Impact of Different Thermal Processing Techniques on the Phytochemical Composition, Antioxidant Capacity, and DNA-Protective Properties of Broccoli

Abstract

Vegetables are usually thermally processed before consumption to improve their flavor and safety. In this work, the effect of boiling (BO), blanching (BL), steaming (ST), air-frying (AF), and pan-frying (PF)on the nutritional value and bioactivity of broccoli (Brassica oleracea var. italica) heads was investigated, including a comparative analysis of the tissue and the cooking water remaining after the treatments. Using spectrophotometric methods, AF broccoli was found to have the highest levels (p ≤ 0.05) of hydroxycinnamic acids (1.58 ± 0.71 mg CAE/g fw), total glucosinolates (3.76 ± 2.09 mg SinE/g fw), carotenoids (6.73 ± 2.89 mg/kg fw), and lycopene (0.91 ± 0.19 mg/kg fw). Steamed and AF broccoli had the highest total phenolics (0.72 ± 0.12 mg GAE/g fw and 0.65 ± 0.15 mg GAE/g fw, respectively; p ≤ 0.05). ST broccoli also had the highest levels of soluble sugars (11.04 ± 2.45 mg SucE/g fw) and total tannins (0.46 ± 0.19 mg GAE/g fw). The water remaining after cooking broccoli (BOW) had the highest total flavonoids (2.72 ± 0.59 mg QE/g fw) and antioxidant capacity (ABTS and FRAP, 57.57 ± 18.22% and 79.34 ± 3.28%, respectively; p ≤ 0.05). The DPPH assay showed that AF (36.12 ± 15.71%) and ST (35.48 ± 2.28%) had the strongest antioxidant potential. DNA nicking assay showed that BOW and BLW were the most effective in preserving plasmid DNA supercoiled form (99.51% and 94.81%, respectively; p ≤ 0.05). These results demonstrate that thermal processing significantly affects the phytochemical composition and functional properties of broccoli, with steaming and air-frying generally preserving the highest nutritional quality. Additionally, cooking water, often discarded, retains high levels of bioactive compounds and exhibits strong antioxidant and DNA-protective effects. To the best of our knowledge, this is the first study to investigate how different thermal processing techniques of vegetables influence their ability to protect plasmid DNA structure. Furthermore, this is the first study to compare the DNA-protective effects of broccoli tissue extracts and the water remaining after cooking broccoli.

1. Introduction

A balanced human diet is one of the most important factors for the health and proper homeostasis of the body, as it provides all the necessary nutrients that play an important role in the biochemical and signaling pathways that influence growth, energy expenditure, and general functioning. According to the pyramid of proper nutrition, grains, fruits, and vegetables form the basis [1]. One of the most important vegetable groups used in the daily diet as a major source of antioxidants—such as flavonoids, fiber, minerals, carotenoids, and vitamins—is vegetables from the Brassicaceae family, with broccoli (Brassica oleracea var. italica) being the most common representative [2]. Broccoli has a high nutritional value [3], is anti-inflammatory and anti-cancer [4], and has antibacterial properties [5].

Eaten raw, e.g., in the form of microgreens, brassica vegetables provide the greatest nutritional value, while various thermal processing methods reduce the content of “healthy” compounds [6]. However, vegetables are usually thermally processed before consumption to improve their flavor and safety [7]. The most commonly used thermal processing techniques today are boiling, blanching, steaming, pan-frying, and air-frying [8]. Each of these techniques significantly affects the nutrient content as many of the bioavailable compounds are heat-sensitive. Therefore, it is important to determine which thermal processing techniques preserve the most bioactive plant compounds for optimal nutritional value [9]. The most commonly used thermal processing techniques are boiling and steaming, which affect the physical and chemical properties of vegetables. Some boiled and steamed vegetables, such as sathkora (Citrus macroptera), pumpkin (Cucurbita maxima), green peas (Pisum sativum), pepper (Capsicum annuum), and spinach (Spinacia oleracea), have been shown to contain more β-carotene than fresh vegetables. However, the total content of polyphenols and flavonoids decreased by up to 70.3% and 82.27%, respectively, during boiling compared to steaming, with a significant reduction in free radical scavenging activity by up to 56.73%, with the exception of carrots [10]. Although hot water was used in both techniques, steaming was more gentle because the vegetables were not immersed in water, and the nutrient loss was far lower than with boiling [11]. Using mature broccoli as an example, boiling caused a greater loss of chlorophyll, vitamin C, and glucosinolates than steaming [12]. Blanching is a technique similar to boiling, with the main difference being the time the vegetables are immersed in boiling water. Blanched white cauliflower had a stronger antioxidant potential with higher levels of total phenolics, protocatechuic acid, quercetin, pyrogallol, vanillic acid, coumaric acid, and kaempferol than boiled cauliflower [13]. In recent years, pan-frying and air-frying have become increasingly popular alternatives for the thermal processing of vegetables, often used for green pepper seeds and mustard. It has been shown that these techniques usually lower the radical scavenging ability [14]. However, the antioxidant potential, measured by ABTS, of pepper seeds and mustard was not significantly affected by pan-frying and air-frying [15,16]. In addition, a study on dried colored bell peppers (Capsicum annuum) showed that the scavenging potential measured with DPPH almost did not decrease, but the ability to scavenge the ABTS radical was drastically reduced [17]. Hwang et al. [18] showed that the antioxidant capacity of red peppers (Capsicum anuum), measured by ABTS and DPPH, decreased after boiling and steaming, while air-frying significantly reduced it. This shows that different thermal processing methods specifically influence the nutritional value of different vegetables. Therefore, it is necessary to evaluate the polyphenol content and antioxidant capacity of each individual vegetable type with no unique “rule of thumb” to assess their nutritional potential after thermal processing [18,19]. Although frying techniques negatively affect polyphenolic content and antioxidant potential of vegetables, they are still a better alternative to boiling [20]. Despite the reduction in the nutritional potential of vegetables during thermal processing, the air-frying technique is considered the least invasive for polyphenolic compounds and it results in the best antioxidant capacity of brassica vegetables such as canola, mustard, kale, broccoli sprouts, red cabbage and green cabbage [14]. In these vegetables, total phenolic and flavonoid content and antioxidant capacity measured by ABTS, DPPH and FRAP assays were the highest, compared to other thermal processing techniques, suggesting that the future of thermal processing of vegetables lies in air-frying.

