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Emulsifying properties of lactose-amines in oil-in-water emulsions
Food Research International 43 (2010) 1111–1115
Contents lists available at ScienceDirect
Food Research International journal homepage: www.elsevier.com/locate/foodres
Emulsifying properties of lactose-amines in oil-in-water emulsions N. Garg a, S. Martini a, D.W. Britt b, M.K. Walsh a,* a b
Nutrition, Dietetics and Food Sciences, Utah State University, Logan, UT 84322-8700, USA Biological and Irrigation Engineering, Utah State University, Logan, UT, USA
a r t i c l e
i n f o
Article history: Received 7 October 2009 Accepted 5 February 2010
Keywords: Lactose-amines Emulsions Emulsion stability Maillard reaction
a b s t r a c t Lactose-amines were synthesized with hexadecyl-amide and lactose via the Maillard reaction and their emulsion stabilization properties were investigated. Lactose-amines were synthesized using two different constant heating (4 and 8 h) and two different heating/cooling cycles (12 and 24 h). Each lactose-amine sample was used as an emulsifier in 20:80 ratio oil-in-water emulsions at four different concentrations (0.01%, 0.05%, 0.1%, and 1%). Emulsion stability was monitored by measuring the oil droplet sizes and the extent of destabilization via clarification over 5 days. At 1% concentrations, emulsions prepared with lactose-amines synthesized for 4, 12, and 24 h were as stable as the whey protein positive control emulsion. The 8 h lactose-amine sample resulted in a less stable emulsion. We assume the difference is related to the amount of heat this sample was exposed to during synthesis, with extensive heat leading to advanced Maillard products, which possessed reduced emulsification properties. Published by Elsevier Ltd.
1. Introduction An emulsion of two immiscible liquids such as oil and water can be formed by applying shear and pressure. With the progression of time, emulsions destabilize by creaming, coalescence and/or flocculation. Emulsifiers, or surface-active agents, can be used to make emulsions stable. The mechanisms of surfactants are based on their amphiphilic properties. Low molecular weight emulsifiers include phospholipids such as lecithin and surfactants such as sugar esters, while high molecular weight emulsifiers are often proteins (caseins, whey proteins, gelatins) (Garti, 1999). Due to the presence of a hemiacetal carbon in glucose, lactose can participate in the Maillard reaction to form an N-lactosylamine in the presence of an amino compound. This compound can rearrange into the Amadori product or ketosamine. Continued heating can result in advanced Maillard products including high molecular weight brown polymers through a series of condensation and polymerization reactions (Liu, Yang, Jin, Hsu, & Chen, 2008; Martins & Van Boekel, 2005; Van Boekel, 2006). Lactose, a by-product of cheese manufacturing, is very economical and yet has low commercial value. The modification of lactose to increase its value is continually being explored. Previous studies have shown that lactose crosslinked to fatty amides or fatty amines results in a novel lactose based hydrogel (Bhattacharya & Acharya, 1999; Dhruv, Draper, & Britt, 2005). Bhattacharya and Acharya (1999) extensively studied the amphiphilic behavior of lactose
* Corresponding author. Tel.: +1 435 797 2177; fax: +1 435 797 2379. E-mail address: [email protected] (M.K. Walsh). 0963-9969/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.foodres.2010.02.003
and maltose coupled to fatty amines and fatty acids. Their synthesized lactose-amines were gels that possessed thermoreversible properties since they were early intermediates in the Maillard reaction. The lactose-amine gels turned clear on applying heat and returned back to an opaque gel with cooling (Bhattacharya & Acharya, 1999; Dhruv et al., 2005). These synthesized polymers, due to their amphiphilic nature may have surfactant properties. The present study used cyclic heating/cooling, and constant heating conditions for the synthesis of lactose-amines from lactose and hexadecyl-amine. Constant heat treatment was used to form intermediate/advanced Maillard products (brown polymers) while the heating/cooling treatment was used to form initial/intermediate Maillard products. The influence of these samples at four different concentrations on the ability to stabilize oil-in-water (o/w) emulsions was investigated. Initial oil droplet sizes and the extent of emulsion destabilization over 5 days were determined and compared to controls to evaluate the effectiveness of lactose-amines synthesized with various heating treatments to act as emulsifiers. 2. Materials and methods 2.1. Materials Lactose was obtained from Hilmar (Hilmar, CA, USA), iso-propanol (90%) and hexadecyl-amine (HCA) (95%) (C16 fatty amine) were purchased from Sigma Aldrich (USA). Whey protein concentrate (WPC) (80% protein, 5% lactose, 6% fat, 3% water, and 6% ash) was obtained from Saputo (St.-Hyacinthe, Quebec). Canola oil was purchased from a local grocery store.
