AN ABSTRACT OF THE THESIS OF
Hamood Khaled Ahmed Al-Makhlafi for the degree of Doctor of
Philosophy in Food Science and Technology presented on
August 1, 1994.
Title: The Influence of Preadsorbed Milk Proteins on
Adhesion of Listeria monocvtocrenes to Silica Surfaces.
Abstract approved by^
Dr,. JtisepA Nfcfeilir^
$ark AA. iD^feschel
iD^jfescl:
Dr.. ferk
P-lactoglobulin (p-Lg), bovine serum albumin (BSA), olactalbumin (a-Lac), and p-casein were adsorbed onto
silanized silica surfaces of low and high hydrophobicity
for 8 h, and P-Lg and BSA for 1 h. The surfaces were
incubated in buffer for 0, 5, 10, or 15 h and then contacted
with Listeria monocytogenes for 3 h. Cell adhesion was
quantified using image analysis. Following 8 h of protein
contact, adhesion to both surfaces was greatest when P-Lg
was present and lowest when BSA was present. Preadsorption
of a-Lac and p-casein showed an intermediate effect on cell
adhesion. Adsorption of P-Lg for 1 h resulted in lower
numbers of cells adhered as compared to the 8 h adsorption
time, while the opposite was observed with BSA, but adhesion
to BSA was observed to decrease slowly with film age to
values comparable to the 8 h tests.
The adsorption of BSA and P-Lg to both surfaces was
also carried out where each protein was allowed to contact
the surface in sequence and simultaneously. In sequential
tests performed with an 8 h contact/protein, cell numbers on
each surface were near that expected for the bare
hydrophobic surface when P-Lg contact preceded introduction
of BSA, whereas adhesion was reduced to values below that
expected for the bare hydrophilic surface when BSA preceded
P-Lg contact. In short-term sequential tests (1 h
contact/protein), adhesion was lower than that recorded on
bare hydrophilic surfaces in each case. Adhesion to each
surface following contact with an equimolar mixture of p-Lg
and BSA was lower than that measured on the bare hydrophilic
surface in each case, with adhesion following 1 h contact
being greater than that following 8 h contact. Adhesion
following competitive adsorption was greater to hydrophobic
than to hydrophilic surfaces. These results were explained
with reference to the surface passivating character of BSA,
and its ability to rapidly attain a nonexchangeable state
upon adsorption, relative to P-Lg.
The Influence of Preadsorbed Milk Proteins on Adhesion of
Listeria monocytogenes to Silica Surfaces
by
Hamood Khaled Ahmed Al-Makhlafi
A THESIS
Submitted to
Oregon State University
in partial fulfillment of
the requirements for the
degree of
Doctor of Philosophy
Completed August 1, 19 94
Commencement June 1995
APPROVED:
Co-ma/jor Afs^ocWte VProfessor of Bioresource Engineering, and
Food (Sciej/lcf an$ Technology in charge of major
-L^t-
Co-major Associate Professoy of Food Science and Technology
in charge of major
/
He§^of Department of Food* Science and Technology
Dean of Graduate School
Date thesis is presented
Typed by the researcher for
August 1, 1994
Hamood Khaled Ahmed Al-Makhlafi
ACKNOWLEDGEMENTS
The words stand short to express the feeling my heart
bears for my great major professors Dr. Joseph McGuire and
Dr. Mark A. Daeschel, the teachers, and the friends whose
support, guidance, and the continuous encouragement
contributed to the successful completion of this research.
My deepest gratitude.
All respect and thanks to Dr. Henry Schaup, my minor
professor for the special attention he has given me during
my Ph.D. program.
All appreciations and gratitude are due to Dr. David
Williams my teacher for more than one of Food Science
coursework from which I have learned a lot and will become
as I believe a professional future teacher in my home
country.
To Dr. Dale Weber, graduate council representative, all
thanks and respect are due to your time and acceptance of
being one of my committee members.
This work was supported in part by the Western Center
for Dairy Protein Research and Technology, the USDA National
Research Initiative Competitive Grants Program (Food
Safety), and Sana'a University Faculty of Agriculture
Project, funded by USAID.
TABLE OF CONTENTS
CHAPTER 1.
INTRODUCTION
REFERENCES
1
4
CHAPTER 2. LITERATURE REVIEW
6
Protein adsorption
6
Protein exchange on solid surfaces
Protein desorption
Surface-induced conformational changes
Bacterial adhesion
Mechanisms and factors affecting adhesion
Temperature effects
Culture concentration and Culture age
Electrostatic and hydrophobic interactions
Effect of organic macromolecules on adhesion
Substratum effect
Surface energy effects
6
8
9
13
19
20
22
23
28
31
32
The effect of macromolecules on substrate
properties
34
Control of bacterial adhesion
35
REFERENCES
CHAPTER 3. THE INFLUENCE OF PREADSORBED MILK PROTEINS
ON ADHESION OF LISTERIA MONOCYTOGENES TO
HYDROPHOBIC AND HYDROPHILIC SILICA SURFACES
40
48
ABSTRACT
49
INTRODUCTION
50
MATERIALS AND METHODS
51
Surface modification
52
Protein adsorption
52
Culture and adhesion
54
Rinsing
54
Criteria used for rinsing unattached bacterial
cells
55
Image analysis
Statistical analysis
RESULTS AND DISCUSSION
Rinsing of unattached bacterial cells
Adhesion following protein contact for 8 h
Hydrophobic surfaces
Hydrophilic surfaces
Adhesion following protein contact for 1 h
Preadsorption of P-Lg
Preadsorption of BSA
REFERENCES
55
56
56
56
57
57
61
64
' 65
66
75
CHAPTER 4. ADHESION OF LISTERIA MONOCYTOGENES TO MODEL
FOOD CONTACT SURFACES FOLLOWING SEQUENTIAL
AND COMPETITIVE ADSORPTION OF BOVINE SERUM
ALBUMIN AND p-LACTOGLOBULIN
79
ABSTRACT
80
INTRODUCTION
82
MATERIALS AND METHODS
84
Surface modification
Protein adsorption
Sequential adsorption
Competitive adsorption
Culture and adhesion
Rinsing
Image analysis
Statistical analysis
RESULTS AND DISCUSSION
Adhesion following sequential protein
adsorption (8 h contact time)
Adhesion following sequential protein
adsorption (1 h contact time)
Adhesion following competitive
adsorption for 8 h
Adhesion following competitive adsorption
for 1 h
REFERENCES
CHAPTER 5.
SIGNIFICANT FINDINGS
84
84
84
85
86
87
87
88
88
88
92
94
95
101
104
BIBLIOGRAPHY
107
APPENDICES
12 0
Appendix A
12 0
Appendix B
144
Appendix C
154
LIST OF FIGURES
Figure
Page
3. 1. Schematic of the flow cell used to rinse
loosely adhered Listeria monocytogenes
from the surfaces.
68
3. 2. Effect of flow rates on residual adherent cells
of Listeria monocytogenes.
69
3. 3. The effect of preadsorbed protein type and film
age on adhesion of Listeria monocytogenes to
hydrophobic silica surfaces.
70
3. 4. The effect of preadsorbed protein type and
film age on adhesion of Listeria monocytogenes
to hydrophilic silica surfaces.
71
3. 5. The effect of p-Lg adsorption time and film
age on adhesion of Listeria monocytogenes to
hydrophobic and hydrophilic silica surfaces.
72
3. 6. The effect of BSA adsorption time and film age
on adhesion of Listeria monocytogenes to
hydrophobic and hydrophilic silica surfaces.
74
4. 1. Adhesion of L. monocytogenes to hydrophobic
silica and hydrophilic silica following
sequential adsorption of P-Lg and BSA (8 h
contact/protein).
97
4. 2. Adhesion of L. monocytogenes to hydrophobic
silica and hydrophilic silica following
sequential adsorption of P-Lg and BSA (1 h
contact/protein).
98
4. 3. Adhesion of L. monocytogenes to hydrophobic
and hydrophilic silica following competitive
adsorption of p-Lg and BSA for 8 h.
99
4. 4. Adhesion of L. monocytogenes to hydrophobic
and hydrophilic silica following competitive
adsorption of p-Lg and BSA for 1 h.
100
LIST OF TABLES
Table
3. 1. The mass of BSA and p-Lg (ug/cm2) remaining on
hydrophobic and hydrophilic surfaces
following 1 h of protein contact and
incubation in bufferfor different times.
Page
73
LIST OF APPENDICES FIGURES
Figure
Page
A. 1. Nisin adsorption isotherms constructed for
hydrophilic and hydrophobic silicon surfaces.
The mass of nisin remaining on each type of
surface after a 12 hour exposure to pure
•buffer is indicated by the closed (hydrophobic)
and open (hydrophilic)circles. The curves drawn
through the data follow equation [2].
137
A. 2. Nisin activity manifested by inhibition zones
around the periphery of hydrophilic silicon
plates. Numbers 1-10 indicate the concentration
of nisin solutions (100 to 1000 mg/1) that had
contacted the surfaces. C denotes surfaces that
were not exposed to nisin. The indicator
bacterium is Pediococcus
pentosaceus FBB-61-2.
Grid squares are 2 cm2.
138
A. 3. Detection of the activity of nisin desorbed
from hydrophilic surfaces with 1% w/w
Tween 80. Numbers 1-10 indicate the
concentration of nisin solutions
(100 to 1000 mg/1) that had contacted the
surfaces. Nis 1 and Nis 2 are standard
concentrations (100 and 50 U/ml respectively)
of nisin. C denotes surfaces that were not
exposed to nisin. The indicator bacterium is
Pediococcus pentosaceus FBB-61-2. Grid
squares are 2 cm2.
139
B. 1. The sequential adsorption of BSA and P-Lg to
hydrophobic surfaces. P-Lg was adsorbed first
for 4 h followed by rinsing for 30 min. then
BSA was introduced for another 4 h.
144
B. 2. The sequential adsorption of BSA and P-Lg to
hydrophobic surfaces. BSA was adsorbed first
for 4 h followed by rinsing for 3 0 min. then
P-Lg was introduced for another 4 h.
145
B. 3. The sequential adsorption of BSA and P-Lg to
hydrophilic surfaces. P-Lg was adsorbed first
for 4 h followed by rinsing for 30 min.
BSA was introduced for another 4 h.
then
146
B. 4. The sequential adsorption of BSA and p-Lg to
hydrophilic surfaces. BSA was adsorbed first
for 4 h followed by rinsing for 30 min. then
P-Lg was introduced for another 4 h.
147
B. 5. The sequential adsorption of BSA and P-Lg to
hydrophobic surfaces. p-Lg was adsorbed first
for 1 h followed by rinsing for 30 min. then
BSA was introduced for another 1 h.
148
B.
6.
The sequential adsorption of BSA and P-Lg to
hydrophobic surfaces. BSA was adsorbed first
for 1 h followed by rinsing for 3 0 min. then
P-Lg was introduced for another 1 h.
149
B. 7. The sequential adsorption of BSA and P-Lg to
hydrophilic surfaces. p-Lg was adsorbed first
for 1 h followed by rinsing for 3 0 min. then
BSA was introduced for another 1 h.
15.0
B. 8. The sequential adsorption of BSA and p-Lg to
hydrophilic surfaces. BSA was adsorbed first
for 1 h followed by rinsing for 3 0 min. then
p-Lg was introduced for another 1 h.
151
B.
9. The competitive adsorption of BSA and P-Lg to
hydrophobic surfaces. Adsorption was performed
from an equimolar mixture of both proteins
for 8 h.
152
B. 10. The competitive adsorption of BSA and p-Lg to
hydrophilic surfaces. Adsorption was performed
from an equimolar mixture of both proteins
for 8 h.
153
LIST OF APPENDICES TABLES
Table
Page
C. 1. The adsorbed mass (yg/cm2) following sequential
adsorption of BSA and P-Lg for 1 and 4 h
to hydrophilic and hydrophobia surfaces. The
tabulated adsorbed mass for the first protein
is recorded immediately after rinsing and before
introduction of the second protein. The final
adsorbed mass as well as the percent increase
in the total adsorbed mass are tabulated after
the second adsorption time of the second
protein and before rinsing.
154
THE INFLUENCE OF PREADSORBED MILK PROTEINS ON ADHESION OF
LISTERIA MONOCYTOGENES TO
SILICA SURFACES
CHAPTER 1: INTRODUCTION
Protein adsorption at an interface is of paramount
importance in several areas including medicine, food and
pharmaceutical processing, and biotechnology. In the food
processing industry, proteins have been found to play a
major role in fouling of heat exchange surfaces because they
are found in high content in some fluid foods and they are
heat denaturable. This fouling results in reduction of heat
transfer efficiency leading to either insufficient heat
treatment of the processed food or increased energy costs.
Increased cleaning and labor costs as well as lost
production during periods of downtime are other aspects of
protein adsorption to such surfaces. In addition, it has
also been found (3) that protein adsorption can mediate
bacterial biofilm development on solid surfaces. Platelet
adhesion to blood-contacting devices and thrombogenisis are
initiated by protein adsorption to biomaterials. Surfaces
such as tooth enamel, mucosa, and the gum are examples of
substrates that are exposed to different proteins found in
saliva. These salivary proteins are suspected in mediating
bacterial colonization leading to gum and dental diseases
(8) .
Adsorbed protein molecules can alter substrate surface
properties significantly. This will substantially affect
subsequent events including the extent of cell adhesion,
thrombus formation and biofilm development.
The extent of protein adsorption as well as cell
adhesion has been attributed in part to the hydrophobicity
of the surface and the adhering entities themselves. Some
proteins adsorb at higher surface concentrations to
hydrophobic surfaces as compared to hydrophilic surfaces.
On adsorption to hydrophobic surfaces, it has been observed
(1) that an adsorption plateau occurs for each protein
studied, increasing with protein hydrophobicity.
Listeria monocytogenes is a pathogenic bacterium that
can contaminate contact surfaces used in food processing (6,
7). Colonization and growth of this microorganism on food
processing surfaces represents a serious obstacle to
consistently providing healthful, high quality products. The
fact that L. monocytogenes can attach to such surfaces at
refrigeration temperatures makes this problem of even
greater concern, particularly to the dairy industry (6).
Control of this problem seems best accomplished by focusing
attention on the early events that take place at the food
contact interface which lead to bacterial attachment and
biofilm formation.
Microbial adhesion to surfaces is, to some extent,
dependent on the presence, composition and conformational
state of a preadsorbed protein film. Characteristics of this
film are dependent on molecular properties of the individual
protein as well as those of the bare contact surface.
Adsorbed protein may change its conformation over time,
and the rate of conformational change would vary among
different proteins and from one surface to another (2, 4, 5,
9). Such surface-induced conformational changes should be
expected to influence protein and surface reactivity with
bacterial cells and spores just as the conformation of blood
serum proteins affects platelet and whole cell adhesion to
implanted biomedical materials. It is the purpose of this
research to evaluate the influence of adsorbed milk proteins
(a-lactalbumin (a-Lac), p-lactoglobulin (p-Lg), p-casein,
and bovine serum albumin (BSA)) on the extent of bacterial
adhesion measured at hydrophilic and hydrophobic silica
surfaces. The specific objectives are
1- prepare adsorbed, single component (or mixed) protein
film(s) of known mass and surface residence time on
silanized silicon surfaces, and expose the surfaces,
with and without preadsorbed films, to L. /nonocytogenes
to document the extent of resulting bacterial adhesion;
and
2- describe the extent of bacterial adhesion as a
function of contact surface hydrophobicity and the
nature of the preadsorbed films.
REFERENCES
Absolom, D., w. zingg, and A. Neumann. 1987. Interactions
of proteins at solid-liquid interfaces: Contact angle,
adsorption, and sedimentation volume measurements, p.
401-421. In J. Brash, and T. Horbett (eds.), Proteins
at Interfaces: Physicochemical and Biochemical Studies.
ACS Symp. Ser. 343, Washington, DC.
2. Andrade, J., V. Hlady, and R. wagenen. 1984. Effects of
plasma protein adsorption on protein conformation and
activity. Pure Appl. Chem. 56: 1345-1350.
3. Bryers, James D. 1987. Biologically active surfaces:
Processes governing the formation and persistence of
biofilms. Biotech. Progr. 3:57-68.
4. Elwing, H., S. Welin, A. Askendahl, and I. Lundstrdm.
1988. Adsorption of fibrinogen as a measure of the
distribution of methyl groups on silicon surfaces.
