Chemistry
Chemistry,1970-2014-Chemistry-2012.jsonl.gz.gz
Chemistry
a63a1274cfd31b0d6091c8068f49a11bae415cad
Physicochemical Parameters for Biological and Synthetic Surfactants Used as Detergents

: Biological and synthetic surfactants were compared in terms of their ability to reduce interfacial tension, change the thermodynamic characteristics of a pre-conditioned surface, and to modify the rheological properties of their respective formulations at two different temperatures. Both classes of surfactants were able to reduce the interfacial tension of their formulations to a similar level. However, the biosurfactants were more effective than the synthetics surfactants. Biosurfactants also altered the surface properties of stainless steel, rendering it hydrophilic. Microbial adhesion to stainless steel conditioned with biosurfactants was found to be thermodynamically unfavorable for all microbial strains tested. A linear relationship between shear stress and shear rate was obtained across a range of experimental conditions for all surfactant mixtures, indicating that all formulations behaved as Newtonian fluids.


Introduction
The contamination of surfaces by spoilage and pathogenic microorganisms is a concern in the food industry. The development of biofilms in food processing environments results in product spoilage and possible risks to public health, in addition to create a number of serious problems for industrial fluid processing operations [1][2][3]. The presence of organisms such as Salmonella spp., Listeria monocytogenes and Escherichia coli in food processing environments can be a persistent source of contamination [4].
To reduce or eliminate microorganisms found on food contact surfaces, cleaning and disinfection procedures using physical and chemical methods have been extensively used over the years [5]. An interesting strategy is the pre-treatment of surfaces using surface-active compounds, also known as surfactants.
Surfactants can be classified into two main groups: synthetic surfactants and biosurfactants. Synthetic Nitschke et al. [13] investigated the effect of the biosurfactant surfactin on the adhesion of the food pathogens Listeria monocytogenes, Enterobacter sakazakii and Salmonella Enteritidis to stainless steel and polypropylene surfaces. The pre-conditioning of stainless steel surfaces with surfactin caused a reduction in the number of adhered cells of Enterobacter sakazakii and Listeria monocytogenes. The most significant result was obtained with L. monocytogenes, where the number of adhered cells was reduced by 10 2 CFU/cm 2 . On polypropylene surfaces, surfactin significantly decreased the adhesion of all strains.
In the literature, there are no references that describe the thermodynamic properties of surfaces that have been pre-conditioned with surfactants. This work aimed to compare biological and synthetic surfactants in terms of their ability to reduce interfacial tension, change the hydrophobicity of pre-conditioned surface and alter the rheological properties of their respective formulations at two different temperatures.

Materials
A rhamnolipid biosurfactant, consisting of a mixture of mono-and di-rhamnolipids from the glycolipid group produced by Pseudomonas aeruginosa was obtained from Jeneil Biosurfactant Co. (Saukville, Winconsin, USA) [14]. The molecular formulas for mono-and di-rhamnolipids are C 26 H 48 O 9 and C 32 H 58 O 13, respectively ( Fig. 1). The critical micelle concentration (CMC) was found to be 0.035 g/L by conductivity measurements performed at various dilutions [15].
The synthetic surfactant used was dodecylbenzene sodium sulfonate, and its CMC is 0.4 g/L.

Detergent Formulations
The detergent formulations used in the following experiments are shown in Table 1. The sodium carbonate concentration was selected to match the concentration typically used in the food industry for cleaning procedures. The concentrations chosen for other surfactants were above their respective CMCs to ensure the formation of micelles.

Interfacial Tension Measurements of the Detergent Formulations
The interfacial tension of each detergent formulation was measured by the pendant drop method using a goniometer (DSA 100, Kruss ® , Germany). The surface tension measurement was carried out at 25 ± 1 °C. To calibrate the instrument, the surface tension of pure distilled Milli-Q water (Millipore, Illinois, USA) was measured before each set of experiments. Coupons (10 mm  10 mm  0.05 mm) of stainless steel #4 were first cleaned by washing with a neutral liquid detergent and water, followed by rinsing with distilled water. Subsequently, they were put in a muffle furnace for 2 h at 200 °C to remove organic materials. After that, the coupons were immersed in detergent formulations (Table 1) for 24 h at 20-25 °C. Control coupons without treatment were maintained in distilled water under the same conditions.
Surface hydrophobicity was determined based on contact angle measurements of the stainless steel with water, formamide and α-bromonaphthalene using a goniometer. The total Gibbs free energy of interaction (∆G sws TOT ) and the total free energy of adhesion between stainless steel and different bacteria (∆G adhesion ) were determined by appropriate formulas [17].

