## 3 Results and Discussion
### Antimicrobial Activity against Candida Strains
#### 3.6.1 Growth Inhibition Properties of _Y. lipolytica_ CMGB32 Biosurfactant  
The antimicrobial activity of the biosurfactant was determined by monitoring the growth dynamics of the two _C. krusei_ strains in the presence of the biosurfactant in concentrations of 40 mg/mL and 20 mg/mL, followed by determining the percentage of microbial growth at different key moments of the growth curve. According to Figure 8(a1,a2), the concentrated biosurfactant successfully inhibited the growth of the laboratory strain _C. krusei_ CMGB94 and had a limited effect on the pathogenic strain _C. krusei_ CMGB-Y8. During the logarithmic growth phase, the presence of the biosurfactant in the culture medium reduced the microbial growth of _C. krusei_ CMGB94 by more than 50%. Moreover, after 8 h of incubation, the percentage of microbial growth recorded for the two biosurfactant concentrations was 30.14% and 36.41%, respectively, which indicates that the biosurfactant delays the entry of yeast into the logarithmic phase of growth (Figure 8(b1)). During the 21 h of incubation, the inhibitory effect of the biosurfactant was constant, but the upward trajectory of the growth curve indicated that it most likely intervenes in the basal metabolism of the cells without having an obvious fungicidal/fungistatic effect. In the case of the pathogenic strain, the antimicrobial effect was not obvious; the biosurfactant had no significant effect on the growth of the pathogenic strain regardless of the key moment analyzed. This result is not surprising, considering that the pathogenic _C. krusei_ strains are described as having additional mechanisms for dealing with stress conditions, which most likely allows them to quickly adapt to the stress represented by the presence of biosurfactants in low concentrations in the culture medium [79]. In general terms, it is considered that a biosurfactant can destabilize microbial membranes or interfere with their cell cycles. Moreover, it seems that its effect is directly linked to the chemical composition of the cell wall and membrane, since many studies reported that Gram-positive bacteria and yeasts are more sensitive to the biosurfactants than Gram-negative bacteria or filamentous fungi [80, 81, 82]. According to a study conducted by [PERSON] et al. [25], the biosurfactant, rufisan, produced by _C. lipolytica_ UCP0988 (currently known as _Y. lipolytica_), was effective against a wide range of microbial pathogens, including _Streptococcus mutans, S. oralis_, and _S. sanguis_, and it had a rather limited effect on pathogenic _C. albicans_ strains. Although many studies regarding biosurfactants have been conducted, their role as anti-adherence agents and the mechanisms involved in the process are not well described in the literature. The proposed mechanism of action relies on their amphiphilic characteristics, and the adsorption of biosurfactants onto specific surface modifies their hydrophobicity, thus altering the adhesion process [87; 88]. Also, although unproved yet, biosurfactants might modify the expression profile of specific _Candida_ genes involved in genetic dimorphism or ones that are directly linked to microbial adherence. This hypothesis is supported by a study conducted by [89], in which it was demonstrated that the biosurfactant produced by _Lactobacillus reuteri_ alters the expression profile of _gtfB_, _gtfC_, and _fft S. mutans_ genes, which are involved in the initial adherence of cells to dental surfaces. Nevertheless, the results published so far suggest that these molecules are a viable strategy for combating microbial infections. Sophorolipids, which are one of the types of biosurfactants produced by _Starmerella bombicola_ strains, were investigated for their antimicrobial properties and biofilm disruption against _Cupriavidus__nector_, _B. subtilis_, and _S. aureus_ reference strains [87]. Similarly, a mixture of biosurfactants produced by _Y. lipolytica_ showed high efficiency in preventing _S. enterica_ biofilm development [90].