JoumalofFood Engineering22 (1994) 409-419

0 1994 Elsevier Science Limited

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ELSEVIER

Specific Solute Effects with Special Reference to

Staphylococcus aurew

Jorge Chirife

Departamento de Industrias, Facultad de Ciencias Exactas y Naturales, Universidad de

Buenos Aires, Argentina

Today it is more or less established that the water activity (a,) of the
medium is not the only determining factor regulating the biological
response of a microorganism subjected to reduced water activity; the
nature of the a~-~on~olling solute play.~ a role. TRILLS,the so-called
specific solute fleet’ is discussed in detail for Staphylococcus aureus.
The influence of changes in the physical properties of the medium (e.g.
viscosity, oxygen solubility, oxygen difisivity, etc.) brought about by
solute/s dissolution was considered as a possible cause of specific solute
eflects. However, it was concluded that, within the ranges studied here,
there is not a clear re~atio~lship between the S. aureus response and the
modification of certain physical properties of the medium. The inhibitoq
eflects of solutes such as sodium chloride and sucrose, which are those
most often present in low a,-preserved foods, are primarily related to their
ability to lower water activity. However, for other solutes such as ethanol,
propylene glysol, bu~lene glycol and various poi}}et~lyleneglycob, specify
al~tiba~terial eflects are important. These anribacterial effects may be attri-
buted mainly to eflects of these molecules on membrane enzymes respon-
sible of peptidoglycan synthesis.

INTRODUCTION

Water activity (a,) has become an increasingly useful determinant of

food stability with respect to microbial growth because measured values
of the medium correlated well with the potential for growth (Troller &
Christian, 1978). As noted by Christian (198 1) the application of the
concept of a, to the water relations of microorganisms makes one major
assumption: when the aqueous solution in the microorganism’s environ-
409
410 Jorge Chirife

ment is concentrated by the addition of solutes the consequences for

microbial growth result solely from the change in a,. Today it is known
that the microbial response differs at a particular a, when the latter is
obtained with different solutes, and it is more or less established that the
CX,of the medium is not the only deter~ng factor regulating the bio-
logical response but that the nature of the a, controlling solute also plays
a role (Christian, 198 1; Gould, 1988). The fact that the specific effects of
different solutes may vary greatly led some authors (Franks, 1991) to
question the role of water activity as a credible measure of physiolo~cal
viability. Other authors (Gould, 1988), whilst r~co~izing that the con-
cept of water activity has been very valuable in physiological studies of
microorganisms, suggested that the uncritical use of a, as a determinant
of cell activity had the effect of steering attention away from some more
fundametal aspects of the water relations of cells. Gould (1985)
acknowledged that in some instances solute effects may depend on the
ability of the solute to permeate the cell membrane. Glycerol, for
example, readily permeates the membrane of many bacteria and so does
not initiate the same osmoregulatory response as nonpermeant solutes
such as sodium chloride and sucrose, and therefore has a different
‘inhibitory’ water activity. Figure 1 compares the minimal water activity
supposing growth of various pathogenic bacteria when sodium chloride

a 0.9
E 0.89
:g 0.88
I 0.87
0.88
0.85

Fig. 1. Comparison of the minimal alp supporting growth when NaCl or glycerol is
used to adjust the water activity (from data reported by Baird-Parker & Freame, 1967;
Emodi & Lechowich, 1969; Kang et al., 1969; Beuchat, 1974; Jakobsen, 1985; Daza et
d, 1991).
 Specific solute effects 411

or glycerol are used to control the water activity. In all cases glycerol is
less inhibitory than sodium chloride. It is noteworthy that in the case of
Stuphylococcus aureus (to be discussed later) the reverse is true, e.g.
sodium chloride is less inhibitory than glycerol, When solutes such as
sodium chloride and sucrose - and to some extent glucose and potas-
sium chloride - which are most often present in low a,-preserved foods,
are used to control a,, specific solute effects are less evident. (Emodi &
Lechowich, 1969; Jackobsen, 1985; Briozzo et al., 1986; Daza et ai.,
1991; Ballesteros et al., 19926).

STA~~~~C~CCUS AUREUS AND SPECIFIC SOLUTE

EFFECTS

Early in 1953, Scott noted that the minimal water activity which would
allow growth of S. aureus was independent of the solutes employed to
adjust the water activity of the medium (Scott, 1953). However, it was
later observed that some solutes were far more inhibitory than predicted
from the simple a, relationship. For some solutes such as sodium
chloride and sucrose the minimal a, supporting growth was in the
vicinity of 0+36; however, the minimal a,,,allowing growth was well above
0+36 when solutes such as alcohols, diols or polyethylene glycols were
used to control water activity (Plitman et al., 1973; Shaper0 et al., 1978;
Vaamonde et al., 1982, 1984). Vaamonde et al. ( 1984) also noted that
cells of S. aureus at in~bitory values died relatively rapidly during incu-
bation in the presence of these solutes at 30°C.

