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The effects of Al(III) on surface properties and lactate accumulation by Bifidobacterium thermophilum were investigated. Bacteria were treated with Al(III) at 37 degrees C and 4 degrees C, then exposed to free radicals or nisin. When exposed to Al(III) at 37 degrees C, the organism exhibited spreading on hydrophobic surfaces and showed high susceptibility to free-radical alteration as indicated by Fe(III) binding, but showed little effect on lactate production in the presence or absence of nisin, even after washing with 2 mM EDTA. At 4 degrees C, there was no increased surface spreading or binding of Fe(III), but protection against nisin action was present. This, however, was abolished after washing with EDTA. It was concluded that membrane fluidity is required to affect membrane lipid rearrangement, resulting in surface spreading and increased susceptibility to peroxidation, whereas only loose binding of Al(III) to membrane surfaces is sufficient to prevent transmembrane channel formation by nisin.

The effects of nisin on lactate accumulation, growth, and Fe(III) binding by Bifidobacterium thermophilum (ATCC 25866) and Bifidobacterium breve (ATCC 15700) were investigated. Nisin inhibited lactate production by B. thermophilum at concentrations of less than 1 microg/ml, but this effect could be largely eliminated by pretreatment of the organism with 100 to 400 microM Al(III) or La(III). Nisin also inhibited the growth of B. thermophilum at concentrations of 2 to 3 microg/ml, with lower concentrations showing lag periods and/or slower rates of growth. However, Al(III) could not negate these effects, most likely because of Al(III) chelation by the trypticase-proteose-yeast extract medium. Nisin was able to increase instantaneous Fe(III) binding by both B. thermophilum and B. breve, though prolonged-time experiments (up to 120 min) with B. thermophilum indicated no difference in total Fe(III) bound. Nisin was thus able to increase the free radical reaction rate with bifidobacteria and the resultant rate of Fe(III) binding. It was concluded that nisin will normally inhibit the metabolic activity of B. thermophilum along with that of certain bacterial pathogens; however, this effect may in some instances, be abated by a pretreatment with Al(III). Moreover, by accelerating free radical action and the binding of iron by bifidobacteria, nisin may be able to potentiate their normal probiotic action.

Bifidobacterium thermophilum (ATCC 25866) was incubated with 100-120 microM (59)FeSO(4) and 300 microM excess of H(2)O(2) for up to 120 min in the presence and absence of glucose. Samples were withdrawn after 5, 30, 60, and 120 min. These were protoplasted and (59)Fe(III) measured in the supernatant fraction (cell walls) and protoplasts (cell membranes). These experiments were also repeated in the presence of 400 microM Al(III), which, in whole cells, caused an increase in Fe(III) binding. The amount of iron in the cell wall fraction was constant regardless of time of incubation, was unaffected by Al(III), and was reduced by approximately 20% by glucose. On the other hand, the amount of iron on the protoplasts increased with time and was affected by both Al(III) (upward) and glucose (downward). Scatchard plots indicated that the number of Fe(III) binding sites on the cell walls was 37.6 nmol/mg of dry cell weight at zero time, whereas that of cell membranes was (1)/(10) of that. It was concluded that Fe(III) binding by bifidobacterial cell walls was instantaneous and marginally dependent on free radical action. That of the cell membranes was time-dependent and was due to lipid peroxidation initiated by free radicals. Bifidobacteria can therefore function in the intestinal tract as probiotics by making Fe(OH)(3) unavailable to pathogens and by moderating free radical activity in the intestinal tract.

Ferrous iron uptake was investigated in Bifidobacterium thermophilum (B. thermophilum) in the presence of Mg2+ and Ca2+ with the following findings: 1. Mg2+ inhibited Fe2+ accumulation in the cells in a dose-dependent manner at 37 degrees, but not at 0 degrees. Removal of Mg2+ from the medium resulted in a resumption of rapid iron uptake. 2. Mg2+ had no effect on the binding of Fe2+ by B. thermophilum protoplasts, its cellular particulate fraction, or distribution between the particulate and soluble fractions. 3. Ca2+ exerted a stimulatory effect on iron uptake by B. thermophilum, but was not able to reverse the inhibitory effects of Mg2+. 4. It was concluded that Mg2+ has no effect on the binding of iron on the surface or interior of B. thermophilum and that it affected the Fe2+ transport mechanism (permease) in a reversible manner. It is possible that iron and magnesium share the same permease in this microorganism.

Protoplasts of Bifidobacterium thermophilum were prepared by a combination of lysozyme and protease digestion, and ferrous iron uptake studies were carried out. Little, if any, iron was internalized by the protoplasts, although large amounts of iron were bound to the protoplast surface. This binding was much greater than that of intact cells, which prefer to internalize iron by an energy-dependent process. It was also found that the binding of iron by protoplasts of cells grown in an iron-deficient medium was much more extensive than that of cells grown in an iron-sufficient medium. Soluble and particulate fractions of protoplasts were prepared by grinding them in a glass homogenizer, and the particulate fraction was also subjected to iron binding studies. The amount of iron bound was the same as that in intact protoplasts, indicating that the particulate fraction membrane fragments bound iron on their outer surface only. Nevertheless, when iron-preloaded cells were protoplasted and their surface cleared of iron, their particulate fraction contained considerable amounts of iron, indicating that the inner surface of the membranes is capable of binding iron as long as the cell is intact. The amount of iron so bound was dose-dependent on the amount of iron entering the cell. The failure of the outer and inner surface iron pools to mix was confirmed by the fact that when iron-preloaded protoplasts were incubated with additional iron, only the latter (surface-bound) was elutable with nonradioactive 2 mM FeSO4. It is concluded that increasing bifidobacterial iron load increases the amount of iron bound to the inner surface of the membrane; the procedure, which is effective in forming bifidobacterial protoplasts, destroys their iron transport mechanism while uncovering surface iron-binding sites; and that such iron-binding sites may be of significance in the cellular iron metabolism processes.

Fe(OH)3 was produced from Fe(II) and H2O2 by methods associated with high and low free radical activity. More Fe(OH)3 became bound to Bifidobacterium thermophilum when the former type of method was used, and protoplasting experiments indicated that binding to both the cell walls and membranes was enhanced. Binding to cell walls was instantaneous, whilst that by the membranes increased with time using both methods. Al (III), a lipid peroxidation enhancer, increased Fe(OH)3 binding by membranes only. It was concluded that B. thermophilum is able to abate increased activity of free radicals not only by scavenging them, but also by removing iron from their environment. [ABSTRACT FROM AUTHOR]