*3.3. Proximate Composition for Biscuits with RFSF*

The proximate composition of the biscuits with RFSF (B0, B10, B25, B40) is summarized in Table 4 Incorporation of the RFSF in biscuits has a significant influence on the major components (*p* < 0.05) and a decrease of the moisture content was observed while fat, ash, protein and fiber content of composite flour mixes increased. RFSF had a significant (*p* < 0.05) effect on the moisture content of the biscuits compared to the control with a decrease in moisture content from 7.34% to 4.92%. Similar patterns were previously noticed by Khouryieh and Aramouni [55] and Kaur et al. [26] who found that as the flaxseed flour concentration increased in the blend, the moisture content of cookies decreased. These results may be due to low moisture content (1.64%) of RFSF. Moreover, a high amount of fat (42.50%) was recorded in RFSF, which also contributed to the hydration of the dough, thus reducing the moisture of the biscuits. Furthermore, the low moisture content of the biscuits allows a longer preservation period, due to minimal microbial or chemical activity.

**Table 4.** Proximate characteristics of the biscuits samples.


\* Values represent mean of three independent determinations ± SD; fw-fresh weight; Mean values followed by the same superscript alphabet in the row are not significantly different at *p* < 0.05 according to Duncan comparison test.

Ash content increased from 1.23% (control biscuits) to 2.60% (sample B40). This might be due to the higher mineral content of flaxseed flour [56,57]. Also, the protein content increased significantly (*p* < 0.05) from 8.35 g/100 g in B0 to 10.77 g/100 g in B40 and fat from 15.19 g/100 g in B0 to 28.34 g/100 g in B40. Moreover, the addition of 40% RFSF registered a positive influence on the fibre content of the final baked biscuits, increasing its value about 6.7-fold compared to the control sample. Improving the nutritional value of pastry products by substituting wheat flour with other fiber-rich sources was also recommended by others [58,59]. This increase could be explained by the fact that roasted flaxseed flour is far higher in fat, protein and crude fiber content compared with wheat flour as mentioned in the proximate analysis section of this article.

The total carbohydrate content was significantly decreased (*p* < 0.05) in biscuits substituted with flaxseed from 61.76% to 48.32% compared to control biscuits (67.14%). These results are similar with the results obtained by Ahmed et al. [60] El-Demery et al. [61] mentioned that as the level of substitution with flaxseed flour increased, all compounds increased except total carbohydrate. Other authors also revealed that the substitution of wheat flour with flaxseed flour resulted in a considerable improvement in protein, crude fiber and ash of biscuit samples [12,16,24,26,50,59,62].

TPC and DPPH of biscuit samples increased with the substitution level of RFSF to WF in the blends. The highest increase in DPPH activity and TPC was exhibited by sample B40 (32.03% RSA and 78.82 mg GAE/100 g) and the lowest (13.67% RSA and 63.06 mg GAE/100 g) was shown by the control sample. Similar results have been obtained by Kaur, et al. [26,33]. This increase can be explained by the fact that flaxseed possesses a very powerful antioxidant system, being particularly rich in lignans, e.g., secoisolariciresinol diglucoside (SDG), which are also present in flaxseed oil. [63]. Besides

lignans, flaxseed contains high amounts of phenolic compounds, such as ferulic acid, syringic acid, cinnamic acid, vanillic acid, *p*-coumaric acid and gallic acid [64]. However, although flaxseed enriches the material basis for exerting its antioxidant activity, previous studies have observed differences in the quality characteristics of flaxseed varieties from different regions of the world, indicating some geographical and varietal specificity [64]. Deng et al. [64] reported that the total phenolic contents in studied flaxseed varieties ranged from 109.93 mg GAE/100 g to 246.88 mg GAE/100 g, and the DPPH values ranged from 32.56 mg TE/100 g to 46.22 mg TE/100 g. Even if the roasting process of flaxseeds could produce a slight decrease in the antioxidant activity, the baking process resulted in a significant increase in antioxidative activity due to the Maillard pigments recognized as having a high antioxidative capacity [59].

#### *3.4. Mineral Content of Biscuits*

The results of the evaluation of the mineral content of biscuits are displayed in Table 5. The addition of RFSF increased the P, K, Zn, Fe, Ca and Mg contents, meanwhile Cu and Mn could not be identified in the biscuit samples.


**Table 5.** The concentration of macro and microelements in biscuit samples.

\* Values represent mean of three independent determinations ± SD; fw-fresh weight; Mean values followed by the same superscript alphabet in the row are not significantly different at *p* < 0.05 according to Duncan comparison test. N.D.—not determined.

