Physico-chemical and hygienic property modifications of stainless steel surfaces induced by conditioning with food and detergent

The effect of repeated conditioning procedures (25 runs), consisting of soiling (milk and meat products) and cleaning steps, on the hygienic status, physico-chemical properties and surface chemical composition of stainless steel (SS) surfaces, was investigated. Five SSs differing in grade and finish were used. Both soiling and surface cleaning/conditioning procedures resulted in a similar increase in the surface contamination with carbon, while the changes in the basic component of the surface free energy depended on the conditioning procedure. The passive film was also affected, the Fe/Cr ratio in particular. The hygienic status was also changed, especially with milk as shown by monitoring the number of residual adhering Bacillus cereus spores after contaminating the surface with spores followed by cleaning. The results show that in food environments, the presence and the nature of conditioning molecules play a major role in the hygienic status of SS surfaces.     

Potential occurrence of adhering living Bacillus spores in milk product processing lines

AIMS: The hygienic risk associated with microbial soil on surfaces of milk processing lines was evaluated, based on experimental results. METHODS AND RESULTS: From a panel of Bacillus spores isolated from milk products, B. cereus CUETM 98/4, was found to be highly resistant to heat (D100=3.32 min in whole milk) and oxidant disinfectant (70% lethality of adherent spores with Ikalin 2%). From adhesion trials, up to 1.1 x 10(7) spores cm(-2) were found to be adherent to solid surfaces when suspended in saline or in custard (10(5) and 10(7) cfu ml(-1)), and over 10% of these adherent spores would resist the cleaning procedure. CONCLUSION: A highly contaminated milk (10(5) cfu ml(-1)) subjected to a current sterilization process (8 log reduction) led to a residual contamination of less than 1 cfu in the representative processing line after a complete production run. SIGNIFICANCE AND IMPACT OF THE STUDY: This study highlighted the fact that under appropriate processing conditions (efficient sterilization and cleaning procedures), even disinfection would be sufficient to eliminate any contamination risk. Conversely, the disinfection procedure becomes an essential step under inappropriate processing conditions.     

Influence of physicochemical properties on the hygienic status of stainless steel with various finishes

The relative hygienic status of 16 stainless steel surfaces, characterised by topography and surface free energy was investigated. B. thuringiensis spores suspended in Bechamel sauce was chosen as the test fouling suspension. Surface topography was assessed using 10 standardised roughness parameters, along with scanning electron microscope observations. The number of residual adhering spores after a fouling and cleaning in place procedure was found to be influenced by the topography of the stainless steel surface, but not by the surface free energy. Among the various roughness parameters, R_A, R_RR R_PK and R_VK were shown to be related to the hygienic status. Microscopic observations demonstrated the influence of the shape and size of surface irregularities on the level of residual soil after cleaning. This confirms that the use of only one roughness parameter, usually R_A, is not sufficient in defining the hygienic status of stainless steel surfaces.     

Adhesion of Bacillus spores and Escherichia coli cells to inert surfaces: role of surface hydrophobicity

The ability of bacterial spores and vegetative cells to adhere to inert surfaces was investigated by means of the number of adherent spores (Bacillus cereus and Bacillus subtilis spores) and Escherichia coli cells and their resistance to cleaning or rinsing procedures (adhesion strength). Six materials (glass, stainless steel, polyethylene high density (PEHD), polyamide-6, polyvinyl chloride, and Teflon) were tested. Slight differences in the number of adherent spores (less than 1 log unit) were observed between materials, but a higher number of adherent E. coli cells was found on the hydrophobic materials PEHD and Teflon. Conversely, the resistance of both B. cereus and B. subtilis spores to a cleaning procedure was significantly affected by the material. Hydrophobic materials were harder to clean. The topography parameter derived from the Abbott-Firestone curve, RVK, and, to a lesser extent, the widely used roughness parameters RA (average roughness) and Rz (maximal roughness), were related to the number of adherent cells. Lastly, the soiling level as well as the adhesion strength were shown to depend largely on the microorganism. The number of adhering B. cereus hydrophobic spores and their resistance to a cleaning procedure were found to be 10 times greater than those of the B. subtilis hydrophilic spores. Escherichia coli was loosely bound to all the materials tested, even after 24 h biofilm formation.     

