Food Research International 45 (2012) 713–721

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Food Research International

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Organic acids as antimicrobials to control Salmonella in meat and poultry products

E. Mani-López a, H.S. García a, A. López-Malo b,⁎
a
UNIDA-Instituto Tecnológico de Veracruz, M.A. de Quevedo # 2779, Col. Formando Hogar, Veracruz, Ver. 91897, Mexico
b
Departamento de Ingeniería Química, Alimentos y Ambiental, Universidad de las Américas Puebla, Cholula, Puebla 72810, Mexico

a r t i c l e i n f o a b s t r a c t

Article history: Salmonella, a food-borne pathogen, has a recurrent incidence in meat and poultry products. Currently, cases of
Received 15 February 2011 salmonellosis represent very important economic losses in many countries. An alternative for the prevention
Received in revised form 14 April 2011 of Salmonella outbreaks due to consumption of meat and poultry products, are organic acids (acetic, citric,
Accepted 18 April 2011
lactic, malic, propionic, and tartaric among others). Organic acids have been utilized for many years for
decontamination of beef, pork and poultry products from several bacteria including Salmonella. Current
Keywords:
Organic acids
reports describe new treatments with organic acids using the hurdle technology approach to inhibit
Salmonella Salmonella. Also, preventive actions should be taken in order to decrease the appearance of resistant strains of
Meat products Salmonella as a result of insufficient or sub-lethal treatments with organic acids in meat and poultry products.
Poultry products This review presents general insights on the use of the organic acids to control Salmonella in meat and poultry
products. Also, information of the action mechanism, types and doses of treatments, as well as how resistance
can occur from improper application of organic acids.
© 2011 Elsevier Ltd. All rights reserved.

1. Introduction 1.1. Salmonella classification

Salmonella is an important pathogen that causes major problems of
morbidity and mortality around the world. The pathogen is generally
associated with contaminated foods like peanut butter, sprouts,
almonds, meat, eggs, and meat products. This pathogen can have a
large socioeconomic impact due to illness, medical costs, lost of
productivity, disability, deaths, litigation, and recalls due to contam-
inated products (Buzby, Frenzen, & Rasco, 2001). The incidence of
food-borne illness caused by Salmonella has remained unchanged
during 2005–2009, and Salmonella is consistently the most common
bacterial pathogen in laboratory confirmed food-borne illness cases
(Centers for Disease Control and Prevention [CDC], 2005, 2006, 2007,
2008, 2009). Several technologies and treatments have been
developed and applied for many years in order to control spoilage
and pathogenic microorganisms. Chemical decontamination was first
used in the 1960s (Acuff, 2005), and contributed to the control of food
pathogens including Salmonella. Today, Salmonella continues as an
important target pathogen for the food industry (particularly for meat
and poultry). The objective of the review is to describe general
insights on the use of organic acids to inhibit Salmonella in meat and
poultry products.
⁎ Corresponding author. Tel.: + 52 222 2292409; fax: + 52 222 2292727.
E-mail address: aurelio.lopezm@udlap.mx (A. López-Malo).
Salmonella is a genus of the family Enterobacteriaceae. These
enteric (i.e., associated with the intestine) bacteria are Gram-
negative, usually motile, facultative anaerobic, and non spore-forming
bacilli. It grows at temperatures between 8 and 45 °C in a pH range of
4–9 and require water activities (aw) above 0.94 (Guthrie, 1992).
Salmonellae are heat-sensitive and are generally killed at tempera-
tures of 70 °C or above. Salmonellae are resistant to drying and may
survive for years in dust and dirt. Salmonella may actually contain only
two lineages that have diverged considerably from each other during
evolution (Bäumler, 1997; Bäumler, Tsolis, Ficht, & Adams, 1998).
These lineages represent two distinct species, designated Salmonella
enterica (Le Minor, Vernon, & Popoff, 1982) and Salmonella bongori
(Reeves, Evins, Heiba, Plikaytis, & Farmer, 1989). The serotype name
remains the main taxonomic descriptor for Salmonella isolates (Foley
& Lynne, 2008). Serotyping is performed by mixing a suspension of
Salmonella with series of antisera specifics for a variety of O and H
surface antigens (Brenner, Villar, Angulo, Tauxe, & Swaminathan,
2000; Voogt, Wannet, Nagelkerke, & Henken, 2002).
Salmonellosis can manifest in a number of disease syndromes
including gastroenteritis, bacteremia, typhoid fever, and focal in-
fections (Darwin & Miller, 1999). The incubation period for develop-
ing gastroenteritis is 6–72 h following ingestion of contaminated food
or water (Guthrie, 1992). Fever of 38–39 °C is common, and there may
be an initial chill. Abdominal pain is frequent and may cause mild to
severe discomfort. In uncomplicated cases, the acute stage usually
resolves within 48 h. However, illness is occasionally more protracted,
with persistent diarrhea and low-grade fever for 10–14 days (Hanes,

