Survival of Salmonella in Home-Style Mayonnaise and Acid Solutions as Affected by Acidulant Type and Preservatives

Abstract

Mayonnaise made from contaminated eggs has been linked to outbreaks of Salmonella infections. This study was undertaken to determine the fate of salmonellae in home-style mayonnaise and acid solutions with or without chemical preservatives. Egg yolks were inoculated with different levels of a three-serotype (Typhimurium, Heidelberg, and Enteritidis [untypeable phage type]) mixture of Salmonella or a three-phage-type (4, 8, and 13) mixture of Salmonella Enteritidis. The inoculated yolks were used to make mayonnaise with 2, 3, or 4 teaspoons of a commercial wine vinegar or lemon juice. The mayonnaise was sampled for salmonellae over a 15-day period at 4°C, and negative samples were tested further by a three-tube most-probable-number assay. The same Salmonella mixtures were respectively inoculated into six acid solutions including wine vinegar, lemon juice, and acetic or citric solutions with or without chemical preservatives. The Salmonella populations of the Salmonella Enteritidis mixture were more persistent than those of the other Salmonella mixture in mayonnaise. Both Salmonella mixtures survived longer in mayonnaise made with vinegar than with lemon juice during storage at 4°C. In the acid solutions, however, the populations of the two Salmonella mixtures were not significantly different. The numbers of the two Salmonella mixtures in acetic or citric acid solutions with the preservatives were significantly lower than those in vinegar, lemon juice, and the solutions without the preservatives. Results suggest that Salmonella in contaminated egg yolks could survive the mayonnaise-making process. The inhibition of Salmonella by vinegar and lemon juice is due to the hurdle effect of organic acids and chemical preservatives.

Full text

Survival of Salmonella in Home-Style Mayonnaise and Acid
Solutions as Affected by Acidulant Type and Preservatives
JUNLI ZHU,
1
JIANRONG LI,
1
AND JINRU CHEN
2
*
1
Research Institute of Food Science and Biotechnology, Zhejiang Gongshang University, 149 Jiao Gong Road, Hangzhou, 310035 People’s Republic of
China; and
2
Department of Food Science and Technology, The University of Georgia, 1109 Experiment Street, Griffin, Georgia 30223-1797, USA
MS 11-373: Received 8 August 2011/Accepted 28 October 2011
ABSTRACT
Mayonnaise made from contaminated eggs has been linked to outbreaks of Salmonella infections. This study was
undertaken to determine the fate of salmonellae in home-style mayonnaise and acid solutions with or without chemical
preservatives. Egg yolks were inoculated with different levels of a three-serotype (Typhimurium, Heidelberg, and Enteritidis
[untypeable phage type]) mixture of Salmonella or a three-phage-type (4, 8, and 13) mixture of Salmonella Enteritidis. The
inoculated yolks were used to make mayonnaise with 2, 3, or 4 teaspoons of a commercial wine vinegar or lemon juice. The
mayonnaise was sampled for salmonellae over a 15-day period at 4uC, and negative samples were tested further by a three-tube
most-probable-number assay. The same Salmonella mixtures were respectively inoculated into six acid solutions including wine
vinegar, lemon juice, and acetic or citric solutions with or without chemical preservatives. The Salmonella populations of the
Salmonella Enteritidis mixture were more persistent than those of the other Salmonella mixture in mayonnaise. Both Salmonella
mixtures survived longer in mayonnaise made with vinegar than with lemon juice during storage at 4uC. In the acid solutions,
however, the populations of the two Salmonella mixtures were not significantly different. The numbers of the two Salmonella
mixtures in acetic or citric acid solutions with the preservatives were significantly lower than those in vinegar, lemon juice, and
the solutions without the preservatives. Results suggest that Salmonella in contaminated egg yolks could survive the mayonnaise-
making process. The inhibition of Salmonella by vinegar and lemon juice is due to the hurdle effect of organic acids and chemical
preservatives.