Influence of surface chemistry on the hygienic status of industrial stainless steel

Coupons of fourteen different stainless steels were investigated in terms of surface chemistry and ease of cleaning. Steel surfaces were exposed to Bacillus cereus spores in static saline solution for 2 h. Surfaces were rinsed and then covered with whole milk and allowed to dry. Surfaces were then cleaned in an experimental flow system that mimics an industrial application. After cleaning, remaining spores were released by sonication, spores cultured and colony forming units determined. Surfaces with higher levels of Fe in the outer surface of the passive film cleaned more easily. There was a relation between the polar component and ease of cleaning. The higher the polar component the more easily the surface cleaned. The cleaning mechanism involves dissolution of Fe enriched hydroxide films on the surface.     

Influence of Surface Chemistry on the Hygienic Status of Industrial Stainless Steel

Coupons of fourteen different stainless steels were investigated in terms of surface chemistry and ease of cleaning. Steel surfaces were exposed to Bacillus cereus spores in static saline solution for 2?h. Surfaces were rinsed and then covered with whole milk and allowed to dry. Surfaces were then cleaned in an experimental flow system that mimics an industrial application. After cleaning, remaining spores were released by sonication, spores cultured and colony forming units determined. Surfaces with higher levels of Fe in the outer surface of the passive film cleaned more easily. There was a relation between the polar component and ease of cleaning. The higher the polar component the more easily the surface cleaned. The cleaning mechanism involves dissolution of Fe enriched hydroxide films on the surface.     

Assessment of the potential suitability of selected commercially available enzymes for cleaning-in-place (CIP) in the dairy industry

The potential suitability of 10 commercial protease and lipase products for cleaning-in-place (CIP) application in the dairy industry was investigated on a laboratory scale. Assessment was based primarily on the ability of the enzymes to remove an experimentally generated milk fouling deposit from stainless steel (SS) panels. Three protease products were identified as being most suitable for this application on the basis of their cleaning performance at 40 °C, which was comparable to that of the commonly used cleaning agent, 1% NaOH at 60 °C. This was judged by quantification of residual organic matter and protein on the SS surface after cleaning and analysis by laser scanning confocal microscopy (LSCM). Enzyme activity was removed/inactivated under conditions simulating those normally undertaken after cleaning (rinsing with water, acid circulation, sanitation). Preliminary process-scale studies strongly suggest that enzyme-based CIP achieves satisfactory cleaning at an industrial scale. Cost analysis indicates that replacing caustic-based cleaning procedures with biodegradable enzymes operating at lower temperatures would be economically viable. Additional potential benefits include decreased energy and water consumption, improved safety, reduced waste generation, greater compatibility with wastewater treatment processes and a reduction in the environmental impact of the cleaning process.     

Assessment of the potential suitability of selected commercially available enzymes for cleaning-in-place (CIP) in the dairy industry

The potential suitability of 10 commercial protease and lipase products for cleaning-in-place (CIP) application in the dairy industry was investigated on a laboratory scale. Assessment was based primarily on the ability of the enzymes to remove an experimentally generated milk fouling deposit from stainless steel (SS) panels. Three protease products were identified as being most suitable for this application on the basis of their cleaning performance at 40°C, which was comparable to that of the commonly used cleaning agent, 1% NaOH at 60°C. This was judged by quantification of residual organic matter and protein on the SS surface after cleaning and analysis by laser scanning confocal microscopy (LSCM). Enzyme activity was removed/inactivated under conditions simulating those normally undertaken after cleaning (rinsing with water, acid circulation, sanitation). Preliminary process-scale studies strongly suggest that enzyme-based CIP achieves satisfactory cleaning at an industrial scale. Cost analysis indicates that replacing caustic-based cleaning procedures with biodegradable enzymes operating at lower temperatures would be economically viable. Additional potential benefits include decreased energy and water consumption, improved safety, reduced waste generation, greater compatibility with wastewater treatment processes and a reduction in the environmental impact of the cleaning process.     

