A pulsed electric field procedure for promoting Maillard reaction in an asparagine–glucose model system

Abstract

Effects of pulsed electric field (PEF) on pH, intermediate products, asparagine and glucose content, browning value, reducing power as well as antioxidant activity of an asparagine–glucose solution were explored in this paper. Results showed that the solution’s UV–Vis absorbance at 294 nm and 420 nm was significantly increased from 0 to approximately 1.14 and 0.74, respectively, at PEF intensity of 40 kV cm⁻¹ for 7.35 ms treatment. The temperature of PEF treated samples were overall lower than 40 °C. It was also detected that the antioxidant activity of treated sample was correspondingly increased by 20.33%. Meanwhile, 14% reduction of asparagine content and 66% reduction of glucose content were observed. It was demonstrated from high performance liquid chromatography analysis that Maillard reaction in the model system had been enhanced by PEF treatment as no 5-hydroxymethyl-2-furaldehyde was generated. This study indicates that pulsed electric field treatment, especially with higher intensities, is a means to significantly promote the Maillard reaction in an asparagine–glucose solution.

Introduction

Generally the Maillard reaction between proteins or amino acids and reducing sugars, occurs in thermal processed foods (Carabasa-Giribet & Ibarz-Ribas, 2000). Series of complex compounds formed in this process are called as Maillard reaction products (MRPs) (Mastrocola & Munari, 2000). The Maillard reaction efficiency depends upon various factors including chemical composition (e.g. proteins, amino acids, reducing sugars or carbohydrates) (Giusti et al., 2008; Karoui et al., 2008; Lertittikul et al., 2007; Morales & Jimenez-Perez, 2001; Yeboah et al., 1999), time, temperature, reactant concentration (Jing & Kitts, 2002) as well as pH (Ajandouz et al., 2001). It was reported that the Maillard reaction is of great potential to become an effective method to generate pigment, aroma and efficacious antioxidant compounds, which can be widely used in food industry (Amarowicz, 2009). However, the food security issue of the currently produced MRPs is always a focus point concerned by most consumers for its high temperature long time process technology (over 70 °C) (Michail et al., 2007).

It was validated that MRPs have the function of inhibiting oxidation for food preservation (Osada & Shibamoto, 2006; Karoui et al., 2008; Nicoli et al., 1997). Its browning and reducing power might be correlated with its antioxidant activity (Morales & Jimenez-Perez, 2001). However, recent study shows that MRPs have also been demonstrated to cause toxicological effects or health problems (Giusti et al., 2008; 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 present technology of manufacturing MRPs needs to be improved.

Pulsed electric field (PEF) technology is a non-thermal food processing method, which can kill most pathogenic micro-organisms and inactivate enzymes, and minimise the loss of colour, taste, nutrients, texture, and heat labile functional components of foods (Jeyamkondan et al., 1999). In the past two decades, it has been successfully applied to deal with various liquid foods, such as starch suspension, milk, wine, and fruit juice, etc., for sterilisation or enhancing processing (Zhong et al., 2009; Sampedro et al., 2007; Torregrosa et al., 2006; Aguilo-Aguayo et al., 2009). Recent studies show that PEF could efficiently retain polyphenols, catechins and original colour of green tea and cause a significant increase in the total free amino acids of green tea infusions (Zhao et al., 2009).

As the technique of applying PEF has the advantages of high intensity (over 10 kV cm⁻¹), short treatment time (by millisecond), applying treatment evenly and fastly, as well as low heat generation, it should have some positive effect on enhancing Maillard reaction. Meanwhile, up to now, the reports about PEF’s treament on chemical reactions, especially on Maillard reactions, is very scarce in international journals. This study aims to investigate the effect of PEF treatment on characteristics and antioxidant activity of MRPs. An asparagine–glucose model system was used for simplifying the reaction system, and investigating the reaction mechanism in the future.

