Technological properties of a bacteriocin

producer strain of Lactoccocus lactis isolated

from a traditional cheese
Marcia Costa, Clia Silva, Susana Ribeiro, Lurdes Dapkevicius , Henrique Rosa
CITA-A, Department of Agricultural Sciences, University of the Azores, Angra do Herosmo, Portugal

INTRODUCTION
Bacteriocins are antimicrobial peptides produced by a large number of bacteria, including
lactic acid bacteria (LAB), normally acting against disease-causing Gram-positive
pathogens. Since Lactoccocus sp are generally regarded as safe (GRAS) organisms, their
bacteriocins have attracted particular attention in an attempt to develop their potential
applications in food systems regarding safety, quality and human health.

MATERIAL AND METHODS

RESULTS AND DISCUSSION

Bacteriocin characterization
The antimicrobial activity of L. lactis NCFS revealed a minimal
inhibitory concentration (MIC) of 800 AUml -1 (Fig 1).
NCFS was inactivated by all proteolytic enzymes tested with
exception of -amilase indicating that the inhibitory compound(s) are
Fig 1 MIC of L. lactis against L.
monocytogenes defined as the
non-glycosilated protein(s).
reciprocal of minimal dilution showing
a visible zone of inhibition.
In general, most of the surfactants had no influence in bioactivity.
Bacteriocin(s) produced by L. lactis were highly thermostable (121C for 15min) and remain
active over a wide pH range (2 - 12).
Evaluation of strain coexistence demonstrated that L. lactis inhibited the growth of all the
isolates tested.
Technological characterization
L. lactis grew well at 15-30C and support higher temperatures (Fig 2). It was able to grow at
2% of salt, but higher percentages of NaCl substantially decrease the isolate growth.
L. lactis exhibited slow acidifier capacity requiring 48h to drop the pH below 5 (Fig. 3).
No proteolysis and lipolysis activity was detected, but the isolate presented high levels of
diacetyl production and high degree of autolysis after 24 h (Table1).
The pattern of enzymatic activities for the L. lactis in the APIZYM gallery system is shown in
Table 2. L. lactis exhibited high esterase and phosphatase activities, but lower peptidase
activities and lacked enzymatic activity for 8 of the studied enzymes (Table 2).

Antimicrobial screening

7.00

Lactococcus lactis was isolated from an Azorean traditional cheese - Pico cheese.
The neutralized cell-free supernatant (NCFS) of an overnight culture of L. lactis (L3A21M1) growth in MRS broth, was

6.00
pH

obtained by centrifugation at 4,000g for 10 min at 4C. The supernatant was neutralized (pH 6.5-7.0) with 1 M NaOH

5.00

pH drop in UHT milk

pH drop in skim milk

4.00

and filter-sterilized through 0.22 m pore size. Antimicrobial activity of the NCFS was determined against L.

3.00

monocytogenes by the critical dilution method.

Bacteriocin characterization
The effect of proteolytic enzymes on the antimicrobial activity of L. lactis was assayed by incubating the CSF with

24

48

Time (h)

Identification of strain
L. lactis was identified by 16S rRNA sequencing analysis.

12

Fig. 2 Growth of L. lactis at different temperatures.

Fig. 3 Acidification activity of L. lactis in UHT milk and

skim milk.

Table 1 Technological properties of bacteriocin producing strains.

proteinase K, trypsin, -chymotrypsin, -amylase and chymosin at 1 mg ml-1.

Resistance to surfactants was tested using SDS, Tween 80, Triton X-100 and urea. Surfactants were added to the CFS
at 1% (v/v) and incubated at 30C for 5h.
To test pH influence on bacteriocin activity, CFS was adjusted to various pH values and allowed to stand at room
temperature for 2 h. After incubation pH was re-adjusted to 6.5 and antimicrobial activity was tested.
Thermal stability of bacteriocins was checked by heating the cell free supernatant (CFS) of the isolates for 30 min and
60min at 100C and 15min at 121C.
Coexistence among the isolates was examined by a cross-streak method.