The aim of this study was to identify the most suitable classical thermal processing technique for preserving the bioactive compounds and antioxidant potential in broccoli heads, while also evaluating the nutritional value of the residual cooking water to emphasize its potential for dietary use rather than disposal. In this sense, the study compares the effects of five different thermal processing techniques (boiling, blanching, steaming, air-frying, and steaming followed by pan-frying) on the phytochemical composition, antioxidant capacity, and DNA-protective potential of mature broccoli heads, including an analysis of both the processed tissue and the residual cooking water, where applicable. In particular, the effects on total phenols, flavonoids, hydroxycinnamic acids, total tannins, proanthocyanidins, intact glucosinolates, soluble sugars, hydrogen peroxide content, and photosynthetic pigments were analyzed, as well as the effect on its antioxidative potential measured by ABTS, DPPH, and FRAP method. Additionally, using DNA nicking assay, the influence of broccoli thermal processing on the potential of its extracts to protect plasmid DNA structure from oxidative damage caused by Fenton’s reagent was analyzed. The results showed that among the tissues, air-fried broccoli had the highest proportion of preserved bioactive compounds, while among the water remaining after processing, the one after cooking had the highest concentration of flavonoids and proanthocyanidins, and the one after blanching showed the strongest protective effect against plasmid DNA oxidation.

2. Materials and Methods

2.1. Plant Material

Broccoli (Brassica oleracea var. italica) was obtained from the Konzum store in Zagreb, Croatia. Heads of broccoli were separated, thermally processed, homogenized by maceration and extracts were prepared in different solvents (70% ethanol, 80% methanol or 80% acetone) for different methods at a final concentration of 300 mg/mL. After techniques of boiling, blanching, and steaming, the remaining water in pot was also collected, filtered through Whatman^® qualitative filter paper, Grade 1 (Merck KGaA, Darmstadt, Germany) and analyzed, while broccoli tissue was used as is. Three biological replicates were prepared along with four technical replicates of each sample per method.

2.2. Thermal Processing Techniques of Broccoli Heads

Five different thermal processing techniques were conducted: boiling (BO), blanching (BL), steaming (ST), air-frying (AF), and blanching followed by pan-frying (PF). These cooking methods were selected based on the most commonly used broccoli preparation techniques found in the literature [12,14,21,22,23], with appropriate adjustments to cooking duration reflecting local culinary practices in Croatia. Boiling (BO) of broccoli was conducted in 100 °C water (boiling water) in a metal pot for 30 min followed by filtration to separate broccoli and the remaining water (compared to broccoli usually being boiled for 5 min [12]). Blanching (BL) was conducted for 3 min (compared to 30–180 s according to the literature [21]) in the boiling water in a metal pot followed by filtration to separate broccoli and the residue water. Steaming (ST) of the broccoli was performed for 3 min in a metal pot above boiling water (usually steamed for 5 min [12,21]). Air-frying of broccoli (AF) was conducted for 4 min at 200 °C in an air fryer EAF7SB (Electrolux d.o.o., Zagreb, Croatia) as compared to 160 °C for 10 min [14]. Pan-frying of blanched broccoli (PF) was performed for 5 min on a Teflon pan with sunflower oil in a ratio 10:1 of vegetable-to-oil since broccoli is usually blanched prior to frying in everyday cooking (often it is stir-fried for 2 min–3 min 30 s [12,23]). After boiling, blanching, and steaming of broccoli, each time at a concentration of 300 mg of broccoli/mL water, the remaining water was collected as an aqueous extract for further analysis and denoted as water after boiling (BOW), water after blanching (BLW), and water after steaming (STW), while thermally processed broccoli tissue was stored at −20 °C for further preparations of extracts and analyses.

2.3. Measurement of Polyphenolic Bioactive Compounds

Polyphenolic bioactive compounds were measured in 70% (v/v) ethanol extract of BO, BL, ST, AF, and PF samples, and in water remaining after boiling, blanching, or steaming, which was filtered (BOW, BLW, and STW). Both types of extracts were prepared at a concentration of 300 mg fresh weight (fw)/mL. The absorbances were measured on a microplate reader Fluostar Optima (BMG LABTECH, Ortenberg, Germany).

Total phenolics was assessed using the Folin–Ciocâlteu method, as described in reference [24]. Absorbance was recorded at 740 nm. A calibration curve was prepared with gallic acid standards ranging from 0.02 to 1.25 mg/mL. Results were expressed as milligrams of gallic acid equivalents per gram of fresh broccoli weight (mg GAE/g fw).

To evaluate total flavonoid levels in broccoli samples, a colorimetric assay involving complex formation with aluminum chloride was applied, following the protocol outlined by [25]. Quercetin served as the reference compound for quantification, and a standard curve was established across the concentration range of 0.08 to 1.25 mg/mL. The absorbance of the resulting complexes was recorded at 520 nm. Flavonoid concentrations were calculated and expressed as quercetin equivalents per gram of fresh weight (mg QE/g fw).

Hydroxycinnamic acid content was determined using the method previously established in our laboratory [24]. A calibration curve was prepared using caffeic acid standards ranging from 0.08 to 1.25 mg/mL. Absorbance measurements were taken at 355 nm. The concentrations were calculated and expressed as caffeic acid equivalents per gram of fresh broccoli weight (mg CAE/g fw).

The determination of total tannin content was carried out following the method described by Galvão et al. [26]. Absorbance readings were taken at 740 nm. A standard curve was generated using gallic acid across concentrations ranging from 0.08 to 1.25 mg/mL. Results were expressed as milligrams of gallic acid equivalents per gram of fresh broccoli weight (mg GAE/g fw).

