Application Of Broccoli Leaf Powder in Gluten-Free Bread: An Innovative Approach To Improve Its Bioactive Potential And Technological Quality

Abstract: In comparison to conventional bread, gluten-free bread (GF) shows many post-baking defects and a lower nutritional and functional value. Although broccoli leaves are perceived as waste products, they are characterized by a high content of nutrients and bioactive compounds. The present study evaluated the nutritional value, technological quality, antioxidant properties, and inhibitory activity against the formation of advanced glycation end-products (AGEs) of GF enriched with broccoli leaf powder (BLP). Compared to the control, gluten-free bread with BLP (GFB) was characterized by a signifificantly (p < 0.05) higher content of nutrients (proteins and minerals), as well as improved specifific volume and bake loss. However, what needs to be emphasized is that BLP signifificantly (p < 0.05) improved the antioxidant potential and anti-AGE activity of GFB. The obtained results indicate that BLP can be successfully used as a component of gluten-free baked products. In conclusion, the newly developed GFB with improved technological and functional properties is an added-value bakery product that could provide health benefits to subjects on a gluten-free diet. 

Keywords: Brassica; vegetable by-product; technological properties; texture parameters; antioxidant activity; anti-ages; gluten-free diet; coeliac disease 

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1. Introduction 

Bread is a staple food that is willingly consumed all over the world every day [1]. However, for some individuals suffering from celiac disease and other gluten-related disorders (wheat allergy and non-celiac gluten sensitivity), the consumption of conventional wheat bread and other gluten-containing products is harmful [2]. In those patients, the dietary gluten proteins or, specifically, the gliadin fraction of wheat and the prolamins from barley (hordeins) and rye (scaling) can lead to deleterious health risks and complications. Nowadays, the only available treatment for gluten-related disorders is adherence to a gluten-free diet. 

Gluten-free breadmaking is a process that varies substantially from conventional breadmaking—in particular, in the ingredients used, batter rheological behavior, and overall quality of the final product [3]. Due to the absence of the continuous three-dimensional gluten network that is responsible for the rheological properties of the dough and the development of high-quality bread, gluten-free breadmaking is challenging [4]. Therefore, the production of gluten-free bread (GF) requires complex formulations, consisting of a mixture of non-gluten basic ingredients and various additives mimicking the viscoelastic properties of gluten [5], as well as diverse technological solutions. In comparison with conventional bread, a GF shows many post-baking defects, such as an unattractive appearance (irregular crust surface and pale color), poor mouthfeel and flavor, and a shorter shelflife. Over the last decade, considerable advances were made to improve the technological and sensory quality of GF and to prolong its shelf-life [6]. However, recently, a growing number of consumers are interested in gluten-free products characterized by improved nutritional and health-promoting quality. 

Numerous studies have shown that the fruits and vegetables-based by-products contain a substantial amount of nutrients (proteins, vitamins, and minerals), as well as functional (dietary fiber) and bioactive compounds (carotenoids, phenolic compounds, and glucosinolates) [7]. Among them, phytochemicals evince important biological activities, such as antioxidant and antimicrobial properties, and thus could play a role in the prevention and treatment of noncommunicable human diseases. The beneficial effects of polyphenols and the glucosinolate derivative on the organism, including the prevention of civilization diseases such as cardiovascular pathologies, type 2 diabetes, some types of cancer, and neurodegenerative diseases, were widely discussed in the literature [8–10]. For that reason, the increasing number of research focuses on the application of by-products in gluten-free products as low-cost sources of nutrients and bioactive compounds [11–13]. Recently, Littardi et al. [14] evaluated the impact of the addition of ground coffee parchment to GF and indicated that this by-product was able to improve the color of this bakery product together with a signifificant enhancement in the antioxidant capacity and oxidative stability. 

