Furthermore, the species-specific effects on microbial growth suggests that Tween 80 in cosmetics and food products could affect the composition of skin and gut microbiota, and the effect of emulsifiers on the human microbiome should therefore be explored to uncover potential health effects.
Tween 80 polysorbate 80, polyoxyethylene sorbitan monooleate is a non-ionic surfactant that is widely used as an emulsifier in cosmetics, pharmaceuticals and food products. The effects were not seen in germ-free mice and were therefore ascribed to changes in the gut microbiota after intake of Tween With the increasing awareness of correlations between gut microbiota and human health, it is of great importance to understand how food additives affect the human microbiome.
Staphylococcus aureus is a Gram-positive pathogen that causes many acute and chronic infections. It is commonly associated with foodborne illnesses Seow et al. Therefore, extensive research has been undertaken to understand the factors that affect the life cycle and virulence of S. Listeria monocytogenes is likewise a Gram-positive pathogen associated with foodborne illnesses Listeriosis Pan et al. We hypothesize that Tween 80 affects bacterial growth by directly affecting 1 viability and growth rates, and 2 biofilm formation.
Earlier studies have shown that addition of Tween 80 reduced bacterial adhesion and inhibited biofilm formation of Pseudomonas sp. However, in all cases, Tween 80 was added before the attachment phase of biofilm development, and therefore reflects its effect on bacterial attachment to abiotic surfaces.
This study adds to existing knowledge by investigating how Tween 80 affects planktonic growth and biofilm development of S. Tween 80 may affect food pathogens directly, or it may affect food safety in general by altering the efficiency of antimicrobials added as preservatives.
We therefore also investigated if Tween 80 affected the antimicrobial potency of two hydrophobic antimicrobial compounds. Staphylococcus aureus DSM and L.
Three biological replica inoculated from individual colonies were prepared for each experiment. Overnight cultures of S. Transfer of the peg lid to TSB with Tween 80 was either done immediately after inoculation, or after biofilms had been allowed to develop for 24 or 48 h.
At the end of the incubation, the biofilm biomass was quantified by crystal violet staining. Finally the optical density the extracted crystal violet was measured at nm. Three biological replicas and three technical replicas were prepared for each experiment. More detailed measurements were performed for all three bacterial strains to observe the biomass development with 12 h intervals in the presence or absence of 0.
At 12 h intervals, incubations were stopped and the biofilm biomass was quantified by crystal violet staining as described above. The liquid was removed by aspiration and the wells were washed twice with PBS. Next, the liquid above the biofilm was carefully replaced with PBS, ensuring that the biofilm did not dry out in the process.
Quantitative image processing was done with ImageJ using the plugin Comstat 2. To investigate whether Tween 80 could change the antimicrobial activity of the two hydrophobic antimicrobials rifampicin and isoeugenol, we determined the minimum biofilm eradication concentration MBEC in the presence and absence of 0.
The stock solution was used as the first concentration in the dilution series. DLS is a technique that uses the fluctuations in scattered laser light to calculate a hydrodynamic radius of particles in suspension.
Samples were transferred to clear disposable zeta cells and mounted in a Nano-ZS Malvern Instruments. Triplicate measurements of each sample were recorded and results are presented as the average of these. Sizes are given as the Z -average value.
The solution was shaken until the solids were dissolved. Next, chloroform was evaporated with nitrogen gas creating a thin film on the walls of the glass vessel. A 70 mM calcein Fluka stock solution was prepared in TSB, and 1 mL was transferred to the glass vessel with the thin film and vortexed thoroughly.
The content was then immersed into liquid nitrogen freezing and thawed again in warm water twelve times. In order to obtain membrane vesicles with uniform size, the solution was extruded 12 times through a 0.
Finally, the solution was run through a PD desalting column GE Healthcare equilibrated with TSB to separate membrane-enclosed calcein from free calcein. Calcein leakage was measured as an increase in fluorescence intensity, as the fluorescence of calcein in the vesicles is initially low due to quenching, and would increase as calcein leaked through the membrane.
Fluorescence intensity was measured with VarioSkan Flash fluorometer Thermo Scientific at nm excitation and nm emission. The background fluorescence from calcein F 0 was measured, and Tween 80 was added in final concentrations between 0. The fluorescence was recorded for triplicate samples 1 h after addition of Tween Finally, Triton X Fluka was added to a final concentration of 0.
Results are given as percentage of calcein release relative to the release obtained with Triton X:. Growth with Tween 80 increased the growth rate for planktonic S.
The lag phase also appeared to be shorter, but this could simply appear to be the case due to the faster growth rate, resulting in these cultures arriving faster at the cell density required for spectrophotometric detection. The growth rate of P.
Growth rates and lag times are included. We measured the biomass of S. Tween 80 has a critical micelle concentration of 0. In the first experiment, Tween 80 was included from the beginning of the 48 or 72 h incubation to determine the effect of Tween 80 on the establishment of biofilms.
This resulted in no or very little biofilm formation for all organisms at all concentrations of Tween 80 Figure 2. In the second experiment, we let the biofilms grow for either 24 or 48 h in TSB before replacing the media with TSB containing Tween 80, and allowing for 24 h subsequent growth.
These experiments would determine the effect of Tween 80 on already established biofilms. Addition of Tween 80 to a 24 h old biofilm had either no effect or impaired further biofilm development for all three organisms. If the biofilm was grown for 48 h before addition of Tween 80, we saw a concentration dependent stimulation of S.
