*3.1. Antioxidant Properties of Biscuits' Ingredients*

A low-cost and simple DPPH assay based on spectrophotometric mixed-mode (having both electron transfer (ET) and hydrogen atom transfer (HAT) mechanisms) was proposed as the most suitable method to determine the AC of ingredients used in the preparation of functional biscuits. This assay was chosen due to the fact that DPPH radical is known to work well with lipophilic (rather than hydrophilic) antioxidants in alcohol solvents [22]. The AC of RPC, WF, rapeseed oil, margarine, and coconut oil was determined by the modified DPPH method after conventional extraction and UAE, and the obtained results are listed in Table 1.


**Table 1.** Antioxidant capacity of biscuits' ingredients.

\* n = 5; SD—standard deviation; Different letters within the same column (a–d) indicate significant differences between DPPH results of biscuits' ingredients. Different letters (x,y) within the same row indicate significant differences between DPPH of each ingredient extract prepared by the conventional extraction (CE) and the ultrasound-assisted extraction (UAE) (one-way ANOVA and Duncan test, *p* < 0.05).

It is noteworthy that the DPPH values of biscuits' ingredients differ significantly from each other (Duncan test). Moreover, DPPH results of WF, margarine, and coconut oil extracts prepared using the classical extraction method over 30 min were significantly lower than those obtained by the UAE for 5 min (Table 1). However, the Duncan test indicated that methanolic extracts from RPC and rapeseed oil after classical extraction, and sonication did not differ significantly in DPPH results.

The UAE permits higher extraction yields in a shorter time, thereby reducing the electrical energy input. An increase in the AC can be explained by the action of the cavitation bubbles generated during the propagation of the acoustic waves. The cavitation bubbles can disrupt the material cell wall, causing solvent diffusion and increasing the release of antioxidant compounds. On the other hand, ultra-sonication contributes to reducing the particle size of the raw material, which increases the surface area. Moreover, the ultrasound waves generate shear force during ultra-sonication thus, the mass transfer of the original material into an extract solution is enhanced [23].

It is evident that the RPC incorporated into the biscuits was the richest source of antioxidants. The methanolic extracts of RPC revealed the highest DPPH values (15,358 and 15,630 μmol TE/100 g for conventional extraction and UAE, respectively). In our previous report the DPPH ranged between 8770 and 33,980 μmol/100 g for ethanolic, methanolic, and aqueous RPC extracts [24]. These differences between the DPPH results for RPC samples can be explained by the influences of genetic, agronomic, environmental, and technological factors, as well as conditions of extract preparation, mainly polarity of the used solvent, which affect the total level of antioxidants.

However, among the evaluated fats, rapeseed oil had the highest DPPH results (424 and 456 μmol TE/100 g after conventional extraction and UAE, respectively), whereas the antioxidant potential of coconut oil determined by the same method was the lowest (87 and 118 μmol TE/100 g after conventional extraction and UAE, respectively) (Table 1).

Also, Casoni et al. [25] observed significantly higher radical scavenging capacity of rapeseed oil (RSC = 35.12%) to scavenge DPPH radical than coconut oils (RSC = 0.46–2.26%). This suggests that rapeseed oil is a rich source of antioxidants, mainly tocopherols (78.51 mg/100 g) and phenolic compounds (5.77 mg/100 g), while coconut oil contains a trace amount of tocopherols (2.90 mg/100 g) and phenolics (less than 0.01 mg/100 g) [26].

Nevertheless, margarine revealed approximately twofold lower DPPH results (171–217 μmol TE/100 g) in comparison with the refined rapeseed oil (DPPH = 424–456 μmol TE/100 g). Although, somewhat lower AC results (126–149 μmol TE/100 g) were determined for WF.

For comparison, WFs used for the preparation of functional shortbread cookies with fruit pomace, and biscuits incorporated with prickly pear peel, revealed significantly higher DPPH values (377–860,000 μmol TE/100 g) [6,14].

### *3.2. Antioxidant Capacity of Biscuits with Rapeseed Press Cake*

The different fats such as rapeseed oil, margarine, and coconut oil with the declared amounts of SAFA at 2.3, 7.8, and 24.9 g, respectively, and increasing RPC powder levels from 0 to 40 g, were used for the preparation of functional biscuits. It can be noted that as the concentration of RPC in the baked products increased, the biscuits became darker in color (Figure 1).

The biscuits fortified with RPC were found to be darker than the control sample without RPC flour. This suggests that RPC flour is a rich source of chlorophyll. Moreover, the darkening of biscuits enriched with RPC can be due to the browning of RPC carbohydrates during baking.

For comparison, with increasing concentrations of by-products, such as olive stone powder (5–15%), white grape pomace (10–30%), goji berry by-product (10–40%), defatted chia flour (5–20%), okara powder (20–40%), prickly pear peel (10–30%), defatted sunflower seed flour (18–36%) in doughs, the color of the enriched biscuits and cookies changed gradually from light brown to brown and finally to dark brown [1,8,10,12–15].

Furthermore, the DPPH values of the baked biscuits increased rapidly with the increasing content of the added RPC (Table 2).

Therefore, the highest DPPH results (8375–10,088 μmol TE/100 g and 4761–7395 μmol TE/100 g) were revealed for methanolic extracts of biscuits with the highest RPC level (40 g) obtained after classical extraction and UAE, respectively. However, the DPPH (535–843 μmol TE/100 g and 598–890 μmol TE/100 g) were the lowest for the studied samples without RPC.