The significant increase of minerals (*p* < 0.05) in biscuits could be justified by the rich minerals content of RFSF. A large body of literature highlighted that flaxseeds are a valuable source of minerals. For instance, Bernacchia et al. [65] showed that P, K, Ca, Mg flaxseed content reached values of 622, 831, 236 and 431 mg/100 g, respectively. Furthermore, Kaur et al. [57] mentioned that flaxseed is rich in Mg which is the second most abundant element in human body, as well as in K, which is the most common macro-mineral with positive effect in reduction of stroke incidence and blood platelets aggregation.

It is important to note that mineral content could vary between flaxseed cultivars and could be influenced by external factors such as soil conditions, fertilizers, water availability, climatic conditions and genetic factors [57].

#### *3.5. Analysis of Volatile Compounds of Biscuit Samples*

A total number of 21 volatile compounds were identified in the RFSF enriched biscuits, by means of ITEX/GC-MS technique as shown in Table 6. In all biscuit samples, the main volatile compound from the aldehydes group was hexanal, ranging from 14.76% to 18.39%, meanwhile β-myrcene and D-limonene were the main volatile compounds from terpenes and terpenoids group, ranging from 8.99% to 15.65% and 2.47% to 8.48%, respectively. Acetophenone was the major volatile compound from the ketones group with values between 2.50% to 10.98%, meanwhile, 4-methyloctane reached a final value of 6.92% in the B40 sample. The presence of 4-methyloctane could be justified by the addition of vanilla essence during the biscuit manufacturing [66]. The amount of D-limonene in RFSF increased linearly with increasing levels of RFSF in the final baked products. D-Limonene is responsible for odour perceptions like citrus and mint. Furthermore, flaxseed represent

a high source of carotenoids [67] which could be correlated with higher amounts of Dlimonene, as reported by Chi¸s et al. [68]. From the terpenes and terpenoids group apart from D-limonene, β-myrcene enhanced the final odour perception through its balsamic, musty and spice perceptions.


**Table 6.** Volatile compounds content of the biscuits with roasted flaxseed flour.

\* Each value was the mean of triplicate measurements; N.D.—not detected. \*\* drawn from [46,47]. Note: a–d different superscripts in a row indicate significant difference within samples (*p* < 0.05) according to Duncan comparison test.

> Acetophenone, from the ketone group, was previously pinpointed as a volatile compound with implication in the overall flavor of flour products which could be formed during Maillard reactions [69]. Its odour perception is pleasant, having musty, flowery and almond characteristics.

> The increased amount of heptan-2-one from the ketone group with increasing yield of RFSF could be explained by the chemical composition of flaxseed, rich in tocopherol and ascorbic acid [65]. In this line, Starowicz et al. [70] showed that tocopherol and ascorbic acid significantly increased the peak area of heptan-2-one.

> Furthermore, a recent study of Hidalgo and Zamora [71] highlighted that benzaldehyde could be formed through chemical reactions such as lipid oxidation. The reaction involved in the first step is a carbonyl-amine reaction followed by a free radical amino-acid degradation, mainly phenylalanine. It was previously shown that flaxseed is a rich source of 6 essential amino-acids, from which phenylalanine amount could vary in the range from 1.44 mg/100 g up to 66.6 mg/100 g, depending on the flaxseed cultivars [57]. Therefore, the

significant differences (*p* < 0.05) between benzaldehyde sample amounts, could be justified by the chemical composition of flaxseed and by lipid oxidation.

It is worth nothing that non-enzymatic Maillard reactions such as the Strecker degradation process could lead to the formation of aldehydes as a result of the reaction of aminoacids with dehydroreductones [54].

Overall, the increased RFSF percentages during biscuits manufacturing, led to significant differences (*p* < 0.05) between aroma volatile compounds, mainly due to the rich chemical composition in lipids, protein, aminoacids, phenols and chemical reactions such as Maillard and lipid oxidation.

#### *3.6. Physical Characteristics of the Biscuits*

The evaluation results of the physical characteristics of the biscuits indicate that there was a significant difference (*p* < 0.05) between the control sample and B40 in thickness, diameter and spread factor (Table 7). However, there was no significant difference in the weight of any type of biscuit although it was lower than the control biscuits.


**Table 7.** Physical characteristics of biscuits.

\* Values represent mean of three independent determinations ± SD. Mean values followed by the same superscript alphabet in the column are not significantly different at *p* < 0.05 according to Duncan comparison test.

