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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
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ProbioticThe probiotics are most commonly defined as live microbial feed supplements that beneficially affect the host by improving its intestinal microbial balance.
Lactobacillus Bifidobacterium
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ProbioticAntimicrobial substances
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•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
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• 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.
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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
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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
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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
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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
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Fig. 2 Bacteriocin activity of L. plantarum against L. casei
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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
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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.
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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%.
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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
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Determination of molecular mass of bacteriocin
Molecular weight(Daltons)
14,437
6,512
3,4961,423
12-14 kDa
1-2 kDa
Inhibition zone
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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
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pH of pasteurized milk during storage
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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
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Scanning Electron Microscope (SEM)
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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
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• 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.
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• 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.
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