Degradation kinetics of bioactive compounds and antioxidant capacity of Brussels sprouts during microwave processing

ABSTRACT

In this study, the effects of microwave (MW) processing of Brussels sprouts on various bioactive compounds, moisture content, and antioxidant capacity were studied. Brussels sprouts was processed at three different MW output powers (460, 600, and 700 W), and then kinetic study for the degradation of the total chlorophyll, vitamin C, total polyphenols, total flavonoids, antioxidant capacity, and two phenolic acids (sinapic and ferulic acid) was conducted. According to the results, MW processing caused significant reductions in analyzed bioactive compounds, moisture content, and antioxidant capacity. It was found that HPLC analysis results confirmed the result of the spectroscopic analysis. Zero-order and first-order kinetic models were fitted to experimental data. The first-order kinetic model was selected as the best according to R², RMSE, and χ². The degradation rate constant (k) for all analyzed properties increased as the MW output power increased from 460 to 700 W, and it could be concluded that the MW output power in which bioactive compounds, moisture content, and antioxidant capacity of Brussels sprouts were best preserved was 460 W.

KEYWORDS:

• Antioxidant capacity
• Brussels sprouts
• Kinetic model
• Microwave
• Polyphenols

Introduction

Vegetable consumption is recommended worldwide because of their richness in nutrients and phytochemicals might provide health benefits like the prevention of free radical-mediated diseases.^[1–4] One of the vegetables that have increasing consumption and popularity is Brussels sprouts. Brassica crops are very nutritive vegetables and containing nutrients and health-promoting phytochemicals like vitamins, carotenoids, phenolic compounds, fiber, soluble sugars, minerals, and glucosinolates. Therefore, the consumption of Brassica species has been directly related to the decrease of the risks of chronic diseases such as cardiovascular diseases and cancer.^[5–7]

In many countries, the tendency toward the consumption of practical foods increased the demand for high-quality dehydrated vegetables. In the whole world, this trend is expected to sustain and even accelerate over the next decade. Dehydration emerges vegetables into a more stable and safe status as it decreases water activity and extends shelf life. Many conventional thermal methods such as air flow drying, vacuum drying, and freeze-drying have long drying times at relatively high temperatures compared to MW drying.^[8–12] This situation causes undesirable thermal degradation of the finished products.^[8,12] MW processing has more advantages than the other conventional heating processes such as definite timing, rapidity, and energy saving.^[13,14] It is possible to improve the final product quality with the decreased heating time as it is 3–5 times faster than the conventional heating methods.^[14]

Thermal processing techniques aim the achievement of commercial sterility because of minimizing changes in sensory properties and nutritional value. On the other hand, thermal processing can increase the formation of reactions that could influence the overall quality of foods. Quality loss involves both of taste changes and nutrients degradation.^[14,15] Many research studies have been devoted to assess the way that MW processing affects the nutrient properties of vegetables such as ready to eat vegetables^[16], broccoli^[17], green beans, broccoli, and asparagus^[18], peas, carrot, spinach, cabbage, cauliflower, and turnips^[19], potato, carrot, onion, broccoli, and white cabbage^[20], Galega kale^[21], brown mustard^[22], and York cabbage.^[14] The results on the effects of MW processing on the various nutrients of vegetables reported by various authors and they differ from each other. It is possible to say that this operation could cause both positive and negative effects on nutrients of vegetables due to the morphological characteristics of them and MW processing conditions used.^[14] MW processing conditions should be optimized with the aim of reducing degradation of nutrients to a minimum level.^[23,24] For this reason, there is a need for the studies of degradation kinetics. In the literature, there is only one study on the effect of MW processing on the kinetic modeling of physiochemical properties of vegetables (i.e. York cabbage).^[14] Given to this background, it is purposed to evaluate the most effective MW output power that might produce high-quality dried Brussels sprout in this research. The effects of MW output powers (460 W, 600 W, and 700 W) on Brussels sprout is determined by the degradation kinetics of some bioactive compounds (chlorophyll, phenolic compounds, and vitamin C content) and total antioxidant capacity. The kinetic of moisture content at MW output powers applied is also explained. This is the first study and will fill the gap in the literature about the kinetic evaluations of bioactive compounds, antioxidant capacity, and moisture content of Brussels sprouts during MW processing.

