Quantitative Adhesion of <i>Staphylococcus aureus</i> on Stainless Steel Coated with Milk

doi:10.4236/fns.2013.43040

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Food and Nutrition Sciences, 2013, 4, 299-304

http://dx.doi.org/10.4236/fns.2013.43040 Published Online March 2013 (http://www.scirp.org/journal/fns)

Quantitative Adhesion of Staphylococcus aureus on

Stainless Steel Coated with Milk

Fatima Hamadi1,2, Hassan Latrache2*, Fatima Asserne2, Soumya Elabed3,4, Ha f i da Zahir2,

Ibnsouda Koraichi Saad3,4, Hafida Hanine2, Jamaa Bengourram5

1Laboratory of Biotechnology and Vaorization of Natural Resources, Department of Biology, Faculty of Science, University Ibn

Zohr, Agadir, Marocco; 2Laboratory of Valorization and Safety of Food Products, Faculty of Sciences and Technics, University Sul-

tan Moulay Slimane, Beni Mellal, Morocco; 3Laboratoy of Microbial Biotechnology, Faculty of Science and Technics, University

Sidi Mohamed Ben Abdellah, Fés-Sáis, Marocco; 4Regional University Center of Interface, University Sidi Mohamed Ben Abdellah,

Fés, Marocco; 5Laboratory of industrial Engineering, Food and Environment, Faculty of Sciences and Technics, University Sultan

Moulay Slimane, Beni Mellal, Marocco.

Email: *[email protected]

Received September 14th, 2011; revised December 9th, 2011; accepted December 16th, 2011

ABSTRACT

The surface energy characteristics of uncoated (clean) and coated stainless steel with UHT milk at various contact time

(5 min, 30 min, 1 hours, 3 hours, 6 hours, 24 hours) were determined using contact angle measurement. Whatever the

contact time, the clean stainless steel coupons became more hydrophobic and more electron acceptor when they are

coated by milk. Inversely, the electron donor character seems to decreasing in this condition. The calculated surface

energy component of coated stainless steel was found to vary with contact time. Its hydrophobicity and its electron ac-

ceptor were minimal after 3 hours of contact, but its electron donor was minimal after 1 hours of contact. Adhesion ex-

periments of Staphylococcus aureus were carried out on uncoated and coated stainless steels at various contact times.

For all contact times, the adhesion results show that milk reduce S. aureus adhesion, and the level of this reduction de-

pend on contact time. This reduction was lower and higher after 1 hour, 5 min and 30 min of contact respectively.

Keywords: Surface Energy Characteristics; S. aureus; Adhesion; Milk; Stainless Steel

1. Introduction

The formation of biofilm creates major problems in the

food industry since it may represent an important source

of contamination for materials or foodstuffs coming into

contact with them, so leading to food spoilage or trans-

mission of diseases. Biofilms are of interest in the dairy

industry, as bacteria within biofilms are more difficult to

eliminate than plank tonic cells, and bacteria detached

from biofilms can contaminate milk and milk products

[1]. This biotransfer may affect hygiene and the com-

mercial value of the product. To control these problems,

it has been recognized that a greater understanding of the

interactions between microorganisms and food—process-

ing surface is required [2-4].

The adhesion of bacteria to surface is the first and es-

sential stage in the formation of biofilm. This adhesion

depends on both physicochemical properties of cell sur-

face and solid surface, and also on characteristics of the

surrounding medium.

Stainless steel is the most frequently used material for

food processing equipment because of its high impor-

tance related to food safety reasons. There are many cir-

cumstances in dairies where substratum surface is either

continuously or periodically in contact with liquids that

contain microorganisms. These conditions could affect

the substratum surface properties and consequently the

adhesion process.

Staphylococcus aureus is a gram positive bacterium,

which is an important food-borne pathogen [5-7]. In food

industry this organism could be able to attach and to

form biofilms on the food-processing surface [5,7,8]. S.

aureus was studied here because little information [5-7]

is available of its adhesion behaviour in dairy industry in

comparison with other organisms such as Listeria mono-

cytognes and bacillus [4,9-12].

The aim of this study was therefore to investigate the

surface properties of stainless steel at various times of

contact with milk. The adhesion of S. aureus to stainless

steel was also examined and discussed in terms of phys-

icochemical properties of cell surface and substratum

surface.

*Corresponding author.