Mayonnaise is one of the most widely consumed
condiments in the world. It is a stable emulsification of oil
and water and contains at least 78.5%fat and 6%egg yolk
(6). Commercial mayonnaise is produced with pasteurized
eggs and does not impose a significant risk to public health
(15). Homemade mayonnaise has, however, been linked to
several outbreaks of infections mainly due to Salmonella
contamination of raw chicken eggs (13). The survival of
Salmonella in mayonnaise prepared with 5%acetic acid or
citric acid has been reported (10, 11, 18). However, few
studies have compared the survival rates of Salmonella in
mayonnaise products made with a commercial wine vinegar
(,5%acetic acid) or lemon juice (,5%citric acid) along
with the chemical preservatives added by vinegar and juice
manufacturers. The objectives of this study were twofold: (i)
to evaluate the fate of salmonellae artificially inoculated into
chicken egg yolks during the mayonnaise-making process
and subsequent storage at refrigeration temperature, and (ii)
to compare the survival of salmonellae in six acid solutions
including a commercial wine vinegar and lemon juice as
well as acetic and citric solutions with or without the
chemical preservatives used by wine vinegar and lemon
juice manufacturers, including sodium metabisulfite, sodi-
um sulfite, sodium benzoate, and potassium metabisulfite.
MATERIALS AND METHODS
Salmonella strains. Two three-strain mixtures of Salmonella
of human and poultry origin were used to inoculate egg yolks in both
phases of the study. The first inoculum contained three different
serotypes of Salmonella including Salmonella Typhimurium 4-1,
Salmonella Heidelberg 4-2, and Salmonella Enteritidis 5-60
(untypeable phage type [PT]), and the other mixture had three
different phage types of Salmonella Enteritidis including PT4 strain
5-55, PT8 strain 5-56, and PT13 strain 5-59. The strains were
retrieved from frozen storage and grown on tryptic soy agar (BD,
Sparks, MD) at 37uC for 24 h. The resulting cultures were
transferred into tryptic soy broth, which was incubated under the
same conditions. Equal volumes of three Salmonella cultures were
pooled, and the cells in the mixtures were harvested by
centrifugation using a bench top centrifuge (Brinkmann Instruments,
Westbury, NY) at 12,000 |gfor 2 min. Harvested cells were
washed twice with, and then resuspended in, sterile distilled water to
an approximate cell concentration of 10
9
CFU/ml. The suspensions
were serially diluted in sterile water to an estimated cell
concentration of 10
3
,10
5
,or10
7
CFU/ml. The numbers of cells in
the suspensions were confirmed by the standard plate count assay.
Mayonnaise preparation. In the first phase of the project,
large, Grade A chicken eggs purchased from a grocery outlet in
* Author for correspondence. Tel: 770-412-4738; Fax: 770-412-4748;
E-mail: jchen@uga.edu.
465
Journal of Food Protection, Vol. 75, No. 3, 2012, Pages 465–471
doi:10.4315/0362-028X.JFP-11-373
Copyright G, International Association for Food Protection
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Griffin, GA, were aseptically broken, and the yolk and albumen
were separated. Each egg yolk was inoculated with 10 mlofa
suspension containing ca. 10
3
,10
5
,or10
7
CFU/ml of Salmonella.
The Salmonella inoculation levels were, therefore ca. 10
1
,10
3
,or
10
5
CFU per yolk. The procedure used to make mayonnaise was
adopted from a Web site (homecooking.about.com/od/saladrecipes/
r/blcon64.htm) with modifications. The ingredients for mayon-
naise including pure lemon juice (Realemon, 5%citric acid) or
white wine vinegar (STAR, 5%acetic acid), sugar, salt, mustard,
and vegetable oil were purchased from a local supermarket. As
preservatives, the lemon juice contained sodium metabisulfite,
sodium sulfite, and sodium benzoate, and the wine vinegar had
potassium metabisulfite. Different amounts (2, 3, or 4 teaspoons
[tsp], equivalent to 10, 15, or 20 ml; http://wiki.answers.com/Q/
How_many_ml_1_tsp) of the lemon juice or wine vinegar were
used to make mayonnaise, and the final concentrations of the acid
in the prepared mayonnaise (,400 g) were calculated as 0.13,
0.19, and 0.25%, respectively. Mayonnaise made with uninocu-
lated egg yolks served as controls. The pH of duplicate
mayonnaise samples was measured at room temperature using a
PICCOLO Plus pH meter and HI1295 Amplified Electrode (Hanna
instrument, Woonsocket, RI). Titratable acidity was determined by
the method of Xiong et al. (19). Water activity (a
w
) was measured
with a Pawkit water activity meter (Decagon Devices, Pullman,
WA). Finished mayonnaise samples were placed into sterile
sample bags (Fisher Scientific, Pittsburgh, PA) and stored at 4uC
for 15 days. The samples were drawn at 3-day intervals and
examined for total aerobic bacteria and Salmonella.
