**4. Anti-adhesive activity of biosurfactant**

Involvement of biosurfactants in microbial adhesion and desorption has been widely described, and adsorption of biosurfactants to solid surfaces might constitute an effective strategy to reduce microbial adhesion and combating colonization by pathogenic microorganisms, not only in the biomedical field, but also in other areas, such as the food industry [36, 37].

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.

250 Practical Applications in Biomedical Engineering

concentrations.

rhamnolipid produced by *P. aeruginosa.* 

**4. Anti-adhesive activity of biosurfactant** 

explanted voice prostheses [35].

industry [36, 37].

isolated from *Lact. paracasei* ssp A20, which completely inhibited the growth of those

The crude biosurfactant showed antimicrobial activity against a broad range of micro-

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

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

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

Involvement of biosurfactants in microbial adhesion and desorption has been widely described, and adsorption of biosurfactants to solid surfaces might constitute an effective strategy to reduce microbial adhesion and combating colonization by pathogenic microorganisms, not only in the biomedical field, but also in other areas, such as the food

microrganisms with concentrations between 25 and 50 mg ml-1) [27] .

organisms, including Gram-positive and Gram-negative bacteria and yeasts.

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


**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 ± standard deviation of results from triplicate experiments

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 values of 91% and 99% with the minimum concentration used. Low inhibitions were observed for *S. epidermidis* and *E. coli*, with values of 27% and 21%, respectively, at the maximum biosurfactant concentration. For the other microorganisms, the anti-adhesive activity was above 45%.

Antimicrobial and Anti-Adhesive Potential of a Biosurfactants Produced by *Candida* Species 253

predators, biocides and extreme conditions [22]. The antiadhesive activity of this biosurfactant was evaluated against a variety of bacterial and fungal strains. The biosurfactant showed antiadhesive activity against most of the microrganisms tested, but the antiadhesive effect depends on the concentration and the microrganism tested (Table 4). This biosurfactant was effective against all the microorganisms tested, albeit to different degree. With regard to the *Lactobacillus* strains, the antiadhesive activity was higher against *Lact. casei* ( 90%), *Lact. casei* 72 (72%), *Lact. reuteri* 104R (55%) and *Lact. reuteri* ML1 (40%). For the pathogenic bacteria studied (*Streptococcus agalactiae*, *Streptococcus mutans*, *Streptococcus mutans* NS, *Streptococcus sanguis* 12, *Streptococcus salivarius* GB and *Echerichia coli*), a complete inhibition of adhesion was also achieved with biosurfactant concentrations of 10 mg ml-1.

Regarding the yeast, a total inhibition of adhesion was also observed for *C. tropicalis* GB at a biosurfactant concentration of 10 mg ml-1. The highest percentages of adhesion inhibition were obtained for *P. aeruginosa* (92%), *Staphylococcus aureus* (92%), *Streptococcus oralis* J22 (97%) and *Rothia dentocariosa* GBJ (72%), while low activity was obtained for *Streptococcus* 

The antiadhesive activity of the crude biosurfactant isolated from *C. sphaerica* completely inhibited the adesion with a concentration of 10 mg ml -1 against *Streptococcus agalactiae, Streptococcus mutans, Streptococcus mutans* NS, *Streptococcus sanguis* 12, *Streptococcus salivarius* GB and *Echerichia coli.* These results were higher to that obtained with the biosurfactants isolated from *Lact. paracasei* ssp A20 [24]. A role of biosurfactants as defense weapons in competition with post-adhesion has been suggested for biosurfactants produced by

Besides possessing *antifungal, antibacterial and antivir*al *activities*, biosurfactants have also proved to be great inhibitors of microbial adhesion and of biofilm formation. For example, the biosurfactant released by *S. mitis* was found to reduce the adhesion of *Streptococcus. mutans* [40]. Similarly, *Lactobacillus fermentum* RC-14 releases surfactant compounds that can

The adsorption of a biosurfactant on surface was found to change its hydrophobicity, which might caused interference in the adhesion and desorption processes [41]. Furthermore, Velraeds et al. [42] reported the inhibition of adhesion of pathogenic enteric bacteria by a biosurfactant produced by *Lactobacillus fermetum* RC-14. The authors suggested the use of this anti-adhesive agent in catheters aiming at *decreasing* biofilm formation*.* Falagas and Makris [37] have proposed the application of biosurfactants isolated from probiotic bacteria to patient care equipments (such as catheters and other medical insertional devices) in hospitals, with the aim of decreasing colonization by microorganisms responsible for nosocomial infections.

In conclusion, in this work we have demonstrated the antimicrobial and anti-adhesive properties of the crude biosurfactant isolated from *C. lipolytica* UCP0988 against several pathogenic and nonpathogenic microorganisms, including bacteria, yeasts and filamentous fungi. The results obtained suggest the possible use of this biosurfactant as an alternative

inhibit the adhesion of uropathogenic bacteria*,* including *Enterococcus faecalis*.

*mutans* HG 985(50%) and *Staphylococcus epidermidis* GB(22%).

*Streptococcus mitis* and *S. mutans* [39].

**5. Conclusion** 

Gudina et al. [24] observed an anti-adhesive activity for the biosurfactant from *Lactobacillus paracasei* against several pathogenic microorganisms such as *S. aureus*, *S. epidermidis* and *S. agalactiae*. However, this biosurfactant showed low anti-adhesive activity against *E. coli*, *C. albicans* and *P. aeruginosa*, in contrast with the antimicrobial activity exhibited against these strains at the same biosurfactant concentrations.