The increase in DPPH values from 20.12 to 30.44%, 3.39–7.55 mmol TE/g, 198–236 mmol TE/g, and 1.5–2.9 mg TE/g was also observed when the biscuit doughs were fortified with increasing amounts of olive stone powder (5–15%), white grape pomace (10–30%), prickly pear peel (10–30%), and defatted sunflower seed flour (18–36%), respectively [1,8,14,15].


**Table 2.** Antioxidant capacity of the studied biscuits.

\* n = 5; SD—standard deviation; Exp.—experimental data; Pred.—predicted values; different letters within the same column (a–g) indicate significant differences between the DPPH of the studied biscuits; different letters (x,y) within the same row indicate significant differences between DPPH of biscuits extracts prepared by the conventional extraction (CE) and the ultrasound-assisted extraction (UAE) (one-way ANOVA and Duncan test, *p* < 0.05).

> Interestingly, biscuits prepared by replacing coconut oil (lower DPPH = 87–118 μmol TE/100 g) with margarine (higher DPPH = 171–217 μmol TE/100 g) and the same content of RPC had a lower level of antioxidants capable of scavenging the free DPPH radical (Tables 1 and 2). Thus, replacement of fat with a richer in SAFA insignificantly increased the DPPH activity of the baked biscuits (Table 2, Duncan test). This can be explained by the fact that SAFA are strongly resistant to deterioration reactions during thermal oxidation, while antioxidants present in the dough are not consumed for the termination of oxidative processes by trapping free radicals.

> Unexpectedly, the DPPH results of fortified biscuits' extracts decreased significantly after ultrasonic treatment (Table 2, Duncan test). A decrease in DPPH values for extracts obtained by UAE indicates that natural antioxidants present in RPC were degraded by free radicals generated upon the ultrasound irradiation [27]. On the other hand, the thickness of the boundary layer (the fortified biscuits–solvent interface) did not sufficiently decrease during mixing by means of ultrasound due to the ultrasonic power dissipation (180 W) used for too short treatment time (5 min × 3 times), and the transfer of antioxidants was limited.

#### *3.3. Hedonic Scale Sensory Evaluation of Biscuits with Rapeseed Press Cake*

It can be noted that there were significant differences in consumer acceptability between 11 biscuit samples containing 0, 20 and 40 g of RPC flour, respectively (Table 3, Duncan test).

Therefore, the samples studied can be divided into three groups according to the mean sensory scores for overall acceptability. The baked biscuits with the highest amount of RPC flour (40 g) had the lowest sensory score values for overall acceptance. The most preferred samples by Polish consumers were biscuits prepared without RPC (Table 3).



acceptabilityratingsystem,anchoringpoint,extremely"and 9—"liked extremely"; ## Purchase intent is based on the 5 scoring scale with anchoring point, 1—"certainly would not buy", 5—"certainly would buy"; Exp.—experimental data; Pred.—predicted values; different letters within the same column indicate significant differences between the studied biscuits' sensory characteristics (one-way ANOVA and Duncan test, *p* < 0.05).

The consumers' results revealed that the mean sensory scores for color, odor, texture, flavor, and overall acceptability of the non-supplemented biscuits prepared by using different types of fats ranged between 5.81 and 6.10, 5.73–6.10, 5.52–6.88, 5.85–6.89, and 6.07–6.79, respectively, and corresponded to the classifications "liked slightly" and "liked moderately" on the hedonic scale where the maximum score is 9. In the context of the proposed products' purchase intent frequency, the biscuits without RPC received the highest scores (3.19–3.33) on a 5-point scale and qualification as "would buy".

On the contrary, with increasing RPC content in the dough, the fortified biscuits' color changed gradually from light (biscuits without RPC) to dark for biscuits with the highest amount of RPC flour (40 g). Therefore, these samples scored the lowest for color (4.45–4.62), odor (3.45–3.93), flavor (3.03–3.52), and overall acceptability (2.89–3.44). The biscuits with the highest RPC level were characterized by a typical intensity of bitter and grassy flavor due to the high concentration of phenolic compounds present in RPC.

Nevertheless, insignificant differences in the mean texture scores were found between control samples (without RPC) and biscuits after the replacement of WF with 20 g of RPC flour (Table 3, Duncan test). However, biscuits incorporated with the highest RPC amount (40 g) scored the lowest for texture (4.93–5.33). For this reason, biscuits formulated with 40 g of RPC flour had the lowest purchase intent scores (1.78–1.90), being largely qualified with "would not buy". The biscuits enriched with 20 g of RPC flour were predominantly qualified as "would probably buy", but there were significant differences with the qualification "would buy" for the control samples without RPC (Table 3, Duncan test).

The Duncan test indicated insignificant differences in scores for color, odor, flavor, overall acceptability, and purchase intent between biscuits obtained from different types of fats with the same content of RPC (Table 3). Only biscuits without RPC and with 20 g of RPC flour prepared by using margarine had significantly lower texture scores than those baked from dough containing rapeseed and coconut oils.

The obtained results suggest that the replacement of WF with varying RPC levels in the recipe affects the overall acceptability of the functional biscuits due to atypical attributes such as bitter and grassy flavor and strange odor. A new component such as chocolate could probably mask the negative attributes and increase the overall acceptability of biscuits fortified with RPC.

#### *3.4. Optimization Process*
