Probiotic Properties of Lactobacilli
3.4. Bile Salt Hydrolase Activity 1. Plate Assay (6)
1. Melt the agar media: MRS agar and MRSBA agar (described in Subheading 2.5.1.) in boiling water. Pour each melted medium separately into sterile Petri dishes (60 × 15 mm).
2. Once solidified, invert the plates and place in an anaerobic chamber for at least 48 h before using.
3. Inoculate each plate on surface with an overnight culture grown in MRS broth by using a 10-µL loop.
4. Incubate the plates at 37°C in anaerobic jars (Systen Oxoid) for 72 h.
5. The bile salt hydrolase activity of the cultures is evidenced by the formation of a white precipitate around the colonies grown in MRSBA agar. This precipitate is not observed in MRS agar (control) without bile salts, where colonies are translucent (seeNote 7).
3.4.2. Colorimetric Assay (7)
1. Inoculate (1%) 20 mL of MRSTT broth with an overnight culture grown in MRS broth and incubate at 37°C for 16 h.
2. Adjust the pH to 7.0 with 1 NNaOH and take to 25 mL with distilled water (see Note 8).
3. Remove the cells by centrifugation at 12,000g at 4°C for 10 min.
4. Adjust 15 mL of the resulting supernatant fluid to pH 1.0 by using 1 NHCl and take to 24 mL with distilled water. (SeeNote 8).
5. Transfer 3 mL of each sample to glass test tubes and add 9 mL of ethyl acetate.
6. Mix the content of each tube and let settle to allow the complete phase separation.
7. Transfer 3 mL of the ethyl acetate layer (upper phase) to a clean test tube and evaporate to dryness at 60°C under nitrogen gas flow.
8. Dissolve the residue formed with 1 mL of 0.01 N NaOH.
9. Carefully add 6 mL of 16N H2SO4to each tube, followed by the addition of 1 mL of 1% furfuraldehyde.
10. Mix the tubes, heat at 65°C for 13 min in water bath, and cool at room temperature.
180 Font de Valdez and Taranto 11. Add 5 mL of glacial acetic acid to each tube, mix the content vigorously, and
read the absorbance at 660 nm (A660) against the reagent blank (seeNote 9).
12. The results are expressed as micromoles (µmol) cholic acid per milliliter.
3.4.3. HPLC Analysis (8)
1. Inoculate (1%) MRSTC broth with an overnight culture grown in MRS broth and incubate at 37°C for 16 h.
2. Remove the cells by centrifugation at 8000g for 5 min and filter the supernatant by using a of 0.22-µm filter membrane.
3. Inject 50 µL of each standard solution, previously filtrated and diluted (1:2).
4. Perform the analysis on a 250 × 4.6-mm C18 Spherisorb 5-µm column.
5. Use the following elution program: isocratic elution with 15% of eluent B, 85%
of eluent A for 10 min, then a 25-min linear gradient to 90% of solvent B.
6. Maintain the mobile-phase composition at 90% of solvent B. The flow rate is 1 mL/min.
7. Perform the detection at 210 nm, and process the data with System GoldTM software.
8. After running the standard, follow steps 3–7for the samples obtained in step 2, which are also filtered and diluted (1:2) (seeNote 10).
4. Notes
1. The range of bile salt concentration used (0.05–0.3%) corresponded to that found in the human intestinal tract; 0.3% bile is the maximun concentration that is present in healthy men.
2. Lactic acid bacteria are anaerobic or microaerophile. Culture growth in liquid and agarized media for evaluation of both cholesterol reduction and bile salt hydrolase activity were performed under anaerobiosis. Thioglycholate and cys- teine chlorhydrate are used to mantain a low redox potential.
3. Methanol and sodium acetate used for HPLC analysis must be of HPLC grade.
4. For determing the bile tolerance of the cells, it is important to use a low inoculum (0.5%, v/v) in order to have a low initial OD560value and to assure that the culture is at an early exponential phase of growth.
5. The comparison of the cultures is based on the time required for each of them to increase the OD560by 0.3 units (generally, this value is found in the early exponen- tial phase of growth) in both MRS and MRSO broths; the difference in time (min) between the culture media is considered as the growth delay (D) (seeFig. 1).
6. The A550is compared with a standard curve to determine the concentration of cholesterol. The same procedure for the samples is used for the standard curve, except that the following amounts of cholesterol (Sigma L-4646) are assayed in place of the samples: 0, 10, 20, 30, 40, 50, 60, 70, 80, 90, and 100 µg. The A550 values are plotted against microgram of cholesterol.
Probiotic Properties of Lactobacilli 181 7. TDCA and GDCA produce the most sharply defined halos (white dense precipitate and diffused halos around colonies, respectively (seeFig. 2), whereas TCA and GCA are slightly less effective. Strains with a high bile salt hydrolase activity for TDCA and GDCA will also release high concentrations of deoxycholic acid, which may inhibit the growth in the plate. In this case, it is suggested to use a lower TDCA or GDCA concentration (2 mM).
8. The pH must be carefully adjusted to 7.0 and 1.0 in each case. In this step, the complete separation of the two forms of the bile acid (i.e., conjugated and unconjugated taurocholic acid) as well as the further extraction of the cholic acid released is performed.
9. The A660 is compared with a standard curve to determine the concentration of cholic acid. The same procedure used for the samples is applied for the standard curve, except that the following amounts of cholic acid are assayed in place of the samples: 0, 0.5, 0.7, 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, and 4.0 µmol. The A660values are plotted against micrograms of cholic acid per milliliter.
10. The early eluting peak (about 0.1 U absorbancy in the elution time 1.6–4 min), corresponds to residues of MRS broth. The elution time for taurocholic, glycocholic, and cholic acids are 4.75, 6.22, and 17.11 min, respectively.
Therefore, the first peak does not interfere with the measurements of interest.
References
1. Hepner, G., Fried, R., Jeor, S., Fusetti, L., and Morin R. (1979) Hypocholesterolemic effect of yogurt and milk. Am. J. Clin. Nutr.32,19–24.
2. Walker, D. K. and Gilliland, S. E. (1983) Relationship among bile tolerance, bile salt deconjugation, and assimilation of cholesterol by Lactobacillus acidophilus.
J. Dairy Sci.76,956–961.
3. Kilara, A. (1982) Influence of in vitrogastric digestion on survival of some lactic cultures.Milchwissenschaft 37, 129–132.
4. De Man, J. C., Rogosa, M., and Sharpe, M. E. (1960) A medium for the cultiva- tion of lactobacilli. J. Appl. Bacteriol.23, 130–135.
5. Rudel, L. L. and Morris, M. D. (1973). Determination of cholesterol using o-phthalaldehyde.J. Lipid Res.14, 364.
6. Dashkevicz, M. P. and Feighner, S. D. (1988) Development of a differential medium for bile salt hydrolase-active Lactobacillus sp. Appl. Environ. Microbiol.55,11–16.
7. Irvin, J. L., Johnson, C. G., and Kopalo, J. (1944) A photometric method of deter- mination of cholates in bile and blood. J. Biol. Chem. 439–457.
8. Scalia, S. (1988) Simultaneus determination of free and conjugated bile acids in human gastric juice by high performance liquid chromatography. J. Chromatogr.
431, 259–269.
Identification of Exopolysaccharide-Producing LAB 183
183
From: Methods in Biotechnology, Vol. 14: Food Microbiology Protocols Edited by: J. F. T. Spencer and A. L. Ragout de Spencer © Humana Press Inc., Totowa, NJ