*3.4. Characterization of the Cookies*

The concentrations of the biosurfactant chosen for this study were defined based on the maximum concentrations recommended for most of the emulsifying additives authorized by both the Brazilian Health Vigilance Agency (Agência Nacional de Vigilância Sanitária, ANVISA) and the US Food and Drug Administration [41,42]. Figure 1 illustrates the cookies before and after baking, and the mean values of their physical properties (weight, diameter, thickness and spread factor) are gathered in Table 5. *Fermentation* **2021**, *7*, 189 9 of 14 diameter and height. Indeed, significant differences in height were found among the formulations as a higher spread factor led to a shorter height.

**Figure 1.** Cookies before (**1**) and after baking (**2**). (**A**): standard formulation; (**B**): formulation with 1% biosurfactant; (**C**): formulation with 0.5% biosurfactant; (**D**): formulation with 0.25% **Figure 1.** Cookies before (**1**) and after baking (**2**). (**A**): standard formulation; (**B**): formulation with 1% biosurfactant; (**C**): formulation with 0.5% biosurfactant; (**D**): formulation with 0.25% biosurfactant.

a, b, c, d: Different letters in same column denote statistically significant differences (*p* ≤ 0.05, Tukey test). The values for each property were compared statistically taking the standard formulation as

A 4.88 ± 0.25 b 48.87 ± 0.40 a 5.79 ± 0.14 b 8.44 ± 0.19 b B 5.87 ± 0.53 a 49.11 ± 0.16 a 6.08 ± 0.04 c 8.08 ± 0.06 c C 5.37 ± 0.14 ba 50.18 ± 0.09 b 6.50 ± 0.16 da 7.72 ± 0.18 da

The physicochemical composition and energy value of the baked cookies are listed in Table 6. This study was necessary to determine whether the addition of the biosurfactant maintained the pre-established standards of identity and quality of the

**Table 6.** Physicochemical composition and energy value of cookies prepared with standard formulation, formulation A (1% biosurfactant), formulation B (0.5% biosurfactant) and formulation

Moisture (%) 6.35 ± 0.04a 5.44 ± 0.06ba 4.87 ± 0.06cb 5.87 ± 1.24cba Ash (%) 1.60 ± 0.08a 1.92 ± 0.07b 1.74 ± 0.17cb 1.66 ± 0.01ca Lipids (%) 11.10 ± 0.13a 13.83 ± 0.82b 11.69 ± 0.69a 11.47 ± 0.31a Proteins (%) 1.07 ± 0.00a 1.02 ± 0.06b 1.07 ± 0.00a 1.07 ± 0.00a

(%) 87.83 ± 0.13a 85.14 ± 0.88b 87.74 ± 0.69a 87.47 ± 0.31a

(cal) 455.52 ± 0.67a 469.17 ± 4.10b 455.97 ± 3.46a 457.33 ± 1.56a

**Formulation Formulation A Formulation B Formulation C** 

C (0.25% biosurfactant) (data expressed as mean ± SD of triplicate determinations).

**Table 5.** Physical properties of the cookies after baking for standard formulation, formulation A (1%

biosurfactant), formulation B (0.5% biosurfactant) and formulation C (0.25% biosurfactant).


**Table 5.** Physical properties of the cookies after baking for standard formulation, formulation A (1% biosurfactant), formulation B (0.5% biosurfactant) and formulation C (0.25% biosurfactant).

a, b, c, d: Different letters in same column denote statistically significant differences (*<sup>p</sup>* <sup>≤</sup> 0.05, Tukey test). The values for each property were compared statistically taking the standard formulation as a reference.

To complement the ANOVA results, the Tukey test was used to evaluate statistically significant differences (*p* ≤ 0.05) between the standard formulation and the formulations containing different concentrations of the biosurfactant with regard to weight, diameter, height and spread factor.

The increase in the concentration of the biosurfactant led to a linear increase in the spread factor. In addition to the benefits regarding the dough homogenization, the addition of the biosurfactant produced by *B. cereus* UCP 1615 promoted an increase in the quantity of lipids in the cookies due to the presence of fatty acids in its non-polar portion, which is a biochemical characteristic of the biomolecule previously described by Durval et al. [15]. This was reflected in the greater spread rate (increase in diameter) during cooking, which was likely related to the increase in the mobility of the system as the lipid fraction melted. It is a well-known fact that the spread rate exerts a direct influence on the diameter and height. Indeed, significant differences in height were found among the formulations as a higher spread factor led to a shorter height.

The physicochemical composition and energy value of the baked cookies are listed in Table 6. This study was necessary to determine whether the addition of the biosurfactant maintained the pre-established standards of identity and quality of the cookie.

**Table 6.** Physicochemical composition and energy value of cookies prepared with standard formulation, formulation A (1% biosurfactant), formulation B (0.5% biosurfactant) and formulation C (0.25% biosurfactant) (data expressed as mean ± SD of triplicate determinations).


a, b, c: Different letters on same line denote statistically significant differences (*<sup>p</sup>* <sup>≤</sup> 0.05, Tukey test). The values obtained for each physicochemical variable in the different formulations were compared statistically taking the standard formulation as a reference.

A 24% reduction in moisture was found in formulation B containing 0.5% biosurfactant compared with the standard formulation. Thus, the use of the biosurfactant was quite promising as the reduction in moisture minimizes the proliferation of microorganisms, thereby enhancing the durability of the product. On the other hand, the ash content only differed (around 20%) when the highest concentration of the biosurfactant was used.

Seventy-eight percent of the fatty acids in the composition of the biosurfactant were essential and unsaturated fatty acids [15], which suggests that its addition should not compromise the nutritional aspects of the cookie. The biochemical composition of the biosurfactant investigated in this study had a direct impact on the quantity of lipids in the

cookies with a statistically significant increase in formulation A containing 1% biosurfactant compared with the other formulations, promoting a proportional increase in the caloric value of the cookies. A cupcake and cookie containing a glycolipid biosurfactant also presented a caloric value increase [22,31]. Conversely, the protein content was unaffected, which may be related to the low protein moiety of the biosurfactant.
