Effects of ultrasound intensities on a glycin–maltose model system – a means of promoting Maillard reaction

Abstract

Effects of ultrasound on pH, intermediate products, browning, maltose content, reducing power and antioxidant activity of a glycin–maltose solution were investigated. Results showed that the ultrasonic treatment at the intensity of 17.83 W cm⁻² for 100 min resulted in the increases of the solution’s absorbance at 294 and 420 nm and the antioxidant activity from approximately 0% to 1.06%, 0.30% and 22.53%, respectively. At the same time, 86.93% reduction in maltose was observed. In addition, little 5-hydroxymethyl-2-furaldehyde was found in HPLC analyses. This study indicated that ultrasound, especially at higher intensities, could potentially be employed as a means to promote the Maillard reaction in the glycin–maltose solution.

Introduction

The Maillard reaction occurs in thermally processed foods (Carabasa-Giribet & Ibarz-Ribas, 2000). The complex series of compounds formed are Maillard reaction products (MRPs) (Mastrocola & Munari, 2000). The Maillard reaction rate depends upon many factors including chemical composition (e.g. proteins, amino acids, reducing sugars or carbohydrates) (Yeboah et al., 1999; Morales & Jimenez-Perez, 2001; Lertittikul et al., 2007; Karoui et al., 2008; Wong et al., 2008), time, temperature, reactant concentration (Jing & Kitts, 2002) and pH (Ajandouz et al., 2001; Noor-Soffalina et al., 2009).

MRPs have been shown to inhibit oxidation during food storage (Nicoli et al., 1997; Osada & Shibamoto, 2006). Their browning and reducing power might correlate with the antioxidant activity (Morales & Jimenez-Perez, 2001). However, recent study shows that MRPs has also been demonstrated to cause toxicological effects or health problems (Baynes & Thorpe, 1999; Lo et al., 2008). For example, asparagine-mediated Maillard reaction is known to lead to the formation of neurotoxic acrylamide (Mottram et al., 2002) in a heating treatment. Methylglyoxal (MG), a major flavour precursor, is a reactive carbonyl compound found in humans and modifies protein residues to form advanced glycation endproducts (AGEs) which are linked to hyperglycaemia and diabetes complications (Singh et al., 2001). Thus the food processing techniques responsible for MRP formation need to be understood and characterised in more detailed.

The characteristics of maltose–amino acid model Maillard reaction has been investigated. It was clearly shown that maltose reacted with methylammoniumacetate in a hot aqueous solution giving a dark brown mixture of products. 1,2-Dimethyl-3-hydroxy-4-pyridone can be isolated from the volatile compounds (Severine & Loidl, 1976). On the other hand, the maltose–glycine Maillard reaction is an effective means to produce melanoidins (Mundt & Wedzicha, 2005).

In the past two decades, ultrasonic cavitation has seen diverse applications in a number of fields, such as synthesis of nanostructured materials (Okitsu et al., 2002), processing of biomass (Stavarache et al., 2005), sonofusion (Taleyarkhan et al., 2004), sonodynamic therapy (Rosenthal et al., 2004; Tiwari et al., 2009), food processing (Zenker et al., 2003; Wu et al., 2008; Adriana et al., 2009), emulsions preparation (Kentish et al., 2008) and sonochemical degradation of pollutants and hazardous chemicals (Okitsu et al., 2005; Nakui et al., 2007). Low frequency ultrasonic field is known to produce unique chemical and physical effects that arise from the collapse of the cavitation bubbles. At high intensity, it was found to promote browning in orange juice (Tiwari et al., 2008). However, report on the ultrasound’s effect on the Maillard reaction exists in the literature is scarce. Thus, this study aims to investigate the characteristics and antioxidant activity of MRPs produced by low frequency ultrasound in a glycin–maltose model system.

Materials and methods

Chemicals

1,1-Diphenyl-2-picryl-hydrazyl (DPPH), glycin, maltose, 5-hydroxymethyl-2-furaldehyde (HMF) and potassium ferricyanide were purchased from Sigma-Aldrich (St Louis, MO, USA). Trichloroacetic acid was obtained from Riedel-deHaen (Seelze, Germany). Other chemicals were chromatographic grade and purchased from Merck (Damstadt, Germany).

