Food Control 14 (2003) 197–200

www.elsevier.com/locate/foodcont

Screening of lactic acid bacteria from Brazilian meats

for bacteriocin formation
E.C.P. De Martinis *, F.Z. Freitas
Faculdade de Ci^
encias Farmac^
euticas de Ribeir~
ao Preto, Departamento de Analises Clınicas, Toxicol
ogicas e Bromatol
ogicas,
Universidade de S~
ao Paulo, Av. do Cafe s/n, CEP 14040-903, Ribeir~ ao Preto, SP, Brazil
Received 2 February 2002; received in revised form 1 May 2002; accepted 7 May 2002

Abstract
Twenty samples of vacuum-packaged meat products from Brazil were screened for the presence of bacteriocin-producing lactic
acid bacteria, using an agar overlay method. Three bacteriocinogenic isolates were obtained and identified as Enterococcus sp. 18
(from bacon), Leuconostoc sp. 20 (from ham) and Lactobacillus sakei 29 (from hot home made ‘‘ling€ uicßa’’). Leuconostoc sp. 20 and
Lactobacillus sakei 29 presented antilisterial activity, inhibiting all the strains of Listeria monocytogenes tested. However, the strains
did not inhibit Brochothrix thermosphacta, Enterobacter sp., Salmonella sp. or Escherichia coli.
Ó 2003 Elsevier Science Ltd. All rights reserved.

Keywords: Lactic acid bacteria; Brazilian meats; Bacteriocins; Listeria monocytogenes

1. Introduction The use of bacteriocins, the organism which produces

them or both is attractive to the food industry because it
The discovery that carcinogenic nitrosamines are is facing both increasing consumer demand for natural
formed from nitrites used as curing agents in meats products and increasing concern about foodborne dis-
initiated a search for nitrite substitutes (Montville & ease (Montville & Winkowski, 1997).
Winkowski, 1997). Thus, recent approaches in the The objective of the present work was to isolate and
preservation of cooked meats and minimally processed identify LAB with potential of use for extending the
refrigerated foods are increasingly directed towards shelf life and/or increasing the microbiological safety of
biocontrol using a protective microflora, usually lactic vacuum-packaged meat products.
acid bacteria (LAB) to inhibit growth of Listeria
monocytogenes and other microorganisms that are
recalcitrant to traditional food preservation methods 2. Materials and methods
(Bredholt, Nesbakken, & Holck, 2001; Montville &
Winkowski, 1997). The products screened for bacteriocin-producing
LAB are considered as food grade organisms that are LAB comprised twenty samples of vacuum-packaged
safe to consume and have a long history of use in food meats: three brands of ‘‘mortadela’’ (a kind of cooked
(Bredholt et al., 2001). They have the potential to be sausage), two brands of smoked sliced lean pork, sliced
used in biopreservation due their ability to produce ham, turkey frank, smoked sliced ‘‘Chester’’ (poultry
several antimicrobial substances including lactic acid, meat), beef frank, cubed bacon, smoked sliced ham,
diacetyl, hydrogen peroxide and bacteriocins (Daeschel, chicken frank, ‘‘Chester’’ frank (poultry meat), spiced
1989; Schillinger, Geisen, & Holzapfel, 1996). Bacte- cubed beef, ‘‘jerked’’ beef (a cured product), beef,
riocins are ribosomally synthesised antimicrobial pep- chicken filet, hot home-made ‘‘ling€ uicßa’’ (a kind of
tides that are not lethal to the producing cells (Montville sausage) and ground beef. All the samples were pur-
& Kaiser, 1993). chased at retail market in Ribeir~ao Preto city, S~ao Paulo
State, Brazil.
The isolation of bacteriocinogenic LAB was per-
*
Corresponding author. Fax: +55-16-6331936. formed according to De Martinis, P ublio, Santarosa,
E-mail address: [email protected] (E.C.P. De Martinis). and Freitas (2001). Briefly, decimal dilution of the
0956-7135/03/$ - see front matter Ó 2003 Elsevier Science Ltd. All rights reserved.
PII: S 0 9 5 6 - 7 1 3 5 ( 0 2 ) 0 0 0 9 2 - 0
198 E.C.P. De Martinis, F.Z. Freitas / Food Control 14 (2003) 197–200

