Antimicrobials from Lactobacillus

plantarum isolated from turmeric

(Curcuma longa linn.) and their applications as biopreservative and in edible film

Melada Supakijnoraset

Food Engineering and Bioprocess Technology, School of Environment, Resources and Development, Asian Institute of Technology

Probiotic

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

Lactobacillus Bifidobacterium

Probiotic Antimicrobial 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 bacteriocin-cassava starch film.

and application of

Bacterial culture and media

• • Probiotic strains

Lactobacillus plantarum

Lactobacillus casei TISTR 1463 MRS medium 37 ° C • Pathogen strains Escherichia coli TISTR 780 • Salmonella typhimurium TISTR 292 • Staphylococcus aureus TISTR 029 Nutrient medium 37 ° C

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) adjust pH to 6.5 and add catalase enzyme Bacteriocin activity test

Bacteriocin activity assay

Table 1 show the inhibition zones by Agar well diffusion assay strain

E. coli Salmonella typhimurium Staphylococcus aureus

Diameter of inhibition zone of L. plantarum (mm) (18 h) Diameter of inhibition zone of L. casei (mm) (24 h) 10.5

10 10 10 10

Purification of bacteriocin

1 L of Lactobacillus plantarum culture in MRS medium at 37 ° C, 18 h Fig 1. Dialyze bacteriocin through dialysis membrane Protein precipitation by ammonium sulphate Dialyze bacteriocin overnight through 1000 Da MWCO membrane Agar well diffusion assay Indicator : L. casei

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

the effect of temperature and pH on bacteriocin activity 40 ° C 60 ° C 10 min 20 min Sensitivity of bacteriocin to heat and pH Heat 80 ° C 30 min 60 min Agar well diffusion test 95 ° C pH pH 3 pH 5 pH 7 2 h Agar well diffusion test pH 9

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

12-14 kDa Molecular weight (Daltons) 14,437 6,512 3,496 1,423 Inhibition zone 1-2 kDa

Shelf-life of pasteurized milk First treatment : without partially purified bacteriocin (Control) Second treatment : 0.5% partially purified bacteriocin Third treatment Forth treatment : 1% partially purified bacteriocin : 2.5% partially purified bacteriocin

pH of pasteurized milk during storage 6,8 6,75 6,7 6,65 6,6 6,55 6,5 6,45 6,4 0 10 20 30

Time (Days)

40 50 Pasteurized milk 0.5% partially purified bacteriocin 1.0% partially purified bacteriocin 2.5% partially purified bacteriocin

Preparation of cassava starch based films

Control film

Mixture of cassava starch, glycerol and water at ratio of 5.0: 2.0: 93.0 to obtain film forming solution

Partially purified bacteriocin film

Mixture 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 film

Mixture 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

Physical properties of cassava starch based film

Scanning Electron Microscope (SEM)

Shelf-life of fermented pork Plastic bag (control) Cassava starch film Cassava starch film incorporated with the whole extract Cassava starch film incorporated with bacteriocin Microbial testing

25 000 20 000 15 000 10 000 5 000 0 0 3 6

Time (day)

9 plastic bag cassava starch film 12 cassava starch film incorporated with the whole extracts Cassava starch film incorporated withpartially purified bacteriocin

• Bacteriocin activity of both

L. plantarum

and

L. casei

showed significant antibacterial activities against indicator strains which were

Escherichia coli Staphylococcus aureus

.

,

Salmonella typhimurium

and •

L. plantarum

can produce more efficient of bacteriocin than

casei

TISTR 1463.

L.

• 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

plantarum

isolated from turmeric approximately 1-2 kDa and 12-14 kDa.

rhizome

L.

is • 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.