Fungistatic and Fungicidal Capacity of a Biosurfactant Extract Obtained from Corn Steep Water

Biosurfactants are surface-active compounds that are produced by microorganisms, which in addition to their surfactant capacity, can possess interesting antimicrobial activities that are used in their incorporation into the agrifood industry. In this work, the preservative capacity of a novel biosurfactant extract obtained from a residual stream of the corn-milling industry was evaluated against two different fungi (Aspergillus brasiliensis and Candida albicans) under different biosurfactant concentrations (0.33–0.99 mg/mL), temperatures (4–40 °C), and incubation times (5–11 days). All the assays started with the same concentration of fungi (2 × 106 CFU/mL). The results showed that temperature played an important role in the bactericidal and fungistatic effects of this biosurfactant extract. It was observed that at a low biosurfactant concentration (0.33 mg/mL) and low or high temperatures in the range tested, this biosurfactant extract possessed an important fungicidal effect (complete inhibition) on A. brasiliensis, while at intermediate temperatures, it achieved a fungistatic effect (50% of inhibition). Regarding C. albicans, it was observed that this strain was more resistant than A. brasiliens, although it was possible to achieve growth inhibitions of 76.3% at temperatures of 40 °C after 8 days of incubation with a biosurfactant concentration of 0.99 mg/mL. This work supports the possible application of biosurfactants extracted from corn steep water as preservatives and antimicrobial agents against fungal contaminations on agrifood products.


Introduction
It is estimated that microbial spoilage is responsible for the loss of almost 33% of the global annual food production [1], resulting in 1.3 billion tons of food waste worldwide [2]. Some of these microbial contaminations are produced by pathogenic fungal strains, among which, some of the most common are Aspergillus and Candida spp. These pathogens can be commonly found in soil, as well as in a wide range of crops, such as maize, lettuce, onion, tomato, and other vegetables [3,4]. Some Aspergillus spp., including A. fumigatus, A. niger, or A. flavus, which can behave as opportunistic pathogens, are known to affect animal and human health. They are responsible for causing food poisoning through the production of mycotoxins, as well as illnesses like aspergillosis, which is a pulmonary disease with symptoms of hemoptysis (coughing up blood) and chronic coughing [5,6].
On the other hand, Candida albicans is an opportunistic pathogen that is widely known for being the most pathogenic yeast species and responsible for causing infections in humans by colonizing oral micellar concentration (CMC) between 100-400 mg/L [22] and a composition of C16 and C18 fatty acids [16].
Therefore, this work aimed to evaluate the fungistatic and fungicidal activity of an extracellular biosurfactant extract, obtained using liquid-liquid extraction with ethyl acetate from CSW, on foodborne pathogens (A. brasiliensis and C. albicans).

Extraction of Biosurfactants from Corn Steep Water
Liquid-liquid extraction with organic solvents was carried out following the methodology described by Vecino et al. [28] to obtain an extracellular biosurfactant from CSW, which was provided by FeedStimulants (Reg. No. NL214247, Lot NL- 2728DK 7). Briefly, the biosurfactant was extracted with ethyl acetate from CSW (CSW solution: ethyl acetate 1:3 (v/v)) at room temperature (25 ± 1 • C) for 60 min. Ethyl acetate was supplied by CARLO ERBA Reagents, S.A.S (Val de Reuil Cedex, France). Then, the organic solvent was evaporated using vacuum distillation and recycled for further extractions, obtaining a multifunctional biosurfactant extract that was dissolved in distilled water at different concentrations (0.33-0.99 mg/mL). Afterward, a 0.22 µm Stericup ® 150 mL Durapore ® polyvinylidene fluoride (PVDF) membrane (EMD Millipore Corporation, Billerica, MA, USA) was used to filter the biosurfactant extract to evaluate its antifungal capacity against A. brasiliensis and C. albicans. All steps of the process were conducted under sterile conditions. The extractive yield for the extracellular biosurfactant extraction from the CSW was determined gravimetrically by weighing a sample before and after drying at 100 • C for 48 h in a stove following the methodology described by Rodríguez-López et al. [12].

