1896

Journal of Food Protection, Vol. 70, No. 8, 2007, Pages 1896–1900

Copyright 䊚, International Association for Food Protection

Effect of Lemon Extract on Foodborne Microorganisms

A. CONTE,1 B. SPERANZA,1 M. SINIGAGLIA,2 AND M. A. DEL NOBILE2*

1Department of Food Science and 2Institute for Research and Biotechnological Applications to Safety and Quality of Typical Food Products,
University of Foggia, Via Napoli, 25-71100 Foggia, Italy

MS 06-350: Received 28 June 2006/Accepted 1 October 2006

ABSTRACT
A quantitative investigation was conducted on the antimicrobial effect of lemon extract against some food spoilage
microorganisms: yeasts, Bacillus species, and lactic acid bacteria. Growth kinetics and dose-response profiles were determined
from experimental data obtained with a suitable macrodilution methodology based on a turbidimetric technique. Growth and
no-growth status of microbial suspensions were expressed in terms of noninhibitory concentration (NIC) and MIC. Lemon
extract was effective in inhibiting the growth of the investigated vegetative cells and spores of microorganisms; effects were
similar for bacteria and yeasts. The NICs for all microorganisms were very small, at around 10 ppm. Based on MICs, among
the Bacillus species, the more resistant was Bacillus licheniformis. For yeasts, Saccharomyces cerevisiae was the least resistant,
and similar results were obtained for Pichia subpelliculosa. Candida lusitaniae had an MIC of more than 100 ppm. Both
Lactobacillus species were more resistant to lemon extract; concentrations necessary to provoke complete inhibition were
approximately 150 ppm.

