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The objective of this study was to evaluate the ability of lactic acid bacteria (LAB) cultures to preserve fresh beef at room temperature, with a view to promoting safety and availability of the product in Nigeria. Two LAB strains, Pediococcus pentosaceus LIV 01 and P. acidilactici FLE 01, were applied as starters (106cfu/g) on sliced fresh beef samples, and were stored for 7days at 30°C. Analyses of microbiological, thiobarbituric acid (TBA) and free fatty acids (FFA) were carried out during storage. Results indicated reduction in the Enterobacteriaceae, Staphylococcus and coliforms in starter inoculated samples. TBA and FFA were lower in starter culture inoculated samples compared to controls during storage. In a challenge experiment against the LAB cultures during a 7-day storage, two sets of meat were inoculated separately with 106cfu/g each of pathogenic organisms Listeria monocytogenes and Salmonella Typhimurium. There was about 1log reduction in the L. monocytogenes on day 1 while counts were below detection limit (<2log) on day 2 in meat samples inoculated with P. pentosaceus alone and in combination with P. acidilactici. Counts of S. Typhimurium showed about 2log reduction in starter inoculated samples during storage while an increase by about 3log was observed in control samples. The protective ability of the LAB strains could be exploited in shelf life extension and control of foodborne pathogens in fresh beef; their use as biological preservatives may help in promoting public health, safety and availability of the product in Nigeria. KeywordsLactic acid bacteria-Foodborne pathogens-Protective ability-Shelf life extension-Public health safety and availability
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ORIGINAL PAPER
Investigation on the potential application of biological agents
in the extension of shelf life of fresh beef in Nigeria
Olusegun A. Olaoye
Abiodun A. Onilude
Received: 19 October 2009 / Accepted: 12 January 2010 / Published online: 6 February 2010
Ó Springer Science+Business Media B.V. 2010
Abstract The objective of this study was to evaluate the
ability of lactic acid bacteria (LAB) cultures to preserve
fresh beef at room temperature, with a view to promoting
safety and availability of the product in Nigeria. Two LAB
strains, Pediococcus pentosaceus LIV 01 and P. acidilac-
tici FLE 01, were applied as starters (10
6
cfu/g) on sliced
fresh beef samples, and were stored for 7 days at 30°C.
Analyses of microbiological, thiobarbituric acid (TBA) and
free fatty acids (FFA) were carried out during storage.
Results indicated reduction in the Enterobacteriaceae,
Staphylococcus and coliforms in starter inoculated sam-
ples. TBA and FFA were lower in starter culture inoculated
samples compared to controls during storage. In a chal-
lenge experiment against the LAB cultures during a 7-day
storage, two sets of meat were inoculated separately with
10
6
cfu/g each of pathogenic organisms Listeria monocyt-
ogenes and Salmonella Typhimurium. There was about
1 log reduction in the L. monocytogenes on day 1 while
counts were below detection limit (\2 log) on day 2 in
meat samples inoculated with P. pentosaceus alone and in
combination with P. acidilactici. Counts of S. Typhimuri-
um showed about 2 log reduction in starter inoculated
samples during storage while an increase by about 3 log
was observed in control samples. The protective ability of
the LAB strains could be exploited in shelf life extension
and control of foodborne pathogens in fresh beef; their use
as biological preservatives may help in promoting public
health, safety and availability of the product in Nigeria.
Keywords Lactic acid bacteria Foodborne pathogens
Protective ability Shelf life extension
Public health safety and availability
Introduction
Food security, the availability of food and its accessibility
to people, has been an important concern in most devel-
oping countries where food preservation techniques have
been very inadequate (Postnote 2006). In Nigeria, this has
adverse effects on beef where losses have been experienced
due to poor storage facilities. Fresh beef is very common in
most countries of West Africa, including Nigeria, Ghana,
Republic of Benin and Cameroon; where it has been one of
the main sources of meat.
Meat is a nutritious, protein-rich food which is highly
perishable and has a short shelf-life unless preservation
methods are used. Shelf life and maintenance of the meat
quality are influenced by a number of interrelated factors
including holding temperature, which can result in detri-
mental changes in the quality attributes of meat. Spoilage
by microbial growth is the most important factor in relation
to the keeping quality of meat (Lambert et al. 1991).
Research findings have suggested that there is increasing
attention on the use of naturally occurring metabolites
produced by selected lactic acid bacteria (LAB) to inhibit
the growth of undesirable microorganisms (Onilude et al.
2002; Kaban and Kaya 2006). LAB growing naturally in
foods produce antimicrobial substances such as lactic and
acetic acids, diacetyl, hydrogen peroxide and bacteriocins
(Olaoye et al. 2008).