The Brassicaceae family includes many vegetables commonly consumed worldwide, not only traditionally for nutrition but, more importantly, for their health-promoting properties [15]. Among them, broccoli (Brassica oleracea var. italica) has acquired considerable relevance in the last few years as a “therapeutic” food, since it contains pharmacologically active substances [16,17]. Many studies have focused on broccoli florets, which represent only 15% of the total aerial biomass [18]. While we were interested in broccoli by-products—in particular, leaves—that are seldom utilized for food. Broccoli leaves, similar to florets, are characterized by a high content of nutrients (proteins, vitamin C, minerals, and trace elements) and bioactive compounds (glucosinolates, phenolic acids, and flavonoids) [19,20]. Although perceived as a waste product, they might be consumed as a valuable fresh product or as sources of phytonutrients, allowing them to obtain added-value baked products [21,22]. Thus, the valorization of broccoli by-products and their application as the ingredient of gluten-free bakery products with potential nutraceutical properties could be one of the alternative strategies to reduce food waste [23,24]. The present study investigated the suitability and functionality of broccoli leaf powder (BLP) as a GF component based on an analysis of the nutritional value, technological quality, antioxidant properties, and inhibitory activity against the formation of advanced glycation end-products (AGEs) of the developed gluten-free bread enriched with BLP (GFB). 

2. Materials and Methods 

2.1. Preparation of Broccoli Leaf Powder

A BLP was prepared as described previously [24]. Brieflfly, undamaged leaves of mature broccoli (Brassica oleracea L. var. italica) donated by the company GEMIX (Olsztyn, Poland) were cleaned of soil residues, washed with water, then blanched shortly (1 min) in hot water to inactivate enzymes and decrease the microbial load. Afterward, petioles and main midribs were removed, and leaf blades were freeze-dried since it is a method that preserves the nutritional and biological value and the color of the raw material [25]. Dry leaves were ground and sieved to obtain homogenous powder (particle size ≤ 0.60 mm). The obtained BLP was packed in a sealed plastic box and kept in a refrigerator for further analysis and application in experimental GF formulation. 

2.2. Preparation of Experimental Gluten-Free Bread

In this study, an optimized GF formula [26] was used as a control (GFC). Corn starch (HORTIMEX, Konin, Poland), potato starch (PPZ “Trzemeszno” Sp. Z o.o., Trzemeszno, Poland), sugar, fresh yeast (Lesaffre Polska S.A., Wołczyn, Poland), pectin (E 440(i), ZPOW Pektowin, Jasło, Poland), rapeseed oil “Kujawski” (ZT “Kruszwica” S.A., Kruszwica Poland), salt, and water were the main ingredients of GFC (Table 1). Previously characterized BLP [24] was incorporated into the GFB by replacing 5% (w/w) of corn starch in the GFC formula. This level of substitution was based on a preliminary study that showed that 5% was the acceptable replacement level that did not affect the sensory properties of bread, whereas the GFB with 7% BLP had too intense a cabbage flavor (data not shown). 

2.3. Characteristics of Experimental Gluten-Free Breads

2.3.1. Determination of Proximal Chemical Composition and Energy Value 

The basic chemical composition was determined in freeze-dried GFs according to the standard method [27]: moisture content was analyzed using the drying method (AOAC 925.10), proteins content was determined with the Kjeldahl method (N × 6.25 for nitrogen to protein conversion) (AOAC 979.09), and fat content using Soxhlet extraction with hexane (AOAC 923.03); total ash was determined using the gravimetric method by burning in a muffle furnace at 550 ◦C for 10 h (AOAC 923.03). The total carbohydrate content was calculated by subtracting the values of the moisture, protein, fat, and ash content from 100. The energy values (kJ) were calculated by multiplying the number of macronutrients by the corresponding conversion factors (17 kJ/g for protein, 37 kJ/g for fat, and 17 kJ/g for carbohydrates) [28]. The conversion factor for calorie calculation is 1 kJ = 0.239 kcal. 

2.3.2. Determination of Physical Parameters

The weight of GFs was evaluated using a digital balance with 0.01-g accuracy. The loaf volume was determined using a modified standard rapeseed displacement method, in which millet seeds were used instead of rapeseed. The specifific volume (SV) was calculated as a loaf volume divided by its weight. Density (D) was calculated as a loaf weight divided by its volume. Bake loss was calculated as indicated in Equation (1). 

where: 

a—the initial weight of batter before baking (g), and 

b—the weight of baked and cooled GFs (g). 

