Journal of Food Processing and Preservation ISSN 1745-4549

ANTIMICROBIAL ACTIVITY OF LACTIC ACID AGAINST

PATHOGEN AND SPOILAGE MICROORGANISMS
SLOBODANKA STANOJEVIĆ-NIKOLIĆ1, GORDANA DIMIĆ1, LJILJANA MOJOVIĆ2, JELENA PEJIN1,
ALEKSANDRA DJUKIĆ-VUKOVIĆ2 and SUNČICA KOCIĆ-TANACKOV1,3
1
Faculty of Technology, University of Novi Sad, Bulevar cara Lazara 1, Novi Sad 21 000, Serbia
2
Faculty of Technology and Metallurgy, University of Belgrade, Belgrade, Serbia

3
Corresponding author. ABSTRACT
TEL: +381 214853699;
FAX: +381 21450413; Preliminary examination of the antimicrobial activity of lactic acid against nine
EMAIL: suncicat@uns.ac.rs bacteria (Escherichia coli, Proteus mirabilis, Salmonella enteritidis, Pseudomonas
aeruginosa, Staphylococcus aureus, Enterococcus faecalis, Listeria monocytogenes,
Received for Publication July 21, 2015
Bacillus cereus and Bacillus megaterium) and three yeasts (Rhodotorula sp., Saccha-
Accepted for Publication October 20, 2015
romyces cerevisiae and Candida albicans) was performed using disc diffusion and
doi:10.1111/jfpp.12679 broth microdilution method. At a concentration of 321 mg/mL, inhibition zones
ranged from 24.0 mm (Es. coli) to 38.3 mm (En. faecalis) for the tested bacteria.
The inhibition zones of the yeasts ranged from 11.3 mm (Sac. cerevisiae) to
14.0 mm (Rhodotorula sp.).
Lactic acid minimal inhibitory concentration for bacteria was ≥1.25 mg/mL, while
minimal biocide concentration was ≥2.50 mg/mL. Minimal inhibitory concentra-
tion for yeasts was ≥12.50 mg/mL, while minimal fungicidal concentration was
≥25.00 mg/mL. The obtained results showed that lactic acid could be used as an
efficient natural antimicrobial agent improving the safety of all-natural foods.

PRACTICAL APPLICATIONS
Consumer perception that synthetic food additives may be associated with poten-
tial toxicological problems has recently generated interest for the use of naturally
derived compounds in the food industry. The use of lactic acid is considered as a
good alternative and may be more acceptable to consumers than synthetic food
additives because of its natural origin, potential antimicrobial activity, as well as
preservative, antioxidant, flavoring and acidifying properties as well as low cost.
For ensuring food safety, combination of lactic acid with other natural antimicro-
bial agents or other preservation methods should be considered. Therefore, there
is a potential for lactic acid usage in the development of eco-friendly technology
which ensures food safety.

agents into food products has been widely applied in the

INTRODUCTION
Pathogenic and food spoilage microorganisms have been
considered as the primary causes of foodborne diseases and
food quality deterioration, which results in many deaths
and significant economic loss each year (Over et al. 2009).
Consequently, there is a considerable interest in ways to
stop this upward trend and reduce the incidence of food
poisoning.
In order to ensure the food safety and to extend the shelf
life of food products, addition of chemical preservative
food industry (Brul and Coote 1999). The need for natural
alternative is due to consumers’ preference for fewer chemi-
cals and more natural foods. Lactic acid has been consid-
ered as a potential alternative. The U.S. Food and Drug
Administration classified lactic acid as “generally recognized
as safe” (GRAS). Lactic acid is a natural substance found in
various fruits and fermented products and exhibits antimi-
crobial activity against foodborne pathogens (Beuchat and
Colden 1989). Moreover, numerous applications for decon-
tamination of meat, fruits and vegetables by lactic acid have