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2.2. Synthesis of lactose-amines For the synthesis of lactose-amines, 250 mM solutions of HCA in 10 ml iso-propanol were added with 250 mM solutions of lactose in 10 ml distilled water according to Dhruv et al. (2005). The treatment groups were 4 h (4H) and 8 h (8H) lactose-amines which were processed for 4 and 8 h of constant heating at 60 °C, while 12 h (12HC) and 24 h (24HC) lactose-amines were processed for 12 and 24 h of cyclic heating at 60 °C followed by cooling at room temperature. For the heating cycle, the solutions were kept in a hot water bath at 60 °C with continuous monitoring of the sample and water bath temperatures. During the heating cycle, when the solutions turned transparent (approximately 30 s), samples were removed from the hot water bath and moved to a room temperature water bath for cooling until they became opaque (approximately 5–10 min). After the synthesis of lactose-amines, the samples were in the form of gels which were frozen to 80 °C. After freezing, the samples were freeze-dried (Dura-Top, FTS Systems, NJ, USA). Dried and ground (grinding was done with mortar and pestle) samples were kept frozen at 4 °C to prevent the possible reverse reactions into lactose and fatty amine. Each lactoseamine sample (4H, 8H, 12HC and 24HC) was synthesized four times and the dried samples were pooled. 2.3. Synthesis of o/w emulsions Emulsions of negative controls and treatment groups were prepared with 80 ml of water and 20 ml of oil. Samples were mixed with a high-speed blender (polytron) (Ultra-Turrax T25, Janke and Kunkel, Staufen, Germany) at 24,000 rpm for 3 min with four different concentrations of lactose, HCA and lactose-amines (0.01%, 0.05%, 0.1% and 1.0%). For the positive control, 80 ml of solution of WPC (2% final protein concentration) and 20 ml of oil were mixed with the polytron as described above. Each solution was homogenized in a microfluidizer (Microfluidics Corporation, Newton, Massachusetts, USA) for 3–5 min at 6900 psi at room temperature. 2.4. Determination of emulsion stability and oil droplet size distribution The droplet size, D(3,2), of the fat globules present in the emulsions was measured using a LS Beckman Coulter droplet size analyzer (LS230, Coulter Corporation, Miami, Florida, USA) with the polarization intensity differential scattering optical module. All measurements were made on two freshly prepared emulsions from each treatment group (4H, 8H, 12HC, and 24HC) at each concentration (0.01%, 0.05%, 0.1%, and 1.0%) and the WPC control, except negative controls (lactose, HCA, and o/w) as they were too unstable to measure. The results for each sample were given in volume (%) of droplet size distribution and droplet size (lm). The oil droplet measurements were taken at angular dependence of the intensity of laser light (k = 632.8 nm) scattered by emulsions, and then the mean oil droplet size was generated as the surface-volume mean particle diameter, using the following equation:
P 3 ni d Dð3;2Þ ¼ Pi¼1 i2 i¼1 ni di where d is the diameter and n is the number of particles. The results were reported as means and standard deviation of D(3,2) and analysis of variance with GLM was conducted to determine significant differences among the concentrations used. The stability of the o/w emulsions with lactose-amines, and both negative and positive controls, was determined using Turbiscan, a vertical scan macroscopic analyzer (TurbiScan MA 2000,
Toulouse, France). About 6 ml of each emulsion was dispensed into glass tubes to measure the percent change in backscattering (DBS%). DBS% was recorded every 15 min over 3 h and then once a day for 5 days. Statistical analysis (two-way factorial with least squares means) was done on the absolute thickness of the clarification layer (0–10 mm at the bottom of the tube) of emulsions formulated with each treatment at each concentration on day 5 data in replicate. 3. Results and discussion 3.1. Synthesis of lactose-amines Each lactose-amine sample possessed a different color due to the extent of heat exposure. Previous research (Dhruv et al., 2005) on the conjugation of lactose to fatty amines provided preliminary information for us to select the treatments that would result in samples with varying degrees of Maillard products. The color of 8H is brown since it was prepared with continuous heating at 60 °C for 8 h and 4H is light brown as its exposure to heat was for 4 h. The resultant brown color of the samples may be the result of dehydration, cyclization, condensation, and polymerization reactions (Liu et al., 2008; Martins & Van Boekel, 2005; Van Boekel, 2006). The 24HC and 12HC samples are white since they were exposed to heat for short times (due to the cooling cycle), i.e. 2–2.5 and 1.5 h, respectively. Based on the progression of the Maillard reaction, it can be assumed that white colored samples (12HC and 24HC) are initial and perhaps early intermediate Maillard products. Studies have shown that there is a series of reversible reactions between a reducing sugar (glucose) and an amino group to form glucosamine, followed by the Amadori rearrangement to yield the Amadori product that can undergo further irreversible reactions of dehydration, condensation and polymerization to yield melanoidins (high and low molecular weight brown nitrogenous polymers and copolymers) known as advanced Maillard products (Liu et al., 2008; Martins & Van Boekel, 2005; Van Boekel, 2006). The light brown color of the 4H sample indicates intermediate/advanced Maillard products, while the dark brown color of the 8H sample may be due to the presence of advanced Maillard products. 3.2. Droplet size measurement of o/w emulsions Table 1 shows the D(3,2) profiles of emulsions formulated with lactose-amines (4H, 8H, 12HC, and 24HC) at various concentrations (0.01%, 0.05%, 0.1%, and 1%) in comparison with WPC on day 0. There is a descending trend of D(3,2) observed from concentrations of 0.01% to 1%, which is statistically significant. Although 12HC shows more variability, with the D(3,2) value at 0.1% higher as compare to other concentrations. The decrease in mean droplet size with an increase in the amount of lactose-amines may be because
Table 1 Droplet mean diameter D(3,2) (lm) of emulsions formulated with lactose-amines compared to whey protein. Treatments
Concentrations 0.01% Average D(3,2)
WPC 4H 8H 12H 24H AB abc
0.48 ± 0.04A 1.09 ± 0.09a 0.81 ± 0.06a 1.01 ± 0.15a 1.17 ± 0.25a
0.05%
0.1%
1.0%
1.08 ± 0.07a 0.72 ± 0.15a 0.90 ± 0.26a 0.77 ± 0.09a
0.70 ± 0.04b 0.57 ± 0.04b 0.73 ± 0.04b 0.62 ± 0.03b
0.51 ± 0.08cA 0.46 ± 0.03cA 0.37 ± 0.03cB 0.33 ± 0.02cB
Means with the same letter are not significantly different. Means with same letter are not significantly different in a row.
