Abstract:
Pregastric lipase (lingual lipase) is the first enzyme involved in milkfat digestion. Results of an initial study on the purification and kinetics of calf pregastric enzymes led to an investigation of the part played by the products of milkfat digestion in the survival of bacteria in the proximal regions of the digestive tract.
A commercial extract from the tongue and epiglottal region of suckling calf has been partially purified to yield a pregastric esterase fraction F2a and a lipase fraction F3b. Fraction F2a has shown that it has esterase activity against 4-nitrophenylalkanoate esters (C2-C12) at 37ºC, pH 7.2, with maximum activity obtained against the decanoate ester. The value of Km for activity against 4-nitrophenylacetate, PNPA, was 0.23 mM. Against 4-nitrophenyldodecanoate, PNPDe, Km was 1.06 µM, and maximum activity was achieved at ca. 1.6 µM, a value equal to the critical micelle concentration of PNPDe. Profiles of pH (5.5-9.0) and temperature (25 - 48ºC) for the esterase activity of fraction F2a were also determined.
The partial hydrolysis of bovine milkfat was catalysed by calf pregastric lipase fraction F3b in a lecithin (0.05%) emulsion at pH 6.5 and 37ºC, and the changes in composition of the hydrolysed milkfat were analysed over time by gas chromatography (GC). As previously determined for lamb and goat pregastric lipases, calf pregastric lipase preferentially releases short chain fatty acids at the sn-3 position, with butyric acid comprising 46% of the released free fatty acids after 1h hydrolysis. A unique feature of calf pregastric lipase is that it preferentially releases unsaturated fatty acids during the hydrolysis of lipids containing long chain fatty acids, and its activity increases with increasing unsaturation in the carbon chain of the long chain fatty acids. These properties were not found for the lamb and goat pregastric lipases. The selectivity characteristics of calf pregastric lipase raise the question of the physiological significance and biological role of this enzyme.
Six enteric and environmental strains of bacteria (2 strains of Escherichia coli, Klebsiella pneumoniae, Enterococcus faecium, Enterococcus faecalis, and Enterococcus casseliflavus) were used during the initial investigations of the antibacterial properties of short chain fatty acids (butyric, caproic, and caprylic acid) at selected concentrations (0-40 mM) and over the stomach's physiological pH range 4.5 to 7.5. All the fatty acids tested showed growth inhibition to the above organisms after overnight (18h) incubation. The inhibitory effect increased with increasing FA concentrations and decreasing pH in the medium, ultimately leading to a bactericidal effect if either of the trends continued. The precise conditions under which bacteria were affected depended on the strains. The highest activity against Gram-negative coliform bacteria occurred with caprylic acid; however, against Gram-positive enterococci, lauric acid was the most effective FA. Using butyric acid as a model, the mechanism of the antibacterial property of FAs was studied through investigation of the effects of changing pH and butyric acid concentration in the medium after growth. The results suggested that the antibacterial action of FAs depends on their acid-ionisation form, and that there could be an energy-linked transport system exporting the fatty acid anions out of the cell.
The antibacterial property of partially hydrolysed milkfat catalysed by calf pregastric lipase was carried out in two types of emulsion systems, containing either Triton X-100 or casein/lecithin, plus 4% (w/w) milkfat in citrate / phosphate buffer (pH 5.0 - 6.0). Both K. pneumoniae and Ent. faecalis grew well in a non-hydrolysed mikfat medium, but could not grow when the milkfat had undergone as little as 9.4% hydrolysis at pH 5.0, confirming the antibacterial properties of the released FAs. The minimum percentages of hydrolysis at which the bacterial growth was affected were determined at different pH values. The analysis of FA and MG compositions in the hydrolysed milkfat media suggested a potent synergy among FAs, MGs, and emulsifying agents against bacterial growth during 18 h incubation. As a result of forming micelles with FAs and MGs, Triton X-100 provided a more compatible medium than did casein/lecithin towards bacterial inhibition of coliforms. The antibacterial activity of milkfat, triggered by hydrolysis catalysed by calf pregastric lipase, may explain in part the role of pre-intestinal lipases in young animals' natural defenses against bacteria in ingested food prior to weaning.