The effects of cleaning and disinfection in reducing Salmonella contamination in a laboratory model kitchen

AIMS: To establish a laboratory model to compare the effectiveness of detergent-based disinfection procedures for reducing cross-contamination risks during handling of contaminated chicken. METHODS AND RESULTS: During handling of chickens, artificially contaminated with Salmonella enteritidis PT4, the organism was widely spread to hands, cloths, and hand- and food-contact surfaces. Hygiene procedures were assessed on the basis of their ability to reduce the number of recoverable salmonellas to <1 CFU. Although detergent-based cleaning using a typical bowl-wash routine without rinsing produced some risk reduction (from 100 to 61.4% of contaminated surfaces), it was insufficient to consistently restore surfaces to a hygienic state. By combining detergent-based cleaning with a rinsing step or with hypochlorite at 500 ppm (of available chlorine) some further reduction in microbial risk was achieved, but was not considered satisfactory for food hygiene purposes. By contrast the risk reduction produced by hypochlorite at 5000 ppm was highly significant and was sufficient to reduce the number of contaminated surfaces to 2.9%. CONCLUSIONS: A key step in achieving a hygienic state through detergent-based cleaning is rinsing but even this will not produce a 'hygienic' result for difficult surfaces such as the chopping board or the dishcloth. Disinfectant compounds should be considered in order to reduce the potential for foodborne cross infection within the home environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Although tests are available to determine the performance of disinfectants, there are no quantitative procedures available to compare the risk reduction achieved by disinfection with that produced by detergent-based procedures. This study describes a reproducible laboratory method which can be used to differentiate the effectiveness of different hygiene procedures for reducing cross-contamination risks during food handling.     

Effect of antimicrobial residues on early adhesion and biofilm formation by wild-type and benzalkonium chloride-adapted Pseudomonas aeruginosa

Antimicrobial residue deposition can change the physico-chemical properties of bacteria and surfaces and thus promote or impair bacterial adhesion. This study focuses on benzalkonium chloride (BC) deposition on polystyrene (PS) surfaces and the influence of this conditioning film on the physico-chemical properties of PS and on early adhesion and biofilm formation by Pseudomonas aeruginosa wild-type and its laboratory BC-adapted strain. The latter readily acquired the ability to grow in BC, and also exhibited physico-chemical surface changes. The existence of residues on PS surfaces altered their hydrophobicity and favoured adhesion as determined by the free energy and early adhesion characterization. Adapted bacteria revealed a higher ability to adhere to surfaces and to develop biofilms, especially on BC-conditioned surfaces, which thereby could enhance resistance to sanitation attempts. These findings highlight the importance of investigations concerning the antimicrobial deposition effect after cleaning procedures, which may encourage bacterial adhesion, especially of bacteria that have been previously exposed to chemical stresses.     

Characterization of spore surfaces from a Geobacillus sp. isolate by pH dependence of surface charge and infrared spectra