0963-9969/$ – see front matter © 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodres.2011.04.043
714 E. Mani-López et al. / Food Research International 45 (2012) 713–721
2003). Most recurrent reports of human isolate infections in the
United States are from 5 serotypes namely S. Typhimurium (19%), S.
Enteritidis (14%), S. Newport (9%), S. Heidelberg (6%) and S. Javiana
(5%)(Braden, 2006; CDC, 2005). S. Typhi is strictly a human pathogen
(Braden, 2006) and causes the typhoid fever.
1.2. Salmonella incidence in meat products
Fresh meat and poultry are highly perishable due to their enriched
nutrient composition, high pH (5.5–6.5) and water activity (0.98–
0.99), which support survival/growth of almost any contaminating
microorganism (Acuff, 2005). Meat and poultry industries are the
main reservoir of Salmonella as a foodborne pathogen. For many years
Salmonella has been associated with poultry and meat because fecal
material and dirt from feathers and the hide, as well as dirt of process
equipment contaminate carcasses during slaughter and packinghouse
operations. Also, specific serotypes are related to animals, such as
swine (S. Choleraesuis), cattle (S. Dublin), and reptiles (S. IV 44:z4z23:-,
formerly known as S. Marina), while S. Gallinarum is an important
chicken pathogen, but rarely causes illness in humans (Braden, 2006).
S. Enteritidis is found primarily in poultry and poultry products. In
many cases Salmonella is freely living in the animals during their
growth. The result is that Salmonella is still present in beef, pork and
broiler products. Inspections of meat and poultry industries show that
Salmonella is commonly positive, (broilers was 7.3%) (USDA-FSIS,
2008). S. Typhimurium was the most common serotype associated
with laboratory-confirmed illness cases (CDC, 2005, 2006, 2007, 2008,
2009). A report from Salmonella test in the verification program by
product class and size establishment from Jul 1, 2007 to Sept 30, 2007
by FSIS showed that 80% of samples of ground chicken, 16.5% of
ground turkey, 10.8% of ground beef, 9.6% of turkey, and 8.2% of
broilers were positive for Salmonella in large establishments (USDA-
FSIS, 2007b). Clearly Salmonella has been a foodborne problem
particularly in meat and poultry products; therefore a conscious
control needs to be applied in these industries.
In the European Union the reports of salmonellosis during the
1990s were related to pork contaminated in Netherlands, Denmark,
and Ireland; documented cases represent 10–15%. Public alerts of
outbreaks caused by Salmonella in contaminated pork rings (USDA-
FSIS, 2004), by S. Newport in fresh ground beef products (USDA-FSIS,
2007a) and by S. Montevideo in varieties of Italian sausage products
including salami (USDA-FSIS, 2010b), are the most recent reports
from Salmonella in the U.S.
In order to eliminate or decrease the counts of pathogens, especially
Salmonella in meat products post-slaughter, many physical treatments
and antimicrobials have been suggested. The most important are thermal
treatments, washing with oxidant agents (hypochlorite, peracetic acid,
and H2O2 among others), application of bacteriocins or lactic acid bacteria,
natural antimicrobials from spices and washing with organic acids (lactic,
acetic, citric, malic, and propionic). In some reports a hurdle technology
approach is applied. Hurdle technology advocates the deliberate
combination of existing and/or novel preservation techniques to establish
a series of preservatives factors to improve the microbial stability and the
sensory quality of food as well as their nutritional and economic
properties (Leistner, 2000; Leistner & Gorris, 1995). The most important
hurdles used in meat and poultry products are temperature, acidity (pH),
Table 1
Organic acids commonly used in meat products as antimicrobials.
Compound(s) Microbial target
Acetic acid, acetates, diacetates, dehydroacetic acid Yeasts, bacteria
Lactic acid, lactates Bacteria
Sodium propionate Molds
a
Food and Drug Administration designations in Title 21 of the Code of Federal Regulations. Food antimicrobials approved by the U.S. Department of Agriculture's Food Safety and
Inspection Service for use in meat products are listed in sections 424.21 and 424.22 of Title 9 of the CFR.
antimicrobials, packaging, and vacuum packaging. Novel treatments to
eradicate Salmonella have been proposed like supercritical CO2 and
prevention of Salmonella infections at farm level are also suggested (Choi,
Kim, Kim, Kim, & Rhee, 2009; Vandeplas, Dubois, Beckers, Thonart, & The
Wis, 2010).
1.3. Organic acids in meat products
Application of organic acids on meat surfaces is a common
procedure; acid treatments are cheap, simple and fast, and have
shown clear efficiency (Hinton & Corry, 1999). Further, organic acids
are designated by the FDA as generally recognized as safe (GRAS) for
meat products. Table 1 depicts their applications. Fig. 1 displays
chemical structures of common organic acids used in meat and
poultry products. Moreover, most of them are not limited in the
acceptable daily intake for humans. These characteristics favor their
use in meat products. Several studies have tested the efficacy of
organic acids (Castillo, Lucia, Goodson, Savell, & Acuff, 1998, 1999;
Castillo, Lucia, Mercado, & Acuff, 2001; Ellebracht, Castillo, Lucia,
Miller, & Acuff, 1999; González-Fandos, Herrera, & Maya, 2009; Over,
Hettiarachchy, Johnson, & Davis, 2009; Tamblyn & Conner, 1997a,
1997b; Thomson, Banwart, Sanders, & Mercuri, 1967) but sensory
changes (color and flavor) might be taken in consideration. Organic
acids and their salts are considered weak acids, meaning they do not
fully dissociate in water but do so in a pH-dependent manner.
Consequently, the antimicrobial activity of organic acids is enhanced
as the pH of the food is lowered to that of, or below, the pKa of the
acid. The pKa is defined as the acid dissociation constant. Reduction in
pH results in a greater concentration of protonated acid, decreasing
the polarity of the molecule and increasing diffusion of acid across the
membrane and into the cytoplasm. However, the substitution of the
donable proton with a monovalent (Na+, K+) or multivalent (Ca+ 2)
cation significantly increases the solubility of organic acid in aqueous
systems. Thus, a balance must be made between the need to maintain
acid solubility with the need to achieve maximal activity via pH
reduction (Taylor et al., in press).
Organic acids are considered to affect microbial activity by two
primary mechanisms: by cytoplasmic acidification with subsequent
uncoupling of energy production and regulation, and by accumulation
of the dissociated acid anion to toxic levels (Taylor et al., in press).
Diffusion of an undissociated acid through a microbial membrane in a
food where the pH of the cellular cytoplasm is higher than that of the
surrounding environment favors the establishment of a transmem-
brane gradient (Gould, 1989). As protonated acid diffuses across the
membrane, an alkaline environment is encountered, favoring the
dissociation of the acid into the acid anion and free proton (Eklund,
1983). The cell reacts by working to efflux the proton, exchanging the
proton for some other cation (e.g. Na+, K+); this is the basis of the
chemiosmotic theory (Mitchell, 1961; Mitchell & Moyle, 1969). It is
proposed that the microbial membrane is impermeable to protons,
requiring active transport to efflux protons and maintain pH
homeostasis in the cellular interior (Brul & Coote, 1999; Hirshfield,
Terzulli, & O'Byrne, 2003). Freese, Sheu, and Galliers (1973), and
Freese (1978) revised this theory, suggesting an uncoupling of
electron transport from oxidative respiration. Fig. 2 exhibits a general
action mechanism of the organic acids. Neither theory completely
Primary food applications Title 21 CFR designationa
Dairy products, meats 184.1005, 182.6197, 184.1754, 184.1185, 184.1721, 172.130
Meats, fermented foods 184.1061, 184.1207, 184.1639, 184.1768
Meat products 184.1784
 E. Mani-López et al. / Food Research International 45 (2012) 713–721 715