Bacterial contamination is the most important factor
influencing food quality loss and shelf life reduction; there-
fore, prevention of microbial contamination is highly rele-
vant to food processors. Although synthetic additives have
been widely used in the food industry to inhibit microbial
spoilage, in recent years western societies appear to be ex-
periencing a trend toward ‘‘green’’ consumerism (30, 32),
favoring fewer synthetic food additives and products that
have a smaller impact on the environment. Consequently,
in the last decade the research on natural compounds has
notably increased (1, 22). Because of their flavorings and
antimicrobial properties, herbs and spices historically have
been used either to enhance the aroma of foods or to in-
crease the shelf life of packaged foods (8, 15). Natural com-
pounds from edible plants such as grains, oilseeds, spices,
fruit, and vegetables have been studied as food preserva-
tives and possible replacements for chemical additives be-
cause of the presence of volatile oils and their safety for
human consumption (2, 5, 28, 29). The antifungal proper-
ties of oregano oil against Candida albicans recently have
been assessed (23). Oils of oregano, thyme, bay, and clove
are very effective against Escherichia coli (11, 31). The
antimicrobial activity of these essential oils is due to the
presence of high concentrations of phenol derivatives such
as thymol, eugenol, and carvacrol, and there is evidence
that minor components play a significant role in the com-
plete antimicrobial effect (18, 20, 24, 26).
Citrus fruits are an important source of bioactive com-
pounds, in particular flavonoids and vitamin C. The main
flavonoids found in citrus species are hesperidine, narirutin,
naringin, and eriocitrin (25, 27). In addition to antimicrobial
activity, these naturally occurring flavonoids contain anti-
* Author for correspondence. Tel: (⫹39) 881 589 242; Fax: (⫹39) 881
740 211; E-mail: ma.delnobile@unifg.it.
inflammatory and antitumor compounds that can be used
as effective therapeutic agents with low toxicity (7, 13, 19,
21). Among the citrus fruit extracts, one of the most prom-
ising is lemon extract. Belletti et al. (3) assessed the anti-
microbial activity of lemon essence against a Saccharo-
myces cerevisiae strain and verified the role of vapor pres-
sure on the bioactivity of this essence. The results obtained
indicated that the most effective compounds were charac-
terized by terpenes, such as citral and p-cymene. Fernan-
dez-Lopez et al. (10) reported that the presence of func-
tional dietary fibers and antioxidants in citrus by-products
allow their application in processing of cooked and dry-
cured sausage to obtain healthy products.
The aim of this study was to investigate the efficacy
of purified lemon extract as a natural preservative against
some yeasts and lactic acid bacteria, typical acid juice spoil-
age organisms (14). Because mild heat treatments are used
to extend the shelf life of foods, the effect of lemon extract
on some spore-forming bacteria also was evaluated. For this
study, we chose Bacillus cereus, a well-known and ubiq-
uitous foodborne pathogen, and two common spoilage or-
ganisms, Bacillus licheniformis and Bacillus subtilis. The
inhibitory activity of lemon extract against B. cereus spores
also was investigated.
MATERIALS AND METHODS
Bacterial strains and media. The microbial strains used in
this work and their source of isolation are listed in Table 1. Most
of the selected microorganisms (culture collection, Laboratory of
Applied Microbiology, Department of Food Science, University
of Foggia) were isolated from fresh and spoiled foods. B. cereus,
B. subtilis, and Saccharomyces cerevisiae were purchased from a
public collection. Lactobacillus curvatus and Lactobacillus plan-
tarum were maintained at ⫺20⬚C in deMan Rogosa Sharpe (MRS)
broth (Oxoid, Milan, Italy) with the addition of 30% glycerol.
J. Food Prot., Vol. 70, No. 8
TABLE 1. Microbial strains, sources, and optimal growth con-
ditions
Media and growth
Strain Source conditions
Lactobacillus cur- “Caciocavallo Cala- MRS broth, 30⬚C,
vatus brese” cheese 24 h
L. plantarum Wine MRS broth, 30⬚C,
24 h
Pichia subpellicu- Minimally processed Sabouraud dextrose
losa apples agar, 25⬚C, 48 h
Candida lusitaniae Minimally processed Sabouraud dextrose
apples agar, 25⬚C, 48 h
Saccharomyes cere- DAL CIN S.p.A. Saboraud dextrose
visiae (Milan, Italy) agar, 25⬚C, 48 h
Bacillus cereus ATCC 10876 Plate count agar,
30⬚C, 24 h
B. subtilis ATCC 6633 Plate count agar,
30⬚C, 24 h
B. licheniformis Minimally processed Plate count agar,
apples 30⬚C, 24 h
Pichia subpelliculosa, Candida lusitaniae, S. cerevisiae, B. cere-
us, B. subtilis, and B. licheniformis were grown on slants of ap-
propriate media (Table 1) and stored at 4⬚C as stock cultures.
Preparation of spore concentrates. Cells of B. cereus were
grown at 30⬚C for 24 h in plate count broth (5 g/liter tryptone, 1
g/liter glucose, and 2.5 g/liter yeast extract), spread onto plate
count agar (PCA) in petri plates, and incubated at 30⬚C for 7 days
until at least 80% of cells sporulated as determined by micro-
scopic examination. Spores were harvested by depositing 1 to 2
ml of sterile water onto the surface of PCA culture plates and
gently rubbing the surface with a sterile swab to dislodged the