O. A. Olaoye (&)
Food Technology Department, The Federal Polytechnic,
PMB 420, Offa, Kwara State, Nigeria
e-mail: olaayosegun@yahoo.com
A. A. Onilude
Microbial Physiology and Biochemistry Unit,
Department of Botany and Microbiology,
University of Ibadan, Ibadan, Nigeria
123
World J Microbiol Biotechnol (2010) 26:1445–1454
DOI 10.1007/s11274-010-0319-5
In fresh meat, many useful strains of LAB can bring
about a mild fermentation process which does not produce
changes in sensory characteristics because of the low car-
bohydrate content and the strong buffering capacity of
meat. In fermented meat the addition of sugar enhances the
growth of LAB thereby lowering the pH of the meat and
causing the characteristic changes in flavour and texture
(Aymerich et al. 1998). Some of the LAB associated with
starter cultures in fermented meat products are Pediococ-
cus acidilactici, P. pentosaceus, Lactobacillus plantarum,
Lb. sake and Lb. curvatus among others (Rodriguez et al.
1995; Aymerich 1996). Meat cannot be pasteurized prior to
the addition of a LAB starter culture, therefore a culture for
its fermentation and biopreservation must compete with the
natural microflora, making the antagonistic products very
important.
LAB have a GRAS (generally regarded as safe) status
and have been widely used as starters in the industrial
preservation of meats (Simpson et al. 2002; Onilude et al.
2002). The ability of LAB to inhibit the growth of unde-
sirable bacteria has been reported and inhibition may be
due to the production of organic acids, hydrogen peroxide,
carbon dioxide, acetaldehyde, diacetyl or bacteriocins
(Helander et al. 1997).
In Nigeria fresh beef forms a significant proportion of
meat intake. It is either eaten cooked or processed into
other forms to avoid associated spoilage. Due to limited
research on the control of spoilage and pathogenic organ-
isms associated with fresh beef in the country, intense
efforts are required to extend shelf life and promote health
and safety among consumers of the product. In the present
study, we investigated the ability of selected strains of
LAB to extend the shelf life of fresh beef at room tem-
perature. Some microbiological and biochemical quality
biomarkers were evaluated on the meat during a 7-day
storage.
Materials and methods
Sources of microbial isolates used and their growth
conditions
The LAB strains, Pediococcus pentosaceus LIV01 and
P. acidilactici FLE01, used as protective cultures on the
fresh beef samples, have been isolated from beef in a
previous study (Olaoye et al. 2008). A non-starter strain
P. acidilactici FLE04, isolated in the same study, was used
as negative control starter. They were cultivated on deMan
Rogossa Sharpe (MRS) at 30°C. Two pathogenic organ-
isms of meat sources, Listeria monocytogenes and Salmo-
nella Typhimurium, were used to challenge the LAB
cultures in situ in the meat during storage. The organisms
were wild types, obtained from the culture collection unit,
University of Ibadan. The two pathogens were grown in
Brain Heart Infusion (BHI, Oxoid, UK) prior to their
inoculation onto the beef samples. Listeria Oxford formu-
lation (Oxoid, UK) was used as selective medium for the
enumeration of L. monocytogenes at 30°C in the beef while
Xylose lysine deoxycholate agar (XLD, Oxoid, UK) was
used for S. Typhimurium (37°C). Bacterial isolates were
maintained in growth broth media containing 20% glycerol
at -20 and -80°C, as working and long-term storage
cultures respectively.
Screening for pediocin production
The cells of the Pediococcus strains (n =
21) were grown
in MRS broth (containing the following in g/l: Dextrose,
20; Beef extract, 8; Yeast extract, 4; Ammonium citrate, 2;
Magnesium sulphate, 0.2; Bacteriological peptone, 10;
Sodium acetate, 5; Dipotassium phosphate, 2; Tween-80, 1;
and Manganese Sulfate, 0.05) for 24 h at 30°C; the broth
cultures were centrifuged at 5,0009g for 15 min (Centri-
fuge Falcon 6/300 series-CFC Free, Lower Sydenham-
London UK). The cell free supernatants (CFS) were col-
lected and pH adjusted to 6.5 (using 1.0 M NaOH) and
treated with 300 U/ml (final concentration) of catalase
(Sigma–Aldrich) to obtain crude pediocin (CP). The CP
was tested for activity against sensitive L. monocytogenes
strain, using the paper disc (6 mm) assay method (Gurira
and Buys 2005).
Production of lactic and acetic acids
Prior to selection of the two isolates as starter cultures, the
LAB strains were evaluated for production of lactic and
acetic acids, using the methods of Olaoye et al. (2008). The
two LAB strains showed considerable production of lactic
and acetic acids of approximately 17 and 10 g/l respec-
tively, which represent 1.7 and 1.0% (w/v); these factors
were used as criteria prior to their choice for further
screening as starters for the fresh beef preservation. How-
ever, the non-starter P. acidilactici FLE04 produced less
than 1 g/l (0.1%, w/v) of each acid.
PCR detection and nucleotide sequencing of pediocin
encoding gene
The P. pentosaceus LIV01 and P. acidilactici FLE01 were
screened for the presence of the gene encoding pediocin
production, although only the former was shown to pro-
duce a bacteriocin-like substance in vitro, DNA was
extracted by a modification of the boiling method described
by Suwanjinda et al. (2007). PCR amplification was carried
out with specific primers targeting 332 bp of the pediocin
1446 World J Microbiol Biotechnol (2010) 26:1445–1454
123
operon in the Pediococcus strain. This was carried out in a
50 ll reaction volume containing 1.25 units of Taq DNA
polymerase (ABgene, Thermo Fischer, UK), 2.5 mM
magnesium chloride (Promega, Southampton, Southampt-
onshire, UK), 0.2 mM dNTPs (Promega), 0.1 ll of each
reverse (5
0
-CTACTAACGCTTGGCTGGCA-3
0
) and for-
ward primer (5
0
-GGTAAGGCTACCACTTGCAT-3
0
), 5 ll
PCR buffer and 5 ll of DNA template. Volume was made
up with sterile deionised water.