Quantitative Adhesion of Staphylococcus aureus on Stainless Steel Coated with Milk

300

2. Materials and Methods

2.1. Bacterial Strains, Growth Conditions and

Preparation of Microbial Suspension

The bacterial strain used in this study was Staphylococ-

cus aureus ATCC 25923. The strain was cultured in Lu-

ria Burtani broth at 37˚C for 24 h after culture, the cells

were harvested by centrifugation for 15 min at 8400 xg

and were washed twice with and resuspended in KNO3

solution with ionic strength 0.1 M. The physicochemical

properties of this strain were measured by contact angle

measurements. The results are presented in Table 1 [13].

2.2. Cleaning of Stainless Steel Coupons

The solid support selected for this study was stainless

steel 304. Before being coated with milk, the steel was

cut into 1cmx1cm coupons and cleaned by soaking for 15

min in ethanol solution. The coupons were then rinsed

with distilled water and autoclaved at 120˚C for 15 min.

2.3. Treatment of Stainless Steel Coupons with

UHT-Milk

The cleaned stainless steel coupons were placed into a

Petri dish and 10 ml of ultrahigh-temperature (UHT)-

treated milk was added. The steel was allowed to contact

milk for 5 min, 30 min, 1 h, 3 h, 6 h and 24 h at 4˚C. Af-

ter each contact time, the coupons of steel were rinsed

three times with distilled water.

2.4. Contact Angle Measurements

Contact angle measurements were performed using a

goniometer (GBX instruments, France) by the sessile

drop method. One drop of a liquid was deposited onto a

dry stainless steel uncoated and coated by milk at differ-

ent contact times. Three to six contact angle measure-

ments were made on substratum surface for all probe

liquids including water, formamide and diiodomethane.

The Lifshitz-Van der Waals (γLW), electron donor (γ−) and

electron acceptor (γ+) components of the surface tension

of bacteria and for stainless steel were estimated from the

approach proposed by Van Oss et al. (1988) [14]. In this

approach the contact angles (θ) can be expressed as:

cos1 222

LW LW

SLSL SL

The Lewis acid-base surface tension component is de-

fined by:

SSS









The surface hydrophobicity was evaluated through

contact angle measurements and by the approach of Van

Oss [14,15]. In this approach, the degree of hydrophobic-

ity of a given material (i) is expressed as the free energy

of interaction between two entities of that material when

immersed in water (w): ΔGiwi. If the interaction between

the two entities is stronger than the interaction of each

entity with water, the material is considered hydrophobic

(ΔGiwi < 0); conversely, for a hydrophilic material, ΔGiwi

> 0. ΔGiwi is calculated through the surface tension com-

ponents of the interacting entities, according to the fol-

lowing formula:



iwi

LW LW

iwi w

iiww wiiw



   

   





 













2.5. Adhesion Experiments

Ten millimetres of bacterial suspension containing 108

CFU.ml−1 was incubated in a Petri dish containing stainless

steel coupons treated by milk for 3 h at 4˚C. After 3 h of

incubation, the coupons were then rinsed three times with

distilled water to remove the nonadhering bacteria. The

stainless steel coupons were immersed in a test tube con-

taining physiological water (Nacl: 9 g/l). Bacterial cells

were detached from the inert support by using a sonica-

tion bath (ultrasonic) for 5 min. CFUs were counted by

using the serial dilution technique of the bacterial sus-

pension obtained after sonication. Counts were deter-

mined on Luria Burtani agar after incubation for 24 h at

37˚C. Each experiment was performed in duplicate.

3. Results and Discussion

3.1. Surface Free Energy Characteristics of

Stainless Steel Uncoated and Coated

with Milk at Various Contact Times

Contact angles were measured on stainless steel surface

before and after coating with milk using the three test

liquids: water, formamide and diidomethane (Table 2).







 

 



Table 1. Contact angle values, surface energy and their components of S. aureus [13].

Contact angle Surface free energy components (mj/m2)

Water Formamide Diidomethane γLW γ− γ+ γAB γt ΔGiwi

26.45 (1) 30.4 (1) 59.45 (2) 28.85 (0) 51.3 (0.99)2.4 (0) 21.9 (3) 50.75 (2.4) 28.57

Standard deviation is given in parentheses.

Quantitative Adhesion of Staphylococcus aureus on Stainless Steel Coated with Milk 301

Table 2. Contact angle values, surface energy and their components of uncoated (control) and coated stainless steel at differ-

ent contact times.

