These conditions were chosen as they resulted in the formation of a peptide layer that would exhibit the best performance against biofilm formation Maity et al. The next day, the substrates were thoroughly washed to remove any non-adherent peptide remains and then dried under nitrogen flow.
To confirm that the peptide coating modified the surfaces, we compared the water contact angles of the peptide-coated substrates to those of bare substrates. The difference between the water contact angle of the bare and peptide-coated surface indicates that the peptide indeed coated the surface and modified it. This increase in the water contact angle value of the peptide-coated surfaces, in line with previous results, also indicates that the modified surfaces exhibited a more hydrophobic nature. These features might contribute to preventing bacterial accumulation on the modified surfaces Wang et al.
Figure 1. Characterization of the physical properties of the peptide-coated surfaces.
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Water contact angle images of A bare stainless steel, B peptide-coated stainless steel. C Comparison between the contact angles of the coated and uncoated surfaces. XPS confirmed the presence of the peptide on the coated surfaces. Bare surfaces did not display any fluorine signal, whereas the coated surface had 1. The images show a significant difference between the bare and coated substrates. In correlation with previous results Maity et al. The roughness of the peptide-coated surface differed from that of the non-coated one on both peaks and valleys and its averaged value Ra increased from 1.
To test the effect of the peptide coating on biofilm formation, we quantified the amount of viable bacteria on peptide-coated and bare stainless steel surfaces by counting the CFUs of the surface-adhered cells. The results show a reduction of around 2-log in the amount of both B. Figure 2. Inhibition of biofilm development by peptide-coated surfaces. Quantification of the number of bacteria adsorbed onto uncoated and peptide-coated stainless steel surfaces at different incubation times for A B.
In addition, we examined the time-course inhibitory ability of the coated surfaces, by measuring the bacterial counts at different time points during biofilm development — from the initial biofilm formation through its maturation. Each time point represents different stages in the biofilm development. The results show a similar percentage in the decrease of biofilm formation following 18, 42, and 66 h of incubation with peptide-coated surfaces in comparison to uncoated surfaces Figure 2.
To further support our results, we analyzed the surface-adhered bacteria using confocal scanning laser microscopy CSLM Figure 3. The images present a significantly lower number of bacterial cells on the peptide-coated surfaces compared with the bare surfaces. This trend was similar for both B. Figure 3. Fluorescent microscopy analysis of inhibition of biofilm establishment using peptide-coated surfaces. CLSM images of biofilms on uncoated and peptide-coated stainless steel surfaces developed at different incubation times. Viable bacterial cells are stained with SYTO 9 green fluorescent nucleic acid stain and dead bacterial cells are stained in red with propidium iodide PI.
On the left panel is biofilm formed by B. To rule out any cytotoxic effect of the peptide on the bacteria, we tested the peptide-coated surfaces for their effect on bacterial growth using a growth curve analysis Figure 4. The curves clearly show similar growth in the peptide-coated vessels in comparison to uncoated ones in both bacterial species.
Figure 4. Peptide coating does not affect the growth of bacteria. Growth curves obtained for A B.
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Values were averaged over three repeats. To determine whether exposure of the peptide-coated surfaces to milk impairs the coating stability and anti-biofilm performance, coated and uncoated stainless steel surfaces were incubated in milk overnight prior to their exposure to bacteria. The results show that the biofilm formation was inhibited notably, following bacterial incubation on peptide-coated surfaces compared to uncoated surfaces, regardless of the pre-incubation in milk Figures 5A , B.
Figure 5. The effect of milk on the anti-biofilm properties of the peptide coating. Peptide-coated stainless steel surfaces exhibit a similar reduction in the number of surface-adhered bacteria with and without pre-incubation in milk. Results for A B. One of the requirements of the modified surfaces is to prove that their exposure had no influence on either the quality of milk or its products.
Biofilms in the Dairy Industry
Therefore, using an Optigraph instrument, we examined the effect of a peptide-coated surface on milk clotting parameters such as the starting time of clotting minutes and the curd firmness V. The results indicate that the duration of the clotting period of milk exposed to peptide-coated surfaces, The strength of the curd obtained under these two conditions was also similar Figures 6A , B.
