bile salt deconjugation and cholesterol

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saltsandremove cholesterol from growth media.Thus thepresentstudy investigated the deconjugation of bile salts and removal of cholesterol in vitro by the 12 Lactobacillus strains. Information onthis will be useful, since, to the best of our knowledge, this is therst study on cholesterol-removing abilities of lactobacilli isolatedfrom chickens.

MATERIALS AND METHODSSource and maintenance of Lactobacillus strains The 12 Lactobacillus strains used in this study were the same asthose isolated from the gastrointestinal tract of local chickensand identied and described by Jin etal .4 They were obtainedfrom the culture collection at the Lifestock Microbial TechnologyUnit, Institute of Bioscience, Universiti Putra Malaysia. Recently,the 12 strains were re-identied using molecular characteristics, 18

and the new identities were used in this study (see Tables 1and 2). The strains were maintained by routine subculturing in deManRogosaSharpe (MRS) broth (Oxoid Ltd, Basingstoke, UK)using a 10 mL L 1 inoculum froman overnightculture. Incubationwas performed under anaerobic conditions (90130 mL L 1 CO2)using anaerobic jars (Oxoid Ltd) with gas-generating kits (OxoidLtd) at 39 C. The cultures were stored in 150 mL L 1 glycerolat 80 C between transfers. Prior to experimental use, eachLactobacillus strain was subcultured at least three times to ensurethat it was physiologically active.

Deconjugation of bile saltsDeconjugation of bile salts by the 12 Lactobacillus strains wasquantiedusing high-performance liquid chromatography (HPLC)as described by Corzoand Gilliland 19 based on the disappearanceof sodium glycocholate (GCA) and sodium taurocholate (TCA)from the growth medium. The medium used for the assaywas MRS broth (50 mL) supplemented with 1 mmol L 1 TCA(Sigma-Aldrich, St Louis, MO, USA) and 1 mmol L 1 GCA (Sigma-Aldrich). The medium was inoculated separately with 10 mL L 1

of overnight culture of each of the 12 Lactobacillus strains. The inoculated medium was incubated at 39 C for 24 h underanaerobic conditions (as described above), after which cells wereremoved by centrifugation (Avanti J-251, Beckman, Fullerton,CA, USA) at 10 000 g for 10 min at 4 C and the supernatantwas ltered through a 0 . 45 m polyethersulfone membrane(Whatman,Maidstone,UK).ThesolutionwasloadedthroughaSep-Pak C18 cartridge (Waters, Milford, MA, USA) and the conjugatedbile acids were eluted from the cartridge with pure methanol,which was then evaporated to dryness with nitrogen gas. Theresidue was dissolved in 1 mL of mobile phase, then passed

through a 0.

45 m nylon lter (Whatman) and analysed using ahigh-performanceliquid chromatograph(HPLC, Waters)equippedwith a variable wavelength UV detector, an automated injectorand a exible-walled reverse phase column (100 mm 8 mm i.d.)packedwith4 m Nova-Pak C 18 (Waters).As mobile phase, 700 mLof methanol and 300 mL of 0.02 mol L 1 acetic acid were usedand the pH was adjusted to 5.6 with 5 mol L 1 NaOH. HPLC-gradesolvents were used throughout the study. The sample or the bilestandard (TCA and GCA, 0 800 g mL 1) was mixed with50 L of internal standard (0.2 mg mL 1 dexamathasone, Sigma-Aldrich),then 20 L of the mixture was passed through a 0 . 45 m nylonlter (Whatman) and injected into the HPLC. UV detection wasperformed at 205 nm and 0.2 absorbance unit. Peak area wascalculated by Millennium software (Waters). The experiment was

carried out three times, and two replicates were made for eachtreatment.