Microbiological sampling. Twenty-five grams of each
mayonnaise sample was mixed with 225 ml of sterile 0.1%
peptone water and homogenized at normal speed for 30 s in a
Stomacher 400 Laboratory Blender (Steward Company, London,
England). The pH of the samples was adjusted to 6.8 with sterile
1 N NaOH. The homogenates or appropriate serial dilutions
(0.1 ml) in 0.1%peptone water were plated in duplicate onto plate
count agar (BD) and bismuth sulfate agar (BD). A three-tube assay
was used to determine the most-probable numbers (MPN) of
Salmonella in samples having ,10 CFU of the pathogen per gram.
Specifically, three consecutive dilutions from 10
21
to 10
23
were
made in sterile 0.1%peptone water, and 1 ml of each dilution was
transferred to test tubes containing 9 ml of lactose broth (BD).
After incubation for 24 h at 37uC, 0.1 ml of the resulting cultures
was inoculated into 10 ml of Rappaport-Vassiliadis broth (BD),
and another 1 ml into 10 ml of tetrathionate broth (BD). The
Rappaport-Vassiliadis broth was incubated at 42uC for 24 h and the
tetrathionate broth at 37uC for 24 h. Ten-microliter volumes of the
cultures in Rappaport-Vassiliadis and tetrathionate broths were
inoculated on bismuth sulfate agar, and the inoculated plates were
incubated at 37uC for 24 h. Samples that tested negative for
Salmonella by standard plating (,10 CFU/ml) were also enriched
by the standard Salmonella enrichment procedures (16).
Survival of Salmonella in different acidulants. In a second
phase of the research, 9.5-ml volumes of the aforementioned two
Salmonella inocula at 10
7
CFU/ml were respectively mixed with
0.5 ml of each of the following acidic solutions: a commercial
lemon juice, 5%citric acid solution, 5%citric acid solution
supplemented with 0.01%sodium metabisulfite, 0.01%sodium
sulfite, and 0.05%sodium benzoate, as well as a wine vinegar, 5%
acetic acid solution, and 5%acetic acid solution supplemented
with 0.015%potassium metabisulfite. The final acid concentration
was 0.25%, which is equivalent to the acid concentration in
mayonnaise made with 4 tsp of wine vinegar or lemon juice in the
first phase of the project. The chemical preservatives were
purchased form Sigma-Aldrich (St. Louis, MO) and organic acids
from Fisher Scientific. The cultures were incubated at 4 and 25uC,
respectively, for 2 h. Following the incubation, the cultures (1 ml)
were serially diluted in Dey-Engley neutralizing broth (BD), and
the diluted cultures were plated in duplicate on plate count agar.
The acidulants were replaced with 0.85%NaCl in the control
samples.
Statistical analysis. Three independent trials were conducted
for the experiments in both phases of the study. Data were
subjected to multiple analysis of variance using Fisher’s least
significance design and the general linear model of the statistical
analysis software (2003, SAS Institute, Cary, NC). Overall
statistical analyses were also performed using Tukey’s honestly
significant difference test. Significant differences in the average
cell populations of Salmonella in various samples at different
sampling intervals were determined based on a 95%confidence
level.
RESULTS
Physical properties of the mayonnaise. Based on
ingredient analysis, the mayonnaise prepared in the present
study had 85%oil, 8%egg yolk, 2.5 to 5.0%lemon juice or
wine vinegar, and 3.75%water. The average pH, percentage
of acid, and a
w
of the mayonnaises made with different
amounts of wine vinegar or lemon juice are shown in
Table 1. The pH of mayonnaise prepared with wine vinegar
was relatively higher than that with lemon juice. The
titratable acidity correlated to the pH of the mayonnaise
samples. The a
w
of the mayonnaise was not affected by the
type of acid used in the study.