Journal of Food Processing and Preservation •• (2015) ••–•• © 2015 Wiley Periodicals, Inc. 1
ANTIMICROBIAL ACTIVITY OF LACTIC ACID
been previously described (Dickson and Anderson 1992;
Greer and Dilts 1995; Calicioglu et al. 2002; Gill and Badoni
2004; Vasantha Rupasinghe et al. 2006; Alvarado-Casillas
et al. 2007; Park et al. 2011; In et al. 2013). Besides its anti-
microbial activity, lactic acid acts as a flavor enhancer and
an antioxidant, preventing lipid oxidation by decreasing the
pro-oxidative effect of NaCl (Paelinck and Szczepaniak
2005).
Lactic acid is lethal to microorganisms via undissociated
molecules that flow through the cell membranes and ionize
inside. The acidic pH inside the cell causes deformation and
damage to enzymatic activities, proteins and DNA struc-
ture, thereby damaging the extracellular membrane
(Mani-Lopez et al. 2011). In another mechanism, changes in
the permeability of the cell membrane hinder substrate
transport, while changes in the pH inside the cell suppress
NADH oxidation; this affects the electron transport system
and leads to the death of the microorganism (Kong et al.
2001).
Many studies showed the antimicrobial activity of lactic
acid bacteria supernatant in which the predominant antimi-
crobial agent is lactic acid (Magnusson and Schnürer 2001;
Aslim et al. 2005; Nardi et al. 2005; Zdolec et al. 2007; Rouse
et al. 2008; Polak-Berecka et al. 2009; Wongsuttichote and
Nitisinprasert 2009; Voulgari et al. 2010; Al Askari et al.
2012; Köhler et al. 2012; Sungsri et al. 2012). However, there
are only a few studies reporting the antimicrobial activities
of lactic acid against Enterococcus faecalis, Proteus mirabilis,
Bacillus megaterium, Candida albicans, Rhodotorula spp. and
other toxic and spoilage microorganisms (Lind et al. 2005;
Broberg et al. 2007; Pundir and Jain 2011).
Therefore, the aim of this study was to determine the in
vitro antimicrobial activity of lactic acid against growth of
nine bacteria and three yeasts that are frequently encoun-
tered in food.
MATERIALS AND METHODS
Preparation of Lactic Acid Solutions
L-(+)-lactic acid (Sigma-Aldrich Co. LLC, Taufkirchen,
Germany) solutions were prepared with sterile distilled
water. Using acetate membrane filters (Sartorius SA,
Göttingen, Germany), pore size 0.2 μm, the solutions were
sterilized and stored at 4C up to 30 days.
Test Microorganisms, Culture Conditions and
Preparation of Suspension
Bacteria used in this study were Escherichia coli (ATCC
10536), Proteus mirabilis (ATCC 12453), Salmonella enteriti-
dis (ATCC 13076), Pseudomonas aeruginosa (ATCC 10145),
2 Journal of Food Processing and Preservation •• (2015) ••–•• © 2015 Wiley Periodicals, Inc.
S. STANOJEVIĆ-NIKOLIĆ ET AL.
Staphylococcus aureus (ATCC 1163), Enterococcus faecalis
(ATCC 29212), Listeria monocytogenes (ATCC 19115), Bacil-
lus cereus (isolated from cheese), and B. megaterium (iso-
lated from cheese) as well as the yeasts Rhodotorula sp.
(isolated from air), Saccharomyces cerevisiae (ATCC 2601)
and Candida albicans (ATCC 10231). Cultures were main-
tained on nutrient agar (NA) (bacteria) (Merck, Darmstadt,
Germany) and on Sabouraud Dextrose agar (SDA) (yeasts)
(Merck) at 4C as a part of the collection of the Laboratory
for Food Microbiology at the Faculty of Technology, Univer-
sity of Novi Sad, Serbia.
Bacterial strains were cultured on an NA for 24 h at 37C
(or 30C for B. cereus and B. megaterium), while yeasts were
grown on SDA for 48 h at 25C. The suspensions were pre-
pared in a sterile saline (0.85%; 10 mL) and adjusted (with
sterile saline) to give a final concentration of 106 cfu/mL
using McFarland Standard (bioMérieux SA, Marcy I’Etoile,
France) and McFarland densitometer (Biosan SIA, Riga,
Latvia). Microorganism suspension concentration was also
determined using agar plate method (using NA medium for
bacteria and SDA medium for yeasts).
Disc Diffusion Assay
Preliminary examination of the lactic acid antimicrobial
activity was performed by a disc diffusion method (Leboffe
and Pierce 2005). A sterile swab was immersed in the
culture suspension (106 cfu/mL) and used to inoculate the
entire surface of a Müller–Hinton agar (MHA) (Merck) (for
bacteria) and SDA (Merck) (for yeasts). Inoculated plates
were left for 15 min at a room temperature to allow good
absorption. Three or four 6-mm sterile filter paper discs
(Himedia, Mumbai, India) were placed equidistant apart on
the inoculated agar surface of the plates and certain
amounts of lactic acid (15.0, 20.0 and 25.0 μL of lactic acid
solution for concentrations of 102, 209 and 321 mg/mL, and
1.5, 2.5, 5.0 and 10.0 μL of pure lactic acid) were immedi-
ately added. Sterile filter paper disc placed on the center of
the inoculated agar surface of the plates in which sterile
water was added was used as a control.
After 24 h of incubation at 37C (or 30C) for bacteria and
48 h at 25C for yeasts, the inhibition zones were measured
in millimeters.
Broth Microdilution Assay
Minimal inhibitory concentration (MIC), minimal bacteri-
cidal concentration (MBC) and minimal fungicidal concen-
tration (MFC) were determined according to the modified
method of Marcello et al. (2003) and Barbour et al. (2004).
Müller–Hinton broth (MHB) (Merck) for bacteria and
Sabouraud Dextrose broth (Merck) for yeasts (100 μL) were
S. STANOJEVIĆ-NIKOLIĆ ET AL. ANTIMICROBIAL ACTIVITY OF LACTIC ACID