Hydrophobicity of the Bacterial Strains
One milliliter of each bacterial culture was stored at -80 °C in a nutrient broth (Merck, São Paulo, Brazil) containing glycerol (80:20). A working culture of each strain, containing approximately 10 7 CFU/mL, was prepared by inoculating 10 mL of Brain Hearth Infusion (BHI, Merck, São Paulo, Brazil) with 100 µL of frozen culture, followed by incubation at 32 °C for 24 h. The culture was sub-cultured three times before use. The number of microorganisms in each suspension was obtained by total count using Plate Count Agar (PCA, Merck, São Paulo, Brazil) at 32 °C for 24 h.
Contact angles for microorganism surfaces were determined on a layer of vegetative cells using the drop method [18]. First, the strains were activated twice in BHI broth (Merck, São Paulo, Brazil), creating suspensions with approximately 10 7 CFU/mL. The suspensions were centrifuged at 12,000 g for 10 min and washed three times in 0.1 M phosphate buffered saline (PBS). The cell pellet was suspended in the same buffer, and the suspension was filtered using a cellulose acetate membrane (0.45 µm pore size, 27 mm in diameter, Millipore, Brazil) with negative pressure. During the filtration, 30 mL of pure water (Milli-Q) was added. The membranes were transferred to Petri plates containing 1% (v/v) of agar and 10% (v/v) of glycerol. The membranes were cut into three parts for contact angle measurements.
Each surface was classified as hydrophobic or hydrophilic according to the determination of the total free energy of interaction (∆G sws TOT ). The total free energy of interaction among molecules of a surface (s) immersed in water (w) was determined by the sum of the apolar and polar free energies of interaction, ∆G sws LW and ∆G sws AB , respectively: Here, γ LW is the interfacial tension arising from Lifshitz-van der Waals forces; γ + is the interfacial tension arising from the electron acceptor component of the acid-base interaction; γis the interfacial tension of the electron donor component of the acid-base interaction; and s and w indicate surface and water, respectively [17].
When ∆G sws TOT > 0, the surface is considered hydrophilic; conversely, if ∆G sws TOT < 0, then the surface is considered hydrophobic. Bacteria were used to determine the total Gibbs free energy of adhesion (∆G adhesion ) between conditioned stainless steel and each strain.

Determination of the Total Gibbs Free Energy of Adhesion (∆G adhesion )
From the values of the individual interfacial tension components, it is possible to determine the ∆G adhesion between two surfaces: microbial cells (b) and food processing surfaces (s): Because the free energy is related to the interfacial tension, ∆G adhesion can be represented by the following: Here,  bs is the interfacial tension between the bacterial surfaces and the adhesion surface;  bl is the interfacial tension between the bacterial surfaces and the liquid; and  sl is the interfacial tension between the adhesion surfaces and the liquid.
The ∆G adhesion values allow for an evaluation of the thermodynamics of the adhesion process: if ∆G adhesion < 0, then the process is favorable; if ∆G adhesion > 0, then the process is unfavorable.
All measurements were performed in duplicate with three repetitions.

Rheological Measurements of the Detergent Formulations
Rheological measurements were performed using a Modular Advanced Rheometer System (HAAKE MARS, Thermo Electron Corp., Germany). The experiments were carried out at 25 °C and 70 °C for the different surfactant formulations, which correspond to manual cleaning temperatures and cleaning-in-place temperatures, respectively. Steady rheological measurements were performed with double gap cylinder system (DG41) geometry, using a gap size set to 0.8 mm. Flow curves were evaluated by measuring the shear rates from 0 to 1,200 s -1 . N ew samples were used for measurements at each experimental temperature. Rheological parameter models were calculated from data using ThermoHaake RheoWin Pro 297 Software by the least squares method.