N. Garg et al. / Food Research International 43 (2010) 1111–1115
the total droplet surface area that could be stabilized by the lactose-amines increased and the rate of oil droplet coverage increased limiting droplet coalescence (Surh, Ward, & McClements, 2006). D(3,2) values of all lactose-amine samples at a 1% concentration are less than or equal to the WPC control. The reported values of D(3,2) of WPC emulsions varies depending on the amount of protein used and the homogenization pressure and ranges from about 0.4 lm to 1 lm (Demetriades, Coupland, & McClements, 1997; Fomuso, Corredig, & Akoh, 2002; Onsaard, Vittayanont, Srigam, & McClements, 2005; Osborn & Akoh, 2004; Surh et al., 2006) which is similar to the D(3,2) presented here, i.e. 0.48 ± 0.04 lm. The D(3,2) values of 24HC and 12HC at the 1.0% concentration are less than WPC, i.e. 0.32 ± 0.002 and 0.37 ± 0.028, respectively. These D(3,2) values are similar to values reported for emulsions stabilized with 0.5% sucrose fatty acid esters (Osborn & Akoh, 2004). Fig. 1 shows the initial oil droplet size distribution (the size distribution of oil droplets in percent volume) of representative emulsions formulated with two concentrations of lactose-amines compared to WPC. The graphs show the oil droplet distribution in relation to volume % with respect to droplet diameter (lm). WPC has 14% of the volume of oil droplets in the range of 0.1– 1 lm and approximately 4.5% of the volume droplets in the range of 1–8 lm. The bimodal distribution of droplets may be due to a limited amount of flocculation or coalescence, which is observed in some WPC stabilized emulsions and is related to pH and salt concentration. Other researchers have shown that WPC stabilized emulsions generally have the majority of the oil droplets in the
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range of 0.1–1.0 lm (Demetriades, Coupland, & McClements, 1997; Fomuso, Corredig, & Akoh, 2002). Emulsions prepared with 1% of each lactose-amine sample follow a similar droplet size distribution as WPC, while lactoseamines at concentrations of 0.01% show the highest volume % of droplets with diameters greater than 1 lm. Specifically, for 24HC, 12HC, and 4H, the droplet size distribution of 0.01% amines shows a broad range of diameters from approximately 1 to 50 lm. The droplet size distribution for 8H is different from the other lactose amines with sharp droplet diameters at both 5 and 10 lm. From the data presented in Fig. 1, we can conclude that each lactoseamine sample used at a 1% concentration in o/w emulsions stabilized the emulsions similar to WPC with respect to the volume % of droplets in the ranges of 0.1–1 lm and 1–8 lm. There is also a concentration-dependent ability to stabilize emulsions, with concentrations of 0.01% of each lactose-amine resulting in more oil droplets with diameters greater than 1 lm. This may be due to the surface area of the oil droplets being more completely covered with higher concentrations of lactose-amines, resulting in smaller oil droplets and more stable emulsions, while at lower concentrations there is considerable droplet aggregation. This data reinforces the results of Table 1 in that a higher concentration of lactoseamines results in smaller oil droplet diameters. 3.3. Destabilization of o/w emulsions Descending trends in oil droplet size measurements with an increase in concentration of lactose-amines implies a more stable
Fig. 1. Droplet distribution of emulsions formulated with (A) 24HC, (B) 12HC, (C) 4H, (D) 8H lactose-amines at concentrations of 0.01% (d) and 1.0% (j) compared to WPC (s) at day 0.
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emulsion was formed at the highest concentrations tested and the emulsions’ stability was similar to the WPC stabilized emulsion. The stability of emulsions over 5 days was monitored to confirm this observation. Selected Turbiscan data of the percent changes in backscattering (DBS%) over the length of the tubes containing the emulsions over 5 days is given in Fig. 2. In Fig. 2F, the DBS% profile of an emulsion formulated with lactose shows clarification (lines below the zero mark on the y axis) at the bottom of the tube (0–5 mm) and an increase in oil droplet size over the tube length (5–45 mm) over time (lines below the zero mark on the y axis) with creaming (lines above the zero mark on the y axis) and clarification (lines below the zero mark on the y axis) at the top of the tube (45–60 mm). This emulsion is considered unstable since it has both clarification at the bottom of the tube and creaming and clarification at the top of the tube and an increase in oil droplet size. The 0.01% lactose-amine samples showed unstable emulsion patterns similar to the lactose emulsions (data not shown). The WPC sample (Fig. 2E) shows much less clarification at the bottom of the tube (0–5 mm) compared to the lactose stabilized emulsion, constant oil droplet size over the length of the tube over time (5– 45 mm), with clarification at the top of the tube (50–60 nm). This emulsion is more stable than the lactose stabilized emulsion, but does show some destabilization which may be due to flocculation. At a 1.0% concentration of lactose-amines (Fig. 2A–D) there is clarification at the bottom of the tubes to that seen with WPC stabilized emulsions (0–5 mm) with constant droplet size over 5 days and some creaming and/or clarification at the top of the tubes. Specifically, emulsions containing 4H and 24HC show destabilization via clarification at the top of the tubes while emulsions containing 12HC and 8H show destabilization via creaming at the top of the tubes. Overall, the stability of 1% lactose-amine emulsions is similar to the WPC emulsions but does show some creaming and/or clarification at the top of the tubes. A change in the DBS% of an emulsion at the bottom of the tube (0–10 mm) is related to the destabilization of emulsions via clarification. The absolute thickness (AT) of the clarification layer over time can be calculated to follow the extent of destabilization. Fig. 3 shows AT of the clarification layer at the bottom of the tubes over the 5 days tested for representative emulsions. In Fig. 3B, the emulsions containing lactose and just o/w emulsions show high AT values that remain at a value of 8 over the 5 days. These emulsions are unstable, with a quickly forming clarification layer. Emulsions
containing the HCA were initially stable with a low AT value, but became unstable after 2 days. The emulsions containing lactoseamines at 1% concentration, and the WPC emulsion, maintained similar low AT values over the 5 days, implying these emulsions are stable over this time frame. The AT of the clarification layer of lactose-amines at concentrations of 0.01% compared to the controls is shown in Fig. 3A. In this figure, the only stable emulsion formed was that containing WPC, all other emulsions and controls reached high AT values rapidly, indicating that all emulsions except those with WPC were unstable. Statistical analysis was conducted on the AT values from day 5 data (Table 2). The bottom rows of Table 2 shows AT values for the controls. AT values for the o/w emulsion is approximately 8 mm, while the value for the positive control, WPC, is approximately 2. The lactose and fatty amide controls show a slight increase in AT with respect to an increase in concentration; both with AT values significantly higher than the WPC stabilized emulsion. Each of the lactose-amine samples shows significantly increasing emulsification activity (decrease in AT values) with increased concentrations. At concentrations of 0.05% and higher, each lactose-amine sample has a significantly lower AT value than the negative controls, showing that the lactose-amines possessed some emulsification capacity at 0.05%. The 4H and 12HC samples have AT values that are not significantly different than the WPC control at concentrations of 0.1% and 1%. The 24HC sample is not significantly different from the WPC only at the highest concentration used. The 8H sample is significantly different from the WPC control at all concentrations. Each of the lactose-amines synthesized possess some level of emulsification properties, with the greatest difference in the 8H sample, which is always significantly different from the positive WPC control. This sample experienced the most sever heat treatment during synthesis, and therefore may contain advanced Maillard products that have some emulsion stabilizing properties as observed in Fig. 2 and Table 2, but are not as effective as the other lactose-amine samples. A recent study (Liu, Yang, Jin, Hsu, & Chen, 2008) on the kinetics and anti-oxidative activity of a galactose/glycine model system at different temperature treatments concluded that Maillard products produced in the early stages tended to be small molecules that possessed pro-oxidative activity. Extensive heating resulted in polymerization of these small molecules into higher molecular weight compounds with anti-oxidative activity.