pipetted into each well of a sterile 96-well microtiter plate

(Greiner Bio-One GmbH, Kremsmünster, Austria). After
that, in the first column of wells in each microtiter plate
100 μL of the initial concentration of lactic acid (5, 10, 20,
50, 75, 102 mg/mL for bacteria and 20, 50, 75, 102, 209,
321 mg/mL for yeasts) was transferred. After mixing by
pipetting, 100 μL of the mixture was transferred to the next
column of wells in a process of 1 : 1 serial dilution until the
column number 12 (ranging from 0.15 to 6.37 mg/mL for
bacteria and from 0.62 to 20.06 mg/mL for yeasts). Each
well was then inoculated with 10 μL of the tested microor-
ganism suspension (106 cfu/mL).
Three controls were used: first control contained appro-
priate broth and lactic acid, second control contained broth
and suspension of tested microorganism, while third
control contained only broth. Incubation of microtiter
plates was performed at the appropriate temperature for
24 h (for bacteria) or 48 h (for yeasts). After incubation,
wells that contained clear broth suspension were used for
inoculation of predried MHA and SDA plates.
The MIC was determined as the lowest lactic acid con-
centration that inhibited the growth of the well (clear broth
suspension), but still showed slightly visible growth on the
plate. MBC and MFC were determined at the lowest lactic
acid concentration that inhibited the growth of the well and
showed no visible growth on the plate.
Statistical Analysis
The tests were performed in three replications. MS Excel
(Microsoft Corporation, Redmond, WA) was used to calcu-
late the means and standard deviations.
RESULTS AND DISCUSSION
Lactic Acid Antimicrobial Activity by Disc
Diffusion Method
The results of the lactic acid antimicrobial activity obtained
by the disc diffusion method are given in Table 1. The
results showed that lactic acid has a potential to inhibit the
growth of all tested bacteria and yeasts. Test microorgan-
isms showed different susceptibility.
The results of the antimicrobial activity of the different
lactic acid solution concentrations (102, 209 and 321 mg/
mL) on the growth of the test microorganisms obtained by
disc diffusion method are given in Table 2. The inhibition
area increased with the increase in lactic acid concentration.
In general, lactic acid showed the strongest inhibitory
activity against bacteria. Lactic acid was more effective
against gram-positive bacteria than gram-negative bacteria.
Among gram-positive bacteria, the most susceptible was En.
faecalis, while Ps. aeruginosa was the most susceptible gram-
negative bacteria. Similar susceptibility was obtained for
Es. coli, Sal. enteritidis and Sta. aureus.
Some studies showed the antimicrobial effect of lactic
acid against Es. coli, L. monocytogenes, Salmonella Gaminara
(Eswaranandam et al. 2004), Sal. enteritidis (Anang et al.
2007), Sta. aureus (Ho et al. 2010; Sudershan et al. 2011),
also against resistant clinical isolates of Ps. aeruginosa
(Jamalifar et al. 2011) and spore-forming bacteria (Jamilal
et al. 2008; Tharmaray and Shah 2009; Ho et al. 2010).
Moreover, Ibrahim et al. (2008) found that lactic acid even
at low concentration (2 mg/mL) inhibits the growth of Sal-
monella spp. and Es. coli O157:H7. Similarly, Yesillik et al.