J. Colloid Interface Sci. 123:306-308.
Krisdhasima, v., P. Vinaraphong, and J. McGuire. 1993.
Adsorption kinetics and elutability of o-lactalbumin,
p-casein, p-lactoglobulin and bovine serum albumin at
hydrophobic and hydrophilic interfaces. J. Colloid
Interface Sci. 161:325-334.
6. Mafu, A., D. Roy, J. Goulet, and P. Magny. 1990.
Attachment of Listeria monocytogenes to stainless
steel, glass, polystyrene, and rubber surfaces after
short contact times. J. Food Protec. 53:742-746.
7. Mafu, A., D. Roy, J. Goulet, and L. Savoie. 1991.
Characterization of physicochemical forces involved in
adhesion of Listeria monocytogenes to surfaces. Appl.
Environ. Microbiol. 57:1969-1973.
8. Mortensson, J., H. Harwin, L. Lundstrdm, and Th.
Ericson. 1993. Adsorption of lactoperoxidase on
hydrophilic and hydrophobic silicon dioxide surfaces
An ellipsometric study. J.Colloid Interface Sci
155:30-36.
9. soderquist, M. and A. Walton. 1980. Structural changes
in protein adsorbed on polymer surfaces. J. Colloid
Interface Sci. 75:386-397.
CHAPTER 2
LITERATURE REVIEW
Since the research conducted here deals with the
influence of protein adsorption on bacterial adhesion, it
seems logical to summarize relevant literature in both
protein adsorption and bacterial adhesion.
Protein adsorption
Protein adsorption to solid surfaces has been studied
for decades, and several thorough reviews are available (2,
27, 46). However, important features of protein adsorption
relevant to the work presented here will be reviewed.
Protein exchange on solid surfaces
Protein exchange on solid surfaces depends on surface
properties, types of adsorbed protein molecules,
concentration and types of protein in solution, and
incubation time. Elwing et al. (15) studied protein exchange
on solid surfaces using a wettability gradient method. The
sequential adsorption of human y-globulin (HGG) and human
fibrinogen (HFG) to silicon surfaces wettability gradient
7
was performed for 1 h. The surfaces then further incubated
for 4 h in HFG or HGG.
They found that protein exchange occured more readily
on hydrophilic than on hydrophobic surfaces. To explain the
occurrence of the exchange reactions, it has been assumed
that an adsorbed protein molecule is attached to the surface
by multiple binding sites. These binding sites are dynamic
in nature so they would disappear and rearrange
continuously. However, it is unlikely that all the binding
sites of the protein would disappear momentarily. Therefore,
the rate of complete desorption is small (15).
Hunter et al.
(28) studied p-casein and lysozyme
adsorption at the air/water interface. Their results
indicated that electrostatic interaction had little role in
determining exchange behavior and that the flexibility of
the adsorbed and displacing molecules were more important
than intermolecular interactions in such behavior.
Enckevort et al. (17) studied the adsorption of bovine
serum albumin at the stainless-steel/aqueous solution
interface using radiochemical techniques. They found
that the adsorbed protein was essentially nonexchangeable
and adsorption was irreversible upon dilution.
Protein desorption
The desorption process of a protein depends on the
incubation time of the protein with the substrate. It has
been found (53) that the longer the incubation time, the
slower the desorption. The desorbed material however,
experiences a change in structure as compared to that of the
protein molecule before adsorption. For example, desorbed
BSA is essentially denatured. However, the conformation of
the desorbed molecule is a function of the time spent in the
adsorbed form. Since the desorbed protein is denatured and
is less able to undergo entropic processes favoring
adsorption, protein slowly desorbs more or less irreversibly
(53). The desorption process is very slow, at least after
long incubation times, compared with the adsorption process.
The rate of desorption for each conformational state will be
different and should depend on the incubation time.
Soderquist and Walton (53) have postulated three steps
in which protein adsorption/desorption on or from polymeric
surfaces takes place. In the first step, they proposed that
fairly rapid and reversible adsorption occurs in a minute or
so, reaching pseudoequilibrium. Up to 50-60% surface
coverage has a random arrangement of adsorbed molecules, but
some form of surface ordering takes place thus allowing
further adsorption. In the second step, each molecule on the
surface undergoes a structural transition as a function of
time to optimize protein-surface interaction. As the
proteins increase their interaction with the surface (and
their entropy), desorption becomes less likely. In the third
step, proteins slowly desorb, although the probability of
desorption decreases with increased residence time.
Surface-induced conformational chances
Protein conformational changes can take place once
protein adsorbs to a surface. The presence of multiple
states of an adsorbed protein has been suggested by several
researchers. For example, rinsing a surface after protein
has been adsorbed does not remove all of the protein,
indicating the presence of weakly and tightly adsorbed
protein. Some of the remaining film is removable with
further incubation in buffer. Small molecular weight surface
active substances such as sodium dodecylsulfate, can remove
some of the protein adsorbed (33) though its ability to
remove the film decreases with increasing surface-protein
contact times. In general, greater amounts of adsorbed
proteins are found on hydrophobic surfaces than on
hydrophilic surfaces. Increased hydrophobic interaction is
indicative of a more extended structure of the molecule
which allow it to cover a larger area of the contacting
10
surface. This has been taken as an indication that a
conformational change has taken place on the surface, since
hydrophobic residues of a protein are generally found in the
interior of the molecule.
Adsorption may lead to an increase or decrease in
titratable groups, subsequently affecting pH values.
Titration data can thus be interpreted in terms of
conformational changes. Norde and Lyklema (47) analyzed
proton titration curves for bovine pancreas ribonuclease
(RNase) and human plasma albumin (HPA) adsorbed to
negatively charged polystyrene lattices. They found that the
charge of the protein molecules varied by adjusting the pH
of the solution. However, upon adsorption of the proteins, a
small change of pH was observed. The plateau values of
adsorbed protein indicated that RNase is less sensitive to
pH than those for HPA. The adsorption of HPA caused
substantial structural changes, which were dependent on pH
and had a minimum change taking place at the isoelectric
point of the protein.
Haynes et al. (25) found that the plateau values of the
globular proteins lysozyme and a-Lac adsorbed to
microspheres of negatively charged polystyrene latex showed
a complex dependence on pH with a maximum occurring at the
isoelectric point of the protein/sorbent complex. They found
that the adsorbed-state titration curves differed markedly
11
from the corresponding dissolved state curves. For o-Lac,
their data suggested that the protein molecule underwent
substantial changes in secondary and tertiary structure upon
adsorption (25). The average of the pKa data for the two
proteins suggested to the authors that a fraction of the
charged carboxyl groups were in close proximity to the
adsorbent and that the adsorbed protein was largely
denatured (25).
Several other techniques have been employed to study
protein conformational changes. Morrissey and Stromberg (43)
used infrared difference spectroscopy to study protein
conformational states on silica particles. This technique
permits deduction of conformational states by determining
the fraction of carbonyl groups bound by direct interaction
with the surface. The authors found that the interaction of
fibrinogen with the surface occured through hydrogen
bonding, and was influenced by concentration. More hydrogen
bonding to the surface was found at low solution
concentrations than high concentrations. However, they found
that solution concentration has no effect on prothrombin and
BSA indicating that the internal bonding of these two
proteins is adequate to prevent changes in their structure
while adsorbed.
Intrinsic ultraviolet total internal reflection
spectroscopy is another technique that indirectly provides
12
information on the local environment of tryptophan residues
of an adsorbed protein. Andrade et al. (3) have used this
technique to study conformational changes of human plasma
fibronectin (HPF) adsorbed to hydrophilic and hydrophobic
silica. The authors found that the adsorbed protein
exhibited fluorescence maxima at 326 and 321 nm on
hydrophobic and hydrophilic respectively, however, the
fluorescence maximum exhibited by unadsorbed protein in
solution was 321 nm. These results indicate that the
adsorbed HPF experienced some conformational changes on the
hydrophobic surface.
Jonsson et al. (32) performed adsorption procedures
using HPF and immunoglobulin G (IgG) either by a single shot
or a successive addition method. When they compared the
isotherms generated on hydrophobic silica, they found them
different. These results suggested that protein adsorption
was irreversibile because the surface-dependent
conformational changes take place over time. On hydrophilic
silica, the successive addition of HPF resulted in an
isotherm that was similar to that obtained by a single
addition of the protein. At long equilibration times, the
isotherms were observed to coincide. The authors proposed
that the HPF which adsorbed to hydrophilic silica tended to
experience conformational changes to a lesser degree than
HPF adsorbed to hydrophobic silica.
13
The previous observations are consistent with results
obtained by investigators using ellipsometry as another
technique for studying protein conformational changes. These
authors (16) believe that protein complement factor 3
undergoes a greater degree of conformational change on
hydrophobic silicon surfaces than on hydrophilic silicon
surfaces, because greater adsorbed mass was found to occur
on hydrophobic silicon surfaces.
Bacterial adhesion
Bacterial adhesion to inert surfaces is of great
concern, as their presence may result in biofouling and act
as a likely source of contamination (26). In a food
processing facility, bacterial adhesion to food and food
contact surfaces is important as transmission of disease or
product losses due to spoilage may be the subsequent result
of bacterial adhesion (26).
The development of microbial biofilms on surfaces
results also in bacterial growth, division, and production
of metabolites within the biofilm. Continuing development of
biofilms results in increased turbulence, which in turn
increases fluid frictional resistance. The resultant energy
loss and reduced performance, as well as the need for
14
regular cleaning of pipe surfaces, cost hundreds of millions
of dollars per year on a worldwide scale (38).
Perhaps the most striking phenomenon is the resistance
of adhered bacterial cells to sanitizers and disinfectants.
This phenomenon has been related to several contributing
factors and a detailed summary of the literature follows.
Initially a clean surface is exposed to an aqueous
environment to become conditioned by the chemical
constituents present. On these conditioned surfaces, the
cell will adhere and firmly attach. The activity of cell
population will then depend on the metabolism and growth of
each member species under local surface conditions. These
metabolic activities can vary from substrate consumption,
cellular growth and replication, and synthesis of
exopolymers (8).
The isolation of encapsulated bacteria from chlorinated
drinking water have been reported by several investigators
which led to a general conclusion that the production of
extracellular capsule helps to protect bacteria from
chlorine. However, this was not the case in a study reported
by LeChevallier et al. (35). These investigators have shown
that encapsulated and uncapsulated strains of Klebiella
pneumonia used in their study showed no significant
difference in their resistance to disinfection. But the
growth of strains in low nutrient medium increased bacterial
15
resistance to free chlorine three-fold. This has been
related to changes in the capsule material and because the
uncapsulated strains lacked other defense mechanisms, it was
speculated that the cell membrane changes were responsible
for the observed resistance. However, this contradicts what
is known about the function of the capsular structure. By
understanding the nature of the cell components and their
functions, it may be possible to speculate about the
resistance characteristic exhibited by microorganisms
adhered to surfaces. It has been reported that the
extracellular capsule of excreted polymers is polysaccharide
in nature, although some other species such as Bacillus
species, form a polypeptide capsule. The main function of
the capsule is to adsorb useful substrate molecules and ions
and adsorb toxic agents, preventing them from penetrating
into the cytoplasm (29). The same material can also be
positively involved in the adhesion process (29). The
formation of films around or above the original biofilm
might provide a protecting means against toxic agents.
Assuming that such films react with the incoming toxic agent
to a degree where the residuals also remain high. But these
residuals will not be effective since probably a more
complex layer has been formed and does not allow the toxic
molecule to penetrate further to reach the cytoplasm where
it can exert its toxic effect.
16
LeChevallier et al.
(35) have also demonstrated that
attachment to surfaces has an impact on the bacterial
resistance to disinfection. Theoretically, the physical
hindrances of a surface could affect the ability of a
disinfectant to approach the cell membrane. A freely
suspended organism is susceptible to a disinfectant from all
sides and at all angles, while an organism attached to a
surface is susceptible only from one side (35) . This is
supported by findings reported by Frank and Koffi (23) who
reported that removing adherent cells of L. monocytogenes
from the surface increased their susceptibility to
sanitizers equivalent to that of planktonic cells. Another
effect observed was an increase in the resistance as the age
of the biofilm increased. LeChevallier et al. (35) observed
that biofilms grown for two days are less chlorine resistant
than biofilms grown for seven days under identical
conditions. This was related to the possible physiological
changes in population, such as starvation effects, or
coalescence of the biofilm which may have made the cells
more resistant.
The literature with respect to L. monocytogenes
resistance to sanitizers is somewhat confusing. In a study
reported by Mustapha and Liewen (45), they indicated that L.
monocytogenes was more resistant to sodium hypochlorite when
incubated on steel surfaces for 1 h than those incubated on
17
the same surfaces for 24 h. They related that to the
presence of moisture on steel surfaces at the short time (1
h) . However, in a recent study, Lee and Frank (36) reported
different results showing that L. monocytogenes adhered to
stainless steel surfaces were more resistant when they were
incubated for even longer times (8 days). However, Lee and
Frank (36) related the increased resistance of the cells
after 8 days to the increased microcolony formation as
opposed to the 4 h population which would have had little
chance to produce microcolonies. This could be a valid
assumption since it is known that the increase in cell
density causes other effects such as resistant to heat.
Moreover, the results of Lee and Frank are consistent with
LeChevallier et al. (35) who reported that biofilms grown
for two days are less resistant to chlorine than biofilms
grown for seven days under identical conditions. In a
previous study by Frank and Koffi (23), the authors stressed
the protection against sanitizers provided by attachment of
microorganisms. Growth after attachment resulted in
multilayer formation, which prevented penetration of
sanitizers, such as quaternary ammonium compounds (QAC)
beyond the first layer of cells. Since QAC are hydrophilic
cationic molecules, they would penetrate the hydrophilic and
negatively charged cell surface. Gram positive bacteria
however, produce lipoteichoic acid which is lipophilic (29)
18
and can prevent penetration of sanitizers (23). The effect
of glycocalyx is confirmed by Mosteller and Bishop (44).
They showed that L. monocytogenes cells were buried under
the biofilm layer, and the glycocalyx was clearly shown to
exist on the teflon surface. Moreover, their results showed
that iodophor, hypochlorite, acid anionic, QAC and other
sanitizers failed to reduce attached bacteria in sufficient
numbers in most of the cases indicating that the glycocalyxcovered cells were less susceptible to sanitizers used at
normal concentrations (44).
In another study by Krysinski et al. (34), the
mechanism of resistance was reported to be surfacedependent. They found that the resistance of L.
monocytogenes was dependent on the type of surface to which
they were attached. Stainless steel was observed to be more
easily cleaned and sanitized than polyester or
polyester/polyurethane surfaces. This conclusion was reached
when scanning electron microscopy revealed no significant
differences among surfaces used in the study in terms of
deep cracks which may provide protection from chemicals.
The mechanism by which bacteria in biofilms resist
antibiotics seems similar to the mechanism of resisting
sanitizers. Anwar et al. (4) reported that old biofilm cells
of Staphylococcus aureus are very resistant to tobramycin
and cephalexin. They observed that a much higher antibiotic
19
concentration was required to eradicate old biofilm cells.
The mechanism of resistance of old biofilm was proposed to
involve changes in penetration of antibiotic across the cell
envelope, the production of antibiotic-degrading enzymes,
and changes of molecular targets of antibiotics. The
presence of cells under the glycocalyx makes them less
susceptible to antibiotics. Moreover, the exopolysaccharides
produced by the bacteria may bind the antibiotics and
prevent penetration (4). These concepts are supported by
another study reported by Vergeres and Blaser (60) who found
the same behavior when studying the effect of amikacin and
other antibiotics on suspended and adherent Pseudomonas
aeruginosa and Staphylococcus epidermidis.
Mechanisms and factors affecting adhesion
Much work has been conducted to determine the
parameters affecting bacterial and cell adhesion. Some of
these factors that have been studied to some extent include
temperature, concentration and age of culture, electrostatic
and hydrophobic interactions, pH, effect of macromolecule
adsorption to the solid surface, and the surface
characteristics of the material onto which adhesion takes
place. The following is a short summary of the literature
concerning these factors.