A procedure reported in Weidner et al. [27] was used for the determination of total proanthocyanidins. Absorbance was measured at a wavelength of 485 nm and a catechin solution at a concentration range of 0.47–7.50 mg/mL was used for the calibration curve. Results are shown as milligrams of catechin equivalents per gram of fresh weight (mg CatE/g fw) of broccoli.

2.4. Measurement of Soluble Sugars

Soluble sugars were determined following the method described by Dubois et al. [28]. Absorbance readings were taken at 485 nm, and a sucrose standard curve was prepared using concentrations ranging from 0.06 to 1.00 mg/mL. The sugar content was expressed as milligrams of sucrose equivalents per gram of fresh weight (mg SucE/g fw) of broccoli.

2.5. Measurement of Total Intact Glucosinolates

Total intact glucosinolates was measured as described in Mawlong et al. [29]. The extracts were prepared in 80% methanol at a final concentration of 300 mg/mL. Absorbance was recorded at 425 nm, using sinigrin solutions ranging from 0.30 to 1.00 mg/mL to construct the standard curve. Results were expressed as milligrams of sinigrin equivalents per gram of fresh weight (mg SinE/g fw) of broccoli.

2.6. Measurement of Hydrogen Peroxide Content

The quantification of hydrogen peroxide (H₂O₂) followed the protocol established by Junglee et al. [30]. Spectrophotometric readings were taken at 405 nm, and a calibration curve was generated using H₂O₂ standards ranging from 0.12 to 1.50 mM. Data were reported as the concentration of H₂O₂, in millimoles, per gram of fresh broccoli tissue (mM H₂O₂/g fw).

2.7. Determination of Chlorophyll, Carotenoids, and Porphyrins

The assessment of photosynthetic pigments was carried out based on the procedure by Sumanta et al. [31]. Pigments including chlorophylls, carotenoids, and porphyrins were extracted using 80% acetone, with the sample concentration adjusted to 300 mg/mL. Absorbance was recorded at 470, 575, 590, 628, 647, and 663 nm using a Thermo Scientific Nanodrop 2000c spectrophotometer. Chlorophyll a and b, carotenoids, porphyrins, β-carotene, and lycopene content was determined according to the following equations [32]:

$$Chla=12.25{Abs}_{663}-2.79{Abs}_{647}$$

$$Chlb=21.50{Abs}_{645}-5.10{Abs}_{663}$$

$$Car={\displaystyle \frac{\left(1000{Abs}_{470}-1.82Chla-85.02Chlb\right)}{198}}$$

$$\begin{array}{ll}Porphyrins=& {\displaystyle \frac{\left(12.25{Abs}_{663}-2.55{Abs}_{647}\right)}{892}}+{\displaystyle \frac{20.31{Abs}_{647}-4.91{Abs}_{663}}{906}}\\ & +\left(196.25{Abs}_{575}-46.6{Abs}_{590}-58.68{Abs}_{628}\right)\\ & +\left(61.81{Abs}_{590}-23.77{Abs}_{575}-3.55{Abs}_{628}\right)\\ & +\left(42.59{Abs}_{628}-34.32{Abs}_{575}-7.25{Abs}_{590}\right)\end{array}$$

$$\beta -Carotene=0.216{Abs}_{663}-1.22{Abs}_{647}-0.304{Abs}_{505}+0.452{Abs}_{453}$$

$$Lycopene=-0.0458{Abs}_{663}+0.204{Abs}_{647}-0.304{Abs}_{505}+0.452{Abs}_{453}$$

where Abs = absorbance [29].

2.8. Determination of Antioxidant Capacity

The antioxidant capacity of the extracts was evaluated using a Fluostar Optima microplate reader, employing miniaturized versions of the ABTS, DPPH, and FRAP assays [24]. Calibration curves were established with Trolox standards spanning concentrations from 0.09 to 3.13 mg/mL. Results are shown as a percentage of inhibition for ABTS and DPPH, while FRAP results are shown as a percentage of reduction according to the following equations:

$$\%\mathrm{inhibition}={\displaystyle \frac{{Abs}_0-{Abs}_t}{{Abs}_0}}\times 100$$

$$\%\mathrm{reduction}={\displaystyle \frac{{Abs}_t-{Abs}_0}{{Abs}_t}}\times 100$$

where Abs_t = absorbance of the extract and Abs₀ = absorbance of control (water or 70% ethanol).

2.9. Extraction of Plasmid DNA

Plasmid pSgM1_HNF1A (kindly gifted by Prof. Dr. Vlatka Zoldoš) was isolated from transformed Escherichia coli bacterial strain NEB^® Stable Competent E. coli (high efficiency) with NucleoBond Xtra Maxi kit for transfection-grade plasmid DNA (Macherey-Nagel) commercial kit which is based on affinity filters binding to DNA plasmids with electrostatic bonds [33,34,35]. Plasmid has spectinomycin resistance cassette as a selection marker.

2.10. DNA Nicking Protection Assay Using Fenton’s Reagent

DNA nicking protection assay was performed using pSgM1_HNF1A (400 ng/µL) plasmid of 2854 bp in size which was combined with 70% ethanol or water extracts (300 mg/mL) and Fenton’s reagent (0.33 mM FeSO₄ × 7 H₂O, 0.323 M H₂O₂ in 1 × PBS, pH 3.5), according to Andonova et al. [36]. Samples were incubated for 30 min at 37 °C. Trolox at a concentration of 30 mg/mL served as a positive control, while negative control consisted of the plasmid mixed with pure Fenton’s reagent. DNA samples were separated on 1.5% agarose gel in Tris-Borate EDTA buffer (TBE) for 2 h and 30 min at 80 V and stained with fluorescent dye GelRed^®. Relative quantification of band density was performed using ImageJ software version 1.54g (Fiji). A standard curve was generated by plotting pixel density (y-axis) against known DNA mass (x-axis), which was then used to calculate DNA mass in the samples.