Cholesterol removal from growth mediaA 10 mL aliquot of MRS broth containing 3 g L 1 bile saltfrom chicken (Sigma-Aldrich) and cholesterol with 10 mL L 1

pleuropneumonia-like organism (PPLO) serum fraction (Difco,

Sparks, MD, USA) was inoculated separately with 10 mL L 1

of overnight culture of each of the 12 Lactobacillus strains. Theinoculated bottles were incubated at 39 C under anaerobicconditions for 24 h. The bacterial cells were removed fromthe culture broth by centrifugation (Avanti J-251, Beckman) at12 000 g for 15 min at 4 C and the supernatant was useddirectly for measuring cholesterol. Total cholesterol was analysedusing an enzymatic procedure, Sigma Diagnostics Procedure No.352 (Sigma-Aldrich), which is a modication of the method of Allain etal .20 The amount of cholesterol removed from the growthmedium wasexpressedas a percentage of the initial concentrationintheuninoculatedbrothmedium.Theexperimentwascarriedoutthree times, andtwo replicates were made foreach treatment. ThepH of the growth medium wasmeasured (Cyberscan 500, ThermoFisher, Waltham, MA,USA)before andafter24 h of incubation witheach of the Lactobacillus strains.

CalculationDeconjugation of bile salts (%) = (amount of TCA or GCAdeconjugated/total amount of TCA or GCA) 100.

Statistical analysisSPSS Version 16 (SPSS Inc., Chicago, IL, USA) was used for thestatistical analyses. For testing mean differences, the analysisof variance (ANOVA) procedure was used. In all analyses the P values for the test of homogeneity were less than 0.05. Hence theGamesHowel post hoc test was used to identify signicant meandifferences. Pearsons bivariate correlation procedure was used tostudy the associations between deconjugation of TCA or GCA andcholesterol removal by the 12 Lactobacillus strains.

RESULTS AND DISCUSSIONAll 12 Lactobacillus strains were able to deconjugate GCA(16.87100%)and TCA(1.6957.43%), butthe level of deconjuga-tion differed signicantly ( P < 0 . 05) among the strains (Table 1).Except for L. salivarius I 24, which showed equally low deconju-gating activity on GCA (16.87%) and TCA (16.65%), the other 11Lactobacillus strains could deconjugate GCA better than TCA. Thethree L. reuteri strains (C 1, C 10 and C 16) and two L. gallinarum

strains (I 16 and I 26) showed high capacity for deconjugat-ing GCA (81.17100%) but moderate ability to deconjugate TCA(35.1657.43%). Of theve L. brevisstrains, I 23 could deconjugatea large amount of GCA (83.37%), while the other four strains (I 12,I 25, I 211 and I 218) could only deconjugate moderate amounts(44.7866.78%). However, all ve strains showed low activity to-wards TCA (1.693.28%). L. panis C 17, like most of the L. brevisstrains, deconjugated a moderate amount (69.82%) of GCA and alow amount (4.19%) of TCA. Among the 12 Lactobacillus strains,positive correlation between deconjugation of TCA and GCA wassignicant ( P < 0 . 05) only in L. reuteri C 1 (r = 0 . 84) and L. brevisI25 (r = 0 . 87) (Table 1).

The ability of the 12 Lactobacillus strains to deconjugatebile salt is not surprising, as the strains were isolated from

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Table 1. Deconjugation of sodium taurocholate (TCA) and sodiumglycocholate (GCA) bile salts by 12 Lactobacillus strains a

Deconjugation (%) bLactobacillusstrain TCA GCA r value c P value

L. reuteri C 16 42. 09 1 . 95b 100 . 00 0 . 01a 0.05 0.93L. reuteri C 10 50. 23 0 . 98b 100 . 00 0 . 01a 0.64 0.17L. reuteri C 1 35. 16 1 . 30b 100 . 00 0 . 01a 0.84 0.04L. brevisI 23 3. 28 0 . 71d 83 . 37 5 . 52b 0.83 0.04L. brevisI 12 1. 69 0 . 37d 57 . 25 2 . 35d 0.34 0.51L. brevisI 25 1. 74 0 . 95d 44 . 78 1 . 87d 0.87 0.03L. brevisI 211 2. 59 0 . 86d 66 . 78 1 . 42c 0.23 0.66L. brevisI 218 2. 79 0 . 50d 65 . 98 2 . 14c 0.43 0.39L. salivarius I 24 16. 65 0 . 65c 16 . 87 0 . 78e 0.12 0.82L. gallinarum I 16 51. 45 2 . 35a 81 . 17 2 . 27b 0.25 0.63L. gallinarum I 26 57. 43 3 . 80a 89 . 15 4 . 49a 0.37 0.47L. panis C 17 4. 19 1 . 05d 69 . 82 2 . 24c 0.69 0.129

Data aremean SE of three experiments, eachduplicated. Means withdifferent letters within a column differ signicantly ( P < 0 . 05).a Lactobacillus strains were grown in MRS broth supplemented with