Aims: The surfaces of spores from a Geobacillus sp. isolated from a milk powder production line were examined to obtain fundamental information relevant to bacterial spore adhesion to materials. Materials and Results: The surfaces of spores were characterized using transmission electron microscopy and infrared spectroscopy. Thin sections of spores stained with ruthenium red revealed an exosporium with a hair‐like nap around the spores. Attenuated total reflection infrared spectra of the spores exposed to different pH solutions on a ZnSe prism revealed that pH‐sensitive carboxyl and phosphodiester groups associated with proteins and polysaccharides contributed to the spore’s negative charge which was revealed by our previous zeta potential measurements on the spores. Lowering the pH to the isoelectric point of spores resulted in an increase in intensity of all spectral bands, indicating that the spores moved closer to the zinc selenide (ZnSe) surface as the charged surface groups were neutralized and the spore surface polymers compressed. The attachment of spores to stainless steel was threefold higher at pH 3 compared with pH 7. Conclusions: This research showed that spore attachment to surfaces is influenced by electrostatic interactions, surface polymer conformation and associated steric interactions. Significance and Impact of the Study: The adhesion of thermophilic spores is largely controlled by functional groups of surface polymers and polymer conformation.     

Defouling and cleaning using nanobubbles on stainless steel

The present work demonstrates that nanobubbles can be used as cleaning agents on stainless steel (SS) surfaces. Cleaning efficiency has been quantified. Using an Atomic Force Microscope (AFM), it was demonstrated that nanobubbles can be produced by electrochemical treatment on a SS surface either with or without adsorbed bovine serum albumin (BSA). After allowing adsorption on SS overnight, radio-labeled BSA was removed by electrochemically generated nanobubbles, and then the remaining BSA on the surface was quantified by radioactivity measurement. The results indicate that nanobubbles can remove >10% of the protein in each 3-min electrochemical treatment while in a control group, washing with water and electrolyte resulted in no more than 3% of the protein being removed each time. Cleaning of conducting surfaces by nanobubbles is promising in any system where fouling occurs in biomedia.     

Effect of different Bacillus subtilis lipopeptides on surface hydrophobicity and adhesion of Bacillus cereus 98/4 spores to stainless steel and Teflon

Various lipopeptides produced by Bacillus subtilis were examined for their ability to modify the surface hydrophobicity of two substrata, stainless steel (SS) and Teflon. These modifications were evaluated by water contact angle measurements. The effects depended on the lipopeptide, its concentration, and the tested substratum. Treatment of SS with different concentrations of surfactin S1 showed an increase of the hydrophobicity between 1 and 100 mg l^?1. On the same substratum, fengycin increased hydrophobicity up to its critical micelle concentration (6.25 mg l^?1). With higher concentrations of fengycin, hydrophobicity decreased. Surfactin, mycosubtilin, and iturin A decreased hydrophobicity on Teflon. The different effects of these three families of lipopeptides were related to their structural differences. A good correlation was shown between hydrophobicity modifications of surfaces and the attachment of B. cereus 98/4 spores. Enhancement in the hydrophobicity of the surfaces increased the number of adhering spores.     

The measurement of Bacillus mycoides spore adhesion using atomic force microscopy,simple counting methods,and a spinning disk technique

An atomic force microscope has been used to study the adhesion of Bacillus mycoides spores to a hydrophilic glass surface and a hydrophobic-coated glass surface. AFM images of spores attached to the hydrophobic-coated mica surface allowed the measurement of spore dimensions in an aqueous environment without desiccation. The spore exosporium was observed to be flexible and to promote the adhesion of the spore by increasing the area of spore contact with the surface. Results from counting procedures using light microscopy matched the density of spores observed on the hydrophobic-coated glass surface with AFM. However, no spores were observed on the hydrophilic glass surface with AFM, a consequence of the weaker adhesion of the spores at this surface. AFM was also used to quantify directly the interactions of B. mycoides spores at the two surfaces in an aqueous environment. The measurements used "spore probes" constructed by immobilizing a single spore at the apex of a tipless AFM cantilever. The data showed that stretching and sequential bond breaking occurred as the spores were retracted from the hydrophilic glass surface. The greatest spore adhesion was measured at the hydrophobic-coated glass surface. An attractive force on the spores was measured as the spores approached the hydrophobic-coated surface. At the hydrophilic glass surface, only repulsive forces were measured during the approach of the spores. The AFM force measurements were in qualitative agreement with the results of a hydrodynamic shear adhesion assay that used a spinning disk technique. Quantitatively, AFM measurements of adhesive force were up to 4 x 10(3) times larger than the estimates made using the spinning disk data. This is a consequence of the different types of forces applied to the spore in the different adhesion assays. AFM has provided some unique insights into the interactions of spores with surfaces. No other instrument can make such direct measurements for single microbiological cells.     