MINIMAL WATER ACTIVITY FOR GROWTH OF

STA~HY~~C~CC~SA~REUS

As mentioned before, a manifestation of ‘specific solute effects’ on S.

aureus cells subjected to reduced water activity was that the minimal
water activity allowing growth was dependent on the solutes used to
adjust it, This effect is observed in Fig. 2 which shows the minimal a, for
growth of S. aureus at near optimum temperature (30/35”C). Values
range from as high as 0.975 for ethanol to as low as 0.86 for solutes such
as sucrose and sodium chloride, which are the solutes most often present
in low a,-preserved foods. It is noteworthy that solutes such as alcohol,
diols and polyethylene glycols are able to inhibit S. aweus growth at
much higher water activity than sucrose or sodium chloride.
412

Sfapbylcccccus aureus
Solute (or solid)
Ethanol
1.3 Butylene alyool
Propylene (Ilyool
PEG-200
PEQ-400
t3iyCtVOi
Na-Acetate
Dried meat
Qlucose*aelta
Sucrose
SUCrOBB”8alts
Salts mixture
Dried milk
Driad soup
N&l

OS8 0.85 069 0,96 1

Minimal Water Activity
Fig. 2. Minimal water activity supporting growth of Staphybmccm c~ureus at near
optimum temperature (frQm SeVeral literature sources; mainly Ballesteros &id., 199%).

SOME P~~IC~-C~E~CAL CHANGES FR~~~CED BY

SOLUTE/S DISSOLUTION IN THE GROWTH MEDIU&I

Gould ( 1985) and Franks ( 1991) suggested that microbial growth is sub-
jected not only to water activity but also to specific molecular and/or
ionic interactions. They asked whether there is some other parameter, or
set of parameters, that would form the basis of a criteria of cell activity as
influenced by water and the aqueous environment. At a first approxima-
tion, this can be analysed by considering other modifications brought
about by dissolution of solutes, in addition to lowering of water activity
and specific interactions between cell and solute, as shown in Fig. 3.
Ballesteros et al. ( 19928) noted that changes in the physical properties of
the medium may eventually influence - in addition to specific interac-
tions and lowered water activity - the bacterial response. For example, a
really substantial increase in viscosity must eventually slow down oxygen
transfer and other transport processes between cell and the aqueous
environment sufficiently to have observable biological effects. Balles-
teros et al. ( 19923) calculated the modifications produced by solute dis-
solution (to lower a,) in some physical properties, namely, viscosity,
dielectric constant, oxygen solubi~ty and diffusivity. Some of their results
are shown in Figs 4 and 5. They concluded that there is not a clear
relationship between the S. aweus response to solute dissolution and the
modification of the above physical properties of the medium in the range
studied.
 Specific solute effects 413

Physico-Chemical
I

I
l Viscosity

l Oxygen solubility
/Lowering of\
1Oxygen diffusivity
\ \ %- //
Dielectric constant

Fig. 3. Some physico-chemical changes produced I solute/s dissolutior 1 in the growth

medium.

8 lll-
5 lOl-
5 Ql-
81.
'5;
AI
g 3:-
51-
41
31
21
11
1
0.8 0.82 0.84 0.88 0.88 0.9 OS2 0.94 0.M 0.98 1
Water Activity
Fig. 4. Effect of the solute used to adjust the water activity on the relative viscosity
(20/25”C) of aqueous solutions (from Ballesteros et al., 19926).

ELECTRON MICROSCOPY OBSERVATIONS

Since physical changes in the aqueous medium did not seem to be

responsible for the antibacterial effect of some solutes as compared to
others, Ballesteros et al. (19923) investigated the behaviour of S. aUreUs
cells through electron microscopy. Figure 6 shows electron micrographs
of normal cells of S. aweus (a) and of cells subjected to sodium chloride
(b) and sucrose (c), solutions having a, = 045. Neither of these solutes
414 Jorge Chirife

110

0
0.76 0.6 0.64 0.66 0.92 0.96
Water Activity
Fig. 5. Effect of the solute used to adjust the water activity on the relative oxygen solu-
bility (25°C) in aqueous solutions (from Ballesteros et al., 1992b).