Acetic acid (C2H4O2) Citric acid (C6H8O7) Lactic acid (C3H6O3) Propionic acid (C3H6O2)
MW =60.05 g/mol MW =192.13 g/mol MW = 90.08 g/mol MW = 74.08 g/mol

Malic acid (C4H6O5) Succinic acid (C4H6O4) Tartaric acid (C4H6O6)

MW = 134.09 g/mol MW = 118.09 g/mol MW = 150.09 g/mol
MW = Molecular weight

Fig. 1. Chemical structure of organic acids commonly used in meat and poultry products.

explains the mode of action of the organic acids, as in any system
where organic acids are used there is always some concentration of
dissociated anion, and the presence of an uncoupling effect suggests
the potential for other inhibitory effects to occur following the
diffusion of the undissociated acid across the cell membrane.
Cherrington, Hinton, Mead, and Chopra (1991) identified possible
modes of inhibition by organic acids resulting from accumulation of
the acid anion in the microbial cytoplasm. In addition to shifting the
internal pH out of range for optimal enzymatic activity, protein and
DNA/RNA synthesis are adversely affected by the presence of organic
acids at elevated levels (Cherrington, Hinton, & Chopra, 1990). Shelef
(1994) reported that sodium lactate exerted antimicrobial activity not
only through the lowering of aw. Russell (1992) reported accumula-
tion of the acid anion to be the driving force for inhibition of cells, as
the accumulation of the anion eventually hinders the proton motive
force (PMF) and inhibits the microbe's ability to re-alkalinize its
cytoplasm. Others have reported that lactic acid permeabilized the
Gram-negative membrane and released lipopolysaccharides (LPS)
from the outer membrane (Alakomi et al., 1999). Furthermore,
chelating properties or intercalation with the outer membrane of
Salmonellawas demonstrated for citric and malic acids (Alakomi et al.,
2007; Helander & Mattila-Sandholm, 2000). New insights pointing
out that hydrophobic organic compounds (such as sorbic acid)
increase the permeability of the membrane as well as cause
interference with membrane proteins in molds (Stratford, Plumridge,
Nebe-von-Caron, & Archer, 2009); suggesting alternative modes of
actions of some organic acids. Most recently, research has demon-
strated that the interplay of all these mechanisms likely drives
inhibition of microbes by organic acids (Koczon, 2009).
1.3.1. Acetic acid and acetates
Acetic acid is a monocarboxylic acid with a pungent odor and taste,
which limits its use in foods. It is the principal component of vinegars
and as such is primarily used for its flavoring abilities. It is highly soluble
in water. It is found in pickled products such as sausages and pigs' feet.
Acetic acid is generally regarded as safe (GRAS) for miscellaneous and
general-purpose usage (21 CFR 184.1005). The acceptable daily intakes
for humans for acetic acid and its derivatives are listed in Table 2. The
data are scarce for the use of peracetic acid in food products. Liao,
Shollenberger, and Phillips (2003) investigated the lethal and sub-lethal
effects of acetic acid on 6 strains of Salmonella; cells were exposed to
various concentrations (0.06% to 3.0%) of acetic acid for different periods
of time (0 to 7 min) when grown on Brain Heart Infusion Agar (BHIA).
Susceptibility of Salmonella to acetic acid action is greatly affected by the

Fig. 2. Action mechanism of organic acids on microbial cells. The undissociated form of organic acid (HA) is diffusing through the microbial membrane when the pH of the cellular
cytoplasm is higher than that of the surrounding environment. In order to maintain the internal pH, active transport to efflux protons (H+) is required. Also acid pH in the internal
cell, damages or modifies the functionality of enzymes, structural proteins, and DNA. *Few organic acids (malic and citric acids) have been shown to efficiently destabilize the outer
membrane by chelation or intercalation.
716 E. Mani-López et al. / Food Research International 45 (2012) 713–721

Table 2 at 52 °C than at 14.4 °C (Anderson et al., 1987). It is possible that acid