Fig. 2. Change in back scattering of o/w emulsions formulated with lactose-amines or lactose at concentrations of 1% (A) 4H, (B) 8H, (C) 12HC, (D) 24HC, (E) WPC, (F) lactose. Arrows show the change in backscattering from day 0 to day 5.
N. Garg et al. / Food Research International 43 (2010) 1111–1115
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Fig. 3. Absolute thickness (at the bottom of the tube from 0 to 10 mm) of the clarification layer of emulsions formulated with lactose-amines 4H (), 8H (h), 12HC (N), and 24HC (j), negative controls lactose (s), HCA (d), and o/w (4) at (A) 0.01%, (B) 1.0% concentrations and WPC (}).
Table 2 Mean absolute thickness of emulsion clarification layers of controls and lactoseamide samples on day 5. Treatments
4H 8H 12HC 24HC Lactose Fatty amides Oil-in-waterd WPCe
Concentration 0.01%
0.05%
0.1%
1%
6.87 ± 0.75Aa 7.73 ± 0.28Aa 6.24 ± 1.27Aa 7.85 ± 0.09Aa 6.62 ± 0.54Aa 6.49 ± 1.40Aa 7.82 ± 0.32Aa 1.81 ± 0.19Ba
3.45 ± 0.08Ab 3.32 ± 0.16Ab 2.82 ± 0.36Ab 3.92 ± 0.61Ab 7.24 ± 0.13Ba 8.4 ± 0.20Bb 7.82 ± 0.32Ba 1.81 ± 0.19Ca
2.18 ± 0.26Ac 2.88 ± 0.25Bc 2.26 ± 0.25Ac 3.22 ± 0.33Bb 8.03 ± 0.33Ba 8.45 ± 0.24Bb 7.82 ± 0.32Ba 1.81 ± 0.19Aa
1.45 ± 0.54Ad 2.61 ± 0.52Bc 2.17 ± 0.50ABc 1.38 ± 0.44Ac 8.35 ± 0.10Cb 8.55 ± 0.15Cc 7.82 ± 0.32Ca 1.81 ± 0.19Aa
ABC
Means with the same letter are not significantly different in each column. Means with same letter are not significantly different in a row. d Oil-in-water emulsions are not at different concentrations, but the same value repeated to show statistical differences. e WPC emulsions contain 2% protein but are repeated to show statistical differences.
4. Conclusion Lactose-amines synthesized with various heating conditions resulted in samples with emulsification activity. There was a concentration-dependent ability of each lactose-amine sample to stabilize o/w emulsions, with the highest concentration resulting in the most stable emulsion. In general, the least severe heat treatment and the most severe heat treatment during synthesis resulted in less emulsion activity as compared to intermediate heat treatment. We propose this may be due to the types of Maillard products (early, intermediate, or advanced) formed. Acknowledgements
abc
This research was supported by Dairy Management Inc., the Western Dairy Center, and the Utah State University Agricultural Research Center. Approved as Journal Paper No. 8146. References
Therefore, the application of extended heat, even to model systems, can result in products with very diverse properties. In this case, the 8H sample may contain various high and low molecular weight compounds that have properties different from the other lactose-amine samples. The 12HC sample did show emulsification properties that were not different from the WPC control, but were also not different from the 8H sample at 1% concentration. This sample experienced the least amount of heating (1.5 h) and therefore may have formed predominately initial Maillard products, lactosylamines and the Amadori product. Previous studies have shown that lactose-amines prepared with cyclic heating possess low molecular weight surfactant properties, but can also be reversed back into lactose and fatty amines on prolonged storage (Bhattacharya & Acharya, 1999). Therefore, the 12HC sample may not have extended emulsification properties suitable for long-term storage of emulsions. With the heating treatments of the 4H and 24HC samples, initial and intermediate Maillard products could have formed that have similar structures to sugar esters which are used as emulsifiers. These two samples also showed similar DBS% profiles in Fig. 2. Compared to the other lactose-amine samples synthesized in this study, the 4H and 24CH samples show emulsification properties similar to WPC with respect to emulsion stability over 5 days.