Materials and methods

Chemicals

1,1-Diphenyl-2-picryl-hydrazyl (DPPH), asparagine, glucose, 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 asparagine–glucose solution

Asparagine–glucose solutions were prepared according to the method of Ji-Sang & Young-Soon, (2009) with a slight modification. 0.02 mol asparagine and 0.02 mol glucose were mixed in 90 mL de-ionised water. The pH of the solution was adjusted to 9 with 6 mol L⁻¹ NaOH and the electric conductivities of samles were adjusted to 4.0 ± 0.1 mS cm⁻¹ by NaCl solution at 25 °C. It was then brought to 100 mL with de-ionised water. Before subjected PEF treatments, all samples were pre-cooled in a refrigerator to about 4 °C.

As a contrast, thermal treated samples were prepared under 40 ± 1 °C for different time (up to 15 min) with the same asparagine–glucose mixture (control tests). Followingly, the thermal sample was immediately cooled down to 4 °C to terminate the Maillard reaction as well. A blank sample was performed by treating 0.1 mol L⁻¹ glucose solution without asparagine addtion (pH 9) with the same PEF condition as introduced above (blank tests). All tests were carried out in triplicate. All samples were kept at 4 °C and chemically analysed within 24 h.

PEF system and PEF treatments

A continuous PEF system (SCUT PEF Team, the South China University of Technology, China) was applied with the following operation parameters: squarewave form, unipolar; pulse frequency (f), 1 kHz; pulse duration (τ), 20 μs; the number of pulses (n), 73.5; electrode diameter, 3.0 cm; electrode gap, 0.30 cm; the flow volume in the treatment chamber was 2.20 mL; and sample flow rate, 30 mL min⁻¹. The single treatment time (τ × n) of the liquid subjected PEF treatment is 1.47 ms. In this experiment, the total treatment time was chosed from 1.47 to 7.35 ms by adjusting the treatment times. The intensity of the PEF was changed from 0 to 40 kV cm⁻¹.

The treatment chamber of the PEF system is consisted of two parallel stainless steel plate electrodes and a tubular insulator body which was made of Teflon. For PEF experiments, the asparagine–glucose mixed solution was pumped (Watson Marlow 323E/D Pump, New Jersey, NJ, USA) to the treatment chamber to subject high intensity electric pulses. A rotameter (Model FM-01, Ningbo Jiutian Meter Company, Ningbo, China) was used to calculate and control the flow rate and a digital oscilloscope was applied to monitore the voltage and the current of PEF system. Two type K thermocouples were inserted at the inlet and outlet of treatment chamber to measure the sample temperature. Before and after each treatment, the PEF system was cleaned and disinfected with 75% (v/v) ethanol solution and rinsed with sterile distilled water (Zhong et al., 2009). The intensity of applied electric pulses was changed from 0 to 40 kV cm⁻¹. The total treatment time was adjusted by placing different PEF treatment circle times. For one circle PEF treatment, the time of sample subjected electric pulses is about 1.47 ms which is calculated by pulse number and pulse width. In this experiment, up to five circles were performed on the asparagine–glucose solution thus the longest PEF treatment time was 7.35 ms. For each single circle experiment, the highest temperature of PEF treated sample was 40 ± 1 °C when the most severe PEF condition was applied (40 kV cm⁻¹). In order to mininise the effect of thermal accumulation among various PEF circles, after each circle of PEF treatment the sample was cooled to 4 °C within 5 s by placing heat-exchange with cool water (4 °C) through a 200 mL jacketed vessel.

Analysis

Measurements of UV–Vis absorbance

According to the method introduced by Ajandouz et al., (2001), the browning value of Maillard reaction products was detected by its UV–Vis absorbance at 420 nm and its intermediate products was detected at 294 nm by applying a spectrophotometer (UV-2550 spectrophotometer, Shimadzu, Kyoto, Japan). A ten-fold dilution of the treated samples was performed for UV absorbance tests at 294 nm.