Acceptable daily intake for humans. penetration of the cells is accelerated at higher temperatures where
Acid Limitations Reference lactic and acetic acids have been shown to be more effective
(mg/kg body weight) (Anderson et al., 1987; Anderson & Marshall, 1989, 1990; Anderson,
Unconditional Conditional Marshall, & Dickson, 1992; Conner, Scott, & Bernard, 1990; Greer &
Dilts, 1995), causing rapid damage that is not related to any acid
Acetic Not limited FAO (1973)
Acetate, Ca2+, K+, Na+ Not limited FAO (1963, 1973) adaptation brought on by a shock response. Incorporation of pulsed
Sodium diacetate 0–15 FAO (1973) power electricity and a 2% acetic acid spray led to improved reduction
Citric Not limited FAO (1973) of inoculated E. coli O157:H7 and S. Typhimurium on beefsteaks
Citrate, Ca2+, K+, Na+ Not limited FAO (1963) (Tinney, Miller, Ramsey, Thompson, & Carr, 1997). Dickson (1992)
Lactic Not limited FAO (1973)
DL-Lactic 0–100a FAO (1973)
contaminated lean and beef tissue surfaces with S. Typhimurium
Lactate, Ca2+, K+, NH4+, Na+ Not limited FAO (1973) followed by treatment with 2% acetic acid; however, S. Typhimurium
Malic Not limited FAO (1973) was reduced only by 0.5 to 0.8 log CFU/cm2; similar results were
Propionic Not limited FAO (1973) reported by Anderson and Marshall (1990) when they tested a
Propionate, Ca2+, K+, Na+ Not limited FAO (1973)
mixture of acetic, lactic, citric, and ascorbic acids on lean tissue to
Tartaric 0–30 FAO (1973)
Tartrate, K+, Na+ 0–30 FAO (1973) reduce S. Typhimurium.
a
Poultry scald water containing 0.1% acetic acid at 52 °C decreased
Content of DL-lactic acid.
levels of S. Typhimurium and Campylobacter jejuni by 0.5 to 1.5 logs
(Okrend, Johnston, & Moran, 1986). Tamblyn and Conner (1997a)
treated broilers with S. Typhimurium; a concentration of acetic acid
physiological state of the bacteria tested. The relative sensitivity of these greater than 4% was needed to reduce by 2 logs the populations of
6 serovars to acetic acid action was found to be in the following order: S. Salmonella, probably because of the possible attachment to broiler
Bareilly N S. TyphimuriumN S. MontevideoN S. PoonaN S. Mbandaka N S. chicken skin. Adjustment of chill water to pH 2.5 with acetic acid was
Stanley. Zhou et al. (2007) evaluated the synergistic effect of thymol and the most effective antimicrobial treatment followed by other acids in
carvacrol with acetic acid on Mueller-Hinton broth (MHB) and observed descending order of effectiveness: adipic, succinic, citric, fumaric, and
strong synergistic activities. In the presence of acetic acid, the lactic (Mountney & O'Malley, 1965). Dickens and Whittemore (1994)
antibacterial activity from the combinations of thymol (100 mg/l) plus exposed broilers to 0.3% and 0.6% acetic acid under the same
acetic acid (0.10%) and of carvacrol (100 μl/l) plus acetic acid (0.10%) conditions, with and without the use of air injection to agitate the
was enough to inhibit S. Typhimurium. These combination treatments chill water. Authors observed decreased Enterobacteriaceae counts by
could achieve the desired antibacterial effect at concentrations low 0.86 log MPN/ml for the 0.3% acid and 2.35 log MPN/ml for the 0.6%
enough to minimize undesirable changes in flavor. Álvarez-Ordóñez, acid solutions (Dickens & Whittemore, 1994). There were no
Fernández, Bernardo, and López (2010) studied the ability of S. significant differences in texture or sensory characteristics between
Typhimurium cultivated in Brain Heart Infusion (BHI) to grow under the treatments, although the skin of the 0.6% acetic acid-treated
acidic conditions at different temperatures, it was affected by the type of broilers was darkened or yellowed (Dickens, Lyon, Whittemore, &
acid used to acidify the growth medium. Authors observed that acetic Lyon, 1994). Water pockets occurred under the skin of broilers with
acid is the best antimicrobial against S. Typhimurium (decreasing order air-agitated samples (Dickens & Whittemore, 1994).
on effectivity: acetic N lactic N citric N hydrochloric). Moderately low Several derivatives of acetic acid are currently in use as
temperatures (10 °C) markedly decreased the acid resistance and antimicrobial agents. The salt form, however, requires different
increased the growth pH boundary of S. Typhimurium suggesting the handling and utilization procedures than the acid. The sodium and
convenience to control the temperature during food processing as a calcium salts are sometimes used in foods and would be expected to
strategy to prevent the growth and survival of this pathogenic have the same antimicrobial properties as acetic acid at the same pH
microorganism (Álvarez-Ordóñez et al., 2010). values (Hoffman, Schweitzer, & Dalby, 1939). Mendonca, Molins,
Several reports probed the acetic acid efficiency to decontaminate Kraft, and Walker (1989) used a dip of sodium acetate in combination
meat and meat products (beef, pork, and poultry); some authors with potassium sorbate (10%) and phosphates (10%) to extend the
search for the optimal conditions among those producing the lowest shelf life of pork chops. Sodium diacetate reduced the pH of the meat
counts of bacteria and pathogens (like Salmonella) and retain sensory slightly. A surface application of 1 to 3 mg/cm2 (2 to 6 g/chicken)
attributes. Bell, Marshall, and Anderson (1986) treated beef cubes sodium diacetate powder extended the shelf life of chickens about
with acetic acid (0.6% or 1.2%) and formic acids (0.046%) for 10 s, and 4 days when held at 2 °C. The powder dissolved in the surface