Bhattacharya, S., & Acharya, S. N. G. (1999). Pronounced hydrocolloids gel formation by the self-assembled aggregates of n-alkyl disaccharide amphiphiles. Chemistry of Materials, 11(12), 3504–3511. Demetriades, K., Coupland, J. N., & McClements, D. J. (1997). Physical properties of whey protein stabilized emulsions as related to pH and NaCl. Journal of Food Science, 62(2), 342–347. Dhruv, H. D., Draper, M. A., & Britt, D. W. (2005). Role of lactose in modifying gel transition temperature and morphology of self-assembled hydrogels. Chemistry of Materials, 17(25), 6239–6245. Fomuso, L. B., Corredig, M., & Akoh, C. S. (2002). Effect of emulsifier on oxidation properties of fish oil-based structured lipid emulsions. Journal of Agriculture and Food Chemistry, 50, 2957–2961. Garti, N. (1999). What can nature offer from an emulsifier point of view: Trends and progress. Colloids and Surfaces A: Physicochemical and Engineering Aspects, 152(1–2), 125–146. Liu, S., Yang, D., Jin, S., Hsu, C., & Chen, S. (2008). Kinetics of color development, pH decreasing, and anti-oxidative activity reduction of Maillard reaction in galactose/glycine model systems. Food Chemistry, 108(2), 533–541. Martins, S. I. F. S., & Van Boekel, M. A. J. S. V. (2005). A kinetic model for the glucose/ glycine Maillard reaction pathways. Food Chemistry, 90(1–2), 257–269. Onsaard, E., Vittayanont, M., Srigam, S., & McClements, D. J. (2005). Properties and stability of oil-in-water emulsions stabilized by coconut skim milk proteins. Journal of Agriculture and Food Chemistry, 53, 5747–5753. Osborn, H. T., & Akoh, C. C. (2004). Effect of emulsifier type, droplet size, and oil concentration on lipid oxidation in structured lipid-based oil-in-water emulsions. Food Chemistry, 84, 451–456. Surh, J., Ward, L. S., & McClements, D. J. (2006). Ability of conventional and nutritionally-modified whey protein concentrates to stabilize oil-in-water emulsions. Food Research International, 39(7), 761–771. Van Boekel, M. A. J. S. V. (2006). Formation of flavor compounds in the Maillard reaction. Biotechnology Advances, 24(2), 230–233.
Contents lists available at ScienceDirect
Food Research International journal homepage: www.elsevier.com/locate/foodres
Emulsifying properties of lactose-amines in oil-in-water emulsions N. Garg a, S. Martini a, D.W. Britt b, M.K. Walsh a,* a b
Nutrition, Dietetics and Food Sciences, Utah State University, Logan, UT 84322-8700, USA Biological and Irrigation Engineering, Utah State University, Logan, UT, USA
a r t i c l e
i n f o
Article history: Received 7 October 2009 Accepted 5 February 2010
Keywords: Lactose-amines Emulsions Emulsion stability Maillard reaction
a b s t r a c t Lactose-amines were synthesized with hexadecyl-amide and lactose via the Maillard reaction and their emulsion stabilization properties were investigated. Lactose-amines were synthesized using two different constant heating (4 and 8 h) and two different heating/cooling cycles (12 and 24 h). Each lactose-amine sample was used as an emulsifier in 20:80 ratio oil-in-water emulsions at four different concentrations (0.01%, 0.05%, 0.1%, and 1%). Emulsion stability was monitored by measuring the oil droplet sizes and the extent of destabilization via clarification over 5 days. At 1% concentrations, emulsions prepared with lactose-amines synthesized for 4, 12, and 24 h were as stable as the whey protein positive control emulsion. The 8 h lactose-amine sample resulted in a less stable emulsion. We assume the difference is related to the amount of heat this sample was exposed to during synthesis, with extensive heat leading to advanced Maillard products, which possessed reduced emulsification properties. Published by Elsevier Ltd.
1. Introduction An emulsion of two immiscible liquids such as oil and water can be formed by applying shear and pressure. With the progression of time, emulsions destabilize by creaming, coalescence and/or flocculation. Emulsifiers, or surface-active agents, can be used to make emulsions stable. The mechanisms of surfactants are based on their amphiphilic properties. Low molecular weight emulsifiers include phospholipids such as lecithin and surfactants such as sugar esters, while high molecular weight emulsifiers are often proteins (caseins, whey proteins, gelatins) (Garti, 1999). Due to the presence of a hemiacetal carbon in glucose, lactose can participate in the Maillard reaction to form an N-lactosylamine in the presence of an amino compound. This compound can rearrange into the Amadori product or ketosamine. Continued heating can result in advanced Maillard products including high molecular weight brown polymers through a series of condensation and polymerization reactions (Liu, Yang, Jin, Hsu, & Chen, 2008; Martins & Van Boekel, 2005; Van Boekel, 2006). Lactose, a by-product of cheese manufacturing, is very economical and yet has low commercial value. The modification of lactose to increase its value is continually being explored. Previous studies have shown that lactose crosslinked to fatty amides or fatty amines results in a novel lactose based hydrogel (Bhattacharya & Acharya, 1999; Dhruv, Draper, & Britt, 2005). Bhattacharya and Acharya (1999) extensively studied the amphiphilic behavior of lactose
* Corresponding author. Tel.: +1 435 797 2177; fax: +1 435 797 2379. E-mail address: [email protected] (M.K. Walsh). 0963-9969/$ - see front matter Published by Elsevier Ltd. doi:10.1016/j.foodres.2010.02.003
and maltose coupled to fatty amines and fatty acids. Their synthesized lactose-amines were gels that possessed thermoreversible properties since they were early intermediates in the Maillard reaction. The lactose-amine gels turned clear on applying heat and returned back to an opaque gel with cooling (Bhattacharya & Acharya, 1999; Dhruv et al., 2005). These synthesized polymers, due to their amphiphilic nature may have surfactant properties. The present study used cyclic heating/cooling, and constant heating conditions for the synthesis of lactose-amines from lactose and hexadecyl-amine. Constant heat treatment was used to form intermediate/advanced Maillard products (brown polymers) while the heating/cooling treatment was used to form initial/intermediate Maillard products. The influence of these samples at four different concentrations on the ability to stabilize oil-in-water (o/w) emulsions was investigated. Initial oil droplet sizes and the extent of emulsion destabilization over 5 days were determined and compared to controls to evaluate the effectiveness of lactose-amines synthesized with various heating treatments to act as emulsifiers. 2. Materials and methods 2.1. Materials Lactose was obtained from Hilmar (Hilmar, CA, USA), iso-propanol (90%) and hexadecyl-amine (HCA) (95%) (C16 fatty amine) were purchased from Sigma Aldrich (USA). Whey protein concentrate (WPC) (80% protein, 5% lactose, 6% fat, 3% water, and 6% ash) was obtained from Saputo (St.-Hyacinthe, Quebec). Canola oil was purchased from a local grocery store.