spores. Pooled suspensions from 15 plates were centrifuged at
3,000 rpm for 20 min at 4⬚C, and the pellet was resuspended in
sterile water and centrifugation again at the same speed and tem-
perature for 10 min. This step was repeated four times. The final
pellet was resuspended in 10 ml of sterile distilled water and heat
shocked at 80⬚C for 10 min. This spore suspension (approximately
107 to 108 spores per ml as enumerated on PCA) was stored at
4⬚C until use.
Growth conditions and culture methods. Lemon extract
was provided by Spencer Food Industrial (Amsterdam, The Neth-
erlands). Stock solutions of the active compound were prepared
in distilled water and sterilized by filtering through membranes
(0.20-␮m pore size; Minisart, Sartorius, Goettingen, Germany).
An aliquot of selected stock solutions was added to microbial
suspensions to obtain lemon extract concentrations ranging from
5 to 1,000 ppm (vol/vol).
For the growth experiments, MRS broth, Sabouraud broth
(Biolife, Milan, Italy), and plate count broth containing the active
solution with an appropriate concentration of lemon extract were
inoculated (1:10) with standardized cultures to yield one active
test for each microorganism under study. These standardized cul-
tures were obtained by allowing each strain to grow in an appro-
priate liquid medium (Table 1) at its optimal temperature and by
diluting these cultures with a physiological saline solution (0.9%
NaCl) to obtain approximately 105 CFU/ml for bacteria and 106
CFU/ml for yeasts. To ensure reproducibility in the inoculated
preparation, cell counts were standardized through the direct plate
count technique (12). To determine the effect of lemon extract on
spores, test tubes containing 10 ml of sterile plate count broth and
ANTIMICROBIAL ACTIVITY OF LEMON EXTRACT 1897
the appropriate concentration of lemon extract were inoculated
with B. cereus spores at a final concentration of 105 spores per
ml.
Acquisition of growth data. To acquire growth data in the
presence of lemon extract, turbidimetric experiments were con-
ducted. Microbial growth was quantified by reading the absor-
bance changes at 420 nm with a spectrophotometer (UV 1601,
Shimadzu, Duisburg, Germany) at regular intervals. Inoculated
media without lemon extract were used as positive controls (PC)
to evaluate the microbial growth under optimal conditions. The
media containing only active solution were used as negative con-
trols (NC) to evaluate the absorbance changes due to the different
lemon extract concentrations and to take into account any possible
changes in liquid media during growth experiments. Each growth
experiment was conducted twice.
Modeling of growth data. To assess the antimicrobial activ-
ity (biostatic or biocidal) of lemon extract, growth kinetics and
dose-response curves were obtained. The Gompertz equation as
modified by Zwietering et al. (33) was used to obtain the growth
kinetics for each microorganism:
冢 冦[
Ā(t) ⫽ K ⫹ A·exp ⫺exp (␮ max·2.7182)·
␭⫺t
A
⫹1
] 冧冣 (1)
where Ā(t) is the normalized absorbance at time t obtained by
dividing the absorbance at time t by the initial absorbance, K is
the initial value of Ā(t) and, as expected, is always about 1, A is
the greatest increase in the normalized absorbance, ␮max is the
increase rate (expressed as 1/s), and ␭ is the delay time (in sec-
onds). Equation 1 was fitted to the experimental absorbance data
for each selected microorganism, and the value of the integral
curve was calculated for each fit. All values related to the area of
each growth curve were used to calculate the growth index (GIt)
(16) as a time-dependent parameter ranging from 0 to 1, where 0
indicates no cell growth during experiments and 1 indicates an
antimicrobial effect of the active compound. A GIt value greater
than 1 indicates that the active compound stimulates microbial
growth. The GI was defined as
AAT ⫺ ANC
GIt ⫽ (2)
APC ⫺ ANC
where t is the contact time, AAT is the area under the growth curves
corresponding to the active test (i.e., the inoculated medium con-
taining the active solution), and APC and ANC represent the area
under growth curves corresponding to the positive and negative
controls, respectively. The subtraction of absorbance relative to
the negative controls eliminates the interference of both the active
compound and solvents from the active test behavior. By using
all inhibitory data, including those relative to the no-growth status,
it was possible to obtain a specific inhibition profile for each test
microorganism. Growth and no-growth status of microbial sus-
pensions containing the active extract were expressed as two crit-
ical values, the noninhibitory concentration (NIC), which is the
threshold value under which any effect on microbial growth can
be observed, and the MIC, which is the threshold concentration
above which the active compound exerts complete inhibition.
When a turbidimetric technique is used to obtain inhibitory data,
MICs indicate that there are no reproducing cells able to cause
turbidity changes of microbial suspensions. To describe the dose-
response behavior of each microorganism against the inhibitor
compound, a simple exponential decay model for estimating the
Lambert parameters, P1 and P2, was proposed (17, 18):
1898 CONTE ET AL. J. Food Prot., Vol. 70, No. 8