20
Temperature effects
The environmental temperature is an important factor
controlling bacterial growth and reproduction, and as such,
it should have an influence on bacterial adhesion to
surfaces as well. Some bacteria can grow within a wide range
of temperatures. L. monocytogenes, for example, can grow at
refrigeration temperatures up to 45 0C. Herald and Zottola
(26) have investigated the influence of temperature on
adhesion of L. monocytogenes to stainless steel. Three
temperatures were used, 35, 21, and 10 0C. It has been
observed that L. monocytogenes Scott A and and L.
monocytogenes Jalisco attached at 35 0C as single cells ,
cell clumps, or microcolonies. Attached cells had multiple
flagella, but in less populated areas a single flagellum was
observed. At an incubation temperature of 21 0C, attachment
occurred only as single cells with 2-3 flagella per attached
cell. The incubation temperature 10 0C resulted in few
adhering cells but each had several nonpolar flagella.
Different temperatures affected the number of cells
adhered and the number of flagella which formed. The
formation of flagellum was postulated to aid adhesion to
such surfaces. However, as the authors noted these flagella
were not always observed when preparations with various pH
levels were studied. Therefore, there should be other
21
surface or physiological characteristics involved.
Temperatures below these studied by Herald and Zottola (26)
have been observed to reduce attachment of other bacterial
species.
Fletcher (19) reported that adhesion of a marine
pseudomonad to polystyrene was dependent on temperature. A
temperature of 3 0C was found to decrease the proportion of
stationary phase cells attached as compared with cells at 20
0
C. The reduced adhesion with lower temperature was ascribed
to an increased medium viscosity, an increase in bacterial
surface polymer, or a change in bacterial physiology (19).
Fletcher and Marshall (21) have also observed that the
decrease in adhesion to hydrophobic petri dishes (PD) and
hydrophilic tissue culture dishes (TCD) at 15 0C was lower
by factors of 0.52 and 0.70, respectively, than that at 20
to 25 0C (21). The effect of temperature seems to depend
upon the bacterial strain. While adhesion was lower at low
temperatures than that at higher temperature for some
species (19, 26), other species were found to adhere at
higher numbers at low temperatures. Belas and Colwell (9)
found that adhesion of Vibrio parahaemolyticus, a common
contaminant of shellfish (31), to chitin correlated well
with the Langmuir adsorption isotherm at temperatures of 15,
25, 37, and 45 0C. Saturation of the chitin was reached and
further increase in cell concentration did not affect
22
adhesion. However, adhesion at 4 0C did not follow the
Langmuir adsorption isotherm, that is; adhesion was directly
proportional to cell concentration in the bulk environment
(9) .
Culture concentration and culture age effects
Fletcher (19) found that the adhesion of a marine
pseudomonad to polystyrene increases with increasing culture
concentration and time of attachment as well as the age of
the culture. A steep increase in the number of cells adhered
was observed to occur at the lower cell concentrations.
Higher cell concentration did not increase adhesion much and
the adhesion leveled off as surfaces became saturated. The
effect of the age of the culture was reflected by adhesion
being greatest when the culture was in the log phase
followed by the stationary phase, and the death phase was
the lowest. High cell concentrations and long incubation
times would allow an increased number of bacterial
collisions with the surface, thereby increasing the
opportunity for adhesion (19). Two factors were postulated
to contribute to the influence of the culture age on
adhesion, changes in cell motility, and changes in cell
surface polymers. Motility increases the chance that a
bacterium may encounter a potential adhesion site while the
23
nonmotile bacterium depends only on Brownian motion or the
water currents movement toward the surface (19). Fletcher
(19) observed that the motility of the studied bacteria
changed with culture age in which the log phase culture had
the largest proportion of motile cells. The cells motility
decreased with the onset of the stationary phase. Cell
surface polymers also play a role in pseudomonad adhesion,
and are affected by culture age (19).
Electrostatic and hvdrophobic interactions
A variety of solid surfaces, as well as bacteria, are
negatively charged (59), a characteristic that results in a
repulsive electrostatic interaction between cells and solid
surfaces. This repulsive interaction is dependent on the
surface potentials and thickness of the electrical double
layer. The thickness of the double layer is inversely
proportional to the square root of the ionic strength (59).
Hence at high electrolyte concentration, or in the presence
of polyvalent counterions, the electrostatic interaction
will be reduced and cell adhesion will be decreased (59).
The influence of electrolytes on adhesion to solid
surfaces was studied by McEldowney and Fletcher (40) in
order to determine the effect of electrostatic interaction.
It was found that the concentration and type of electrolyte
24
influenced bacterial adhesion to PD and TCD surfaces. This
influence varied with the bacterial species and the solid
surface (40). Marshall et al. (39) found that the number of
achromobacteria reversibly adhered, increased with
increasing electrolyte concentration or as the thickness of
the electrical double layer decreased. The bacteria were
observed to be reversibly adsorbed at lower concentration of
the divalent electrolyte MgSC^, than of the monovalent
electrolyte NaCl. The concentrations at which complete
repulsion occurred were approximately 5 x 10~4 M for NaCl
and approximately 5 x 10"5 M for MgS04. The difference was
related to the greater compression of the double-layer in
the divalent system at comparable concentration, with both
types of electrolyte, bacterial repulsion was observed when
the theoretical thickness of the defuse double-layer
exceeded about 2 00 A (39).
Husmark and Ronner (30) observed that adhesion of
Bacillus cereus spores to hydrophobic and hydrophilic
surfaces was influenced by pH. It has been observed that
adhesion was maximum at pH 3 which corresponds to the
isoelectric point of the spore (30). The differences in
adhesion at different pH values were related to the altered
charges of the spore surface. This surface charge alteration
may change the electrostatic interactions between the glass
surface and/or the conformation of the spore surface (30).
25
At pH values above the isoelectric point of the spores,
adhesion was less than that at the isoelectric point and the
relative decrease was larger for the strongly negatively
charged hydrophilic surface than for the more weakly charged
hydrophobic surface. This was related to the electrostatic
repulsion between the spore surface and the negatively
charged hydrophilic glass surface. A similar explanation was
offered when adhesion was observed to decrease at pH values
below the isoelectric point of the spores (30).
Hydrophobic interactions between surfaces largely
depend on the unique properties of water itself. The
hydrophobic moieties, immersed in aqueous phase, are
surrounded by structured layers of water. The water
molecules in such shells have limited freedom to undergo
hydrogen bonding, and are at a higher energy level than
molecules in the bulk solution (51). Assuming that the
hydrophobic moiety can not interact with water molecules,
then the energy required to introduce this hydrophobic
entity into the water phase is analogous to that required to
make a cavity in the water which have the size of the
immersed hydrophobic moiety. This energy is also similar to
that required to increase the surface area of water.
Structured water molecules must assume a constrained
conformation, and when they are proximal to apolar moieties,
they must assume a configuration of higher energy. When two
26
polar moieties or surfaces approach each other, the
constrained water molecules can be freed into the bulk
aqueous phase. This is an energetically favorable process.
It is associated with an increase in entropy (51).
Therefore, adhesion mediated by hydrophobic interaction can
be seen as a process of exclusion from the water phase, i.e.
minimizing an unfavorable interface (51).
Results obtained from several studies concerning the
influence of hydrophobic interaction on adhesion of bacteria
to solid surfaces are in line with the previous argument.
Dahlback et al. (13) have reported a correlation between the
accumulation of the bacteria at an interface and the degree
of hydrophobicity of the accumulating bacteria. Hydrophobic
interaction can dominate under specific experimental
conditions. Meinders et al. (42) have found that in adhesion
experiments carried out at pH 2, the electrostatic
interactions were virtually absent because the substrata
used had a zero zeta potential. Van Loosdrecht et al.
(57)
characterized bacterial cell hydrophobicity by measuring
water contact angle and electrophoretic mobility. It was
concluded that cell surface hydrophobicity was the major
determining factor in adhesion to negatively charged
polystyrene. When the relative hydrophobicity declined,
electrophoretic mobility had more influence on adhesion.
Husmark and Ronner (30) studied the forces involved in
27
adhesion of Bacillus cereus spores to hydrophobic and
hydrophilic glass surfaces under different environmental
conditions. The spores were adhered from media with
different polarities as well as different pH and ionic
concentrations. When increasing ethanol concentrations were
applied to the media, the polarity of the media was observed
to decrease and the predominant force of adhesion was found
to be hydrophobic in nature (30) . This was concluded when
adhesion was observed to decrease with decreasing polarity
of the medium which is in agreement with theories regarding
the formation of hydrophobic interactions (30). When the
less polar ethanol is introduced to the medium, it is
expected that H-bond formation will be reduced. This would
in turn reduce the hydrophobic effect. Results obtained on
hydrophobic glass indicated that adhesion was markedly
decreased with increasing ethanol concentrations. Moreover,
using the highest ethanol concentration solution tested, the
adhesion to both hydrophobic and hydrophilic glass surfaces
was the same (30). This indicates the importance of
hydrophobic interactions for bacterial adhesion and the fact
that hydrophobic interactions are less dominant in less
polar solutes (30). However, when the hydrophobicity of the
spores of different Bacillus species was determined using
hydrophobic interaction chromatography (HIC)
(50), it was
found that more hydrophobic spores adhered to hydrophobic
28
surfaces than to hydrophilic surfaces. Moreover, HIC
measurements indicated that there are large variations in
surface hydrophobicity between the spores of different
species tested due to the large differences in chemical and
morphological characteristics of the spores (50). For
example, one of the species tested was B. cereus. These
spores have a loosely associated exosporium as the outermost
layer. This layer has a structure composed mainly of
different proteins (up to 52%), lipids (13%), and
phospholipids(6%). Such structure has been implicated to
contribute to the high hydrophobicity and degree of adhesion
to hydrophobic surfaces (50).
Effect of organic macromolecules on adhesion
Adsorption of soluble macromolecules onto solid
substrates may affect adhesion. Soil surfaces, in aquatic
environment for example, will quickly adsorb organic
molecules forming a conditional film. Proteins, as organic
molecules, readily adsorb to a variety of surfaces too. Some
microorganisms, including Listeria, are capable of
synthesizing polymeric exudates which can serve to anchor
them to contact surfaces (26) . Though adhesion is often
mediated by these polymeric exudates, it is likely that such
exudates still have to adhere to some preadsorbed film (5,
29
7). Several studies have directly investigated the effect of
preadsorbed protein films on adhesion and results obtained
were, in some cases, protein- and surface dependent. Tamada
and Ikada (55) investigated the effect of preadsorbed BSA,
bovine yglobulin (IgG) , and plasma fibronectin (FN) on L
cell (the L-form cells are common cells that do not or have
lost the ability to produce the peptidoglycan layer of their
cell walls) adhesion to fourteen polymer surfaces of various
wettabilities. They found that BSA preadsorption entirely
inhibited L .cell adhesion, independent of substrate
wettability. IgG yielded results similar to those of BSA. On
the other hand, when FN was preadsorbed to the surfaces, L
cells were able to adhere to all substrates with the
exception of poly(vinyl alcohol) and an acrylamide-grafted
polyethylene. The inhibition of L cell adhesion to the
substrates precoated with BSA and IgG was related to the
ability of these proteins to render the surfaces
sufficiently "nonartificial" so as to prevent cell adhesion.
Preadsorption of FN apparently provided the surfaces with
ligands for the receptor sites of cells. Fletcher (18)
investigated the effect of BSA and several other proteins on
adhesion of a marine pseudomonad to polystyrene Petri
dishes. She found that BSA, gelatin, fibrinogen and pepsin
impaired cell attachment through adsorption to the dish
30
surface. Protamine and histone were found not to markedly
inhibit adhesion.
Meadows (41) studied the effect of four proteins on
adhesion. The attachment of Aeromonas liquifaciens,
Escherichia coli and Pseudomonas fluorescens to glass slides
was followed after their suspension in phosphate buffer of
pH 7.0 containing the proteins salmine, BSA, casein or
gelatin. The results indicated that the presence of salmine
and BSA markedly reduced the number of cells adhered, as
compared to adhesion from buffer. However, casein and
gelatin promoted adhesion except for the effect of gelatin
on E. coli where adhesion was reduced. The effects were
related to the glass, bacterial surface and molecular
properties of each protein. The glass and the bacterial
surfaces carry a net negative charge, however, the basic
protein salmine is positively charged at pH 7.0. Therefore,
salmine is expected to reduce adhesion by reducing the
negative charge through its adsorption to both glass and
bacterial cells. The proteins casein and gelatin have
isoelectric points below pH 7.0 and therefore promote
adhesion, probably by a converse mechanism. BSA, however,
inhibited adhesion even though it has an isoelectric point
below pH 7.0 (41). Clearly, the effect of each protein on
bacterial adhesion varied according to the type of protein
present (41).
31
Substratum effect
Fletcher and Loeb (20) studied the influence of
substratum characteristics on attachment of a marine
Pseudomonad to solid surfaces. The hydrophobicity of the
substrates was measured by contact angle method. The
substrates were divided into three groups in relation to
their hydrophobicity. The first group included nonpolar
polymers PTFE (teflon), PE, and PS. The second group
represents polymers with polar groups, such as nylon 6.6,
epoxy resin, and PET. The third group include inorganic,
hydrophilic materials such as glass, mica, germanium
discharge treated in air and platinum. The results showed a
linear relationship between bacterial attachment and water
wettability in the series of polymers. Cell adhesion
increased with increasing contact angle. However, adhesion
to hydrophilic surfaces were inconsistent. The authors
related that to the surface characteristics. For example,
glass and mica had a low number of adhered bacteria. This
was related to the negative charge of the surfaces as
determined by electrokinetic measurement. On the other hand,
platinum, which had a higher number of attached bacteria was
found to be electrokinetically positive in organic-depleted
seawater. Such data illustrate the importance of charge on
32
hydrophilic substrata and the importance of hydrophobic
interaction on polymers on bacteria adhesion (20).
Surface energy effects
Several studies have tried to correlate between
adhesion of macromolecules and bacterial cells with surface
characteristics such as surface free energy, surface
wettability and critical surface tension (1, 6, 7, 11, 14,
49). There is a zone where adhesion is minimal and it is
usually referred to as nonadhesive zone despite the presence
of an abundant but loosely organized overcoating material or
conditioning film. This zone is observed to range between 20
to 30 dynes/cm (7). Tenacious bioadhesion is correlated with
critical surface tensions converging in the zone between 3 0
and 40 dynes/cm, as a result of the tighter, more coherent
overlying films bound to such surfaces (6, 7). When a
variety of solid surfaces were exposed to a warm coastal
seawater for periods of times ranging between 2 0 to
384 h, the number of attached cells was a function of
existing critical surface tension (11) . More cells adhered
to high energy surfaces, than to low energy surfaces. From
such observations, it was concluded that adsorbed organic
layers change initial values of critical surface tension
33
toward a narrow range of values that appear to promote
bacterial adhesion (11).
Absolom et al.
(1) developed a thermodynamic model that
predicts adhesion to surfaces at various conditions of free
energies of the substrate, the bacterial cell surface, and
the suspending medium. The model predicts that the change of
free energy of adhesion (AFadh) would increase as the
substrate surface free energy increases provided that the
suspending medium has a surface tension less than that of
the bacterial cell surface. The AFadh would decrease with
increasing substrate surface free energy if the suspending
medium has a greater surface tension than the bacterial cell
surface. Experimental data obtained by using various
substrates and various bacterial strains both with various
surface tensions, including L. monocytogenes, agreed with
the predicted model. L. monocytogenes, a hydrophilic
bacterium (contact angle of 26.1° and a surface tension of
66.3 ergs/cm2) , has shown that it would adhere in greater
amounts to substrates with low surface free energy, and
adhesion was less to substrates with higher surface free
energy in a suspending medium of constant surface tension
(i.e., at surface tension of 72.8 ergs/cm2) . Adhesion of
this bacterium was also observed to go down as the substrate
free energy increased using suspending mediums with various
surface tensions (1).
34
In a previous study, Gerson and Scheer (24) found a
linear relationship between the logarithm of the number of
cells adhered and the free energy of adhesion of
Staphylococcus aureus to a variety of hydrophobic surfaces.
The effect of macromolecules on substrate properties
Immediately following immersion of a solid surface into
an aquatic environment, macromolecules and other low
molecular weight substances, start to adsorb to the surface
forming conditioning films which change the charge and free
energy of the surface (38). Sometimes changing the surface
properties does not alter the course of adhesion (6). It is
not clear whether bacterial macromolecules interact directly
with the conditioning films or whether they probe through
them and interact directly with original solid surface (38).