Table 1 Table 2
Spectrum of inhibitory activity of LAB isolates, measured as the width Source of isolation and identification of bacteriocinogenic LAB
of the inhibition halo (mm), from the border of the producer culture to Isolate Source Identification
the border of inhibition zone
LAB 18 bacon Enterococcus sp. 18
Indicator strain LAB 18 LAB 20 LAB 29 LAB 20 ham Leuconostoc sp. 20
Lactobacillus sakei ATCC 15521 08 11 12 LAB 29 hot home made ‘‘ling€
uicßa’’ Lactobacillus sakei 29
Listeria monocytogenes 184A – 07 14
Listeria monocytogenes 186A – 08 11
Listeria monocytogenes 197A – 08 13
Cell morphology and biochemical tests were used to
Listeria monocytogenes 198A – 08 09
Listeria monocytogenes 211A – 08 13 identify the isolates and the results are shown in Tables 3
Listeria monocytogenes 212A – 07 09 and 4. The results obtained with rapid test API 50 CH
Listeria monocytogenes 226A – 07 09 from bioMerieux (France) allowed the identification of
Listeria monocytogenes 244A – 11 12 LAB 18 as Lactobacillus paracasei, with good correla-
Listeria monocytogenes 250A – 07 10
tion at the genus level. However, considering the cell
Leuconostoc mesenteroides A13B – 07 –
Lactobacillus plantarum 33C – 08 07 morphology, the API identification was not acceptable.
Brochothrix thermosphacta C21B – – – LAB 18 was finally identified according to Stiles and
Brochothrix thermosphacta D18D – – – Holzapfel (1997). The characteristics were compatible
Salmonella sp. IALRPE – – – with the genus Enterococcus: Gram positive, catalase
Enterobacter sp. IALRPE – – –
negative, grew at 10 and 45 °C, grew in the presence of
Escherichia coli ATCC 29522 – – –
6.5% sodium chloride, produced only the L -isomer of
A ¼ isolated from chicken meat; B ¼ isolated from beef; C ¼ isolated
lactate and survived heating at 60 °C for 30 min. En-
from ground beef; D ¼ isolated from irradiated beef; E ¼ clinical iso-
lates. terococci may be used as starter cultures in some foods
and are commercially available as probiotic cultures for
preventing and treating intestinal disorders in animals
samples were prepared and plated on surface in MRS
and humans (Stiles & Holzapfel, 1997). However, this
agar plates in duplicate and incubated at 25 °C, for 72 h.
genus may also be negatively associated with foods due
One of the replicate plates (with up to 100 UFC) was
to its possible implication as an indicator of faecal
overlayed with semi-solid BHI (Oxoid) seeded with L.
contamination (Franz, Holzapfel, & Stiles, 1999).
sakei ATCC 15521. The plates showing inhibition zones
In relation to strain 20, the results for API 50 CH
were replicated to tryptic soy agar plus 0.6% yeast ex-
yielded an unacceptable profile. Nonetheless, with the
tract (Oxoid) and incubated under anaerobiosis. Inhib-
use of the schemes of Schillinger and L€ ucke (1987) for
itory activity of selected colonies was confirmed with the
classification of LAB, it could be assigned to the genus
spot-on-the-lawn assay (Lewus, Kaiser, & Montville,
Leuconostoc. Cells of LAB 20 were Gram positive,
1991). Inhibition due to lytic bacteriophages and due to
produced gas from glucose, did not hydrolyse arginine
hydrogen peroxide production were ruled out using the
and produced only D -lactate.
technique described by Lewus et al. (1991). The protei-
naceous nature of the inhibitor was demonstrated by its
sensitivity to proteases type XIV from Streptomyces
Table 3
griseus (Sigma) and a-chymotrypsin, as described by De Morphological and biochemical characteristics of LAB isolates
Martinis et al. (2001).
Test LAB 18 LAB 20 LAB 29
The bacteriocin-producing isolates were identified
Catalase – – –
based on carbohydrate fermentation patterns obtained Morphology cocci cocci rods
with API 50 CH (bioMerieux, France), complemented Gram þ þ þ
with the tests recommended by Schillinger and L€ ucke, KOH reaction – – –
1987 and Stiles and Holzapfel (1997). Gas from glucose – þ –
The spectrum of inhibitory activity was studied using Growth at 4° C – þ þ
Growth at 10° C þ þ þ
the spot-on-the-lawn assay according to Lewus et al. Growth at 15° C þ þ þ
(1991) with the indicators shown in Table 1. Growth at 45° C þ – –
Growth at pH 3.9 – – –
Growth at NaCl 7.0% þ þ þ
3. Results and discussion Growth at 10% – – –
Slime from sucrose – þ –
H2 S formation þ – þ
The methodology applied led to the isolation of three Arginine hydrolysis þ – –
strains with confirmed inhibitory activity due to bacte- D / L -lactate L- D- D/L-
riocin production. LAB 18 was isolated from bacon, Survival at 60 °C/30 min. þ ND ND
LAB 20 from ham and LAB 29 from hot home made Acetoin production þ – þ
‘‘ling€
uicßa’’ (Table 2). ND ¼ not determined.
 E.C.P. De Martinis, F.Z. Freitas / Food Control 14 (2003) 197–200 199

