Bifidobacterium bifidum Is Due to Their Bile

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1 PPLIED ND ENVIRONMENTL MICROIOLOGY, pr. 1993, p /93/4112-5$2./ Copyright 1993, merican Society for Microbiology Vol. 59, No. 4 The ssumed ssimilation of Cholesterol by Lactobacilli and ifidobacterium bifidum Is Due to Their ile Salt-Deconjugating ctivity FRNK. M. KLVER' ND ROELOF VN DER MEER2* Departments of Microbiology' and Nutrition,2 Netherlands Institute for Dairy Research (NIZO), P. O. ox 2, 671 Ede, The Netherlands Received 25 September 1992/ccepted 26 January 1993 To study the mechanism of the proposed assimilation of cholesterol, we cultured various strains of LactobaciUlus acidophilus and a ifidobacterium sp. in the presence of cholesterol and oxgall. During culturing, both cholesterol and bile salts were precipitated. ecause of bacterial bile salt deconjugation, no conjugated bile salts were observed in either the culture fluids or the pellets. During incubation, the cell count and optical density decreased. The degree of precipitation of bile salts and of cholesterol was dependent on the culture conditions. If L. acidophilus RP32 was cultured under acidifying conditions, the degree of precipitation of deconjugated bile salts was higher than if the ph was maintained at 6.. Under acidifying conditions, cholesterol was coprecipitated with the bile salts, whereas in ph-controlled cultures, no coprecipitation of cholesterol was observed. From control experiments with different mixtures of bile salts, it appeared that coprecipitation of cholesterol during culturing was a result of formation of deconjugated bile salts, which have a decreased solubility at ph values lower than 6.. It is concluded that the removal of cholesterol from the culture medium by L. acidophilus RP32 and other species is not due to bacterial uptake of cholesterol, but results from bacterial bile salt-deconjugating activity. Lactobacillus acidophilus and ifidobacterium species are frequently associated with health-promoting effects in the human and animal intestinal tract. For this reason, there is increasing interest in the incorporation of these species in fermented milk products. These so-called probiotic effects are generally related to inhibition of pathogenic species (5, 6, 1, 12), reducing the risk of colon cancer (5, 6, 1, 12), increasing the immune response (14, 18), and decreasing serum cholesterol level (5, 7, 9). Gilliland et al. (7) have suggested that the hypocholesteremic effect of L. acidophilus is due to the capacity of bile-resistant strains to assimilate cholesterol. In in vitro experiments, a decrease of the cholesterol level was observed in culture supernatants of L. acidophilus incubated in the presence of bile salts (7). t the same time, an increase in the cholesterol level of the cell pellet was observed. Gilliland et al. (7) referred to this phenomenon as cholesterol assimilation (or uptake) by the cells. Presently, the cholesterol assimilation model is frequently used to explain in vivo hypocholesteremic effects and is considered a relevant property of potentially probiotic species. However, the experimental setup used to prove the cholesterol assimilation theory did not take into account the effects of bacterial deconjugation of bile salts. Deconjugated bile salts have a greatly reduced solubility, which in turn affects the solubility of cholesterol (16). Therefore, we hypothesize that the assumed cholesterol assimilation may not be the result of uptake by L. acidophilus or other bacterial cells, but rather may be due to the coprecipitation of cholesterol with deconjugated bile salts. This paper addresses the in vitro experimental evidence on the role of bile salt deconjugation by lactobacilli and bifidobacteria in relation to cholesterol assimilation. * Corresponding author. 112 MTERILS ND METHODS Origin and maintenance of the bacterial strains. L. acidophilus RP32 was obtained from Oklahoma State University (Stillwater). Lactobacillus casei MUH117, L. acidophilus MUH79, L. acidophilus MUH41, and ifidobacterium bifidum MUH8, originating from commercial starters for fermented dairy products, were obtained from the collection of Mona Research Division (Woerden, The Netherlands). L. acidophilus CH1 originated from the collection of Netherlands Institute for Dairy Research (NIZO). Strains were routinely stored at - C after.5% (wt/vol) inoculation in sterile skim milk containing.5% (wt/vol) yeast extract. Inocula were prepared by incubation of a stock culture for 2 h at 37 C and subsequently subcultured (2 h at 37 C) in MRS medium (E. Merck G, Darmstadt, Germany). Culture conditions. Culturing was performed anaerobically in static culture for approximately 2 h at 37 C in MRS medium, to which.5% (wt/vol) cysteine HCl was added for. bifidum MUH8. fresh inoculum was prepared for every experiment and was added to the medium at 1% (vol/vol). To the final culture medium,.5% (wt/vol) bile salts (oxgall; Difco Laboratories, Detroit, Mich.) and, in some cases5 1% (vol/vol) pleuropneumonia-like organism serum fraction (PPLO, obtained from RIVM, ilthoven, The Netherlands) were added. MRS medium plus oxgall and PPLO contained approximately 6.4 mmol of predominantly conjugated bile salts per liter and 82,umol of cholesterol per liter, whereas MRS medium plus oxgall contained approximately 5.6 mmol of bile salts per liter (more than 97% in conjugated form) and 68,umol of cholesterol per liter. ll medium components were autoclaved (3 min, 12'C), with the exception of cysteine HCl and PPLO, which were sterilized by filtration (.22-,um-pore-size filter; Millipore, Molsheim, France). Culturing with ph control at ph 6. was performed in a