they observed changes in color (discoloration) when using acetic acid moisture of the raw chicken to form acetic acid and sodium acetate
alone, but beef treated with the mixture of acids did not presented and produced a surface pH of about 4.8. Enterobacteriaceae counts
changes in flavor from untreated beef. Hamby, Savell, Acuff, dropped 1000-fold over 6 days at 2 °C (Moye & Chambers, 1991).
Vanderzant, and Cross (1987) used intermittent sprays of 1% acetic
acid or lactic acids, which resulted in significant reductions in aerobic 1.3.2. Citric acid and citrates
plate count (1.8 to 4.3 log/cm2). An application of 2% acetic acid Citric acid (CAS No. 77-92-9) is a hydroxy tricarboxylic acid
reduced the incidence of Salmonella on pork cheek meat in addition to produced naturally by various plants. It is water soluble, approved for
significantly reducing aerobic plate and coliform counts (Frederick, direct addition to multiple foods, is affirmed as GRAS (21CFR184.1033),
Miller, Thompson, & Ramsey, 1994). The treatment of buffalo steaks and is approved for use in the manufacture of fresh and processed meats
with 3% solutions of acetic and lactic acid had no adverse effect on and poultry at concentrations specific to its purpose (USDA-FSIS,
surface color (Surve, Sherikar, Bhilegaonkar, & Karkare, 1991). More 2010a). Acceptable daily intake for humans is presented in Table 2.
than one treatment sometimes helps on the bacterial reduction and Citric acid is known to inhibit cells through metal chelation, in some
produces lesser effects on food quality. A report by Anderson and instances resulting in enhanced pathogen inhibition versus the
Marshall (1989) demonstrated that application of acetic acid at 70 °C monocarboxylic acids like lactic acid (Miller, Call, & Whiting, 1993).
was most effective in sanitizing beef semitendinous muscle inoculat- Several studies point out the effectiveness of citric acid and its salts in
ed with E. coli, S. Typhimurium, or manure slurry. Microbial loads on various food systems. S. enterica serovar Typhimurium generation times
beef carcasses subjected to machine washing and sanitization with in BHI containing citric acid (pH values of 6.4, 5.4 or 4.5) increased from
1.5% acetic acid were reduced more when treatments were conducted 0.36 h to 0.54 h, and finally to 0.75 h, respectively (Álvarez-Ordóñez,
 E. Mani-López et al. / Food Research International 45 (2012) 713–721
Fernández, Bernardo, & López, 2009). Minimum inhibitory concentra-
tions of various organic acid salts were determined in chicken juice for
S. Typhimurium and were 1.25% for sodium citrate and sodium lactate at
37 and 42 °C (Milillo & Ricke, 2010). Tamblyn and Conner (1997a)
compared antimicrobial effects of multiple organic acids applied at
points along the poultry slaughter continuum for the reduction of
S. Typhimurium on chicken skin. Citric acid (4%) in a simulated chiller
tank resulted in a 1.9 log reduction compared to controls for attached
S. Typhimurium cells. By comparison, 4% lactic acid application resulted
in 0.7 log reduction under similar conditions (Tamblyn & Conner,
1997a). Scannell, Hill, Buckley, and Arendt (1997) evaluated sodium
citrate at 1.5% on fresh pork sausage and observed a reduction of 0.3 logs
in S. Kentucky. Spray application of the commercial antimicrobial
intervention Chicxide (Birko Corp., Denver, CO), containing blended
lactic and citric acids, to surfaces of broiler carcasses resulted in a 1.3 log
CFU/ml reduction of inoculated salmonellae, whereas immersion of
broiler carcasses in the antimicrobial intervention for up to 20 s resulted
in 2.3 log CFU/ml reduction in inoculated Salmonella (Laury et al., 2009).
Citric acid at 3% did not produce unacceptable odors and color
acceptability was retained for longer periods compared to untreated
controls (González-Fandos et al., 2009). Over et al. (2009) evaluated
different acids in boneless/skinless chicken breast meat (cubes)
inoculated with 108 CFU/g of S. Typhimurium, vacuum infusion
(63.5 cm Hg for 20 min at 4 °C) with 150.0 mM citric acid, malic acid
and tartaric acid were tested; the authors observed reduced counts of
S. Typhimurium to near undetectable levels by day 6 of storage
(100 CFU/g) and to undetectable levels after day 9 of storage at 4 °C.
Citric acid inhibited S. Typhimurium as much as acetic acid (Zhou et al.,
2007). Citric acid and its salts have demonstrated efficacy for pathogen
control in both fresh and processed meat and poultry, but their usage is
potentially limited by possible negative sensory impact and the need for
low pH maintenance for optimum antimicrobial activity.
1.3.3. Lactic acid and lactates
Lactic acid (2-hydroxypropanoic acid) is a monocarboxylic acid
(pKa 3.79) produced during anoxic respiration or by fermentation by
several bacterial microorganisms, including lactic acid bacteria, [LAB]
(Axelsson, 1998). It occurs in two isomeric forms (D-, L-); it has been
reported that L isomer is more effective for pathogen inhibition
(McWilliam-Leitch & Stewart, 2002a, 2002b). Lactate is used as a
flavoring agent, color stabilizer, and can be useful for its effects on
retardation of lipid oxidation and subsequent off-odor development
(Papadopoulos et al., 1991a, 1991b). It is allowed for direct addition to
various foods and is affirmed as GRAS (21CFR184.1061). Lactic acid is FDA
approved as an antimicrobial for application to animal carcasses (pre- and
post-chilling; b5% acid solution), sub-primal cuts and trimmings (2–3%
acid, b55 °C), and to beef heads and tongues (2.0–2.8% in washing