The original solid surface properties clearly have an
influence on the extent and type of adhering bacteria, as
well as their strength of adhesion. This influence could be
from direct interactions between the surface and the
bacteria, or indirectly through the orientation of the
adsorbed macromolecules (38) . However, it has been found
that some of these macromolecules contributed to reduced
adhesion, possibly by their ability to change the surface
properties of the substrate. The changes that this causes in
35
surface hydrophobicity, can be measured by contact angle
methods.
Some studies indicated that adhesion would be reduced
if surface contact angle were reduced by contact with
protein. Fletcher and Marshall (21) have examined the effect
of several proteins on surface contact angle and subsequent
bacterial adhesion to such surfaces. The contact angle
measured on bare PD and TCD were 90 and 29°, respectively.
Increasing concentrations of bovine glycoprotein (BGP) gave
progressively diminishing contact angles on both surfaces
and at 100 \ig/ml rendered the TCD surface completely
wettable (<150) and the PD moderately wettable (64°). BSA
and fatty acid-free BSA (BSA-FAF) had a similar effect,
which was more pronounced with BSA-FAF, in which the bubbles
failed to make contact with the TCD at 1.0 ug/ml and with
the PD at 100 ug/ml (21) . This resulted in decreased
bacterial adhesion to such surfaces. BGP, BSA or BSA-FAF
inhibited adhesion regardless of the time allowed for
adsorption (21).
Control of bacterial adhesion
Over the years, several studies have been conducted to
test different surfaces and surface treatments, but these
have not generated adequate control procedures to prevent
36
microbial fouling of heat transfer surfaces. The ability of
microorganisms, especially pathogenic and food spoilage
causing microorganisms, to attach to inert surfaces is a
serious problem and continues to be the focus of
concentrated investigation in several fields. Microorganisms
that are adhered to surfaces have been found to be much less
susceptible to the killing effect of sanitizers commonly
used in the food industry (44) . Therefore, alternative
approaches need to be developed which will prevent initial
adhesion of microbial contaminants to food contact surfaces
rather than trying to detach them once they are adhered.
Some materials, when impregnated with biocides or
antibiotics, resist bacterial colonization for as long as
the antibacterial agents are released from the surfaces. For
example, antifoulant paints have been used to protect the
hulls of ships from fouling. These paints prevent organisms
from adhering to surfaces by releasing small amounts of
active ingredients (12).
Some surface-active compounds have also proven
effective in preventing microbial adhesion. Whitekettle
(61), treated stainless steel and wood surfaces with nonionic or anionic compounds of differing chemical nature.
With stainless steel, the results indicated an excellent
efficacy with many of the non-ionic surfactants, i.e., the
DP-1200 series dispersants. These materials all gave better
37
than 90% inhibition of microbial adhesion as compared to
untreated controls. The anionic compounds did not inhibit
adhesion. Cationic compounds such as quaternary ammonium
salts, also resulted in excellent inhibition of adhesion;
but it was due to its toxicity against planktonic cells
(61) .
A variety of polymer plastics are susceptible to
biodegradation by bacteria and fungi that metabolize
plasticizers. Attempts to prevent plastic degradation was
made by incorporating some toxic and nontoxic inhibitors.
Price et al. (48) have studied the effect of an insoluble
antimicrobial quaternary amine complex in plastics against
bacteria and fungi adhesion. The compound called Intersept®,
a relatively nontoxic inhibitor, was processed into the
matrices of ethylene vinyl acetate (EVA), polystyrene and
low-density polyethylene (LDPE). The EVA-LDPE containing 3%
Intersept was found to significantly reduce adherence of
Pseudomonas aeruginosa as compared to the EVA-LDPE polymer
without Intersept. Statistical differences in degree of
adherence of P. aeruginosa to the polymers with less than 2%
Intersept was not observed. The observed effect was related
partially to the possible changes in the hydrophobicity of
the plastics with incorporation of the inhibitor, as well as
the biocidal effect on the adhered bacteria (48).
38
Surface modification was suggested by several authors.
Ronner et al. (50) studied adhesion of Bacillus spores to
glass and stainless steel with relation to the spore
hydrophobicity. The spores were found to be hydrophobic and
therefore adhered to both hydrophobic and hydrophilic
surfaces more than did the vegetative cells. Since stainless
steel surfaces are quite hydrophobic (50) and they are most
often used in the dairy industry where B. cereus is a
causative agent for microbial contamination, the authors
suggested that steel surfaces may be replaced or treated so
that they yield more hydrophilic surfaces and thereby
minimi-ze the risk of colonization by Bacillus spores (50).
However, since dairy foods may contain different bacteria
with different hydrophobicity and hydrophilicity
characteristics it is difficult to change stainless steel
surfaces to be just hydrophilic. For example, van der Mei et
al.
(56) characterized the cell surface properties of the
thermophilic dairy streptococci and they found them to be
hydrophilic. The authors suggested that to prevent fouling
in the pasteurization process, the heat exchanger plates of
the pasteurizer should be rendered more hydrophobic since
the hydrophilic strains should adhere minimally to
hydrophobic surfaces (56).
Since adhesion of bacteria and other cells are
generally observed to occur to surfaces with preconditioning
39
films, and since these conditioning films most often are
organic molecules including proteins, several attempts to
prevent adsorption of these conditioning films have been
made. One of these attempts was in the biomedical field
where the search for developing blood compatible materials
for implantation in the vascular system is ongoing.
Selective adsorption of some proteins to surfaces could
prevent the adsorption of other proteins and consequently
decrease cell adhesion. This was based on the hypothesis
that adsorbed albumin provides passivation of blood
contacting surfaces since albumin layers interact minimally
with platelets. Selective adsorption of albumin is based on
the natural affinity of the protein for lipid-like materials
such as long chain fatty acids. Therefore, polyurethane
surfaces have been grafted with chains of alkyl groups of
different lengths. These surface modifications resulted in
significant increase of albumin retention in contact with
plasma as compared to control (underivatized) surfaces (10).
40
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48
CHAPTER 3
THE INFLUENCE OF PREADSORBED MILK PROTEINS ON ADHESION OF
LISTERIA MONOCYTOGENES TO HYDROPHOBIC AND HYDROPHILIC SILICA
SURFACES*
HAMOOD AL-MAKHLAFI, JOSEPH MCGUIRE AND MARK DAESCHEL
*This chapter is currently in press in Applied and
Environmental Microbiology.
49
ABSTRACT
The adsorption of p-lactoglobulin, bovine serum
albumin, a-lactalbumin and p-casein for 8 h, and plactoglobulin and bovine serum albumin for 1 h at silanized
silica surfaces of low and high hydrophobicity, followed by
incubation in buffer and contact with Listeria
monocytogenes, resulted in different numbers of cells
adhered per unit surface area. Adhesion to both surfaces was
greatest when p-lactoglobulin was present and was lowest
when bovine serum albumin was present. Preadsorption of alactalbumin and p-casein showed an intermediate effect on
cell adhesion. Adsorption of p-lactoglobulin for 1 h
resulted in a generally lower number of cells adhered as
compared to the 8 h adsorption time, while the opposite
result was observed with respect to bovine serum albumin.
The adhesion data were explainable in terms of the relative
rates of arrival to the surface and post-adsorptive
conformational change among the proteins, in addition to the
extent of surface coverage in each case.
50
INTRODUCTION
Listeria monocytogenes is a pathogenic bacterium that
can adhere to the types of contact surfaces used in food
processing (22, 23). Colonization and growth of this
microorganism on food processing surfaces would represent a
serious obstacle to consistently providing healthful, high
quality products. The fact that L. monocytogenes can attach
to such surfaces at refrigeration temperatures makes this
problem of even greater concern, particularly to the dairy
industry (22). Better understanding and hence optimal
control of this problem seems best accomplished by focusing
attention on the early events that take place at the food
contact interface which lead to bacterial attachment and
biofilm formation.
Microbial adhesion to surfaces is, to some extent,
dependent on the presence, composition and conformational
state of a preadsorbed protein film. Characteristics of this
film are dependent on molecular properties of the individual
protein as well as those of the bare contact surface.
Contact surface hydrophobicity or hydrophilicity, and its
influence on protein adsorption as well as cell adhesion,
have received much attention for several decades (2, 9, 12,
19, 25) .
51
Adsorbed protein may change its conformation over time,
and the rate of conformational change would vary among
different proteins and from one surface to another (4, 9,
16, 29). Such surface-induced conformational changes should
be expected to influence protein and surface reactivity with
bacterial cells and spores just as the conformation of blood
serum proteins affects platelet and whole cell adhesion to
implanted biomedical materials. It is the purpose of this
research to begin to evaluate the influence of adsorbed milk
proteins (o-lactalbumin (o-Lac) , p-lactoglobulin (P-Lg) , |3casein, and bovine serum albumin (BSA)) on the extent of
bacterial adhesion measured at hydrophilic and hydrophobic
silica surfaces.
MATERIALS AND METHODS
Monocrystalline and polished silicon plates (hyperpure,
type N, phosphorus doped, 1-0-0 orientation, resistivity =
0.8-2 ohm cm) were obtained from Wacker-Chemitronic GMBH
(Germany). Xylene, and sodium phosphate (mono and dibasic),
were of analytical grade. The bovine milk proteins P-Lg,
which contained the genetic variants A and B (3 x
crystallized and lyophilized, lot No. 98F8080), o-Lac (type
III, lot No. 128F8140), BSA (lot No. 15F-0112), and p-casein
(lyophilized, essentially salt free, lot No. 40H9510) were
52
obtained from Sigma Chemical Co. (St. Louis, MO).
Dichlorodimethylsilane (DDS)
(lot No. 11905cx) was obtained
from Aldrich Chemical Company, Inc. (Milwaukee, WI).
Surface modification
Silicon wafers were cut into plates of approximately 1
cm2 with a tungsten pen. The silanizations were slightly
modified from the method described by Jonsson et al.
(14).
Surfaces were silanized by reaction with 0.01 or 0.1% DDS in
xylene as described previously (15) . These surface
derivitizations are stable, and exhibit a low or high
hydrophobic character, respectively, as shown by contact
angle analysis (15). Surfaces silanized with 0.01% DDS
exhibit a partial negative charge in addition to low
hydrophobicity, and we will refer to them as hydrophilic
surfaces.
Protein adsorption
Equimolar solutions of protein (equivalent to 1 mg/ml
P-Lg) were prepared by first dissolving each in 0.01 M
phosphate buffer (pH 7.0) while stirring for 20 min. Each
protein solution was filtered through a preassembled,
presterilized Nalgene™ Bottle Top Filter (0.2 vim)
(Nalge
53
Company, Rochester, NY). From this solution, 10 ml was
pipetted into small Petri dishes (60 X 15 mm style
disposable polystyrene, Falcon 1007, Becton Dickinson and
Company, Lincoln Park, NJ) and surfaces were then introduced
to the protein solution. Surfaces were allowed to contact
protein solution for 8 or 1 h, after which they were rinsed
sequentially in three, 300 ml volumes of distilled,
deionized water. Each individual surface was rinsed for
about 15 s in each step. Protein films formed on surfaces
were then aged in 20 ml buffer for 0, 5, 10, or 15 h, then
surfaces were rinsed again as above and introduced to the
medium with bacterial cells. Great care was taken not to
allow drying of the adsorbed protein films. For the purpose
of quantifying the amount of protein desorbed during
incubation in buffer, the same tests were performed for P-Lg
and BSA adsorption for 1 h but in the absence of any cell
contact. Protein films in this case were dried after the
final rinse by blowing the surfaces with nitrogen gas and
then were kept in a dust-free desiccator for 24 h. The
amount of protein remaining on the surface was then
determined using ellipsometry (7).
54
Culture and adhesion
Listeria monocytogenes Scott A was grown in 50 ml
protein-free defined medium and incubated for 24 h at 37 0C
to reach the stationary phase. The defined medium was made
up of 20.8 g/1 RPMI-1640 medium (lot No 119F-4645-1, Sigma).
The medium was modified by the addition of 1% glucose, and
2% casamino acids (Difco Laboratories, Detroit, MI). Before
use, this medium was filter-sterilized using a preassembled,
presterilized Nalgene™ Bottle Top Filter (0.2 urn). Culture
was diluted (in Butterfield1s buffer; a phosphate buffer of
pH 7.0; 0.00426% potassium phosphate) to a cell density of
107 - 109 cfu/ml. Ten ml of this diluted culture was
transferred to small Petri dishes. Hydrophilic or
hydrophobic silica surfaces, either bare or coated with a
protein film, were introduced to the Petri dishes and
allowed to contact the culture for 3 h.
Rinsing
After contact with cells, surfaces were affixed to a
microscope glass slide using Elmer's Stix.All glue (Borden
Inc. Columbus, OH) without allowing the biofilm to dry.
Rinsing to remove the loosely adhered cells was done by
passing 1200 ml of distilled water through a flow cell into
55
which the glass slides with the affixed surfaces had been
inserted. The flow cell (volume 87.5 ml; a generous gift
from The Swedish Institute for Food Research) is shown in
Fig. 3. 1.
Criteria used for rinsing unattached bacterial cells
Unmodified silica surfaces were allowed to contact
cells for 3 h. Contact with cells was terminated and the
surfaces were rinsed with distilled water at different flow
rates for different periods of time. The flow rate and rinse
time chosen for all subsequent experiments were those for
which little change in the number of attached cells was seen
with increasing time and/or flowrate.
Image analysis
Following rinsing, surfaces were quickly covered by a
cover slip and images of bacterial cells adhered to surfaces
were taken using an incident light microscopy-image analysis
system (IAS). The IAS is composed of a 486 IBM compatible
computer interfaced to a Sony monitor and a Reichert Epistar
incident light microscope to which a video camera (COHU
solid state camera Model 4815-2000/0000, Cohu Inc., San
Diego CA) is attached. Into the computer, two programs were
56
installed. Image-Pro Plus Version 2.0 (Media Cybernetics®,
Silver Spring, MD) for recording images and Visionplus-AT®
(Imaging Technology Inc., Bedford, MA) for image processing.
Images were recorded under the 100/1.25 oil objective. Eight
to 25 images from each surface were digitized and saved for
later analysis. The number of cells in a 2500 urn2 field in
each image was determined.
Statistical analysis
Data of the completely randomized design with two
replications were evaluated by analysis of variance and
least significant difference at the 95% significance level
using Statistical Analysis System (SAS) statements PROC
ANOVA and PROC GLM (26) .
RESULTS AND DISCUSSION
Rinsing of unattached bacterial cells
The procedure for rinsing unadhered cells was
established by contacting unmodified silica surfaces with
Listeria monocytogenes, then rinsing at different flow rates
for different periods of time. In Fig. 3. 2, the number of
cells that remained on the surface after rinsing is plotted
57
against rinsing time at flow rates of 100, 300, and 550
ml/min. For subsequent experiments, rinsing was performed
with a flow rate of 300 ml/min for 4 min. A one way analysis
of variance (LSD, 95% confidence level) indicated that there
is no significant difference (p > 0.05) between rinsing at a
flowrate of 3 00 ml/min for 4 min, and at 550 ml/min for 4 or
10 min. Although the rinsing protocol was established with
reference only to adhesion to bare surfaces, the aim in
these experiments was simply to assist selection of a method
that could be consistently applied in each adhesion
experiment.
Adhesion following protein contact for 8 h
Hvdrophobic surfaces
Figure 3. 3 illustrates the effect of film age on the
number of cells adhered to hydrophobic silica surfaces
following contact with protein for 8 h. It also shows the
extent of adhesion measured on bare hydrophobic and
hydrophilic silica surfaces. Fig. 3. 3 indicates that the PLg film encouraged adhesion more than did the other protein
films (p < 0.05), although the extent of adhesion to
preadsorbed P-Lg remained lower than that measured on bare
hydrophobic surfaces. While P-Lg had the greatest effect on
58
adhesion in terms of the number of cells adhered per unit
area, preadsorbed BSA reduced the number of cells adhered to
values well below that of the bare hydrophilic surfaces (P <
0.05). The other two proteins, p-casein and a-Lac, showed an
intermediate effect.
The effects of protein preadsorbed to these hydrophobic
surfaces are consistent with previous observations. Proteins
adsorbed to solid surfaces alter the original interfacial
properties (6, 27, 31, 33). Yang et al.
(33) used contact
angle methods to measure the change in hydrophilic/
hydrophobic balance exhibited by a number of different
materials following adsorption of P-Lg. They found that
adsorption of P-Lg rendered hydrophilic surfaces more
hydrophobic, and hydrophobic surfaces more hydrophilic.