2 VOL. 59, 1993 LCTOCILLI ND. IFIDUM PRECIPITTE CHOLESTEROL 1121 fermentor (DI12; pplikon, Schiedam, The Netherlands) by automatically dosing 12.5% (wt/vol) ammonia into a continuously stirred culture. During ph-controlled fermentation, the headspace of the fermentor was flushed with nitrogen to maintain anaerobiosis. ile salt and cholesterol precipitation at different ph values. MRS medium was brought to different ph values ranging from 4.5 to 7. by the addition of 6 M HCl. To aliquots of MRS medium of the different ph values were added either oxgall, a mixture of individual bile salts of the same concentration as present in oxgall (i.e., 2.65 mmol of glycocholate per liter, 2.88 mmol of taurocholate per liter,.2 mmol of cholate per liter,.18 mmol of glycochenodeoxycholate per liter,.15 mmol of taurochenodeoxycholate per liter,.58 mmol of glycodeoxycholate per liter, and. mmol of taurodeoxycholate per liter; as sodium salts obtained from Sigma Chemical Co., St. Louis, Mo.), or a mixture of the individual deconjugated bile salts at the concentrations present in oxgall after complete deconjugation (5.73 mmol of cholate per liter,.43 mmol of chenodeoxycholate per liter, and 1.18 mmol of deoxycholate per liter; as sodium salts from Sigma). Together with the last two mixtures, the same concentration of cholesterol as present in oxgall (,umol/ liter; DH, Poole, United Kingdom) was also added to MRS. fter 15 min of incubation at 37 C, samples were taken, centrifuged, and analyzed in the same way as described for the culture fluids. Measurement of bacterial growth. acterial growth was monitored by measuring the culture optical density (OD) at 4 nm by the method described in reference 11 and by determining the colony counts on MRS agar (Merck) after 2 days of anaerobic incubation (L GasPak; ecton Dickinson, Cockeysville, Md.) at 37 C. ile salt and cholesterol analyses. 5-ml sample of culture was centrifuged (1 min, 1, x g). Supernatants were used directly for analyses, and the pellets first were resuspended in 5 ml of demineralized water. The concentration of cholesterol in supernatants and petroleum-ether extracts (1) of the pellets was determined enzymatically with the CHOD- PP kit (Monotest Cholesterin; oehringer GmbH, Mannheim, Germany). The total bile salt concentration (conjugated plus deconjugated bile salts) was assayed enzymatically with the Sterognost-3a Flu kit (Nyegaard Diagnostics, Oslo, Norway). nalysis of individual bile salts was performed by high-pressure liquid chromatography (HPLC) as described by Dekker et al. (3). From the data obtained from the analyses, the relative amount of deconjugated bile salts and the relative amount of precipitated cholesterol and bile salts in the pellets were calculated. RESULTS ile salt and cholesterol precipitation by different strains. L. acidophilus RP32 was cultured under acidifying conditions in MRS medium plus oxgall, exactly as described in reference 7. The effect on precipitation of cholesterol and bile salts is shown in Table 1. Cholesterol and bile salts were significantly precipitated by L. acidophilus RP32. The recovery of cholesterol in supematants plus pellets was identical (12% + 6%) to its total concentration, which indicates that precipitated cholesterol is not metabolized. Cultures which were grown in the presence of PPLO gave very similar results (data not shown). ile salts were also completely recovered from the supernatants and pellets. nalysis of the individual bile salts by HPLC showed that only deconjugated bile salts were present after incubation, TLE 1. Degree of precipitation of cholesterol and bile salts and the degree of deconjugation in pellets of different strains after 24 h incubation in MRS medium plus oxgalla Precipitated Precipitated Deconjugated Strain cholesterol bile salts bile salts (% (% of total) (% of total) of total bile L. acidophilus RP32 49 (±7) 54 (+8) 1 (+) L. acidophilus MUH L. acidophilus MUH bifidum MUH L. acidophilus CH1 L. casei MUH117 a The degree of precipitation of cholesterol and of bile salts is expressed as a percentage of the initial concentration in the medium, and the degree of deconjugation is expressed as a percentage of the initial amount of bile salts in the medium. Figures in parentheses represent standard deviation (SD) values, n = 3. whereas before incubation more than 97% of the bile salts were conjugated. This effect is demonstrated in Fig. 1, showing the HPLC chromatogram of the individual bile salts before and after 24 h of incubation of L. acidophilus RP32. The same experiments were performed with other strains of L. acidophilus and bifidobacteria. The results with these strains are also given in Table 1. Effects similar to those.2.2 " GC TC DC Time FIG. 1. Chromatograms showing the bile salts composition before () and after () 24 h of incubation of L. acidophilus RP32 under acidifying conditions in MRS medium containing oxgall and PPLO (deconjugated bile salts: C, cholate; DC, deoxycholate; conjugated bile salts: GC, glycocholate; TC, taurocholate; GCDC, glycochenodeoxycholate; GDC, glycodeoxycholate; TCDC, taurochenodeoxycholate; TDC, taurodeoxycholate). (min) ~~~~

