**4.4 Microemulsions**

352 Biomedical Science, Engineering and Technology

nanoparticles using the bacteria *Bacillus subtilis.* Gold nanoparticles were synthesized both intra- and extracellularly, while silver nanoparticles were exclusively formed extracellularly (Reddy et al., 2010). According to the Authors the nanoparticles were stabilized by the surface-active molecules i.e., surfactin or other biomolecules released into the solution by *B.* 

Surfactin produced by *Bacillus amyloliquefaciens* KSU-109 was also used for the synthesis of cadmium sulfide nanoparticles which remained stable up to six months without compromising their functionality (Singh et al., 2011). This kind of nanoparticles works as semiconductors with unique optical properties and tunable photo-luminescence allowing potential applications in solar energy conversion, nonlinear optical, photoelectrochemical cells and heterogeneous photocatalysis (Singh et al., 2011). In addition, surfactin produced by strain KSU-109 was easily extracted and used without further purification for nanoparticles stabilization under ambient conditions (Singh et al., 2011). Such simple, inexpensive and environmental friendly procedure of obtaining surfactin offers a further advantage of use in

nanobiotechnology for the large-scale production of highly stable metal nanoparticles.

Both rhamnolipids and sophorolipids have also been successfully used for the synthesis and stabilization of metal-bound nanoparticles. Purified rhamnolipids from *P. aeruginosa* strain BS-161R were used to synthesize silver nanoparticles which exhibited good antibiotic activity against both Gram-positive and Gram-negative pathogens and *Candida albicans*, suggesting their broad spectrum antimicrobial activity (Kumar et al., 2010). In another work, a glycolipid biosurfactant produced from sponge-associated marine bacteria *Brevibacterium casei* MSA19, using agro-industrial and industrial waste as substrate, were used as a "green" stabilizer for the synthesis of stable and uniform silver nanoparticles (Kiran et al., 2010b). The biosurfactant acted as stabilization agent and prevented the

Palanisamy & Raichur, (2009) also described a simple and eco-friendly method for synthesizing spherical nickel oxide nanoparticles by microemulsion technique using rhamnolipids as alternative surfactant. The synthesized nanoparticles were found to be fully crystalline and spherical in shape with uniform distribution and increasing the pH of the solution decreased the size of the nanoparticles. Xie et al., (2006) were also able to synthesize silver nanoparticles in rhamnolipid reverse micelles while in another study rhamnolipids were used as capping agents for the synthesis of ZnS nanoparticles in aqueous medium

Sophorolipids were also tested for use in nanoparticles synthesis and reported to be good reducing and capping agents for cobalt and silver particles (Kasture et al., 2007, 2008, as cited in Van Bogaert & Soetaert, 2010). Singh et al., (2009) demonstrated the antibacterial activity of sophorolipid-coated silver and gold nanoparticles against both Gram-positive and –negative bacteria. They also verified that sophorolipid-coated gold nanoparticles were more cyto and geno-compatible with respect to silver nanoparticles (Singh et al., 2010). They also plan to investigate these nanoparticles suitability for medical and diagnostic

Recently, methodologies for the biological synthesis of metal nanoparticles using microbes have also been described (Narayanan & Sakthivel, 2010; Kalishwaralal et al., 2010; Reddy et al., 2010). In addition Smyth et al., (2010b) reported on the production of selectively deuterated rhamnolipids and sophorolipids using deuterated substrates. The production of such deuterated biosurfactants, in particular, or other bioactive microbial products in general, in which distinct pattern of labeling could be achieved resulting in varing molecular

*subtilis*.

formation of aggregates.

(Narayanan et al., 2010).

applications.

Microemulsions are thermodynamically stable, isotropic dispersions of oil, water and surfactant (Rosen, 1989, as cited in Nguyen et al., 2010). Microemulsion systems produce high solubilization capacity and ultralow interfacial tensions of oil and water, making them desirable in practical applications such as enhanced oil recovery, drug delivery, food and cosmetic applications (Bourrel & Schechter, 1988, Kogan & Garti, 2006, Komesvarakul et al., 2006, Lawrence & Rees. 2000, Vandamme, 2002, Yuan et al., 2008, as cited in Nguyen et al., 2010). Xie et al., 2005 demonstrated that rhamnolipids could be successfully used to form microemulsions using medium chain alcohols as cosurfactant. Subsequently, the same Authors observed that the phase behavior and microstructure of these microemulsions were rational to the conformational changes of rhamnolipid molecules at the interface of oil/water (Xie et al., 2007). Microemulsion technique using oil–water–surfactant mixture has also emerged as a promising method for nanoparticle synthesis and can be used to synthesize different types of particles (Eastoe et al., 2006, as cited in Palanisamy & Raichur, 2009). Palanisamy & Raichur, (2009), for example, successfully used rhamnolipids as the surfactant to synthesize spherical nickel oxide nanoparticles by microemulsion technique. In another work, Nguyen & Sabatini, (2009) were able to formulate alcohol-free microemulsions using rhamnolipid biosurfactant and rhamnolipid mixtures.