Surface Activity and Critical Micellar Concentration Determination-Wilhelmy Plate Assay
The determination of the surface tension (ST) and CMC of the extracellular biosurfactant extracted with ethyl acetate from the CSW was achieved by using a Krüss K20 EasyDyne tensiometer with a 1.9 cm platinum Wilhelmy plate (Krüss GmbH, Hamburg, Germany). They were prepared using several dilutions in distilled water to determine the CMC of the biosurfactant extract from the CSW. All measurements were carried out in triplicate at room temperature and all the results are shown as the mean value and standard deviation of all three measurements.

Elemental Analysis of the Extracellular Biosurfactant Obtained from the Corn Steep Water
Elemental analysis was performed to determine the C, H, N, and S composition of the biosurfactant extract obtained from the CSW using an elemental analyzer (Fisons Carlo Erba EA-1108 CHNS-O, LabX, Midland, ON, Canada). To obtain the amount of N, a correlation with the protein content was conducted by multiplying it by a factor of 6.25, based on a previous study [30].

Strains and Standard Culture Conditions for the Antimicrobial Assay
The antimicrobial activity of the extracellular biosurfactant obtained from the CSW was assessed against two pathogenic fungal strains obtained from the Spanish Type Culture Collection (CECT) (Valencia, Spain). The strains selected were the following: Aspergillus brasiliensis CECT-2574 (ATCC-16404) and Candida albicans CECT-1392 (ATCC-2091). Both strains were cultivated in potato dextrose broth (PDB) medium at 22 • C for 5 days in aerobic conditions in 250 mL Erlenmeyer flasks at 150 rpm. The composition of the PDB medium was 4 g/L potato peptone and 20 g/L of glucose.

Antimicrobial Assay
The antimicrobial activity of the biosurfactant extracted with ethyl acetate from the CSW against two pathogenic fungal strains of A. brasiliensis and C. albicans was determined by measuring the optical density at 600 nm in 96-well plates in a microplate reader (MultiSkan GO Microplate Photometer, ThermoFisher Scientific, Waltham, MA, USA) following the methodology described by Vecino et al. [20].
Briefly, PDB medium containing different concentrations of the biosurfactant extract from the CSW (0.33-0.99 mg/mL) were prepared in 10 mL sterile tubes with a final sample volume of 2 mL. An inoculum of 20 µL of the selected fungal pathogen, with a final concentration of microorganisms of 2 × 10 6 colony-forming units (CFU)/mL, was used for each of the experiments. All samples were prepared in triplicate. Likewise, positive controls consisted of PDB medium containing the fungal strain, whereas the negative control was formulated with only PDB medium (in the absence of the biosurfactant extracted from the CSW and the pathogenic strain). Every tube was then rinsed and incubated at three different temperatures of 4, 22, and 40 • C. After 5, 8, and 11 days, a volume of 250 µL of each of the samples and controls was placed into the columns of 96-well microplates and then the optical density was measured at 600 nm.
Percentages of growth inhibition of the fungal strains at different concentrations of the biosurfactant extracted from the CSW were calculated using Equation (1): where A c represents the absorbance of the samples at the different concentrations of the biosurfactant extract from the CSW and A 0 represents the absorbance of the positive control well in the absence of the biosurfactant extract. All the results are shown as the mean value and standard deviation of all three measurements of the triplicate.

Experimental Design
An incomplete Box-Behnken factorial design [31] was carried out to assess the antifungal activity of a biosurfactant extracted with ethyl acetate from the CSW against A. brasiliensis and C. albicans under different conditions of biosurfactant concentration, temperature, and incubation time.
Independent and dependent variables assessed in this work, along with their ranges established in the experiments, are shown in Table 1. The coded dimensionless independent variables used were defined as x 1 (biosurfactant concentration), x 2 (temperature), and x 3 (time) with limits of variation between −1 and 1. The dependent variables were defined as y 1 (% of growth inhibition of A. brasiliensis) and y 2 (% of growth inhibition of C. albicans). To determine the relationship between the independent and dependent coded variables of this study within the ranges of variation established in Table 1, linear equations were applied (Equation (2)): where ∆z i represents the distance between the real value obtained at the central point and the real value obtained at the upper or lower level of each variable, β d is the major coded limit value in the matrix for each variable, and z i 0 is the real value in the central point.
Values of −1, 0, and +1 were assigned to each coded variable, which represent the minimum, central, and maximum values, respectively, for each variable of the experiment within the ranges established in Table 1.
The fungistatic and fungicidal capacity of the biosurfactant extracted from the CSW was determined for each of the two pathogenic strains as the lowest concentration of the biosurfactant that inhibited 50% of the fungal growth and produced complete inhibition (100%), respectively.