TABLE 1. PURE LACTIC ACID ANTIMICROBIAL

Pure lactic acid
ACTIVITY ON TESTED MICROORGANISMS
OBTAINED BY DISC DIFFUSION METHOD Amount (μL/disc) 1.5 2.5 5.0 10.0
Gram-negative bacteria
Escherichia coli 14 ± 1.11* 18 ± 0.00 24 ± 0.44 32 ± 0.66
Salmonella enteritidis 10 ± 0.53 16 ± 0.98 16 ± 0.98 26 ± 0.00
Proteus mirabilis 8 ± 0.89 11 ± 1.70 11 ± 1.70 20 ± 0.00
Pseudomonas aeruginosa 17 ± 0.55 35 ± 0.76 40 ± 0.87 50 ± 0.75
Gram-positive bacteria
Staphylococcus aureus 19 ± 0.00 20 ± 0.30 24 ± 0.00 30 ± 0.45
Enterococcus faecalis 18 ± 0.34 33 ± 0.88 33 ± 1.32 38 ± 0.32
Listeria monocytogenes 32 ± 0.12 37 ± 1.21 38 ± 0.45 40 ± 0.54
Bacillus cereus 20 ± 0/44 21 ± 0.00 33 ± 0.22 34 ± 0.12
Bacillus megaterium – 10 ± 0.00 16 ± 0.40 33 ± 0.22
Yeasts
Rhodotorula sp. – – 7 ± 0.12 12 ± 0.00
Candida albicans – – 7 ± 0.23 9 ± 0.34
Saccharomyces cerevisiae ci ci ci ci

* Standard deviation.
ci, complete inhibition; –, not sensitive.

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 ANTIMICROBIAL ACTIVITY OF LACTIC ACID S. STANOJEVIĆ-NIKOLIĆ ET AL.

(2011) showed that lactic acid at a concentration of

24.0 ± 0.00
27.6 ± 1.15
28.3 ± 0.57

25.0 ± 1.00
28.3 ± 0.57

38.3 ± 1.53
36.0 ± 0.00

14.0 ± 0.00
12.6 ± 0.57
11.3 ± 0.57
29.6 ± 1.15
30.0 ± 0.00
9 mg/mL inhibits the growth of Salmonella typhimurium

25.0
(inhibition zone of 22.6 mm) and Ps. aeruginosa (inhibition
zone of 22.5 mm), while against Es. coli and Sta. aureus
lactic acid was ineffective.
23.6 ± 1.15
23.0 ± 0.00
22.6 ± 1.15

24.0 ± 0.00

13.0 ± 1.00
10.3 ± 0.57
10.6 ± 1.15
24.3 ± 0.57

36.0 ± 1.00
31.0 ± 1.00
26.6 ± 0.57
28.3 ± 0.57
Several authors suggested that lactic acid is more efficient
antibacterial agent than acetic, citric and propionic acid
20.0

(Bjornsdottir et al. 2006; Pundir and Jain 2011; Daskalov

2012). Other studies indicate higher efficiency of sorbic and
benzoic acid than lactic acid (Yuk et al. 2007; Ho et al.
22.6 ± 1.15
21.6 ± 1.15
18.6 ± 1.15

20.6 ± 1.15
23.0 ± 1.00

30.3 ± 0.57
25.6 ± 0.57

11.0 ± 1.00
23.0 ± 0.00
21.3 ± 1.15
2010).
The obtained results showed that lactic acid is more effec-
15.0

0
0
321

tive against gram-positive bacteria than gram-negative bac-

teria, which is in agreement with the results obtained by
other authors (Houtsma et al. 1993; Yuk et al. 2007;
23.6 ± 1.15
24.3 ± 0.57
24.6 ± 1.15

