The effect of frying on browning, acrylamide and 5-hydroxymethylfurfural formation on Malaysian curry puff skin treated with l-asparaginase (2024)

Curry puff skin preparation and frying conditions

Three types of curry puff skin samples were produced: (i) control (without asparaginase enzyme, Acrylaway® L), (ii) treated with E 100 U/kg flour of enzyme (Acrylaway® L, supplied by Novozymes, Switzerland), and (iii) treated with E500 U/kg flour Acrylaway® L. The main ingredients for curry puff skin sample were flour (61.45%), distilled water (23.41%), cooking oil (13.40%), and margarine (1.75%). All the ingredients underwent mixing at room temperature for 10min by using a dough mixer. The dough was made to rest for 15min at room temperature to allow Acrylaway® L activity. Then the dough was shaped by using a curry puff, moulded at a thickness of approximately 0.50cm thickness and 7.00cm diameter without any fillings. Curry puff skin was deep-fried in a deep fryer at 180°C, 190°C, and 200°C for 2min, 3min, 5min and 7min (Table1).

Colour measurement

The colour measurement of the fresh and frozen curry puff skin samples was evaluated by using a chromameter (Konica Minolta CS-150 Chroma Meter, China) according to the CIE Lab (the L* a* b* colour space). The CIE Lab system gave three readings of colour components: luminosity L* (− black to + white components) and chromaticity coordinates, a* (+ red to − green components) and b* (+ yellow to − blue components). The colour difference (ΔE) was evaluated by comparing the results of different frying temperatures and times by using the equation:

where, ΔL* is the brightness difference, Δa* the redness difference, and Δb* the yellowness difference.

Moisture content analysis

Moisture content analysis was determined according to the method from AOAC International (2005), whereby the samples were oven dried in the drying oven (Memmert UF 30 230V, Cole-palmer, US) at 105°C with a holding time until the samples reached a constant weight for three consecutive readings.

Water activity analysis

Water activity (a_w) was determined by methods from Landrock and Proctor (1951). The analysis was performed by using AQUALAB 4TE Benchtop Water Activity Meter from Meter Group Inc., USA.

Textural profile analysis (TPA)

The texture analysis of the curry puff skin was performed by using a texture analyser (TA-XT2, Stable Micro Systems Ltd., UK) equipped with a cylindrical probe with a diameter of 36mm, test speed of 1.0mm/s and compression load of 25kg. The crust of the samples was removed, and the samples were sliced into equal squares (2cm × 2cm). The textural profiles of the samples were determined by using Texture Expert 1.05 software (Stable Micro Systems). The parameters observed were hardness, firmness, and springiness. Each texture profile analysis was duplicated.

Quantification of acrylamide by using high-performance liquid chromatography (HPLC)

The method used for acrylamide determination was based on Nur Fatihah and Razinah (2018) with slight modifications. The fried curry puff skin was grounded prior to the extraction process. One gram of the ground sample was placed in a 50mL Erlenmeyer flask, added with 20mL of deionised water at 50°C and 5mL of petroleum ether to remove fats in the sample. The mixture was agitated by using an orbital shaker at 250rpm for 30min. Next, the mixture was centrifuged at 3000rpm for 20min. The mixture formed a fat layer, whereby the layer was removed. The remaining layer was added with 1mL each of Carrez I and Carrez II solution. The mixture was re-agitated for 30min and centrifuged at 3000rpm for 20min. Lastly, three layers were formed, protein layer (upper), acrylamide layer (middle) and sediment layer. The middle layer was purified using 1mL each of Carrez I and Carrez II. Once again, the fat layer was removed, and the remaining layer was added with Carrez I and Carrez II. The sample was filtered through a 0.45µm filter and was sonicated before being analysed.

The acrylamide content was measured by using a high-performance liquid chromatography (HPLC) (Prominence LC-20A, Shimadzu Scientific Instruments, Europe) system equipped with a Diode Array Detector and analytical column C18 steel. A total of 7% (v/v) acetonitrile in deionised water was used as the mobile phase. The quantification of acrylamide was performed by measurement of peak area at wavelength 215nm and the acrylamide retention time of 7.5min. The amount of acrylamide content was determined by comparison with the calibration curve as the injection volume was set at 10 μL and flow rate at 1mL/min.