Adsorption of each of the four proteins studied to
hydrophobic surfaces is entropically driven (17, 18) and
probably accompanied by orientation of negatively charged,
hydrophilic regions to solution, yielding a less hydrophobic
interface. Considering only the general observation that
Listeria adhesion is greater on hydrophobic as opposed to
hydrophilic surfaces (1), it is thus expected that adhesion
of Listeria to such film-covered surfaces would result in
lower numbers of adhered cells relative to those measured at
a bare hydrophobic surface.
59
The previous argument, however, cannot explain the
differences in adhesion responses evoked by P-Lg and BSA.
BSA has a net charge of -18 at pH 7.0 while that of p-Lg is
-5 (32), and would be expected to take part in a more
repulsive electrostatic interaction with a bacterium, pcasein, on the other hand, is a linear amphiphile, with
essentially all of the protein's net charge (-12 at pH 7.0)
found on the first 43 residues of the N-terminal domain
(10). Upon adsorption at a hydrophobic surface, the Nterminal domain is oriented toward the solution. This
mechanism has been used to explain the observation that a
preadsorbed layer of P-casein prevents a sequential
adsorption of P-Lg (24). Nevertheless, the difference
between the effect of a preadsorbed film of p-casein and
that of o-Lac (net charge -3 at pH 7.0) on Listeria adhesion
was found to be statistically insignificant.
A more comprehensive treatment of these results would
account for relative rates at which each of the four
proteins is able to adopt a "passivating" conformation at
the hydrophobic surface.
One such treatment begins with reference to a model for
interactions between adsorbed protein and protein in
solution. Lundstrom and Elwing (21) considered an
experimental situation in which adsorption to a solid
surface is allowed to occur from a single-component protein
60
solution, followed by incubation in buffer, after which a
second, dissimilar protein is added. They showed that the
fraction of originally adsorbed protein that is not
exchangeable with dissolved protein is related to rate
constants governing conversion of the originally adsorbed
protein to a nonremovable form, and exchange of adsorbed
protein by dissimilar protein introduced to the solution.
Krisdhasima et al. (16) adapted this concept to the sodium
dodecylsulfate-mediated removal of a-Lac, P-Lg, BSA and pcasein from silica surfaces silanized according to the
procedure used here for hydrophobic surfaces. They were able
to rank the rate constants governing arrival of each protein
to the surface (kj) and surface-induced conversion to a
nonremovable state (sj . Results of that ranking showed
Si, BSA > Sj, p-caSein > s^ a_Lac > s^ p.Lg. This finding is consistent
with the adhesion results of Fig. 3. 3, as it indicates that
once adsorbed, BSA would offer greater resistance to removal
by an incoming adsorbate than would p-Lg. So assuming that
each protein would have some capacity to passivate the
surface, P-Lg would be more likely to leave the interface
upon exposure to surface active species (in this case,
Listeria) than would BSA, exposing a greater area of bare,
hydrophobic silica to adhering bacteria.
61
Hvdrophilic surfaces
Figure 3. 4 depicts the effect of
preadsorption of
each protein on adhesion to hydrophilic surfaces. It also
indicates the extent of adhesion expected on bare
hydrophilic and hydrophobic surfaces. P-Lg and BSA again
behaved most dissimilarly, with P-Lg evoking much greater
adhesion than BSA, while p-casein and o-Lac exhibited an
intermediate effect. However, adhesion mediated by p-Lg
preadsorbed to hydrophilic silica was greater than that
exhibited on hydrophobic silica (P < 0.05). Adsorption of pLg in this case would involve interaction between regions of
negative charge on the surface and positive charge on the
molecule, in addition to the entropically-driven attraction
described earlier, as these surfaces were silanized as well.
Adsorption occurring via favorable electrostatic attraction
would orient hydrophobic regions of the molecule away from
the surface. Indeed, the presence of these hydrophobic
domains leads to a higher water contact angle than that
measured on bare hydrophilic surfaces (33). Moreover,
Arnebrant et al. (5) reported that adsorption of p-Lg to
hydrophilic chromium oxide surfaces results in a protein
bilayer, where the second layer, bound via hydrophobic
association to the partially unfolded first layer, can be
partially removed by rinsing. This was not observed for p-Lg
62
adsorption to hydrophobic surfaces. Concerning the results
of Fig. 3. 4, any amount of a second layer removed by
rinsing would leave a more hydrophobic interface for
bacterial adhesion.
The BSA-mediated inhibition of bacterial adhesion on
hydrophilic surfaces was similar in order of magnitude, but
more extensive than that observed on hydrophobic surfaces (P
<0.05). Slightly less adhesion measured for BSA-coated
hydrophilic surfaces may simply be due to any removal of
adhered molecules being accompanied by exposure of
hydrophilic, as opposed to hydrophobic, interface. That the
passivating effect of preadsorbed BSA was similar on each
type of surface, and more pronounced in each case than that
of the other preadsorbed proteins is not surprising. BSA
adsorption to both hydrophobic and negatively-charged
hydrophilic interfaces has been postulated to take place
according to a similar mechanism (3). In particular, BSA
consists of three similarly-sized globular domains arranged
in series. One end domain is neutral at pH 7.0 while the
remaining domains carry a high net negative charge.
Adsorption with the neutral domain oriented toward the
surface in each case accompanied by structural rearrangement
of that domain to minimize free energy, yields a more or
less stable, hydrated and negatively-charged interface that
is probably not favorable for bacterial adhesion. This
63
thinking is consistent with the facts that, unlike the other
proteins, both BSA adsorption kinetic (16) and adsorption
equilibrium behavior (30) have been observed to be similar
at hydrophobic and hydrophilic silica surfaces. Moreover,
human serum albumin shows great homology with BSA and is
known to passivate biomedical material surfaces to
fibrinogen adsorption and platelet adhesion (8). In fact,
retention of albumin on blood-contacting implants to reduce
thrombogenesis remains a major focus in biomedical materials
research (13).
Other studies
have directly identified BSA as capable
of passivating surfaces against cell adhesion. Tamada and
Ikada (31) investigated the effect of preadsorbed BSA,
bovine Y~9l0kulin (IgG), and plasma fibronectin(FN) on L
cell adhesion to fourteen polymer surfaces of various
wettabilities. They found that BSA preadsorption entirely
inhibited L cell adhesion, independent of substrate
wettability. Fletcher (11) investigated the effect of BSA
and several other proteins on adhesion of a marine
pseudomonad to polystyrene Petri dishes. She found that BSA,
gelatin, fibrinogen and pepsin impaired cell attachment
through adsorption to the dish surface. Protamine and
histone were found not to markedly inhibit adhesion.
Figs. 3. 3 and 3. 4 would suggest a real decrease in
the number of cells adhered with film age for a-Lac, while
64
Fig. 3. 4 shows an increase in adhesion with film age for pcasein. For o-Lac, the decrease was observed most clearly at
hydrophilic surfaces. Krisdhasima et al. (16) found a-Lac to
exhibit a very low affinity for hydrophilic silica and ot-Lac
desorption occurring with longer incubation time might be a
contributing factor to the low adhesion observed in that
case. At hydrophobic surfaces, the decrease is consistent
with slow conformational adaptations exposing a more
hydrophilic interface to incoming bacteria.
The increased number of cells adhered to p-caseincovered hydrophilic surfaces with film age could be related
to the flexibility of p-casein. p-casein is more flexible
than the globular proteins and this might contribute to
conformational adaptations continuing to occur over the time
scale of these experiments such that the hydrophilic surface
is rendered more hydrophobic with time.
Adhesion following protein contact for 1 h
Since P-Lg and BSA exhibited the most dissimilar
effects on adhesion, with little dependence on film age,
adhesion following a shorter preadsorption period was
monitored. In this way, the possible impact of timedependent conformational changes on adhesion could be better
resolved.
65
Preadsorption of p-La
Fig. 3. 5 shows the effect of preadsorbed P-Lg on
adhesion at hydrophilic and hydrophobic surfaces, where
preadsorption was allowed to occur for 1 h. Relevant data
from Figs. 3. 3 and 3. 4 is included for comparison. At
hydrophobic surfaces, cell adhesion was lower than that
observed for 8 h preadsorption, for film ages of 0 and 5 h.
It is important to note that after 1 h (and in the absence
of rinsing), the surface coverage of P-Lg is only about 79%
of that measured after 8 h of contact, and well below that
expected for a monolayer (15, 16). The higher amount of
interfacial area per molecule may allow p-Lg to adopt either
a more energetically favorable orientation at the interface,
or more rapidly adopt a more passivating conformation as
described earlier. As film age is increased, cell numbers
increase, probably due to desorption of p-Lg. To verify
whether P-Lg desorbs from hydrophobic silica after
preadsorption for 1 h, similar tests were run over the 15 h
period but in the absence of cell contact. Between film ages
of 0 and 15 h, the adsorbed mass of p-Lg decreased about 20%
(Table 3. 1). The increased exposure of bare hydrophobic
silica to incoming cells would facilitate adhesion.
At hydrophilic surfaces, the opposite behavior was
observed in the sense that initially, cell numbers were
66
comparable to those observed after the 8 h preadsorption,
then decreased to values consistent with those expected for
a bare hydrophilic surface. Thus, the same argument would
serve to explain the results in this case. After 1 h (and in
the absence of rinsing), the surface coverage of p-Lg is
only about 74% of that measured after 8 h of contact, again
well below that expected for a monolayer (15, 16). The
increased area per molecule at the interface may have
facilitated an energetically favorable orientation with
concomitant exposure of hydrophobic regions to the solution.
Decreasing cell numbers with increasing film age are
consistent with p-Lg desorption which, between film ages of
0 and 15 h was observed to decrease the initial value of
adsorbed mass by about 25%. The importance of available
interfacial area, i.e., area not occupied by protein, has
been well documented with reference to models (20) and
experimental interpretation (28).
Preadsorption of BSA
The adhesion response evoked on BSA-coated surfaces
following a 1 h preadsorption is shown in Fig. 3. 6, along
with relevant data from Figs. 3. 3 and 3. 4. Krisdhasima et
al. (16) showed that a rate constant describing initial
arrival of BSA to a hydrophobic silica surface involving
67
"end-on" orientation with its neutral domain adjacent to the
surface, was lower than the arrival rate constants of the
other three milk proteins used in this study. Based on that,
it is not surprising that the amount of cell adhesion was
higher than that observed following an 8 h preadsorption. At
hydrophobic surfaces, although the adsorbed mass of BSA
after 1 h is about 86% of that attained after 8 h (16),
kinetic analysis would indicate that the population of BSA
molecules tightly adsorbed in the "passivating" orientation
may be relatively low. Little desorption was observed for
BSA between 0 and 15 h, which suggests that the passivating
orientation characteristic of the 8 h data was not attained,
even after 15 h. The relatively small decrease in cell
numbers with increasing film age suggests that breaking
intermolecular and protein-surface associations to reorient
in the passivating orientation was a rather slow process.
At hydrophilic surfaces, the same argument holds for
explanation of the response evoked immediately after protein
contact (i.e., film age = 0 h) but the decrease in adhesion
with increasing film age is substantial in comparison to
that observed at the hydrophobic surface. In this case, the
high negative charge of the molecule may have served to
facilitate molecular reorientation and conformational
adaptation on the surface with increasing age of the film.
68
E
E
o
50 mm
Figure 3. 1. Schematic of the flow cell used to rinse
loosely adhered Listeria monocytogenes from the
surfaces.
69
eu
D)
C
-H
C
-H
e
1-1
u
u
01
Time, mm
Figure 3.2. Effect of flow rates on residual adherent cells
of Listeria monocytogenes. O 100 ml/min, • 300 ml/min,
□ 550 ml/min.
70
10'
bare hydrophobic
0)
u
10 7 j.
0)
0)
o
bare hydrophilic
10
6.
3
s
O-
10'
T
0
10
5
15
Film age, h
Figure 3. 3. The effect of preadsorbed protein type and film
age on adhesion of Listeria monocytogenes to
hydrophobic silica surfaces. The bars indicate the
standard deviation of the means. O BSA, • p-Lg,
A P-casein, A o-Lac.
71
10'
,bare hydrophobic
eu
t/1
0)
o
10
0)
u
s:
o
10 6.
bare hydrophilic
d
2
10'
0
T
—i—
5
10
15
Film age, h
Figure 3. 4. The effect of preadsorbed protein type and film
age on adhesion of Listeria monocytogenes to
hydrophilic silica surfaces. The bars indicate the
standard deviation of the means. O BSA, • p-Lg,
A P-casein, A o-Lac.
72
10'
'E
o
en
bare hydrophobic
bare hydrophilic
10'
—i—
0
5
10
15
Film age, h
Figure 3. 5. The effect of p-Lg adsorption time and film age
on adhesion of Listeria monocytogenes to hydrophobic
and hydrophilic silica surfaces.
• Adhesion following adsorption to hydrophobic
surfaces for 8 h.
■ Adhesion following adsorption to hydrophobic
surfaces for 1 h.
O Adhesion following adsorption to hydrophilic
surfaces for 8 h.
D Adhesion following adsorption to hydrophilic
surfaces for 1 h.
73
Table 3.1. The mass of BSA and p-Lg (ug/cm2) remaining on
hydrophobic and hydrophilic surfaces following 1 h
of protein contact and incubation in buffer for
different times.
Film age,
h
adsorbed mass of BSA, pg/cm2
hydrophobic
surface
hydrophilic
surface
adsorbed mass of p-Lg, pg/cm2
hydrophobic
surface
hydrophilic
surface
0
0.375
0.356
0.297
0.279
5
0.356
0.311
0.301
0.227
10
0.373
0.315
0.250
0.197
15
0.365
0.324
0.256
0.217
74
1087
bare hydrophobia
eu
en
Q)
o
0
l-i
0)
T3
n3
O
3
s
10
-i
0
1
5
1—
10
15
Film age, h
Figure 3. 6. The effect of BSA adsorption time and film age
on adhesion of Listeria monocytogenes to hydrophobia
and hydrophilic silica surfaces.
• Adhesion following adsorption to hydrophobia
surfaces for 8 h.
■ Adhesion following adsorption to hydrophobia
surfaces for 1 h.
O Adhesion following adsorption to hydrophilic
surfaces for 8 h.
D Adhesion following adsorption to hydrophilic
surfaces for 1 h.
75
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Oss, and A. Neumann 1983. Surface thermodynamics of
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Absolom, D., w. zingg, and A. Neumann. 1987. Interactions
of proteins at solid-liquid interfaces: Contact angle,
adsorption, and sedimentation volume measurements, p.
401-421. In J. Brash, and T. Horbett (eds.), Proteins
at Interfaces: Physicochemical and Biochemical Studies.
ACS Symp. Ser. 343, Washington, DC.
3. Andrade, J., V. Hlady, A. Wei, and C. G61ander. 1990. A
domain approach to the adsorption of complex proteins:
Preliminary analysis and application to albumin.
Croatica Chemica Acta 63:527-538.
4. Andrade, J., V. Hlady, and R. wagenen. 1984. Effects of
plasma protein adsorption on protein conformation and
activity. Pure Appl. Chem. 56: 1345-1350.
Arnebrant, T., B. ivarsson, K. Larsson, I. Lundstrdm, and
T. Nylander. 1985. Bilayer formation at adsorption of
proteins from aqueous solutions on metal surfaces.
Progr. Colloid Polymer Sci. 70:62-66.
Baier, R. E. 1980. Substrata influences on adhesion of
microorganisms and their resultants new surface
properties, p. 59-104. In G. Bitton, and K. Marshall
(eds.), Adsorption of Microorganisms to Surfaces. John
Wiley & Sons, Inc., New York.
7. Daeschel, M., J. McGuire, and H. Al-Makhlafi. 1992.
Antimicrobial activity of nisin adsorbed to hydrophilic
and hydrophobic silicon surfaces. J. Food Protec. 55:
731-735.
8. Eberhart, R. C, M. S. Munro, J. R. Frautschi, and v. I,
Sevastianov. 1987. Organization of albumin on polymer
surfaces, p. 378-400. In J. Brash, and T. Horbett
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(eds.), Proteins at Interfaces, Physicochemical and
Biochemical Studies. ACS Symp. Ser. 343, Washington,
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9. Elwing, H., S. Welin, A. Askendahl, and I. Lundstrttm.
1988. Adsorption of fibrinogen as a measure of the
distribution of methyl groups on silicon surfaces.