2.11. Statistical Analysis

Statistical evaluation of the data was carried out using Statistica version 14.1.0.8 (TIBCO, Cloud Software Group, San Ramon, CA, USA). Differences among groups were assessed through one-way ANOVA, followed by Duncan’s New Multiple Range Test (DNMRT) for post hoc comparisons, with significance defined at p ≤ 0.05. Most analyses were based on three biological replicates and four technical replicates per sample. Exceptions included measurements of soluble sugars in boiled broccoli and in the cooking water, as well as glucosinolate content in pan-fried broccoli, which were analyzed using two biological replicates, each with four technical replicates.

3. Results and Discussion

3.1. Effect of Thermal Processing on the Phytochemical Content of Broccoli Extracts

Different thermal processing methods of broccoli heads resulted in significantly different phytochemical composition of the extracts (

Table 1. Effect of different thermal processing techniques on different groups of polyphenolics in broccoli extracts.

	TP (mg GAE/g fw)	TF (mg QE/g fw)	THCA (mg CAE/g fw)	TT (mg GAE/g fw)	TPAN (mg CatE/g fw)
BO	0.37 ± 0.18 bc	0.54 ± 0.13 c	0.72 ± 0.40 bc	0.18 ± 0.09 cd	0.13 ± 0.03 b
BOW	0.39 ± 0.20 bc	2.72 ± 0.59 a	0.92 ± 0.50 abc	0.29 ± 0.05 bc	0.96 ± 0.89 a
BL	0.47 ± 0.21 b	0.86 ± 0.25 c	0.50 ± 0.35 c	0.22 ± 0.10 bcd	0.15 ± 0.06 b
BLW	0.25 ± 0.16 c	0.74 ± 0.25 c	0.64 ± 0.38 bc	0.10 ± 0.08 d	0.41 ± 0.45 b
ST	0.72 ± 0.12 a	1.49 ± 0.47 b	1.19 ± 0.56 ab	0.46 ± 0.19 a	0.31 ± 0.04 b
STW	0.43 ± 0.34 bc	0.75 ± 0.25 c	1.50 ± 0.64 a	0.27 ± 0.23 bc	0.07 ± 0.05 b
AF	0.65 ± 0.15 a	1.71 ± 0.70 b	1.58 ± 0.71 a	0.27 ± 0.10 bc	0.42 ± 0.13 b
PF	0.39 ± 0.20 bc	1.42 ± 0.66 b	0.98 ± 0.37 abc	0.35 ± 0.15 ab	0.21 ± 0.06 b

Values represent average ± standard deviation of three biological and four technical replicates. Different letters indicate a significant difference among the values in a row (one-way ANOVA, Duncan’s test, p ≤ 0.05). fw = fresh weight; BO = broccoli boiled; BOW = water after boiling broccoli; BL = broccoli blanched; BLW = water after blanching broccoli; ST = broccoli steamed; STW = water after steaming broccoli; AF = broccoli air-fried; PF = broccoli blanched and then pan-fried; TP = total phenolics; GAE = gallic acid equivalent; TF = total flavonoids; QE = quercetin equivalent; THCA = total hydroxycinnamic acids; CAE = caffeic acid equivalent; TPAN = total proanthocyanidins; CatE = catechin equivalent; TT = total tannins.

). The amount of total phenolic content was the highest in steamed and air-fried broccoli (0.72 ± 0.12 mg GAE/g fw and 0.65 ± 0.15 mg GAE/g fw, respectively), while boiled and blanched broccoli contained lower amounts (0.37 ± 0.18 mg GAE/g fw and 0.47 ± 0.21 mg GAE/g fw, respectively). This suggests that steamed and air-fried broccoli encapsulates these biochemicals more efficiently than boiled or blanched. A considerable amount of phenols was dissipated in water, as evidenced by the presence of phenols in the water remaining after boiling, blanching, and steaming. The same phenomenon was observed for all other polyphenolic compounds (total flavonoids, hydroxycinnamic acids, tannins, and proanthocyanidins). All these phytochemicals exhibit limited (proanthocyanidins) [37] to moderate (polar aglycone flavonoids) water solubility [38], which is improved by heat [39]. Therefore, a considerable amount of these chemicals can be found in the hot water remaining after thermal processing. The fact that steaming does not include direct immersion of broccoli into the boiling water is the most likely reason why steamed broccoli has a higher total phenolic content than boiled or blanched. In addition, boiling and blanching are more “aggressive” methods, as more cells are damaged and the polysaccharide fibers are destroyed, which can lead to oxidation of the molecules [17,40]. Similar results were found in Brussel sprouts and pumpkin [41], where a higher phenolic content was observed after air steam treatment, compared to water steaming. This could be due to the degradation that occurs in normal steaming treatment compared to the air steaming treatment due to the presence of air by oxidizing the antioxidant molecules [17]. In agreement with our results, Ahmed and Ali [13] showed that the total amount of phenolic compounds was also significantly lower in cooked and blanched white cauliflower compared to steamed and stir-fried. Flavonoids were also significantly reduced, with the greatest reduction occurring after boiling, followed by blanching, while stir-frying showed the best results [13]. Similar phenomenon was also recorded in air-fried eggplants with the highest polyphenolics content after air-frying [42]. In general, boiling and blanching are considered as very disruptive methods of vegetable thermal processing because phenolic compounds disintegrate during long heat exposure, and this was observed in green pea, pepper, beet and eggplant [43], as well as broccoli, cabbage, colored peppers, leek, peas, spinach, squash, and tomatoes [43,44,45,46,47]. The highest amount of phenolics retained by air-frying compared to other cooking methods was also noticed in red cabbage, green cabbage, broccoli sprouts, Brussel sprouts, and kale [14]. In some cases, certain thermogenic compounds such as canolol can even be formed during air-frying compared to other cooking methods [15,16].