Preparation of glycin–maltose solution and the ultrasound tests

The experimentation employed a SCIENTZ Electronics ultrasound device (JY98-3, Ningbo, China) at a frequency of 25 kHz and a maximum nominal power of 1600 W with the manual on–off control. The glycin–maltose solution was prepared by mixing 0.01 mol glycin with 0.01 mol maltose in 90 mL de-ionised water. The pH of the solution was adjusted to 10 with 6 mol L⁻¹ NaOH. It was then brought to 100 mL with de-ionised water. After thorough mixing, the solution was ready for the ultrasound tests. Various intensity levels (i.e. 10.19, 12.74, 15.29 and 17.83 W cm⁻²) and treatment time (i.e. 0, 20, 40, 60, 80 and 100 min) of the ultrasound with a 5 s-on-and-5 s-off pulsation were performed on the glycin–maltose solution. For each test, 100 mL glycin–maltose solution was placed in a 200 mL jacketed vessel with a constant flow of 4 ±2 °C circulation water at a rate of 0.6 L min⁻¹ to maintain a sample temperature below 50 °C, and the time/temperature profile was shown in Fig. 1. A digital thermometer was used to detect the sample temperature. The 2.0 cm-diameter ultrasound probe was submerged in the solution 2.5 cm below the surface. In order to expatiate the real impact of ultrasound on the formation of Maillard products in the present experiments, a control is necessary, and the mixed glycin–maltose solution was heated to 50 °C for different time (i.e. 0, 20, 40, 60, 80 and 100 min). All tests were carried out in triplicate. After ultrasound or thermal treatment, 10 mL of the solution was immediately placed in iced water for 30 min to stop the reaction. Samples were kept at 4 °C before chemical analysis within 24 h.

pH measurement

The pH value of system is always changed during Maillard reaction (Lertittikul et al., 2007). pH is, therefore, an important parameter that reflects the extent of the Maillard reaction. A pH meter (Model SP-71; Suntex Inc., Taipei, Taiwan) was used for the determination.

Measurements of UV-absorbance and browning

UV-absorbance and browning of MRPs were measured according to the method of Ajandouz et al. (2001) with a slight modification to detect the colour changes due to the Maillard reaction. The upper limit of the spectrophotometer (UV-2550 spectrophotometer; Shimadzu, Kyoto, Japan) was 4.00 on the scale. Therefore, a thirty fold dilution of the treated solution was made using distilled water for the determinations of the absorbance at 294 nm, as well as the browning intensity at 420 nm.

HPLC analysis of maltose content

Each solution was filtered through a Millex-HN nylon clarification kit, 0.45 μm pore size (Millipore, Bedford, MA, USA), and then analysed using an HPLC-Refractive Index Detector (RID) system. The system contains a Sugar-pak1 6.5 × 300 mm Ion Exchange Chromatography (Waters Co., Milford, MA, USA), a Waters 600 pump and a Waters 2414 Refractive Index Detector (Waters Co.). The injection volume was 20 μL, and the mobile phase was a 50 mg L⁻¹ EDTA–Ca water solution over 30 min at a flow rate of 0.5 mL min⁻¹. Column temperature was 90 °C.

HPLC analysis of HMF content

HPLC analysis of HMF was determined according to the method of Sanz et al. (2003) with a slight modification. Each treated sample was filtered through a Millex-HN nylon clarification kit of 0.45 μm pore size (Millipore), and then analysed by an HPLC-Diode Array Detector (DAD) system, which consisted of a 5 μm Waters XTerra RP18 150 × 4.6 mm column (Waters Co.), a Waters 600 pump and a Waters 996 diode array detector (Waters Co.). The injection volume was 20 μL. The mobile phase was a linear gradient of 100% water to 100% methanol over 60 min at a flow rate of 1.0 mL min⁻¹. Column temperature was set at 30 °C. Absorption spectra, in the range of 200–500 nm, were recorded. The chromatographic peak of the compound was compared with the retention time of a HMF standard compound to allow confirmation.

