**3. Antimicrobial activity of biosurfactant**

Several biosurfactants which exhibit antimicrobial activity against various microorganisms have been previously described. They include surfactin and iturin produced by *Bacillus subtilis* strains [20], rhamnolipids from *Pseudomonas* species [21], mannosylerythritol lipids from *C. antarctica* [22] and biosurfactants produced by some fungi [23].

Among the genus *Bacillus, B. subtilis* produces a broad spectrum of bioactive lipopeptides which have a great potential for biotechnological and biopharmaceutical applications. The characteristic structure of lipopeptides is a fatty acid combined with an amino-acid moiety. Several lipopeptides have potent antibiotic activity and have been the subject of several studies on the discovery of new antibiotics. The list includes surfactin, produced by *B. subtilis*, the most powerful of biosurfactant known to date. These compounds have many pharmacological activities: antibacterial, antifungal, antiviral, and antimycoplasma properties; inhibition of the fibrin clot formation and hemolysis; formation of ion channels in lipid bilayer membranes [24]; antitumour activity against Ehrlich's ascites carcinoma cells; and inhibition of the cyclic adenosine 3,5-monophosphate phosphodiesterase [25].

Antimicrobial and Anti-Adhesive Potential of a Biosurfactants Produced by *Candida* Species 249

concentration of 12 mg/l or 4xCMC. Non-pathogenic species associated with the oral cavity of *Streptococcus* were used (*S. mutans* HG – 64.9%; *S. oralis* J22 – 62.8%; *S. mutans* – 58%; *S. sanguis* 12 – 48%; *S. mutans* NS – 46%). On the other hand, the biosurfactant did not show an effective antimicrobial activity against the *Lactobacillus* strains studied. It inhibited only

The growth of the other microorganisms tested was poorly inhibited. Percentages of 5%, 5%, 15%, 16% and 18% were observed for *C. albicans, E. coli*, *S. aureus, P. aeruginosa* and *S.* 

The antimicrobial activity of the biosurfactant isolated from *Candida sphaerica* was determined by measuring the growth inhibition percentages obtained for several

**Table 2.** Percentages of growth inhibition obtained with the crude biosurfactant Lunasan isolated from

The tested biosurfactant presented antimicrobial activity against all microorganisms used, although, depending on the microorganism, the biosurfactant presents different effective concentrations. The highest concentration of biosurfactant tested (10 mg ml-1) showed high percentages of inhibition for *Streptococcus oralis* J22 (68%)*, C. albicans* (57%) *and Staphylococcus epidermidis (*57.6%). The antimicrobial activity of the crude biosurfactant isolated from *Candida sphaerica* with concentrations between 5 and 10 mg ml1 against *C. albicans, Staph. aureus* and *Staph. epidermidis* was less to that obtained with the biosurfactants

*Candida sphaerica* at different concentrations (mg/l). Results are expressed as means ± standard

deviations of values obtained from triplicate experiments

32.1% of the growth of *L. reuteri* ML1 at the maximum concentration tested (12 mg/l).

*epidermidis*, respectively.

microorganisms (Table 2).

The evaluation of the antimicrobial activity of these compounds was carried out against 29 bacteria. *Enterococcus faecalis* (11 strains), *Staphylococcus aureus* (6 strains) and *Pseudomonas aeruginosa* (7 strains) and *Escherichia coli* CI 18 (1 strain) displayed a profile of well defined drug resistance. All strains were sensitive to the surfactants, in particular *Enterococcus faecalis*. The results demonstrated that lipopeptides have a broad spectrum ofaction, including antimicrobial activity against microorganisms with multidrug-resistant profiles [26].

The antimicrobial activity of the crude biosurfactant isolated from *Candida lipolytica* UCP 0988 was determined by measuring the growth inhibition percentages obtained for several microorganisms (Table 1).


