Braz. J. Food Technol., Campinas, v. 13, n. 1, p. 18-22, jan./mar. 2010
DOI: 10.4260/BJFT2010130100003
Scientific note:
Antagonistic activity of lactic acid bacteria isolated from artisan italian salami
Nota científica:
Atividade antagônica de bactérias ácido lácticas isoladas de salame artesanal tipo italiano
Autores | Authors
Sidney Emílio BORDIGNON
JUNIOR
Universidade do Oeste de Santa Catarina
Curso de Pós-Graduação em Controle de
Qualidade em Alimentos
e-mail: [email protected]
Daiana Jaqueline GATTI
Vinhos Iomerê
e-mail: [email protected]
Jane Mary Lafayette Neves
GELINSKI
Universidade do Oeste de Santa Catarina
Núcleo de Biotecnologia
Laboratório de Microbiologia de Alimentos
Bloco K, Rua Paese, 198
Bairro Universitário
CEP: 89560-000
Videira/SC - Brasil
e-mail: [email protected]
Autor Correspondente | Corresponding Author
Recebido | Received: 03/02/2009
Aprovado | Approved: 25/03/2010
Summary
The aim of this research was to select strains of lactic acid bacteria with
antagonistic activity against bacteria of importance in food. One hundred and fifty
three lactic acid bacteria strains were isolated from Italian artisan salami elaborated
without the use of starter culture. Two tests were used to verify the antagonistic
activity of the isolated strains: the indirect inhibition method and the well diffusion
method. Three species of lactic acid bacteria (Lactobacillus brevis; Lb. plantarum;
and Lb. delbrueckii subsp. delbrueckii) showed antagonistic activity for at least one
of the indicator microorganisms: Listeria monocytogenes, Staphylococcus aureus
ATCC 25923 and Leuconostoc mesenteroides subsp. cremoris CCT 0672. It was
shown that strains of lactic acid bacteria of the same specie, mainly Lb. brevis,
presented differences with respect to the pattern of carbohydrate assimilation. The
products analyzed presented very low numbers of lactic acid bacteria (approx.
103 CFUg-1 per sample). It was concluded that in the region where the Italian salami
was made there was a convenient source of lactic acid bacteria with antagonistic
activity against undesirable microorganisms in food products.
Key words: Lactobacillus; Antagonism; Cured meat product.
Resumo
O objetivo da pesquisa foi selecionar cepas de bactérias lácticas com
atividade antagônica a outros micro-organismos de importância em alimentos.
Foram isoladas cento e cinquenta e três cepas de bactérias lácticas a partir
de amostras de salames elaborados artesanalmente sem adição de culturas
iniciadoras de fermentação. Dois tipos de ensaios foram usados para verificar a
atividade inibitória das cepas isoladas: método de inibição indireta e por difusão
em ágar. Três espécies de bactérias lácticas (Lactobacillus brevis, Lb. plantarum
e Lb. delbrueckii subesp. delbrueckii) apresentaram atividade antagônica
aos micro-organismos indicadores: Listeria monocytogenes, Staphylococcus
aureus ATCC 25923 e Leuconostoc mesenteroides subesp. cremoris CCT 0672.
Verificou-se que cepas de bactérias lácticas da mesma espécie, principalmente
Lb. brevis, apresentaram diferenças no padrão de assimilação de carboidratos.
Os produtos analisados apresentaram cerca de 103 UFC g-1 de bactérias ácido
lácticas, valor esse que, embora possa ser considerado baixo, permite concluir
que, na região em que os salames foram elaborados, existe uma fonte conveniente
de bactérias lácticas com atividade antagônica a micro-organismos indesejáveis
em alimentos.
Palavras-chave: Lactobacillus; Antagonismo; Produto cárneo curado.
www.ital.sp.gov.br/bj
Scientific note:
Antagonistic activity of lactic acid bacteria isolated from artisan italian salami
BORDIGNON JUNIOR, S. E. et al.
1 Introduction
Lactic acid bacteria have extensive applications in
a great variety of foods, such as products derived from
milk, vegetables and meats. These bacteria produce
substances that have antagonistic activity against
pathogenic and spoilage bacteria found in food products
and can be used as starter cultures for cured meat
products.