Materials and methods

Sample

Fresh Brussels sprouts were purchased from a local market in Izmir, Turkey. They were stored in a plastic bag at refrigerated condition (4 ± 1ºC) until the experiments. Brussels sprouts divided perpendicular to the fruit axis into approximately equally sized two parts manually with a knife and dried in MW immediately.

MW processing

MW processing was carried out using a programmable MW oven (Arçelik, MD 674, Turkey) with a maximum output of 700 W. The used MW oven was run by a control terminal, which controlled both emission time and MW power level. MW heating was performed at 460, 600, and 700 W. Selected MW power levels are the most commonly used powers in food processing applications. In the MW processing, Brussels sprouts were placed inside the MW oven. For all the power levels studied, samples (38 ± 0.5 g) were taken from the MW oven every 0.75 min up to 3.75 for 700 W, up to 4.5 min for 600 W, and up to 5.25 min for 460 W. The total drying time was determined as the passing time until no discernible weight change for each sample was observed in each MW power level. The processed material was cooled to room temperature and kept in a plastic bag (20 × 25 cm) until the analysis. All analyses were performed on the same day and carried out in duplicates.

Moisture content

For the determination of the moisture content, 3–4 g of raw or processed homogenized Brussels sprouts were dried using a convection oven at 105°C until the weight of samples reached a constant weight.^[3] The results were expressed as g water of sample per g dry basis (DB) of the sample.

Preparation of extracts

For the preparation a 2 g of sample was extracted using 20 mL of 80% (v/v) methanol into the flasks. Flasks were kept in a shaking water bath (Memmert, Germany) at 50°C for 90 min. The polyphenol extracts were filtered with Whatman # 1 and the volumes of them were completed to 25 mL with 80% methanol.^[25] The chlorophyll compounds were extracted from 0.6 g of samples with 6 mL of 80% (v/v) acetone at 0ºC. After centrifugation at 4500 rpm for 20 min at 0ºC, the volumes of the aliquots were completed to 10 mL with 80% acetone.^[26,27] A 1 g of sample was treated with 8 mL of 3% (w/v) oxalic acid at 4ºC for obtaining vitamin C extracts. After centrifugation at 4500 rpm for 20 min at 4ºC, the extracts were vortexed for 1 min and their volumes were completed to 10 mL with 3% oxalic acid.^[28]

The spectroscopic analyses

The total phenolic content (TPC) was quantified by a modified Folin-Ciocalteu method^[29,30] and results were expressed as mg gallic acid equivalent (GAE)/100 g DB. Meanwhile the aluminum chloride colorimetric method described by Heimler et al.^[31] was used for determining total flavonoids contents (TFC) and results were expressed as (+)-catechin equivalents ((+)-CEA)/100 g DB. The ability of the polyphenol extracts to scavenge 2,2-diphenil-1-picrylhydrazyl (DPPH) free radical was measured and evaluated as % of antioxidant activity as described by the modified methods of Chu et al. and Cheung et al..^[32,33] The ferric reducing antioxidant power (FRAP) assay was applied and the obtained values were expressed as mg of reduced iron equivalents (FeSO₄) per 100 g DB according to the modified methods of Guo et al. and Xu et al.^[34,35] The total chlorophyll (chlorophyll a and chlorophyll b) concentrations were determined from the chlorophyll extracts by using the methods of Lichtenthaler and Viňa^[26,27] and the results were expressed as mg chlorophyll (Chl)/g DB. The vitamin C contents were measured by using vitamin C extracts with Pearson and Cox^[36] method and evaluated as mg vitamin C/100 g DB.

