Cheddar cheese and Mozzarella cheese used in their study were obtained from retail super market (Nilgiris), Chennai. A well vacuum packed cheese was obtained.
Materials and Methods
- Sample collection – Cheddar cheese & Mozzarella cheese
- Serial dilution and spread plate method
- Isolation of Lactic acid bacteria in MRS medium
- Selective Isolation of Leuconostoc from other LAB
- Addition of vancomycin to MRS medium
- Leuconostoc was isolated and maintained at 4oC
- Species identification
- Biochemical characterization
Sample collection: Cheddar cheese and Mozzarella cheese used in their study were obtained from retail super market (Nilgiris), Chennai. A well vacuum packed cheese was obtained.
Bacterial Strains and Culture
Isolation of Leuconostoc
Culture Media: Leuconostoc are fastidious chemo-organotrophic bacteria. Therefore, culture media must be rich in nutrients such as carbohydrates as energy providers, amino acids as nitrogen compounds, salts and vitamins. The most widely used medium was MRS medium (Man Rogosa Sharpe). The supplements studied in MRS medium are glucose, peptone, yeast extract, beef extract, dipottasium hydrogen phosphate, ammonium citrate, magnesium sulphate, manganese sulphate sodium acetate, and tween 80. Tween 80 usually increases the growth of Leuconostoc by providing oleic acid incorporated into the cell membrane.
Composition of MRS medium Media gm/lit
- Glucose – 1.0gm
- Peptone – 1.0gm
- Beef extract – 0.8gm
- Yeast extracts - 0.5gm
- Di-potassium hydrogen phosphate
- (K2HPo4) – 0.2gm
- Magnesium sulphate (MgSo4) – 0.2gm
- Manganese sulphate (MnSo4) – 0.005gm
- Ammonium citrate – 0.2gm
- Sodium acetate – 0.5gm
- Tween – 80 0.1gm
- Agar – 2.0gm
- pH – 6.8gm
The medium was sterilized at 121oC for 15mins. Leuconostoc was enumerated by spread plate method. Serial dilutions were made from the master dilutions containing 1gm of cheddar cheese in 99ml of distilled water which gives a dilution of 10-1 (serial dilutions were made upto 10-1). 0.1ml of each dilution was spreaded on MRS medium, and incubated at 37oC for 24hrs.
From food sample, all LAB are counted since the complex nutrient requirements and optimal conditions for the growth are roughly comparable for lactobacillus, Pediococcus and Leuconostoc. So, for selective isolation of Leuconostoc from other lactic acid bacteria (vancomycin sensitive), 30mg/ml of vancomycin solution was added to the MRS medium. The Leuconostoc strain isolated was maintained by sub-culturing in MRS medium.
Indicator bacterial strains
- Salmonella typhi, ATCC-21563
- Escherichia coli, ATCC-1023
- Shigella flexinerrae, ATCC-11461
were received from NCIM and maintained by sub-culturing in nutrient agar slants for further biochemical tests. The incubation period for the test organisms was 37oC for 24hrs. All the cultures were stored at 4oC.
Media used for cultural characterization of Leuconostoc
- MacConkey agar medium
- Blood agar medium
Biochemical characterization of Leuconostoc for species identification
- Gram’s staining
- Motility test – hanging drop method
- Capsule staining – negative staining
- Spore staining – Scaffer-fulton method
- Methyl red
- Voges Proskauer
- Arginine hydrolysis
Carbohydrate fermentation test
All the above biochemical tests were performed and the results were noted to differentiate Leuconostoc species. The differentiating test among Leuconostoc species are catalase test, arginine test and carbohydrate fermentation tests.
Separate biochemical test were performed for the indicator organisms to confirm the strains and results were noted.