Rapid screening of surfactant and biosurfactant surface cleaning performance

https://doi.org/10.1016/j.colsurfb.2009.03.015Get rights and content

Abstract

Surface Plasmon Resonance (SPR) and rubisco protein stain were used as tools to screen the effectiveness of detergent formulations in cleaning a protein stain from solid surfaces. Surfactant and biosurfactant-based formulations, with and without added protease, were screened for cleaning performance. Enzyme-free detergent formulations at 1500Ā ppm total surfactant were insufficient to cause complete surface cleaning, despite the high concentration of surfactant. The cleaning performance of a ā€œhome-madeā€ formulation containing 2Ā ppm subtilisin A (SA) and 2Ā ppm sodium dodecyl benzyl sulphonate (SDOBS) was as efficient as the best amongst the three enzyme-free 1500Ā ppm formulations. The cleaning performance of 2Ā ppm SA in the absence of SDOBS was less effective than the combined formulation, even though 2Ā ppm SDOBS alone did not cause any protein removal. The observed synergistic performance was attributed to the cooperative mechanisms (chemical and physical attack) by which these two agents act on a rubisco stain. Replacing SDOBS in the enzyme-surfactant formulation with the same amount of surfactin biosurfactant (2Ā ppm) gave the best rubisco removal of all formulations examined in this study, irrespective of the surface chemistry underlying the protein film. It was found that 75% and 80% of immobilised rubisco stain could be removed from hydrophobic and hydrophilic surfaces, respectively, by the biosurfactant-SA formulation (compared with 60% and 65%, respectively, using the SDOBS-SA formulation). Our results suggest that it may be possible to generate fully renewable biochemical-based cleaning formulations that have superior cleaning performance to existing technologies. In developing optimised formulations, there is a pressing need for chip-based tools similar to that developed in this research.

Introduction

Protein-fouled surfaces that require cleaning occur ubiquitously. For example, protein fouling on surfaces of food and process equipment, surgical and dental tools, fabrics, dishes, and contact lenses all present a challenge to cleaning technologies. Surface-bound protein stains can be removed, to different extents, by surfactants, enzymes, or surfactant-enzyme mixtures. The performance of cleaning agents differs according to the degree of the mutual interactions between the system components (solid surface, protein, enzyme and surfactants). Understanding such differences might aid in optimising protein removal from solid surfaces, and hence it is necessary to screen and assess the performance of the cleaning agents on different stain-surface systems. Such screening studies require relevant quantitative tools (e.g., a relevant model protein stain and suitable technique), as developed in this study.

Rubisco is found in all photosynthetic leaves and represents up to 65% of the total soluble proteins in leaf extracts [1], making it a good representative of the class of grassy stains. It also forms a naturally tough stain since it irreversibly adsorbs at the air-liquid [2] as well as the solidā€“liquid interfaces. Rubisco has received little research attention and only few published studies have considered it [3], [4], [5], [6]. Kirchman et al. [3] studied the adsorption of rubisco from seawater on different surfaces and found that adsorption increased as surface hydrophobicity increased. They pointed out that surface hydrophobicity controls adsorption in seawater, consistent with the phenomenon of charge screening in high-salt solutions. When the authors adsorbed rubisco from low saline buffer, they found no significant relationship between adsorption and surface hydrophobicity, and concluded that some other interactions, probably electrostatic, controlled protein-surface interaction. Electrostatic interactions can dominate protein-surface interactions when the surface and the protein are oppositely charged [7]. Taylor et al. [4] studied rubisco adsorption from seawater, at different concentrations, on different surfaces. The authors reported that rubisco accumulation on solid surfaces is concentration dependent. At low concentrations, the protein exhibited higher affinity for hydrophobic surfaces, which is in agreement with the findings reported by Kirchman et al. [3]. However, at higher concentrations, rubisco accumulated more on hydrophilic surfaces [4]. Such behaviour was attributed to an unknown variation in the physical state of the protein upon adsorption.

Currently, surfactants used in detergent formulations are commonly alkyl aromatic anionic surfactants [8], for example sodium dodecyl benzyl sulphonate (SDOBS). These chemical surfactants are produced from petrochemical precursors, which are limited and non-renewable, resulting in steadily increasing cost inputs. Additionally, environmental restrictions on carbon-depleting chemical manufacture will further increase the production cost of chemical surfactants, necessitating the exploration of alternatives. Biosurfactants are an interesting alternative to chemical surfactants in a wide range of applications [9]. Biosurfactants are produced from renewable resources and occur in several classes, e.g., glycolipids, lipopeptides, and designed peptide surfactants [9]. Surfactin (an anionic cyclic lipopeptide biosurfactant [10]) is one of the most interesting biosurfactants components due to its superior surface activity [9], [10]. However, very little work about the detergency (cleaning) performance of surfactin has been published.