Chromatographic separation of phenolic acids in Brussels sprouts

Some of the phenolic acids in Brussels sprouts were determined by using an Agilent 1200 LC system (Agilent, Santa Clara, CA) equipped with a diode array detector (DAD) and the software Chemstation (Agilent, Santa Clara, CA), which generated a 3-dimensional data set (absorbance, retention time, and wavelength). Phenolics in the injected extracts (20 μL) were quantified using an ACE C18 (5 μm, 100 Å, 250 × 4.6 mm) column (Advanced Chromatography Technologies Ltd, UK) enclosed in an oven maintained at 25°C. The mobile phase, consisted of (A) 1% phosphoric acid in water and (B) 1% phosphoric acid in methanol, used a linear gradient starting with 30% B and increasing to 50% B at 8 min, reverting to 30% B for 2 min and keeping in 30% B during 10 min. The constant flow rate as 1 mL/min was utilized. DAD data acquisition was detected in the range of 190–400 nm and peak areas were monitored at the maximum absorbance of the compounds, i.e., 280 nm for cinnamic acid and 320 nm for chlorogenic acid, caffeic acid, sinapic acid, and ferulic acid. The used chromatographic conditions were taken based on the modified method of Belguidoum et al.^[37]

The chromatographic peaks obtained were identified by matching the retention times and UV spectra of samples with standards and using standard addition technique. Two phenolic acids (ferulic acid and sinapic acid) were identified in the polyphenol extracts of samples. The quantitation of these phenolics was done with using a six-point regression curve (r ≥ 0.997) for each standard. The range of standards’ concentrations in calibration curves was between 3.13 to 62.50 mg/L, approximately. The results were expressed as mg/100 g DB.

According to the results of analytical method validation, the LOD (limit of determination) values were 0.06 mg/L for ferulic acid and 0.08 mg/L for sinapic acid, when the LOQ (limit of quantification) values were 0.20 mg/L for ferulic acid and 0.28 mg/L for sinapic acid. The recovery capacities of the method were determined by adding the original concentrations defined in the samples of phenolic acids in sample extracts. The values of recovery were evaluated as 98.02% for the ferulic acid and 91.36% for the sinapic acid.

Mathematical models and kinetic analysis

The experimental data was used for the kinetic modeling to determine the degradation kinetics of total phenolic content, total flavonoid content, and antioxidant capacity determined by DPPH and FRAP methods, two phenolic acids (sinapic acid and ferulic acid) content and moisture change of Brussels sprouts during MW processing. In the present study, a zero-order (Eq.1) and a first-order kinetic model (Eq. 2) were employed to find the best fit between the model and experimental data. Kinetic modeling was performed on three MW powers (460, 600, and 700 W) for evaluating the change of each compound. Reaction rate and the relevant coefficients were also described as below:

(1)

(2)

where C_t is the parameter at predefined time t, C₀ is the initial value of the parameter, k is the reaction rate constant at MW output power (W), and t is the MW processing time (min⁻¹).

Statistical analysis

All experiments were performed in duplicates. The results calculated as the mean ± standard deviation. Statistical evaluation and non-linear regression analyses were carried out using SPSS v.20.0 statistical package. The statistical criteria applied to discriminate between a zero-order and a first-order kinetic model were the coefficient of determination (R², the higher the better), root mean square error (RSME, the lower the better), chi-square (χ², the lower the better), and standard error (std. error) for each coefficient. Among these criteria RSME and χ² values were calculated by Microsoft Excel 2010 program, while R² and std. error were determined by the SPSS v.20.0 program. The confidence level used to detect statistical significance was 95%.

Results and discussion

Effect of MW processing on moisture content, bioactive compounds, and antioxidant capacity

A decrease in all analyzed properties of Brussels sprouts had occurred under all MW output powers with the time factor. The initial moisture content of samples was 6.71 g water/g DB and it was reduced by almost 99.49–99.61% at the end of the MW processing (Figure 1a). Similar trends in decrease of moisture content with increasing MW output power were also observed by Lüle and Koyuncu.^[38] Again, it had also been observed in this study that the drying time decreased as the MW output power increased. In MW processing, MWs spread from a source in all directions. During the drying process, foodstuffs absorb this energy that is carried by these waves. This energy converts to heat by polar molecules. Water is the common polar molecule that is a component of foods. Water molecules convert MW energy to heat during the drying process and then, they start to evaporate, hence, foodstuffs start to dehydrate.^[39,40] In MW processing, the drying rate of the sample increased with an increase of MW output power. It is known that after a short heating period, the time needed during constant rate period changes by MW output powers used.^[40]

Figure 1. Effect of MW output power [460 (

), 600 (

), and 700 W (

)] applied on (a) moisture content, (b) total Chl content, (c) vitamin C content, (d) TPC, (e) TFC, (f) sinapik asit content, (g) ferulik asit content, (h) DPPH scavenging capacity and (i) FRAP scavenging capacity of Brussels sprouts.