Contact angle Surface free energy components (mj/m2)

Contact time

Water Formamide DiidomethaneγLW γ− γ+ γAB γt ΔGiwi

Control 64 (2) 68 (1.8) 60 (5) 28.1 (2.6)32.5 (2) 0.1 (0) 3.16 (2) 31 (5) 11.5

5 min 127 (5) 104 (1.13) 79 (2) 18 (3) 0.5 (0) 0.7 (0) 0.75 (0.3) 18.8 (4) −73.53

30 min 129 (2) 107 (4) 75 (4) 19.65 (2.1)0.45 (0) 1.15 (0.6)0.85 (1) 20.5 (3) −69.76

1 h 129 (0.7) 112 (0.9) 72 (4) 21.45 (2)0.05 (0) 3.25 (0.4)0.4 (0) 21.9 (1.9) −62.68

3 h 115 (0.45) 72 (0.9) 51 (0.6) 33.3 (0.3)3.9 (0.1) 0.6 (0.1) 3 (0.37) 36.5 (0.6) −42.10

6 h 127 (4) 96 (4) 63 (2) 26.6 (1.1)1.6 (1) 1.11 (0.2)2.3 (1.2) 28.9 (2.4) −67.60

24 h 115 (1.7) 73 (3) 56 (0.7) 31.19 (0.6)3.1 (0.7) 0.7 (0.4) 2.9 (1.1) 34.13 (1.8) −64.46

Standard deviation is given in parentheses.

The contact angle data were then used to calculate the

surface energy components of all samples (Table 2). The

results show that uncoated stainless steel surface was

hydrophilic with ΔGiwi = 11.50 mj/m2. Regardless of

contact time, stainless steel coated with milk alters sig-

nificantly its surface hydrophobicity. The uncoated

stainless steel surface hydrophobycity ranged from hy-

drophilic character (positive value of ΔGiwi) to hydro-

phobic character (negative value of ΔGiwi). It is known

that milk is a complex biological fluid composed by sev-

eral components including proteins, fats and calcium

phosphate. According to Mittelman (1998) [16], the ad-

sorption of milk and its components on substratum sur-

face occurs within 5 s to 10 s.

The effect of proteins hydrophobicity of solid surface

is reported by some works [17,18]. Yang et al. (1991)

[17] have found that the adsorption of β-lactoglobulin

onto substratum surface could render hydrophilic sur-

faces more hydrophobic and hydrophobic surfaces more

hydrophilic. Barnes et al. (1999) [5] reported that the fat

components are likely to interact with hydrophobic sur-

face of stainless steel. Other works [19], have reported

that surface energy characteristics of a solid surface in-

fluence the extent and rate of protein adsorption. In our

work the observed increasing hydrophobicity of coated

stainless steel could be due to the adsorption of proteins

and/or fat components to substratum surface. The order

of deposition of milk components should be related to

initial surface energy characteristic of substratum.

Harnett et al. (2006) [20] have calculated the surface

energy of various materials coating a series of proteins of

collagen, and fibroncetin and they found that these pro-

teins affect significantly the electron donor and the elec-

tron acceptor of some substratum surfaces. To our knowl-

edge, the effect of proteins or other components of milk

on electron donor/electron acceptor properties of sub-

stratum surface were not examined previously. Stainless

steel coated with milk has a very lower electron donor

compared to stainless steel uncoated with very high elec-

tron donor property (Table 2). In opposite, the electron

acceptor of stainless steel was not markedly affected by

the presence of milk (Table 2). The variation of hydro-

phobicity, electron donor and electron acceptor proper-

ties of stainless steel pretreated with milk as a function of

contact time are presented in Figure 1.

The surface hydrophobicity decreases from 5 min to 3

h and increases from 3 h to 24 h (Figure 1(a)). This hy-

drophobicity achieved the minimum at 3 h of contact.

Figures 1(b) and (c) show that contact time affect mark-

edly the electron donor and electron acceptor properties

of coated stainless steel. The electron donor and electron

acceptor properties achieved the maximum at 3 h of con-

tact and 1h of contact respectively.

Kim and Lund (1997) [21] have found that the adsorp-

tion process for β-lactoglobulin on stainless steel was

very rapid in the first 5 min and essentially reached equi-

librium within 10 min. These authors have also reported

that the precipitation of calcium phosphate onto the stain-

less steel surface was very slow compared to monolayer

deposition of β-lactoglobulin. Addesso and Lund (1997)

[19] show that protein adsorption onto a surface depends

on protein concentration. The random observed variation

of physicochemical properties of stainless steel as a func-

tion of contact time should be related to a nature and an

amount of milk components adsorbed onto substratum

surface and its kinetic deposition.