Cleanability in relation to bacterial retention on unused and abraded domestic sink materials

H olah , J.T. & T horpe , R.H. 1990. Cleanability in relation to bacterial retention on unused and abraded domestic sink materials. Journal of Applied Bacteriology 69 , 599–608.
The relative Cleanability of stainless steel, enamelled steel, mineral resin and polycarbonate domestic sink materials was assessed by comparing the number of organisms remaining on surfaces after cleaning. In unused condition all materials, other than one enamelled steel, were equally cleanable. Stainless steel, abraded artificially or impact damaged to a similar degree as stainless steel subjected to domestic wear, retained approximately one log order less bacteria after cleaning than the other materials subjected to the same treatments. Little difference in Cleanability was recorded between the abraded surfaces of the other materials although enamelled steel surfaces were less cleanable than mineral resin or polycarbonate after impact damage, because of the greater susceptibility of enamelled steel to damage by this treatment. When cleaning time was extended beyond 10 s for the abraded and impact damaged materials, their Cleanability was not enhanced as compared with stainless steel. Changes in surface finish after abrasion were assessed by surface roughness measurement and scanning electron microscopy. Surfaces with poor Cleanability before and after abrasion were characterized by pitting, crevices or jags. These surfaces are likely to retain more bacteria because of increased numbers of attachment sites, a larger bacterial/material surface contact area and topographical areas in which applied cleaning shear forces are reduced. Materials that resist surface changes, e.g. stainless steel, will remain more hygienic when subjected to natural wear than materials which become more readily damaged.     

Biomolecules in multilayer film for antimicrobial and easy-cleaning stainless steel surface applications

Microorganisms are able to attach to, grow on, and ultimately form biofilms on a large variety of surfaces, such as industrial equipment, food contact surfaces, medical implants, prostheses and operating rooms. Once organized into biofilms, bacteria are difficult to remove and kill, which increases the risk of cross-contamination and infection. One way to address the problem may thus be to develop antibacterial, anti-adhesion, ‘easy cleaning’ surfaces. In this study, stainless steel (SS) surfaces with antibacterial properties were created by embedding several antimicrobial peptides in a multilayer film architecture. The biocidal effect of these surfaces was demonstrated against both Gram-positive and Gram-negative bacteria according to two ISO tests. Also, coating SS surfaces with either mucin or heparin led to a reduction of S. epidermidis adhesion of almost 95% vs the bare substratum. Finally, by combining both antibacterial and anti-adhesion biomolecules in the same multilayer film, SS surfaces with better cleanability were produced. This surface coating property may help to delay the buildup of a dead bacterial layer which is known to progressively reduce exposure of the coating, leading to an undesirable decrease in the antibacterial effect of the surface.     

Possible overestimation of surface disinfection efficiency by assessment methods based on liquid sampling procedures as demonstrated by in situ quantification of spore viability