Several approaches for screening the cleaning performance of detergent formulations have been presented. The main differences between these approaches lie in the methodology of creating the protein or peptide stain [11], [12] and the technique used to evaluate formulation cleaning performance. The stain can be created by protein immobilisation onto a relevant solid support, the chemistry of which can be engineered to different characteristics. Processes based on the self-assembly of monolayers (SAMs) offers wide scope for engineering the chemistry of solid supports [13]. Stain immobilisation on solid supports can be achieved via physical (e.g., adsorption or spin-coating) or chemical (covalent linking of the stain into the solid support) methods [11], [12], [14]. Physical and chemical methods can be used together through, for example, stain spin-coating and cross-linking [15]. Several techniques can be used to probe the immobilisation of the stain and its subsequent removal by enzyme and detergent formulations. For example, Wolff et al. [16] used reflectance measurements to evaluate the cleaning performance of proteases in the presence and absence of detergent, and vice versa. In that study the authors used cotton fabric swathes, soiled with different protein stains. Brode and Rauch [11] studied the removal (hydrolysis) of immobilised peptide substrate on aminopropyl controlled pore glass by subtilisin BPNā€² using a standard p-nitroaniline (pNA) assay, which relies on colour production due to the release of pNA from the hydrolysis of pNA-containing substrate [17]. Esker et al. [12] used the Brode and Rauch approach [11] to study the removal of immobilised peptide substrate under a wide range of experimental conditions.

In this study we use a highly surface sensitive technique, Surface Plasmon Resonance (SPR) [18], capable of detecting adsorbed materials at solidā€“liquid interfaces down to a sub-nanometre level. SPR has proven to be a useful technique in studying protein adsorption [19]. It is an optical label-free sensor based on the change in the refractive index of very thin layers of material in contact with a thin metal film [20], [22], [23] (the optimum thickness is āˆ¼50Ā nm [18], [20], [21]), typically gold or silver [24]. The amount of the adsorbed material is linearly proportional to the shift in the SPR angle [24], [25], [26], [27]. Furthermore, the shift in the SPR angle is linearly proportional to the change in the refractive index [24], [28]. At a fixed incident angle, the change in the reflectivity is linearly proportional to the change in the film thickness [29], [30], [31]. Therefore, surface events, e.g. stain immobilisation and removal, can be followed quantitatively in real time. Advantageously, SPR enables decoupling of the various adsorptionā€“desorption processes, and kinetics, which is not always possible using previously reported approaches.

In this work, rubisco and SPR were used as tools to study the cleaning performance of different detergent formulations. We also report the effects of using renewable biosurfactant on the removal of immobilised protein stain from solidā€“liquid interfaces. The effect of surface chemistry (hydrophobicity and charge) on rubisco adsorption and its subsequent removal were also examined. Furthermore, the effects of enzyme concentration and surfactant addition on the extent and rate of rubisco removal from solid surfaces were quantified. The results suggest SPR is a valuable detergent-screening technology able to identify sustainable detergent formulations having superior cleaning performance.

Section snippets

Materials and methods

All reagents and chemicals used were of analytical grade. Water (MQW) was obtained from a Milli-Q (Millipore, Sydney, Australia) system with a 0.22Ā Ī¼m filter and had a resistivity ofĀ >Ā 18.2Ā MĪ©Ā cm.

Results and discussion

Screening the cleaning performance of detergent formulations might lead to the optimisation (through the selection/development of the most efficient formulations) of the cleaning process of protein-stained surfaces. However, systematic and quantitative screening studies, particularly using low levels of cleaning agent formulations, are not fully addressed in the literature. The first step in such studies is the immobilisation of a protein stain onto a suitable solid surface. The chemistry of

Conclusion

Rubisco adsorption at solidā€“liquid interfaces is irreversible, making it an excellent candidate for screening detergent performance since any observed protein removal will be mainly due to the effect of the detergent cleaning action. Protein removal by surfactant is due to the displacement and/or solubilisation mechanism, and therefore, the extent of protein removal depends on surfactant penetration through the immobilised protein network. The results show that surfactant-enzyme mixtures

Acknowledgement

The authors acknowledge the financial support from Procter and Gamble Co (Cincinnati, OH). Anton Middelberg acknowledges support from the Australian Research Council in the form of a Federation Fellowship (grant FF0348465).

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