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2.2. Synthesis of lactose-amines For the synthesis of lactose-amines, 250 mM solutions of HCA in 10 ml iso-propanol were added with 250 mM solutions of lactose in 10 ml distilled water according to Dhruv et al. (2005). The treatment groups were 4 h (4H) and 8 h (8H) lactose-amines which were processed for 4 and 8 h of constant heating at 60 °C, while 12 h (12HC) and 24 h (24HC) lactose-amines were processed for 12 and 24 h of cyclic heating at 60 °C followed by cooling at room temperature. For the heating cycle, the solutions were kept in a hot water bath at 60 °C with continuous monitoring of the sample and water bath temperatures. During the heating cycle, when the solutions turned transparent (approximately 30 s), samples were removed from the hot water bath and moved to a room temperature water bath for cooling until they became opaque (approximately 5–10 min). After the synthesis of lactose-amines, the samples were in the form of gels which were frozen to 80 °C. After freezing, the samples were freeze-dried (Dura-Top, FTS Systems, NJ, USA). Dried and ground (grinding was done with mortar and pestle) samples were kept frozen at 4 °C to prevent the possible reverse reactions into lactose and fatty amine. Each lactoseamine sample (4H, 8H, 12HC and 24HC) was synthesized four times and the dried samples were pooled. 2.3. Synthesis of o/w emulsions Emulsions of negative controls and treatment groups were prepared with 80 ml of water and 20 ml of oil. Samples were mixed with a high-speed blender (polytron) (Ultra-Turrax T25, Janke and Kunkel, Staufen, Germany) at 24,000 rpm for 3 min with four different concentrations of lactose, HCA and lactose-amines (0.01%, 0.05%, 0.1% and 1.0%). For the positive control, 80 ml of solution of WPC (2% final protein concentration) and 20 ml of oil were mixed with the polytron as described above. Each solution was homogenized in a microfluidizer (Microfluidics Corporation, Newton, Massachusetts, USA) for 3–5 min at 6900 psi at room temperature. 2.4. Determination of emulsion stability and oil droplet size distribution The droplet size, D(3,2), of the fat globules present in the emulsions was measured using a LS Beckman Coulter droplet size analyzer (LS230, Coulter Corporation, Miami, Florida, USA) with the polarization intensity differential scattering optical module. All measurements were made on two freshly prepared emulsions from each treatment group (4H, 8H, 12HC, and 24HC) at each concentration (0.01%, 0.05%, 0.1%, and 1.0%) and the WPC control, except negative controls (lactose, HCA, and o/w) as they were too unstable to measure. The results for each sample were given in volume (%) of droplet size distribution and droplet size (lm). The oil droplet measurements were taken at angular dependence of the intensity of laser light (k = 632.8 nm) scattered by emulsions, and then the mean oil droplet size was generated as the surface-volume mean particle diameter, using the following equation:
P 3 ni d Dð3;2Þ ¼ Pi¼1 i2 i¼1 ni di where d is the diameter and n is the number of particles. The results were reported as means and standard deviation of D(3,2) and analysis of variance with GLM was conducted to determine significant differences among the concentrations used. The stability of the o/w emulsions with lactose-amines, and both negative and positive controls, was determined using Turbiscan, a vertical scan macroscopic analyzer (TurbiScan MA 2000,
Toulouse, France). About 6 ml of each emulsion was dispensed into glass tubes to measure the percent change in backscattering (DBS%). DBS% was recorded every 15 min over 3 h and then once a day for 5 days. Statistical analysis (two-way factorial with least squares means) was done on the absolute thickness of the clarification layer (0–10 mm at the bottom of the tube) of emulsions formulated with each treatment at each concentration on day 5 data in replicate. 3. Results and discussion 3.1. Synthesis of lactose-amines Each lactose-amine sample possessed a different color due to the extent of heat exposure. Previous research (Dhruv et al., 2005) on the conjugation of lactose to fatty amines provided preliminary information for us to select the treatments that would result in samples with varying degrees of Maillard products. The color of 8H is brown since it was prepared with continuous heating at 60 °C for 8 h and 4H is light brown as its exposure to heat was for 4 h. The resultant brown color of the samples may be the result of dehydration, cyclization, condensation, and polymerization reactions (Liu et al., 2008; Martins & Van Boekel, 2005; Van Boekel, 2006). The 24HC and 12HC samples are white since they were exposed to heat for short times (due to the cooling cycle), i.e. 2–2.5 and 1.5 h, respectively. Based on the progression of the Maillard reaction, it can be assumed that white colored samples (12HC and 24HC) are initial and perhaps early intermediate Maillard products. Studies have shown that there is a series of reversible reactions between a reducing sugar (glucose) and an amino group to form glucosamine, followed by the Amadori rearrangement to yield the Amadori product that can undergo further irreversible reactions of dehydration, condensation and polymerization to yield melanoidins (high and low molecular weight brown nitrogenous polymers and copolymers) known as advanced Maillard products (Liu et al., 2008; Martins & Van Boekel, 2005; Van Boekel, 2006). The light brown color of the 4H sample indicates intermediate/advanced Maillard products, while the dark brown color of the 8H sample may be due to the presence of advanced Maillard products. 3.2. Droplet size measurement of o/w emulsions Table 1 shows the D(3,2) profiles of emulsions formulated with lactose-amines (4H, 8H, 12HC, and 24HC) at various concentrations (0.01%, 0.05%, 0.1%, and 1%) in comparison with WPC on day 0. There is a descending trend of D(3,2) observed from concentrations of 0.01% to 1%, which is statistically significant. Although 12HC shows more variability, with the D(3,2) value at 0.1% higher as compare to other concentrations. The decrease in mean droplet size with an increase in the amount of lactose-amines may be because
Table 1 Droplet mean diameter D(3,2) (lm) of emulsions formulated with lactose-amines compared to whey protein. Treatments
Concentrations 0.01% Average D(3,2)
WPC 4H 8H 12H 24H AB abc
0.48 ± 0.04A 1.09 ± 0.09a 0.81 ± 0.06a 1.01 ± 0.15a 1.17 ± 0.25a
0.05%
0.1%
1.0%
1.08 ± 0.07a 0.72 ± 0.15a 0.90 ± 0.26a 0.77 ± 0.09a
0.70 ± 0.04b 0.57 ± 0.04b 0.73 ± 0.04b 0.62 ± 0.03b
0.51 ± 0.08cA 0.46 ± 0.03cA 0.37 ± 0.03cB 0.33 ± 0.02cB
Means with the same letter are not significantly different. Means with same letter are not significantly different in a row.