HPLC of asparagine and glucose contents

Each solution was filtered through a Millex-HN nylon clarification kit of 0.45 μm pore size (Millipore, Bedford, MA, USA), and then analysed using an high performance liquid chromatography (HPLC)-Refractive Index Detector (RID) system. The system consisted of 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. The regression equations for asparagine and glucose standards are:

where C₁= asparagine concentration, mol L⁻¹; C₂=glucose concentration, mol L⁻¹; a*=peak area of asparagine; and, b*=peak area of glucose.

HPLC of 5-hydroxymethyl-2-furaldehyde content

HPLC analysis of HMF was determined according to the method of Szu-Chuan et al. (2007) 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 Atlanstis T3 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 water/methanol (10:90, v/v) solution. HPLC analysis was achieved at a flow rate of 1.0 mL min⁻¹ for 30 min. Column temperature was set at 30 °C. Absorption wavelength was set at 284 nm. The compound was compared its retention time with HMF standard 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. 1 mL of a five-fold dilution of the MRP sample was mixed with 1 mL 0.2 mol L⁻¹ sodium phosphate buffer (pH 6.6) and 1 mL 1% potassium ferricyanide. The mixture was incubated in a water bath at 50 °C for 20 min, added with 1 mL 10% trichloroacetic acid, and followed by centrifugation at 750 × g for 10 min at room temperature using a Mikro 20 centrifuge (Hettick zentrifugen, Tuttlingen, Germany). 1 mL of the supernatant was added with 1 mL de-ionised water and 200 μL 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 ten-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 and A_c is the absorbance value of the control.

Statistical analysis

All experiments were conducted in triplicate and each analytical determination was carried out once. Significant differences between the results were calculated by analyses of the variance (Anova). One-way Anova was calculated, and differences at P < 0.05 were considered to be significant. A least significant difference (LSD) test was applied to indicate the samples between which there were differences. All statistical analyses were performed using a Spss package (Spss 10.0 for windows, Spss Inc., Chicago, IL, USA).

Results and discussion

Changes in pH

The changes in pH of asparagine–glucose solutions after various PEF treatments are shown in Table S1. It was demonstrated that the pH was decreased slightly when the applied electric field was lower than 20 kV cm⁻¹ regardless of the reaction time. However, significant decrease of pH was investigated as the PEF intensity was increased to 30 and 40 kV cm⁻¹ at all reaction times (P < 0.05). At the same time, little change of pH was found in the blank sample as well as control sample (Table S1), regardless of the PEF intensities applied.

The result revealed that the PEF treatment with higher intensity over 30 kV cm⁻¹ had generated some MRPs and resulted in decrease of pH in the model solution. The decrease of pH is 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 PEF intensities of 30 and 40 kV cm⁻¹. This result is accorded with the phenomenon observed by hydrothermal method by Morales & Jimenez-Perez (2001) who found that during the Maillard reaction, the pH frequently decreases as the heating time increases.

Changes in A₂₉₄

The relationship with PEF intensity, treatment time and UV absorbance value at 294 nm which reflects the amount of intermediate products of MRPs is shown in Table S2. Apparently, a significant increase of A₂₉₄ value of the asparagine–glucose solution can be observed within the first 5.88 ms of PEF treatment under 40 kV cm⁻¹ and within all treatment times under 30 kV cm⁻¹ (P < 0.05) (Table S2). However, at lower PEF intensities (i.e. 10 and 20 kV cm⁻¹), the A₂₉₄ change was slight. As contrast, nearly no change was observed in the blank and control tests (Table S2).