**Table 1.** Percentages of growth inhibition obtained with the biosurfactant Rufisan isolated from *Candida lipolytica* at different concentrations (mg/l). Results are expressed as means ± standard deviations of values obtained from triplicate experiments

The biosurfactant was effective against the microorganisms tested, albeit to different degrees. The highest anti-adhesive percentages were obtained for a biosurfactant concentration of 12 mg/l or 4xCMC. Non-pathogenic species associated with the oral cavity of *Streptococcus* were used (*S. mutans* HG – 64.9%; *S. oralis* J22 – 62.8%; *S. mutans* – 58%; *S. sanguis* 12 – 48%; *S. mutans* NS – 46%). On the other hand, the biosurfactant did not show an effective antimicrobial activity against the *Lactobacillus* strains studied. It inhibited only 32.1% of the growth of *L. reuteri* ML1 at the maximum concentration tested (12 mg/l).

248 Practical Applications in Biomedical Engineering

microorganisms (Table 1).

characteristic structure of lipopeptides is a fatty acid combined with an amino-acid moiety. Several lipopeptides have potent antibiotic activity and have been the subject of several studies on the discovery of new antibiotics. The list includes surfactin, produced by *B. subtilis*, the most powerful of biosurfactant known to date. These compounds have many pharmacological activities: antibacterial, antifungal, antiviral, and antimycoplasma properties; inhibition of the fibrin clot formation and hemolysis; formation of ion channels in lipid bilayer membranes [24]; antitumour activity against Ehrlich's ascites carcinoma cells;

The evaluation of the antimicrobial activity of these compounds was carried out against 29 bacteria. *Enterococcus faecalis* (11 strains), *Staphylococcus aureus* (6 strains) and *Pseudomonas aeruginosa* (7 strains) and *Escherichia coli* CI 18 (1 strain) displayed a profile of well defined drug resistance. All strains were sensitive to the surfactants, in particular *Enterococcus faecalis*. The results demonstrated that lipopeptides have a broad spectrum ofaction, including

The antimicrobial activity of the crude biosurfactant isolated from *Candida lipolytica* UCP 0988 was determined by measuring the growth inhibition percentages obtained for several

and inhibition of the cyclic adenosine 3,5-monophosphate phosphodiesterase [25].

antimicrobial activity against microorganisms with multidrug-resistant profiles [26].

**Table 1.** Percentages of growth inhibition obtained with the biosurfactant Rufisan isolated from *Candida lipolytica* at different concentrations (mg/l). Results are expressed as means ± standard

The biosurfactant was effective against the microorganisms tested, albeit to different degrees. The highest anti-adhesive percentages were obtained for a biosurfactant

deviations of values obtained from triplicate experiments

The growth of the other microorganisms tested was poorly inhibited. Percentages of 5%, 5%, 15%, 16% and 18% were observed for *C. albicans, E. coli*, *S. aureus, P. aeruginosa* and *S. epidermidis*, respectively.

The antimicrobial activity of the biosurfactant isolated from *Candida sphaerica* was determined by measuring the growth inhibition percentages obtained for several microorganisms (Table 2).


**Table 2.** Percentages of growth inhibition obtained with the crude biosurfactant Lunasan isolated from *Candida sphaerica* at different concentrations (mg/l). Results are expressed as means ± standard deviations of values obtained from triplicate experiments

The tested biosurfactant presented antimicrobial activity against all microorganisms used, although, depending on the microorganism, the biosurfactant presents different effective concentrations. The highest concentration of biosurfactant tested (10 mg ml-1) showed high percentages of inhibition for *Streptococcus oralis* J22 (68%)*, C. albicans* (57%) *and Staphylococcus epidermidis (*57.6%). The antimicrobial activity of the crude biosurfactant isolated from *Candida sphaerica* with concentrations between 5 and 10 mg ml1 against *C. albicans, Staph. aureus* and *Staph. epidermidis* was less to that obtained with the biosurfactants

isolated from *Lact. paracasei* ssp A20, which completely inhibited the growth of those microrganisms with concentrations between 25 and 50 mg ml-1) [27] .