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the total droplet surface area that could be stabilized by the lactose-amines increased and the rate of oil droplet coverage increased limiting droplet coalescence (Surh, Ward, & McClements, 2006). D(3,2) values of all lactose-amine samples at a 1% concentration are less than or equal to the WPC control. The reported values of D(3,2) of WPC emulsions varies depending on the amount of protein used and the homogenization pressure and ranges from about 0.4 lm to 1 lm (Demetriades, Coupland, & McClements, 1997; Fomuso, Corredig, & Akoh, 2002; Onsaard, Vittayanont, Srigam, & McClements, 2005; Osborn & Akoh, 2004; Surh et al., 2006) which is similar to the D(3,2) presented here, i.e. 0.48 ± 0.04 lm. The D(3,2) values of 24HC and 12HC at the 1.0% concentration are less than WPC, i.e. 0.32 ± 0.002 and 0.37 ± 0.028, respectively. These D(3,2) values are similar to values reported for emulsions stabilized with 0.5% sucrose fatty acid esters (Osborn & Akoh, 2004). Fig. 1 shows the initial oil droplet size distribution (the size distribution of oil droplets in percent volume) of representative emulsions formulated with two concentrations of lactose-amines compared to WPC. The graphs show the oil droplet distribution in relation to volume % with respect to droplet diameter (lm). WPC has 14% of the volume of oil droplets in the range of 0.1– 1 lm and approximately 4.5% of the volume droplets in the range of 1–8 lm. The bimodal distribution of droplets may be due to a limited amount of flocculation or coalescence, which is observed in some WPC stabilized emulsions and is related to pH and salt concentration. Other researchers have shown that WPC stabilized emulsions generally have the majority of the oil droplets in the
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range of 0.1–1.0 lm (Demetriades, Coupland, & McClements, 1997; Fomuso, Corredig, & Akoh, 2002). Emulsions prepared with 1% of each lactose-amine sample follow a similar droplet size distribution as WPC, while lactoseamines at concentrations of 0.01% show the highest volume % of droplets with diameters greater than 1 lm. Specifically, for 24HC, 12HC, and 4H, the droplet size distribution of 0.01% amines shows a broad range of diameters from approximately 1 to 50 lm. The droplet size distribution for 8H is different from the other lactose amines with sharp droplet diameters at both 5 and 10 lm. From the data presented in Fig. 1, we can conclude that each lactoseamine sample used at a 1% concentration in o/w emulsions stabilized the emulsions similar to WPC with respect to the volume % of droplets in the ranges of 0.1–1 lm and 1–8 lm. There is also a concentration-dependent ability to stabilize emulsions, with concentrations of 0.01% of each lactose-amine resulting in more oil droplets with diameters greater than 1 lm. This may be due to the surface area of the oil droplets being more completely covered with higher concentrations of lactose-amines, resulting in smaller oil droplets and more stable emulsions, while at lower concentrations there is considerable droplet aggregation. This data reinforces the results of Table 1 in that a higher concentration of lactoseamines results in smaller oil droplet diameters. 3.3. Destabilization of o/w emulsions Descending trends in oil droplet size measurements with an increase in concentration of lactose-amines implies a more stable
Fig. 1. Droplet distribution of emulsions formulated with (A) 24HC, (B) 12HC, (C) 4H, (D) 8H lactose-amines at concentrations of 0.01% (d) and 1.0% (j) compared to WPC (s) at day 0.
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emulsion was formed at the highest concentrations tested and the emulsions’ stability was similar to the WPC stabilized emulsion. The stability of emulsions over 5 days was monitored to confirm this observation. Selected Turbiscan data of the percent changes in backscattering (DBS%) over the length of the tubes containing the emulsions over 5 days is given in Fig. 2. In Fig. 2F, the DBS% profile of an emulsion formulated with lactose shows clarification (lines below the zero mark on the y axis) at the bottom of the tube (0–5 mm) and an increase in oil droplet size over the tube length (5–45 mm) over time (lines below the zero mark on the y axis) with creaming (lines above the zero mark on the y axis) and clarification (lines below the zero mark on the y axis) at the top of the tube (45–60 mm). This emulsion is considered unstable since it has both clarification at the bottom of the tube and creaming and clarification at the top of the tube and an increase in oil droplet size. The 0.01% lactose-amine samples showed unstable emulsion patterns similar to the lactose emulsions (data not shown). The WPC sample (Fig. 2E) shows much less clarification at the bottom of the tube (0–5 mm) compared to the lactose stabilized emulsion, constant oil droplet size over the length of the tube over time (5– 45 mm), with clarification at the top of the tube (50–60 nm). This emulsion is more stable than the lactose stabilized emulsion, but does show some destabilization which may be due to flocculation. At a 1.0% concentration of lactose-amines (Fig. 2A–D) there is clarification at the bottom of the tubes to that seen with WPC stabilized emulsions (0–5 mm) with constant droplet size over 5 days and some creaming and/or clarification at the top of the tubes. Specifically, emulsions containing 4H and 24HC show destabilization via clarification at the top of the tubes while emulsions containing 12HC and 8H show destabilization via creaming at the top of the tubes. Overall, the stability of 1% lactose-amine emulsions is similar to the WPC emulsions but does show some creaming and/or clarification at the top of the tubes. A change in the DBS% of an emulsion at the bottom of the tube (0–10 mm) is related to the destabilization of emulsions via clarification. The absolute thickness (AT) of the clarification layer over time can be calculated to follow the extent of destabilization. Fig. 3 shows AT of the clarification layer at the bottom of the tubes over the 5 days tested for representative emulsions. In Fig. 3B, the emulsions containing lactose and just o/w emulsions show high AT values that remain at a value of 8 over the 5 days. These emulsions are unstable, with a quickly forming clarification layer. Emulsions