Two sets of species-specific primers were used. The first
was made up of 5
0
-TAA AAA GAT ATT TGA CCA AAA-
3
0
(Reverse) and 5
0
-AAA ATA TCT AAC TAA TAC TTG-
3
0
(Forward), targeting the 711 bp of the pediocin operon
of P. acidilactici strain (Mathys et al. 2007). The second
was 5
0
-CTACTAACGCTTGGCTGGCA-3
0
(Reverse) and
5
0
-GGTAAGGCTACCACTTGCAT-3
0
(Forward), target-
ing 332 bp of the pediocin operon of P. pentosaceus strain
(Suwanjinda et al. 2007).
Electrophoresis of the PCR products was performed on
the Bio-Rad Contour-Clamped Homogenous Electric Field
(CHEF) DRII electrophoresis cell (Hemel Hempstead,
UK). Agarose gel (Biogene Kimbolton Cambs, UK), 1.5%
(w/v), stained with 0.5 lg/ml ethidium bromide, was used
in 19 TAE (Tris–Acetate EDTA) buffer at 84 V for 1.5—
2 h. The molecular size markers used include 100 bp and
1 kb (Promega G210A) ladders.
Sequencing of the pediocin genes was done by resolving
40 ll of the PCR products in 1% Agarose gel. Amplicons
were excised from gel, purified using the Wizard PCR
Preps DNA Purification System (Promega) and sent to
Germany (MGW-Biotech, Germany) for sequencing. The
nucleotide sequences were used in the GenBank database
using BLAST at the website, http://www.ncbi.nlm.nih.
gov/blast/, to determine the closest known relatives of the
pediocin gene sequences.
Source of fresh beef samples
Fresh beef samples (already deboned) were purchased from
a Butcher’s shop in the city of Nottingham, United King-
dom. They were carried to the laboratory on ice cubes for
immediate processing and analysis.
Inoculation with starter cultures
The two LAB strains, P. pentosaceus LIV01 and
P. acidilactici FLE01, were subcultured three times in
MRS broth at 30°C for 24 h. The beef samples were cut
into pieces (6.5 9 4 9 0.4 cm), each weighing 15 ± 1.5 g.
They were dipped in filter sterilised glucose solution
(10% w/v) for 10 min, expected to yield a final 3% w/w
concentration in the meat to stimulate LAB growth
(Guerrero et al. 1995).
The beef samples were inoculated with starter cultures
(10
6
cfu/g) in four treatments: PEN, Inoculation with
P. pentosaceus LIV01; PED, Inoculation with P. acidi-
lactici FLE01; MIX, Inoculation with mixed cultures of
P. pentosaceus LIV01 and P. acidilactici FLE01; CONT,
Uninoculated control samples; CONT-NEGATIVE LAB,
Inoculation with non-starter P. acidilactici FLE04. Storage
was carried out at 30°C for 7 days, during which period,
samples were taken for microbiological and biochemical
evaluations every 24 h. The abilities of the LAB starters to
control L. monocytogenes and S. Typhimurium were tested
by inoculating meat samples with 1 9 10
6
cfu/g each of
the pathogens, grown in Brain Heart Infusion agar (BHI,
Oxoid- Hampshire, UK). Sterilised meat samples (by
autoclaving) were used to set a baseline for the microbial
counts of the pathogens.
Evaluation of microbiological quality
Beef samples (10 g) were homogenized in standard stom-
acher bag (BA 6141, Seward, West Sussex, UK) containing
90 ml maximum recovery diluent (MRD) for 3 min at
230 rpm, using a Seward stomacher (model 400 circulator,
P/4/518, 50–60 Hz, UK). One millilitre of homogenate was
serially diluted in 9 ml of MRD producing 10 fold dilu-
tions; 0.1 ml (1 ml for Enterobacteriaceae) samples of
appropriate dilutions were spread plated (poured plated for
Enterobacteriaceae) in replicates on selected agar media.
The media used included deMan Rogosa Sharpe (MRS,
Oxoid) for LAB, incubated at 30°C for 48 h; Rose Bengal
Chloramphenicol Agar (RBCA, Oxoid) for yeast and
moulds at 25° C for 72 h; Mannitol salt phenol red agar,
MSPRA (Sigma–Aldrich, St. Louis, Missouri, USA) for
Staphylococcus at 37°C for 24 h; MacConkey Agar (Ox-
oid) for coliforms at 37°C for 24 h; Violet red bile glucose
agar (VRBGA, Oxoid) for Enterobacteriaceae,at30°C for
48 h; Xylose lysine deoxycholate (XLD, Oxoid) for Sal-
monella at 37°C for 24 h; and Oxford formulation (Oxoid)
for Listeria monocytogenes at 30°C for 48 h. Emerging
colonies were counted and the results expressed in loga-
rithmic scale of colony forming units per gram (log cfu/g)
of meat.