On the other hand, the water remaining after cooking (boiling) had significantly higher content of total flavonoids (2.72 ± 0.59 mg QE/g fw) and proanthocyanidins (0.96 ± 0.89 mg CatE/g fw) than all other samples. Among the tissues, air-fried and pan-fried broccoli had the highest amount of preserved flavonoids (1.71 ± 0.70 mg QE/g fw and 1.42 ± 0.66 mg QE/g fw, respectively), while air-fried broccoli had the highest amount of preserved proanthocyanidins (0.42 ± 0.13 mg CatE/g fw). Similar results were also recorded in broccoli florets [22], where the loss of flavonoids could be due to thermal degradation and “leaching” into cooking water. Steaming also proved to be a better cooking method than boiling and blanching in terms of preserving polyphenolics in green vegetables as well [48]. In addition, the content of flavonoids in garden spinach leaf, Indian spinach leaf, and green leaf amaranth was higher after frying compared to boiling [49] which is due to a higher availability of the compounds trapped in fibers [50]. Additionally, higher levels of some proanthocyanidins were shown to protect the vegetable quality in deep frying oil by inhibiting oxidation [51], which would explain higher percentage of most phytochemicals in pan-fried broccoli.

The total hydroxycinnamic acid content was highest in the water remaining after steaming broccoli (1.50 ± 0.64 mg CAE/g fw) and in air-fried broccoli (1.58 ± 0.71 mg CAE/g fw). Steamed broccoli tissue had the highest total tannin content (0.46 ± 0.19 mg GAE/g fw). Although frying reduces the amount of phytochemicals compared to raw vegetables due to the high temperatures, it was shown that hydroxycinnamic acids from fried red skin onion had a higher bioavailability after digestion compared to other types of baking and grilling [52], which indicates that frying had a less negative effect on these compounds. Unfortunately, no comparisons were found between fried and boiled/steamed/blanched onion to determine whether there are even greater losses of hydroxycinnamic acids when being cooked in other ways. Similarly, potatoes, still retain a high amount of hydroxycinnamic acids, despite the high temperatures reached during frying in oil, suggesting that frying does not necessarily have a negative impact on phytochemical integrity [53,54].

Although most of the mentioned phytochemicals remain preserved in broccoli when air-fried or pan-fried, total tannin content was the highest in steamed broccoli (0.46 ± 0.19 mg GAE/g fw), while in air-fried and pan-fried broccoli, their levels were lower (0.27 ± 0.10 mg GAE/g fw and 0.35 ± 0.15 mg GAE/g fw, respectively). The tannin composition of different vegetables varied depending on the type of thermal processing. For example, the tannin content was higher in boiled kale, spinach, beans, carrot, and tomatoes compared to air-fried and microwaved vegetables [55]. A significant loss of tannins was also found in microwaved spinach, cabbage, cauliflower, and yellow turnip [56]. This indicates that the type and length of a thermal processing technique is very important for optimal tannin concentration as tannins are extremely sensitive to heat.

In general, steamed and air-fried broccoli showed higher content of the measured polyphenolics compared to other types of thermally processed broccoli. Phenolic compounds remain in broccoli probably because steaming inactivates oxidative enzymes [57]. Regarding the lower content of phytochemicals after frying broccoli in a pan than in air, it is hypothesized that, due to the fact that oil can achieve much higher temperatures than water, oil increases the permeability of plant membranes significantly more [58] and, consequently, also the degradation of the bioactive components. In addition, oil extracts the lipophilic components from vegetables and, therefore, makes vegetables depleted of lipophilic nutrients. Ultimately, thermal processing in an air fryer requires less time than traditional pan-frying, resulting in a shorter exposure of phytochemicals to high temperatures.

3.2. Effect of Thermal Processing on Hydrogen Peroxide Level in Broccoli

It is known that H₂O₂ is formed in cooked vegetables such as beans, broccoli, onion, and leek due to heat stress [59]. In our study, the H₂O₂ content was highest in air-fried broccoli (6.79 ± 0.33 mM H₂O₂/g fw), while water extracts had the lowest concentrations (Figure 1). This might be due to the fact that hot air can disrupt the polysaccharides in the cell wall [60] and that the extracellular matrix has an important role in the generation of H₂O₂ [61]. These results could additionally be explained by the loss of activity of peroxidases, which neutralize H₂O₂, in plants during cooking. More than 95% of peroxidase activity was lost in sweet corn after hot water, steam, and microwave blanching, with hot water blanching being the most severe and microwaving being the least detrimental to peroxidase activity [62]. However, the activity of peroxidases depends not only on the cooking technique of vegetables, but also on the duration and temperature of thermal processing [63]. Similar results were found for carrot, sathkora, pumpkin, green peas, peppers, and spinach where boiling, steaming, and microwave cooking affected different physiochemical properties and levels of phytochemicals. Although H₂O₂ was not measured directly in these vegetables, it is reasonable to assume that these cooking methods can induce oxidative stress in vegetables by altering specific phytochemicals and thus increase the H₂O₂ content [10].