24.0 ± 1.00

11.6 ± 1.15
10.0 ± 0.00
10.0 ± 0.00
24.6 ± 1.15

28.0 ± 1.00
20.3 ± 0.57
23.3 ± 1.15
29.3 ± 0.57

Tharmaray and Shah 2009). One reason for this is the dif-
ferent structural and chemical compositions of the cells’
TABLE 2. LACTIC ACID ANTIMICROBIAL ACTIVITY AGAINST TESTED BACTERIA AND YEASTS OBTAINED BY DISC DIFFUSION METHOD

25.0

outer layers (Nikaido and Vaara 1985) and acid tolerance of

each microorganism. In this sense, Nikaido (1996, 2003) has
reported that the resistance mechanisms in gram-negative
22.3 ± 0.57
21.6 ± 1.00
19.0 ± 0.00

21.6 ± 1.53

10.0 ± 0.00
23.6 ± 0.57

26.0 ± 0.00
17.6 ± 1.15
22.0 ± 1.00

7.6 ± 1.15
26.6 ± 0.57

bacteria are more complicated than those present in gram-

positive bacteria as the former is surrounded by the outer
20.0

membranes or a membrane that has hydrophobic nature,

which may block the entry of hydrophilic molecules of low
molecular mass such as monosaccharides, amino acids,
19.6 ± 1.73
19.0 ± 0.00
16.6 ± 1.15
18.6 ± 1.15

23.6 ± 0.57
18.6 ± 0.57

15.3 ± 0.57
20.6 ± 0.57
19.6 ± 1.15

8.0 ± 0.00

nucleosides and alkali or alkaline ions, which can only pass

through water-filled channels formed by transmembrane
15.0

0
0
209

proteins (porins) embedded into the lipid bilayer that

permits hydrophilic transport. On the one hand, the gram-
positive cell wall contains only a thick peptidoglycan layer
16.6 ± 1.15
15.6 ± 0.57
13.6 ± 1.15
17.0 ± 0.00

19.0 ± 0.00
16.0 ± 1.00

18.0 ± 0.00
20.3 ± 0.57
17.3 ± 0.57

10.3 ± 0.57
Diameter of inhibition zone (mm) (mean ± SD)

and a lipid bilayer. That fact would explain the greater sensi-
25.0

0
0

tivity of En. faecalis (gram-positive) to lactic acid than

gram-negative bacteria. On the other hand, Sta. aureus was
more resistant to lactic acid than En. faecalis. This is in
13.3 ± 0.57
14.0 ± 0.00
12.0 ± 0.00
16.6 ± 1.15

18.3 ± 0.57
15.3 ± 0.57

16.0 ± 1.00
18.0 ± 1.00
15.6 ± 0.57

8.3 ± 0.57

accordance with the results reported by other authors

(Miller and Kaspar 1994; Arnold and Kaspar 1995;
20.0

0
0

Benjamin and Datta 1995; Lin et al. 1996) who indicated

that acid resistance is a typical characteristic of the tested
microorganism.
11.3 ± 0.57
12.3 ± 0.57
10.3 ± 0.57
13.6 ± 1.15

15.6 ± 1.15
11.0 ± 1.73

13.6 ± 0.57
15.3 ± 0.57
12.0 ± 1.00

However, some authors found that effectiveness of the

organic acids can vary depending on its molecular weight.
15.0

0
0
0
102

Eswaranandam et al. (2004) indicated that undissociated

smaller molecules of lactic (90.08 Da) acid may enter into
the bacterial cells easily and change the internal pH of the
Lactic acid concentration (mg/mL)

microorganism, thus showing higher antimicrobial activity

than undissociated larger molecules of citric (192.13 Da)
Pseudomonas aeruginosa

Saccharomyces cerevisiae
Listeria monocytogenes
Staphylococcus aureus

and tartaric (150.09 Da) acids, which may not enter to the
Salmonella enteritidis
Gram-negative bacteria

Enterococcus faecalis

SD, standard deviation.

Gram-positive bacteria

Bacillus megaterium

cell interior effectively. That fact would explain the consid-

Candida albicans
Proteus mirabilis

Rhodotorula sp.
Escherichia coli

erable sensitivity of some gram-negative bacteria

Amount (μL/disc)

Bacillus cereus

(Ps. aeruginosa) to lactic acid.

