Listeria Presentation (1)

Synergistic anti-microbial effects of nisin and cinnamaldehyde

against Listeria innocua on pears

Presented by: Joseph Nguyen & Tanveer Syeda

Introduction• L. inncocua

– gram+ rod aerobic bacterium– Found in soil, water, vegetation

and animal faeces– Associated with processed fruits

and vegetable.– Analogue for L. monocytogene

• Fever, headaches, diarrhea gastrointestinal problems and sometimes death

– Health hazard for infants, elderly, and immunodeficient

Introduction Cont’d

• Chlorine is the current sanitizer

• Not effective against L. monocytogene

• Effects health, quality of foods and environment

• Carcinogenic with combined with organic matter

Introduction Cont’d

Nisin

• Lactococcus lactis• Effective against Gram

+’ve

• Interacts with lipids on cell membrane

Cinnamaldehyde

• Essential Oil• Effective against bacteria

and fungi

• Interacts with free amino groups and disrupts protein

• Objective– Find optimal concentration of nisin and

cinnamaldehyde– Combine the optimal concentrations of both to

observe any synergistic activity– Cultural enumeration and well diffusion

• Hypothesis– Greater antimicrobial effect with the

combination of nisin and cinnamaldehyde

Methodology

• Listeria innocua ATTC #33090

• Isolated a colony• Incubate in TSB 37ºC/48

hours

• Serial dilution target 10-7

• Initial concentration is 3.2x108 cfu/ml

Biochemical Test Results

Catalase PositiveBubbles

MR PositiveRed colour

VP NegativeYellow colour

Phenol Red with Dextrose

PositiveTurbidity

Gram stain Gram positivePurple rods

Cultural Enumeration

• Nisin – 0.02%, 0.04%, 0.06% and 0.08%

• Cinnamaldehyde – 0.3%, 0.5% and 0.8%

• Anjou Pear

• Washed with 70% alcohol and rinsed with sterile dH2O

• 9 slices were 5-7 grams and 5mm

• 0.1ml of L. innocua inoculated for 30mins

• Treated with agents for 25secs

• Incubated on BHI for 37°C/48 hours

ResultsPlate Results

0.08% Nisin ++

0.06% Nisin +++

0.04% Nisin +++

0.02% Nisin +++

0.08% Cinnamaldehyde +++

0.05% Cinnamaldehyde ++++

0.03% Cinnamaldehyde ++++

Positive Control (Inoculated Pear) ++++

Negative Control (Non-inoculated Pear) No growth

Media Control No growth

Scale: ++++ = Lots of growth + = Little growth

Well Diffusion method

• Well diffusion method is mostly used to study the anti-bacterial activity of plant extracts like oils (eg: nisin, cinnamaldehyde, eugenol etc..)

Disc Diffusion Method

• A disc of wafer infused with known anti-biotic is placed on to culture to see the susceptibility of the bacteria against the anti-biotic.

Methods

• A spread plate for L. innocua was prepared on the BHI plates

• Appropriate wells were prepared on these plates.

• Each well was filled with two drops of different concentration of nisin (0.08%,0.06%, 0.04%,0.02%) and Cinnamaldehyde (0.8%, 0.5%, 0.3%).

Well diffusion method

• A disc of chloramphenicol was placed on the BHI plated with L.innocua.

• These all plates were incubated at 35 c for ⁰48 hours.

• Zone of inhibitions were measured after.

• Well diffusion results for anti-microbial activity of Nisin against L.innocua.

• 0.08% of Nisin was selected for the synergistic activity with cinnamaldehyde

BHI plates Zone of Inhibition

L. innocua +0.02% nisin (inc: 35 c for 48 hours)⁰

22mm

L. innocua +0.04% nisin (inc: 35 c for 48 hours)⁰

23.88mm

L. innocua +0.06% nisin (inc: 35 c for 48 hours)⁰ 22.38mm

L. innocua +0.08% nisin (inc: 35 c for 48 hours)⁰

24.25mm

• Well and disc diffusion results for anti-microbial activity of cinnamaldehyde and chloramphenicol and against L.innocua.

• No zone of inhibition is seen in plates with cinnamaldehyde whereas for chloramphenicol it was seen.

BHI plates zone of inhibition

L.Innocua + 0.3% cinnamaldehyde (inc: 35 c ⁰for 48 hours)

no zone of inhibtion is seen

L.Innocua + 0.5% cinnamaldehyde (inc: 35 c ⁰for 48 hours)

no zone of inhibtion is seen

L.Innocua + 0.8% cinnamaldehyde (inc: 35 c ⁰for 48 hours)

no zone of inhibtion is seen

L.Innocua + disc of 30ug chloramphenicol (inc: 35 c ⁰for 48 hours)

26.5mm

Conclusion

From this study it can be concluded that cinnamaldehyde did not showed any synergistic activity with Nisin instead it antagonized the activity of Nisin. This can be due to less effectiveness of cinnamaldehyde against Gram-positive L. innocua and also due to inappropriate concentrations of these combined agents.

A graph representing the zone of inhibition of nisin and cinnamaldehyde against

L.innocua

Current research

• Current research trends in food and pharm industries focus on use of natural anti-microbial compounds as a preservatives that are active against organisms like L. moncytogenes, P. aerugenosa and other harmful bacteria’s.

Questions?