containing the HCA were initially stable with a low AT value, but became unstable after 2 days. The emulsions containing lactoseamines at 1% concentration, and the WPC emulsion, maintained similar low AT values over the 5 days, implying these emulsions are stable over this time frame. The AT of the clarification layer of lactose-amines at concentrations of 0.01% compared to the controls is shown in Fig. 3A. In this figure, the only stable emulsion formed was that containing WPC, all other emulsions and controls reached high AT values rapidly, indicating that all emulsions except those with WPC were unstable. Statistical analysis was conducted on the AT values from day 5 data (Table 2). The bottom rows of Table 2 shows AT values for the controls. AT values for the o/w emulsion is approximately 8 mm, while the value for the positive control, WPC, is approximately 2. The lactose and fatty amide controls show a slight increase in AT with respect to an increase in concentration; both with AT values significantly higher than the WPC stabilized emulsion. Each of the lactose-amine samples shows significantly increasing emulsification activity (decrease in AT values) with increased concentrations. At concentrations of 0.05% and higher, each lactose-amine sample has a significantly lower AT value than the negative controls, showing that the lactose-amines possessed some emulsification capacity at 0.05%. The 4H and 12HC samples have AT values that are not significantly different than the WPC control at concentrations of 0.1% and 1%. The 24HC sample is not significantly different from the WPC only at the highest concentration used. The 8H sample is significantly different from the WPC control at all concentrations. Each of the lactose-amines synthesized possess some level of emulsification properties, with the greatest difference in the 8H sample, which is always significantly different from the positive WPC control. This sample experienced the most sever heat treatment during synthesis, and therefore may contain advanced Maillard products that have some emulsion stabilizing properties as observed in Fig. 2 and Table 2, but are not as effective as the other lactose-amine samples. A recent study (Liu, Yang, Jin, Hsu, & Chen, 2008) on the kinetics and anti-oxidative activity of a galactose/glycine model system at different temperature treatments concluded that Maillard products produced in the early stages tended to be small molecules that possessed pro-oxidative activity. Extensive heating resulted in polymerization of these small molecules into higher molecular weight compounds with anti-oxidative activity.
Fig. 2. Change in back scattering of o/w emulsions formulated with lactose-amines or lactose at concentrations of 1% (A) 4H, (B) 8H, (C) 12HC, (D) 24HC, (E) WPC, (F) lactose. Arrows show the change in backscattering from day 0 to day 5.
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Fig. 3. Absolute thickness (at the bottom of the tube from 0 to 10 mm) of the clarification layer of emulsions formulated with lactose-amines 4H (), 8H (h), 12HC (N), and 24HC (j), negative controls lactose (s), HCA (d), and o/w (4) at (A) 0.01%, (B) 1.0% concentrations and WPC (}).
Table 2 Mean absolute thickness of emulsion clarification layers of controls and lactoseamide samples on day 5. Treatments
4H 8H 12HC 24HC Lactose Fatty amides Oil-in-waterd WPCe
Concentration 0.01%
0.05%
0.1%
1%
6.87 ± 0.75Aa 7.73 ± 0.28Aa 6.24 ± 1.27Aa 7.85 ± 0.09Aa 6.62 ± 0.54Aa 6.49 ± 1.40Aa 7.82 ± 0.32Aa 1.81 ± 0.19Ba
3.45 ± 0.08Ab 3.32 ± 0.16Ab 2.82 ± 0.36Ab 3.92 ± 0.61Ab 7.24 ± 0.13Ba 8.4 ± 0.20Bb 7.82 ± 0.32Ba 1.81 ± 0.19Ca
2.18 ± 0.26Ac 2.88 ± 0.25Bc 2.26 ± 0.25Ac 3.22 ± 0.33Bb 8.03 ± 0.33Ba 8.45 ± 0.24Bb 7.82 ± 0.32Ba 1.81 ± 0.19Aa
1.45 ± 0.54Ad 2.61 ± 0.52Bc 2.17 ± 0.50ABc 1.38 ± 0.44Ac 8.35 ± 0.10Cb 8.55 ± 0.15Cc 7.82 ± 0.32Ca 1.81 ± 0.19Aa
ABC
Means with the same letter are not significantly different in each column. Means with same letter are not significantly different in a row. d Oil-in-water emulsions are not at different concentrations, but the same value repeated to show statistical differences. e WPC emulsions contain 2% protein but are repeated to show statistical differences.
4. Conclusion Lactose-amines synthesized with various heating conditions resulted in samples with emulsification activity. There was a concentration-dependent ability of each lactose-amine sample to stabilize o/w emulsions, with the highest concentration resulting in the most stable emulsion. In general, the least severe heat treatment and the most severe heat treatment during synthesis resulted in less emulsion activity as compared to intermediate heat treatment. We propose this may be due to the types of Maillard products (early, intermediate, or advanced) formed. Acknowledgements
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This research was supported by Dairy Management Inc., the Western Dairy Center, and the Utah State University Agricultural Research Center. Approved as Journal Paper No. 8146. References
Therefore, the application of extended heat, even to model systems, can result in products with very diverse properties. In this case, the 8H sample may contain various high and low molecular weight compounds that have properties different from the other lactose-amine samples. The 12HC sample did show emulsification properties that were not different from the WPC control, but were also not different from the 8H sample at 1% concentration. This sample experienced the least amount of heating (1.5 h) and therefore may have formed predominately initial Maillard products, lactosylamines and the Amadori product. Previous studies have shown that lactose-amines prepared with cyclic heating possess low molecular weight surfactant properties, but can also be reversed back into lactose and fatty amines on prolonged storage (Bhattacharya & Acharya, 1999). Therefore, the 12HC sample may not have extended emulsification properties suitable for long-term storage of emulsions. With the heating treatments of the 4H and 24HC samples, initial and intermediate Maillard products could have formed that have similar structures to sugar esters which are used as emulsifiers. These two samples also showed similar DBS% profiles in Fig. 2. Compared to the other lactose-amine samples synthesized in this study, the 4H and 24CH samples show emulsification properties similar to WPC with respect to emulsion stability over 5 days.
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