Determination of Mechanism of Action for Reduction of Listeria

monocytogenes by LactiguardTM Diego Casas1,2, David Campos2, Ashley Orange2, Lacey Guillen Ph.D2, Mindy Brashears Ph.D2.

1. SOWER Scholar ICFIE, Texas Tech University

2. Department of Animal & Food Science, Texas Tech University

Methodology

Results

For both LAB treatments (washed cells and freeze-dried product),

after 24 hours the pH ranged approximately from 3.88-4.29 with the

control samples having a pH of 4.64. Both LAB treatments with and

without enzyme at 37° C resulted in less L. monocytogenes by

approximately log 5.675 CFU/ml when compared to the control. At

7oC, the pH after 5 days was higher, ranging from 5.11-5.98 for both

LAB treatments with the control being at 6.08. For both treatments,

after 5 days, there was more than a 2 log reduction of L.

monocytogenes in samples without added enzymes and with catalase.

This indicates that the addition of catalase to inactivate hydrogen

peroxide did not result in changes in the inhibitory capacity. However,

in all samples treated with the other enzymes, there was no significant

reduction in the pathogen after 5 days.

Objective

To evaluate the mechanisms of inhibition on L. monocytogenes by a

4-strain cocktail of LAB (LactiguardTM: NP51, NP28, NP7 and NP3)

at different temperatures.

References:

Lewus CB, Kaiser A, Montville TJ. 1991. Inhibition of foodborne bacterial

pathogens by bacteriocins from lactic acid bacteria isolated from meat. Appl

Environ Microbiol 57(6):1683–1688.

O’Sullivan L, Ross R.P, Hill C. 2002. Potential of bacteriocin producing lactic

acid bacteria for improvements in food safety and quality. Biochimie, 84(12):

593–604.

Shalini M, Rameshwar S. 2005. Antibiotic resistance in food lactic acid

bacteria—a review, International Journal of Food Microbiology. 105(15): 281-

295.

Tyler HL, Triplett EW. 2008. Plants as a habitat for beneficial and/or human

pathogenic bacteria. The Annual Review of Phytopathology, 46(21): 53-73.

Wilderdyke MR, Smith DA, Brashears MM. 2004. Isolation, identification,

and selection of lactic acid bacteria for competitive inhibition of food-borne

pathogens on alfalfa sprouts. J of Food Prot, 67(5): 947-951.

Introduction

•The Center for Disease Control and Prevention estimates foodborne

illnesses are responsible for the sickness of 48 million U.S. citizens,

from which 128,000 are hospitalized and 3000 die.

•Listeria monocytogenes is held responsible for 19% of the

foodborne infections acquired in the U.S.

•Listeria monocytogenes is a pathogen that is ubiquitous in nature

which is associated with soil, plants, animal products, and food

processing environments.

•Lactic acid bacteria has proven effective at inhibiting foodborne

pathogens, including Shiga Toxin producing Escherichia coli

(STEC), Salmonella, and Listeria monocytogenes, in culture media

and/or food products.

•Lactic acid bacteria has the ability to form an antagonistic

environment through the production of organic acids, hydrogen

peroxide, and bacteriocins.

0

1

2

3

4

5

6

0 hour

24 hour

Lis

teri

a m

on

ocy

tog

enes

(lo

g C

FU

/ml)

Lactic acid Bacteria enzyme treatments

Figure 1. Freeze dried Lactic Acid Bacteria Mechanism

at 37°C

0

1

2

3

4

5

Day 0Day 5

Lactic acid bacteria enzyme treatments

Lis

teri

a m

on

ocy

tog

enes

(lo

g C

FU

/ml)

Figure 4. Washed Lactic Acid Bacteria Mechanism at

7°C

0

1

2

3

4

5

Day 0Day 5

Lis

teri

a m

on

ocy

tog

enes

(lo

g C

FU

/ml)

Lactic acid bacteria enzyme treatments

Figure 3. Freeze Dried Lactic Acid Bacteria Mechanism

at 7°C

0

1

2

3

4

5

6

0 hour

24 hour

Lis

teri

a m

on

ocy

tog

enes

(lo

g C

FU

/ml)

Lactic acid bacteria enzyme treatments

Figure 2. Washed Lactic Acid Bacteria Mechanism

at 37°C

Conclusions

•The mechanism of action to inhibit Listeria monocytogenes was

temperature dependent, where inhibition at 37°C was primarily due to

a drop in pH due to the production of lactic acid.

•At 7°C the mechanism of action of the inhibition was primarily due to

the production of protein based compounds, where there was no

reduction when enzymes inactivated the proteins.

•The amount of catalase added (1 mg/ml) was found not to be enough

to inactivate hydrogen peroxide to an extent where it affected the

mechanism of action of LAB in the reduction of Listeria

monocytogenes.

pH Freeze Dried Cells Treatments 24 hour Day 5 Control 4.64 6.08 No Enzyme 4.16 5.23 Catalase 4.21 5.21 Trypsin 4.10 5.23 Pepsin 4.01 5.98 Protease 14 4.15 5.24 Proteinase K 4.08 5.15

pH Washed cells

Treatments 24 hour Day 5

Control 4.64 6.08

No Enzyme 4.16 5.16

Catalase 4.29 5.20

Trypsin 4.02 5.18

Pepsin 3.88 5.57

Protease 14 4.13 5.17

Proteinase K 4.08 5.11

Listeria monocytogenes growth in R&F agar

Lactic acid bacteria growth in MRS agar Table 1. pH of Freeze dried cells Table 2. pH washed cells