The diameter and thickness of biscuit samples (B10, B25 and B40) increased slightly with increasing substitution percentage of RFSF compared with control biscuit (B0). Sample B40 presented the maximum diameter and thickness (6.26 and 0.89 cm). The spread factor is the ratio that depends on the values of the thickness and diameter of the cookies and it is used to determine the quality of flour for producing cookies [26]. The results of the spread ratio of biscuits revealed a significant reduction (*p* < 0.05) in spread ratio from 7.90 to 7.03 cm for B40. With the increase in the concentration of RFSF, the spread factor of biscuits gradually decreased. These results are in the line with the findings of other authors [16,32] who stated that proteins and dietary fibers have more water-binding power. Ganorkar and Jain [16], argue that the presence of more water in the dough, leads to higher dissolution of sugar during mixing and this lowers the initial dough viscosity and the cookie is able to spread at a faster rate during cooking. Moreover, in the opinion of the same authors, an inverse correlation is obtained when the flour components absorb large quantities of water and, as a consequence, reduce the amount of water that is available to dissolve the sugars in the formula. Therefore, the initial viscosity is higher and the biscuits spread less during baking.

#### *3.7. Instrumental Analysis of Texture*

The results of the instrumental analysis of texture are presented in Table 8. Instrumental analysis of hardness showed statistically significant differences (*p* < 0.05) between control (B0) and B40. Hardness decreased as the amount of RFSF increased, mean values of 10,655 g (control sample) and 5714 g (B40), respectively. Similar patterns were noticed for the load required to reach the hardness work value. The values of hardness work (mJ) decreased with increasing RFSF from 262.73 mJ to 122.30 mJ. These results are similar to those obtained by Ganorkar and Jain [16] and Omran et al. [32] who reported that the textural parameters were found to decrease with increasing flaxseed flour incorporation. This might be due to an increase in dietary fiber and protein, high water absorbing capacity components as well as due to the high level of fat (42.50%) found in RFSF. These factors

contributed to a sticky dough which reduced the extensibility of dough. Previous studies claim that with an increase in fat content, the gluten network gets interrupted thus the physical properties of biscuits are changed and become less hard. At very high fat content the lubricating function is high and a soft texture is obtained. Hence the hardness gradually decreases forming softer biscuits with an increased level of flaxseed flour [16,32,72].

**Table 8.** Instrumental analysis of texture.


Values represent mean of three independent determinations ± SD. Mean values followed by the same superscript alphabet in the row are not significantly different at *p* < 0.05 according to Duncan comparison test.

#### *3.8. Sensory Analysis*

Before a product becomes commercially available, many tests are conducted with consumers to evaluate the product acceptability. Nowadays, consumers are more health conscious then before and functional foods have an ascending trend on the market. This context brings new challenges upon sensory analysis, transforming it into a more proactive role in producing unique, innovative and functional products. Austria et al. [73] hypothesised that foods that contain significant quantities of flaxseed will be well tolerated by the public.

The mean of hedonic scores and their standard deviation are presented in Table 9. The control sample scored the highest for appearance, hardness, chewiness and aftertaste.


**Table 9.** Sensory evaluation of the biscuits.

Values represent hedonic scores calculated as mean ± SD (*n* = 52). Mean values followed by the same superscript alphabet in the column are not significantly different at *p* < 0.05 according to Duncan comparison test.

> For the attribute of appearance no significant difference was encountered between the control sample and the B10. However, all tested samples which contained flaxseed flour (B10, B25 and B40) scored progressively lower than the control (B0). This can be explained by the fact that evaluators were able to visually discriminate the samples solely based on their color and degree of irregularities which could be seen on the surface of the biscuit. The decrease of the hedonic appearance values may be due to the progressive decrease amount of moisture and total carbohydrates in the biscuit samples (B0 > B10 > B25 > B40 as can be seen in Table 4) which caused the depreciation of biscuits' surface and darker color during baking. There was a positive correlation between appearance and moisture (0.97, *p* < 0.05), and between appearance and total carbohydrates (0.99, *p* < 0.05). Moreover, the appearance negatively correlated with ash (−0.99, *p* < 0.05), fat (−0.99, *p* < 0.05) and crude fiber (−0.98, *p* < 0.05). The decrease of appearance perception with the increase of the flaxseed content was also recorded previously in studies performed on biscuits [29,32,74] as well as on other bakery products, like cookies [19,75] muffins [76,77] and bread [13].