In this study the most susceptible yeasts to lactic
Yeasts

acid was Rhodotorula sp., while similar susceptibility was

obtained for C. albicans and Sac. cerevisiae at all applied

4 Journal of Food Processing and Preservation •• (2015) ••–•• © 2015 Wiley Periodicals, Inc.
S. STANOJEVIĆ-NIKOLIĆ ET AL.
TABLE 3. MINIMAL INHIBITORY CONCENTRATION (MIC) AND
MINIMAL BACTERICIDAL CONCENTRATION (MBC) OF LACTIC ACID
FOR THE TESTED MICROORGANISM
Microorganisms MIC (mg/mL)
Gram-negative bacteria
Pseudomonas aeruginosa 1.25
Salmonella enteritidis 1.25
Proteus mirabilis 2.50
Escherichia coli 2.50
Gram-positive bacteria
Listeria monocytogenes 1.25
Staphylococcus aureus 2.50
Enterococcus feacalis 2.50
Bacillus megaterium 12.50
Bacillus cereus 18.75
Yeasts
Rhodotorula sp. 12.50
Candida albicans 18.75
Saccharomyces cerevisiae 37.50
concentrations. Some authors suggest that lactic acid is not
sufficiently effective as an antifungal agent (Woolford 1975;
Wongsuttichote and Nitisinprasert 2009; Muck 2010), while
other studies indicate that lactic acid has the potential anti-
fungal activity (Lind et al. 2005; Broberg et al. 2007). Some
yeasts can grow at lower pH values (Beales 2004) and at
lower lactic acid concentrations (Narendranath et al. 2001),
while others may use lactic acid as a nutrient (Moon 1983).
Some yeasts (Sac. cerevisiae and C. albicans) use organic
acids by decarboxylation (Stratford et al. 2007).
The variability in the sensitivity to weak organic acid pre-
servatives, among the yeast species, may be related to their
capacity to change the cell metabolism in response to acid
stress conditions and physiological differences such as cell
permeability, enzyme structure, and induction or suppres-
sion of genes that effect susceptibility to weak acids (Warth
1991). Some yeasts possess enzymes able to degrade acids
and reduce diffusion coefficient of preservatives across the
membrane, resulting in a reduced ability of weak acids to
enter the cell (Brul and Coote 1999). Reducing diffusional
entry of the acid into the cell is probably a key mechanism
of yeasts’ resistance (Piper et al. 2001). Phenotypically
acquired resistance to acid preservatives is well documented
for yeasts, and this results from the enhanced ability of cell
to catalyze energy-dependent extrusion acids (Warth 1985).
Piper et al. (1998) showed evidence of an efflux system that
removes accumulated anions from inside the cell.
Lactic Acid MIC, MBC and MFC
The results of lactic acid MICs, MBCs (for bacteria) and
MICs, MFCs (for yeasts) against the tested microorganisms
are given in Table 3. Lactic acid MIC for bacteria ranged
Journal of Food Processing and Preservation •• (2015) ••–•• © 2015 Wiley Periodicals, Inc.
ANTIMICROBIAL ACTIVITY OF LACTIC ACID
from 1.25 mg/mL (Sal. enteritidis, Ps. aeruginosa and
L. monocytogenes) to 18.75 mg/mL (B. cereus), while for
yeasts MIC ranged from 12.50 mg/mL (Rhodotorula sp.) to
MBC (mg/mL) 37.50 mg/mL (Sac. cerevisiae). Lactic acid MBC ranged
from 2.50 to 37.50 mg/mL, while MFC ranged from 25.00
2.50 to 75.00 mg/mL (Table 3).
2.50 Lactic acid showed high inhibitory effect against
5.00 L. monocytogenes, with a MIC of 1.25 mg/mL. The results of
5.00
MIC and MBC showed that two gram-negative bacteria
(Sal. enteritidis and Ps. aeruginosa), with a MIC concentra-
2.50
5.00 tion of 1.25 mg/mL, were more sensitive to lactic acid than
5.00 most gram-positive bacteria (Table 3).
25.00 Spore-forming bacteria were more resistant to the lactic
37.50 acid effect than the other test bacteria. MIC and MBC for
B. megaterium and B. cereus were significantly higher than
25.00
those of the tested non-sporulating bacteria. Russell (1991)
37.50
75.00
reported that organic acids added as food acidulants are
almost equally effective in inhibiting gram-positive and
gram-negative bacteria, while spore-forming bacteria are
usually more resistant to preservatives than are non-
sporulating bacteria (Gauld 1985; Russell 1990). The resis-
tance of spore-forming bacteria to preservatives is a
property of the spore coats, although the cortex may be a
contributing factor (Knott et al. 1990).
Higher lactic concentrations, which are necessary for