FIGURE 2. Inhibitory profile of Bacillus subtilis vegetative cells

FIGURE 1. Inhibitory profile of Bacillus cereus vegetative cells
as a function of lemon extract concentrations. The curve is the fit
of equation 6 to the GI data.
GIt* ⫽ exp ⫺
[冢 冣]
[C]
P1
P2
(3)
where GIt* is GIt calculated at a given level of exposure time (t*),
[C] is the inhibitor concentration, and P1 determines the position
of the inflexion point of the inhibition profile and is defined as
the log of the inhibitor concentration for which GIt* is numerically
equal to 1/e. Low P1 values indicate high microbial sensitivity to
the active compound. P2 represents a shape parameter describing
the abruptness in the GI change of the dose-response curve. Once
the Lambert parameters are estimated, it is possible to evaluate
NIC and MIC with the following equations:
as a function of lemon extract concentrations. The curve is the fit
of equation 6 to the GI data.
ment of the dose-response curves. To evaluate the potential
biostatic or biocidal activity of the lemon extract, growth
kinetics were obtained for each of these microorganisms.
To estimate microbial growth in the presence of the active
compound, absorbance measurements have been used as a
rapid and inexpensive technique (4, 17). The obtained ex-
perimental data were modeled by fitting the Gompertz func-
tion as modified by Zwietering et al. (33), and the integral
value was calculated for each fit. The obtained area values
for each growth curve were used to calculate the GI (16).
However, because of the insufficient precision of Gompertz
parameters estimated from absorbance data (6), the results

冢P2冣 and
1 for the antimicrobial activity of the natural preservative
NIC ⫽ P1·exp (4)
were converted to NIC and MIC. The t* was set to 283 h,
the time necessary to reach the stationary phase for all test-
MIC ⫽ P1·exp 冢
P2 冣
1⫺e
(5) ed microorganisms. Equation 6 was fitted to all GI283 data
as a function of the log of the lemon extract concentrations,
The main disadvantage in using the above approach in a suscep- and the inhibition profile was obtained for each microor-
tibility study is the impossibility of estimating the confidence in- ganism. The inhibition profiles of all tested microorganisms
terval of both NIC and MIC because they do not compare ex- are shown in Figures 1 to 4. To describe the susceptibility
plicitly in equation 3. In this work, an advance in the Lambert to lemon extract, each dose-response curve was subdivided
and Pearson model (17) was used by rearranging equation 3 as
into three primary regions defined by NICs and MICs. The
follows:
first region, lying below the NIC, defined a range of lemon
⎡ ⎤ extract concentrations that did not significantly affect mi-
⎞⫺e/{ln[ln(NIC) /ln(MIC)]}

⎢ ⎜⎢ ⎥
⎛ [L]

⎥⎟
(6) crobial growth. Between the MIC and the NIC, there was
⎡ ln(MIC) ⎤
a sigmoid trend in the inhibitory profile, whereas the third
GIt* ⫽ exp ⎢⫺ ⎧ ⎫ ⎟ ⎥

⎢ ⎜⎢
⎜
⎢
exp ⎨
⎪⫺
[ ]
⎪ ln ln(NIC) ⎪ ⎥
ln(MIC) ⎬
⎪ ⎥⎟ ⎥
region (above the MIC) defined the no-growth status. Data
reported in each figure suggest that the susceptibility of the
⎣ ⎝ ⎣ ⎩ e ⎭⎦ ⎠ ⎦

where [L] is the lemon extract concentration. Specified contact

time (t*) is supposed to be long enough to reach the stationary
phase of the growth cycle in the presence of the lemon solutions.
Therefore, by fitting equation 6 to the GIt* data, it was possible
to directly estimate the NICs and MICs with their confidence in-
tervals.

