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Lactate Acid Inhibition of Salmonella typhimurium in Yogurt
HOWARD E. RUBIN t, THOMAS NERAD ~, and FRIZELL V A U G H A N
Department of Environmental and Industrial Health
School of Public Health
The University of Michigan
Ann Arbor 48109
ABSTRACT dissociated because the pHi was higher
We determined how lactic acid inhibits than the external pH. The dissociated
growth of Salmonella typhimurium in moiety accumulated because it could n o t
yogurt. This inhibition was demonstrated leave the cell in this form, consequently
by microscopic examination not to be due lowering the pH i. Thus, inhibition of S.
to bacteriolysis. Neither growth nor meta- typhimurium in yogurt is from the intra-
bolic activity could be initiated after cells cellular dissociated moiety of lactic
were washed in phosphate buffer and acid.
exposed to 1.5% lactic acid for 1 h at
3 7°C, indicating that lactic acid inhibition INTRODUCTION
is irreversible. Thegrowth rate of S. typbi- In yogurt, lactic acid is the main inhibitor of
murium at 3 7°C was computed at various Salmonella typbimurium, the most prevalent of
combinations of pH and lactic acid the milk-borne pathogens (9). However, the
concentrations, and the intracellular mechanism of acid inhibition is unknown (3).
conditions (pH and lactic acid con- Arnott et al. (2) reported that over 50% of all
centration) at bacteriostasis subsequently commercial yogurts tested were contaminated
were extrapolated. Cellular death resulted with various microorganisms. Therefore, an
when these intracellular bacteriostatic understanding of the mechanism of lactic acid
conditions were surpassed. Thus, growing inhibition of bacteria could help determine how
cells could be used indirectly to determine yogurt might be improved to ensure consumer
intracellular conditions at the time of health.
death. Intracellular pH (pH i) and inhibi- Once cellular death occurs, the intracellular
tion of the growth rate were unrelated. microenvironment is drastically altered and is
Also, bacteriostasis was observed when hy- probably different from that in cell-free extracts.
drochloric acid was used to lower the pH i Thus, viable microorganisms have been used in
of Salmonella to 5.5 whereas a bactericid- this research to study intracellular conditions
al effect was observed when the pH i was at the moment of lactic acid-induced death.
lowered to 5.5 with lactic acid. The Analytical techniques have not been de-
lactate anion, rather than the hydrogen veloped to allow intracellular pH (pH i) to be
ion, exerted the inhibitory effect against measured directly. However, these variables
S, typhimurium. When the pH i became can be measured by indirect determinations.
less than 5.3, inhibition was from the Intracellular pH can be measured from the
hydrogen ion concentration. Thus, lactic concentration and pK of a weak acid. The
acid inhibition was a complex and variable acid concentration taken up by the cell mass
mechanism in relationship with pH i . can be measured. If concentration of the
Lactic acid entered the cell in the un- undissociated species inside the cell is the
dissociated state. Once inside the cell, it same as concentration of the undissociated
species outside the cell, pH i can be determined
(5).
Lactic acid could be inhibitory toward S.
typhimurium directly by acting on unknown
Received January 23, 1981. metabolic processes and indirectly by lowering
t Laboratory of Soil Microbiology, Department
of Agronomy, Cornell University, Ithaca, NY 14853.
the pH i . As the main inhibitor of Salmonella in
2American Type Culture Collection, 12301 Park- yogurt (9), lactic acid was studied to determine
lawn Drive, Rockville, MD 20852. its bactericidal properties. Additionally, the
1982 J Dairy Sci 65:197-203 197
198 RUBIN ET AL.
roles that pH and the undissociated and dis- growth was followed by viable count on brilliant
sociated species of lactic acid play in the green agar spread plates. To determine genera-
inhibition of S. typhimurium are elucidated. tion time, the following equation was used:
M A T E R I A L S A N D METHODS G t -- T/3.3 log (B/b) [a]
Organisms and Media where:
S. typhimurium was obtained from the T is time
culture collection of the Microbiology Depart- B is number of cells at T h
ment, The University of Michigan, Ann Arbor,
b is initial number of cells
and was maintained by weekly transfer on
trypticase soy agar slants incubated overnight at
Cultures were grown until approximately 9.2 x
35°C.
108 cells/m1 were obtained. Cultures then were
Brilliant green agar (Difco) spread plates
split into two aliquots and washed twice with
were used for enumeration. Salts-glucose
phosphate buffer. One portion was used for
medium was used as in (9).
calculation of internal pH and the other for
determination of internal lactic acid con-
Preparation of Inoculum
centration.