3.2. Adhesion of S. aureus to Stainless Steel

Treated with Milk under Different Contact

Time. Kinetic Evolution of S. aureus

Adhesion on Stainless Steel

Pretreated by Milk

Several works [4,5,11,12,22-24] have studied the effect

of milk or proteins milk on bacterial adhesion. In this

study, we are interested to examine the adhesion kinetic

of S. aureus to stainless steel coated with UHT milk. The

results of S. aureus adhesion on coated and uncoated

stainless steel are presented in Figure 2.

Coated stainless steel with UHT milk was shown to

reduce the attachment of S. aureus whatever contact time.

Quantitative Adhesion of Staphylococcus aureus on Stainless Steel Coated with Milk

302

(a)

(b)

(c)

Figure 1. Variation of physicochemical properties of stai nl e ss

steel coated with milk as a function of contact time. (a) Hy-

drophobicity; (b) Electron donor property; (c) Electron

acceptor property.

The role of milk or components of milk in inhibiting

bacterial adhesion was reported previously by several

works. Barnes et al. (1999) [5] have reported that the

pretreatment of stainless steel with skim milk was found

to reduce S. aureus adhesion. According to Hood and

Figure 2. Number of S. aureus cells adhered to uncoated

(control) and coated stainless steel at different contact times.

Zottola (1997) [4], the attachment of Listeria monocyto-

genes and Salmonella typhimurium to stainless steel was

inhibited by preconditioning with whole and chocolate

milk and was enhanced when using diluted milk.

To our knowledge, the surface physicochemical prop-

erties have not been considered in interpreting the effect

of milk on bacterial adhesion results despite the clear

change in substratum surface physicochemical properties

after contact with milk or milk components.

The adhesion results obtained here were discussed and

interpreted in terms of hydrophobicity and electron do-

nor/electron acceptor properties of both surfaces (cell

surface, stainless steel surface). The electrostatic interac-

tions were neglected since our experience was performed

in a solution with high ionic strength [25,26]. Since S.

aureus is hydrophilic (Table 1) and uncoated stainless

steel surface is also hydrophilic (Table 2), the S. aureus

adhesion on this substratum was increased. In the other

hand, the adhesion of hydrophilic S. aureus was reduced

on hydrophobic stainless steel coated with milk. These

results are in accord with the hypothesis that the hydro-

phobic cells tend to attach to a hydrophobic substrate and

the hydrophilic cells tend to attach to a hydrophilic sub-

strate. On the other hand, the difference in level adhesion

between stainless uncoated and coated stainless steel

could be related to the contribution of acid-base interac-

tions; these interactions seem to be lower in the case of

uncoated stainless steel since its electron donor was very

low in comparison with the electron donor of coated

stainless steel.

From Figure 2, we also observe that the level of S.

aureus adhesion changes as a function of contact time.

The S. aureus adhesion was much reduced at 30 min

comparatively for other times of contact. This variation is

not completely explained by physicochemical interac-

tions. However, others interactions between cell surface

and milk or milk components adsorbed on surface could

be contribute in bacterial adhesion at different contact

times. The difference in nature of proteins adsorbed for

each contact time and the faster conformational rear-

Quantitative Adhesion of Staphylococcus aureus on Stainless Steel Coated with Milk 303

rangement undergone by one protein at surface relative

to that of other proteins could be the origin of the varia-

tion observed in adhesion results. Barnes et al. (1999) [5]

have found that the pre-treatment of stainless steel with

the individual milk proteins α-, β- and



casein and

α-lactalbumin at equal concentration reduce attachment

of S. aureus and this reduction was marked with β casein.

McEldowney and Fletcher (1987) [27] observed that hy-

drated layers of polymers and proteins that form on inert

surfaces can either facilitate or reduce bacterial adhesion.

Al Makhlafi et al. (1994) [22] examined the effect of

competitive adsorption of bovine serum albumin (BSA)

and β-lactoglobulin on Listeria monocytogenes adhesion

to silica, and they found that the film formed by the ad-

sorption of β-lactoglobulin followed by BSA encouraged

adhesion more than the film formed by the adsorption of

BSA followed by β-lactoglobulin.

4. Conclusion

The results obtained here show that the physicochemical

properties including hydrophobicity and electron donor-

electron acceptor properties of stainless steel surface were

markedly affected by treatment by milk. The adhesion

results show that whatever the contact time, the pre-

treatment of substratum by milk reduce the adhesion

level. This reduction is random with the contact time.

This research suggests that it is very important to take

into account the contact time between the substratum and

milk in the cleaning and sanitizing process.

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