Variable Nomenclature Units
(c) Dependent variables studied Growth inhibition of A. brasiliensis y 1 % Growth inhibition of C. albicans y 2 %

Statistical Analysis
The experimental data obtained in this study were analyzed using the response surface method with Design-Expert ® Version 12 (Stat-Ease, Inc., Minneapolis, MN, USA) by fitting the results obtained to a quadratic function shown in Equation (3): where y is the dependent variable of each experiment (% of growth inhibition of A. brasiliensis or C. albicans, respectively); β i represents the regression coefficients, which were calculated from experimental data by performing multiple regressions using the least-squares method; and x i represents the independent variables of this study (biosurfactant concentration, temperature, and time of incubation). This equation predicts the percentage of growth inhibition of A. brasiliensis and C. albicans within the range of the independent variables considered in the Box-Behnken factorial design shown in Table 1. For the experiment conducted in this work, unlike a linear equation, a quadratic equation produces a parabola that begins at a single point, called the vertex, and extends upward or downward in the y-direction. In this particular design, there is no one-to-one relationship between x and y, because it results in two values of x for any given value of y, except for the y value corresponding to the vertex point; this is in contrast with a linear equation, where this relationship is one-to-one since each x value produces only one y value [31].

Biosurfactant Characterization
Regarding its physico-chemical characteristics, the biosurfactant extract from the CSW revealed its ability to reduce the surface tension of water to a minimum value of 43.8 mN/m, which is in concordance with the results shown in previous studies [12,15,21]. Additionally, the CMC obtained for the biosurfactant extract was 306.5 mg/L. Regarding the elemental analysis, the N, C, H, and S content showed that C was the element present in the highest proportion with 43.67%, while the contents of N, H, and S were 0.77%, 5.89%, and less than 0.30%, respectively, all of which are in the range of values of previous analysis performed on biosurfactants from CSW [21].

Antimicrobial Activity
The antimicrobial activity of the biosurfactant extracted with ethyl acetate from the CSW was determined by carrying out an incomplete Box-Behnken factorial design to assess the effect of different conditions (biosurfactant concentration x 1 , temperature x 2 , and time x 3 ) to measure the growth inhibition percentages against two pathogenic fungal strains of A. brasiliensis and C. albicans. The experiments were conducted under static conditions to simulate real situations and to test the reproducibility of the study in a real setting. The biosurfactant concentrations used in the factorial design were chosen based on previous experiments of the evaluation of the bactericidal effect of a biosurfactant extract from CSW [21]. The range of temperatures was selected to be between refrigeration storage (4 • C) and a maximum temperature outside on a hot day (40 • C). The time for incubation was chosen based on the minimum days of incubation needed for fungal strains to grow, which was 5 days, up to a maximum of 11 days. The dependent variables evaluated were the growth inhibition percentages (y 1 , y 2 ) of A. brasiliensis and C. albicans, as shown in Table 1. The experimental data obtained for the growth inhibition of A. brasiliensis (y 1 ) and C. albicans (y 2 ) in the 15 experiments carried out in triplicate at different concentrations of biosurfactant (x 1 ), temperature (x 2 ), and time of incubation (x 3 ) are exhibited in Table 2. The initial concentrations of A. brasiliensis and C. albicans were 2.0 × 10 6 CFU/mL. Table 2. Operational conditions used in this study, expressed as coded dimensionless and uncoded independent variables: concentration of the biosurfactant (x 1 ), temperature (x 2 ), and incubation time (x 3 ); and the results obtained for the dependent variables: y 1 (% of growth inhibition of A. brasiliensis) and y 2 (% of growth inhibition of C. albicans). (−1, minimum value of the variable within the range; 0, central value of the variable within the range; 1, maximum value of the variable within the range).