Total plate counts. The total plate counts varied from
2.00 to 3.49 log CFU/g in the mayonnaise over the 15 days
of storage at refrigeration temperature (data not shown).
Fate of Salmonella during mayonnaise-making
process and refrigeration storage. By use of the standard
enumeration assay, Salmonella was recovered from the
mayonnaise freshly made with egg yolks inoculated with
10
5
CFU of different serotypes of Salmonella and 2 or 3 tsp
of lemon juice or 2, 3, or 4 tsp of wine vinegar (Table 2) as
TABLE 1. pH, titratable acidity, and a
w
of mayonnaise made
with different amounts of lemon juice or wine vinegar
a
Type of
acidulant in
mayonnaise
Amt of acidulant in
mayonnaise (tsp
b
)
Mayonnaise
pH
Acid concn
(%, wt/vol) a
w
Lemon juice 2 4.21 0.18 0.92
3 3.94 0.23 0.92
4 3.80 0.29 0.93
Vinegar 2 4.29 0.18 0.92
3 4.08 0.24 0.92
4 3.96 0.30 0.92
a
Values for pH, acidity, and a
w
are means of two independent
measurements.
b
1 tsp ~5 ml.
466 ZHU ET AL. J. Food Prot., Vol. 75, No. 3
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TABLE 2. Numbers or MPNs of different serotypes of Salmonella in mayonnaise made with lemon juice or wine vinegar and stored for 15 days at 4
u
C
a
Amt of acid
(tsp)
Salmonella
inoculation level
(CFU/yolk)
No. or MPN of different serotypes of Salmonella organisms, after indicated storage period, in mayonnaise made with:
Lemon juice Wine vinegar
0 days 3 days 6 days 9 days 12 days 15 days 0 days 3 days 6 days 9 days 12 days 15 days
210
1
—
b
—————,0.48 a B———— —
10
3
,0.48 a B,0.48 a B,0.48 a A———,0.48 a B,0.48 a C,0.48 a C,0.48 a A,0.48 a A,0.48 a A
10
5
2.30 aA2.15 aA,0.48 b A,0.48 b A,0.48 b A—2.69 aA2.15 aA2.00 aA,0.48 b A,0.48 b A,0.48 b A
310
1
——————,0.48 a B———— —
10
3
,0.48 a B—————,0.48 a B,0.48 a C,0.48 a C,0.48 a A,0.48 a A—
10
5
2.39 aA,0.48 c B————2.24 aA2.00 aA1.35 bB,0.48 c A,0.48 c A,0.48 c A
410
1
——————————— —
10
3
——————,0.48 a B,0.48 a C,0.48 a C—— —
10
5
,0.48 c B—————2.15 aA1.35 bB,0.48 c C,0.48 c A,0.48 c A,0.48 c A
a
Values in italics are plate count results (log CFU per gram); values in roman type are MPNs (log MPN per gram; ,3 MPN/g). Means in the same row not followed by the same lowercase letters are
significantly different (P,0.05) with regard to storage time and type of acid used; means in the same column not followed by the same uppercase letters are significantly different (P,0.05) with
regard to the amount of acid and inoculation level of Salmonella used.
b
—, samples with negative enrichment results (,1 CFU/25 g or 0.04 CFU/g).