The results suggested that MRPs were produced to a great extent under higher PEF intensities (i.e. 30 and 40 kV cm⁻¹). With extended reaction time, some intermediate products might polymerise together which resulted in only a small amount of intermediate products was remained in the final products. For example, little change of the A₂₉₄ was observed when the treatment time was longer than 5.88 ms at 40 kV cm⁻¹. It appeared that when the treatment time was longer than 5.88 ms (at PEF intensity of 40 kV cm⁻¹), some intermediate products turned into new polymers, leaving a reduced amount of intermediate products. Meanwhile, asparagine and glucose continued to react (Tables S3 and S4) producing new intermediate products. For example, at 40 kV cm⁻¹, asparagine and glucose reduced from approximately 0.174 mol L⁻¹ to 0.172 mol L⁻¹; and 0.074 mol L⁻¹ to 0.068 mol L⁻¹, respectively, as treatment time was extended from 5.88 ms to 7.35 ms. The phenomena detailed above proved that asparagine and glucose continued to react and new MRPs were produced. And the rates of formation and polymerisation of the intermediate products might become equal. Thus, the change of A₂₉₄, as shown in Table S2, was slight after 5.88 ms treatment at 40 kV cm⁻¹. However, the mechanism and the rate of the MRPs formation affected by the PEF intensity need further study. This result was in accordance with Moreno et al. (2003) who found that a higher pH of the glucose-lysine model system was related with a higher A₂₉₄. Moreover, Ajandouz et al. (2001) also found that a higher pH value gave a higher A₂₉₄ in the fructose–lysine system.

Changes in browning intensity

Brown colour development (A_420 nm) is the easiest measurable consequence of the Maillard reaction because it offers a visual estimate. Its intensity is often used as an indicator of the extent to which the Maillard reaction took place in foods and it symbolises an advanced stage of the Maillard reaction (Morales & Jimenez-Perez, 2001; Ji-Sang & Young-Soon, 2009). A₄₂₀ of the asparagine–glucose solution treated with different PEF intensities was a measure of the browning effect. As shown in Table S5, the A₄₂₀ increased significantly at 30 and 40 kV cm⁻¹ between 1.47 and 7.35 ms (P < 0.05). However, at lower PEF intensities (i.e. 10 and 20 kV cm⁻¹), the A₄₂₀ changed only slightly at all reaction times. At the same time, no significant changes were observed in the blank and control tests (Table S5).

The brown pigment development, indicated by the A₄₂₀, was coincided with the colourless intermediate formation, which was evidenced by the increased A₂₉₄ value. Such correlation suggested that the formation of brown pigments was positively related with the generation of intermediate products. On the other hand, the reaction rate became high after 4.41 ms at the intensity of 30 kV cm⁻¹, the asparagine and glucose concentration in the solution were 0.187 and 0.131 mol L⁻¹ and pH at 8.71, which means that the Maillard reaction could continue despite the polymerisation of the intermediate products and formation of the brown pigments. 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. This result was also in agreement with that of Lu et al. (2005) who reported that diglycine–glucose reaction mixtures have a higher degree of browning, followed by glycine–glucose, and triglycine–glucose reaction mixtures.

Changes of asparagine and glucose contents

The HPLC peaks were identified to be asparagine and glucose by comparing the retention times between them and standard compounds.

As shown in Tables S3 and S4, at lower PEF intensities (i.e. 10 and 20 kV cm⁻¹), no significant change on the asparagine or glucose content was found. However, as the intensity was raised to 30 and 40 kV cm⁻¹, asparagine and glucose in the solution decreased significantly at all reaction times (P < 0.05). However, slight change of asparagine and glucose contents was observed in the blank and control tests (Tables S3 and S4). On the other hand, the behaviour, much greater loss of glucose compared to the amino acid, is also a feature of the normal heat induced Maillard reaction. Although the reports in the literature is scarce, our investigations (not only in asparagine–glucose by PEF treatment, but also in other amino acid-reducing sugar model systems by the normal heat treatment or ultrasonic) found that a much greater loss of glucose or other reducing sugar compared to the amino acid occurs during Maillard reaction.