3 1122 KLVER ND VN DER MEER PPL. ENVIRON. MICROIOL. TLE 2. Growth characteristics of different strains after 24 h of incubation at 37 C in MRS medium plus oxgall (plus in some cases plus PPLO) and in MRS medium Strain MRS medium + oxgall MRS medium OD ph OD ph L. acidophilus RP32a.3 (±.17) (±.28) 3.8 L. acidophilus MUH L. acidophilus MUH bifidum MUH L. acidophilus CH L. casei MUH Figures in parentheses represent SD values, n = ,- -, = &I K % ---o / I _ % * _ = = = = obtained with L. acidophilus RP32 on cholesterol and bile salt precipitation were observed for strains L. acidophilus MUH41, L. acidophilus MUH79, and. bifidum MUH8. These strains also completely deconjugated bile salts during incubation. L. acidophilus CH1 and L. casei MUH117 did not deconjugate bile salts, and no bile salts or cholesterol were present in the pellet after incubation of these strains. ll strains which showed deconjugation of bile salts showed low biomass concentrations after incubation in the presence of oxgall compared with the amount of biomass obtained in MRS medium without oxgall (Table 2). Influence of culture conditions on bile salt and cholesterol precipitation. To monitor deconjugation of bile salts and precipitation of bile salts and cholesterol during the course of incubation, we performed additional fermentation experiments with L. acidophilus RP32, under both acidifying conditions and ph-controlled (ph 6.) conditions. The results on biomass and on bile salt deconjugation and precipitation are shown in Fig. 2 and 3, respectively. During incubation, complete deconjugation of bile salts was observed under both acidifying and ph-controlled conditions. Significant precipitation of bile salts was only observed after deconjugation had occurred. In parallel with bile salt precipitation, a decrease in biomass was found. The degree of precipitation of bile salts was higher under acidifying conditions than during ph-controlled incubation (i.e., approximately 5% versus 3%). Under the acidifying conditions, about 5% of the cholesterol was found in the pellet factions. In contrast, no cholesterol was found in the pellets throughout the entire incubation period under ph-controlled conditions. Similar experiments, with and without ph control, were performed with the bile salt-deconjugating strain. bifidum MUH8. The results with this strain were similar to those described for L. acidophilus RP32 (data not shown). Influence of ph on solubility of bile salt and cholesterol mixtures. To study the influence of ph on the degree of precipitation of bile salts and cholesterol, we performed experiments in the absence of bacterial cells. Oxgall, consisting of over 97% conjugated bile salts, together with cholesterol was added to MRS with ph values ranging from 4. to 7.. lso, mixtures of bile salts were composed corresponding to those present in oxgall but in either conjugated or deconjugated form and used in a similar way. Precipitation of deconjugated bile salts and of cholesterol was clearly dependent on the ph of the medium (Fig. 4). No precipitation of bile salts and cholesterol was observed over this ph range for oxgall or for the mixture of conjugated bile salts. Precipitation of bile salts occurred in the mixture of deconjugated bile salts at ph values lower than 6.. Simultaneously, precipitation of cholesterol in this latter mixture only occurred at ph values lower than ' 8-2 oi I 'p-- Og -- _,oyo,,i lt7 -- l _ FIG. 2. and the degree of deconjugation () and precipitation of bile salts () and cholesterol () () during incubation of L. acidophilus RP32 in MRS medium plus oxgall. The degree of deconjugation and the degree of precipitation are expressed as a percentage of the original concentration of bile salts or cholesterol present in the medium. OD (@), colony count (log CFU/ml, ), and ph () () DISCUSSION, high level of serum cholesterol in humans is generally considered to be a risk factor for coronary heart disease (13). Much interest now exists to find ways to decrease the levels of serum cholesterol. Some reports have suggested the possibility of decreasing the serum cholesterol levels after ingestion of L. acidophilus (5-7, 9). In the studies of Gilliland et al. (7), the mechanism by which the in vivo serum cholesterol level was lowered after pigs were fed with