Total Chl content was decreased by almost 79.82 to 91.23% depending on the MW output powers used (Figure 1b). Armesto et al.^[21] determined that reduction of total Chl content in Galega kale (Brassica oleracea L. var. acephala) ranged between 47.34 and 52.67% in MW application at 900 W. The reason of Chl content reduction is due the fact that Chl are degraded to undesirable gray–brown compounds like pheophorbide or pheophytin and further metabolizes to colorless compounds because of heat treatment.^[41,42] The temperature rise in the product because of enhanced MW power caused an increase in the rate of degradation. The findings of this study is in line with the result obtained by Nawirska-Olszańska^[43] who reported Chl a and b contents of pumpkins decreased to a greater extent when dried under 250 W compared to 100 W.

Vitamin C content of samples decreased at MW processing and reached to a final loss of 90.97–92.72% at the end of the process (Figure 1c). Vitamin C is a heat-sensitive compound.^[44] Heat is the major factor affecting the oxidation of L-ascorbic acid to L-dehydroxy-ascorbic acid, which could transform irreversibly to 2,3-deketogulonic acid by oxidation. 2,3-deketogulonic acid has no vitamin C biological activity.^[45] Under MW processing conditions, an increase in absorbed power causes an increment in temperature of the sample and a greater rate of vitamin C loss occurred accordingly.^[46] In addition, the loss of vitamin C could be increased due to the exposure duration of the sample to atmospheric oxygen.^[47,48] The change of degradation rate in vitamin C of samples related to MW output power used correlated with the results of Khraisheh et al.^[46] In contrast to Ozkan et al.^[49], who conducted MW drying treatments in spinach, and generally, significant increases were determined in the ascorbic acid values when MW power changed from 90 W to 1000 W. Severini et al.^[50] reported that microwave blanching induced a considerable increase in ascorbic acid content of broccoli. Between the results obtained in this study and the published works of Ozkan et al.^[49] and Severini et al.^[50] could be because of differences in MW processing conditions in addition to the sample type.

TPC in the final samples dried with different MW output power was approximately 67.19–73.51% lower than the fresh samples, while TFC in samples at the end of the same MW processing was approximately 76.71–81.08% lower than fresh samples (Figure 1d and 1e). These data show that the flavonoids in the samples are more degradable than the phenolic acids. Sinapic acid and ferulic acid, known as phenolic acids, were decreased by 77.66–80.64% and 64.56–69.36%, respectively (Figure 1f and 1g). The fact that sinapic acid degradation is higher than ferulic acid reveals sinapic acid is a more sensitive compound to heat than atmospheric oxygen and ferulic acid. Additionally, also seems that the results of HPLC-DAD confirm the findings of the spectroscopic method (TPC). The change of the MW output powers in the MW processing leads to the change in the temperature of the product. During MW processing, the temperature of samples increased and their polyphenols contents decreased.^[51–56] The increase of temperature could be attributed to oxidative processes during MW drying. The degradation of polyphenols is affected by cellular destruction, which occurred with long drying times, high temperatures, and the moisture content of sample based on food material. Water plays a critical role since it allows polyphenols to dissolve and then to be drifted to the surface.^[56] This phenomenon accelerates the degradation of polyphenols with the result of oxidation reaction. This trend in samples depending on the MW output power applied was determined to be in agreement with previous findings that İçier et al.^[57] for black olive slices and Arslan and Özcan^[48] for onion slices. In addition, Igual et al.^[58], Śledź et al.^[59], Alean et al.^[56], and Fouad and Rehab^[60] have reported similar findings to these observations in the present study that polyphenols content of the final product is lower than the fresh ones at the end of the MW processing.