The use and potential commercial applications of biosurfactants in the medical field has increased considerably in the last years. Their antimicrobial and anti-adhesive properties make them relevant molecules for use in combating many diseases and infections and as therapeutic agents [18].


**Table 4.** Anti-adhesive properties of crude biosurfactant isolated from *Candida sphaerica*. 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

Adhesion to surfaces and subsequent biofilm formation consist in a surviving strategy used by microorganisms in several hostile environments, protecting them from dehydration, predators, biocides and extreme conditions [22]. The antiadhesive activity of this biosurfactant was evaluated against a variety of bacterial and fungal strains. The biosurfactant showed antiadhesive activity against most of the microrganisms tested, but the antiadhesive effect depends on the concentration and the microrganism tested (Table 4).

This biosurfactant was effective against all the microorganisms tested, albeit to different degree. With regard to the *Lactobacillus* strains, the antiadhesive activity was higher against *Lact. casei* ( 90%), *Lact. casei* 72 (72%), *Lact. reuteri* 104R (55%) and *Lact. reuteri* ML1 (40%). For the pathogenic bacteria studied (*Streptococcus agalactiae*, *Streptococcus mutans*, *Streptococcus mutans* NS, *Streptococcus sanguis* 12, *Streptococcus salivarius* GB and *Echerichia coli*), a complete inhibition of adhesion was also achieved with biosurfactant concentrations of 10 mg ml-1.

Regarding the yeast, a total inhibition of adhesion was also observed for *C. tropicalis* GB at a biosurfactant concentration of 10 mg ml-1. The highest percentages of adhesion inhibition were obtained for *P. aeruginosa* (92%), *Staphylococcus aureus* (92%), *Streptococcus oralis* J22 (97%) and *Rothia dentocariosa* GBJ (72%), while low activity was obtained for *Streptococcus mutans* HG 985(50%) and *Staphylococcus epidermidis* GB(22%).

The antiadhesive activity of the crude biosurfactant isolated from *C. sphaerica* completely inhibited the adesion with a concentration of 10 mg ml -1 against *Streptococcus agalactiae, Streptococcus mutans, Streptococcus mutans* NS, *Streptococcus sanguis* 12, *Streptococcus salivarius* GB and *Echerichia coli.* These results were higher to that obtained with the biosurfactants isolated from *Lact. paracasei* ssp A20 [24]. A role of biosurfactants as defense weapons in competition with post-adhesion has been suggested for biosurfactants produced by *Streptococcus mitis* and *S. mutans* [39].

Besides possessing *antifungal, antibacterial and antivir*al *activities*, biosurfactants have also proved to be great inhibitors of microbial adhesion and of biofilm formation. For example, the biosurfactant released by *S. mitis* was found to reduce the adhesion of *Streptococcus. mutans* [40]. Similarly, *Lactobacillus fermentum* RC-14 releases surfactant compounds that can inhibit the adhesion of uropathogenic bacteria*,* including *Enterococcus faecalis*.

The adsorption of a biosurfactant on surface was found to change its hydrophobicity, which might caused interference in the adhesion and desorption processes [41]. Furthermore, Velraeds et al. [42] reported the inhibition of adhesion of pathogenic enteric bacteria by a biosurfactant produced by *Lactobacillus fermetum* RC-14. The authors suggested the use of this anti-adhesive agent in catheters aiming at *decreasing* biofilm formation*.* Falagas and Makris [37] have proposed the application of biosurfactants isolated from probiotic bacteria to patient care equipments (such as catheters and other medical insertional devices) in hospitals, with the aim of decreasing colonization by microorganisms responsible for nosocomial infections.

### **5. Conclusion**

252 Practical Applications in Biomedical Engineering

strains at the same biosurfactant concentrations.

activity was above 45%.

therapeutic agents [18].

values of 91% and 99% with the minimum concentration used. Low inhibitions were observed for *S. epidermidis* and *E. coli*, with values of 27% and 21%, respectively, at the maximum biosurfactant concentration. For the other microorganisms, the anti-adhesive

Gudina et al. [24] observed an anti-adhesive activity for the biosurfactant from *Lactobacillus paracasei* against several pathogenic microorganisms such as *S. aureus*, *S. epidermidis* and *S. agalactiae*. However, this biosurfactant showed low anti-adhesive activity against *E. coli*, *C. albicans* and *P. aeruginosa*, in contrast with the antimicrobial activity exhibited against these

The use and potential commercial applications of biosurfactants in the medical field has increased considerably in the last years. Their antimicrobial and anti-adhesive properties make them relevant molecules for use in combating many diseases and infections and as

**Table 4.** Anti-adhesive properties of crude biosurfactant isolated from *Candida sphaerica*. Negative controls were set at 0% to indicate the absence of biosurfactant. Positive percentages indicate the

Adhesion to surfaces and subsequent biofilm formation consist in a surviving strategy used by microorganisms in several hostile environments, protecting them from dehydration,

reductions in microbial adhesion when compared to the control

In conclusion, in this work we have demonstrated the antimicrobial and anti-adhesive properties of the crude biosurfactant isolated from *C. lipolytica* UCP0988 against several pathogenic and nonpathogenic microorganisms, including bacteria, yeasts and filamentous fungi. The results obtained suggest the possible use of this biosurfactant as an alternative

antimicrobial agent in the medical field for applications against microorganisms responsible for diseases and infections, making it a suitable alternative to conventional antibiotics.

Antimicrobial and Anti-Adhesive Potential of a Biosurfactants Produced by *Candida* Species 255

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