S. typhimurium was grown statically on Intracellular pH was determined spectro-
salts-glucose medium for 18 h at 37°C. Cells photometrically by measuring the amount of
were harvested and washed twice with sterile 2,4-dinitrophenol (DNP) concentrated intra-
phosphate buffer (1). The washed cell pellet cellularly at a particular external pH (7). The
was resuspended in phosphate buffer to a DNP was added to salts-glucose medium similar
turbidity that, when added to the test medium, to the medium in which the culture was grown.
gave the initial concentration of cells desired. incubation lasted for 5 rain at 37°C before
DNP determinations. This method relied on the
Lactic Acid Inhibition
Henderson-Hasselbach equation [b] to compute
S. typhimurium was incubated at 37°C for 1 the concentration of the undissociated (HA)
h in salts-glucose medium containing 1.5% and dissociated (A) moieties of DNP:
lactic acid at a pH of 4.5. The culture then was
washed twice with phosphate buffer to remove pH -- PKDN p = log [ A ] / [ H A l [b]
lactic acid. Aliquots were used to inoculate
trypticase soy broth, trypticase soy agar, where:
brilliant green broth, and brilliant green agar for The pH is the negative log of the hydrogen
48 h at 37°C. ion concentration o f the medium
pKDN P is the dissociation constant of DNP
Determination of pH
(4.1)
All pH measurements were accomplished
with a Beckman pH meter equipped with a glass By equation [b], the ratio of dissociated to
combination electrode. undissociated species of DNP in the medium at
the end of the incubation period was calculated.
Lactic Acid Determinations The ratio of dissociated to undissociated species
Lactic acid was measured spectrophoto- of DNP in the medium and total concentration
metrically with a Guilford Model 240 Spectro- of DNP in the medium are used to determine
photometer by the method of Davidson (6). the concentration of each species of DNP.
Because the undissociated species within the
Determination of Intracellular Conditions cell is assumed to be equal to the undissociated
S. typhimurium was grown in salts-glucose species outside the cell at equilibrium, the
medium containing each possible combination concentration o f the dissociated species of DNP
of the following pH's and lactic acid con- within the cells can be assessed by equation [c] :
centrations: pH 7.0, 6.5, 6.0, and 5.5; lactic
acid 1.0%, .75%, .50%, and .25%. Resulting cell [A] = [DNP]initial-- 2[HAl . - - [ A I . [c]
Journal of Dairy Science Vol. 65, No. 2, 1982
LACTATE INHIBITION OF S. TYPHIMURIUMIN YOGURT 199
wh ere: bacteriostasis after exposure to the .acid.
[A] i is concentration of dissociated DNP However, cells inhibited by 1.5% lactic acid at
within cells pH 4.5 showed neither an increase in turbidity
or acidity nor a decrease in pH in trypticase soy
[DNP]initial is total concentration at the
broth or brilliant green broth after 48 h. No
beginning of incubation
colony formation was found on either trypticase
[ H A ] . is concentration of undissociated soy agar or brilliant green agar plates at the end
DNP outside cells at the end of incubation of 72 h. This indicated that the effect was
[A] , is concentration of the dissociated bactericidal and probably was irreversible
species of DNP outside cells at the end of because of the inability of the cells to reinitiate
incubation growth and cessation of metabolic activity.
Next, an attempt was made to determine if
The following equation can be used to the end result of lactic acid inhibition of S•
determine the pH i (intracellular pH) of the cells typbimurium was via bacteriolysis. The numbers
as [A] . a n d [Ali can be computed: obtained by direct count after incubating in
phosphate buffer and 1.42% lactic acid had not
pH i = p H . + log [A]i/IA] o [d] changed substantially at the end of 24 h of
incubation as seen in Table 1. No viable counts
were observed on brilliant green agar at the end
where:
of 72 h. This indicated that S. typbimurium
pH o= pH of medium
was killed and no appreciable amount of lysis.
Equation [d] is derived by subtracting the When optical density was measured at 540 nm,
Henderson-Hasselbach equation [b] for cal- absorbancy had increased by 20.8% by the end
culation of external species of DNP at equili- of 24 h.
brium from the equation for calculation of
Intracellular pH
internal species of DNP [c]. After rearrange-
ment, this yields equation [d]. The method to measure pHi is based on the
The washed cell pellet was added to salts- assumption that the intracellular and extra-
glucose medium at a pH and lactic acid con- cellular undissociated concentrations of DNP
centration identical to those in which it was are equal as DNP acts as a weak acid. To test
grown. This suspension was incubated for 1 h at this assumption, a washed cell pellet of S.