In MW processing, the antioxidant capacities of samples decreased 15.05–75.18% at 460 W, 24.21–81.54% at 600 W, and 28.56–81.86% at 700 W for DPPH and FRAP scavenging capacities, respectively (Figure 1h and 1i). Different MW output powers caused different results in the degradation of DPPH and FRAP scavenging capacities of the samples. The higher MW power applied, the lower DPPH and FRAP scavenging capacities were observed. The compounds responsible for FRAP scavenging capacity were highly sensitive to MW processing and only almost 20–25% activity left at the end of the process. During MW processing antioxidant components of Brussels sprouts such as polyphenols, vitamin C, and Chl have been shown to be lost heavily. For this reason, the result of antioxidant capacity is parallel to other analysis results obtained in the current research. These data support the statement of Jaiswal and Abu-Ghannam^[14], who reported that the DPPH and FRAP scavenging capacities of York cabbage decreased at different MW powers applied. Subudhi and Bhoi^[22] determined that microwave cooking reduced the DPPH and FRAP scavenging capacities of brown mustard (Brassica juncea). Contrary to it, Igual et al.^[58] noted a significant increase of antioxidant capacity in MW-dried apricot and suggested that the reason of increased antioxidant capacity could be due to the sampling type.

Kinetic analysis of moisture content, bioactive compounds, and antioxidant capacity

Kinetics of the bioactive compounds, antioxidant capacity, and moisture content change was modeled by using a zero-order Eq. (1) and a first-order Eq. (2) kinetic model. The values of R², RMSE, and χ² were used for comparison of the experimental and predicted data from models. The first-order kinetic model has fitted the experimental data with higher R² values with lower RMSE and χ² values at all MW powers applied (Table 1). Experimental and predicted (first-order kinetic model) data for moisture content change and bioactive compounds and antioxidant capacity degradations of samples that were processed at the three MW power levels (460, 600, and 700 W) are presented in Figure 2a-i. It seemed that the degradation rate constant (k) increased as the MW output power increased from 460 to 700 W. The highest changes in k values were observed in moisture content and DPPH radical scavenging capacity of the sample. They increased 1.07 to 1.91 times, respectively with an increase in MW output power from 460 to 700 W. Because of the MW power increment, the k values related with vitamin C content (0.61 times), total Chl content, and TPC (0.54 times), TFC (0.41 times), and sinapic acid content (0.42 times) were increased. The lowest changes in k values occurred in ferulic acid content and FRAP radical scavenging capacity of sample. They increased 0.39 and 0.32 times, respectively with an increase in MW output power from 460 to 700 W.

Table 1. Kinetic parameter estimated (k) and statistical values of first-order models of bioactive compounds degradation in Brussel sprouts due to microwave processing at different output powers.

		C^0	k (min-1)
	Power (W)	(std. error)	(std. error)	R^2	RMSE	χ2
Moisture content	460	6.56	−0.934	0.979	0.044	0.003
		(0.223)	(0.062)
	600	6.707	−1.315	0.996	0.026	0.001
		(0.106)	(0.046)
	700	6.699	−1.934	0.997	0.053	0.004
		(0.104)	(0.081)
Total chlorophyll content	460	6.087	−0.438	0.97	0.069	0.006
		(0.212)	(0.027)
	600	5.537	−0.458	0.921	0.091	0.011
		(0.302)	(0.046)
	700	5.665	−0.674	0.968	0.070	0.007
		(0.228)	(0.968)
Vitamin C content	460	361.894	−1.347	0.921	0.168	0.038
		(22.766)	(0.188)
	600	367.958	−1.967	0.941	0.178	0.044
		(21.489)	(0.314)
	700	368.646	−2.173	0.947	0.172	0.045
		(21.886)	(0.381)
Total polyphenol content	460	908.902	−0.261	0.952	0.057	0.004
		(29.205)	(0.017)
	600	926.2	−0.307	0.99	0.022	0.001
		(14.505)	(0.010)
	700	913.994	−0.403	0.979	0.043	0.003
		(23.936)	(0.021)
Total flavonoid content	460	309.006	−0.309	0.998	0.011	0.000
		(2.096)	(0.004)
	600	305.094	−0.329	0.997	0.017	0.000
		(2.624)	(0.006)
	700	312.289	−0.435	0.998	0.013	0.000
		(2.669)	(0.007)
DPPH radical scavenging capacity	460	94.552	−0.034	0.952	0.013	0.000
		(0.570)	(0.002)
	600	92.289	−0.065	0.905	0.032	0.001
		(1.376)	(0.006)
	700	92.909	−0.099	0.923	0.033	0.002
		(1.665)	(0.009)
Ferric reducing antioxidant power (FRAP)	460	1770.445	−0.303	0.986	0.033	0.001
		(33.622)	(0.011)
	600	1800.563	−0.365	0.997	0.011	0.000
		(18.374)	(0.007)
	700	1855.369	−0.4	0.982	0.044	0.003
		(46.287)	(0.020)
Sinapic acid	460	17.215	−0.449	0.899	0.115	0.018
		(1.027)	(0.047)
	600	17.557	−0.581	0.881	0.130	0.024
		(1.257)	(0.075)
	700	17.279	−0.637	0.823	0.151	0.034
		(1.586)	(0.109)
Ferulic acid	460	0.437	−0.21	0.921	0.059	0.005
		(0.016)	(0.017)
	600	0.448	−0.265	0.951	0.052	0.004
		(0.014)	(0.019)
	700	0.451	−0.291	0.92	0.064	0.006
		(0.019)	(0.029)