Quantification of HMF by using ultraviolet–visible (UV–Vis) spectrophotometer

The HMF content was measured by using spectrophotometric method based on Zappala et al. (2005) without modifications. The fried samples were finely ground for analysis. Five grams of samples were dissolved in 25mL of distilled water. Then, it was transferred into a 50mL volumetric flask. In the flask, a total of 0.5mL of Carrez I and 0.5mL of Carrez II were added. Next, distilled water was added to make a 50mL solution. The solution was filtered by using a 0.45µm filter paper and it rejected the first 10mL of the filtrate. Finally, 5mL of the remaining filtrates was added into 5mL of distilled water (sample solution) and pure deionised water was used as a reference solution.

A stock solution of HMF was prepared at a concentration of 1000µg/kg. The absorbance of the solutions was determined by using a VARIAN mod. UV–VIS (UV2600, Shimadzu Scientific Instruments, Europe) at the wavelength of 336nm. The HMF was quantified by using the formula obtained from the standard calibration curve.

Statistical analysis

The raw data obtained were analysed by using a statistical analysis method. In this study, all the analyses were done in replications. For statistical purposes, the software used was Minitab 2019 and the method of analysis used was ANOVA—Analysis of Variance (Fit General Linear Model) to determine the mean and standard deviation. The significance level is set at p < 0.05.

The effect of asparaginase on the colour of curry puff skin

Colour development is one of the positive effects of the Maillard reaction. The crust of the curry puff skin turned brownish after the frying process. The browning of crust indicated the presence of melanoidins in samples (Izydorczyk, 2005). Fried curry puff skin with and without asparaginase were cooled and directly measured for colour changes. Colour difference (ΔE*) was used instead of ΔL* due to the chromic coordinates and was easily described in the browning process during frying (Ciesarová et al., 2009). In this work, the addition of enzyme reduced the colour difference of curry puff skins. Based on Fig.1, the increase in frying time and frying temperature showed an increase in the ΔE of curry puff skin (Fig.1). The colour changes were apparent for the curry puffs without treatment with enzyme suggesting that the Maillard reaction was uninterrupted throughout frying process. The colour difference increased as the increase in frying temperature due to the decrease in lightness (L*) value. Capuano et al. (2009) observed similar finding with the decrease of the L* value as frying temperature increased. The colour development of fried food products was the results of moisture loss, oil migration and the Maillard reaction, which depend on the number of amino acids and reducing sugars at the surface of the products as well as the temperature and length of frying time (Krokida et al., 2001). Curry puff skin treated with asparaginase enzyme was observed to have a similar trend, however, with lower ΔE value as opposed to the control sample. The a* and b* values from the colour measurement showed small increment during the heating process at each temperature setting. Furthermore, curry puff skin treated with asparaginase enzyme at 500 U/kg showed the most reduction ofΔE. Both frying temperature and time of 200°C at 2min and 200°C at 5min respectively, were observed to be significant (p < 0.05). The reduction suggests that the breakdown of asparagine in dough during fermentation by asparaginase enzyme which reduced the capacity of the Maillard reaction leading to less browning effect. At elevated time of frying i.e. 7min, the effect of the enzyme was insignificant as the browning effect was similar with the control. At long exposure to heat, caramelisation contributed to the browning effect of the curry puff skin which represented the progressive Maillard reaction.

