Antimicrobials from Lactobacillus plantarum isolated from turmeric

(Curcuma longa linn.) and their applications as biopreservative

and in edible film

Melada SupakijnorasetFood Engineering and Bioprocess Technology,

School of Environment, Resources and Development, Asian Institute of Technology

ProbioticThe probiotics are most commonly defined as live microbial feed supplements that beneficially affect the host by improving its intestinal microbial balance.

Lactobacillus Bifidobacterium

ProbioticAntimicrobial substances

•User-friendly and eco-friendly

•The films can be consumed with the packaged product

•Replace the polyehtylene packages and reduce the environmental pollution

•The films can function as carriers for antimicrobial and antioxidant agents

• Compare the efficiency of bacteriocin production between Lactobacillus plantarum and Lactobacillus casei.

• Extraction and purification of the bacteriocin from probiotic bacteria from tumeric rhizome and study the effect of temperature and pH to activity of bacteriocin and determination of molecular mass of bacteriocin.

• To study the application of bacteriocin as a food biopreservative.

• Preparation, characterization and application of bacteriocin-cassava starch film.

Bacterial culture and media

MRS medium37°C

Nutrient medium37°C

Probiotic strains• Lactobacillus plantarum• Lactobacillus casei TISTR 1463

Pathogen strains• Escherichia coli TISTR 780•Salmonella typhimurium TISTR 292•Staphylococcus aureus TISTR 029

Antimicrobial and bacteriocin activity assay

L. plantarum and L. casei were cultured in MRS broth

The culture of both strain were taken every 2 h (from 16 to 26 h)

Bacteriocin activity test

adjust pH to 6.5 and add catalase enzyme

Bacteriocin activity assay

Table 1 show the inhibition zones by Agar well diffusion assay

strain Diameter of inhibition zone of L. plantarum (mm) (18 h)

Diameter of inhibition zone of L. casei (mm) (24 h)

E. coli 10.5 10

Salmonella typhimurium 10 -

Staphylococcus aureus 10 10

Purification of bacteriocin

1 L of Lactobacillus plantarum culture in MRS medium at 37°C, 18 h

Protein precipitation by ammonium sulphate

Dialyze bacteriocin overnight through 1000 Da MWCO membrane

Agar well diffusion assayIndicator : L. casei

Fig 1. Dialyze bacteriocin through dialysis membrane

Fig. 2 Bacteriocin activity of L. plantarum against L. casei

the effect of temperature and pH on bacteriocin activity

Sensitivity of bacteriocin to heat and pH

pHHeat

40°C 60°C 80°C 95°C

10 min 20 min 30 min 60 min

Agar well diffusion test

pH 3 pH 5 pH 7 pH 9

Agar well diffusion test

2 h

The effect of temperature on bacteriocin activity

Bacteriocin activity stable at 95°C for 10 min.

The activity decreases with the increasing time in 95°C.

The effect of pH on bacteriocin activity

Bacteriocin activity stable at pH 5 and 7. At pH 3 and pH 9, bacteriocin activity decrease 50%.

Determination of molecular mass of bacteriocin

Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SDS-PAGE is a technique widely used to separate proteins according to their electrophoretic mobility

Determination of molecular mass of bacteriocin

Molecular weight(Daltons)

14,437

6,512

3,4961,423

12-14 kDa

1-2 kDa

Inhibition zone

Shelf-life of pasteurized milk

First treatment : without partially purified bacteriocin (Control)

Second treatment : 0.5% partially purified bacteriocin

Third treatment : 1% partially purified bacteriocin

Forth treatment : 2.5% partially purified bacteriocin

pH of pasteurized milk during storage

Preparation of cassava starch based films

Control filmMixture of cassava starch,

glycerol and water at ratio of 5.0: 2.0: 93.0 to obtain film

forming solution

Partially purified bacteriocin filmMixture of cassava starch, glycerol, bacteriocin and water at ratio of 5.0: 2.0: 1.0: 92.0 to obtain film forming

solution

The whole extracts filmMixture of cassava starch, glycerol,

supernatant of L. plantarum culure at 18 h and water at ratio of 5.0: 2.0: 46.5: 46.5 to

obtain film forming solution

Scanning Electron Microscope (SEM)

Shelf-life of fermented pork

Microbial testing

Plastic bag (control)

Cassava starch filmCassava starch film

incorporated with the whole extract

Cassava starch film incorporated with

bacteriocin

• Bacteriocin activity of both L. plantarum and L. casei showed significant antibacterial activities against indicator strains which were Escherichia coli, Salmonella typhimurium and Staphylococcus aureus.

• L. plantarum can produce more efficient of bacteriocin than L. casei TISTR 1463.

• The activity of partially purified bacteriocin from L. plantarum is 5,120 AU/ml.

• The bacteriocin activity of L. plantarum was not affected when treated at 95°C for 10 min, and stable at pH 5 and pH 7.

• The molecular weight of bacteriocin extracted from L. plantarum isolated from turmeric rhizome is approximately 1-2 kDa and 12-14 kDa.

• The partially purified bacteriocin from L. plantarum has ability to prolong the shelf-life of product.

• The surface of all type of edible film based on cassava starch was wrinkled with small folding.

• The cassava starch incorporated with the whole extract has ability to prolong the shelf-life of product.