The above results suggested that analogous studies should be carried out on Helicobacter pylori, a worldwide pathogen which colonises the stomach of over 50% of the human population, and is causally linked to gastritis, dispepsia, stomach and duodenal ulcers, and gastric cancer. The susceptibility of H. pylori towards FAs and MGs was examined in Iso-Sensitest broth with the aim of detecting a quick lethal effect (40-min incubation) at neutral pH 7.4. It was found that fatty acids with 12 equivalent carbon number were the most potent: eg lauric acid was the most potent saturated FA with a minimal lethal concentration of 1 mM and myristoleic and linolenic acid were the most potent unsaturated FAs with a minimal lethal concentration of 0.5 mM. It was also observed that unsaturated fatty acids were more potent than their saturated counterparts, and their activity increased with increasing unsaturation in the carbon chain. These results support the hypothesis that the bactericidal effect of unsaturated FAs is mediated by a peroxidative process involving hydrogen peroxide and bacterial iron, and was also affected by their strong surfactant action on the cell membrane. Investigation of the effect of pH on the potencies of several saturated FAs showed that at a certain value of pH bacterial survival became critical, and a 0.5-pH unit decrease caused transition from a non-lethal to a lethal effect. This result suggests that the action of saturated FAs against the survival of H. pylori depends on their acidic form, and that the same mechanism as was discussed for FA action against coliforms and enterococcis might be applicable.
Examination of the bactericidal action of monoglycerides (from MG C8 to MG C18) showed that they were more potent than their fatty acid derivatives. Monolaurin was the most potent MG with a minimal lethal concentration of 0.5 mM. The bactericidal action of monolaurin was not pH-dependent, suggesting a different mechanism for its killing action on H. pylori from that operating for fatty acids, a change possibly due to the strong surfactant action of monolaurin on the bacterial cell wall.
Tests on possible synergy between each potent FA and MG demonstrated that the combined activity of FAs and MGs was only additive. However, synergy was observed between FAs and the surfactant Tween 80. The presence of 0.05% Tween 80 in the medium decreased the concentration of myristic acid required for an antibacterial effect from 3 mM to 0.5 mM. In contrast, the presence of 0.05% lecithin in the medium attenuated the antibacterial action of myristic acid.
A special milkfat fraction, termed Alsoil, whose triglycerides were enriched in short and medium fatty acids, was used to examine the antibacterial effect of hydrolysed triglycerides from milkfat on H. pylori (40-min exposure). Normal bovine milkfat was also run, in parallel, for comparison. Alsoil, which contains 9.4% FFAs, exhibited a quick lethal effect on H. pylori even before it was hydrolysed. After 1h-hydrolysis, catalysed by calf pregastric lipase, its quick bactericidal action on H. pylori was maintained even after 1:8 dilution, while the bactericidal effect of 1h-hydrolysed bovine milkfat disappeared with as little as 1:2 dilution.
One of the ways in which H. pylori protects itself from the acidity in the stomach is through the production of urease. The effect of urea and urease on the bactericidal action of FAs was examined by incubating H. pylori in a medium containing both urea and caprylic acid. The results showed that the presence of urea was not deleterious to the bactericidal action of caprylic acid. A test on the resistance of H. pylori to the antibacterial effect of FAs, using 6 cycles of exposure to 0.7 mM lauric acid, also showed that H. pylori does not develop resistance to the bactericidal action of FAs. These results clarified the misunderstanding about the function of the milk diet for the treatment of gastric ulcer in the 1950s-1960s, and support the potential for future application of potent FAs and MGs to the treatment of H. pylori infection.