RESULTS AND DISCUSSION

The antimicrobial effectiveness of lemon extract on
cells of C. lusitaniae, P. subpelliculosa, S. cerevisiae, L.
plantarum, L. curvatus, B. subtilis, B. licheniformis, and
both the vegetative cells and spores of B. cereus was eval- FIGURE 3. Inhibitory profile of L. plantarum as a function of
uated. The wide range of lemon extract concentrations, lemon extract concentrations. The curve is the fit of equation 6 to
from 5 to 1,000 ppm, used in this work allowed develop- the GI data.
J. Food Prot., Vol. 70, No. 8 ANTIMICROBIAL ACTIVITY OF LEMON EXTRACT 1899

TABLE 2. MICs and NICs and their difference obtained by fitting

equation 6 to the GI dataa
MIC ⫺ NIC
Microorganism NIC (ppm) MIC (ppm) (ppm)

Bacillus cereus 8.4 ⫾ 1.6 53.9 ⫾ 19.8 45.5

vegetative cells
B. cereus spores 6.8 ⫾ 8.3e⫺3 24.5 ⫾ 6.6e⫺2 17.7
B. subtilis 13.1 ⫾ 1.6 76.4 ⫾ 33.4 63.3
B. licheniformis 10.0 ⫾ 2.4 100.8 ⫾ 67.8 90.8
Lactobacillus 13.9 ⫾ 2.7 150 ⫾ 55.2 136.1
curvatus
L. plantarum 9.9 ⫾ 2.2 169.9 ⫾ 69.1 160
Saccharomyces 20.4 ⫾ 0.3 27.7 ⫾ 0.3 7.3
FIGURE 4. Inhibitory profile of Candida lusitaniae as a function cerevisiae
of lemon extract concentrations. The curve is the fit of equation Candida lusitan- 14.1 ⫾ 1.8 133.2 ⫾ 36.9 119.1
6 to the GI data. iae
Pichia subpelli- 12.7 ⫾ 1.8 45.6 ⫾ 10.7 32.9
culosa
microorganisms to lemon extract was not linearly related to a Values are means ⫾ the standard deviation.
the concentration of the active compound. This nonlinear
dose-dependent increase in antimicrobial activity was evi-
dent in the second region of the inhibition profile. Measur-
inhibition (the MIC) was the lowest. For L. plantarum,
able bacterial growth was not detectable within the third
which was characterized by the highest resistance to the
region of the inhibition profile.
lemon extract, the value below which no effect on microbial
Microbial sensitivity to the active compound was re-
growth was observed was very low. For this reason, a new
lated to the NICs, whereas the microbial resistance was
parameter, the difference between the MIC and the NIC
related to the MICs. The values of the fitted parameters
(MIC ⫺ NIC) was used to quantify the differences in mi-
with their confidence intervals are reported in Table 2.
crobial susceptibility to lemon extract. This parameter bet-
There were differences in both microbial sensitivity and
ter described microbial behavior in the presence of lemon
resistance to lemon extract among tested microorganisms.
extract. As can be inferred from the data in Table 2, L.
The NICs ranged from 9 to 20 ppm, and the MICs ranged
plantarum and L. curvatus were the most resistant micro-
from 27 ppm for one of the three investigated yeasts to 170
organisms, whereas S. cerevisiae was the most susceptible.
ppm for one of the two tested Lactobacillus species. For
The other two yeasts, in particular C. lusitaniae, appeared
the B. cereus spores, both the NICs and MICs were lower
to be less sensitive to the lemon extract. For the Bacillus
than those for all other vegetative cells. The antimicrobial
species, susceptibility to the lemon extract was in the fol-
effects of other essential oils, such as those of oregano and
lowing order: B. licheniformis ⬎ B. subtilis ⬎ B. cereus.
thyme, on bacterial spores has been assessed (4, 26). Lower
The B. cereus spores appeared to be very sensitive to the
concentrations of essential oils were required to inhibit the
lemon extract action, with one of the lowest difference val-
germination of the spores compared with those necessary
ues.
to suppress cell multiplication, probably because of the hy-
The results of this study suggest that lemon extract
drophobic nature of the spores. In the present study, the
could be a valuable natural antimicrobial compound for the
two Lactobacillus species were more resistant to the action
food industry because it exerts its activity even at very low
of lemon extract than was the spore-forming species. These
concentrations. The high effectiveness of this compound
results are in agreement with those obtained by Falcone et
could allow a decrease in the amount of additives used in
al. (9), who investigated the antimicrobial properties of the
industrial applications, with effects on both economical and
thymol at the same concentrations against the same micro-
safety aspects of food preservation.
organisms. All tested bacteria had NICs of less than 100
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