Water activity and moisture content relation during frying

The availability of free water in curry puff skin may influence the Maillard reaction. Moisture content and water activity were measured to determine the availability of water after frying. The frying process allows migration of water to vapour from the inner side of the curry puffs creating a crispy skin. Based on Table ​Table1,1, the released of free water was observed to increase at elevated temperature and time. The decrease in both moisture content and water activity was significant (p < 0.05) for curry puff skin without asparaginase enzyme. These results were in correspondence with results reported by Capuano et al. (2009) and Ciesarová et al. (2009), Hass et al. (1976), whereby moisture content of common pastries such as bread ranged between 35 and 45% after undergoing heat treatment at very high temperature. Furthermore, the reduction of moisture content and water activity had a direct implication on colour difference (ΔE), in which the (ΔE) increased when the amount of the free water depleted. A similar trend was observed for curry puff skin treated with enzyme at both concentrations of 100 U/kg and 500 U/kg. The reduction was not as significant for curry puff skin without enzyme. At 2min of frying (180–200°C), the changes were insignificant for moisture content but the decrease in water activity for both 100 U/kg and 500 U/kg of enzyme led to the decrease in lightness (L*) value. Interestingly, the addition of asparaginase enzyme enables the retention of water within the curry puff skin. Doneva et al. (2015) showed that the addition of proteases, such as papain and bromelain, allowed the increase in water retention due to partial hydrolysis of protein which was the affinity for water. That would indicate the improved texture of curry puff skins for being crispy on the outside yet soft on the inside. Due to this, the moisture effect observed in this study suggested that the availability of free water and asparaginase works simultaneously in developing desirable process parameter, sensory acceptability and most importantly, safe for consumption. Therefore, the measurement of acrylamide and HMF were crucial to determine the effectiveness of enzyme.

Acrylamide in curry puff skin

Curry puffs with and without asparaginase enzyme were fried at different temperatures and times. Then, the samples were cooled and analysed for their acrylamide content by using HPLC. Ahn et al. (2002) had reported that the limit of detection (LOD) for acrylamide detection in toasted bread, fried chips, grilling and baking of potatoes was 2500µg/kg. The addition of enzyme in curry puff skin dough allows the reduction of acrylamide (Fig.2). At frying temperature of 180°C for 2min, the acrylamide content was significantly reduced (p < 0.05) from 3277 to 2675μg/kg. As the frying time increased, the acrylamide content remained marginal in the range of 2500–2600μg/kg. Interestingly, the acrylamide in control started to decrease suggesting initial decomposition. At 7min of frying, the acrylamide for both control and with enzyme showed similar content which may be due to further decomposition of acrylamide in the control sample. Increasing the enzyme concentration showed no improvement in acrylamide reduction. At higher temperature (190°C and 200°C), the addition of asparaginase enzyme showed no reduction in the acrylamide content. The amount remained marginal at 2500μg/kg for both control and sample with enzyme, suggesting that at higher temperature, the possibility of acrylamide reduction may be due to the dual effect of the enzyme and the decomposition effect. The National Institute of Occupational Safety and Health (NIOSH, 2020) described that the minimum decomposition temperature for acrylamide was from 175 to 300°C. Based on this information, the decomposition of acrylamide may be plausible at the studied frying temperature.

In past studies, Tareke et al. (2002) had reported that the acrylamide content in French fries ranges between 2200 and 3700µg/kg, whereas the World Health Organisation (WHO) in 2005 had declared that acrylamide in French fries could have a maximum concentration of 12,800µg/kg. Besides, the European Food Safety Authority (EFSA) also had listed the acrylamide content in several food products which are processed at a very high temperature. According to the list, biscuits are found to have the highest amount of acrylamide that could reach the maximum level of 4200µg/kg and followed by several other products that contain up to 4700µg/kg and potato crisps at 4180µg/kg of acrylamide. In conjunction with these studies, Tardiff et al. (2009) had reported that the tolerable intake of acrylamide for human consumptions was 2600µg per kilogram bodyweight per day. The results of this research have shown that the level of acrylamide formed in both fresh and frozen curry puff samples is still in the safe range for human consumption.

Hydroxymethylfurfural (HMF) in curry puff skins

HMF is also a product of the Maillard reaction and giving toxic effect when consumed by humans (Miao et al., 2013). Therefore, HMF is commonly placed as a chemical marker of the Maillard reaction in a high temperature cooking process. The presence of HMF was detected in all samples. The discovery of acrylamide and HMF in food urged the researchers to investigate various factors involved in the formation process, including frying times (Gokmen et al., 2006). The Commission Regulation (EU) established the benchmark level of acrylamide and HMF in flour-based product with 350mg/kg and 40mg/kg, respectively.

According to Fig.3, the increase time and temperature leads to the higher formation of HMF. The HMF content in control showed the most significant amount (p < 0.05) due to the free reducing sugar and amino acids, including asparagine. The addition of asparaginase enzyme suggested that by reducing the availability of overall amino acids limited the Maillard reaction which reduced the HMF formation. At frying temperature of 180°C, the initial HMF content at 2min for all samples was similar ranging from 187 to 217μL/L. As the exposure time increased to 7min, the HMF content became apparent as the 500 U/kg curry puff skin showed the lowest (227μL/L) significantly (p < 0.05). At higher temperature (190°C and 200°C), the trend was similar with higher amount of HMF. Therefore, the use of asparaginase enzyme reduced the HMF formation with 500 U/kg showed the lowest formation at all tested frying temperatures.