Determination of reducing power

The reducing power of MRPs was determined according to the method of Lertittikul et al. (2007) with a slight modification. One millilitre of a fivefold dilution of the MRP sample was mixed with 1 mL of 0.2 mol L⁻¹ sodium phosphate buffer (pH 6.6) and 1 mL of 1% potassium ferricyanide. The mixture was incubated in a water bath at 50 °C for 20 min, added with 1 mL of 10% trichloroacetic acid, and followed by centrifugation at 750× g for 10 min at room temperature using a Mikro 20 centrifuge (Hettick zentrifugen, Germany). One milliliter of the supernatant was added with 1 mL of de-ionised water and 200 μL of 0.1% FeCl₃. The blank was prepared in the same manner as described above, except that 1% potassium ferricyanide was replaced by distilled water. The absorbance at 700 nm was measured for the sample solutions. The reducing power was expressed as an increase in A₇₀₀ over the blank.

Determination of DPPH radical-scavenging activity

DPPH radical-scavenging activity was determined according to the method of Lertittikul et al. (2007). To 400 μL of a twenty fold dilution of the MRPs sample, 2 mL of 0.12 mmol L⁻¹ DPPH in ethanol was added. The mixture of MRPs and DPPH–ethanol solution was then allowed to stand at room temperature in the dark for 30 min. The absorbance of the mixture was measured at 517 nm using a UV-2550 spectrophotometer. The control was prepared in the same manner, except that de-ionised water was used instead of MRPs sample. DPPH radical-scavenging activity was calculated by the following formula (Singh & Rajini, 2004):

where A_s is the absorbance value of sample; A_c is the absorbance value of the control of the DPPH radical-scavenging activity test.

Statistical analysis

All experiments were carried out in triplicates. Means and standard deviations of the data were calculated for each treatment by using Excel software (Microsoft Office 2003). Analysis of variance (Anova) was carried out to determine any significant differences (P < 0.05) among the applied treatments by a Spss package (Spss 10.0 for windows; SPSS Inc., Chicago, IL, USA).

Results and discussion

Changes in pH

The changes in pH of the glycin–maltose solution after treatment with different ultrasonic intensities are shown in Fig. 2. The pH changed little at the control (heating at 50 °C) and the intensity of 10.19 W cm⁻² regardless of the reaction time. Slight changes were observed at the intensity of 12.74 or 15.29 W cm⁻², especially during the last 60 min of the treatment. However, significant changes in pH appeared as the ultrasonic intensity was increased to 17.83 W cm⁻² at all reaction times (P < 0.05). For example, at ultrasonic intensity of 17.83 W cm⁻², the pHs reduced 7.03%, 8.77%, 11.23% and 12.83% at 40, 60, 80 and 100 min, respectively.

The result revealed that only ultrasound intensity affects the rate of reaction, but not interaction between time and temperature (the result of control in Fig. 2). On the other hand, ultrasound at higher intensities could produce MRPs and reduce the pH in the model solution. The pH reduction might be due to the decrease in amino acids and the formation of organic acids, such as formic and acetic acid (Van Boekel & Martins, 2002) at the ultrasonic intensity of 17.83 W cm⁻². The result was in agreement with that obtained under hydrothermal method by Morales & Jimenez-Perez (2001) who found that the pH value of glucose–lysine model solution reacted for 24 h reduced from about 7 to 4.33.

Changes in A₂₉₄

The A₂₉₄ of the glycin–maltose solution after being treated by the ultrasonic intensity at 17.83 W cm⁻² showed a significant increase within the last 60 min (P < 0.05) (Fig. 3). At the ultrasonic intensity of 17.83 W cm⁻², the A₂₉₄ increased from approximately 0 to 0.25, 0.54, 1.01 and 1.06 at 40, 60, 80 and 100 min, respectively. Slight changes were observed at the intensity of 12.74 or 15.29 W cm⁻². On the other hand, at lower ultrasonic intensities (i.e. 10.19 W cm⁻²) and in the control tests, the A₂₉₄ changes were not significant.