The standard test methods used to assess the efficiency of a disinfectant applied to surfaces are often based on counting the microbial survivors sampled in a liquid, but total cell removal from surfaces is seldom achieved. One might therefore wonder whether evaluations of microbial survivors in liquid-sampled cells are representative of the levels of survivors in whole populations. The present study was thus designed to determine the "damaged/undamaged" status induced by a peracetic acid disinfection for Bacillus atrophaeus spores deposited on glass coupons directly on this substrate and to compare it to the status of spores collected in liquid by a sampling procedure. The method utilized to assess the viability of both surface-associated and liquid-sampled spores included fluorescence labeling with a combination of Syto 61 and Chemchrome V6 dyes and quantifications by analyzing the images acquired by confocal laser scanning microscopy. The principal result of the study was that the viability of spores sampled in the liquid was found to be poorer than that of surface-associated spores. For example, after 2 min of peracetic acid disinfection, less than 17% ± 5% of viable cells were detected among liquid-sampled cells compared to 79% ± 5% or 47% ± 4%, respectively, when the viability was evaluated on the surface after or without the sampling procedure. Moreover, assessments of the survivors collected in the liquid phase, evaluated using the microscopic method and standard plate counts, were well correlated. Evaluations based on the determination of survivors among the liquid-sampled cells can thus overestimate the efficiency of surface disinfection procedures.     

The detection of food soils on stainless steel using energy dispersive X-ray and Fourier transform infrared spectroscopy

Organic soiling is a major issue in the food processing industries, causing a range of biofouling and microbiological problems. Energy dispersive X-ray (EDX) and Fourier transform infra red spectroscopy (FT-IR) were used to quantify and determine the biochemical groups of food soils on stainless steel surfaces. EDX quantified organic material on surfaces where oily based residues predominated, but was limited in its usefulness since other food soils were difficult to detect. FT-IR provided spectral 'fingerprints' for each of the soils tested. Key soiling components were associated with specific peaks, viz. oils at 3025 cm(-1)-3011 cm(-1), proteins at 1698 cm(-1)-1636 cm(-1) and carbohydrates at 1658 cm(-1)-1596 cm(-1), 783 cm(-1)-742 cm(-1). High concentrations of some soils (10%) were needed for detection by both EDX and FT-IR. The two techniques may be of use for quantifying and identifying specific recalcitrant soils on surfaces to improve cleaning and hygiene regimes.     

Observations on the Value of a Swab Technique for Determining the Bacteriological State of Milking Equipment Surfaces

The reproducibility and efficacy of a swab technique for milking equipment in normal use was studied. Reproducibility was satisfactory when swabbing was done by trained personnel. It was confirmed that swabs pick up only a proportion of the microorganisms present on a surface; this proportion varies with the consistency of the contaminating material, but not with the type of surface on which it occurs. It is concluded that swabs allow the estimation of the maximum ability of a surface to contaminate the milk.
Rigid smooth surfaces of the milking machine are recontaminated by microorganisms originating from joints and complex parts of the machine during rinsing after cleaning. A procedure to evaluate the efficacy of cleaning these surfaces is proposed.     

Comparison of adhesion of the food spoilage bacterium Shewanella putrefaciens to stainless steel and silver surfaces

AIMS: To compare the number of attached Shewanella putrefaciens on stainless steel with different silver surfaces, thus evaluating whether silver surfaces could contribute to a higher hygienic status in the food industry. METHODS AND RESULTS: Bacterial adhesion to three types of silver surface (new silver, tarnished silver and sulphide-treated silver) was compared with adhesion to stainless steel (AISI 316) using the Malthus indirect conductance method to estimate the number of cfu cm(-2). The number of attached bacteria on new silver surfaces was lower than on steel for samples taken after 24 h. However, this was not statistically significant (P > 0.05). The numbers of attached bacteria were consistently lower when tarnished silver surfaces were compared with stainless steel and some, but not all, experiments showed statistical significance (P < 0.05). Treating new silver with sulphide to reproduce a tarnished silver surface did not result in a similar lowering of adhering cells when compared with steel (P > 0.05). CONCLUSIONS: New or tarnished silver surfaces caused a slight reduction in numbers of attached bacteria; however, the difference was only sometimes statistically significant. SIGNIFICANCE AND IMPACT OF THE STUDY: The lack of reproducibility in differences in numbers adhering to the different surfaces and lack of statistical significance between numbers of adhered viable bacteria do not indicate that the tested silver surfaces can be used to improve hygienic characteristics of surfaces in the food industry.