37°C, and then 2 ml was removed to determine typbimurium was suspended in 6 ml of the
the dry weight. Samples of medium were minimal salts solution without glucose at pH
removed both before addition of the cell pellet 3.7 and containing .335 mM DNP. After 5 rain
and after incubation. The amount of lactic acid of incubation, the cells were filtered onto a .45
taken up by the cell mass was determined by /am filter. The concentration of DNP in the
the difference in the medium before and after filtrate was .085 mM. Therefore, the con-
incubation.
The cell dry weight was measured by passing
2 ml of the suspension through a preweighed
TABLE 1. Effect of 1.42% lactic acid and pH 3.85
.45/am filter and then evaporating at 105°C for in phosphate buffer on Salmonella typbimurium.
at least 6 h. The lactic acid concentration then
could be calculated by the following formula: Time
Measurement Initial 24 h
/ag lactic acid/mg cell dry wt --
2(/ag lactic acid/ml)/mg cell dry wt [el Absorbance a .57 .72
Direct countb 2.4 × 109 2.5 X 109
C
R ESU LTS Plate countb 4.2 × 108
Lactic Acid Inhibition a540 nm.
Lactic acid inhibition of S. typbimurium in bCells/ml.
yogurt could be from either cellular death or CLess than 1 colony/ml.
Journal of Dairy Science Vol. 65, N~. 2, 1982
200 RUBIN ET AL.
centration of DNP within the cell mass was 2.5 centration in Figure 2. Since fermenting yogurt
mM. The pH i then was calculated as 4.6. had a lactic acid concentration of .34% at the
The cells retained on the filter were re- end of bacteriostasis (9), the pHi at bacteriostasis
suspended in minimal salts at pH 3.7 for 5 rain in fermenting yogurt was approximately 5.3.
longer and again were filtered through a .45/am It is possible that the pH i was bactericidal
filter. The DNP concentration in the filtrate independent of lactic acid. Growth ensued
was .067 raM. The extracellular undissociated when the pHi of Salmonella was lowered
DNP concentration then was .048 raM. If it is to 5.5 with hydrochloric acid. When sufficient
valid that extracellular and intracellular un- hydrochloric acid was added to the medium to
dissociated DNP concentrations are equal, then obtain a pHi of about 5.2, cellular death
the pH i computed after the second incubation occurred. When a pHi of 5.3 is reached, the
should be close to 4.6. The p h i was 4.55; the hydrogen ion concentration may be sufficient
assumption is valid. to cause cellular death. Thus, intracellular lactic
Viable cells were used to determine the pHi
at bacteriostasis. Figure 1 shows the relationship
between pHi and the reciprocal of the generation
time (generation t i m e - l ) . The higher the
concentration of lactic acid, the greater was S.
typbimurium's generation time. As lactic acid
concentration increased, generation time in-
creased for a given pH i. The pH i at bacteriostasis
for each lactic acid concentration was deter- 1.50
mined by extrapolating each curve to 0 genera-
tion time - t (bacteriostasis). These then were
plotted against the extemal lactic acid con-
,.41
1.2
I--
o
1.0o
J
tO I
#
t/
• //
81 /
.50 ¸
/
r'
/ /
/ •
External Lactic Acid Concen~rotion
5.0 60
PH i
-
o . . . .
-
6-
-
o
-
- - -a,
2.5%
.50
175%
1.00%
%
"LO
o PH z
I
Figure 2. Effect of pHi on extracellular lactic acid
Figure 1. Effect of intracellular pH (pHi) on concentration (% lactic acidexternal) ors. typhimurium
growth of S. typbimurium at 37°C. at 37°C.
Journal of Dairy Science Vol. 65, No. 2, 1982
LACTATE INHIBITION OF S. TYPHIMURIUMIN YOGURT 201
acid at a pH i of 5.3 or above is probably the external pH (Figure 4). Since the pH of fer-
inhibitory factor. The hydrogen ion concen- menting yogurt was 4.5 at bacteriostasis, the
tration might be sufficient at a pHi lower than intracellular lactic acid concentration was about
5.3 to be bactericidal. 195 #g/mg dry wt. This was about 30% of the
total weight of the cellular mass.