Technological characterization
MRS broth inoculated with L. lactis was incubated at 4C, 15C, 30C and 45C for 48h. Bacterial growth was
measured by following OD at 630 nm.
To test NaCl resistance, bacterial growth at 2%, 6% and 10% NaCl was evaluated by OD at 630nm every two hours,
during 48h.
Acidification activity was measured by the change in pH during time and evaluated in UHT milk and Skim milk.
Extracellular proteolytic activity was determined by streaking L. lactis suspensions onto the surface of Skim Milk Agar
and incubated at 30C. After 72 h, plates were flooded with 1% HCl.
Lipolytic activity was evaluated by streaking the L. lactis onto the dried surface of Tributyrin Agar plates, followed by
incubation at 30C for 72 h.
Autolysis of the cells was measured as described by Mora et al. (2003).
Diacetyl production was determined according to King (1948).
The API-ZYM system was used to determine the enzyme activity profile of the strains under study, following to the
manufacturers instructions.

Safety evaluation
Haemolysis was evaluated on Tryptose Blood Agar plates enriched with sheep blood according to Asteri et al. (2009).
Ability to produce histamine was tested using a differential medium according to Joosten and Northolt (1989).
For detection of DNase, L. lactis was cultured on DNase agar at 37C for 48 h.
Gelatinase production was tested using gelatine agar plates as described by Terzic-Vidojevic et al. (2009).
Antibiotic discs were used to determine the susceptibility of the isolates to 21 antibiotics.

Sensory evaluation
Fresh cheeses were made from pasteurized (73C/16s) milk with 0.02% CaCl2, 1% NaCl and rennet (LMF 1/15.000,
0,2g/L). Control cheeses were made without any starter culture. Test cheeses were made adding L. lactis L3A21M1
(1%) previously incubated at 30 C in skim UHT milk for 48 h. A non-trained panel of 52 tasters evaluated the acidity,
salt content, firmness, aroma and global appreciation of the fresh cheeses.

Table 2 Enzymatic activity detected using API-ZYM system of L lactis. Enzymatic activity (approximate values)
expressed as arbitrary units of substrate hydrolysed.

Enzymes

L. lactis (L3A21M1)

Alkaline phosphatase
Esterase (C4)
Esterase lipase (C8)
Lipase (C14)
Leucine arylamidase
Valine arylamidase
Cystine arylamidase
Trypsin
-chymotrypsin
Acid phosphatase

5
20
10
5
10
5
5
5
5
20

Enzymes
Naphthol-AS-BIphosphohydrolase
-galactosidase
-galactosidase
-glucuronidase
-glucosidase
-glucosidase
N-acetyl--glucosaminidase
-mannosidase
-fucosidase

L. lactis (L3A21M1)
20
0
0
0
0
0
0
0
0

Safety evaluation
The isolate was negative for haemolysis, histamine and DNase production, but positive for
gelatinase production.
L. lactis was sensitive to the majority of antibiotics tested (15 of 22), being resistant to
ofloxacin, piperacillin, streptomycin, rifampicin, trimethoprim/sulfamethoxazole and nalidixic
acid.
Sensory evaluation
No significant differences were found on sensory evaluation of the fresh cheese inoculated with
L. lactis compared to control.

CONCLUSIONS

Lactococcus lactis isolate presented important technological properties for practical application
as protective culture in fresh cheese production. The use of bacteriocin-producing L. Lactis
isolate as starter/adjunct culture may be beneficial on dairy products and provide therapeutic
potential in the intestinal environment.

Acknowledgments
This work is financed by National founds from FCT Fundao para a Cincia e Tecnologia, Project
PTDC/AGR-ALI/104385/2008. S.R. thanks the FRC Fundo Regional para a Cincia, Azores, Portugal for
the Ph.D. Grant M3.1.2/F/011/2011.