J. Colloid Interface Sci. 123:306-308.
10. Farrell, H. 1988. Physical equilibria: Proteins, p. 461510. In N. P. Wong, R. Jenness, M. Keeney and E. Marth
(eds.), Fundamentals of Dairy Chemistry. Van Nostrand
Reinhold, New York.
11. Fletcher, M. 1976. The effects of proteins on bacterial
attachment to polystyrene. J. Gen. Microbiol. 94:400404.
12. Fletcher, M., and J. Pringle. 1985. The effect of
surface free energy and medium surface tension on
bacterial attachment to solid surfaces. J. Colloid
Interface Sci. 104:5-14.
13. Horbett, T. 1988. Molecular origins of the surface
activity of proteins. Protein Eng. 2:172-174.
14. JQnsson, U., M. Malmqvist, I. Rdnnberg, and L. Berghem.
1982. Adsorption behavior of fibronectin on wellcharacterized silica surfaces. J. Colloid Interface
Sci. 90:148-163.
15. Krlsdhasima, v., J. McGuire, and R. Sproull. 1992.
Surface hydrophobic influences on p-lactoglobulin
adsorption kinetics. J. Colloid Interface Sci. 154:337350.
16. Krlsdhasima, v., P. Vinaraphong, and J. McGuire. 1993.
Adsorption kinetics and elutability of a-lactalbumin,
P-casein, p-lactoglobulin and bovine serum albumin at
hydrophobic and hydrophilic interfaces. J. Colloid
Interface Sci. 161:325-334.
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17. Lee, R. J., and s. w. Kim. 1974. Adsorption of proteins
onto hydrophobic polymeric surfaces: Adsorption
isotherms and kinetics. J. Biomed. Mater. Res. 8:251259.
18. Lee, H. S., and E. Ruckenstein. 1988. Adsorption of
proteins onto polymeric surfaces of different
hydrophilicities- A case study with bovine serum
albumin. J. Colloid Interface Sci., 125:365-379.
19. Lu, D., and K. park. 1991. Effect of surface
hydrophobicity on the conformational changes of
adsorbed fibrinogen. J. Colloid Interface Sci. 144:271
281.
20. Lundstrdm, I. 1985. Models of protein adsorption on
solid surfaces. Progr. Colloid Polymer Sci. 70:76-82
21. Lundstrdm, I., and H. Elwlng. 1990. Simple kinetic
models for protein exchange reactions on solid surface
J. Colloid Interface Sci. 136:68-84.
22. Mafu, A., D. Roy, J. Goulet, and P. Magny. 1990.
Attachment of Listeria monocytogenes to stainless
steel, glass, polystyrene, and rubber surfaces after
short contact times. J. Food Protec. 53:742-746.
23. Mafu, A., D. Roy, J. Goulet, and L. Savoie. 19 91.
Characterization of physicochemical forces involved in
adhesion of Listeria monocytogenes to surfaces. Appl.
Environ. Microbiol. 57:1969-1973.
24. Nylander, T., and N. M. wahlgren. 1994. Competitive and
sequential adsorption of p-casein and p-lactoglobulin
on hydrophobic surfaces and the interfacial structure
of p-casein. J. Colloid Interface Sci. 162: 151-162.
25. Pringle, J., and M. Fletcher. 1983. Influence of
substratum wettability on attachment of freshwater
bacteria to solid surfaces. Appl. Environ. Microbiol.
45: 811-817.
78
26. SAS institute. 1988. The SAS procedures guide. Release
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27. Schakenraad, J., and H. Busscher. 1989. Cell-Polymer
interactions: The influence of protein adsorption.
Colloids Surf. 42:331-343.
28. Slack, s. M., and T. A. Horbett. 1989. Changes in the
strength of fibrinogen attachment to solid surfaces: An
explanation of the influence of surface chemistry on
the Vroman effect. J. Colloid Interface Sci. 133: 148165.
29. Soderquist, M., and A. Walton. 1980. Structural changes
in protein adsorbed on polymer surfaces. J. Colloid
Interface Sci. 75:386-397.
30. Suttiprasit, P., and J. McGuire. 1992. The surface
activity of o-lactalbumin, p-lactoglobulin, and bovine
serum albumin. J. Colloid Interface Sci. 154:327-336.
31. Tamada, Y., and Y. ikada. 1993. Effect of preadsorbed
proteins on cell adhesion to polymer surfaces. J.
Colloid Interface Sci. 155:334-339.
32. Wahlgren, M. C, M. A. Paulsson, and T. Arnebrant. 1993
Adsorption of globular model proteins to silica and
methylated silica surfaces and their elutability by
dodecyltrimethylammonium bromide. Colloids Surf. 70:
139-149.
33. Yang, J., J. McGuire, and E. Kolbe. 1991. Use of
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79
CHAPTER 4
ADHESION OF LISTERIA MONOCYTOGENES TO MODEL FOOD CONTACT
SURFACES FOLLOWING SEQUENTIAL AND COMPETITIVE ADSORPTION OF
BOVINE SERUM ALBUMIN AND
P-LACTOGLOBULIN*
HAMOOD AL-MAKHLAFI, JOSEPH McGUIRE AND MARK DAESCHEL
* To be presented for publication in Applied and
Environmental Microbiology.
80
ABSTRACT
The adsorption of bovine serum albumin and (3lactoglobulin to silanized silica surfaces of low and high
hydrophobicities was carried out where each protein was
allowed to contact the surface in sequence or
simultaneously. Adsorption in both the sequential and
competitive modes was followed by incubation in buffer and
contact with Listeria monocytogenes for 3 h. In sequential
tests performed with an 8 h contact/protein, cell numbers on
each surface were near that expected for the bare
hydrophobic surface when p-lactoglobulin contact preceded
introduction of bovine serum albumin, whereas adhesion was
reduced to values below that expected for the bare
hydrophilic surface when BSA preceded p-lactoglobulin
contact. In short-term sequential tests (1 h
contact/protein), adhesion was lower than that recorded on
bare hydrophilic surfaces in each case. Adhesion to each
surface following contact with an equimolar mixture of plactoglobulin and bovine serum albumin was lower than that
measured on the bare hydrophilic surface in each case, with
adhesion following 1 h contact being greater than that
following 8 h contact. Adhesion following competitive
adsorption was greater to hydrophobic than to hydrophilic
surfaces. These results were explained with reference to the
81
surface passivating character of bovine serum albumin, and
its ability to rapidly attain a nonexchangeable state upon
adsorption, relative to p-lactoglobulin.
82
INTRODUCTION
Adsorption of soluble macromolecules such as proteins to
solid surfaces may affect adhesion of microorganisms and
other cells (11). Adsorbed proteins can inhibit or
facilitate attachment of subsequently arriving
microorganisms, or be displaced by other surface active
species (12). The presence and conformational state of
adsorbed protein molecules depends on both protein and solid
surface properties. The conformation of an adsorbed protein
is believed to be different on different surfaces for a
number of proteins (2, 6, 8, 10, 20), and this surface
conformation is related to its binding strength (10).
Proteins bound by a greater number of noncovalent contacts
per unit area would be expected to experience little
desorption by rinsing and slower exchange with molecules in
solution (5, 10, 23).
Most of the literature available on the nature of
adsorption in protein mixtures concerns experiments
conducted with blood proteins. It has been observed that
higher molecular weight proteins are usually adsorbed in
preference to smaller ones. This was explained by existence
of a greater number of anchoring segments characteristic of
larger molecules (18), although substrate properties
influence these phenomena as well.
83
Several studies concerning adsorption of milk proteins
to various surfaces are available (9, 21, 24), most having
focused on adsorption from single-component solutions. Other
studies have focused on adsorption of milk proteins from
binary mixtures as well as their sequential adsorption (3,
15, 16). A number of authors have described the effect of
adsorbed bovine serum albumin (BSA) on adhesion of bacteria
and other cells (7, 13, 22). Recently, Al-Makhlafi et al.
(1) reported the effect of four milk proteins (o-lactalbumin
(o-Lac), p-lactoglobulin (p-Lg), p-casein, and BSA) on the
adhesion of Listeria monocytogenes to silica surfaces
exhibiting either high or low hydrophobicity. The proteinspecific effects on adhesion were different, with p-Lg and
BSA evoking the most dissimilar response. Little is known in
a quantitative sense regarding bacterial adhesion to
surfaces onto which selected proteins had spontaneously
adsorbed from a mixture (competitive adsorption), or had
been introduced one at a time, in a selected sequence
(sequential adsorption). The importance of such study is
practically quite relevant as processes involving contact
between surfaces and biological fluids can involve many
different surface active species. The purpose of this study
was to evaluate bacterial adhesion following adsorption of
selected milk proteins in sequence and from mixtures to
surfaces exhibiting high and low hydrophobicities.
84
MATERIALS AND METHODS
All the materials used in this study were described
previously (1).
Surface modification
Silicon wafers were cut into plates of approximately 1
cm2 with a tungsten pen. Surfaces were silanized by reaction
with 0.01 or 0.1% dichlorodimethylsilane (DDS) in xylene as
described previously (9). These surface derivitizations are
stable, and exhibit a low or high hydrophobic character,
respectively, as shown by contact angle analysis (9).
Surfaces silanized with 0.01% DDS exhibit a partial negative
charge in addition to low hydrophobicity, and we will refer
to them as hydrophilic surfaces.
Protein adsorption
Sequential adsorption
Single-component solutions of P-Lg and BSA (each
equivalent to 1 mg/ml P-Lg) were prepared by first
dissolving each in 0.01 M phosphate buffer (pH 7.0) while
stirring for 20 min. Each protein solution was filtered
85
through a preassembled, presterilized Nalgene™ Bottle Top
Filter (0.2 \im)
(Nalge Company, Rochester, NY). From this
solution 10 ml were pipetted to small Petri dishes (60 X 15
mm style disposable polystyrene. Falcon 1007, Becton
Dickinson and Company, Lincoln Park, NJ) and surfaces were
then introduced to the protein solution. Surfaces were
allowed to contact the first protein solution for 8 or 1 h
after which time they were rinsed sequentially in three, 300
ml volumes of distilled, deionized water. Each individual
surface was .rinsed for about 15 s in each step. Surfaces
were then introduced to the second protein solution.
Surfaces were allowed to contact protein solution for
another 8 h if preceded by 8 h contact with the first
protein solution, or 1 h if preceded by 1 h contact with the
first protein. They were then rinsed as described above.
Films formed on surfaces were then aged in 20 ml buffer for
0, 5, 10, or 15 h, then surfaces were rinsed again as above
and introduced to the medium with bacterial cells. Great
care was taken not to allow the adsorbed protein films to
dry throughout these experiments.
Competitive adsorption
Equimolar amounts of P-Lg and BSA (concentration of each
protein equivalent to 1 mg/ml p-Lg) were dissolved in 0.01 M
86
phosphate buffer (pH 7.0) while stirring for 20 min. The
protein solution was then filtered as described above. From
this solution, 10 ml was pipetted into small Petri dishes
and surfaces were then introduced to the protein solution.
Surfaces were allowed to contact the protein solution for 8
or 1 h after which time they were rinsed as described above.
Protein films formed on surfaces were then aged in 20 ml
buffer for 0, 5, 10, or 15 h, then surfaces were rinsed
again and introduced to the medium.
Culture and adhesion
Listeria monocytogenes Scott A was grown each time in 50
ml protein-free defined medium and incubated for 24 h at 37
0
C to reach the stationary phase. The defined medium was
made up of 20.8 g/1 RPMI-1640 medium (lot No 119F-4645-1,
Sigma) . The medium was modified by the addition of 1%
glucose and 2% casamino acids (Difco Laboratories, Detroit,
MI). Before use, this medium was filter-sterilized using a
preassembled, presterilized Nalgene™ Bottle Top Filter (0.2
urn). Culture was diluted in Butterfield's buffer (a
phosphate buffer of pH 7.0; 0.00426% potassium phosphate) to
a cell density of 107 - 109 cfu/ml. Ten ml of this diluted
culture was transferred to small Petri dishes. Hydrophilic
or hydrophobic silica surfaces, either bare or with a
previously adsorbed protein film, were introduced to the
Petri dishes and allowed to contact the culture for 3 h.
Rinsing
After contact with cells, surfaces were glued to a
microscope glass slide using a Krazy Glue® Pen (Borden Inc.,
HPPG Columbus, OH) without allowing the biofilm to dry.
Rinsing to remove loosely adhered cells was done by passing
distilled water through a flow cell into which the glass
slides had been inserted. The flow cell structure and the
conditions used for rinsing were described previously (1).
Image analysis
Following rinsing, surfaces were quickly covered by a
cover slip and images of bacterial cells adhered to surfaces
were taken using an incident light microscopy-image analysis
system (IAS). The IAS is composed of a 486 IBM compatible
computer interfaced to a Sony monitor and a Reichert Epistar
incident light microscope to which a video camera (COHU
solid state camera Model 4815-2000/0000, Cohu Inc., San
Diego CA) is attached. Image-Pro Version 2.0 (Media
Cybernetics®, Silver Spring, MD) was used for reading
images, and Visionplus-AT® (Imaging Technology Inc.,
88
Bedford, MA) was used for image processing. Images were
recorded under the 100/1.25 oil objective. Twenty five
images from each surface were digitized and saved for later
analysis. The number of cells in a 2500 urn2 field in each
image was determined.
Statistical analysis
Data of the completely randomized design with two
replications were evaluated by analysis of variance and
least significant difference at the 95% significance level
using Statistical Analysis System (SAS) statement PROC ANOVA
(17) .
RESULTS AND DISCUSSION
Adhesion following sequential protein adsorption (8 h
contact time)
Fig. 4. 1 illustrates the effect of film age on the
number of cells adhered to hydrophobic and hydrophilic
silica surfaces following sequential contact with proteins
for 8 h. It also shows the extent of adhesion measured on
bare hydrophobic and hydrophilic silica surfaces. Fig. 4. 1
indicates that the film formed by adsorption of p-Lg
89
followed by BSA (P-Lg/BSA) encouraged adhesion more than did
the film formed by adsorption of BSA followed by p-Lg
(BSA/p-Lg)
(p < 0.05), although the extent of adhesion to p-
Lg/BSA remained lower than that measured on bare hydrophobic
surfaces. Moreover, BSA/p-Lg reduced the number of cells
adhered to values well below that evoked by the bare
hydrophilic surfaces, with BSA/p-Lg inhibition of cell
adhesion on hydrophilic surfaces being more extensive than
that observed on hydrophobic surfaces (P <0.05).
The mechanisms by which these two proteins may affect
Listeria adhesion to both hydrophobic and hydrophilic silica
surfaces when each adsorbs from a single-component solution
for 1 and 8 h were discussed previously (1). The surfaces
treated with p-Lg/BSA evoked an adhesion response similar to
that measured following P-Lg adsorption alone, while that
treated with BSA/p-Lg evoked a response closer to that
measured following adsorption of BSA alone though a little
increase was observed following BSA/p-Lg contact (1).
Based only on the relative rate at which each of these
proteins, once adsorbed, achieves a nondisplaceable (or
otherwise more tightly bound) state, we would expect P~Lg to
experience more resistance in exchanging with adsorbed BSA,
than BSA would experience in exchanging with adsorbed P-Lg
(1, 10). Fig. 4. 1 suggests that 8 h is sufficient for
rendering the first protein effectively nonremovable; i.e..
90
kinetics associated with adoption of a more tightly bound
form are not relevant after 8 h of protein-surface contact.
Previous studies have described the ability of BSA to
displace proteins that had adsorbed to a surface (4, 16,
19). Generally, the ability of BSA to displace such proteins
depends on the solution conditions. Ruzgas et al.(16)
studied the sequential adsorption of Y~interferon (Y~1NF)
and BSA on hydrophobic silicon surfaces. Y~1NF is similar to
P-Lg in that it is a dimer, with molecular weight of the
monomer equal to about 16.8 kDa; however, it has an
isoelectric point of 9.5. When BSA adsorbed from phosphate
buffer (0.01 M, pH 7.2) to surfaces, following a 30 min
preadsorption of Y~INF from the same buffer, BSA displaced
34% of the Y-INF- The displacement of Y^NF by BSA was
observed to increase with decreasing electrostatic
interaction between the two proteins.