1 mmol L

1 GCA and 1 mmol L

1 TCA and incubated at 39C for 24 h.b Deconjugation (%) = (amount of TCA or GCA deconjugated/total

amount of TCA or GCA) 100.c Pearson correlation.

the gastrointestinal tract of chickens where bile is a commonoccurrence. In an earlier study, Knarreborg etal .17 also reportedthat two Lactobacillus strains isolated from chicken intestine wereable to deconjugate a taurine-conjugated bile salt. Tanaka etal .7

studied more than 300 lactic acid bacterial strains and reportedthat BSH activity was found primarily in organisms isolated fromthe gastrointestinal tracts of mammals, while organisms isolated

from fermented milk preparations and vegetables did not exhibitBSH activity. The studies indicate that BSH-active organisms aremost often isolated from the intestines or faeces where theenvironment is rich in bile acids. 21 The reason for the varyingability to deconjugate bile among the 12 Lactobacillus strainsin this study is not known. However, recent microbial genomeanalyses suggest that the organisation and regulation of genesencoding BSH differ between species and genera 8 and evenamong strains. 7 Begley etal .21 reported that several lactobacillipossess more than one BSHhomologue, which are notnecessarilyidentical, and that the bsh gene may also have different geneticregions, causing variability in their deconjugating ability.

Bile from chicken generally contains glycine conjugates in a

higher proportion than taurine conjugates, and this could havecontributed to the higher afnity for glycine conjugates shownby most of the Lactobacillus strains used in the present study. 22

The higher deconjugating activity of the Lactobacillus strains onglyco-conjugates may be of considerable signicance for thesurvival of these Lactobacillus strains in the gut. The glyco-conjugates are far more toxic than the tauro-conjugates; thismay be attributed to the difference in dissociation constant of taurine and glycine conjugates (p K a values of GCA and TCA are3.9 and 1.0 respectively). 11,23 Unlike glycine, taurine metabolismcontributes to production of hydrogen sulde, which is highlytoxic to the host after deconjugation. 8 Therefore strains that areable to deconjugate mostly glycine but little or no taurine may beuseful as probiotics (Table 1).

Although lactobacilli are able to deconjugate GCA and TCA tounconjugatedprimary bileacids, theydo notfurther transformtheunconjugatedbileacids intodeoxycholate (secondarybile acids). 16

This is a good probiotic trait, because formation of secondary bileacids, which areusually producedby intestinalbacteria, may causeundesirable effectson thehost. 21 Probiotic lactobacillimay protectthe host, as they have been found to assimilate deconjugated bileacids (cholic acid) in vitro,8 and the majority of the primary bileacids produced through their BSH activity are precipitated andexcreted in faeces. 21

All 12 Lactobacillus strains in the present study were capableof removing cholesterol from the growth medium after 20 h of incubation, but the percentage of cholesterol removal variedconsiderably among the strains (Table 2). The pH changes inthe broth media of the 12 Lactobacillus strains during growthwere quite similar (data not shown). The pH of the medium wasinversely proportional to growth; that is, as growth increased, thepH of the medium decreased from 5.96 (initial pH) to 4.05 whengrowth was maximum. Lactobacillus reuteri C 10 was the mostactive ( P < 0 . 05) in removing cholesterol (85.41%). A signicant(P < 0 . 05) correlation was observed between cholesterol removal

and deconjugation of TCA ( r = 0 . 83) among the L. reuteri strains(C1, C10 and C16) (Table 2). The L. brevis strains (I 12, I 23, I25, I 211 and I 218) removed cholesterol by 31.15 65.53%,and signicant ( P < 0 . 05) correlations were observed betweencholesterol removal and deconjugation of both TCA ( r = 0 . 38)and GCA (r = 0 . 70) (Table 2). Previous studies have also reportedcorrelations between removal of cholesterol and deconjugationof bile salts by L. acidophilus16,2426 and L. casei 26 isolated fromdairy products or of human origin. Klaver and Van der Meer 25

hypothesised that cholesterol removal by L. acidophilus or otherbacterial cells is due to the co-precipitation of cholesterol withdeconjugated bile salts as the pH of the medium drops ( < 6)with increased acid production during growth. This model hasbeen frequently used to explain in vivo hypocholesterolaemiceffects andis considered a relevant property of potentialprobioticspecies.