Confirmation of colonies on the selective media
by polymerase chain reaction (PCR)
Isolates emerging from the various selective media, used
for microbial enumeration of the fresh beef samples during
storage, were subjected to confirmation by PCR. Genomic
DNA extraction, PCR amplifications and reactions of 16S
rDNA genes, variable 4 (V4) regions, were performed as
previously described for pediocin gene above. Universal
eubacterial primers, targeting V4 region (*400 bp) of the
World J Microbiol Biotechnol (2010) 26:1445–1454 1447
123
16S rDNA genes, were used; V4-Forward, 5
0
-CAGCAG
CCGCGGTAATAC-3
0
and V4-Reverse, 5
0
-CCGTCAATT
CCTTTGAGTTT-3
0
(Yu and Morrison 2004). Electro-
phoresis of the PCR products and 16S rDNA nucleotide
sequencing were performed as above. The molecular size
markers used were same for the pediocin gene. Isolates
were confirmed in the GenBank database, using their
nucleotide sequences of the 16S rDNA genes to determine
the closest known relatives.
Determination of thiobarbituric acid (TBA)
Thiobarbituric acid values were determined for the meat
samples as described by Brewer et al. (1992) with some
modifications. Beef samples (10 g) were blended with
15 ml of cold extracting solution containing 9% perchloric
acid. The resulting slurries were transferred to 100 ml
volumetric flasks and made up to 50 ml each with distilled
water (DW). The slurries were filtered through Whatman
no. 2 filter paper. Fifty millilitre of each of the filtrates were
transferred to test tubes and 5 ml of 0.02 N TBA reagent
was added into each and mixed thoroughly. The tubes were
kept in the dark for 17 h and the absorbance read at 530 nm
with a spectrophotometer (Spectronic 20, Thermo Fisher,
Waltham, USA) against a blank containing DW and TBA
solution. Malonaldehyde (MDA) concentrations were cal-
culated from a standard curve using solutions of 1,1,3,3-
tetraethoxypropane (TEP). The MDA standard stock solu-
tion (10 mM) was prepared by acid hydrolysis of 239 ml
TEP (97%, Fisher Scientific, UK) in 100 ml of 1% sul-
phuric acid (Fisher Scientific, UK) for 2 h at room tem-
perature. This was diluted with DW to 100 lM and used as
working standard solution, from which appropriate dilu-
tions (0.1–50 lM) were further made. The absorbance
readings of the MDA standard dilutions were used in con-
structing calibration curve from which MDA in the samples
were quantified, using the equation obtained from the linear
regression of the standard curve (R
2
= 0.993) as follows:
TBA (mg MDA/kg sample) = [(0.0276 9 A - 0.1672) 9
43 9 67], where A is the absorbance. The samples and
standard solutions used for the calibration were taken
through the TBA procedure at the same time. Preparations
were made in three replicates and TBA values were
expressed as mg MDA/kg sample.
Determination of free fatty acids (FFA)
This was determined by comminution of replicated beef
samples and lipid extraction followed by alkali titration
using standard AOAC (1990) procedures. Fat extraction
was carried out by homogenising 20 g of sample in 100 ml
of chloroform followed by filtration. Twenty millilitre of
water were added to the filtrate and the mixture was
agitated and decanted while the chloroform phase was
collected. Then one gram of Na
2
SO
4
was added, agitated
and filtered. The determination of total FFA was done by
titration of 10 ml of the lipid extract dissolved in 20 ml of
ethanol/diethyl-ether (1/1), with an ethanolic solution of
0.02 M KOH, using phenolphthalein as indicator. Result
was expressed as mg KOH/g lipid.
pH measurement
pH of the fresh beef samples was monitored during storage
by taking 10 g of sample and homogenized in standard
stomacher bags (BA 6141, Seward, UK) containing 100 ml
sterile DW, using a Seward stomacher (model 400 circu-
lator, P/4/518, 50–60 Hz, Leighton Buzzard, UK). The pH
was then measured by a pH meter (pH 212 Microprocessor,
Hanna Instruments, USA) using the method of Marugg
et al. (1992).
Statistical analysis
Results which depend on starter cultures and storage times
were analyzed according to a completely randomized
design with three replicates. Data were subjected to vari-
ance analyses and differences between means were evalu-
ated by Duncan’s multiple range test using SPSS statistic
programme, version 10.01 (SPSS 1999). Significant dif-
ferences were expressed at p \ 0.05.
Results and discussion
Some technological features of the LAB used as starters
The LAB strains, P. pentosaceus LIV01 and P. acidilactici
FLE01, used as starter cultures in this study, were among
the strains isolated from fresh beef in a previous study
(Olaoye et al. 2008). The study reported the identification of
the strains by their 16S rDNA—V3 region nucleotide
sequences which were submitted to the GenBank under
accession numbers EU667381 and EU667382, respectively.