TABLE 3. Numbers and MPNs of Salmonella Enteritidis organisms in mayonnaise made with lemon juice or wine vinegar and stored for 15 days at 4
u
C
a
Amt of acid
(tsp)
Salmonella
inoculation level
(CFU/yolk)
No. or MPN of different phage types of Salmonella Enteritidis, after indicated storage times, in mayonnaise made with:
Lemon juice Wine vinegar
0 days 3 days 6 days 9 days 12 days 15 days 0 days 3 days 6 days 9 days 12 days 15 days
210
1
,0.48 a C—
b
————,0.48 a C,0.48 a C,0.48 a C—— —
10
3
0.56 a B,0.48 b C,0.48 b A— — — 0.56 a B,0.48 b C,0.48 b C,0.48 b B,0.48 b A,0.48 b A
10
5
2.82 aA2.15 bA,0.48 c A,0.48 c A,0.48 c A—2.92 aA2.80 aA2.39 abA2.00 bA,0.48 c A,0.48 c A
310
1
,0.48 a — — — — — ,0.48 a C,0.48 a C,0.48 a C—— —
10
3
0.56 a B,0.48 b C— — — — 0.56 a B,0.48 b C,0.48 b C,0.48 b B,0.48 b A—
10
5
2.87 aA1.15 cB,0.48 d A———2.87 aA2.75 aA2.15 bB,0.48 d B,0.48 d A,0.48 d A
410
1
——————————— —
10
3
,0.48 b C— — — — — 0.56 a B,0.48 b C,0.48 b C,0.48 b B——
10
5
2.81 aA—————2.69 aA1.00 bB,0.48 c C,0.48 c B,0.48 c A,0.48 c A
a
Values in italics are plate count results (log CFU per gram); values in roman type are MPNs (log MPN per gram; ,3 MPN/g). Means in the same row not followed by the same lowercase letters are
significantly different (P,0.05) with regard to storage time and type of acid used; means in the same column not followed by the same uppercase letters are significantly different (P,0.05) with
regard to the amount of acid and inoculation level of Salmonella used.
b
—, samples with negative enrichment results (,1 CFU/25 g or 0.04 CFU/g).
J. Food Prot., Vol. 75, No. 3 SALMONELLAE IN MAYONNAISE AND ACID SOLUTIONS 467
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well as from the mayonnaise freshly made with egg yolks
inoculated with 10
3
or 10
5
CFU of Salmonella Enteritidis
and 2 or 3 tsp of lemon juice or 2, 3, or 4 tsp of wine vinegar
(Table 3). Some samples that tested negative by direct
plating were found to contain ,0.48 MPN of Salmonella
per g (Tables 2 and 3).
The results of statistical analysis revealed that the
number of Salmonella Enteritidis organisms was signifi-
cantly higher than the number of organisms of the other
Salmonella mixture during mayonnaise preparation and
storage (Table 4). However, the differences between the
surviving populations of the two mixtures were small. Both
Salmonella mixtures survived longer in mayonnaise made
with wine vinegar than in mayonnaise made with lemon
juice during storage at 4uC (Table 4). The average
population of Salmonella in mayonnaise prepared with
wine vinegar was significantly higher than the Salmonella
population in mayonnaise made with lemon juice under the
same storage conditions (P,0.05). Cells of different
serotypes of Salmonella were not detected by the standard
enrichment assay approximately 6 days into storage at 4uC
in mayonnaise made with lemon juice except for those that
were made with egg yolk inoculated with 10
3
and 10
5
CFU
of Salmonella and 2 tsp of lemon juice (Table 2). The same
Salmonella mixture was detected in mayonnaise made with
wine vinegar by the enrichment procedures until the end of
the 15-day storage period with only a few exceptions.
Similar trends were noticed with Salmonella Enteritidis
(Table 3). In general, Salmonella viability decreased as the
amounts of vinegar and lemon juice in mayonnaise
increased (Table 4).
Survival of Salmonella in the acid solutions. The
fates of the salmonellae in the six different acid solutions are
shown in Table 5. Results of statistical analysis revealed
that there was no significant difference in the pathogen
populations of the two Salmonella mixtures at 25 and 4uC
(Table 6). The effectiveness levels of the six acidulants on
Salmonella Enteritidis inactivation were, in decreasing
order, as follows: 0.25%citric acid solution supplemented
with preservatives, commercial lemon juice, 0.25%citric
acid solution, 0.25%acetic acid solution supplemented with
preservatives, 0.25%acetic acid solution, and commercial
wine vinegar (Table 6). The effects of the six acidulants on
different serotypes of Salmonella were slightly different and
were, in decreasing order, as follows: 0.25%citric acid
solution supplemented with preservatives, 0.25%citric acid
solution, commercial lemon juice, 0.25%acetic acid
solution supplemented with preservatives, 0.25%acetic
acid solution, and commercial wine vinegar (Table 6). The
average Salmonella Enteritidis population (6.24 CFU/ml) at
4uC was significantly higher than that (4.97 CFU/ml) at
25uC. Similar results were obtained with the mixture of
different serotypes of Salmonella. The average populations
of both Salmonella mixtures were significantly different at
various sampling intervals except for the counts of
Salmonella at the 0- and 10-min sampling intervals. The
Salmonella counts in the six acid solutions decreased as the
length of incubation time increased.