3.3. Effect of Thermal Processing on Total Glucosinolates in Broccoli

The total amount of glucosinolates was significantly higher in air-fried broccoli (3.76 ± 2.09 mg SinE/g fw) than in all other samples (Figure 2). The lowest amount was detected in pan-fried broccoli (0.14 ± 0.10 mg SinE/g fw), which contained about 28 times less glucosinolates than air-fried broccoli. However, the amount in pan-fried broccoli was not significantly different from the amount in boiled, blanched, and steamed broccoli. Total glucosinolates were not measured in water extracts due to their low water solubility. One of the reasons why air-fried broccoli had the highest amount of glucosinolates might be the fact that at high temperatures, their degradation increases in parallel with the amount of water [63]. The high standard deviations observed in the samples may be attributed to the broccoli heads being harvested in different seasons, potentially affecting the concentration of glucosinolates. This is because myrosinase activity is regulated in response to abiotic and biotic factors such as temperature, light intensity, photoperiod, and pathogens [64], and this might significantly affect the concentration of glucosinolates in the same developmental stage in different seasons. Similar studies were conducted on Brussel sprouts, white and green cauliflower, broccoli, and curly kale, which showed a significant loss of total glucosinolates after boiling and blanching, 72.4% and 30%, respectively [65]. A decrease in glucosinolates was also observed in rutabaga, green and purple cauliflower after cooking. It was observed that indole glucosinolates are more sensitive to cooking than other types of glucosinolates [66]. A similar decrease in glucosinolates due to cooking was also found in broccoli, Brussel sprouts, cabbage, and cauliflower and the authors claim that 80% of the loss was due to leaching into water, while 20% was due to high temperature [67]. However, in broccoli, Brussel sprouts, cauliflower, and green cabbage, the loss of total glucosinolates was significantly lower during steaming, microwaving, and stir-frying than during cooking [68]. Microwaving even showed a beneficial effect on the amount of total glucosinolates in red cabbage, with an increase of 78% compared to raw vegetables [69], as well as in Brussel sprouts, with an increase of 86% [70]. One of the theories is that the inactivation of enzymes such as myrosinase occurs during prolonged cooking, which could contribute to the stability of glucosinolates [67]. However, the degradation of glucosinolates depends not only on myrosinase, but on multiple other factors [71]. Nevertheless, indole glucosinolates have been shown to be more sensitive to boiling (51% loss) than aliphatic ones (38% loss) [72]. Finally, the loss of glucosinolates during different cooking methods could be due to the presence of certain plant components which could further influence the stability of glucosinolates depending on the cooking method, temperature, and cooking time [66].

3.4. Effect of Thermal Processing on Soluble Sugars in Broccoli

Steamed broccoli had the highest soluble sugars content (11.04 ± 2.45 mg SucE/g fw), and the content was high in air-fried broccoli as well (8.23 ± 4.96 mg SucE/g fw) (Figure 3). Water from steaming broccoli contained only 3% soluble sugars compared to associated tissue, as the tissue was not in direct contact with water. A previous study on bamboo shoots showed that the loss of soluble sugars was up to 14.39% during boiling and up to 17.42% during stir-frying [73]. The addition of oil contributes to the decrease in soluble sugar content by increasing the temperature and inducing Maillard reaction, which is consistent with our results (air-fried broccoli had 2.7 times more soluble sugars than pan-fried broccoli in oil). Since air-frying does not involve oil, as compared to pan-frying, the Maillard reaction is weaker, so more soluble sugars are present. Blanched broccoli had a higher soluble sugars content than broccoli which was blanched and then pan-fried, suggesting that pan-frying had a negative effect, which is consistent with the theory that the Maillard reaction degrades soluble sugars. Yuan et al. [12] obtained similar results. They found that steamed broccoli had the highest soluble sugars content, while the lowest retention was observed in boiled and stir-fried broccoli.

3.5. Effect of Thermal Processing on Photosynthetic Pigments in Broccoli Extracts

The concentration of chlorophyll a, chlorophyll b, porphyrins, and β-carotene did not differ significantly between the five thermal processing techniques investigated (

Table 2. Effect of different thermal processing techniques on photosynthetic pigments in broccoli extracts.

mg/kg fw	BO	BL	ST	AF	PF
Chlorophyll a	7.27 ± 2.59 a	12.26 ± 2.05 a	11.93 ± 1.58 a	10.53 ± 4.99 a	13.20 ± 5.48 a
Chlorophyll b	4.18 ± 3.25 a	3.32 ± 0.54 a	5.84 ± 4.80 a	4.20 ± 1.08 a	15.93 ± 11.22 a
Carotenoids	3.65 ± 0.03 ab	4.02 ± 0.92 ab	3.53 ± 1.17 ab	6.73 ± 2.89 a	1.77 ± 0.43 b
Porphyrins	31.73 ± 25.84 a	23.70 ± 5.41 a	43.86 ± 37.87 a	53.38 ± 6.38 a	125.85 ± 83.70 a
β-Carotene	0.43 ± 0.03 a	0.51 ± 0.14 a	0.54 ± 0.02 a	0.56 ± 0.26 a	0.17 ± 0.00 a
Lycopene	0.47 ± 0.22 b	0.81 ± 0.16 ab	0.80 ± 0.04 ab	0.91 ± 0.19 a	0.72 ± 0.17 ab

Values represent average ± standard deviation of three biological replicates. Different letters indicate a significant difference among the values in a row (one-way ANOVA, Duncan’s test, p ≤ 0.05). fw = fresh weight; BO = broccoli boiled; BL = broccoli blanched; ST = broccoli steamed; AF = broccoli air-fried; PF = broccoli blanched and then pan-fried.