> The texture plays a key role in assessing consumers' acceptance of a food product. The complex changes of the texture during the process of eating (mouth behaviour) can influence the acceptance or rejection of the product. Although most of the times it is

unconscious, the consumer's preference toward a product is often due to the texture. The biscuits were evaluated by three different textural attributes: hardness, crispiness and chewiness. Each attribute was clearly explained to the participants and a brief description of them was inserted in the evaluation sheet next to the hedonic scale as stated in the method section. The participants were instructed to evaluate the crispiness as the force and noise created when the sample breaks during chewing the sample between the molar teeth. It was noted that the biscuits with higher amount of RFSF presented lower amount of moisture (Table 4). Negative correlation (*p* < 0.05) was found between the consumer's perception of crispiness and the amount of moisture and total carbohydrates of the biscuits (−0.85 and −0.82, respectively). Similar findings were published by Hussain et al. [19] and Marpalle et al. [13]. This result can be explained by flaxseed's capacity to bind water, which influences the textural properties of the final product [76].

Taste and aroma improved with the increasing amount of the flaxseed in the biscuits. The flaxseed has a unique nutty flavour (Tables 3 and 7) which was found pleasant by the consumers, therefore the taste and aroma characteristics of samples with RFSF scored higher than the control (B0 > B10 > B25 > B40). Moreover, the taste and aroma of the samples was positively correlated with the biscuits' content in protein (0.99, *p* < 0.005), ash (0.99, *p* < 0.005), fat (0.99, *p* < 0.005) and crude fiber (0.99, *p* < 0.001) and negatively correlated with moisture (−0.99, *p* < 0.005) and total carbohydrates (−0.99, *p* < 0.01). Some authors mention a bitter taste in samples prepared with high amounts of flaxseed flour [78]. However, in this study, the nutty flavour was appreciated by the consumers which are accustomed to the taste and flavor of different types of traditional biscuits and cookies prepared with various amounts of nuts.

The aftertaste correlated negatively with the biscuit's content in protein (−0.97, *p* < 0.05), ash (−0.95, *p* < 0.05), fat (−0.96, *p* < 0.05) and crude fiber (−0.95, *p* < 0.05) and correlated positively with moisture (0.97, *p* < 0.05) and total carbohydrates (0.96, *p* < 0.05).

Although the sensory attributes of the samples with RFSF were decreased compared to the control, the aroma and crispiness increased and the significant differences between samples in appearance, hardness, chewiness and aftertaste did not reflect on the overall appreciation of the biscuits.

#### **4. Conclusions**

The results of this study reveal the effect of roasted flaxseed flour addition on the physico-chemical parameters, volatile profile, and sensory acceptability of biscuits. The high incorporation of RFSF advantageously influenced the nutritional properties of biscuits as evidenced by significant increases in the fibre contents (about 6.7-fold higher than in control biscuits), proteins and minerals. Furthermore, the addition of RFSF increased the total phenolic content and radical scavenging activity amount, reaching final values for biscuits manufactured with 40% RFSF such as 78.82 mg GAE/100 g and 32.03%, respectively. With respect to the volatile profile of biscuits, the addition of RFSF leads to the formation of aroma compounds such as hexanal, β-myrcene, D-limonene, acetophenone and 4-methyloctane, having odor perceptions such as grass, balsamic, must, citrus mint and alkane. Sensory evaluation indicated that the overall biscuits appreciation was not affected in a significant way (*p* < 0.05) by RFSF addition, meanwhile the aftertaste intensity decreased. Considering the nutritional, textural and sensorial analysis of the final baked goods, we can conclude that 25% RFSF could be successfully used in biscuit manufacturing.

**Author Contributions:** Conceptualization, S.M.M. and M.S.C.; methodology, V.M., S.A.S., L.S. and A.P. (Anamaria Pop); software, M.S.C., L.S.; validation, S.A.S. and A.P. (Adriana Păucean); formal analysis, M.S.C., S.A.S., S.M.M. and L.S.; writing—original draft preparation, S.M.M., M.S.C. and L.S.; writing—review and editing, A.P. (Adriana Păucean) and L.S.; supervision, L.S., A.P. (Adriana Păucean) and S.M.M.; project administration, S.M.M., S.M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** This work was supported by a grant of Romanian Ministry of Research and Innovation, CNCS—UEFISCDI, project number PN-III-P4-ID-PCE2020-1847, within PNCDI III.

**Conflicts of Interest:** The authors declare no conflict of interest.