During our studies, we discovered that the commercial Tween 80 solution did contain some oleic acid. Oleic acid has been shown to inhibit S. However, the authors also showed that oleic acid could stimulate S. It was therefore relevant to determine if the effect we observed were caused by Tween 80 or oleic acid. We therefore compared the effect of Tween 80 and oleic acid by quantifying biofilm biomass after replacing the overlying media of 24 or 48 h old biofilms with TSB containing either 0.
Tween 80 stimulated the biomass of S. Addition of Tween 80 to L. Development of biomass over time measured by crystal violet staining OD measurement for S. We visualized S. We did not observe any apparent differences in biofilm morphology.
The differences were statistically significant for the three first images in the z-stack, corresponding to 1. Confocal laser scanning microscopy images of three replicate S. Occupied area in percentage per layer in z-stacks from confocal laser scanning microscopy images. Each layer corresponds to 0. Since Tween 80 is a surfactant, it can form micelles and emulsions encapsulating hydrophobic substances. We therefore suspected that Tween 80 might influence the antibacterial efficacies of hydrophobic antimicrobials.
Essential oils have gained much attention recently for their potential use as natural food preservatives. Since essential oils are hydrophobic, emulsifiers such as Tween 80 are often added to ensure dispersion. We therefore investigated if Tween 80 could affect the antibacterial efficacy of the essential oil isoeugenol, and of the hydrophobic antibiotic rifampicin. We observed that addition of Tween 80 to both antimicrobials increased the concentration required to eradicate S. We hypothesized that Tween 80 could change the nutrient availability by changing the size of nutrient particles in media.
We therefore measured the particle size distribution of TSB with and without Tween Another way for Tween 80 to affect nutrient availability could be through membrane permeabilization. We will discuss this issue in more detail in the discussion section. The concentration used in bacterial assays, 0. Permeabilization of a model membrane after 1 h treatment with Tween 80, measured by the leakage of calcein from vesicles suspended in TSB.
Leakage is measured as fluorescence intensity, as vesicles become increasingly fluorescent as the calcein concentration within decreases. Tween 80 is a commonly used emulsifier both in the laboratory and in industry. It is often included in laboratory experiments without any comments or evaluations of possible side effects. In this study, we show that Tween 80 affects microbial growth either positively or negatively. These effects cannot be ignored, and the use of Tween 80 should be reconsidered.
We hypothesized that Tween 80 affects both planktonic and biofilm growth, and we tested our hypothesis on three different bacteria. Tween 80 stimulated the growth of S. Several other studies have investigated the effect of Tween 80 on biofilm formation. Toutain-Kidd et al. For S. All the isolates that were not inhibited showed high levels of lipase activity, which could result in cleavage of Tween 80 at its ester bond, producing oleic acid, and polyethylene Banin et al.
However, the authors seem to have only examined addition of Tween 80 from the beginning of biofilm formation, though it is not clearly stated in the article. We observed similar trends for addition of Tween 80 at the onset of biofilm formation, which also suggests that lipase activity did not affect the results in our study.
However, we also show that Tween 80 can stimulate biofilm growth for S. We suggest that Tween 80 can affect planktonic bacteria and biofilms through multiple mechanisms. The surfactant may affect nutrient availability in three ways: i by reducing the size of nutrient particles, which indicates dispersal of, e.
Above a critical concentration, this would be detrimental to the cell but below the critical concentration it might have a growth-promoting effect by slightly stressing the cell and possibly allowing for higher nutritional uptake from the media Taoka et al.
Application of electric fields can be utilized to create membrane permeabilization. It has been shown that moderate electric fields can result in reduced lag time of Lactobacillus acidophilus Loghavi et al.
Hence, some degree of permeabilization can be growth promoting. We show that Tween 80 can permeabilize a model membrane, and although this system is much simpler than a real bacterial membrane, the result demonstrates a potential for membrane perturbation. The permeabilizing properties of Tween 80 will depend on the specific lipid composition of the membrane both inner and outer for Gram-negative bacteria , on membrane proteins, and on the properties of the peptidoglycan layer and surrounding extracellular polymeric substances.
Therefore, this effect will be strain dependent and may explain the different effects seen for different strains in this study. Ions take part in many cell processes, such as signaling and enzyme activity. Potassium has recently been shown to mediate electrical signals within Bacillus subtilis biofilms, which in turn was correlated with coordinating metabolism Prindle et al.
Divalent cations, such as magnesium, calcium, and iron, are important for biofilm cohesiveness. If these ions are removed by chelating agents such as EDTA, the bacteria in the biofilm will detach and some will be killed Banin et al. We can imagine that Tween 80 could transport and concentrate divalent cations in the biofilm matrix.
Finally, Tween 80 has been shown to interact with lipases Liu et al. It is also widely utilized in biochemical applications, including:. G-Biosciences offers a variety of products and supplies for your biochemical and life science experiments, including research-grade Tween 20 and Tween Visit our website to claim yours today! Topics: Detergents.
What is the Difference Between Tween 20 and Tween 80? Tween 20 is a common component in the following applications: The pre-extraction of membranes to remove peripheral proteins Membrane-based immunoassays Lysing mammalian cells PCR polymerase chain reaction ELISA enzyme-linked immunosorbent assay Western blot analysis Due to its drying property, Tween 80 is typically present in ice cream.
Some antibodies and other proteins bind tightly to nitrocellulose and dissociation of these proteins by Tween 20 is barely detectable. In contrast, other proteins are nearly completely stripped from the nitrocellulose by the same detergent.
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