Gram-positive lactic acid bacteria are aerobic,
micro-aerobic or facultative anaerobic microorganisms
with variable metabolic characteristics, such as the
production of diacetyl, hydrogen peroxide, lactic acid
and others. They are divided into two groups based on
their carbohydrate metabolism: those which ferment
sugar by a homolactic pathway, producing only lactic
acid as an end-product (via Embedeb-Meyehof), and
those which ferment by a heterolactic pathway, producing
similar amounts of lactic acid, ethanol and carbon dioxide
via the pentose or monophosphate hexose pathway
(ROSS et al., 2002). Lactic acid bacteria are widely
studied and used in the food industry (KONINGS et al.,
2000; AYAD et al., 2004). They are responsible for
many of the microbial transformations found in several
products, such as wines, beer, bread and cured meats,
amongst others (ROSS et al., 2002). In general, the
lactic bacteria are important because they can produce
substances with pleasant sensory characteristics during
meat processing (HUGAS, 1998). Moreover, the action
of various antimicrobial compounds produced during the
fermentation process, such as lactic acid, acetic acid and
propionic acid, may result in an unfavourable environment
for the development of spoilage and pathogenic
microorganisms (DE VUYST, VANDAMME, 1993 apud
HELANDER et al., 1997). These microorganisms, such
as members of the families Enterobacteriaceae and
Pseudomonadaceae (ALAKOMI et al., 2000), may occur
in cured meat products when the hygienic conditions are
poor during the manufacturing process.
The lactic acid bacteria are efficient in producing
substances with inhibitory activity against microorganisms
present in foods (DE MARTINIS and FREITAS, 2003).
Currently the major interest in using lactic bacteria is
that the undesirable microorganisms in foods may be
inhibited by one of the bacteriocin-producing lactic acid
bacteria. Bacteriocins are microbial compounds of a
proteic nature that have a bactericidal or bacteriostatic
effect on other closely related species (KLAENHAMMER,
1993; STILES, 1996). Many types of bacteriocins have
been characterized and they have considerable potential
for application in foods, aiming at the quality and safety
of these foods (DE MARTINIS et al., 2002). According
to Nascimento et al. (2008) bacteriocins can be used in
three different ways in fermented foods: in situ production
Braz. J. Food Technol., Campinas, v. 13, n. 1, p. 18-22, jan./mar. 2010
by the addition of a bacteriocinogenic lactic culture as a
co-culture or by the direct addition of the bacteriocin.
In this study, lactic acid bacteria were previously
isolated from artisan Italian salami with the aim of selecting
lactic acid bacteria strains with inhibitory activity against
bacteria of importance in foods.
2 Material and methods
2.1 Sampling and microbiological analysis
Ten different types of artisan Italian salami products
were obtained from local markets in cities in the State of
Santa Catarina, Brazil. All the products were made without
the intentional addition of starter cultures. The expiry
date for each product was not less than 15 days and not
greater than 30 days after production. The samples were
collected, processed aseptically and the pH registered.
2.2 Isolation of lactic acid bacteria
The salami samples were serially diluted (1:10) in
a sterile saline water solution (0.85%) and plated on MRS
agar (Oxoid, England), and incubated at 30 ºC for 48 h
under microaerobic conditions using a CO2 generator
(Anaerogen™, Oxoid). The isolates were selected based
on their Gram staining patterns and submitted to the
catalase test. The selected colonies were sub cultured
in TSB-Tryptic Soy Broth (Oxoid, England) and stored at
–20 ºC in MRS broth supplemented with 20% glycerol.
When necessary, the lactic bacteria were revived from
the frozen glycerol stocks and inoculated into MRS broth
containing 0.5% glucose for use.
2.3 Antagonistic activity
Two assays (indirect inhibition and well diffusion
methods) were tested to evaluate the antagonistic
activity of lactic bacteria against four aerobic indicator
microorganisms: Listeria monocytogenes Scott A
(FOEGEDING et al.,1992), Salmonella Typhimurium
(GELINSKI, 2003), Escherichia coli ATCC 25922,
Staphylococcus aureus ATCC 25923 and one facultative
anaerobic microorganism: Leuconostoc mesenteroides
subsp. cremoris CCT 0672 (from the André Tosello
Foundation, Brazil):
1)The previously isolated lactic acid bacteria
were cultured onto MRS agar plates and
incubated under micro-aerobic conditions for
48 h (Anaerobic System Anaerogen, Oxoid) at
30 ºC. Visible colonies were overlayed with soft
agar (TSB-Tryptic Soy Broth plus 0.9% agar
[Oxoid, England]) which contained approx. 107
CFU.mL–1 of an overnight culture of an indicator
microorganism. The new agar plates were
incubated at 30 or 37 ºC under aerobic or micro19
www.ital.sp.gov.br/bj
Scientific note:
Antagonistic activity of lactic acid bacteria isolated from artisan italian salami
BORDIGNON JUNIOR, S. E. et al.