Interfacial Tension of Detergent Formulations
We observed that all surfactant formulations had interfacial tensions that were reduced relative to the control. However, formulations B, C and D produced greater reductions in interfacial tension than formulation A (Table 2). Although there is not a significant difference (P > 0.05) among the interfacial tensions of formulations B, C and D, biosurfactants are clearly advantageous as they are effective at low concentrations. It is important to emphasize that formulations C and D employed biosurfactant concentrations 50-fold and 10-fold lower, respectively, than the synthetic surfactant concentration in formulation B. Normally, sodium carbonate is used in the food industry to clean surfaces. In this work, we verified that the addition of a biosurfactant to a sodium carbonate formulation reduced its interfacial tension, thereby making the hygiene procedure easier and more efficient.
The results of this experiment were similar to the findings of Pornsunthorntawee et al. [6]. These researchers found that, in the case of the crude biosurfactant produced by Pseudomonas aeruginosa, the surface tension rapidly decreased as the concentration of biosurfactants increased. Furthermore, they found that a minimum surface tension of 29.0 mN/m was obtained at an initial surfactant concentration greater than 0.02%. In our experiment formulation D with a rhamnolipid concentration of 0.02% (Table 2) decreased the interfacial tension to 28.09 mN/m (46%) when compared with formulation A.

Hydrophobicity of Conditioned Stainless Steel Surfaces
The conditioning of stainless steel changed its hydrophobicity. The coupons of stainless steel that were immersed in water (control formulation) were classified as hydrophobic because the ∆G sws TOT value was negative, whereas coupons of stainless steel conditioned with the detergent formulations were considered hydrophilic (∆G sws TOT > 0). In particular, treatment with a 0.004% formulation of rhamnolipid (Formulation C) resulted in a more hydrophilic surface (42.48 MJ/m 2 ) ( Table 3). According to thermodynamic principles, when TOT sws G  is negative, there is a net cohesive attraction between the molecules of a surface when immersed in water. Thus, for a hydrophobic material, TOT sws G  has a negative sign. In other words, molecules of a hydrophobic surface, when immersed in water, prefer to interact with each other rather than form an interface with water, whereas a positive value of TOT sws G  shows that the molecules of a surface prefer to interact with adjacent water molecules rather than with themselves. Such a surface is hydrophilic [19].
The contact angle measurements are interpreted directly in terms of the surface energy of the adsorbed layer and provide access to equilibrium or steady-state adsorption because angle measurements do not perturb adsorption dynamics. The treatment of stainless steel with biosurfactants causes its surface properties to change [13]. The addition of a synthetic or biological surfactant alters the wettability of solids because of its adsorption at the solid/liquid interface. Adsorption profiles suggest that the hydrocarbon chains of biosurfactants attach to hydrophobic surfaces and that their polar groups project toward aqueous solutions. This combination of events makes a surface more hydrophilic. The orientation of surfactant molecules at the solid/liquid interface determines the ability of the surfactant to improve the wetting ability of the liquid. A phenomenon involving a change in orientation of the adsorbed molecules could also be important in the kinetics of the wetting process [20].

Total Gibbs Free Energy of Adhesion
The adhesion process was thermodynamically favorable (∆G adhesion < 0) for all tested strains on stainless steel surfaces conditioned with formulation B or a control formulation (Table 4). In contrast, the adhesion process was unfavorable (∆G adhesion > 0) on stainless steel surfaces conditioned with formulations C or D. According to thermodynamic theory, adhesion is favorable only if the process results in a decrease in total free energy. Therefore, the rhamnolipid biosurfactant may potentially be incorporated into detergent formulations to reduce or avoid microbial adhesion and subsequent biofilm formation.

Rheological Characteristics of the Detergent Formulations
A linear relationship between shear stress and shear rate was obtained across a range of experimental conditions for all surfactant mixtures, indicating that all formulations behaved as Newtonian fluids (Figs. 2 and  3). Penott-Chang et al. [21] observed similar As expected, an increase in temperature decreased the viscosity of all tested solutions. In an industrial context, a decreased viscosity can lead to more efficient cleaning procedures. More specifically, by decreasing the absolute viscosity (µ) at elevated temperatures, solvent flow across cleaning surfaces may be increased, thereby reducing pump power consumption and therefore energy costs.

Surfactant
formulations containing low concentrations of rhamnolipids achieved reductions in interfacial tension comparable to a formulation containing a higher concentration of a synthetic surfactant. The pre-conditioning with biosurfactant converted microbial adhesion to stainless steel into a thermodynamically unfavorable process. Moreover, at   room temperature, a detergent formulation containing a low rhamnolipid concentration exhibited a reduced viscosity relative to a control. This formulation potentially offers a great advantage for cleaning procedures within the food industry. Therefore, a biosurfactant can be superior to a synthetic surfactant for preventing or delaying microbial colonization of food contact surfaces.