Table 4 The results of the evaluation of the inhibitory activity

Carbohydrate fermentation pattern of LAB isolates of the bacteriocin-producing LAB isolates towards a
Carbohydrate 18 20 29 panel of microorganisms are presented in Table 1.
Control – – – Leuconostoc sp. 20 and Lactobacillus sakei 29 presented
Glycerol þ – – antilisterial activity. All the Gram negative indicators
Erythritol – – –
used were not inhibited by any of the bacteriocins pro-
D -arabinose – – –
L -arabinose – – þ duced. This result is not surprising and this ineffective-
Ribose þ ? þ ness of bacteriocin-producing LAB in inhibiting Gram
D -xylose – – – negative organisms has been reported previously by
L -xylose – – – Mathieu, Suwandhi, Rekhif, Milliere, and Lefebvre
Adonitol – – –
(1993) and Hechard, Derijard, Letellier, and Cenati-
b-methyl-D -xyloside – – –
Galactose þ – þ empo (1992). It may be attributed to the lipopolysac-
D -glucose þ þ þ charide layer of the cell wall, which protects the cell
D -fructose þ þ þ membrane, the site of action of bacteriocins (Stevens,
D -mannose þ ? þ Sheldon, Klapes, & Klaenhammer, 1991).
L -sorbose – – –
Two strains of Brochothrix thermosphacta were also
Rhamnose – – –
Dulcitol – – – used as indicators: the wild type B. thermosphacta 21
Inositol – – – and a radioduric strain B. thermosphacta 18. Neither
Mannitol þ – – strain was inhibited by our LAB isolates. Daba et al.
Sorbitol þ – – (1991) reported similar results when studying bacterio-
a-methyl-D -mannoside – ? –
cin-producing Leuconostoc mesenteroides UL5. A clini-
a-methyl-D -glucoside – ? –
N-acetyl-glucosamine þ – þ cal isolate of Enterobacter sp. was not affected by any of
Amygdalin þ – ? the LAB strains. Leuconostoc mesenteroides A13 and
Arbutin þ – þ Lactobacillus plantarum 33 were inhibited only by bac-
Esculin þ þ þ teriocin-producer Leuconostoc sp. 20.
Salicin þ – þ
The LAB isolates are currently under further in-
Cellobiose þ ? ?
Maltose þ – – vestigation to implement their application in meat
Lactose – – – systems.
Melibiose – – þ
Sucrose þ þ þ
Trehalose þ þ –
Inulin – – –
Acknowledgements
Melezitose þ – –
D -raffinose – – –
Starch – – – This work was supported by a research grant from
Glycogen – – – Fundacß~ao de Amparo a Pesquisa do Estado de S~ ao
Xylitol – – – Paulo (1998/10759-2). The authors are grateful to Prof.
b gentiobiose þ ? –
Maria Teresa Destro, Ph.D. and Prof. Mariza Landgraf,
D -turanose – þ –
D -lyxose – – – Ph.D., from Faculdade de Ci^encias Farmac^euticas,
D -tagatose þ – – Universidade de S~ao Paulo, for donating some indicator
D -fuccose – – – strains used in this study.
L -fuccose – – –
D -arabitol – – –
L -arabitol – – –
Gluconate þ ? ? References
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