aerobic conditions (according to the indicator
microorganism) for 48 h until growth or a visible
inhibition zone; and
2)Colonies that had shown inhibitory activity on
TSA were transferred from the master plate
to a new MRS agar plates and subsequently
assayed using the well diffusion agar technique
(HARRIS et al., 1989). The lactic bacteria
cultures were cultivated in MRS broth under
micro-aerophilic conditions at 30 ºC until the
end of the log phase. The cultures were then
harvested by centrifugation (4.470 x g/10 min)
and the pH of each cell-free supernatant adjusted
to 6.0 with 1.0 M NaOH and filtered through a
0.22 µm pore size sterile filter (Millipore, U.S.A).
Forty microliters of sterile cell-free supernatant
were added to one of the 5 mm diameter wells
in the agar plates (TSB plus 1% agar and 1%
of the indicator culture). Specifically in the
case of Listeria monocytogenes Scott A, 0.6%
yeast extract was also added to the soft agar. A
positive control was prepared by adding 40 µL of
a 100 IU.mL–1 nisin solution (Nisaplin®, Danisco,
England). The negative control was represented
by a well containing 40 µL of sterile TSB. The agar
plates were incubated for 24-48 h at 37 ºC under
aerobic or micro-aerobic conditions (according
to the indicator microorganism). Inhibitory
activity was detected by the formation of an
inhibition zone and measured from the centre of
the well containing the culture supernatant to the
periphery of the inhibition zone. Strains of lactic
bacteria responsible for some type of inhibition
were recovered from the corresponding mother
plate and sub cultured into MRS agar plates and
identified using Gram staining, catalase test and
KOH reaction. All of the cultures were maintained
at –20 ºC in MRS broth supplemented with 20%
glycerol until use.
manufactured without the addition of starter cultures
are more susceptible to spoilage and to pathogenic
microorganisms. Lactic acid and other final products of
their metabolism act as bio-preservatives increasing the
shelf-life of the food (DEEGAN et al., 2006), and reducing
the risks of foodborne diseases (MACIEL et al., 2003).
Thus the presence of lactic acid bacteria may confer
desirable qualities and increase the safety of fermented
products (ADAMS and NICOLAIDES, 1997).
The five colonies of lactic bacteria isolated from
samples of artisan Italian salami showed a milky, rounded
aspect and were white in colour, with an average diameter
of 2.0 mm. The isolates Lactobacillus brevis (two strains),
L. delbrueckii subsp. delbrueckii (one strain), and
L. plantarum (two strains) were Gram positive, catalase
negative, KOH negative rods and did not produce gas
from glucose. All the isolates only grew at 15 ºC, except
L. delbrueckii subsp. delbrueckii, which only grew at
45 ºC.
Of the 153 strains showing some inhibitory activity
in the indirect inhibition assay only the five strains of
lactobacilli maintained the same pattern of inhibitory
activity in the well diffusion assay with at least one of
the following indicator microorganisms: Staphylococcus
aureus 25923, Leuconostoc mesenteroides subsp.
cremoris and the pathogenic microorganism Listeria
monocytogenes (Table 1). In the indirect inhibition method,
growing colonies of the microorganism were inoculated
under a layer of indicator microorganisms. In this method
the action of various inhibitory substances produced by
bacteria (lactic acid, diacetyl, hydrogen peroxide etc)
may occur, while in the well diffusion assay the pH of the
supernatant of the lactic culture is neutralized (pH 5.5-6.0).
Therefore the action of lactic acid was not considered
in these experiments. The activity of hydrogen peroxide
was also not considered because all the lactic cultures
were maintained under micro-aerobic conditions for
the production of cell-free supernatants containing the
inhibitory substance.
I n t h e c u r re n t re s e a rc h s t r a i n s o f l a c t i c
bacteria showing antimicrobial activity to one or more
microorganisms considered important as contaminants
and/or spoilage of foods, were isolated. A very low number
of lactic bacteria (approx. 103 CFU.g–1 per sample) were
detected, which could be considered unfavourable
from the point of view of the artisan products. Products
Table 1. Results for the antagonistic activity of lactic acid
bacteria strains isolated from artisan Italian salami.