In the present study, P. pentosaceus LIV01 strain was
confirmed to encode gene for pediocin production by PCR,
of approximately 330 bp in size; the same strain also
exhibited production of bacteriocin-like substance (BLS) in
vitro. The strain P. acidilactici FLE01 was not confirmed
to encode gene for pediocin production. This was not
surprising because the strain did not show production of
BLS in vitro. Millette et al. (2008) have reported similar
detection of the bacteriocin in Pediococcus strains. The use
of bacteriocin producing LAB as biopreservatives may
have major applications in improving safety in food
products including meat (Cintas et al. 2001).
1448 World J Microbiol Biotechnol (2010) 26:1445–1454
123
Among the 21 strains tested, the two Pediococcus
strains, P. pentosaceus LIV01 and P. acidilactici FLE01,
exhibited production of considerable concentrations of
organic acids in vitro (Olaoye et al. 2008); about 1.7 and
1.0 % (w/v) were measured as lactic and acetic acids
respectively in the two strains. These concentrations could
bring about antagonism against unwanted organisms that
are usually associated with food spoilage (Djenane et al.
2005). Higher concentration of the acids (C3%) in meat
has been reported as undesirable because it can impart
negatively on sensory qualities (MRNL 1986; Ammor and
Mayo 2007). Production of bacteriocin and organic acids is
known to a play vital role in antagonism against spoilage
and pathogenic organisms (Aymerich et al. 1998).
Confirmation of colonies on the selective media
by PCR
Coupled with their phenotypic and characteristic features,
emerging colonies on the different selective media were
confirmed by PCR. The universal eubacterial primers, used
to target V4 region of 16S rDNA genes in the various
bacteria, produced amplicons of about 400 bp in size
(Fig. 1). The bacterial isolates on respective media had
above 80% homology to those in the GenBankDatabase.
Specifically, the colonies of L. monocytogenes and S. Ty-
phimurium, enumerated on respective Oxford and XLD
selective media, had above 90% homology to similar
strains of accession numbers FJ774256.1 and X80681.1
respectively in the database.
Microbial dynamics during storage of fresh beef
Detection limits estimated for each of the indicator group
of organisms monitored during storage were 2 log, except
Enterobacteriaceae (1 log). During storage of the fresh
beef samples, there was an increase in the numbers of LAB
with time during storage (Fig. 2): up to a 3.5 log increase
was recorded. Kaban and Kaya (2006) reported similar
observation in sucuk, a traditionally fermented sausage in
Turkey. In the present study, LAB counts were generally
higher in inoculated samples than controls; maximum
values were observed in most samples on day 3 of storage,
after which counts declined.
The initial coliform counts (day 0) ranged between 2
and 2.5 log in the meat samples (Fig. 3). The counts
decreased with time of storage in starter inoculated samples
and were below detection limit in samples treated with only
1 4 7 9 11
750bp
500bp
250bp
1500bp
1000bp
500bp
2 3 12
1086 5
Fig. 1 Typical PCR amplifications of the 16S rDNA genes from
colonies on the various selective media. (1) 100 bp ladder; (2)a
colony from VRBGA; (3) a colony from MSPRA; (4) a colony from
MacConkey; (5) a colony from XLD; (6) a colony from Oxford; (7)a
colony from VRBGA; (8) a colony from MSPRA; (9) a colony from
MacConkey; (10) a colony from XLD; (11) a colony from Oxford;
(12) 1 kb ladder
2.00
4.00
6.00
8.00
10.00
12.00
01234567
Storage days
Log cfu/g
Fig. 2 Lactic acid bacteria counts of fresh beef during storage. PEN
(
) Inoculation with Pediococcus pentosaceus LIV01; PED
(
) Inoculation with P. acidilactici FLE01; MIX ( ) Inocu-
lation with mixed cultures of Pediococcus pentosaceus LIV01 and
P. acidilactici FLE01; CONT (
) Uninoculated control samples;
CONT- NEGATIVE LAB (
), Inoculation with non-starter
P. acidilactici FLE04. For each day of storage, values are means of
three replicate samples
2.00
3.00
4.00
5.00
6.00
0246
Storage days
Log cfu/g
Fig. 3 Coliform counts of fresh beef during storage. PEN ( );
PED (
); MIX ( ); CONT ( ); CONT- NEGATIVE LAB
(
). For each day of storage, values are means of three replicate
samples
World J Microbiol Biotechnol (2010) 26:1445–1454 1449
123
P. pentosaceus LIV01 starter on day 1. Counts were also
below detection limit for meat samples inoculated with
only P. acidilactici FLE01 starter on day 3 while the same
observation was made for those treated with combination
of both starters on day 2. For uninoculated control (CONT)
and non-starter P. acidilactici FLE04 inoculated samples
(CONT- NEGATIVE LAB), coliform counts increased to
about 5 and 4.5 log respectively from day 4 of storage,
indicating a reduction in the starter inoculated meat sam-
ples compared to the controls. The decrease in counts in
starter inoculated samples suggests the protective ability of
the starter cultures against coliforms. In the report by
Kalalou et al. (2004) on meat inoculated with LAB cultures
in Morocco, a reduction in coliform counts during storage
was noted. The researchers concluded that the LAB used as
protective cultures could have generated some antimicro-
bials that were effective for the control of coliforms.