L. acidophilus was explained by a direct action of L. acidophilus on cholesterol which was found under in vitro conditions. The experimental results presented here confirm the experiments of Gilliland et al. (7), in that cholesterol was partially removed from the medium after culturing of L. acidophilus RP32 in the presence of oxgall as the source of bile salts. This phenomenon was explained by these researchers to be due to uptake by L. acidophilus and is therefore referred to as cholesterol assimilation in subsequent publications (5, 1, 15). However, Gilliland et al. (7) did not investigate the fate of bile salts during incubation, but stated that the removal of cholesterol was in some way related to bile salt resistance. From our results, it is clear that precipitation of bile salts occurred during incubation. Moreover, bile salts in the pellets as well as in the supernatants after culturing were only present in the deconjugated form. ile salt deconjugation has been reported earlier for

4 VOL. 59, 1993 LCTOCILLI ND. IFIDUM PRECIPITTE CHOLESTEROL i- -., O.-. "'Q~.,- o- ' - - *6 1 s precipitated bile salts (% of total) 2 O/ a 1 12,x, S 6 7 ph 1X soe 2C *// /- I o - o e FIG. 3. OD (), colony count (log CFU/ml, ), and ph () () and the degree of deconjugation () and precipitation of bile salts () and cholesterol () () during incubation of L. acidophilus RP32 with ph control (ph 6.) in MRS medium plus oxgall. The degree of deconjugation and the degree of precipitation are expressed as a percentage of the original concentration of bile salts or cholesterol present in the medium. lactobacilli (2, 8). We also found the presence of this property in some other strains tested (L. acidophilus MUH41, L. acidophilus MUH79, and. bifidum MUH8) along with precipitation of cholesterol. The nondeconjugating strains L. acidophilus CH1 and L. casei MUH117 did not show precipitation of bile salts or precipitation of cholesterol. The precipitation of cholesterol in culture fluids appears to be related to deconjugation of bile salts and their subsequent precipitation at low ph. This is supported by the results of experiments in which deconjugation and precipitation of bile salts and precipitation of cholesterol were compared under acidifying and under ph-controlled culture conditions. Under both conditions, complete deconjugation of bile salts occurred, but the degree of precipitation of deconjugated bile salts was lower for ph-controlled cultures than for acidifying cultures. Cholesterol precipitated under acidifying conditions, but not under ph-controlled conditions. The solubility of cholesterol is clearly dependent on the solubility of bile salts. This is in agreement with earlier observations on the bile salt dependency of cholesterol solubility (16). Further support for the coprecipitation hypothesis is obtained from the observation that a considerable loss of biomass occurs especially during incubation under acidifying conditions, which indicates that the precipitation of cholesterol is not a direct result of metabolic activity of the bacteria. Our control experiments with mixtures of bile salts in -O * precipitated cholesterol of total) (%,.ip. _ et o toal" FIG. 4. Precipitation of bile salts () and precipitation of cholesterol () at different ph values in solutions of oxgall (-), a mixture of conjugated bile salts (), and a corresponding mixture of deconjugated bile salts (). The amount of precipitated bile salts or cholesterol is expressed as a percentage of the initial concentration added. conjugated or nonconjugated form provided additional proof for coprecipitation of cholesterol, which occurred only in the presence of deconjugated bile salts at ph values lower than 6.. On this basis, we conclude that, also in the absence of bacterial cells, cholesterol is precipitated as a result of reduced solubility of deconjugated bile salts. Thus, assimilation or uptake of cholesterol by bacterial cells is not required for this effect. It remains unclear whether and how microbial bile deconjugation properties are related to the hypocholesteremic effects observed in vivo (7), especially since the physiological ph in the intestinal tract of humans is usually neutral to alkaline (4). Moreover, deconjugation can potentially result in the formation of cytotoxic secondary bile salts (17). Therefore, the net effect of such properties on health cannot be predicted satisfactorily. ph k CKNOWLEDGMENTS We thank F. Kingma, H. T. de Vries, and R. Dekker for their excellent technical support. We are grateful to S. E. Gilliland (Oklahoma State University, Stillwater) for providing L. acidophilus RP32 and R. ngulo (RIVM, ilthoven, The Netherlands) for providing PPLO. We thank R. de oer and F. M. Driessen of Campina Melkunie V for their stimulating and critical discussions. We thank. H. Weerkamp (NIZO, Ede, The Netherlands) for critically reviewing the manuscript. This work was partly financed by The Mona Research Division of Campina Melkunie V, Woerden, The Netherlands.

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