Results from our own laboratory on the sequential
adsorption of these two proteins studied with ellipsometry
indicate that an increase in adsorbed mass of about 18 and
29% occurs upon introduction of BSA to a P-Lg film
(following a 4 h contact of p-Lg) at hydrophobic and
hydrophilic surfaces, respectively (14, Appendices B and C).
This could suggest that BSA adsorbs to the previously
adsorbed P-Lg layer, as the value of adsorbed mass attained
upon introduction of the second protein is well above that
91
expected for a monolayer of either protein (i.e., a
monolayer of BSA is about 0.155 and 0.140 ug/cm2 at
hydrophobic and hydrophilic silica, respectively). BSA
molecules adsorbed in this outer layer may readily leave the
interface upon rinsing and/or collision with cells during
contact with media. BSA may also displace some amount of 0Lg, but Fig. 4. 1 indicates the extent of displacement is
not enough to reduce cell adhesion.
Similarly, ellipsometric data for P-Lg adsorption
following 4 h of BSA contact show an increase in adsorbed
mass of about 16% on hydrophobic surfaces, and about 3 0% on
hydrophilic surfaces. Again, the increase in adsorbed mass
was to a level greater than that expected for a monolayer of
either protein. Here, Fig. 4. 1 would suggest that the outer
layer of p-Lg is readily desorbed upon rinsing and/or
contact with Listeria, leaving only BSA and leading to a low
extent of Listeria adhesion.
Finally, the fact that greater inhibition of Listeria
adhesion is seen at hydrophilic surfaces for the BSA/p-Lg
tests is consistent with the facts that: (i) less adhesion
would be expected on exposed hydrophilic silica than on
exposed hydrophobic silica; and (ii) while BSA shows roughly
equal affinity for each surface, p-Lg shows greater affinity
for hydrophobic surfaces and may exchange more readily with
BSA adsorbed to hydrophobic surfaces than to hydrophilic
92
surfaces (10). These two points explain the increase in
adhesion measured at both surfaces following BSA/p-Lg
contact over that measured following adsorption of BSA alone
(1) .
Adhesion following sequential protein adsorption (1 h
contact time)
Fig. 4. 2 illustrates the effect of preadsorbed pLg/BSA and BSA/p-Lg on adhesion of L. monocytogenes, where
adsorption was allowed to occur for 1 h for each protein. At
both surfaces, cell adhesion to p-Lg/BSA as well as to
BSA/p-Lg was lower than that observed at bare hydrophilic
surfaces. Fig. 4. 2 would suggest that adsorbed P-Lg was
readily exchanged with BSA while adsorbed BSA resisted
exchange with p-Lg, at least to the extent that the surfaces
evoked an adhesion response similar to that expected for a
BSA film-covered surface following 8 h contact (1).
Earlier we reported adhesion following a 1 h contact
with BSA to be greater than that measured in the present
tests (1). It was argued that due to its low value of k1 (1,
10) relative to that of P-Lg, the population of BSA
molecules tightly adsorbed in the passivating orientation
would be relatively low. Apparently, the presence of P-Lg in
this case facilitated BSA adsorption in the most passivating
93
orientation. Concerning the case where BSA was adsorbed
first, the results of Fig. 4. 2 would suggest that after 2
h, the BSA film is more similar to one formed during 8 h of
contact than to one formed during 1 h of contact, when
formed from a single-component solution at either surface
(1) •
Ellipsometric data recorded for the p-Lg/BSA sequential
adsorption (1 h contact/protein)
(14, Appendices B and C)
show an increase in adsorbed mass upon introduction of BSA
of about 21.5 and 48% on hydrophobic and hydrophilic silica,
respectively. But even though such a substantial increase in
adsorbed mass was recorded, the final value of adsorbed mass
was reasonably close to that expected for a monolayer of BSA
at each surface. When BSA adsorption preceded P-Lg
introduction, a relatively small increase in adsorbed mass
was recorded (about 7 and 14% on hydrophobic and hydrophilic
surfaces, respectively)
(14, Appendices B and C).
Ellipsometry alone cannot distinguish one protein from
another, but the thought that BSA displaces adsorbed p-Lg
while p-Lg does not displace adsorbed BSA is consistent with
Fig. 4. 2 and at least not contradicted by the ellipsometric
data.
94
Adhesion following competitive adsorption for 8 h
Fig. 4. 3 illustrates the effect of adsorption from a
mixture of P-Lg and BSA on adhesion to hydrophobic and
hydrophilic silica. Adhesion to protein-coated hydrophobic
silica is greater than that observed on protein-coated
hydrophilic silica (p < 0.05) but it remains below that
recorded on bare hydrophilic silica. This adhesion behavior
is similar to that observed in the case of BSA adsorbed
alone at each surface (1). The reduced number of cells in
the case of the hydrophilic surfaces could be related to the
faster conformational rearrangement undergone by BSA at that
surface relative to p-Lg (5, 10), as well as less adhesion
expected on regions of exposed hydrophilic than hydrophobic
surfaces.
The concentrations of these two proteins in solution
are equal, and greater than that corresponding to diffusionlimited adsorption. Due to its higher kj (1, 10) it is
expected that p-Lg would initially bind to the surface
faster than BSA, but it would readily exchange with BSA as
Si BSA >
s
i p-Lg- Adsorbed BSA on the other hand may be much
more difficult to displace. Ellipsometric data concerning
competitive adsorption (14, Appendix B) show attainment of
an adsorbed mass greater than that of a monolayer of either
BSA or P-Lg. However, the initial adsorption rates are more
95
closer to those observed following BSA adsorption alone.
Smith and Sefton (19) investigated the adsorption of
BSA and thrombin to polyvinyl alcohol (PVA), heparin-PVA
hydrogels and polyethylene (PE). They found that thrombin
can adsorb to PVA and PE. However, thrombin could be
displaced from PE during BSA adsorption, and thrombin
adsorption to PE was prevented by prior or simultaneous
exposure to BSA.
Adhesion following competitive adsorption for 1 h
Fig. 4. 4 shows the effect of adsorption from a mixture
of p-Lg and BSA on adhesion to hydrophobic and hydrophilic
surfaces. On both surfaces, the number of cells adhered is
greater than that recorded after contact with the protein
mixture for 8 h (Fig. 4. 3), but remained lower than that
recorded on the bare hydrophilic surface. In this case,
arguments supporting the results of Fig. 4. 3 would apply to
explain the surface passivation. The higher number of cells
adhered in the present case compared to Fig. 4. 3 could be
related to the lower surface coverage of protein after 1 h
contact leaving bare hydrophobic and bare hydrophilic
regions which would drive the cell numbers up relative to
the 8 h protein contact. The adhesion response shown in
96
Fig. 4. 4 is lower than that recorded following BSA contact
from single-component solution for 1 h (1). The reason for
this is not obvious, but the finding is analogous to that
associated with the P-Lg/BSA tests of Fig. 4. 2. The results
of both Figs. 4. 2 and 4. 4 suggest that the presence of pLg at the interface may facilitate adsorption of BSA in a
favorable, passivating orientation.
97
10'
bare hydrophobic
ao
0)
S io7
bare hydrophilic
0)
i-l
0)
x:
rO
O
U
6 ■•
0)
e
i ■
10
0
5
10
15
Film age, h
Figure 4. 1. Adhesion of L. monocytogenes to hydrophobic
silica (closed squares and circles) and hydrophilic
silica (open squares and circles) following sequential
adsorption of P-Lg and BSA (8 h contact/protein).
■ P-Lg/BSA, • BSA/P-Lg, □ P-Lg/BSA, O BSA/p-Lg.
98
10'
bare hydrophobic
o
/
0)
o
107:
bare hydrophilic
u
m
o
u
0)
3
s
io-
i
0
5
10
15
Film age, h
Figure 4. 2. Adhesion of L. monocytogenes to hydrophobic
silica (closed squares and circles) and hydrophilic
silica (open squares and circles) following sequential
adsorption of p-Lg and BSA (1 h contact/protein).
■ P-Lg/BSA, •BSA/P-Lg/ □ P-Lg/BSA, O BSA/p-Lg.
99
1081
bare hydrophobia
e
u
en
o lO7-1
-a
bare hydrophilic
:/..
f0
t
3
2
10:
0
10
Film age, h
15
Figure 4. 3. Adhesion of L. monocytogenes to hydrophobia
(•) and hydrophilic (O) silica following competitive
adsorption of p-Lg and BSA for 8 h.
100
10'
bare
hydrophobia
I
eo
en
o
/
10 7.
bare hydrophilic
(U
05
Film age, h
Figure 4. 4. Adhesion of L. monocytogenes to hydrophobic
(•) and hydrophilic (O) silica following competitive
adsorption of p-Lg and BSA for 1 h.
101
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102
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103
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104
CHAPTER 5
SIGNIFICANT FINDINGS
1. Adhesion following 8 h adsorption from a single component
solution
A. P-Lg encouraged adhesion of L. /nonocytogenes to
hydrophilic and hydrophobic surfaces. Adhesion was
significantly greater than that observed following
contact with the other three proteins studied. This
suggested that p-Lg adsorbed in a less passivating
conformation to both types of surfaces.
B. BSA adsorption to both surfaces significantly reduced
the number of cells adhered to values less than that
recorded following contact with P-Lg, p-casein, or ocLac. This implied that BSA adsorbed in a more
passivating conformation.
C. p-casein and a-Lac influenced adhesion to each surface
to extents that were intermediate between p-Lg and BSA.
2. Adhesion following 1 h adsorption from a single
component solutions
A. Adhesion to surfaces contacted with BSA was 2-3 orders
of magnitude greater than that following 8 h contact.
105
Since BSA has a lower kl value, these results indicated
that the population of BSA molecules tightly adsorbed
in the "passivating" orientation might be relatively
low.
B. Adhesion to both surfaces contacted with P-Lg was lower
than that following its 8 h contact and greater than
that following BSA 1 h contact. These results inferred
that because of its low S! value, P-Lg desorption from
surfaces resulted in greater bare areas for adhesion.
3. Adhesion following sequential adsorption (8 or 1 h
contact time/protein)
Adhesion results following sequential adsorption of BSA
and P-Lg were similar to that following contact with each
individual protein for 8 h. This indicated that
neither BSA nor p-Lg was able to displace each other
after 8 h contact. However, in the 1 h tests, adhesion
was reduced significantly suggesting that BSA was able to
displace P-Lg and dominates the surfaces.
4. Adhesion following competitive adsorption for 8 and 1 h
Adhesion results to both surfaces following adsorption
from a mixture of BSA and P-Lg for 8 and 1 h were relatively
106
similar to that observed on surfaces precoated with BSA
alone for 8 h. These results pointed out that because of its
lower Si value, P-Lg would readily exchange with BSA.
However, adsorbed BSA may be much more difficult to
displace.
5. Finally, this research will contribute to a better
understanding of the effects of the proteins studied on
adhesion of L. inonocytogenes to food contact surfaces.
107
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APPENDICES
120
Appendix A
A Research Note
ANTIMICROBIAL ACTIVITY OF NISIN ADSORBED TO HYDROPHILIC AND
HYDROPHOBIC SILICON SURFACES*
MARK A. DAESCHEL, JOSEPH MCGUIRE, AND HAMOOD AL-MAKHLAFI
* This paper was published in Journal of Food Protection,
Vol. 55, No. 9, Pages 731-735 (September, 1992).
121
ABSTRACT
The antimicrobial activity of nisin was studied after
its adsorption to hydrophilic and hydrophobic silicon
surfaces. Adsorption was allowed to occur from buffered
nisin solutions under static conditions, and the adsorbed
mass of nisin was calculated from the resultant film
thickness and refractive index, determined using
ellipsometry. Once adsorbed, nisin was observed to be stable
to buffer rinsing; the amount of nisin adsorbed onto each
type of surface was determined to be of a quantity sufficent
for inhibition of susceptible bacteria. Antimicrobial
activity was maintained both upon silicon surface contact
with microbial media, and after nisin desorption induced by
surfactant displacement.
122
INTRODUCTION
The ability of disease and food spoilage causing
microorganisms to adhere to inert surfaces is widely
recognized as a serious problem and has been the focus of
numerous investigations. It is believed that microorganisms
that are attached to surfaces may be much less susceptible
to the killing effect of sanitizers commonly used in the
food industry. As an analogy it has been observed (6) that
when medical devices such as cardiac pacemakers are
colonized by bacteria, very high levels of antibiotics are
needed to eliminate the bacteria. In industry, increasing
the concentration of chemical sanitizing agents is probably
unacceptable, since increased concentrations of sanitizers
may pose health risks to employees, possibly result in
unacceptable chemical residuals in the product, and increase
production costs. Additionally, higher chemical
concentrations do not necessarily result in greater
sanitation efficiency.
An alternative and novel approach to optimize
sanitation strategies is to inhibit the initial adhesion of
microbial contaminants to food contact surfaces, as opposed
to trying to remove them once they are adhered. A similar
concept has been in use in the way of antifoulant paints to
protect the hulls of ships kept afloat for extended periods
123
from the onset of marine growth. These paints work by
releasing in some controlled manner small amounts of active
ingredients that prevent fouling organisms from adhering to
the surface (5).
Here we describe an approach that would involve
treatment of a surface with a preadsorbed layer of nisin, a
protein that when free in solution exhibits antimicrobial
activity. If nisin activity is maintained in the adsorbed
state, sensitive bacterial cells or spores that attempt to
attach would be killed, thus inhibiting biofilm development
and reducing the incidence of food product contamination by
pathogenic or spoilage causing bacteria. Historically nisin
has been used in foods as a direct additive to inhibit the
growth of gram-positive cells and spores and has recently
been approved (USA) for use with pasteurized cheese spreads.
Nisin can withstand activity loss during thermal processing
and exposure to acidic food environments. These
characteristics and others (16) such as non-toxicity make
its use as an antimicrobial agent attractive in many food
processing situations.
In general, proteins are extremely surface active.
Their strongly amphipathic nature gives them great stability
in the adsorbed state, and protein adsorption is thus
involved in several instances of technical interest.
Protein interactions with solid surfaces have been studied
124
for decades, and several thorough reviews are available (2,
15, 29). Protein adsorption is characterized by the likely
presence of a time dependence in the development of bonds
with the surface, a time dependence in the lateral mobility
of the protein molecules, and time-dependent conformational
changes; it is thus very difficult to quantify. There are,
however, some established principles or "rules of thumb"
that guide explanation of the course of protein adsorption.
In this note we describe the antimicrobial activity of
nisin bound.to hydrophilic and hydrophobic silicon surfaces.
Its stability at these surfaces is discussed with reference
to established principles thought to govern protein surface
activity. Silicon is not used in food contact, but the
hydrophobicity of its surface can be varied in a controlled
manner. As surface hydrophobicity is known to play an
important, sometimes dominant role in protein and whole cell
adhesion to food contact surfaces (2, 15, 29), an approach
allowing experimental observations to be quantitatively
related to contact surface hydrophobicity is quite
attractive; such an approach is afforded by use of modified
silicon surfaces.
125
MATERIALS AND METHODS
Solid surfaces
Optically flat, monocrystalline and polished silicon
wafers (Wacker Siltronic Corp., Portland, OR: 1-0-0
orientation; resistivity = 0.8-2 ohm cm) were cut into
rectangular plates using a tungsten knife. The surface area
varied from about 1 to 2 cm2 per plate. We were unable to
obtain plates with identical dimensions due to difficulties
encountered in cutting the silicon. Preparation of
hydrophilic silicon surfaces by oxidation and hydrophobic
surfaces by silanization with dichlorodimethylsilane was
performed according to Jonsson et al. (17). Surface
modification was verified with contact angle analysis; water
formed a contact angle less than 10° on hydrophilic silicon,
and a contact angle greater than 100° on hydrophobic silicon
(26) .
Adsorption
Hydrophilic silicon samples were each rinsed in
distilled, deionized water, dried with nitrogen, and had
their optical constants measured by ellipsometry immediately
after their preparation, prior to protein contact.
126
Hydrophobic surfaces were rinsed in trichloroethylene
(immediately following contact with dichlorodimethylsilane),
rinsed in acetone and then in ethanol, dried with nitrogen,
and had their optical constants measured prior to protein
contact. A high potency grade of nisin was obtained from
Aplin and Barrett, Ltd. (Dorset, UK). Activity was indicated
as 45.5 x 106 U/g (Lot code 5150J). Sodium phosphate buffer
solutions were prepared using chemicals of analytical grade
and water that was both distilled and deionized. Nisin was
added to 0.01 M monobasic sodium monophosphate (pH 4.5) to
assure complete solubilization. Dibasic sodium monophosphate
(0.01 M) was added to solubilized nisin until a pH of 6.0
and a final nisin concentration of 1 mg/ml was obtained.