systems) (USDA-FSIS, 2010a). Acceptable daily intake for humans is
exhibited in Table 2. Its use in the meat industry is widespread and many
researchers have documented its efficacy for the reduction of enteric
pathogens on the surfaces of carcasses and derived cuts (Baird, Lucia,
Acuff, Harris, & Savell, 2006; Bosilevac, Nou, Barkocy-Gallagher, Arthur, &
Koohmarie, 2006; Castillo et al., 2001; Castillo, Lucia, Goodson, Savell, &
Acuff, 1998, 1999; Delmore et al., 2000; Dixon, Vanderzant, Acuff, Savell, &
Jones, 1987; Ellebracht et al., 1999).
The efficacy of lactic acid and lactates has been studied intensely
with regards to their antimicrobial utility on animal carcasses and
derived cuts. Salmonella was reduced on skins by 3.4 and 2.8 logs
when 10% lactic acid and 55 °C were applied (Carlson, Ruby, Smith,
Sofos, & Bellinger, 2008). Spraying of 2.0 and 4.0% lactic acids on beef
trim surfaces resulted in reductions of E. coli O157:H7 and Salmonella of
~2.0 and 1.5 logs, respectively (Harris, Miller, Loneragan, & Brashears,
2006). Jimenez-Villarreal, Pohlman, Johnson, and Brown (2003) treated
ground beef with 2% lactic acid and the treated beef maintained relatively
similar sensory color and beef odor characteristics as the control. Özdemir
et al. (2006) observed a reduction of 1.2 log CFU/g of S. Typhimurium in
717
beef tissue immersed for 15 s in hot water (82 °C) and lactic acid (2%).
Lactic acid at 150 mM reduced counts of S. Typhimurium by approxi-
mately 2.5 log CFU/g from the initial inoculation count (108 CFU/g) by the
6th day of storage at 4 °C with minimal reduction occurring on days 9 and
12 in vacuum-infusion boneless/skinless chicken breast meat cubes (Over
et al., 2009). A 1.0% to 1.25% concentration of acid sprayed on veal
carcasses, followed by vacuum packaging, lowered microbial counts after
storage for 14 days at 2 °C, but a 2% concentration led to discoloration of
the carcass surface (Smulders & Woolthuis, 1983; Woolthuis &
Smulders, 1985). For the combined treatment of 3% lactic acid and
super critical-CO2 at 12 MPa and 35 °C for 30 min in fresh pork a
reduction of 2.33 log CFU/cm2 for S. Typhimurium was obtained (Choi
et al., 2009). A 1-min vacuum tumbling of deboned chicken meat in a 1%
lactic acid solution has demonstrated a significant decontaminating
effect on Salmonella spp. in fresh sausages (Deumier, 2006). Lactic acid
(1%) added to both chill water (0 °C to 1.1 °C, pH 2.8) and scald water
(54 °C and 2 min) reduced the bacterial level of broilers inoculated with
S. Typhimurium to almost non-detectable numbers (Izat, Colberg,
Thomas, Adams, & Driggers, 1990). Consumer evaluation of chicken
treated with a 0.5% lactic acid/0.5% sodium benzoate dip concluded that
sensory qualities were acceptable (Hathcox, Hwang, Resurreccion, &
Beuchat, 1995; Hwang & Beuchat, 1995). Hwang and Beuchat (1995)
demonstrated that a 0.5% lactic acid/0.05% sodium benzoate solution
was able to reduce Salmonella on chicken wings to non-detectable levels
after 4–6 days at 4 °C.
Alternative use of organic acids to prevent Salmonella in poultry
included their application at farm level. European regulations were
established to force member states to implement programs to
efficiently control Salmonella in poultry. These regulations are based
on an integrated approach and include control measures applied
throughout the poultry production chain (Vandeplas et al., 2010).
Byrd et al. (2001) evaluated the addition of 0.5% acetic, lactic, or
formic acid in drinking water during an 8-h pre-transport feed
withdrawal period and found reduced numbers of S. Typhimurium in
the crop (0.79 versus 1.45 log CFU/g) after treatment with lactic and
formic acids when compared with control birds.
Processors wishing to use lactic acid must not only confirm the
antimicrobial efficacy for the product(s) of interest, but also account
for potential undesirable effects on product quality and sensory
attributes.
1.3.4. Other organic acids
1.3.4.1. Propionic acid and propionates. Propionic acid (21 CFR 184.1081)
and its salts, calcium (21 CFR 184.1221) and sodium propionate (21 CFR
184.1784), are approved as GRAS substances for miscellaneous and
general purpose usage. In addition, calcium and sodium propionates are
listed as antimycotics when migrating from food-packaging material
(21 CFR 181.23). No upper limits are prescribed for use of this additive,
excepting bread and rolls, which conform to standards of identity. The
acceptable daily intake for humans is presented in Table 2. Propionic
acid at pH 5.5 inhibited the growth of Salmonella at a higher pH value
than other acids (pH 5.4, acetic; pH 5.1, adipic; pH 4.6, succinic; pH 4.4,
lactic; pH 4.3, fumaric and malic; pH 4.1, tartaric; pH 4.05, citric)
(Chung & Goepfert, 1970). Cherrington et al. (1991) determined that
0.5 to 0.7 M propionic acid at pH 5.0 was more inhibitory in 60 min
compared to formic acid in 3 h for E. coli K12 and Salmonella. At a
concentration of 5 mM, the rate of RNA, DNA, protein, lipid, and cell-
wall synthesis decreased, which led to an increase in cell mass without
cell division and a decrease in viability (Cherrington et al., 1990).
Inhibitory activity in BHI was evaluated for S. Typhimurium ST-10
supplemented with propionic acid and chloride salts (NaCl and KCl);
reductions of 1.2 and 2.3 log (CFU/ml) were obtained for propionic acid
(50 mM) + NaCl (500 mM) and propionic acid (25 mM) + mixture
salts of KCl–NaCl (375 mM) respectively (Hinton, 1999). Sodium
propionate minimum inhibitory concentration (0.31%) was lower