Results from the present study showed that L. salivarius I 24removed cholesterol (50.16%) to a greater extent ( P < 0 . 05) thanL.gallinarum I 16 (46.88%)and I 26 (45.24%)did. LactobacilluspanisC 17 removed the least ( P < 0 . 05) amount of cholesterol (26.74%).Although L. gallinarum I 16 and I 26 showed high deconjugatingactivity,theirabilitytoreducecholesterolfromthegrowthmediumwas signicantly ( P < 0 . 05) lower than that of L. salivarius I 24,which had low deconjugating ability. Lactobacilluspanis C 17 alsoshowed moderate deconjugating ability ( 70%) butcouldreduceonly a low amount of cholesterol. Statistical analyses also showedno signicant correlation between cholesterol removal and bile

saltdeconjugation in L.salivarius I24,L.gallinarum I16andI26or L. panis C17. Thus thedeconjugation of bile salts may notbe entirelyresponsible for the cholesterol-reducing activity of these strains.Similarly,Usman andHosono 27 reported that 27 outof 28 L.gasseri strains isolated from dairy products were able to remove between29 and 87% of cholesterol from the growth medium, but theyfound no signicant correlation between bile salt deconjugationand cholesterol removal. Other mechanism(s) may be involved inthe removal of cholesterol from growth media. Gilliland etal .28

suggested that the removal of cholesterol by lactobacilli in vitrowas due to an uptake or assimilation of cholesterol by bacterialstrains. 28 Later, it was further demonstrated that a portion of thecholesterol assimilated by Lactobacillus strains was incorporatedinto the cellular membrane. 26,29,30

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Table 2. Removal of cholesterol from growth media by 12 Lactobacillus strains and correlation with deconjugation of sodium taurocholate (TCA)and sodium glycocholate (GCA) bile salts a

Correlation b

Lactobacillus strain Cholesterol removed (%) TCA GCA

L. reuteri C 10 85. 41 2 . 79a r = 0 . 83, P = 0 . 01 r = 2 . 56, P = 0 . 30L. reuteri C 16 57. 21 2 . 05bL. reuteri C 1 44. 04 1 . 73cL. brevisI 23 65. 53 1 . 40b r = 0 . 38, P = 0 . 04 r = 0 . 70, P = 0 . 01L. brevisI 12 39. 47 1 . 32cL. brevisI 25 31. 15 1 . 91dL. brevisI 211 50. 84 2 . 19bL. brevisI 218 43. 39 1 . 46cL. salivarius I 24 50. 16 2 . 85b r = 0 . 73, P = 0 . 10 r = 0 . 34, P = 0 . 52L. gallinarum I 16 46. 88 2 . 02c r = 0 . 11, P = 0 . 73 r = 0 . 52, P = 0 . 08L. gallinarum I 26 45. 24 1 . 81cL. panis C 17 26. 74 0 . 97d r = 0 . 78, P = 0 . 07 r = 0 . 39, P = 0 . 45

Data are mean SE of three experiments, each duplicated. Means with different letters differ signicantly ( P < 0 . 05).a Lactobacillus strains wereincubated in MRSbroth containingcholesterol and 3 g L 1 bilesalt. The cholesterol content of uninoculated medium was212 . 83 g mL 1 .b Intraspecies correlation ( r value via Pearsons bivariate correlation procedure) between removal of cholesterol and deconjugation of TCA or GCA.

Inconclusion,thepresentstudyshowedthatthe12 Lactobacillusstrains isolated from chickens were able to deconjugate bile saltsand remove cholesterol to varying degrees. Except for L. salivariusI24,the Lactobacillus strains weremoreselective in deconjugatingGCA than TCA. The cholesterol removal observed in the L. reuteri and L. brevisstrains may, at least in part, be due to deconjugationof bile salts; however, in L. salivarius I 24, L. gallinarum I 16and I 26and L. panis C 17, cholesterol could have been reduced throughassimilation, as there was no correlation between cholesterolreduction and bile salt deconjugation. Cholesterol removal from

the growth medium by the Lactobacillus strains may be strain-dependent. However, further studies are required to determinethe mechanism(s) involved in the removal of cholesterol by theseLactobacillus strains in vitro.

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bile salt deconjugation and cholesterol

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