DISCUSSION
Mayonnaise made with different amounts of lemon
juice by Xiong et al. (18) had a pH level of 2.90 to 3.77,
which corresponded to a titratable acidity of 0.68 and 0.26,
respectively. This pH range was slightly lower, and the
titratable acidity range was slightly higher, than what is
reported in the present study (Table 1). In a separate report,
the titratable acidity and pH of pasteurized (70uC, 15 min)
or unpasteurized mayonnaise with wine vinegar were 0.24%
and 3.63, respectively (1), which are similar to what is
reported in the present study (Table 1). Worrasinchai et al.
(17) stated that the a
w
of mayonnaise was mainly
TABLE 4. Average Salmonella populations in mayonnaise as affected by the type and amount of acid, inoculation level, and storage
conditions used in the study
a
Treatments
Avg populations (Log CFU/g)
Different serotypes
of Salmonella
Different phage types
of Salmonella Enteritidis
Type of acid in mayonnaise (n~108) Vinegar 0.52 a B0.65 a A
Lemon juice 0.20 b B0.33 b A
Amount of acid (tsp) in mayonnaise (n~72) 2 0.52 a A0.65 a A
3 0.36 b A0.51 b A
4 0.20 c A0.29 c A
Salmonella inoculation level (CFU/yolk) (n~72) 10
1
0.04 a B0.10 a A
10
3
0.22 b B0.27 b A
10
5
0.82 c B1.09 c A
Storage time (days) (n~36) 0 1.21 a A1.22 a A
3 0.59 b A0.71 b A
6 0.33 c A0.47 c A
9 0.15 d A0.26 d A
12 0.15 d A0.15 de A
15 0.10 d A0.10 e A
a
Means in the same column within a same treatment category not followed by the same lowercase letters are significantly different (P,
0.05); means in the same row not followed by the same uppercase letters are significantly different (P,0.05).
468 ZHU ET AL. J. Food Prot., Vol. 75, No. 3
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TABLE 5. Populations of two three-strain mixtures of Salmonella in six different acidulants with or without chemical preservatives at 4 and 25
u
C for 2 h
a
Temp
(uC) Acid used pH
Populations of Salmonella (Log CFU/ml)
Incubation time of different serotypes of Salmonella (min) Incubation time of different phage types of Salmonella Enteritidis (min)
0 10 20 30 60 120 0 10 20 30 60 120
25 Control (0.85%NaCl) 5.70 7.24 a A7.22 a A7.24 a A7.22 a A7.23 a A7.23 a A7.21 a A7.18 a A7.19 a A7.21 a A7.19 a A7.20 a A
Lemon juice 2.70 7.22 a A6.70 b D5.14 c CD 3.52 d D2.60 e FND f G7.19 a A7.03 a B5.58 c C3.54 d GND f HND f H
Citric acid 2.47 7.21 a A6.60 b D4.96 c D3.14 d DE 2.59 ef F2.31 f F7.11 a A6.75 bC4.62 c D3.16 d H2.95 d eE2.69 ef EF
Citric acid with preservatives
b
2.48 7.17 a A6.43 b E3.69 c E2.71 e END f GND f G7.12 a A6.47 b D3.31 d END f IND f HND f H
Vinegar 3.05 7.20 a A7.04 ab BC 7.01 ab AB 6.79 bc AB 6.31 d C5.42 e C7.18 a A7.16 a A6.96 bc AB 6.81 bc BC 6.57 cd B5.47 e C
Acetic acid 2.98 7.19 a A6.97 bc C6.65 cd B6.12 e C2.73 f EF 2.29 g F7.17 ab A7.03 ab B6.75 bcd B6.48 de CD 2.63 fg EF 1.64 h G
Acetic acid with preservative
c
2.98 7.24 a A7.01 a BC 5.36 bC3.34 d D2.39 e F2.21 ef F7.18 a A7.05 a AB 5.51 b C4.55 c F2.15 f GND g H
4 Control (0.85%NaCl) 5.70 7.23 a A7.22 a A7.22 a A7.24 a A7.24 a A7.23 a A7.20 a A7.20 a A7.18 a A7.19 A7.20 a A7.19 a A
Lemon juice 2.70 7.16 a A7.09 ab AB 6.98 bc AB 6.80 c AB 3.72 d D3.51 e D7.14 ab A7.08 ab AB 6.94 bc AB 6.93 bc AB 3.75 d C3.01 f E