The crust and crumb color of GFs was evaluated using a HunterLab ColorFlex (Hunter Associates Laboratory, Inc, Reston, VA, USA). Crust color was determined at the middle point of the top of the loaf crust, while crumb color was analyzed at the middle point of the central 2-cm slice. The measurements were performed through a 3-cm diameter diaphragm containing an optical glass. The color was expressed in accordance with the CIELab system, and the parameters determined were: lightness (L* = 0 (black) and L* = 100 (white) and chromatic components: a* (−a* = greenness and +a* = redness) and b* (−b* = blueness and +b* = yellowness). Values were the mean of at least nine replicates.

To present the appearance of crumb and crust of exemplary GFC and GFB scans of the example central slice of each experimental, GF was made using a flatbed scanner (Epson Perfection V200 Photo) supported by Epson Creativity Suite Software Images (Figure 1). 

Figure 1. The visual appearance of crumb and crust of exemplary control gluten-free bread (A,C) and gluten-free bread with broccoli leaves powder (B,D). 

2.3.3. Evaluation of Textural Properties

The texture profile (TPA test) of fresh (2 h) and stored (for 24 and 72 h after baking)crumbs of GFs were analyzed using a TA.HD Plus Texture Analyser (Stable Micro Systems Ltd., Godalming, UK) is equipped with a 30-kg load cell. The middle bread slices of 25-mm thickness underwent a double compression cycle up to 40% deformation of their original height with a 35-mm flat-end aluminum compression disc (probe P/35). The selected settings were as follows: pre-test/test/post-test speed, 2.0 mm/s, relaxation time, 5 s, force, 10 g, and trigger, mode auto. Each slice was compressed twice to give a two-bite texture profile curve [29], from which the following textural parameters were obtained: hardness, springiness, chewiness, cohesiveness, and resilience, as calculated by the software of the tetrameter. Six replicates were analyzed for each kind of fresh and stored GF. 

2.4. Evaluation of the Antioxidant Capacity of BLP and GFs 

2.4.1. Determination of Total Phenolic Content 

The total phenolic content (TPC) was determined with the use of the Folin–Ciocalteu reagent based on the method described previously by Horszwald and Andlauer [30]. Methanol extracts were obtained from 200 mg of freeze-dried GF and 100 mg of BLP with 1 mL of 67% methanol. Samples were subjected to ultrasonic vibration (30 s) and vortexing (30 s), then were centrifuged for 10 min at 13,000 rpm at 4 ◦C. The above step was repeated fifive times, and the supernatants were collected into a 5-mL measuring flflask. Methanol extracts were prepared in triplicate. The TPC assay was performed in microplates, and aliquots of 15 µL of methanol extracts were placed in microplate wells. Subsequently, 250 µL of the Folin–Ciocalteu reagent (previously diluted with water 1:15, v/v) was added, and the mixture was incubated for 10 min in dark at room temperature. Then, 25 µL of 20% sodium carbonate was added to each well, and the mixture was incubated for 20 min. The microplate was shaken automatically before reading, and absorbance was measured at λ = 755 nm with the Infifinite M1000 PRO plate reader (Tecan Group AG, Männedorf, Switzerland). Gallic acid was used for standard calibration (0.03–1.0 mg L−1 ), and the results were expressed in mg of gallic acid equivalents (GAE) per one gram of dry matter (g DM) of GFs or BLP. 

2.4.2. Trolox Equivalent Antioxidant Capacity by ABTS Assay 

The Trolox Equivalent Antioxidant Capacity (TEAC) by the 2,20 -casino-bis (3-ethylbenzenethiazoline-6-sulfonic acid (ABTS) assay was performed as described by Horszwald and Andlauer [30]. To obtain an ABTS radical cation (ABTS· + ) solution with an absorbance value of 0.70 ± 0.02 at 734 nm, 10 mL of 7-mmoL/L aqueous solution of ABTS and 0.5 mL of 51.4-mmoL/L−1 aqueous solution of K2S2O4 were mixed, then stored in the dark at room temperature for 16 h. Next, the ABTS· + solution (1480 µL) was added to 20 µL of methanol extracts of BLP and GF. For the analysis in the microplates, aliquots of 10 µL of the sample (the methanol extracts of BLP or GF prepared as described above for the TPC assay), standards, or blanks were placed in microplate wells. The reaction and time measurements were started upon the addition of 270 µL of the ABTS· + solution. The reaction was carried out at 30 ◦C in dark for 6 min. After the reaction, the absorbance was measured at 734 nm with a microplate reader. Trolox was used for standard calibrations (0.25–1000 µmol/L−1 ), and the results were expressed in µmol Trolox g−1 DM of GFs or BLP.