Intracellular Lactic Acid The amount of dissociation of intracellular
Intracellular lactic acid did n o t appear to be lactic acid at the end of bacteriostasis could be
metabolized during the experiment because a determined by the Henderson-Hasselbach equa-
linear curve resulted when pH i was plotted tion (equation [b] ) as pH i is also known. The
against the extracellular concentration of lactic dissociated lactic acid concentration was 10
acid (Figure 2). If lactic acid was metabolized #g/mg dry wt. Therefore, the dissociated
by the cells, a curved line would be expected moiety of lactic acid was the predominant
because at higher pHi's, the acid should be intracellular species at the end of bacteriostasis
metabolized more rapidly. in yogurt.
Figure 3 shows the relationship between the
Inhibition by the Dissociated Moiety
reciprocal of generation time and intracellular
lactic acid concentration at various pH. As The intracellular concentrations of the
intracellular lactic acid concentration increased, undissociated and dissociated moieties of lactic
generation time also increased. A t bacteriostasis, acid were calculated for each intracellular
there was an increasing intracellular lactic acid lactic acid concentration and pH (Figure
concentration as pH was lowered. There was an 5A, B). There was no linear correlation between
inverse relationship between intracellular lactic the undissociated species and its effect on gen-
acid concentration and generation time. The eration time of S. typbimurium (Figure 5A).
intracellular lactic acid concentration at bac- However, a direct relationship between the
teriostasis was extrapolated, and plotted against intracellular dissociated species and generation
time can be observed in Figure 5B. This indicat-
ed that the dissociated intracellular lactic acid
moiety was the species responsible for the
bactericidal effect of yogurt toward S.
External pH typhimurium.
*-- --Q 65
-- 6 0
I - - - .Q 5 5
70"
6C
g .B- r,
~ -
5C
6-
x
4.0
x
.2 ! ~ ,.
\ x
5.0 . . . .
oi o go ,6o ,5'o 260
C-
250
0
./ug L A C T I C
too
ACID i /rag
200
DRY W E I G H T
300
OF
400
CELLS
~g LACTIC AC]Di/mg DRY WEIGHT OF CELLS
Figure 4. Effect of intracellular lactic acid (lactic
Figure 3. Effect of intracellular lactic acid (lactic acidi) on external pH (PHexternal) of S. typbimurium
acid i) on growth rate of S. typbimurium at 37°C. at 37°C.
Journal of Dairy Science Vol. 65, No. 2, 1982
202 RUBIN ET AL.
A
7 IO'
W
io 2o 30 40 50 SO ~0 8O 90 ~00 20 40 SO eO K~O IZO 140 ISO mO 2¢0
Figure 5. Effect of intracellular species of lactic acid on growth rate of S. typbimurium at 37°C. A) Intra-
cellular undissociated [HA] i lactic acid. B) Inlxacellular dissociated [A] i lactic acid.
DISCUSSION sociated species. However, Baskett and Hentges
Reports (4, 8) demonstrated that an increase (3) obtained similar amounts of inhibition of
in absorbance is caused by a shrinkage of oxidative metabolism in cell-free extracts of
cellular volume. Thus, although cells of S. Sbigelta flexneri for both moieties of acetic
typbimurium remain contiguous after lactic acid. A direct correlation was demonstrated
acid-induced death as indicated by the absence between inhibition of whole cells of S. typbi-
of bacteriolysis, cells shrink in size, perhaps murium and the intracellular concentration of
from the cell membrane becoming leaky. There dissociated lactic acid, thus agreeing with
was no restoration of either metabolic activity the conclusions of Weiner and Draskoezy
or growth when lactic acid was removed from (10).
the cells, which indicates that lactic acid The internal concentration of lactate anions
inhibition of S, typbimurium in yogurt is and hydrogen ions (pH i ) could be responsible
irreversible. There was an accumulation of for the inhibitory effects of the dissociated
lactic acid inside the cell resulting in a con- moiety against S. typbimurium. There was no
centration greater than that in the external clear relationship between p h i and inhibition of
environment. This showed that the membrane growth rate although bacteriostasis was observed
was intact and at least partially functioning when hydrochloric acid lowered the pHi to 5.5
until death. (Figure 1). However, die-off rate was rapid for
The amount of lactic acid accumulated Salmonella, at the same pHi, when lactic acid
intracellularly increased as pH of the medium was present. This indicated that the lactate
was lowered. Also, there was an increase anion, rather than the hydrogen ion (pH),
in the die-off rate of Salmonella corresponding exerted the inhibitory effect against S, typbi-
with an increase in the intracellular lactic acid murium in yogurt. Inhibition of the micro-
concentration. This evidence suggested that the organism seemed to be from the lactate anion
undissociated species was the form in which when the pHi was 5.3 or above. When the pHi
lactic acid entered the cell and that lactic acid was less than 5.3, inhibition could be from both
was inhibitory when it accumulated intra- increased lactate anion and hydrogen ion con-
cellularly. However, the mechanism of centrations. Thus, lactic acid inhibition is
lactic acid inhibition of S. typbimurium is not probably a complex and variable mechanism in
known. relationship with pHi and lactic acid con-
Weiner and Draskoczy (10) showed that in centration.