Citric acid 2.47 7.13 a A7.03 ab BC 6.88 bc B6.75 c AB 3.12 e E2.63 f E7.13 a A7.02 ab B6.82 bc AB 6.44 d D3.31 e D2.82 f EF
Citric acid with preservatives
b
2.48 7.20 a A7.02 a BC 6.85 ab B6.51 b BC 2.79 d EF 2.36 e EF 7.12 a A7.03 a B6.80 ab AB 6.06 c E2.58 de F2.48 e F
Vinegar 3.05 7.19 a A7.19 a A7.18 a A7.13 a A6.86 b AB 6.15 c B7.16 a A7.12 a AB 7.10 a AB 7.03 ab AB 7.01 ab A6.34 c B
Acetic acid 2.98 7.18 a A7.12 a AB 7.12 a AB 7.02 a AB 6.49 b BC 3.68 c D7.16 a A7.09 a AB 7.02 a AB 7.01 a AB 6.42 b B3.86 c D
Acetic acid with preservative
c
2.98 7.18 a A7.10 a AB 7.07 a AB 6.92 b ABC 6.35 c C3.39 e D7.14 a A7.07 a AB 7.03 ab AB 7.00 b AB 6.27 c B3.64 d D
a
ND, not detected by the standard plate count assay (,10 CFU/ml). Means in the same row not followed by the same lowercase letters are significantly different (P,0.05) with regard to storage
time and type of Salmonella cultures used; means in the same column not followed by the same uppercase letters are significantly different (P,0.05) with regard to treatment temperature and the
amount and type of acidulants used.
b
The preservatives in the citric acid solutions included 0.01%sodium metabisulfite, 0.01%sodium sulfite, and 0.05%sodium benzoate.
c
The preservative in the acetic acid solutions was 0.015%potassium metabisulfite.
J. Food Prot., Vol. 75, No. 3 SALMONELLAE IN MAYONNAISE AND ACID SOLUTIONS 469
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determined by the chemical nature and concentration of
soluble solids in the aqueous phase of mayonnaise. The a
w
of mayonnaise containing 77 to 79%oil was reported as
0.93 (4), similar to what is reported in the present study
(Table 1). Abu-Salem and Abou-Arab (1) reported that
mayonnaise made from chicken eggs had low and stable
total aerobic counts of 2 to 3 log CFU/g during 5 weeks of
storage at refrigeration temperature. Similar results were
observed in the present study (data not shown).
Lemon juice and vinegar are usually used as flavoring
agents and acidulants in sauces and dressings to retard the
growth of microorganisms. Perales and Garcia (11) studied
the behavior of Salmonella in home-style mayonnaise
prepared with wine vinegar or lemon juice. The bactericidal
activity of vinegar was found to be greater than that of
lemon juice, due to the fact that at the same pH level (pH 3.6
to 5) more acetic acid than citric acid is in the undissociated
form (11). In a study conducted by Lock and Board (10), the
number of Salmonella Enteritidis PT4 organisms in
mayonnaise made with 2.64%acetic acid (6%, vol/vol)
quickly declined during storage at 20uC, while a similar
phenomenon was not observed in the mayonnaise made
with the same amount of lemon juice. In the present study,
however, the inhibitory effect of lemon juice was signifi-
cantly stronger (P,0.05) than that of vinegar (Table 4).
This difference in inhibition may be caused by the brand of
acidulants used, the pH and titratable acidity of the products,
and the chemical preservatives in the acidulants. The pH of
mayonnaise made with lemon juice in this study was
relatively lower than that made with vinegar (Table 1).