In addition, we examined how the peptide coating affected the protein levels in raw milk as well as the effect of incorporating proteins into cheese using the Kjeldahl method. Importantly, we could not detect any changes in the amount of protein in milk as well as in the cheese samples Figures 6C , D.
Figure 6. The effects of peptide coating on the technological properties of dairy products. Peptide coating does not induce any change in A clotting time, B curd firmness and the amount of protein in C raw milk and D soft cheese compared to uncoated surfaces. Bacterial adhesion to surfaces and the formation of biofilms in dairy processing equipment are the main source of contamination of dairy products Cappitelli et al.
The use of biocides to eradicate bacterial biofilms is not advised because they could be released to the products Midelet and Carpentier, Therefore, it is desirable to prevent the adhesion of bacteria in advance in order to mitigate subsequent biofilm formation. This study provides evidence of the possibility of successfully modifying milk contact surfaces such as stainless steel to prevent biofilm formation and, thus, to prevent subsequent contamination of milk during its processing.
Unlike early stage biofilms, mature biofilms consist of a developed extracellular matrix and more protected bacterial cells. Therefore, it is important to develop anti-biofilm coatings that can reduce bacterial levels for long durations. Similar to the short incubation time 18 h , a lower number of bacteria were counted and sparse bacteria could be detected for the peptide-coated surfaces at and h time points. As expected, with increasing incubation times, bare stainless steel surfaces exhibited denser biofilms with growing thickness, whereas the density of bacterial cells on peptide-coated surfaces was significantly low and remained constant throughout time Figures 2 and 3.
These results indicate that the peptide-coated surfaces could be effective against biofilm formation and maturation for both Gram-positive and Gram-negative bacterial species. Although biofilm prosperity is compromised in the presence of the peptide coating, apparently the bacteria are not directly affected by it. Growth curves showed that the tested bacterial species were able to thrive despite the presence of the peptide coating. Thus, we concluded that the peptide-coated surfaces were not cytotoxic to the tested bacterial strains.
This conclusion is also supported by CLSM micrographs Figure 3 showing that very few bacteria adhered to the peptide-coated surfaces; however, most of them were found to be alive since they were stained in green. These results support our conclusion of an anti-biofilm mode of action of the peptide coating rather than of its biocidal activity.
The results presented in Figures 5A , B suggest that the peptide coating properties remained intact after incubation with milk, keeping not only the same trend but also the same order of magnitudes in resisting bacterial accumulation. These proteins may adhere to the surface and act as a conditioning film on which bacterial adherence could be promoted Sjollema et al. The process by which milk coagulates into curd might be sensitive to the environment.
Different components in the surroundings may affect the timing of clotting, and curd firmness Munro et al. Since it is possible that the milk components would react with the peptide coating, this could have a drastic impact on the quality of the curd and its nutritious properties Leitner et al.
None of the parameters tested displayed any difference compared to the results obtained from the non-coated samples, implying that the peptide coating did not affect the technological properties of the dairy products.
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More importantly, there was no change in the protein concentration in milk or cheese following the exposure of the raw milk to the coated surface. In this paper, we demonstrated that a modification of the stainless steel surface using a peptide-based coating prevents biofilm formation and subsequent maturation by bacterial species prevalent in the dairy industry. In addition, we found that the peptide coating does not affect the technological properties of dairy products; thus, it can be an attractive solution and can be safely used in the dairy industry or in the manufacture of various associated dairy products.
MS and MR designed and planned the study. SN synthesized the antifouling peptide, coated the stainless steel surfaces, and performed the physical characterization of the peptide-coated surface. AF performed the experiments related to the characterization of the anti-biofilm properties of the surfaces as well as the technological effect of the surfaces following their exposure to raw milk.
MS and AF wrote the initial manuscript. AF and SN contributed equally to this work. All authors integrated the data, discussed the results, and crafted the final manuscript.
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This work was partially supported by the Israel Dairy Board grant number grant. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. We would like to acknowledge Prof.
Doron Steinberg from The Hebrew University in Jerusalem for his supportive suggestions and discussions.
Alves, P. The effects of fluid composition and shear conditions on bacterial adhesion to an antifouling peptide-coated surface. MRS Commun. Austin, J.