Abraham et al. (2011) reported that the highest level of HMF was found in cereals made of wheat such as biscuits, pastries and bread, whereby the maximum level of HMF formed was 503µg/kg and the estimated daily intake of HMF was between 400 and 3000μg per person per day. However, Janzowski et al. (2000) have reported that the estimated intake for HMF was ranged from 30 to 150mg per person.

Sensory analysis (hedonic scale analysis)

Hedonic test was used to determine the acceptability of curry puff skin with and without asparaginase enzyme. The test was based on the use of an untrained panellist of 60 university students and staff with the ages ranging from 20 to 25years old. The panellists were given 3-blinded samples to evaluate based on their preference based on the observed attributes (colour, odor, texture and overall acceptance).

Colour showed a significant difference at frying temperature of 180°C (p < 0.05). The addition of asparaginase enzyme in curry puff skin reduced the acceptability of panels (Fig.4). Although at 100 U/kg of asparaginase enzyme had showed similar ΔE with the control, the panellists preferred the control sample. Interestingly, an increased temperature to 190°C for 5min allowed the increase of the panellists’ liking. Similar mean score for both control and with asparaginase enzyme was observed. The results corroborated with the findings from colour measurement whereby ΔE were similar for all samples. However, the mean score decreased significantly (p < 0.05) at 200°C of frying temperature suggesting that at higher temperature (higher than 190°C), the curry puff skins for both control and with the addition of 100 U/kg enzyme were not able to improve panellist acceptability. On the contrary, the addition of 500 U/kg enzyme maintained the mean score similar to that of curry puff skin produced at 190°C for 5min. At 200°C, the temperature caused an extensive browning effect (Maillard reaction and caramelisation) on the curry puff skin. The addition of 100 U/kg had no effect in reducing the panellist acceptability. At high level of asparaginase enzyme (500 U/kg), the breakdown of asparagine had directly contributed to the increased panellists’ acceptability and decreased colour changes.

Maillard reaction contributes to the formation of colour and odor in curry puff skin. The trend for mean score for odor was similar to that of the colour, suggesting their relation to Maillard reaction. The browning subsequently leads to flavour generation in all control samples. However, at lower temperature (180°C), the flavour released from curry puff skin treated at 100 U/kg and 500 U/kg asparaginase enzyme showed a significant drop on the mean score (p < 0.05) (Fig.4). At higher temperature, the flavour of curry puff skin was well developed, in which the curry puff skins at 190°C showed higher mean score for all samples. At 200°C, the mean score for control and sample was reduced at 100 U/kg enzyme, but at higher enzyme concentration showed improved panellist acceptability.

The frying process allows the migration of water from curry puff skin, creating a crisp crust. Based on Table ​Table1,1, the increased temperature and time enable free water to escape from the curry puff skin and during its exposure time formed crust. Control sample showed the highest mean score as perceived due to its free amino acids and reducing sugar (Fig.4). At 190°C, the formation of crust was observed to be desirable in the panellists with no significant difference as compared to that of the control by lowering the frying temperature (180°C) reduces its acceptability even with the addition of asparaginase enzyme. At 200°C, the temperature started to affect the texture for both control and sample at 100 U/kg enzyme. At 500 U/kg, the limited browning as well as partial degradation of protein allowed the retention of moisture (Table ​(Table1)1) allowing a desirable texture (Fig.4) at high temperature. Although the texture was unaffected at high temperature, this condition would affect the cost of operation. Furthermore, the acrylamide and HMF contents were similar to that of the sample at 190°C. Therefore, with the mean score of the entire attribute tested reflected on the overall acceptance of the panellist (Fig.5). The curry puff skin was preferred at 190°C with mean score for 100 U/kg and was slightly higher above control and 500 U/g, which indicated that the addition of asparaginase enzyme did not temper with the original quality.

Conflict of interest

The authors declare that they have no conflict of interest.

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