Coded Independent Variable Uncoded Independent Variable Dependent Variable
Exp. To reduce the possible effect of systematic errors associated with the experimental data obtained in the present work, a random testing sequence of the 15 experiments was carried out. Regression coefficients were calculated for experiments 1 to 12, whereas for experiments 13 to 15, the evaluated influence of experimental errors represented the central points of the independent variables within the range established in Table 1.
On the other hand, the significance of each coefficient was established using p-values for the variables y 1 and y 2 displayed in Table 3. Using these regression coefficients, equations were created to determine the values of the dependent variables studied within the ranges established before the experiments were undertaken (see Table 1). By replacing the significant coefficients in Equation  In the case of A. brasiliensis, the concentration of the biosurfactant (x 1 ) and temperature (x 2 ) were the most statistically significant variables (p < 0.05) influencing the growth inhibition of the fungal strain (see Table 3). Furthermore, the interaction of these two independent variables produced higher inhibition percentages against A. brasiliensis. Figure 1 shows the variation of the growth inhibition percentage of A. brasiliensis for the above-mentioned independent variables as the most statistically significant, namely the concentration of the biosurfactant (x 1 ) and temperature (x 2 ), with the time of incubation (x 3 ) fixed at 5 (Figure 1a), 8 (Figure 1b), and 11 days (Figure 1c). Among them, the temperature had a more significant effect on the growth inhibition of A. brasiliensis; therefore, when the incubation was carried out at a low temperature (4 • C) with a low biosurfactant concentration (0.33 mg/mL) for the range of storage time evaluated in this work, a complete inhibition (100%) of A. brasiliensis was achieved, as shown in Figure 1. These results showed the efficiency of the biosurfactant extracted from the CSW against A. brasiliensis, mainly at low temperatures; hence, the biosurfactant extract could be applied for the preservation of food products stored in refrigeration conditions. Additionally, at high temperatures (40 • C), a fungicidal effect (82.5% of growth inhibition) was achieved at a biosurfactant concentration of 0.33 mg/L. The factorial design also determined the optimal conditions established by the factorial design for both pathogenic strains at room temperature (25 • C), showing that a biosurfactant concentration of 0.99 mg/mL resulted in a maximum inhibition of 57.1% against A. brasiliensis after 11 days of incubation, achieving a fungistatic effect. These results agree with the results obtained by Rodríguez-López et al. [32], where a biosurfactant extracted from CSW with chloroform displayed a fungistatic effect on A. brasiliensis at a temperature of 22 • C.
Regarding C. albicans, all three independent variables-biosurfactant concentration (x 1 ), temperature (x 2 ), and time (x 3 )-were found to be statistically significant (p < 0.05), influencing the growth inhibition of the pathogenic strain (see Table 3). Additionally, the interaction between the biosurfactant concentration (x 1 ) and incubation temperature (x 2 ) was found to be statistically significant. The maximum growth inhibition achieved from the Box-Behnken factorial design was 76.34%, achieving a fungistatic effect at a biosurfactant concentration of 0.99 mg/mL and a temperature of 40 • C after 8 days of incubation, as shown in Table 2. At room temperature (25 • C), the results showed that the highest inhibition percentage of the fungal strain was 20.0% at a biosurfactant concentration of 0.99 mg/mL after 5 days of incubation. However, at a temperature of 4 • C, the maximum growth inhibition achieved against C. albicans was 17.79%, as shown in Table 2. These results agree with a previous study conducted by Rodríguez-López et al. [32], where the biosurfactant extract obtained from CSW after extraction with chloroform did not achieve a fungistatic effect on C. albicans at a temperature of 22 • C. Based on the results obtained in this work, it can be established that the biosurfactant extracted from the CSW with ethyl acetate was much more effective against A. brasiliensis than C. albicans, showing promising results as an antifungal agent against the Aspergillus family. It can be speculated that low temperatures and the presence of the biosurfactant produced a synergic effect on the Based on the results obtained in this work, it can be established that the biosurfactant extracted from the CSW with ethyl acetate was much more effective against A. brasiliensis than C. albicans, showing promising results as an antifungal agent against the Aspergillus family. It can be speculated that low temperatures and the presence of the biosurfactant produced a synergic effect on the inhibition of A. brasiliensis, while in the case of C. albicans, this effect was observed at high temperatures. This might be because the optimal temperature for the growth of C. albicans (33-38 • C) [33] is slightly higher than the optimal temperature for the growth of A. brasiliensis (24-35 • C) [34].