However, the ability of the pediocin-producing P. pento-
saceus LIV01 to control coliforms in this study may not be
due to its ability to produce bacteriocin, but rather to
production of other antimicrobial agents such organic
acids, diacetyl or hydrogen peroxide (Cintas et al. 2001).
This is because bacteriocins act against species that are
only closely related to the producer organism (Klaenham-
mer 1988; Cleveland et al. 2001). Besides, a similar
reduction was also recorded in samples treated with non-
bacteriocin-producing P. acidilactici FLE01. Moreover,
the ineffectiveness of the non-starter P. acidilactici FLE04
strain to control coliforms in the beef samples could con-
firm that the organic acids produced by the starter culture
strains may have contributed to the control of the
coliforms.
The Enterobacteriaceae counts are presented in Fig. 4.
A decrease was recorded in starter inoculated samples
compared to the control. The PEN samples recorded counts
of below 2 log on day 4 while a similar pattern was
recorded for PED and MIX samples on day 5. However, in
the control samples (CONT and CONT-NEGATIVE
LAB), an increase of about 2–3 log was recorded during
storage. The starter also seemed to have exerted its pro-
tective ability on Staphylococcus in the inoculated samples
(Fig. 5). Except for CONT and CONT-NEGATIVE LAB
samples on day 4, counts were below detection limit
among the fresh beef samples during the 7 day storage.
Counts of between 2.5 and 3.0 log were recorded for the
CONT samples between day 4 and 7, while values of below
2.5 were recorded in the CONT-NEGATIVE LAB samples
over the same period. Staphylococcus counts were recorded
to be higher in CONT and CONT-NEGATIVE LAB
samples compared to their starter inoculated counterparts,
however no significant difference was recorded (p \ 0.05).
In a report of Hammes and Hertel (1996), the ability of
starter LAB to control Staphylococcus was noted. Antag-
onism by LAB against Staphylococcus is majorly mediated
by the production of organic acids and not bacteriocin
(Lucke 2000). Hence the antagonism recorded in the
present study against Staphylococcus may not be due to
pediocin produced by P. pentosaceus LIV01. Kaban and
Kaya (2006) recorded low counts of Enterobacteriaceae
and Staphylococcus in meat samples inoculated with
strains of LAB. In a related study, Kaya and Gokalp (2004)
reported a reduction to below detection level in Staphylo-
coccus counts during ripening of a fermented sausage after
treatment with protective LAB cultures. In another study
Gomo
´
łka-Pawlicka et al. (2004) noted susceptibility of
Staphylococcus to LAB cultures in meat.
Yeast and moulds counts were observed to decrease in
starter inoculated samples compared to the control (Fig. 6).
Counts were lower in the former compared to latter;
1.00
2.00
3.00
4.00
5.00
6.00
7.00
01234567
Storage days
Log cfu/g
Fig. 4 Enterobacteriaceae counts of fresh beef during storage. PEN
(
); PED ( ); MIX ( ); CONT ( ); CONT- NEGA-
TIVE LAB (
). For each day of storage, values are means of
three replicate samples
2.00
2.50
3.00
01234567
Storage days
Log cfu/g
Fig. 5 Staphylococcus counts of fresh beef during storage. PEN
(
); PED ( ); MIX ( ); CONT ( ); CONT- NEGA-
TIVE LAB (
). For each day of storage, values are means of
three replicate samples
1450 World J Microbiol Biotechnol (2010) 26:1445–1454
123
however no significant reduction (p \ 0.05) was recorded.
Casaburi et al. (2007) observed that the growth of yeast and
moulds in Italian style sausages was controlled during
storage after inoculation with LAB starter cultures; they
concluded that it could be due to the antagonistic activities
of the latter. Another study reported similar observations in
a Turkish sausage after inoculation with LAB strains as
protective cultures (Erkmen 2008).
Effect of starter LAB cultures on pathogens
A set of fresh beef samples was initially inoculated with
approximately 10
6
cfu/g of L. monocytogenes to challenge
the starter cultures during storage. The result of the counts
of the pathogen in the fresh beef samples during storage are
shown in Fig. 7. Counts decreased by about 1.4 log on day
1 in PEN and MIX samples and were below detection
limits on day 2 of storage. There was only 1 log reduction
in the count of the pathogen in PED samples, suggesting a
potential lethal effect by pediocin producing P. pentosac-
eus strain in the inoculated meat samples. An increase of
about 3 log was recorded in the uninoculated control
samples from day 0 to day 7 of storage. Susceptibility of
the foodborne pathogen to class IIa bacteriocin, such as
pediocin, has been reported (Cintas et al. 2001; Djenane
et al. 2005). The organism, L. monocytogenes, is a known
threat to food safety (Colak et al. 2007) and it can con-
taminate meat and products during slaughter, processing
and production. It can also tolerate low and high pH values,
low water activity and refrigeration temperatures and hence
the need for its control, especially through biopreservation,
to safeguard public health (Thevenot et al. 2005).