). Previously, boiling and stir-frying were shown to significantly decrease the chlorophyll content of broccoli [12,73]. On the other hand, these authors reported an opposite effect of steaming on chlorophyll content, Pellegrini et al. [74] reported a decrease, while it remained unchanged in the work of Yuan et al. [12]. In agreement with our results, chlorophyll content did not differ significantly among boiled, steamed (only in Pellegrini et al. [71]), and stir-fried broccoli. However, it should be noted that the values obtained in this study were lower than those reported in their work [12,74]. Pellegrini et al. [74] also showed that the concentration of β-carotene was the same in raw, boiled, and steamed broccoli. Again, the levels of β-carotene obtained in this study were much lower than those reported by Pellegrini et al. [74]. Air-fried broccoli had the highest concentration of carotenoids (6.73 ± 2.89 mg/kg fw) and lycopene (0.91 ± 0.19 mg/kg fw), while pan-fried broccoli had the lowest content of carotenoids (1.77 ± 0.43 mg/kg fw). This might be due to the lipophilicity of the carotenoids, which were absorbed from the tissue by the oil. Carotenoids are also sensitive to heat and the lower content of carotenoids in pan-fried broccoli could also be due to the higher temperature achieved because of the added oil. High temperature can also cause oil to produce hydroperoxide radicals and accelerate the degradation of carotenoids [75]. The stability of carotenoids in vegetables is variable due to both intrinsic and extrinsic factors, such as characteristics of the food matrices (chemical composition, oxygen dissolved, size, physical state of the carotenoid) and type and severity of heat treatment [76]. For broccoli, contrary results were obtained. Pellegrini et al. [77] found a higher content of carotenoids in boiled than in steamed broccoli, while Yuan et al. [12] found a lower content in boiled broccoli than in steamed and stir-fried. In cauliflower, which also belongs to the Brassicaceae family, it was observed that raw vegetables had the highest carotenoid content, followed by blanched, while boiled and stir-fried cauliflower had the lowest carotenoid content [13]. Boiled broccoli had the lowest concentration of lycopene (0.47 ± 0.22 mg/kg fw). As a polyene, lycopene contains unsaturated double bonds and is isomerized and degraded during processing at high temperatures [77]. Interestingly, isomerization contributes to the antioxidant activity of lycopene [78], and the highest concentration of lycopene found in air-fried broccoli was indeed consistent with the highest antioxidant potential (DPPH) of air-fried broccoli (

Table 3. Effect of different thermal processing techniques on antioxidant capacity of broccoli extracts.

	BO	BOW	BL	BLW	ST	STW	AF	PF
ABTS (% inhibition)	26.73 ± 8.01 c	57.57 ± 18.22 a	20.04 ± 7.34 c	21.90 ± 14.97 c	50.17 ± 12.05 ab	20.71 ± 14.84 c	43.04 ± 9.92 b	27.41 ± 21.59 c
DPPH (% inhibition)	27.14 ± 10.43 ab	18.26 ± 12.52 bc	15.51 ± 9.68 c	10.71 ± 8.06 cd	35.48 ± 2.28 a	4.38 ± 3.74 d	36.12 ± 15.71a	17.54 ± 2.74bc
FRAP (% reduction)	47.79 ± 6.34 d	79.34 ± 3.28 a	48.33 ± 10.49 d	35.37 ± 8.86 e	73.87 ± 3.46 ab	14.59 ± 8.30 f	67.79 ± 8.71 b	56.22 ± 8.63 c

Values represent average ± standard deviation of three biological and four technical replicates. Different letters indicate a significant difference among the values in a row (one-way ANOVA, Duncan’s test, p ≤ 0.05). BO = broccoli boiled; BOW = water after boiling broccoli; BL = broccoli blanched; BLW = water after blanching broccoli; ST = broccoli steamed; STW = water after steaming broccoli; AF = broccoli air-fried; PF = broccoli blanched and then pan-fried.

).

3.6. Effect of Thermal Processing on the Antioxidant Capacity of Broccoli Extracts

The antioxidant capacity of water and 70% ethanol extracts from thermally processed broccoli was determined using three different methods (Table 3). The ABTS and FRAP results showed that the water remaining after boiling broccoli had the highest antioxidant potential (57.57 ± 18.22% and 79.34 ± 3.28%, respectively), while the DPPH results indicated the highest antioxidant potential of air-fried (36.12 ± 15.71%) and steamed (35.48 ± 2.28%) broccoli. The ABTS and FRAP data also showed that steaming and air-frying broccoli had a relatively high antioxidant capacity compared to the other thermal processing methods. Using the same antioxidant assays, other authors reported that thermal processing of broccoli increased its antioxidant potential [46,79]. They found that the antioxidant potential of boiled and steamed broccoli was not significantly different (ABTS and DPPH) or that boiled broccoli even had a higher antioxidant potential than steamed broccoli (FRAP). The discrepancy with our results could be due to the difference in cooking time. In our study, cooking took much longer (30 min compared to 5 and 8 min in their study). This could have resulted in more antioxidants being degraded or leached out. Nandasiri et al. [14] investigated the effect of thermal processing methods on the antioxidant potential of broccoli sprouts. Their results showed that, according to the DPPH method, steamed vegetables had the highest antioxidant potential, followed by stir-fried, while air-fried had the lowest. In contrast, in the FRAP method, air-fried vegetables had the highest antioxidant potential, followed by steamed, while the stir-fried had the lowest. In our study, both the ABTS and FRAP methods showed a significantly higher antioxidant capacity of water in which broccoli was cooked (water remaining after boiling) than the cooked tissue itself. Using the DPPH method, no significant difference was found between the water remaining after boiling and the cooked tissue itself (18.26 ± 12.52% and 27.14 ± 10.43%, respectively). These results are consistent with FRAP being more suitable for hydrophilic antioxidants and DPPH being more suitable for lipophilic antioxidants, whereas ABTS reliably detects both types of antioxidants [79,80]. It is likely that a lot of hydrophilic antioxidants were extracted into water during cooking and were detected by the FRAP and ABTS methods. On the other hand, most lipophilic antioxidants were retained in the tissue and detected by the DPPH method. From these results, it can be concluded that the water remaining after boiling broccoli has a higher antioxidant potential than the cooked tissue. Water and tissue from blanched broccoli showed similar antioxidant potential, with the FRAP method indicating that the tissue had a higher potential than water (35.37 ± 8.86% and 48.33 ± 10.49%, respectively). Blanched broccoli had the same (ABTS and FRAP) or lower (DPPH) antioxidant potential than boiled, suggesting that blanching (a shorter thermal processing) does not preserve broccoli better than cooking. Steaming clearly preserved antioxidant potential of the broccoli tissue, while the remaining water had a very low potential. Therefore, steamed broccoli, the water remaining after cooking, and air-fried broccoli had the highest antioxidant potential. The high antioxidant potential of steamed broccoli and water remaining after cooking could be due to the high content of total phenols and other groups of polyphenols (Table 1), which act as antioxidants. In addition to the high content of polyphenols (Table 1), the high levels of glucosinolates (Figure 2) and carotenoids (Table 2) also contributed to the high antioxidant potential of air-fried broccoli. These results suggest that, from the point of view of antioxidant potential, steaming and air-frying are optimal ways of preparing broccoli for consumption.