The results suggested that MRPs were produced to a great extent with higher ultrasonic intensities (i.e. 17.83 W cm⁻²). With extended reaction time, some intermediate products might polymerise resulting in only a small amount of intermediate products. For example, when the treatment time was longer than 80 min (at ultrasonic intensity of 17.83 W cm⁻²), some intermediate products turned into new polymers, leaving a reduced amount of the intermediate products. Meanwhile, maltose continued to be reacted and formed new intermediate products (Fig. 4). The rates of formation and polymerisation of the intermediate products might become equal. Thus, the change of A₂₉₄, as shown in Fig. 3, was slight after 80 min treatment at 17.83 W cm⁻². The mechanism and the rate of the MRPs formation affected by the ultrasonic intensity need further study.

Changes in browning intensity

A  ₄₂₀ of the glycin–maltose solution treated with different ultrasonic intensities was a measure of the browning effect. As shown in Fig. 5, the A₄₂₀ increased significantly at 17.83 W cm⁻² between 40 and 100 min (P < 0.05). At the ultrasonic intensity of 17.83 W cm⁻², the A₄₂₀ increased from approximately 0 to 0.06, 0.11 and 0.30 at the treatment time of 60, 80 and 100 min, respectively. Slight, but not significant, changes were observed at the intensity of 12.74 or 15.29 W cm⁻². On the other hand, at lower ultrasonic intensities (i.e. 10.19 W cm⁻²) and in the control tests, the A₄₂₀ changed only slightly at all reaction times.

The brown pigment development, indicated by the A₄₂₀, coincided with the colourless intermediate formation, which was evidenced by the increased A₂₉₄. The correlation suggested that the brown pigments were formed parallel to the intermediate products generated. At the ultrasonic intensity of 17.83 W cm⁻², when the treatment time reached 80 min the reaction rate became low because of little maltose remaining in the solution (Fig. 4). However, browning intensity was increased significantly at that time. An explanation might be that browning pigment was developed by the polymerisation between intermediate products. On the other hand, little change was found in the control test, which means that interaction between time and temperature/intensity is not a main factor to promote Maillard reaction in glycin–maltose system by using ultrasound treatment. This result was in accordance with Lertittikul et al. (2007), who found that, during the Maillard reaction, intermediate products and browning frequently increased as the reaction time extended.

Changes in maltose content

The HPLC peak was identified to be maltose by comparing the retention times between it and standard compounds. The regression equation for maltose standard is a* = 7 × 10⁷C + 20070; R² = 0.9989, where C = maltose concentration, mol L⁻¹; a* = peak area of maltose. The minimum detection limit of the maltose assay by HPLC–RID is 0.0005 mol L⁻¹ in this test. And the experimental range was 0.0005–0.5 mol L⁻¹.

As shown in Fig. 4, little change was found in the control tests, which suggsets that interaction between time and temperature (lower than 45 °C) is not an effective factor to promote Maillard reaction. On the other hand, at lower ultrasonic intensities (i.e. 10.19 W cm⁻²), no significant change on maltose content was found. Slight changes were observed at the intensity of 12.74 or 15.29 W cm⁻². For example, only 15.77% maltose was reduced after 100 min treatment at 15.29 W cm⁻². However, as the intensity was raised to 17.83 W cm⁻², maltose in the solution decreased drastically at all reaction times (P < 0.05). At 17.83 W cm⁻², it reduced by 19.40%, 43.91%, 79.16% and 86.93% for 40, 60, 80 and 100 min of treatment, respectively.