It is well known that Candida spp. can produce biofilm layers that act as a protective structure for the microorganism [8], making them more resistant than other microorganisms. In this work, the biofilm formation was not investigated, although it can be speculated that if the biosurfactant extract from CSW could inhibit the growth of C. albicans, it would also inhibit the formation of biofilms. Table 4 shows the operational conditions within the parameters established in the Box-Behnken factorial design that achieved fungistatic (50% of growth inhibition) and fungicidal (100% complete inhibition) effects of the biosurfactant extracted from the CSW on A. brasiliensis and C. albicans (at 4 and 25 • C). For A. brasiliensis, concentrations of 0.33 and 0.99 mg/mL of the biosurfactant extract resulted in inhibitions of 50% and 100% at 4 • C during an incubation period of 5 and 10 days, respectively, achieving fungistatic and fungicidal effects on the pathogen depending on the concentration of the biosurfactant extract and the incubation time. Similarly, when the concentration of the biosurfactant extract was increased to 0.99 mg/mL, a 100% inhibition effect on A. brasiliensis was achieved after 10 days of incubation at room temperature (25 • C). Additionally, the results obtained for the optimal conditions to achieve both fungistatic and fungicidal effects on A. brasiliensis within the range of temperatures of the Box-Behnken factorial design were temperatures of 15.5 • C for the 50% growth inhibition and 39.6 • C for complete inhibition using a biosurfactant concentration between 0.97-0.99 mg/mL during incubations of 8.4 and 10.9 days, respectively. On the other hand, for C. albicans, neither fungistatic nor fungicidal effects were achieved at 4 and 25 • C, where the maximum percentages of growth inhibition were 17.9% at 4 • C and 20.0% at 25 • C for the range of biosurfactant concentrations tested, as shown in Table 4. However, it was possible to inhibit 50% of the growth of C. albicans at 36.6 • C with a biosurfactant extract concentration of 0.97 mg/mL after 10.8 days of incubation, showing a fungistatic effect under these conditions. Additionally, the growth inhibition kinetics of A. brasiliensis and C. albicans were evaluated at the maximum biosurfactant concentration tested (0.99 mg/mL) at 40 • C over the incubation time tested (5-11 days), as shown in Figure 2. To obtain these results, the theoretical equation obtained in the factorial design was used. The r 2 values obtained for the factorial design were 0.976 and 0.966 for A. brasiliensis and C. albicans, respectively. As can be observed in Figure 2, within the experimental period, the growth inhibition of A. brasiliensis was increased until it achieved complete inhibition, whereas C. albicans showed a higher resistance with a decrease in the growth inhibition during the period of incubation. For comparative purposes, Table 5 summarizes those results obtained in previous works against A. brasiliensis and C. albicans. Only a few antimicrobial studies have carried out experiments with biosurfactants on A. brasiliensis. Table 5 also includes those microorganisms that can produce biosurfactants with antimicrobial activities against both A. brasiliensis and C. albicans. In most cases, high concentrations of biosurfactants were required to inhibit at least 50% of the growth of the pathogens, except for the study by Rodríguez-López et al. [32], who obtained almost complete inhibition of the growth of A. brasiliensis with concentrations of 1 mg/mL of a biosurfactant extracted from CSW with chloroform after 7 days of incubation. In contrast, for C. albicans, the same authors did not observe any growth inhibition during the whole experiment, which agrees with the results obtained in this work with the biosurfactant extracted from the CSW with ethyl acetate, where a different effect on C. albicans and A. brasiliensis was observed. Some authors have observed that cationic surfactants induce changes in the ionic character of the cell surface membrane from negative to positive, producing an antifungal effect [35]. The antimicrobial activity of the biosurfactant extract from the CSW evaluated in this work can be explained in terms of its amphoteric character, which has been proved in a previous study showing anionic and cationic charges [12].
Basit et al. [36] showed that a biosurfactant from a Bacillus cereus strain showed a fungistatic effect on both fungal pathogens at a concentration of 7 mg/mL, which is higher than the concentrations of the biosurfactant extract obtained from the CSW used in this work to obtain a fungistatic effect. Similar results were achieved for biosurfactants extracted with phosphate buffer saline (PBS) and phosphate buffer (PB) from L. pentosus and L. paracasei on C. albicans [18].
It is interesting to remark that even though the biosurfactant evaluated in this work did not achieve a fungicidal effect on C. albicans, it showed a higher efficiency in terms of the growth inhibition at lower concentrations than other biosurfactant extracts, such as those produced, for instance, from Rhodococcus fascians BD8 [37] (see Table 5). Moreover, in this work, at temperatures higher than 35 °C, it was possible to achieve a fungistatic effect. Additionally, Gudiña et al. [18] produced growth inhibitions over 50% on C. albicans with biosurfactants extracted from L. paracasei ssp. paracasei A20, although at higher concentrations than the ones used in this work (3.12-6.25 mg/mL). Moreover, biosurfactants from L. helveticus showed good fungistatic activity on C. albicans, although at concentrations around 25 mg/mL [19]. Growth inhibition (%)