718 E. Mani-López et al. / Food Research International 45 (2012) 713–721
than sodium citrate and lactate (1.25%) for S. Typhimurium in chicken
juice at 37 and 42 °C (Milillo & Ricke, 2010). Dubal et al. (2004)
observed a complete inhibition of S. Typhimurium (103 CFU/g) on
forequarters of sheep meat when they spray them with a mixture of
acetic and propionic acids (1.5 + 1.5%). Few reports are available
regarding the inhibitory abilities of propionic acid on Salmonella, thus
more studies are necessary. Despite, these few studies show that
sodium propionate is more effective to inhibit Salmonella than currently
organic acid salts (citrate and lactate). Propionic acid and its salts have
promising applications on meat and poultry products.
1.3.4.2. Succinic acid. Succinic acid is a non-hygroscopic, dicarboxylic
acid having low water solubility. It has a slightly bitter taste and serves
as a flavor enhancer. Succinic acid is used to modify the plasticity of
bread dough and in the production of edible fats. It can be
incorporated into gelatin deserts and cake flavorings (Gardner,
1972). Succinic acid is approved as a GRAS substance for miscella-
neous and general-purpose usage (21 CFR 184.1091). No limit has
been set on the acceptable daily intake for humans (Table 2). Succinic
acid successfully lowered microbial loads on poultry carcasses
(Mountney & O'Malley, 1965); however a 3% or 5% concentration
used at 60 °C impaired the appearance of the product (Cox, Mercuri,
Juven, Thomson, & Chew, 1974). Thomson et al. (1967) reduced the
population of inoculated S. Typhimurium by 60% on chicken carcasses
after spraying with 1.0% succinic acid. Succinic acid has not been used
as much as lactic or acetic acid but the previous studies point a
potential antimicrobial for meat and poultry products. More studies
are necessary in order to use this acid in the industry.
1.3.4.3. Tartaric acid, malic acid, and their salts. Tartaric and L-malic
acids are dicarboxylic organic acids found naturally in various fruiting
plants and berries. Both are approved for direct addition to foods and
affirmed as GRAS (21CFR184). Doores (2005) reported that both acids
function as antimicrobials by acidification, though their inhibitory
potential is not as great as other organic acids. In addition to reported
antibacterial activity, both have demonstrated antifungal activity. S.
Typhimurium inoculated and incubated in BHI broth and after 24-h
incubation, was reduced approximately 2 log CFU/ml with L-tartaric
acid at 37.5 mM (Over et al., 2009). L-malic acid and L-tartaric acid at
75 mM reduced bacterial counts of S. Typhimurium by 4 log CFU/ml
within 12 h after inoculation in BHI (Over et al., 2009). Tamblyn and
Conner (1997b) studied various organic acids to remove S. Typhimur-
ium from chicken breast skin and they observed that 0.5% and 1% of
malic acid reduced 1.2 and 2.16 log CFU respectively when the acid
was applied in chiller conditions (0 °C for 60 min), and while scalder
conditions (50 °C for 2 min) reduced 1.62 log (0.5%) and 2.75 log (1%).
Meanwhile tartaric acid had similar results only under scalder
conditions (50 °C for 2 min) and reduced 2.64 and 1.23 log CFU at
0.5 and 1%, respectively (Tamblyn & Conner, 1997b). Over et al.
(2009) also tested the inhibition of S. Typhimurium with 150 mM of
malic acid and tartaric acid vacuum infusion in boneless/skinless
chicken breast meat (cubes) and reported undetectable counts after
9 days of storage at 4 °C. Raybaudi-Massilia, Mosqueda-Melgar,
Sobrino-López, Soliva-Fortuny, and Martín-Belloso (2009) reported
a reduction of 3.5 logs CFU/g of S. Enteritidis when fresh-cut apples
were dipped in antioxidant mixture added with malic acid. An
alternative application for malic acid was proposed when tested as
antimicrobial in a whey protein coating on turkey frankfurter system,
S. Typhimurium was inoculated on the surface of the system, counts
were lowered by 2.6 and 3.3 log for 2 and 3% concentrations,
respectively (Gadang, Hettiarachchy, Johnson, & Owens, 2008).
Tartaric and malic acids have been recently tested as antimicrobial
in poultry products, and good inhibitory action against Salmonella has
been found. Studies in beef and pork are necessary. These acids have a
great potential in the meat and poultry industries because their
efficacy.
1.3.4.4. Fat effects on antimicrobial activity of organic acids. Meat is
defined as the flesh of animals used as food. Meat and poultry flesh
generally have fat. Several studies proposed a favorable role of fat in
bacterial decontamination. In general cells attached to fat tissue were
more sensitive to the effects of the acid. Previous reports (Dickson,
1988, 1992) indicated that S. Typhimurium cells attached to fat tissue
were generally more sensitive to sanitizers than those attached to
lean tissue. S. Typhimurium reductions were greater when the
bacteria were attached to fatty tissue, possibly because this tissue
retained less moisture (20%) than lean tissue (75%) and the reduced
water activity could have enhanced the antimicrobial effects (Acuff,
2005). With less surface moisture, there would be less water to dilute
the acid at the tissue/fluid interface. It is plausible that moisture in the
lean tissue may have either diluted the acid or solubilized components
of the tissue cells, which buffered the acid on the surface. Alternately,
the acid could be absorbed by the tissue cells at a faster rate than by
the bacterial cells, which would account for the increased observed