caused important morphological changes in S. aureus cells. Plasmolysis

is not observable with S. aureus cells since the cytoplasmic membrane is
in continuous close contact with the cell wall. It is known, however, that
non-permanent solutes such as sodium chloride and sucrose, do cause
cell dehydration in S. uureus, as shown by Christian & Waltho (1964).
Figures 7 and 8 show electron micrographs of S. aureus cells after
being placed by various times in solutions of propylene glycol (a = O-92),
1,4 butylene glycol (a, = 0.85) and polyethylene glycol 400 and 1000
(a, = O-85). These solutes caused dramatic morphological modifications
in the cells which may be summarized as follows: (a) the cell wall of S.
uureus seems to be a main target of the antibacterial action of diols and
polyethylene glycols; cells may be observed completely separated or
showing signs of rupture but still in a close contact with the membrane;
(b) intense morphological modifications which included widening of the
septum in daughter cells during the process of division and ‘giant’ cocci
frequently observed in media of water activity adjusted with poly-
ethylene glycols, suggesting substantial weakening of the restraining cell
wall followed by expansion of the cytoplasm or fusion (Ballesteros et d.,
1992b).
Most of these morphological modifications were similar to those
observed by Ballesteros et al. (1992~) on the effect of ethanol on S.
uureus cells. Thus, and it was postulated for ethanol, it was assumed that
a main antibacterial action of diols and polyethylene glycols was the
 Specific solute effects 415

lb)

(4
Fig. 6. (a) Electron micrograph of S. aureus cells grown in BHI’ (control ceils); (b)
electron micrograph of S. aweus cells after being placed in sodium chloride-supple-
mented medium having a, = 0.85; (c) electron micrograph of S. aureus cells 24 h after
being placed in sucrose-supplemented medium having a, = 0.85 (from Ballesteros et al,,
19926).
416 Jorge Chirife

(4

(b)
Fig. 7. (a) Electron micrograph of S. aureus cells 36 h after being placed in propylene-
glycol-supplemented medium having a, = 092. Arrows show cell walls separated from
the cells and walls with signs of rupture but still in close contact with the membrane. (b)
Electron micrograph of S. uureus cells 24 h after being placed in 1,4 butylene-glycol-
supplemented medium having a, = 0.85.

alteration of the biosynthesis of the cell wall of S. aureus through the

inhibition of the enzymes involved in cross-linking of peptidoglycans
(Ingram, 198 1; Ingram and Buttke, 1984). It is likely that ethanol inhibits
cross-linking during peptidoglycan synthesis by decreasing the strength
of hydrophobic interactions (Ingram, 1982). Diols and PEGS are rela-
tively-polar organic molecules which, like ethanol, pertain to the group of
the so-called ‘amphipathic’ molecules. These molecules are charac-
terized by a hydrophobic and a hydrophilic portion consisting of hydro-
xyl groups. Amphipathic molecules are in general easily partitioned into
 417

(b)
Fig. 8. (a) Electron micrograph of S. aareas cells 24 h after being placed in PEG-400-
supplemented medium having a, = @85. Arrows show celf walls with signs of rupture
but still in a close contact with the membrane; (b) electron micrograph of S. aurem cells
after being placed for 24 h in PEG-lOOO-supplemented medium having a, = 0%5 fol-
lowed by 24 h in the same medium but previously diluted to a, = O-98. Arrows show cell
walls with sign of rupture but still in close contact with the membrane.

phospholipid membrane bilayers and cause alterations in membrane

structure and function.
though the general effect of ethanol, propylene glycol, butylene
glycol and PEGS appears to be related to membrane pe~rbation result-
ing in the inhibition of enzymes which assemble peptidoglycan, other
mechanisms such as inhibition of transport processes through the mem-
brane and macromolecular synthesis, among others (Ingram & Buttke,
1984) cannot be ruled out.
 CONCLUSIONS

The following conclusions may be drawn: (1) the inhibitory effects of

sucrose and sodium chloride against S. uureus cells are primarily related
to their ability to lower water activity, specific solute effects are not sig-
nificant; (2) changes in physical properties of the growth medium
brought about by solute addition did not seem to influence the S. aurem
response, in the range of conditions studied here; and (3) the antibac-
terial effects of amphiphatic molecules (ethanol, dials, PEGS) may be
attributed, apart from nonspecific effects on water activity, to specific
effects of these molecules on membrane enzymes responsible of pepti-
doglycan synthesis,

ACKNOWLEDGMENTS

The authors acknowledge financial support from Universidad de

Buenos Aires and Consejo NacionaI de Investigaciones Cientificas y
Tecnicas de Ia Republica ~gen~na.

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