Similarly, the pH of 5%lemon juice or 0.25%citric acid
solution had lower pH than that of 5%white wine vinegar
or 0.25%acetic acid, respectively (Table 6). At extremely
acidic pH (pH ,3), proton leakage across the membrane
proceeds faster than the housekeeping pH homeostasis
systems of bacterial cells can remove them, leading to an
intracellular acidification and subsequent disruptions to
biochemical processes (3). Furthermore, the commercial
lemon juice and vinegar used in the present study contained
different chemical preservatives. The lemon juice had three
preservatives including sodium benzoate, sodium metabi-
sulfite, and sodium sulfite, while wine vinegar contained
only potassium metabisulfite. The sulfiting preservatives are
used primarily in foods to reduce or prevent spoilage and
browning (8), and free sulfur dioxide (SO
2
) liberated from
the preservatives is believed to be responsible for the
antimicrobial actions (2). Free SO
2
is more effective at a
pH of ,5.0, due to the ability of nonionized SO
2
to pass
across the cell membrane and disrupt the normal metabolic
activity of bacterial cells (2, 14). The undissociated
molecules of benzoate acid could change the permeability
of microbial cell membranes by possibly causing a
conformational change in the lipid moieties of these
structures (5). Thus, these preservatives could play a
distinct role against Salmonella in mayonnaise and
acidulant solutions.
The pathogen population of the mixture of Salmonella
Enteritidis was found to be significantly more persistent
than that of the mixture of different serotypes of Salmonella
during the mayonnaise-making process and storage (Ta-
ble 4). A previous study also found that Salmonella
Enteritidis was more tolerant to mild acid treatment than
other serotypes of Salmonella (9). A similar phenomenon
was, however, not observed in the solutions of different
acidulants (Table 6). It is not clear whether this is caused by
the relatively shorter treatment time used in the experiments.
TABLE 6. Average Salmonella populations in the six different acidulants as affected by the type and amount of acids and storage
conditions used in the study
a
Treatments
Avg pathogen populations (Log CFU/ml)
Different serotypes
of Salmonella
Different phage types of
Salmonella Enteritidis
Temp (uC) (n~84) 4 6.26 a A6.24 a A
25 5.11 b A4.97 b A
Acid (n~24) Control 7.23 a A7.20 a A
Lemon juice 5.04 e A4.84 e A
Citric acid 5.03 e A5.06 de A
Citric acid with preservatives
b
4.39 f A4.08 f A
Vinegar 6.79 b A6.82 b A
Acetic acid 5.88 c A5.86 c A
Acetic acid with preservative
c
5.46 d A5.38 d A
Time (min) (n~28) 0 7.20 a A7.15 a A
10 6.98 a A7.02 a A
20 6.38 b A6.34 b A
30 5.80 c A5.67 c A
60 4.31 d A4.15 d A
120 3.46 e A3.31 e A
a
Means in the same column within a same category not followed by the same lowercase letters are significantly different (P,0.05);
means in the same row not followed by the same uppercase letters are significantly different (P,0.05).
b
The preservatives in the citric acid were 0.01%sodium metabisulfite, 0.01%sodium sulfite, and 0.05%sodium benzoate.
c
The preservative in the acetic acid was 0.015%potassium metabisulfite.
470 ZHU ET AL. J. Food Prot., Vol. 75, No. 3
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Cells of Salmonella became undetectable in the lemon
juice and citric acid solutions supplemented with the
preservatives after 60 and 120 min of treatments at 25uC
(Table 5). Similar results have been reported by Gorden and
Small (7). Salmonella counts in acid solutions at 4uC were
significantly higher than those in samples at 25uC (Table 6).
The relatively higher cell membrane permeability at 25uC
may have permitted more-efficient cross-membrane migra-
tion of undissociated organic acid molecules.
In conclusion, the fate of Salmonella during the
preparation and storage of home-style mayonnaise and in
different acid solutions depends upon the type and amount
of inoculated Salmonella as well as the type of acids and
chemical preservatives in the acidulants. The anti-Salmo-
nella activity of the commercial lemon juice was signifi-
cantly stronger than that of the wine vinegar used in the
present study. Findings of this research will assist in control
of the safety of homemade mayonnaise.
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