total microorganism inhibition, may cause changes in food
sensory properties. However, in many cases, a concentration
sufficient to result in growth stasis may be all that is
required to achieve a safe product, provided that the initial
microorganism number is low (de Barros et al. 2012;
Tangwatcharin and Khopaibool 2012). Also, the concentra-
tion required for the growth stasis can be considerably
lower than that necessary for the total inhibition.
Inhibitory activity of the lactic acid against three strains
of foodborne pathogenic bacteria was investigated by Qiao
et al. (2008). In comparison with our study, MIC of lactic
acid for Sta. aureus, B. cereus, and Es. coli ranged from
0.9 mg/mL (Es. coli) to 3.6 mg/mL (B. cereus), while MBC
ranged from 1.8 to 7.2 mg/mL. Hsiao and Siebert (1999)
showed that MIC of lactic acid for B. cereus was 3.48 mg/
mL, while MIC for Es. coli was 3.72 mg/mL. Some factors,
such as bacterial strain and its sensitivity, inoculum volume,
incubation time and temperature, affect the MIC and MBC
values (Zaika 2002; Raybaudi-Massila et al. 2009). More-
over, the method used to assess the antimicrobial activity
could be related to the variations of the experimental results
(Jiang 2011).
In comparison with other tested yeasts, Sac. cerevisiae was
the least sensitive to lactic acid. Strong inhibitory effects
against Sac. cerevisiae require relatively high lactic acid con-
centration (Thomas et al. 2002; Abbott et al. 2008), which is
in agreement with our results. The inhibitory effect of lactic
and acetic acid on the growth of Sac. cerevisiae was investi-
5
ANTIMICROBIAL ACTIVITY OF LACTIC ACID
gated by Narendranath et al. (2001). The results of this
study showed that lactic acid concentration of 25 mg/mL is
required to achieve MIC of Sac. cerevisiae. Thomas et al.
(2002) reported that Sac. cerevisiae were completely inhib-
ited at a lactic acid concentration of 49.4 mg/mL. MIC of
lactic acid at different pH values was investigated by Lind
et al. (2005). These authors found that MIC of lactic acid
for Rhodotorula mucilaginosa ranged from 14.4 mg/mL
(pH 3) to >45.04 mg/mL (pH 5 and pH 7), while MIC of
lactic acid for Pichia anomala was >45.04 mg/mL at differ-
ent pH values.
Lactic acid inhibitory activity on yeast growth is
pH-dependent and is accompanied by changes in the intra-
cellular pH; there are indications that the mechanism
underlying its toxicity differs from other weak acid preser-
vatives (Narendranath et al. 2001) and involves the toxicity
of the lactate ion (Thomsson and Larsson 2005). Although,
at lower pH values of the medium, lactic acid exists substan-
tially in nondissociated form, whereby molecules easily pass
through the cell membrane and causes loss of viability and
cell destruction (Torino et al. 2001). Alakomi et al. (2000)
reported that the antimicrobial activity of lactic acid is
assigned largely, but not totally, to nondissociated lactic acid
form. Sudershan et al. (2011) noted that the antimicrobial
activity of nondissociated form is 100- to 600-fold higher
than the antimicrobial activity of a dissociated form of
lactic acid.
CONCLUSIONS
According to the obtained results, it can be concluded that
lactic acid has an important antibacterial and antifungal
activities. The tested bacterial strains were affected more
than yeasts by lactic acid. Lactic acid antibacterial activity
was stronger against the tested gram-positive bacteria than
the gram-negative bacteria.
Relatively low lactic acid MIC and MBC for some of the
most important pathogens, which cause food poisoning,
show a potential application of the natural preservatives.
Further studies will provide more details on the com-
bined effects of different natural antimicrobial agents with
lactic acid. However, the potential use of lactic acid to
control food pathogens requires a detail investigation of its
activity in a real food system.
ACKNOWLEDGMENTS
The study is the part of the investigations realized with the
scope of the Project No. TR-31017 financially supported by
the Ministry of Education, Science and Technological
Development of the Republic of Serbia.
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