Lecithin-based microemulsions have proven to be desirable in biocompatible formulations due to their tendency to mimic the phospholipid nature of cell membranes (Nguyen et al., 2010). In a recent report Nguyen et al., (2010) formulated and evaluated microemulsions of lecithin/rhamnolipid/sophorolipid biosurfactants with a range of oils. Sophorolipid played an important role as the hydrophobic component in these formulations and the phase behavior of these biocompatible microemulsions did not change significantly with changing temperature and electrolyte concentration, making them desirable for cosmetic and drug delivery applications.

### **4.5 A survey over biotechnological commercial applications and patents of biosurfactants and bioemulsifiers**

Due to their broad-range of functional properties and the diverse synthetic capabilities of microbes, biological surfactants and emulsifiers have been recently used in various industries like detergents and soaps, petroleum, textile, agriculture, cosmetic, medicine and food (Banat et al., 2000, 2010). Due to their environmental acceptability, biodegradability and lower toxicity, they are generally accepted as good candidates to substitute synthetic surfactants. Commercial applications of biosurfactants and bioemulsifiers in the biotechnological field are mainly related to the oil industry, enhanced oil recovery and bioremediation technologies (Desai & Banat, 1997). However, interesting marketable products and patents have been issued in the last few years in the health care and cosmetic industries, reviewed by Shete et al., (2006) and Banat et al., (2010).

Sugar-based biosurfactants, sophorolipids in particular, are very attractive in these fields, because of their good detergency, emulsifying, foaming and dispersing properties (Faivre & Rosilio, 2010). Sophorolipids are better solubilizers than emulsifiers, but their derivatives containing propylene glycol have excellent hygroscopic properties and are applied as moisturizer or softener in cosmetic products (Faivre & Rosilio, 2010). For example, a product containing 1 mol of sophorolipid and 12 mol of propylene glycol has excellent skin compatibility and is used commercially as a skin moisturizer (Yamane, 1987, as cited in Desai & Banat 1997). Sophorolipid is commercially used by Kao Co. Ltd. as a humectant for cosmetic makeup brands such as Sofina. This company has developed a fermentation process for sophorolipid production, and after a two-step esterification process, the product finds application in lipstick and as moisturizer for skin and hair products (Inoue et al., 1979 a, 1979b, as cited in Desai & Banat, 1997). Moreover, sophorolipids are also believed to stimulate the leptin synthesis through adipocytes, in this way reducing the subcutaneous fat overload (Pellecier & André, 2004, as cited in Van Bogaert & Soetaert, 2010).

The French company Soliance (http://www.groupesoliance.com) produces sophorolipidbased cosmetics for the body and skin and the Korean MG Intobio Co. commercializes Sopholine cosmetics (Van Bogaert & Soetaert, 2010). They are also found in cleaning soap mixtures (EcoverTM products). Despite the high number of scientific publications and patents, industrial surfactin applications still remain quite limited (Jacques, 2010). Sold by SIGMA and SHOWA DENKO for analytical or laboratory purposes, the compound is also available in several Japanese cosmetic products.

During the last decades, many patents have been issued worldwide in relation with applications of biosurfactants and bioemulsifiers in the health care field (Shete et al., 2006). Bioemulsifiers produced by *Acinetobacter calcoaceticus*, for examples, have been used in shampoos and soaps against acne and eczema and in personal care products. The skin cleansing cream and lotion containing these bioemulsifiers have, among other properties, the ability to interfere with microbial adhesion on skin or hair (Hayes et al., 1989, 1990, 1991, 1992, as cited in Shete et al., 2006). Viscosin and analogues have been patented as antibacterial, antiviral, antitrypanosomal therapeutic compounds that inhibit the growth of *Mycobacterium tuberculosis*, Herpes simplex virus 2 and/or *Trypanosoma cruzi* (Burke et al., 1999, as cited in Shete et al., 2006). *Lactobacillus* biosurfactants have also been patented as inhibitors of adherence and colonization of bacterial pathogens on medical devices (Reid et al., 2000).

Another interesting patented area is related to antimicrobial biosurfactant peptides produced by probiotic strains able to selectively bind to collagen and inhibit infections around wounds at the site of implants and biofilms associated with infections in mammals (Howard et al., 2002, as cited in Shete et al., 2006). Sophorolipids, in particular have been the object of many patents as moisturizing agents and for the amelioration of skin physiology, skin restructuration and repair (Shete et al., 2006). Sophorolipids are also used for the treatment of skin, as an activator of macrophages, and as agent in fibrinolytic healing, desquamating and depigmenting process (Maingault, 1999 as cited in Shete et al., 2006). A germicidal composition containing fruit acids, a surfactant and a sophorolipid biosurfactant, able to kill in 30 seconds 100% of *E. coli*, *Salmonella* and *Shigella*, has been patented for cleaning fruits, vegetable, skin and hair (Pierce & Heilman, 2001).

Rhamnolipids in comparison have been patented in a process to make some liposomes and emulsions (Ishigami & Suzuki 1997; Ramisse et al., 2000) both important in the cosmetic industry. More recently an activator and anti-aging agent containing MEL as active ingredient has been patented (Suzuki et al., 2010). Another recent invention is directed to polymeric acylated biosurfactants that can self-assemble or auto-aggregate into polymeric micellar structures useful in topically-applied dermatologic products (Owen & Fan, 2010). Another patent has been deposited about a biosurfactant composition produced by a new *B. licheniformis* strain, with anti-adhesion activity against biofilm producer microbial pathogens (Martinotti et al., 2009).