Little HMF was found in HPLC analyses. Thus, maltose was degraded to glucose and then occurred to a simple dehydration of losing three water molecules cannot explain the HMF results (Sanz et al., 2003) in this test. On the other hand, the decreases of maltose paralleled the increase in colourless MRPs and brown pigment formation. Yet, the reaction rates did not relate to each other, especially in the last 20 min of the treatment at 17.83 W cm⁻². For example, when the treatment time was longer than 80 min at 17.83 W cm⁻², only a small amount of intermediate products were generated but a pronounced increase in browning intensity was observed, while maltose continued to decrease. It is postulated that the Maillard reaction in the glycin–maltose solution induced by the ultrasonic treatment produced MRPs that might polymerise and form brown pigments. And, during the entire reaction time, glycin and maltose were reacting at a relative invariable rate.

Changes in reducing power

The reducing power of the glycin–maltose solution treated by the ultrasonic intensity of 17.83 W cm⁻², increased significantly within the last 40 min, as indicated by the absorbance at 700 nm (Fig. 6), from approximately 0.16 to 0.38, 0.74 and 0.90, for 60, 80 and 100 min of treatment, respectively (P < 0.05). However, a slight, but not significant, increase from approximately 0.16 to 0.18, 0.27 and 0.33, was observed at the intensity of 10.19 and 12.74 and 15.29 W cm⁻², respectively, at all reaction times.

The result revealed that MRPs produced from the glycin–maltose model, especially with ultrasonic treatments at relative high intensities, had hydrogen-donating activity. And the hydroxyl groups of MRPs played an important role in the reducing activity. Yoshimura et al. (1997) reported that the reducing power increased linearly with increasing reaction time on glucose–glycine mixtures. The reducing sugars and glycine provided strong reducing materials, which are the key intermediates for the Maillard reaction.

Changes in DPPH radical-scavenging activity

The scavenging activity of glycin–maltose solution on DPPH radical, a molecule containing a stable free radical, was depicted in Fig. 7. The DPPH radical-scavenging activity is due to the hydrogen-donating ability of the antioxidants (Lertittikul et al. 2007). DPPH radical-scavenging activity ratio of the solution increased slightly at the ultrasonic intensities of 10.19 12.74 and 15.29 W cm⁻² or in the control tests. Significant increase was found at 17.83 W cm⁻² after 20 min of reaction (P < 0.05). For example, at 17.83 W cm⁻², DPPH radical-scavenging activity ratio of the glycin–maltose solution increased from nearly 0.33% to 3.50%, 8.18%, 16.80% and 22.53% with the treatment of 40, 60, 80 and 100 min, respectively. However, no significant changes were found in the first 10 min of the treatment, regardless of the ultrasonic intensities applied.

The result indicated that MRPs formed in the glycin–maltose solution with a higher ultrasonic intensity were free radical inhibitors. They could work as an antioxidant. This result is in accordance with that of Lertittikul et al. (2007), who found that the scavenging activity of DPPH frequently increased as the reaction time extended in the porcine plasma protein–glucose Maillard reaction model.

Conclusion

The ultrasonic treatment showed a significant effect on the Maillard reaction model. In particular, at higher intensities (i.e., 17.83 W cm⁻²) ultrasound accelerated MRPs formation in the glycin–maltose solution. Significant increases were found in the intermediate products content, browning intensity and antioxidant activity of the solution. At the ultrasonic intensity of 17.83 W cm⁻², the absorbance at 294 nm, 420 nm and antioxidant activity increased from nearly 0% to 1.06%, 0.30% and 22.53%, respectively, after 100 min of treatment. At the same time, 86.93% of maltose disappeared. The results suggested that ultrasound could potentially be applied as a means for promoting the glycin–maltose Maillard reaction, especially at an intensity greater than 17.83 W cm⁻². However, the duration shown in this study (i.e. 100 min) is not suitable in commercial practice. So further study should be on to relate the ultrasound intensity and duration, and make an appropriate processing technology for commercial practice.

Acknowledgments

This research is supported by the Science Foundation for Province and Ministry CEEUSRO cooperation of Demonstration Base and Innovation Platform Construction of Guangdong Province, China (No. 2008A080403009). All authors appreciate the work of sentence modification in this paper by Professor Frank Huang (Advanced Food Technology, CA, USA) and He-Cheng Meng (South China University of Technology, Guangzhou, China).

References