resistance of the bacteria to acetic acid on lean tissue (Samelis, 2005).
1.4. Salmonella resistance to organic acids
Several benefits and the usefulness of organic acids to inhibit or
decrease counts of pathogens like Salmonella in meat and poultry
products have been presented. However, lethal effects of these weak
acids depend on concentration, pH of the environment and the
dissociation constant of each acid. Sometimes, treatments are not
optimal and sub-lethal conditions can induce the development of
adapted or resistant strains. Sub-lethal conditions are generated by
exposing and adapting the organism to a mildly acidic pH (pH 5.8) for
one generation thus increasing its tolerance to more extreme acid
conditions (pH 3.0) (Foster & Hall, 1990). Salmonella specifically has
the ability to adjust to acid environments (i.e., foods) and survive in
drastic conditions of pH. Salmonella's mechanism for acid resistance
has been established and it is fairly complex. Foster (1995) discovered
the acid tolerance response that protects Salmonella against the lethal
effects of organic acids, especially at low pH values (pH 4.4). Inducible
acid tolerance response (ATR) is a two-stage process involving
overlapping acid protection systems triggered at different levels of
acidity. Encounters with external pH (pH0) 6 is the first stage (pre-
acid shock); second stage (post-acid shock) is engaged once pH0 falls
below 4.5. In this stage approximately 50 acid shock proteins (ASP)
are necessary to repair cell damage (Bearson, Bearson, & Foster, 1997).
Salmonella required different signals to induce the synthesis of acid
shock proteins among internal or external pH, or both (Foster, 1995).
Only adapted cells are able to synthesize ASP. Several inducible amino
acid decarboxylases appear to contribute to emergency pH internal
maintenance in S. Typhimurium. Lysine decarboxylase (CadA)–lysine-
cadaverine antiporter (CadB) and regulatory proteins like RpoS, Fur
and PhoP are elemental in Salmonella survival in emergency pH
homeostasis (Bearson et al., 1997). Álvarez-Ordóñez et al. (2009)
determined the ability of S. Typhimurium to activate its ATR
phenotype following growth in BHI acidified to pH 4.5 with various
acids, including malic acid. Compared to controls, malic acid exposure
resulted in significant (p b 0.05) longer generation times and lag
phase, but not in time to achieve the stationary phase, thus indicating
that the pathogen was able to adapt to the acid.
Interestingly, the pathogen's heat resistance also increased acid
resistance, increasing the time to inactivate 1.0 log from 9.6 min to
N42 min (Álvarez-Ordóñez et al., 2009). Adaptation times to generate
resistance differed according to the tested organic acid (Greenacre,
Brocklehurst, Waspe, Wilson, & Wilson, 2003). For example, at pH 5.5
S. Typhimurium required an adaptation time of only 2 h for the
optimum ATR to be induced by using acetic acid but 3 h when lactic
acid was employed. Samelis, Ikeda, and Sofos (2003) observed that
glucose (1%) can increase resistance in two strains of S. Typhimurium
to lactic acid (pH 3.5 y 3.7) and acetic acid (pH 3.7). Álvarez-Ordóñez
 E. Mani-López et al. / Food Research International 45 (2012) 713–721
et al. (2010) found that growth temperature is an important factor
modifying bacterial cell composition and physiology, and, therefore,
affecting the bacterial resistance to environmental stresses, including
acid resistance. The most acid resistant cells were those grown at their
optimal temperature (37 °C), decreasing the acid resistance at
temperatures both above and below this temperature.
Acid adaptation response has been extensively reported for S.
Typhimurium (Álvarez-Ordóñez et al., 2009; Bearson, Benjamin,
Swords, & Foster, 1996; Bearson, Wilson, & Foster, 1998; Foster & Hall,
1990; Greenacre et al., 2003; Lee, Slonczewski, & Foster, 1994), and
represents a great concern for meat and poultry industries, since during
meat product processing, microorganisms encounter several mildly
acidic conditions which may induce an ATR and may also be responsible
for an increased ability to survive to other lethal stresses, including
otherwise fatal low pH exposures associated with stomach and
intracellular environments (Tosun & Gönül, 2003; Wilde, Jorgensen,
Campbell, Rowbury, & Humphrey, 2000; Wilmes-Riesenberg, Bearson,
Foster, & Curtiss, 1996). In order to minimize chances for chemical meat
decontamination to enhance the development of stress adaptation and
resistance in pathogenic bacteria, it is of vital importance to inactivate
the target bacteria during decontamination. Proper doses, variety of
acids, temperature control and good manufacturing practices are
important processing parameters to adequately treat meat and poultry
products in order to reduce the initial levels of pathogens to, or near to,
undetectable levels.
2. Final remarks
Despite information available and government programs efforts to
reduce the incidence of Salmonella, it continues to be a concern for the
meat and poultry industries. Organic acids are excellent antimicro-
bials against bacteria including Salmonella. Organic acids offer several
advantages as antimicrobials because they are GRAS, have no limited
acceptable daily intake, are low-cost, easy to manipulate, and effect
minor sensory changes on the product. However, it is important to use
these acids according to good manufacture practices in order to avoid
the development of Salmonella strains resistant to acidic conditions.
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