Lactic Acid bacteria
Antagonistic action against
Lactobacillus brevis
Leuconostoc mesenteroides
subsp. cremoris
Lactobacillus brevis
L.mesenteroides subsp.
cremoris
Lactobacillus delbrueckii
L.mesenteroides subsp.
subsp. delbrueckii
cremoris;
Listeria monocytogenes
Lactobacillus plantarum
L. mesenteroides sbsp.
cremoris Staphylococcus
aureus ATCC 25923
Lactobacillus plantarum
Staphylococcus aureus
ATCC 25923
Braz. J. Food Technol., Campinas, v. 13, n. 1, p. 18-22, jan./mar. 2010
20
2.4 Biochemical tests
Only strains of lactic acid bacteria showing
antagonistic activity to one or more microorganisms
were characterized from their carbohydrate fermentation
pattern using API 50 CHL (bioMérrieux, France).
3 Results and discussion
www.ital.sp.gov.br/bj
Scientific note:
Antagonistic activity of lactic acid bacteria isolated from artisan italian salami
BORDIGNON JUNIOR, S. E. et al.
Table 2. Carbohydrate fermentation profile of five lactic acid
bacteria isolates.
Carbohydrate
LAB LAB LAB LAB LAB
1
2
3
4
5
Glycerol
–
–
–
–
–
Erythritol
–
–
–
–
–
D-arabinose
–
–
–
–
–
L-arabinose
+
+
–
+
+
Ribose
+
+
+
+
+
D-xylose
+
–
–
–
–
Adonitol
–
–
–
–
–
b-methyl-D-xylose
–
–
–
+
+
Galactose
+
+
+
–
+
D-glucose
+
+
+
+
+
D-fructose
+
+
+
+
+
D-mannose
–
+
+
+
+
L-sorbose
–
–
–
–
–
Rhamnose
–
–
–
–
–
Dulcitol
–
–
–
–
–
Inositol
–
–
–
–
–
Mannitol
–
–
–
+
+
sorbitol
–
–
–
+
+
α-methyl-D-mannoside
–
–
+
–
–
N-acetyl-glucosamine
+
+
+
+
+
Amygdalin
–
+
–
+
+
Arbutin
–
–
–
+
+
Esculin
+
+
+
+
+
Salicin
–
+
–
+
+
Cellobiose
–
+
–
+
+
Maltose
+
–
–
+
+
Lactose
–
+
–
+
+
Melibiose
+
+
–
+
+
Sucrose
–
+
+
+
+
Threhalose
–
+
+
+
+
Inulin
–
–
–
–
–
Key: + = fermentation; - = no fermentation. LAB 1 = Lactobacillus
brevis; LAB 2 = L. brevis; LAB 3= L. delbrueckii subsp. delbrueckii;
and LAB 4 = L. plantarum; LAB5= L. plantarum. Differences between
strains of the same species are shown in the shaded areas.
The carbohydrate assimilation pattern is shown
in Table 2. The shaded areas show some changes
in carbohydrate assimilation between isolates of
Lactobacillus brevis and a single difference between
isolates of L. plantarum. These differences may result
in different responses in relation to the antagonistic
activity against other microorganisms. In food systems,
it is important to consider that interactions between
different strains exist and this may result in alterations
in the microbial growth rate and metabolic activities
(VINDEROLA et al., 2002).
4 Conclusions
In this study, samples from different artisan
Italian salami products showed very low numbers of
lactic acid bacteria. However, Italian salami can still
be considered as a convenient source of lactic acid
Braz. J. Food Technol., Campinas, v. 13, n. 1, p. 18-22, jan./mar. 2010
bacteria with antagonistic activity against undesirable
microorganisms in foods. Future research will continue
to evaluate the nature of the inhibitory substances as
well the technological properties of the strains obtained
in this study.
It was observed that LAB strains from the same
species may present differences with respect to their
carbohydrate assimilation pattern. Further studies on the
identification of lactic acid bacteria strains of the same
species and their action against pathogens should help
to explain if these differences may result in significant
alterations in the production of inhibitory substances.
Acknowledgements
This work was supported by the Conselho Nacional
de Desenvolvimento Cientifico e Tecnológico-CNPq/
PIBIC-Brazil and the Fundação de Apoio a Pesquisa do
Estado de Santa Catarina-FAPESC.
References
ADAMS, M. R.; NICOLAIDES, L. Review of the sensitivity of
different foodborne pathogens to fermentation. Food Control,
London, v. 8, n. 5-6, p. 227-239, 1997.
ALAKOMI, H. L.; SKYTTA, E.; SAARELA, M.; MATTILASANDHOLM, T.; LATVA-KALA, K.; HELANDER, I. M. Lactic acid
permeabilizes gram-negative by disrupting the outer membrane.
Applied Environmental Microbiology, Washington, v. 66, n. 5,
p. 2001-2005, 2000.
