3.1. Physical Properties of the Dough
shows the firmness (a) and cohesiveness (b) of the control dough produced with five flours of different origins and with the corresponding doughs enriched with 10% (w
) LE. The doughs prepared with different types of flour have different texture properties (firmness and cohesiveness).
It should be noted that the control gluten-free dough without LE (rice and buckwheat), are 20% firmer and 40% less cohesive than the others. This behavior should result from the different composition of these two flours (Table 1
). In these cases, the structuring of the system is essentially achieved by the starch present, although the different types of proteins present can also contribute to the reinforcement of this structure. Thus, the doughs obtained from these two flours have a greater resistance to penetration (high firmness), which is related to more compact doughs. The absence of the gluten matrix decreases the air retention capacity of the system [34
], contributing to firmer doughs. At the same time, a reduction in the cohesiveness associated with a greater disaggregation is observed [35
]. These characteristics are less positive in terms of the technological handling of these doughs.
In the case of rice, the high starch content is relevant, compared to other flours, which has an important impact on structure creation. Regarding the buckwheat, the type of proteins involved could also explain the increase in firmness and decrease in cohesiveness since its proteins are rich in lysine and arginine, unlike the other flours studied [36
]. Complementary studies can be developed, in the future, in order to support this statement.
When 10% (w
) of the flours under study is replaced by LE, a relevant impact on the texture characteristics of the dough is observed. In general, the incorporation of proteins contributes to an increase in dough firmness (Figure 1
a) and a significant (p
< 0.05) reduction of at least 50% in cohesiveness (Figure 1
b). A similar behavior was observed by the other researchers upon the addition of potato peel to cakes [37
], whey protein to cheese [38
] and lupin flour to biscuits [26
It is important to highlight the strong impact of LE addition on the two gluten rich flours—spelt and kamut doughs are about four times firmer (from 2.66 N to 12.19 N, in the case of spelt and from 2.05 N to 9.95 N in the case of kamut) than the corresponding control. This should result from a strong interaction between the main macromolecules present in the system: (i) lupin proteins–flour starch; and (ii) lupin proteins–flour gluten proteins. This type of interactions is strongly dependent on the protein composition of the added protein fraction, as well as on the starch conformation [39
]. More important than the total amount of macromolecules present in the dough, which is similar in all the cases, is the biochemical composition and conformation of these proteins and polysaccharides. A firmer dough should reflect a more effective entangled network developed among these macromolecules [39
], which may be important in terms of the dough stability, but which translates to a less cohesive dough.
The relevant reinforcement on the structure observed for the kamut and spelt doughs, due to the incorporation of LE, allows us to predict that there was a reinforcement in the gluten structure already present in the control doughs, resulting from a synergy between the gluten and the lupin proteins. The firmness increase and cohesiveness decrease resulting from the LE incorporation has a relevant impact in technological terms: the doughs become more difficult to mold, meaning it may be necessary to optimize the cookie production process such as the optimization of the water absorption (e.g., MicrodoughLab procedure) that consists of the quantity of water needed to reach the optimal dough consistency [40
The impact of LE addition on the linear viscoelastic behavior of the cookie’s dough prepared with five different flours can be observed in Figure 2
. These results were obtained from small amplitude dynamic rheological measurements (small amplitude oscillatory system - SAOS) and are related to the degree of dough structuring, reflecting the level of molecular interactions that are established, especially among the macromolecules present.
The evolution of G’ (storage modulus) and G” (loss modulus) over the frequency range tested reveal that both moduli slightly increased with increasing frequency. This weak gel-like rheological behavior is typical of cookie doughs [41
] and other cereal dough products such as bread [42
] and pasta [43
The addition of LE causes the reinforcement of the dough structure for all the flours studied, except for the buckwheat flour. This is evidenced by the higher values of G’ and G” for the formulations enriched with LE, compared to the standard flours. These results are in agreement with the texture results-also in terms of firmness, the buckwheat flour formulation was the only one without significant differences (p > 0.05) due to the addition of LE.
To obtain a more detailed comparison among the linear viscoelastic behaviors of the different formulations, Table 2
shows the G’ values obtained at 1 Hz (G’ 1 Hz) from the three replicates of each test. It turns out that the G’ 1 Hz values were significant higher in rice, spelt and kamut flours, when the lupin incorporation dough was compared with the control without LE. The maximum value for G’ was 8.3 × 105
Pa for the lupin-incorporated rice flour. However, the greatest increment measured in G’ 1 Hz due to the addition of LE was achieved for the kamut flour. In these cases, lupin incorporation increased the degree of dough structuring, which results from the formation of more complex three-dimensional structures among the macromolecules present in the systems, as previously discussed for the dough texture results.
3.2. Physical Properties of Cookies
Characteristic dimensions of the LE incorporation in five different flours are presented in Table 3
. In general, the incorporation of LE had significant differences (p
< 0.001) in all the flours tested, except for oat flour. For spelt, kamut and buckwheat flours, the addition of LE increased the area in relation to the control. However, the rice flour cookies were the only ones with a significant (p
< 0.001) reduction in the cookie area. Therefore, the presence of gluten does not seem to affect the cookie area and no direct relationship can be established with the expansion of the structure. In relation to thickness, the two gluten-free flours (rice and buckwheat) showed a significant increase (p
< 0.05) in the LE-containing cookies, unlike the gluten flours, where it showed a generalized decrease. Similar studies were performed with wheat cookies and Jayasena and Nasar-Abbas [26
] reported no effect in the cookie diameter and an increase in the cookie thickness with the presence of 10% (w
) lupin flour. Nevertheless, Bilgiçli and Levent [44
] demonstrated no effect in the thickness in cookies containing lupin flour, whereas Tsen et al. [45
] showed a reduction in the cookie diameter prepared with soy protein isolates.