3.7. Effect of Thermal Processing on the Potential of Broccoli Extracts to Preserve Plasmid DNA Structure

The main purpose of heat treatment is to ensure microbial safety by eliminating pathogens and to simultaneously alter or improve the health-promoting properties of the food [81]. Thermal processes disrupt the structural integrity of cell walls, thereby promoting the release of bioactive compounds such as polyphenols [82]. Thermal processing resulted in different abilities of broccoli to preserve the plasmid DNA structure (Figure 4). The water remaining after boiling and blanching of broccoli were most effective in preserving the supercoiled DNA form (99.51% and 94.81%, respectively). This suggests that bioactive phytochemicals leached into cooking water by heat processing show high biological activity with relevance to genome protection. Polyphenolics, including flavonoids, are hydrophilic components that are able to leach out from the tissues into the water, which exert a certain level of stability when exposed to high temperatures [57]. Therefore, it is likely that these compounds have contributed to the DNA preservation from oxidative stress. Moreover, it was shown that hydrophilic compounds have markedly higher antioxidant potential than lipophilic [83], which further confirms the best protective effect of the remaining water. Additionally, the DNA-protective activity of the cooking water (BOW and BLW) suggests that the disruption of plant tissue facilitates the release of bioactive compounds into the water, illustrating how structural breakdown influences their availability. This is the first time that the DNA-protecting activity of cooking water, a common cooking byproduct rich in bioactive compounds normally disposed of, is highlighted, emphasizing its possibility for valorization in functional food or nutraceutical purposes. Cooked, blanched, steamed, and pan-fried extracts showed similar levels, among each other, of the preservation of the supercoiled DNA form. Water from steamed broccoli was the least effective in preserving the supercoiled DNA form (32.17%). All extracts, with the exception of the air-fried broccoli and the water remaining after steaming broccoli, were more efficient in protecting DNA from oxidative damage than the standard Trolox at a concentration of 30 mg/mL. The high level of DNA protection of water remaining after boiling broccoli is consistent with its high antioxidant potential (Table 3) and its high content of polyphenols (Table 1). Polyphenols not only act as antioxidants, but they also have the ability to act as chelating agents and form strong complexes with iron [84]. This lowers the concentration of iron ions in the Fenton’s reagent and consequently reduces the concentration of hydroxyl radicals and the extent of oxidative damage. The high level of plasmid DNA protection by water remaining after blanching is probably due to the presence of hydrophilic bioactive components, which were not analyzed in this study. Although the air-fried broccoli exhibited high antioxidant potential, it showed a lower level of preservation of the supercoiled DNA form. This could be due to the high content of endogenous H₂O₂ in the air-fried broccoli (Figure 1). Namely, H₂O₂, if not adequately neutralized, can diffuse into assay systems and participate in Fenton-type reactions, producing hydroxyl radicals that damage DNA. Additionally, antioxidant assays are often non-specific and measure overall electron- or hydrogen-donating capacity, not necessarily protection in a biological context. To the authors’ knowledge, this is the first study to investigate the effect of thermal processing of vegetables on the potential to preserve plasmid DNA structure.

Thermal treatments such as boiling, blanching, steaming, air-frying, and pan-frying, disrupt the plant cell wall matrix to a different level, which reflects on the release, degradation, or transformation of bioactive compounds. The extensive exposure of plant tissues to hot water, combined with prolonged cooking time, contributes to the breakdown of cell walls and the degradation of bioactive compounds, resulting in greater losses of these constituents [85]. Indeed, as shown in Table 1, the share of larger polyphenols and tannins in broccoli was markedly lower in BO, BOW, BL, and BLW samples, suggesting their decomposition to the oligomers and/or monomers. This has also been discussed in the review of Cheng et al. (2006) [86]. In addition, enzymes can be released from the cell wall and denatured by heat [87]. Cellulases are reportedly more stable at high temperatures than other plant cell wall degrading enzymes [88]. The inhibitory activity of plant polyphenols toward gastrointestinal enzymes, such as α-amylase, α-glucosidase, and lipase, can also be regulated by cooking conditions [89]. This is due to the fact that different cooking treatments released different types of polyphenolics, resulting in different inhibitory enzyme activities. During cooking, the concentration of polyphenolics typically decreases because they are polymerized and then oxidized by polyphenol oxidases. These oxidized forms (o-quinones) bind covalently to nitrogen compounds [90]. Due to their stronger molecular bonds, covalent complexes provide superior oxidation resistance, better safeguard polyphenols, and maintain higher thermal stability than non-covalent complexes [91]. Compared to high temperature, steaming inactivates oxidative enzymes, which preserves polyphenolics in vegetables [57].

4. Conclusions

Thermal processing techniques can significantly alter the phytochemical composition and bioactivity of vegetables, thereby affecting their nutritional value and potential health benefits. Among the tested methods, air-frying and steaming proved most effective in preserving or enhancing beneficial compounds, with air-fried broccoli showing the highest levels of glucosinolates, carotenoids, and lycopene, while steamed broccoli was richest in soluble sugars and polyphenolics. The antioxidant capacity varied depending on the assay, with air-fried and steamed broccoli performing best in the DPPH assay, while boiling water residues exhibited the strongest ABTS and FRAP activities. Remarkably, boiling and blanching water residues demonstrated superior protection of plasmid DNA, suggesting that water-soluble antioxidants released during cooking remain bioactive in the cooking medium. This study highlights the importance of choosing appropriate thermal processing techniques to maximize the nutritional and functional qualities of vegetables and is the first to link such processing with plasmid DNA protection. As far as the authors are aware, this is the first study to investigate how different thermal processing methods of vegetables affect their ability to protect the plasmid DNA structure.