Incubation time (days)
A. brasiliensis C. albicans For comparative purposes, Table 5 summarizes those results obtained in previous works against A. brasiliensis and C. albicans. Only a few antimicrobial studies have carried out experiments with biosurfactants on A. brasiliensis. Table 5 also includes those microorganisms that can produce biosurfactants with antimicrobial activities against both A. brasiliensis and C. albicans. In most cases, high concentrations of biosurfactants were required to inhibit at least 50% of the growth of the pathogens, except for the study by Rodríguez-López et al. [32], who obtained almost complete inhibition of the growth of A. brasiliensis with concentrations of 1 mg/mL of a biosurfactant extracted from CSW with chloroform after 7 days of incubation. In contrast, for C. albicans, the same authors did not observe any growth inhibition during the whole experiment, which agrees with the results obtained in this work with the biosurfactant extracted from the CSW with ethyl acetate, where a different effect on C. albicans and A. brasiliensis was observed. Some authors have observed that cationic surfactants induce changes in the ionic character of the cell surface membrane from negative to positive, producing an antifungal effect [35]. The antimicrobial activity of the biosurfactant extract from the CSW evaluated in this work can be explained in terms of its amphoteric character, which has been proved in a previous study showing anionic and cationic charges [12].
Basit et al. [36] showed that a biosurfactant from a Bacillus cereus strain showed a fungistatic effect on both fungal pathogens at a concentration of 7 mg/mL, which is higher than the concentrations of the biosurfactant extract obtained from the CSW used in this work to obtain a fungistatic effect. Similar results were achieved for biosurfactants extracted with phosphate buffer saline (PBS) and phosphate buffer (PB) from L. pentosus and L. paracasei on C. albicans [18].
It is interesting to remark that even though the biosurfactant evaluated in this work did not achieve a fungicidal effect on C. albicans, it showed a higher efficiency in terms of the growth inhibition at lower concentrations than other biosurfactant extracts, such as those produced, for instance, from Rhodococcus fascians BD8 [37] (see Table 5). Moreover, in this work, at temperatures higher than 35 • C, it was possible to achieve a fungistatic effect. Additionally, Gudiña et al. [18] produced growth inhibitions over 50% on C. albicans with biosurfactants extracted from L. paracasei ssp. paracasei A20, although at higher concentrations than the ones used in this work (3.12-6.25 mg/mL). Moreover, biosurfactants from L. helveticus showed good fungistatic activity on C. albicans, although at concentrations around 25 mg/mL [19].

Conclusions
This is the first study concerning the possible application of a biosurfactant extracted from corn steep water with ethyl acetate as fungicidal and fungistatic agents against foodborne pathogens. The results obtained in this work showed the preservative and antimicrobial effects of a biosurfactant extract on pathogenic fungi like A. brasiliensis and C. albicans, which can be found on a wide range of crops and food products, resulting in massive losses due to their microbial contamination of agrifood products. Therefore, the biosurfactant extract could be considered as an additive with multifunctional properties for its application in the formulations of food products and crops. From the two experimental designs carried out in this study, it can be concluded that the biosurfactant extract was more effective against A. brasiliensis than C. albicans, especially at low storage temperatures. Finally, this work takes advantage of a circular economy since the biosurfactant extract was obtained from a secondary raw material of the agrifood industry with an important impact on the production of more biodegradable and sustainable preservatives and pesticides that would help to avoid food poisoning and contaminations.