions stabilized by CH under all the NaCl concentrations tested. In the NaCl concentration range of 0 to 100 mM, the ζ-potential absolute values of CHBS-20 containing emulsions were higher than 12 mV, suggesting that the emulsions were in a relatively stable state. When the NaCl concentration increased to 250 mM and 500 mM, the absolute value of ζ-potential decreased quickly, and thus, the stability of emulsion turned worse. The increase of electrolyte in solution could neutralize a part of the charge of CHBS-20 and decrease its hydration effect, which led to the decline of solubility, cmc and emulsifying capacity of CHBS-20. As shown in Figure 6B, the particle size of the emulsions stabilized by CHBS-20 kept relatively stable as NaCl concentration increased, presumably because the amide groups (NH−CO) of CHBS possess salt tolerance to some extent.30 So, CHBS-20 might be suitable to be used as a emulsifier in the condition of low salt concentration.

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AUTHOR INFORMATION

Corresponding Author

*Tel.: +86 28 85405508. Fax: +86 28 85400356. E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was financially supported by the National Science and Technology Support Program of China (2011BAC06B11), National Natural Science Foundation of China (21276165), Specialized Research Fund for the Doctoral Program of Higher Education of China (20130181120094), and Open Fund for the Key Lab of Sichuan Province (13-R01).

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REFERENCES

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