2.4.3. Trolox Equivalent Antioxidant Capacity by DPPH Assay

The TEAC by 2-diphenyl-picryl-hydroxyl (DPPH) radical scavenging assay was performed according to Horszwald and Andlauer [30]. To obtain the DPPH solution absorbing in the range from 0.95 to 1.10 at λ = 517 nm, 10 mg of DPPH was dissolved in 250 mL of 80% methanol. The DPPH solution was freshly prepared before analysis. For analysis, 20 µL of methanol extracts of BLP and GF (described in Section 2.4.1), blanks or standards were placed into microplate wells, and then, 300 µL of DPPH· solution was added. The reaction was performed at ambient temperature for 30 min in the dark. Trolox was used for standard calibration (0.005–0.75 mM), and the results obtained were expressed as µmol Trolox Equivalents (TE) per g DM of GFs or BLP. 

2.4.4. Photochemiluminescence Assay

A photo chemiluminescence (PCL) assay was performed as described by Zieli ´nski, Zieli ´nska, and Kostyra [31]. This method was used to measure the antioxidant capacity of BLP and freeze-dried GF extracts against superoxide anion radicals generated from the luminol photosensitizer under exposure to UV light in the Photochem apparatus (Analytik Jena, Leipzig, Germany). Antioxidant activity was analyzed with ACW (hydrophilic condition) and ACL (lipophilic condition) kits according to the manufacturer’s protocols. For ACW, a 50-mg sample was extracted with 1 mL of water, and for ACL—a 50-mg sample was extracted with 1 mL of the MeOH and hexane mixture (4:1; v/v). The concentration of the extract solution was adjusted to ensure that the generated luminescence was within the range of the standard curve. Antioxidant capacity was calculated by comparing the delay time of the sample with the Trolox standard curve, and it was expressed in µmol Trolox g−1 DM. 

2.5. Evaluation of Inhibiting Activity Against AGEs 

The inhibiting activity against advanced glycation end-products (AGEs) was assessed using two in vitro model systems: bovine serum albumin (BSA)-glucose and BSA-methylglyoxal (MGO). The extraction and incubation procedures were adopted from Szawara-Nowak et al. [32]. Briefly, 150 mg of freeze-dried sample was extracted with 67% methanol by shaking at 25 ◦C for 40 min using a thermomixer (Thermomixer, Eppendorf, Poland). The supernatant obtained after the centrifugation was evaporated to dryness under nitrogen, and the dry residue was dissolved in phosphate buffer (0.1 M, pH 7.4). 0.5 mL of the obtained solution was incubated with 1 mL of the mixture containing BSA (10 mg/mL) and sodium azide (0.1 mg/mL) in phosphate buffer (0.1 M, pH 7.4) and appropriately D-glucose or MGO. For the measurement, 250 µl of the reaction mixture was placed into wells (microplate 96-wells, black, Porvair). The fluorescent intensity of λexcitation 330 nm and λemission 410 nm (BSA-glucose), and λexcitation 340 nm and λemission 420 nm (BSA-MGO) were measured. For each extract, the test was run in triplicate. A 1 mM of aminoguanidine was used as a positive control. The results were presented as a percentage of AGEs inhibitory activity. 2.6. Statistical Analysis Unless otherwise stated, the data reported in all the tables are mean values and standard deviations of triplicate observations. Generally, the differences between experimental GFs were analyzed with an unpaired t-test with Weich’s correction (p < 0.05), except for the differences between GFs caused by storage time that was analyzed with the one-way ANOVA, using GraphPad Prism version 8.0.0 for Windows, GraphPad Software (San Diego, CA, USA).