AYAD, E. H. E.; NASHAT, S.; EL-SADEK, N.; METWALY, H.;
EL-SODA, M. Selection of wild lactic acid bacteria isolated from
traditional Egyptian dairy products according to production and
technological criteria. Food Microbiology, London, v. 21, n. 6,
p. 715-725, 2004.
DEEGAN, L. H.; COTTER, P. D.; HILL, C.; ROSS, P. Bacteriocins:
biological tools for biopreservation and shelf-life extension.
International Dairy Journal, London, v. 16, n. 9, p. 1058-1071,
2006.
DE MARTINIS, E. C. P.; ALVES, V. F.; FRANCO, B. D. G. M.
Fundamentals and perspectives for the use of bacteriocins
produced by lactic acid bacteria in meat products. Food Reviews
International, Philadelphia, n. 2-3, p. 191-208, 2002.
DE MARTINIS, E. C. P.; FREITAS, F. Z. Screening of lactic acid
bacteria from Brazilian meats for bacteriocin formation. Food
Control, London, v. 14, n. 3, p. 197-200, 2003.
FOEGEDING, P. M.; THOMAS, A. B.; PILKINGTON, D.
H.; KLAENHAMMER, T. R. Enhanced control of Listeria
monocytogenes by in situ-produced pediocin during dry
fermented sausage production. Applied and Environmental
Microbiology, Washington, v. 58, n. 3, p. 884-890, 1992.
GELINSKI, J. M. L. N. Bacteriocina de Lactobacillus sake
2a: potencial de aplicação em combinação com outras
21
www.ital.sp.gov.br/bj
Scientific note:
Antagonistic activity of lactic acid bacteria isolated from artisan italian salami
BORDIGNON JUNIOR, S. E. et al.
substâncias antimicrobianas na inibição de cepas de
Salmonella de origem alimentar. 2003. 98 f. Tese (Doutorado
em Ciência dos Alimentos) - Universidade de São Paulo, São
Paulo, 2003.
HARRIS, L. J.; DAESCHEL, M. A.; KLAENHAMMER, T. R.
Antimicrobial activity of lactic acid bacteria against Listeria
monocytogenes. Journal of Food Protection, Belo Horizonte,
v. 52, n. 4, p. 384-387, 1989.
HELANDER, I. M.; VON WRIGHT, A.; MATTILA-SANDHOLM,
T. M. Potential of lactic acid bacteria and novel antimicrobials
against Gram-negative bacteria. Trends in Food Science and
Technology, London, v. 8, n. 5, p. 146-150, 1997.
HUGAS, M. Bacteriocinogenic lactic acid bacteria for the
preservation of meat and meat products. Meat Science, London,
v. 49, suppl. 1, p. 139-150, 1998.
KLAENHAMMER, T. R. Genetics of bacteriocins produced by
lactic acid bacteria. FEMS Microbiology Reviews, Weinheim,
v. 12, n. 1-3, p. 39-85, 1993.
Braz. J. Food Technol., Campinas, v. 13, n. 1, p. 18-22, jan./mar. 2010
KONINGS, W. N.; KOK, J.; KUIPERS, O. P.; POOLMAN, B.
Lactic acid bacteria: the bugs of the millennium. Ecology and
Industrial Microbiology, London, v. 3, n. 3, p. 276-282, 2000.
MACIEL, J.; TEIXEIRA, M. A.; MORAES, C. A.; GOMIDE, L. A.
M. Antibacterial activity of lactic acid cultures isolated of Italian
salami. Brazilian Journal of Microbiology, São Paulo, v. 34,
suppl. 1, p. 121-122, 2003.
NASCIMENTO, M. S.; MORENO, I.; KUAYE, A. Y. Bacteriocins em
alimentos: uma revisão. Brazilian Journal of Food Technology,
Campinas, v. 11, n. 2, p. 120-127, 2008.
ROSS, R. P.; MORGAN, S.; HILL, C. Preservation and
fermentation: past, present and future. International Journal of
Food Microbiology, London, v. 79, n. 1-2, p. 3-16, 2002.
STILES, M. E. Biopreservation by lactic acid bacteria. Antonie
van Leeuwenhoek, Netherlands, v. 70, n. 2-4, p. 331-345,
1996.
VINDEROLA, C. G.; MOCCHIUTTI, P.; REINHEIMER, J. A.
Interations among lactic acid starter and probiotic bacteria
used for fermented dairy products. Journal of Dairy Science,
Champaign, v. 85, n. 4, p. 721-729, 2002.
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