Roxana Beatriz Medina, Marta Beatriz Katz, and Silvia González
Lactic acid bacteria (LAB) comprise a diverse group of Gram-positive, non-spore-forming microorganisms. These bacteria are widely used in food technology. The species identification of LAB depends mainly on physiological and biochemical criteria. The esterolytic systems of LAB remain poorly characterized. Esterases (EC 184.108.40.206) represent a diverse group of hydrolases catalyzing the cleavage and formation of esters bonds (1) Screening of esterases is usually performed either by employing chromophoric substances (e.g., a- or p-naphthyl esters of short-chain fatty acids). The post-electro-phoretic detection of esterases is a sensitive technique applied in bacterial systems, that mainly provides information on the similarity of strains within the same species or subspecies according to their esterase patterns. This technique is principally used to determine the number and substrate specificity of esterases and lipases, revealing the complexity of lipase and esterase systems (2,3). The present chapter describes the technique of polyacrylamide gel electrophoresis (PAGE; in the absence of sodium dodecyl sulfate [SDS]), in non-denaturing conditions, to find intracellular fractions for strain typing of LAB.
1. de-Man-Rogosa-Sharpe (MRS) broth (4): 10 g/L polypeptone, 10 g/L meat extract, 5 g/L yeast extract, 20 g/L glucose, 5 g/L sodium acetate, 1 g/L Tween-80, 2 g/L ammonium citrate, 2 g/L K2HPO4, 0.2 g /L MgSO4 -7H2O, and 0.05 g/L MnSO4- 4H2O, pH 6.4 ± 0.2. Sterilize in an autoclave at 121°C for 15 min.
2.2. Postelectrophoretic Detection of Esterases
1. Phosphate buffer.
a. Solution A (0.2 M NaH2PO4): weigh 27.6 g NaH2PO4-H2O and make to 100 mL with distilled water (dH2O).
b. Solution B (0.2 M Na2HPO4): weigh 53.05 g of Na2HPO4-7H2O and make to 100 mL with dH2O.
c. To prepare 1 L of 0.1 M phosphate buffer (pH 7.0), mix 195 mL of solution A and 305 mL of solution B and bring to 1000 mL with dH2O.
2. Acrylamide solution.
a. Weigh 29.2 g acrylamide and 0.8 g N,W-methylene-Ws-acrylamide, add about 50 mL of dH2O, stir until disolved, and dilute to 100 mL.
b. Remove insoluble material by filtration.
c. Store at 4°C in a dark bottle (30 d maximum to avoid hydrolysis of acrylamide to acrylic acid).
d. Caution: acrylamide is neurotoxic; repeated skin contact or inhalation is dangerous.
3. Separation gel buffer: dissolve 18.15 g Tris in 80 mL dH2O, adjust to pH 8.8 with HCl, and make up to 100 mL with dH2O. The solution is stable for at least 2 wk when stored at 4°C.
4. Stacking gel buffer: dissolve 6 g Tris in 80 mL dH2O, adjust to pH 6.8 with HCl, and make up to 100 mL with dH2O. The solution is stable for at least 2 wk when stored at 4°C.
5. 10% (w/v) Ammonium persulfate (APS): dissolve 100 mg APS in 1 mL dH2O. Always use a freshly prepared solution.
6. Reservoir buffer: prepare solution by combining 6.05 g Tris-HCl and 28.8 g glycine. Dissolve with dH2O to a final volume of 2 L. Final pH should be about 8.3. Store at 4°C; warm to 30°C before use if precipitation occurs.
7. N,N,N',N'-Tetramethylethyenediamine (TEMED).
8. Sample buffer: prepare solution by combining 4 mL dH2O, 1 mL stacking gel buffer, 0.8 mL glycerol, and 0.002% bromophenol blue. Dilute the sample 1:1 with sample buffer.
9. 1% (w/v) Substrate solution: dissolve 100 mg a- and ß-naphthyl derivate of fatty acids of C2-C10 carbon atoms in 1 mL acetone. Dissolve 100 mg a- and ß-naphthyl derivate of fatty acid of C12 carbon atoms in propanol-2. Always use a freshly prepared solution.
10. Solution substrate for developing color: dissolve 5 mg of Fast Red TR (Sigma) in 10 mL phosphate buffer 0.1 M, pH 7.0, and add 200 ^L substrate solution.
11. Glass beads no. 31/14 (diameter, 0.1-0.11 mm; B. Braun Biotech International, Germany).
12. Cell disruptor (B. Braun Melsungen, Germany).
13. Mini-Protean II electrophoresis cell (Bio-Rad).
3.1. Cultivation of Bacteria
1. Inoculate 2% of the microorganisms in 200 mL MRS broth.
2. Incubate overnight at 30°C (lactococci, leuconostocs, and mesophilic lactobacilli) or 37°C (enterococci and thermophilic lactobacilli) (see Note 1)
3. Collect cells by centrifugation at 10,000g for 10 min at 4°C.
4. Wash the pellet twice with 10 mL of 0.1 M sodium phosphate buffer, pH 7.0.
5. Resuspend at 40% w/v washed cells in the afore-mentioned buffer by vortexing. Be sure that the pellet is completely resuspended before proceeding.
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