Figure 2. Experimental [460 (

), 600 (

), and 700 W (

)] and predicted [(

)] data related with different MW output power applied on (a) moisture content, (b) total Chl content, (c) vitamin C content, (d) TPC, (e) TFC, (f) sinapik asit content, (g) ferulik asit content, (h) DPPH scavenging capacity and (i) FRAP scavenging capacity of Brussels sprouts.

A graph was plotted between MW powers and k values estimated in order to evaluate the relationship between MW powers and degradation of all analyzed compounds (not shown). The results indicated a correlation between MW powers and all analyzed compounds degradation and the R² values obtained from graph were in the interval of 0.733–0.996. The R² values proved that there is a strong relationship between MW power and all analyzed compounds degradation.

Jaiswal and Abu-Ghannam^[14] focused on effects of different MW powers on degradation kinetics of color, texture, polyphenols, and antioxidant capacity of York cabbage. They explained that when the MW power was increased, the k values of bioactive compounds degradation reactions increased significantly as well. In their research, İçier et al.^[57] studied the effect of MW power on TPC and color of black olive slices and determined that the applied MW powers were linearly related to k values of TPC in black olive slices. Khraisheh et al.^[46] examined the quality and structural changes in potatoes during microwave and convective drying. The results showed that the k values related with vitamin C content of potatoes reduced when MW power was increased. Similar with this study, first-order reaction model showed a good fit for the degradation of bioactive compounds in all mentioned studies.

Conclusion

The used MW power could change the temperature and period of exposure to atmospheric oxygen of the product and it could cause adverse effects on some quality properties of the foodstuffs such as the degradations in polyphenols, ascorbic acid, and carotenoids etc. This study indicated that the contents of moisture, Chl, vitamin C, polyphenols, flavonoids, and antioxidant capacity of Brussels sprouts were significantly decreased by MW processing at different MW powers applied. The first-order kinetic model was determined as the most appropriate model to represent the change of bioactive compounds, antioxidant capacity, and moisture content of Brussels sprouts during all MW processing. It is proved that the quality retention of green vegetables like Brussels sprouts during MW processing was directly related to the intensity of MW power applied. The use of relatively low power levels like 460 W may be advisable to reduce the main losses in the contents of Chl, vitamin C, polyphenols, flavonoids, and antioxidant capacity of Brussels sprouts in MW processing. Actually, in industrial applications where the final vegetable volume is desired to be low, it is known that saving a lot of time and electricity energy can be achieved as a result of the use of a low MW power and short processing time. The decrease in MW power used in this study increased the drying time. It has been found that the loss of nutrients was lesser when a lower MW power was used, although the drying time was increased in the meanwhile. This reveals that the value of microwave power used was more effective in comparison to the drying time in preserving nutrients.