In summary, even in cases where statistically significant results were obtained, all the structural alterations resulting from the addition of LE could be neglected, as far as the magnitude was concerned (maximum 20% variation for the area and thickness of buckwheat cookies). This conclusion can be important in terms of technological performance and consumer acceptance.
The texture properties of foods are an important requirement for their acceptance by consumers, especially in what concerns crispy products, such as cookies [46
]. In this sense, the impact of LE addition to different types of cookies was evaluated in both the presence and absence of gluten. Firmness values (N) obtained in week 0 and eight weeks later are presented in Figure 3
It is evident that the cookies prepared with the ancient grains and without LE (spelt and kamut) were firmer than the other control cookies and this observation remained valid after storage (eight weeks). The changes induced by the LE in the cookie structure differed over time, since at week 0 only the spelt flour had no significant difference (p
> 0.05) when compared to the control; however, after eight weeks, the spelt, kamut and rice flours showed no significant differences (p
> 0.05) when the cookie with LE and the control were compared. Additionally, the oat and buckwheat flours were statistically different over time (eight weeks), meaning that the incorporation of lupin clearly modified the texture of the cookies, making them firmer. Hence, it cannot be stated that the differences between the five different flours on one hand and LE addition on the other occurred due to the presence of gluten. Jayasena and Nasar-Abbas [26
], Obeidat, Abdul-Hussain and Al Omari [47
] and Bilgiçli and Levent [44
] reported that cookie hardness increased with the addition of lupin flour in the cookie formulation. This can also be stated for other types of legume seeds, such as chickpeas [48
], green lentils and navy beans [4
]. The different behavior observed between the doughs and the respective cookies is corroborated with other studies [41
]. Indeed, macromolecular structures present in each flour undergo dramatic changes during heat treatment. In spelt and kamut flours, gluten is the main element that accounts for the structure; in oat, the main structural role is played by β-glucans, and in gluten-free flours (rice and buckwheat), the structure is mainly accounted for by starch. When the LE (protein) is added, there is an overall structural rearrangement leading to distinct interactions among these macromolecules, as supported by our results. The interactions among macromolecules and the type of structures which arise are differentially affected by the heat treatment which takes place during cooking.
The impact of LE addition on the color parameters of cookies is summarized in Table 4
. The ΔE* values were calculated to compare the color variation in relation to the cookies without LE. In the same table, the water activity (aw
) and moisture content (H) of the ten formulations studied are also indicated.
The ΔE* values obtained were always higher than 5 for both time periods studied (week 0 and week 8), which means that the color difference between the lupin-enriched cookies and the control is visually distinguishable by the human eye. These differences result mainly from a general decrease in the lightness parameter (L*) in all lupin-containing cookie samples, resulting in a golden-brown color. These results agree with other studies, showing a decrease in cookie lightness with lupin flour at the same concentration level [44
]. The results can be explained by the Maillard reaction, as proteins and sugars initiate a complex cascade of reactions during heating (higher than 100 °C), producing the darker color [49
]. This darkening did not have a negative impact on the characteristics of the final product; on the contrary, the LE cookies presented very appealing colors, as those supported by other studies [26
Cookies are a relatively dry product with a low moisture content and water activity values. These parameters are crucial to predict both the stability and safety of the product, with great impact in conservation, particularly for the maintenance of a crispy texture [50
]. Moisture content values of cookies with and without LE are low (ranging from 1.04 to 5.61%), comparing favorably with other studies on similar cookies and indicating a positive impact in terms of conservation [30
values for lupin-enriched cookies at week 0 are significantly higher (p
< 0.05 or p
< 0.001) than those of the control cookies. After 8 weeks of storage, all the LE cookies had similar (except for rice flour) aw
values, but significantly higher (p
< 0.001) than the controls. Furthermore, all the samples were shown to have an aw
value of less than 0.5 (except lupin-enriched cookies with rice flour at week 0), which means that all cookie formulations (with and without LE) had a low percentage of free water for microbial proliferation, leading to a high stability product [50
]. Such low aw
values are essential to prevent microbial growth on the cookies. Uysal et al. [51
] found an increase in aw
values with the incorporation of apple and lemon fiber in cookies. Batista et al. [28
] also found an increase in aw
values, resulting from the incorporation of microalgae biomass with a high protein content. However, Fradinho et al. [30
] found an opposite effect when Psyllium fiber was added to the cookies similar to those prepared in the present work, resulting from the high-water holding capacity of Psyllium. The differential capacity to retain the water of the molecules present in the formulation had a direct impact on the water activity of the final product. For the LE cookies, the water holding capacity of the protein should be lower than that of the respective flour, justifying the increase in water activity.
Lupin is considered a potential functional food because of its protein content, dietary fiber and more recently discovered bioactivities [20
] that need to be explored in food products in the near future.