Similarly, S. Typhimurium was used to challenge the
starter cultures at the same inoculum count (10
6
cfu/g). The
counts of the bacterium (Fig. 8) showed between 1 and
2 log reductions in starter inoculated samples during stor-
age on day 3. In contrast, about 3.5 log increase was
recorded in the CONT samples, which had the highest
counts on day 3. The antagonistic activity displayed by the
starter on the pathogen could be due to the production of
other antimicrobials besides pediocin. This is because the
bacteriocin of LAB has been reported to be ineffective
against gram-negative organisms (Cintas et al. 2001;
Albano et al. 2007).
Effect of starter cultures versus controls
It was generally observed that the microbial flora being
examined were lower in starter inoculated samples
2.00
4.00
6.00
8.00
01234567
Storage days
Log count/g
Fig. 6 Yeast and mould counts of fresh beef during storage. PEN
(
); PED ( ); MIX ( ); CONT ( ); CONT- NEGA-
TIVE LAB (
). For each day of storage, values are means of
three replicate samples
2.00
4.00
6.00
8.00
10.00
12.00
01234567
Storage days
log cfu/g
Fig. 7 Listeria monocytogenes counts of fresh beef during storage.
PEN (
); PED ( ); MIX ( ); CONT ( ); CONT-
NEGATIVE LAB (
). For each day of storage, values are
means of three replicate samples
2.00
4.00
6.00
8.00
10.00
12.00
01234567
Storage days
Log cfu/g
Fig. 8 Salmonella Typhimurium counts of fresh beef during storage.
PEN (
); PED ( ); MIX ( ); CONT ( ); CONT-
NEGATIVE LAB (
). For each day of storage, values are
means of three replicate samples
World J Microbiol Biotechnol (2010) 26:1445–1454 1451
123
compared to the CONT and CONT-NEGATIVE LAB,
despite the similar counts of LAB recorded in both groups
during storage. This confirms the need for screening of
LAB strains for suitability as starter cultures for meat
preservation, as most of the non-starter LAB noted in the
CONT samples may not have potentials for suitability as
starters cultures (Ammor and Mayo 2007). This observa-
tion was also noted in the samples inoculated with non-
starter LAB strain, P. acidilactici FLE04, which was
shown to be of poor technological features, especially in
terms of lactic and acetic acid production.
Maximum counts (log cfu/g) for total bacteria, Entero-
bacteriaceae and Staphylococcus have been recommended
as 6, 3 and 2 respectively in meat products, while coliform
count should be below 2. Listeria and Salmonella should be
undetectable in 25 and 10 g of meat (USDA 2004). No limit
as maximum count of LAB has been reported; probably
because they have GRAS status (Simpson et al. 2002).
Statistical analysis
Results of statistical analysis of the microbiological pro-
files in the meat samples during storage showed that starter
cultures had a significant effect (p \ 0.05) on the coli-
forms, Enterobacteriaceae, Staphylococcus, L. monocyt-
ogenes and S. Typhymurium in starter inoculated samples
compared to CONT. No significant difference was, how-
ever, recorded in yeast & mould counts. Also, results of the
two LAB cultures were not significantly different from
each other, except on L. monocytogenes, and this could be
as a result of pediocin produced by P. pentosaceus, which
has a lethal effect on the pathogen (Cintas et al. 2001;
Djenane et al. 2005).
pH patterns in the beef samples during storage
pH measurements in the meat samples showed that values
in the starter inoculated samples were lower than 5 from
day 2 of storage (Fig. 9) compared to the CONT samples.
The decrease in pH values of inoculated meat samples may
be due to the production of organic acids by the Lactic acid
bacteria starter cultures. Lowering of pH in food products
inoculated with lactic acid bacteria is an important factor in
the control of undesirable microorganisms (Vermeiren
et al. 2004; Kaban and Kaya 2006). The reduction in pH
recorded in the samples inoculated with LAB may have
contributed to the lower values of microbial counts recor-
ded in those samples compared to the CONT and CONT-
NEGATIVE LAB samples. However, the pH reduction did
not have any significant effect on the reduction of yeast and
moulds, probably because of their broad pH requirement
which allow them to tolerate low pH values below 4. In a
similar report, low pH medium in meat was observed to
produce no significant reduction in the counts of yeast and
moulds (Onilude et al. 2002).
Thiobarbituric acid (TBA) and free fatty acid (FFA)
Results of TBA and FFA contents of the meat samples
during storage showed that lower values were recorded in
the LAB inoculated samples in comparison to the CONT
samples (Table 1). TBA and FFA are measurements of the
breakdown products of fat and lipids, an indication of
quality deterioration in foods. Lipid oxidation gives prod-
ucts that may change the colour, aroma, flavour, texture
and even the nutritive value of the food (Ulu 2004). Con-
trol and monitoring of TBA and FFA during meat pro-
cessing or storage are therefore important due to increased
demand for meat products. With increased period of stor-
age, TBA and FFA values showed corresponding increase,
suggesting possible oxidation of lipids in the meat samples.
The inoculated samples appeared to keep well for the first
3 days of storage, having lower TBA and FFA values
compared to CONT. The values obtained were similar to
those reported by Onilude et al. (2002) on meat products
inoculated with LAB strains. In a related study, Ulu (2004)
reported the occurrence of oxidation of lipids leading to
rancidity during storage in meat samples. Free fatty acids
and TBA have been noted to be an indication of rancidity
development in meat products (Brewer et al. 1992).