From HPLC analysis, it was demonstrated that little HMF was generated during the PEF enhanced Maillard reaction. Therefore, the mechanism of significant glucose reduction cannot be simply resulted from dehydration of glucose by losing three water molecules (Szu-Chuan et al. 2007). On the other hand, the decrease of asparagine and glucose was positively related with the increase of colourless MRPs and brown pigment. Yet, the reaction rates did not relate to each other, especially in the first 1.47 ms of the treatment at 30 and 40 kV cm⁻¹. For example, when the treatment time was less than 1.47 ms at 30 kV cm⁻¹, only a small amount of browning pigment was generated but a pronounced decrease in asparagine and glucose contents was observed. It is postulated that the Maillard reaction in the asparagine–glucose solution induced by the PEF treatment produced MRPs that might polymerise and form brown pigments. And, during the entire reaction time, asparagine and glucose were reacting at a relative invariable rate.

Increase in reducing power

The reducing power of the asparagine–glucose solution treated by the PEF intensities of 30 and 40 kV cm⁻¹, increased significantly within 7.35 ms (P < 0.05), as indicated by the absorbance at 700 nm (Table S6). However, a slight, but not significant, increase was observed at the intensity of 10 and 20 kV cm⁻¹ at all reaction times. Little change in reducing power of the asparagine–glucose solution was observed in the blank and control tests (Table S6).

The result revealed that MRPs produced from the asparagine–glucose model, especially with PEF treatments at relative high intensities, had hydrogen-donating activity. And the hydroxyl groups of MRPs played an important role in the reducing activity. The glucose and asparagine provided strong reducing materials, which are the key intermediates for the Maillard reaction. Additionally, the intermediate reductone compounds of MRPs were reported to break the radical chain by donation of a hydrogen atom (Lertittikul et al. 2007), which might be one of the main reasons to explain that MRPs have the reducing power. Some studies also reported that the reducing power increased significantly with increasing reaction time on glucose–glycine (Yoshimura et al., 1997), xylose–lysine (Yen & Hsieh, 1995), sugar–lysine (Wijewickreme et al., 1999) mixtures.

Changes in DPPH radical-scavenging activity

DPPH is a chromogen-radical-containing compound that can directly react with antioxidants. When the DPPH radical is scavenged by antioxidants through the donation of hydrogen to form a stable DPPH-H molecule, the colour is changed from purple to yellow (Shon et al., 2003). Stable radical DPPH has been widely used for the determination of primary antioxidant activity, that is, the free radical scavenging activities of pure antioxidant compounds, plant and fruit extracts, and food materials (Ji-Sang & Young-Soon, 2009). The scavenging activity of asparagine–glucose solution on DPPH radical, a molecule containing a stable free radical, was depicted in Table S7. The DPPH radical-scavenging activity is due to the hydrogen-donating ability of the antioxidants (Lertittikul et al., 2007). It was explored from Table S7 that the DPPH radical-scavenging activity ratio of the solution was increased slightly when the applied PEF intensity was quite low as 10 and 20 kV cm⁻¹. Meanwhile, significant increase of the DPPH radical-scavenging activity with increasing treatment time was detected when the PEF intensity was increased to 30 and 40 kV cm⁻¹ (P < 0.05). However, slight significant changes were found in the blank (Table S7), regardless of the PEF intensities applied. And little change was observed in the control at all times (Table S7).

The result indicated that MRPs formed in asparagine–glucose solution with a higher PEF intensity were free radical inhibitors. And they can be used as antioxidants. 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. The result is also in accordance with that of Rufián-Henares & Morales (2007) who reported a statistically significant linear relationship between the antioxidant activity measured with the DPPH assay and the iron-binding capability of melanoidins.

Conclusion

A significant positive effect of pulsed electric field treatment on the Maillard reaction in asparagine–glucose solution was investigated. It was found that the formation of MRPs was dramatically enhanced when the applied PEF intensity was higher than 30 kV cm⁻¹. Correspondingly, significant increase of intermediate product content, browning intensity and antioxidant activity of the solution were detected. All the results suggested that PEF could potentially be applied as a means for promoting the Maillard reaction, especially at an intensity greater than 30 kV cm⁻¹.

Acknowledgments

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). This research is supported by the Key S&T project (2009B050400003) and Natural Science Project of Guangdong province, China(9151008901000159).

References