cell-free extracts of Escbericbia coli inhibitory Lactic acid enters the cell in the undissociated
effects of mandelic and hippuric acids could be form (10). Once inside the cell, lactic acid
correlated to the concentration of the dis- dissociated because Salmonella's pH i was higher
Journal of Dairy Science Vol. 65, No. 2, 1982
LACTATE INHIBITION OF S. TYPHIMURIUM IN YOGURT 203
t h a n t h e e x t e r n a l pH. T h e dissociated m o i e t y water and wastewater. 13th ed. APHA, Washington,
c o u l d n o t leave the- cell in this f o r m so it DC.
a c c u m u l a t e d a n d c o n c u r r e n t l y l o w e r e d the pH i . 2 Arnot, D. R., C. L. Duitschaever, and D. H. Bullock.
1974. Microbiological evaluation of yogurt pro-
I n h i b i t i o n o f S. t y p b i m u r i u m was f r o m t h e duced commercially in Ontario. J. Milk Food
i n t r a c e l l u l a r dissociated m o i e t y o f lactic acid Technol. 37:11.
r a t h e r t h a n t h e h y d r o g e n ion c o n c e n t r a t i o n , t o 3 Baskett, R., and D. Hentges. 1973. Sbigella flexneri
a pH i o f 5.3. If pH i was l o w e r t h a n 5.3, t h e inhibition by acetic acid. Infect. Immun. 8:91.
4 Bernheim, F. 1963. Factors which affect the size
i n t r a c e l l u l a r h y d r o g e n ion c o n c e n t r a t i o n p r o b - of the organisms and the optical density of suspen-
ably w o u l d be s u f f i c i e n t to be i n h i b i t o r y . sions of Pseudomonas aeruginosa and Escbericbia
M a n y p o i n t s n e e d t o be clarified. Weiner a n d coll. J. Gen. Microbiol. 30:53.
D r a s k o c z y (10) n o t e d t h a t i n d o p h e n o l blue 5 Caldwell, P. C. 1956. Intracellular pH. Int. Rev.
r e d u c t i o n was i n h i b i t e d b y lactic acid in E. coli, Cytol. 5:229.
6 Davidson, J. 1949. The colorimetric determination
i m p l y i n g t h a t lactic acid m i g h t t e r m i n a t e of lactic acid in milk and milk products. J. Dairy
oxidative m e t a b o l i s m b y i n h i b i t i n g d e h y d r o - Res. 16:209.
genase activity. T h e e f f e c t o f lactic acid o n 7 Kotyk, A. 1961. Uptak e of 2,4-dinitrophenol by
t r a n s p o r t s y s t e m s has n o t b e e n investigated. the yeast cell. Folia Microbiol. 7:109.
8 Mager, J., M. Juczinski, G. Schatzberg, and Y.
Metabolic pathways affected and enzymes
Avi-Dor. 1956. Turbidity changes in bacterial
i n h i b i t e d b y lactic acid s h o u l d be d e t e r m i n e d . suspensions in relation to osmotic pressure. J. Gen.
Also, sites o n the i n h i b i t e d e n z y m e s s h o u l d be Microbiol. 14:69.
elucidated. 9 Rubin, H. E., and F. Vaughan. 1979. Elucidation
of the inhibitory factors of yogurt against Sal-
monella typbimurium. J. Dairy Sci. 62:1873.
10 Weiner, N., and P. Draskoczy. 1961. The effects of
REFERENCES
organic acids on the oxidative metabolism of intact
1 American Public Health Association (APHA). and disrupted E. coll. J. Pharmacol. Exp. Ther.
1971. Standard methods for the examination of 132:299.
Journal of Dairy Science Vol. 65, No. 2, 1982
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