Varying concentrations of nisin solutions were obtained by
dilution with 0.01 M sodium phosphate buffer (pH 6.0).
Sample silicon plates were contacted with nisin solutions
under static conditions by contacting one plate with one
volume (15 ml) of protein solution for 8 h. After contact,
silicon samples were rinsed three times with distilled,
deionized water to remove protein not tightly bound to the
surface, dried in a nitrogen stream, stored in a desiccator
for about 12 h, then removed for ellipsometric analysis.
Triplicate samples were run simultaneously. In desorption
tests, film covered samples were rinsed three times with
127
distilled, deionized water as above, but then immersed in 15
ml pure buffer for 12 h. Samples were then rinsed three
times again, dried with nitrogen, stored in a desiccator for
about 12 h, and removed for ellipsometric analysis.
Duplicate samples were run simultaneously.
The thickness and refractive index of the nisin film
remaining on each surface was ellipsometrically determined.
In each case, readings were made at each of 15 to 20
different surface locations. Ellipsometric measurements were
made using an automated ellipsometer (Model L104B, Gaertner
Scientific Corp., Chicago, IL). The light source was a 1 mW,
helium-neon laser having a beam wavelength of 6328 A. The
angle of incidence was 70°.
Elliosometry
Ellipsometry is an optical technique used to determine
the thickness and refractive index of thin films by analysis
of the change in the state of polarized light that
accompanies reflection from a film-covered surface. The
state of polarization is defined by the phase and amplitude
relationships between the two component plane waves into
which the electric field oscillation is resolved. In
general, reflection causes a change in the relative phases
of these waves and a change in the ratio of their
128
amplitudes. Reflected light is characterized by the angle A,
defined as the change in phase, and the angle 7, the
arctangent of the factor by which the amplitude ratio
changes. Software supplied by Gaertner ("SubCA") was used to
determine the optical constants T and A for bare surfaces as
well as adsorbed films. Resolution of the measured Y and A
for each film-covered surface into values of film thickness
and refractive index was done using a computer program (20)
based on calculation procedures described by McCrackin et
al. (28).
Given the values of film thickness and refractive
index, the adsorbed mass of a film immersed in buffer can be
calculated by an application of the Lorentz-Lorenz
relationship as experimentally verified by Cuypers et al.
(7). If a protein film remaining on a surface after rinsing
is dried, the adsorbed layer can be approximated as a layer
consisting of protein and air. In this case,
T = 0.1 d (Mp/Ap)
(n£2 - l)/(n£2 + 2)
[1]
where F (ug/cm2) is the adsorbed mass of protein; d (run) is
the film thickness; Mp (g/mol) is the molecular weight of
protein; and Ap (cmVmol) is the molar refractivity of
protein. The refractive index n£ refers to that of the film.
For nisin, Mp/Ap = 3.72 g/cm3. Ap was calculated by
appropriately summing individual molar refractivities of the
amino acids in nisin, excluding the dehydro-residues. Molar
129
refractivity values were tabulated for the twenty amino acid
residues by Pethig (30) . The value of Mp was then taken as
the sum of the molecular weights of the amino acids in
nisin, excluding the dehydro-residues.
Construction of adsorption isotherms
The relationship between adsorbed mass of protein and
its equilibrium concentration may be described by more than
one model or equation. A Langmuir-type model of the form
r = rmax ceq / (a + ceq)
[2]
was used to describe nisin adsorption at each surface. Ceci
is the apparent equilibrium concentration (mg/1) ; rmax is the
plateau value of adsorbed mass; and a (mg/1) is a function
constant. The Langmuir adsorption isotherm assumes a
monolayer film, a homogeneous surface, and no lateral
interaction among adsorbed protein molecules. Equation [2]
resembles the Langmuir isotherm, but should not be taken to
imply or assume any of its fundamental premises.
Detecting nisin activity
The activity bioassay for nisin consisted of preparing
150 x .25 mm MRS (Difco, Detroit MI) agar dishes seeded with
a 0.1% vol/vol of a standardized overnight culture of
130
Pediococcus pentosaceus FBB61-2 (8,19). Dried silicon plates
with and without adsorbed nisin were placed face down
directly onto the surface of the seeded agar dishes. Seeded
agar dishes containing silicon plates were held at 40C for
24 hours prior to incubation at 370C for outgrowth of the
indicator. Adsorbed nisin was desorbed from individual
surfaces by agitating 50 ul of 0.01 M phosphate buffer (pH
6.0) containing 1% polyoxyethylene sorbitan (Tween 80) with
a pipette tip over the entire surface. The 50 ul was then
transferred to wells (5.6 mm dia.) that had been made in
seeded agar dishes.
RESULTS AND DISCUSSION
Nisin adsorption to silicon
The effect of silicon surface silanization on nisin
adsorption isotherms is illustrated in Fig. A. 1. Adsorption
to the hydrophobic surface exhibited a steep initial slope
followed by immediate attainment of a plateau. Adsorption to
the hydrophilic surface exhibited a lower surface affinity
at low concentrations, but steadily increased and did not
attain a plateau value in the concentration range 100 to
1000 mg/1. Once adsorbed, a single type of protein can
probably exist in multiple conformational states on a
131
surface (1, 12, 15, 18), and protein molecules are assumed,
in general, to change conformation to a greater extent on
hydrophobic relative to hydrophilic surfaces (12). This is
due to the presence of hydrophobic interactions between the
solid surface and hydrophobic regions in the protein
molecule. This interaction gives the molecule an extended
structure, covering a relatively large area of the surface.
The repulsive force normally acting between native protein
molecules is probably decreased for such conformationally
changed molecules. On a hydrophilic surface, forces acting
between the surface and the molecule may be smaller in
magnitude. The resulting conformational change would likely
be smaller, preserving a greater repulsive force among
adsorbed molecules.
Probably as a result of nisin's unusual chemical
properties, the present results are inconsistent with the
general observation that greater amounts of adsorbed protein
are found on hydrophobic as opposed to hydrophilic surfaces.
Nisin is probably not a very flexible protein, consisting of
only 34 amino acid residues with five thioether
cross-linkages forming internal rings (21). Nisin is,
however, a hydrophobic protein, and its adsorption to
hydrophobic surfaces is consistent with adsorption being
entropically driven in this case. The effective
irreversibility in adsorption with respect to dilution
132
implies that multiple noncovalent interactions serve to hold
the molecule in place, with complete separation of the
molecule from the surface constituting an unlikely event.
Current models of protein adsorption are based on the
assumptions that a protein molecule may not only change
conformation after adsorption, but may also desorb from the
surface (23-25). However, the likelihood of "pure"
desorption is low, especially at long contact times, and if
desorption occurs at all it is most likely through
bulk-surface exchange reactions among proteins. Such
exchange reactions take place more readily on hydrophilic
than on hydrophobic regions of a surface (10, 11).
Adsorption to hydrophilic silicon is describable with
reference to electrostatic interactions. Hydrophilic silicon
is negatively charged, with an isoelectric point of about 2,
and nisin is positively charged at pH 6. Conceivably, at low
nisin concentrations, the favorable electrostatic
interaction between protein and surface could be somewhat
reduced due to "shielding" by counterions in the buffer. At
greater nisin concentrations, with the same (0.01 M sodium
phosphate) buffer, shielding would have a lesser effect
(22). The observation that adsorption to hydrophilic silicon
is only partially irreversible to dilution implies that
although multiple noncovalent "contacts" are probably
involved in adsorption, the electrostatic and hydrogen
133
bonding forces involved are much more readily displaceable
in an aqueous environment than are hydrophobic interactions.
The classification of protein adsorption into monolayer
or multilayer film formation is based on the magnitude of
adsorbed mass with respect to protein dimensions in solution
(9, 27). Values of adsorbed mass recorded in these
experiments are consistent with monolayer coverage, but some
workers have reported that protein can adsorb onto a surface
in more than one layer. For example, Arnebrant et al.
(3)
studied adsorption of P-lactoglobulin and ovalbumin on
hydrophilic and hydrophobic chromium surfaces using
ellipsometry and potential measurements. On clean
hydrophilic surfaces their results showed that a thick,
highly hydrated layer is obtained, which can be partially
removed by aqueous buffer rinsing. They suggested that the
protein adopts a bilayer formation on the surface, with the
bottom layer unfolded and attached by strong polar bonds to
the surface. In the case of protein in contact with a
hydrophobic metal surface, they found recorded values of
adsorbed mass to be consistent with formation of a
monolayer.
134
Antimicrobial activity of adsorbed nisin
The antimicrobial activity of nisin adsorbed to silicon
plates treated to exhibit hydrophilic properties is
represented in Fig. A. 2. The silicon plates numbered 1-10
indicate the concentration range of 100 to 1000 mg/1 of
nisin solution from which the protein was adsorbed.
Inhibition zone size in antimicrobial bioassays is generally
proportional to log10 concentration of the antimicrobial
agent. In this study we did not attempt to quantify the
nisin activity associated with the sample plates because of
their irregular dimensions. Desorption of nisin from the
silicon surfaces was undoubtedly essential in order to
observe antimicrobial activity. Nisin is a small polypeptide
which must traverse through the microbial cell wall to
elicit lysis of the cell membrane. Thus while adsorbed to a
surface, nisin would not be able to interact with microbial
cells.
Although adsorbed nisin was seen to be stable to
rinsing with buffer, it is probable that desorption of nisin
occurred when contacted with the microbial medium in the
bioassay petri plates. Tween 80, a commonly used food
emulsifier and a component of the MRS medium used in this
study has been shown to enhance nisin activity in milk (4,
19). Goff et al. (13) showed that Tween 80 has the ability
135
to displace proteins from the surface of milk fat globules.
We evaluated the ability of Tween 80 in buffer to desorb
nisin from plate surfaces. In Fig. A. 3 the activity of
desorbed nisin (from hydrophilic surfaces) is seen as
inhibition zones in the agar well diffusion assay. The
amount of recovered nisin roughly corresponds to the
relative amount of mass intially adsorbed, similar to the
previously described relationship between Fig. A. 1 and A.
2. No residual nisin was detected on plates after treatment
with Tween 80. The inhibition zone size represented with
well 10 is not valid because most of the desorbed nisin
sample was lost during manipulations. It should be noted
that the amount of nisin adsorbed onto surfaces is of a
quantity sufficent for inhibition of susceptible bacteria.
Commonly, about 100 U/g are used in food products (14) to
prevent the outgrowth of gram-positive bacterial spores and
vegetative cells. The amount of nisin (activity) estimated
to be adsorbed to the silicon surfaces was between 50 and
100 U/cm2. This estimate is based on comparison between
inhibition zone size of standardized amounts and amounts of
nisin recovered from the surfaces (Fig. A. 3).
Basic questions relating to the short- and long-term
stability of adsorbed nisin, how well immobilized nisin can
resist displacement by dissolved solution components, and
what surface concentrations are necessary to inhibit biofilm
136
formation need to be addressed. These issues will contribute
to the subject of future reports.
137
0.5
in
en
n
•a
o
•o
a
■
hydrophobic
D
hydrophilic
1000
1200
nisin concentration, nig/I
Figure A. 1. Nisin adsorption isotherms constructed for
hydrophilic and hydrophobic silicon surfaces. The mass
of nisin remaining on each type of surface after a 12
hour exposure to pure buffer is indicated by the closed
(hydrophobic) and open (hydrophilic) circles. The
curves drawn through the data follow equation [2].
138
Figure A. 2. Nisin activity manifested by inhibition zones
around the periphery of hydrophilic silicon plates.
Numbers 1-10. indicate the concentration of nisin
solutions (100 to 1000 mg/1) that had contacted the
surfaces. C denotes surfaces that were not exposed to
nisin. The indicator bacterium is Pediococcus
pentosaceus FBB-61-2. Grid squares are 2 cm2.
139
Figure A. 3. Detection of the activity of nisin desorbed
from hydrophilic surfaces with 1% w/w Tween 80. Numbers
1-10 indicate the concentration of nisin solutions (100
to 1000 mg/1) that had contacted the surfaces. Nis 1
and Nis 2 are standard concentrations (100 and 50 U/ml
respectively) of nisin. C denotes surfaces that were
not exposed to nisin.The indicator bacterium is
Pediococcus pentosaceus FBB-61-2.Grid squares are 2
cm2.
140
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144
Appendix B
100
Figure B. 1. The
hydrophobia
followed by
for another
200
300
400
Time, min.
500
600
sequential adsorption of BSA and P-Lg to
surfaces. P-Lg was adsorbed first for 4 h
rinsing for 3 0 min. then BSA was introduced
4 h.
145
100
200
300
400
Time, min.
Figure B. 2 The sequential adsorption of BSA and B-Lg to
hydrophobia surfaces. BSA was adsorbed first for 4 h
followed by rinsing for 3 0 min. then B-Lg was
introduced for another 4 h.
146
200
300
400
RDf,
Time, min.
Figure B. 3. The
hydrophilic
followed by
for another
sequential adsorption of BSA and p-Lg to
surfaces. P-Lg was adsorbed first for 4 h
rinsing for 3 0 min, then BSA was introduced
4 h.
147
600
Time, min.
Figure B. 4 The sequential adsorption of BSA and B-La to
fof^H1^ surfaces- BSA was adsorbed fi?st for 4 h
followed by rmsmg for 30 min. then p-Lg was
introduced for another 4 h.
K
y w^b
148
20
40
60
80
100
Time, min.
140
160
FigUr
^^" 5; Khe secJuential adsorption of BSA and B-Lg to
hydrophobia surfaces. p-Lg was adsorbed first fo? 1 h
for'ano^ IT.^
f0r
30 min
-
then BSA was
introduced
149
20
40
60
80
100
Time, min.
120
40
Figure B. 6. The sequential adsorption of BSA and B-Lg to
hydrophobic surfaces. BSA was adsorbed first for 1 h
followed by rinsing for 3 0 min. then (J-Lg was
introduced for another 1 h.
60
150
?n
Figure B. 7. The
hydrophilic
followed by
for another
40
60
80
100
Time, min.
160
sequential adsorption of BSA and B-Lg to
surfaces. p-Lg was adsorbed first for 1 h
rinsing for 30 min. then BSA was introduced
1 h.
151
0.18
60
80
100
Time, min.
160
Figure B. 8. The sequential adsorption of BSA and p-Lg to
hydrophilic surfaces. BSA was adsorbed first for 1 h
followed by rinsing for 3 0 min. then p-Lg was
introduced for another 1 h.
152
6
0.1
0.08
T3
(D
o
O
0.06
0.04
0.02
"SO
HK)
150
200 250 300
Time, min.
350
400
450
500
Figure B. 9. The competitive adsorption of BSA and p-Lg to
hydrophobic surfaces. Adsorption was performed from an
equimolar mixture of both proteins for 8 h.
153
100
150
00
250
300
350
400
450
500
Time, min.
Figure B. 10. The competitive adsorption of BSA and p-Lg to
hydrophilic surfaces-. Adsorption was performed from an
equimolar mixture of both proteins for 8 h.
154
Appendix C
Table C.l. The adsorbed mass (ug/cm2) following sequential
adsorption of BSA and P-Lg for 1 and 4 h to hydrophilic
and hydrophobic surfaces. The tabulated adsorbed mass
for the first protein is recorded immediately after
rinsing and before introduction of the second protein.
The final adsorbed mass as well as the percent increase
in the total adsorbed mass are tabulated after the
second adsorption time of the second protein and before
rinsing.
Adsorption first
second
time, h
adsorbed adsorbed
protein protein
surface adsorbed final
%increase in
type
mass
adsorbed adsorbed
mass
mass
4
BSA
P-Lg
hphobic
0.155
0.180
16
4
BSA
P-Lg
hphilic
0.135
0.176
30
4
P-Lg
BSA
hphobic
0.153
0.181
18
4
P-Lg
BSA
hphilic
0.138
0.178
29
1
BSA
P-Lg
hphobic
0.13 9
0.150
7
1
BSA
P-Lg
hphilic
0.142
0.162
14
1
P-Lg
BSA
hphobic
0.144
0.175
21.5
1
P-Lg
BSA
hphilic
0.106
0.157
48
hphobic = hydrophobic surface
hphilic = hydrophilic surface