SCREENING OF LACTIC ACID BACTERIA FOR BACTERIOCINS ...

AN INNOVATIVE LABORATORY EXERCISE:

SCREENING OF LACTIC ACID BACTERIA
FOR BACTERIOCINS BY
MICROBIOLOGICAL AND PCR METHODS

Duongdearn Suwanjinda, Mahidol University

Chris Eames, University of Waikato

Watanalai Panbangred, Mahidol University

Outline


Introduction


Rationales of the Study


Laboratory Unit Development


Lactic Acid Bacteria (LAB)


Bacteriocins


Polymerase Chain Reaction (PCR)


Science and Education Outcomes


Rationales of the Study



A

need for the integration of current molecular
biology together with conventional methods for
undergraduate laboratory courses



Positive laboratory experiences can encourage
students to continue their education in molecular
biology or provide them an appreciation for how
technology applies to their daily lives.



Rationales of the Study


Topic of selected experiment


Concern for food safety: Lactic acid bacteria (LAB)
are recognized as GRAS. LAB and their products
(Bacteriocins) are used in many foods.


There are many kinds of fermented foods in
Thailand in which LAB can be found.


Experiment on LAB and bacteriocins

Lactic Acid Bacteria (LAB)


LAB are widely used in food fermentation.





LABs produce bacteriocins.


Bacteriocins can inhibit the growth of
undesirable bacteria.



Bacteriocins



Example of bacteriocin application

Nisin produced by
Lactococcus lactis
subsp.
lactis

has been
approved and used widely in food preservation.


Substances

used

in

defense

systems

to

compete

for

growth

and

survival

of

bacteria

against

other

genetically

related

species



Bacteriocin is a protein by nature and there are
many types of bacteriocins.

724aa

Examples of Nisin and Pediocin Operons

IS904

nisA

nisB

P

P

nisT

nisC

nisP

nisR

229aa

682aa

245aa

418aa

600aa

851aa

IR rho
-
independent
terminator

IR
-
mRNA
processing

5’
-
termini
Tn5276
Tn5301

(A) Nisin Operon

174aa

62aa

(B) Pediocin (PA
-
1) Operon

112aa

kb sequenced

5.5 kb

P

nisI

pedA

pedB

pedC

pedD

Fig. 1. Organization of nisin A (A), and pediocin PA
-
1 (B) operons.

Method Development: Microbiology



Isolation of LAB on selective media (MRS).



An overlay method was chosen for testing
bacteriocin activity.



Lactobacillus plantarum

was used as a tester
strain.




Method Development: Microbiology

Nham sample

Serial dilution

Spread on plate

Colonies on plate

Overlay with

Lactobacillus plantarum


Day 1

Day 2

UD

10
-
1

10
-
2

10
-
3

10
-
4

10
-
5

10
-
6

pipette

100
µl

weigh

1 g

30
˚
C

24 h

Method Development:
Bioinformatics

Nucleotide searching and Similarity searching

Fig. 2. Website for nucleotide searching and similarity searching.

Learning bioinformatics through internet

Method Development:
Bioinformatics

Sequence alignment

Fig. 3. Website for sequence alignment.

Learning bioinformatics through internet

Polymerase Chain Reaction (PCR)

Denaturation

Annealing

Extension

Fig. 4. PCR diagram



PCR has been
widely used in
research.




Le
arning PCR principles
has
facilitated students’
understanding of invitro DNA
amplification.

Method Development
: PCR

LAB

Bacteriocin genes

Pediococcus pentosaceus

pediocin

(
ped
)

Enterococcus faecium

enterocin
(
ent
)

Lactococcus lactis

nisin
(
nis
)

Lactic acid bacteria and bacteriocin genes


Three pairs of three bacteriocin gene
-
specific primers were designed and
tested.

Method Development:
PCR



DNA template preparation by simple cell boiling method


TE buffer

Boil

Laboratory Unit



Teaching unit development: Students activities



chose their own food samples (Day1).


screened for bacteriocin
-
producing bacteria (Day1
-
3).


selected bacteria to perform the PCR procedures (Day 3).


learned bioinformatics (Day 3).


performed PCR (Day 4).


PCR analysis by gel electrophoresis (Day 5).


Laboratory Unit




Teaching unit trial



trialed in February 2006 and 2007


students worked in groups of five

Scientific Outcome: Microbiological Results




Fig. 5. MRS agar plate with lactic acid bacterial colonies overlaid with soft agar containing
L. plantarum
. The black
arrow indicates one of seven positive colonies with a clear halo zone around it.


The white arrow indicates a negative
colony with no clear halo zone around it. The photograph was taken from above with the cover of Petri dish removed


Conclusion: Each student group



Several LAB colonies were obtained on
MRS agar.



Students can observe inhibition zone.



Three colonies were picked for PCR.

Scientific Outcome: Microbiological Results

Scientific Outcome: Bioinformatics Experience


Students have an experience on using
free online software to design primers
for the detection of pediocin, enterocin,
and nisin genes.



Students know how to do DNA
sequence alignment.

Scientific Outcome: PCR Results

Fig. 6. Sample agarose gel of PCR products from amplification of pediocin (
ped
), enterocin (
ent
), and nisin (
nis
)
genes.
Lane 1
,

100
-
bp DNA ladder;
Lane 2
, amplicons from DNA isolated from
Pediococcus pentosaceus
,

Enterococcus faecium

and
Lactococcus lactis
;
Lanes 3
-
5
, amplicons from DNA from boiled cells of
Pediococcus
pentosaceus
,

Enterococcus faecium

and
Lactococcus lactis
, respectively;
Lane 6
, amplicons from DNA from a nham
sample spiked with
Pediococcus pentosaceus, Enterococcus faecium

and
Lactococcus lactis
;
Lanes 7
-
8
, amplicons
from DNA from food samples;
Lanes 9
-
11,
amplicons from DNA from colonies producing clear halos in a
Lactobacillus
plantarum

overlay.


Scientific Outcome: PCR Results


Conclusion: Each student group can
detect 608, 412, and 332 bp PCR
products.

Educational Outcome


Questionnaires and Interviews


Students were asked with series of questions to
monitor their perceptions of technical skills,
scientific knowledge, inquiry skills, and
enjoyment of science gained through the lab
unit.


Student’s responses were positive.


Observation


Observation results were corresponded with
the questionnaires and interviews.


Educational Outcome



Findings from the study

Students had gained


•

Technical

skills


•

Scientific knowledge


•

Scientific inquiry skills


•

Enjoyment of science

D
.

Suwanjinda, C
.

Eames, W
.

Panbangred
(
2007
)
Screening of Lactic Acid Bacteria for Bacteriocins by Microbiological

and PCR Methods
,

Biochem. Mol. Biol. Educ.
35
(5),

364
–
369
.



Acknowledgement



Prof. Dr.
Watanalai Panbangred


The institute for Innovation and Development of Learning
Process


The Institute for the Promotion of Teaching Science and
Technology (IPST), Thailand


Promotion of Science and Mathematics Teachers project


A research
grant from Mahidol University


Third year undergraduate biotechnology students


Members of K535
-
539



Questions or Comments?