Antimicrobial and Anti-Adhesive Potential of a Biosurfactants Produced by *Candida* Species 251

Biosurfactants have been found to inhibit the adhesion of pathogenic organisms to solid surfacesor to infection sites, thus prior adhesion of biosurfactant to solid surfaces might constitute a new and effective means of combating colonization by pathogenic microorganisms [12]. Precoating vinyl urethral catheters by running a surfactin solution through them before inoculation with media resulted in a decrease of the amount of biofilm formed by *Salmonella typhimurium*, *Salmonella enteric, Escherichia coli* and *Proteus mirabilis* [38]. Given the importance of opportunistic infections with *Salmonella* species, including urinary tract infections of AIDS patients, these results have great potential for practical applications.

In addition to the antimicrobial properties, the anti-adhesive activity of the biosurfactant was evaluated against a variety of bacterial and fungal strains. The biosurfactant showed anti-adhesive activity against most of the microorganisms tested, but the anti-adhesive effect

**Table 3.** Anti-adhesive properties of crude biosurfactant isolated from *Candida lipolytica*. Negative controls were set at 0% to indicate the absence of biosurfactant. Positive percentages indicate the reductions in microbial adhesion when compared to the control. Results are expressed as means ±

The crude biosurfactant showed anti-adhesive activity against most of the microorganisms tested from the minimum concentration used (0.75 mg/l). The anti-adhesive property was proportional to the concentration of the biosurfactant. For the microorganisms of the *Lactobacillus* anti-adhesive values around 81% were observed at the minor concentration tested (0.75 mg/l). The major anti-adhesive specificity was observed against *L. casei* with

standard deviation of results from triplicate experiments

depends on the concentration and the micro-organism tested (Table 3).

The crude biosurfactant showed antimicrobial activity against a broad range of microorganisms, including Gram-positive and Gram-negative bacteria and yeasts.

Biosurfactants antimicrobial activity has been described, as for example surfactin, a *cyclic*  lipopeptide produced by *Bacillus subtilis* [28]. The antimicrobial activity of surfactin was tested against several microbes. All tested bacteria, except for *B. subtilis*, showed susceptibility to surfactin. *P. aeruginosa* was the most sensitive Gram-negative bacteria, while *E. coli*, *Salmonella choterasius* and *Serratia marcescens* were inhibited in a lower level. Also, the lipopeptide affected the growth of Gram-positive bacteria, especially *Micrococcus luteus* and *Bacillus cereus* [18]. Other examples have been reported by Rodrigues and coworkers [8, 27]. Crude biosurfactants isolated from *Lactococcus lactis* 53 and *Streptococcus thermophilus* A showed antimicrobial activity against *C. troplicalis* GB in low concentrations.

Some biosurfactants are able, even in low concentrations, to destabilize the microorganism's membranes, killing them or disabling their growth [29, 30]. The interest in biosurfactants was first expressed due to its potential antimicrobial properties, being the first reported and actually the most studied biosurfactants, rhamnolipid and surfactin [31]. Gram-positive bacteria are more sensitive to biosurfactants than Gram-negative bacteria, which are weakly inhibited or not inhibited at all [32]. *C. bombicola* and *C. apicola* were reported to produce a glycolipid-type biosurfactant (sophorolipid) that inhibit the growth of *B. subtilis*, *S. epidermidis* and *Streptococcus faecium* in concentrations between 6 and 29 mg/l [33]. Other glycolipids inhibit not only the growth of Gram-positive bacteria, but also Gram-negative ones, such as *E. coli* and *S. marcescens* [31]. Kitamoto et al. [34] reported in their work an antimicrobial activity against *S. aureus*, *E. coli*, *P. aeruginosa* and *C. albicans* for a mannosylerythritol roduced by *C. antarctica*, a sophorolipid produced by *C. apicola* and a rhamnolipid produced by *P. aeruginosa.* 

Several biosurfactants that exhibit antimicrobial activity have been previously described. However, there are few reports about the antimicrobial activity of biosurfactants isolated from *Candida;* only biosurfactants obtained from *S. thermophilus* A and *L. lactis* 53 showed significant antimicrobial activity against several bacterial and yeast strains isolated from explanted voice prostheses [35].