Conclusion
Results obtained in this study suggest that the shelf life of
fresh beef could be extended by 2–4 days at normal
3
4
5
6
01234567
pH
Storage days
Fig. 9 pH patterns of fresh beef samples during storage. PEN
(
); PED ( ); MIX ( ); CONT ( ); CONT- NEGA-
TIVE LAB (
). For each day of storage, values are means of
three replicate samples
1452 World J Microbiol Biotechnol (2010) 26:1445–1454
123
ambient temperature in Nigeria. The protective ability of
the LAB strains could be exploited to improve shelf life
and safety of beef meat. In this study, the LAB strains were
tested on the muscle portion of fresh beef, however their
potentials as starter cultures could be improved upon and
exploited for use on other portions. The ability of the
starter cultures to control L. monocytogenes and S. Typ-
hymurium is very important following the public health
significance of the two pathogens.
Due to concerns over food security in most developing
countries, especially Nigeria, efforts are being required,
through research activities, to promote food availability.
Inadequate storage facilities and preservation techniques
are the major factors that have contributed to food inse-
curity in Nigeria. It is the candid belief of the authors that if
necessary support is provided, the use of biological agents
being proposed in the present study could be exploited in
complementing existing food preservation techniques in
the country.
Acknowledgments The authors are grateful to the School of Bio-
sciences, University of Nottingham, UK, where part of this research
study was carried out.
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a
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a
PED 0.22 ± 0.02
a
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a
MIX 0.19 ± 0.01
a
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a
CONT 0.23 ± 0.02
a
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a
1 PEN 0.23 ± 0.02
a
0.45 ± 0.07
a
PED 0.25 ± 0.01
a
0.51 ± 0.02
a
MIX 0.24 ± 0.02
a
0.46 ± 0.05
a
CONT 0.34 ± 0.01
b
0.61 ± 0.06
b
2 PEN 0.41 ± 0.01
a
0.50 ± 0.01
a
PED 0.43 ± 0.03
a
0.52 ± 0.03
a
MIX 0.41 ± 0.01
a
0.53 ± 0.01
a
CONT 0.62 ± 0.02
b
0.70 ± 0.03
b
3 PEN 0.43 ± 0.02
a
0.55 ± 0.05
a
PED 0.48 ± 0.01
a
0.54 ± 0.02
a
MIX 0.44 ± 0.04
a
0.52 ± 0.01
a
CONT 0.66 ± 0.01
b
0.71 ± 0.03
b
4 PEN 0.45 ± 0.02
a
0.50 ± 0.06
a
PED 0.50 ± 0.02
a
0.60 ± 0.07
b
MIX 0.47 ± 0.01
a
0.55 ± 0.04
a
CONT 0.64 ± 0.02
b
0.71 ± 0.03
c
5 PEN 0.45 ± 0.01
a
0.50 ± 0.05
a
PED 0.54 ± 0.02
a
0.62 ± 0.05
b
MIX 0.51 ± 0.02
a
0.50 ± 0.03
a
CONT 0.69 ± 0.01
b
0.83 ± 0.02
c
6 PEN 0.40 ± 0.02
a
0.45 ± 0.03
a
PED 0.47 ± 0.03
a
0.62 ± 0.03
b
MIX 0.43 ± 0.01
a
0.53 ± 0.04
a
CONT 0.65 ± 0.04
b
0.77 ± 0.03
c
7 PEN 0.40 ± 0.02
a
0.47 ± 0.03
a
PED 0.41 ± 0.01
a
0.62 ± 0.07
a
MIX 0.43 ± 0.04
a
0.50 ± 0.03
a
CONT 0.64 ± 0.03
b
0.81 ± 0.02
b
For each day of storage, values are means of three replicates
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letters are significantly different (p \ 0.05)
MDA Malonaldehyde
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... Brings about two-fold reduction in the growth of the pathogenic organisms those are associated with the degradation of meat [34] Sausages of meat P. acidilactici-associated production of bacteriocin L. monocytogenes Three-fold reduction in the targeted organism [16] Sucuk sausages L. plantarum-mediated production of bacteriocin L. monocytogenes Brings about marked reduction in the growth of the microorganims [35] Emulsion of goat meat ...
... Brings about 2-to 3-fold reduction in the targeted organism [36] Natural casings of sheep Bacteriocins produced by LAB Clostridium sporogene Brings about marked reduction in the Clostridium sp. [37] Still, the reduction in the pathogenic organism was found to be more when there was the combinatorial organism of P. pentosaceus and P. acidilactici was applied [34]. Higher temperatures, pH, and free fatty acids facilitate the protective activity exhibited by LAB [34]. ...
... [37] Still, the reduction in the pathogenic organism was found to be more when there was the combinatorial organism of P. pentosaceus and P. acidilactici was applied [34]. Higher temperatures, pH, and free fatty acids facilitate the protective activity exhibited by LAB [34]. The maintenance of pH below 5 by the LAB group is responsible for preventing the growth of pathogenic organisms in the meat. ...
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