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The fermented cereal-based gruel togwa is used as weaning food for children in Tanzania. Togwa is rich in minerals but these are often not available for uptake in the human intestine due to natural inhibitors, such as phytate (IP(6)). The yeasts Pichia kudriavzevii TY13, Hanseniaspora guilliermondii TY14 and TY20, isolated from Tanzanian togwa, and selected for high phytase activity in complex yeast medium YPD, were now studied regarding their ability to degrade IP(6) in maize-based model togwa. A modified constitutively high-phytase producing Saccharomyces cerevisiae BY80 and commercial Aspergillus ficuum phytase were included for comparison. In addition, a strain of Lactobacillus plantarum was included in the model-togwa set-up. All yeasts in the study grew and reached final cell density 1.5-2 log units higher than the start value. S. cerevisiae BY80 degraded 85% of the IP(6) in 48 h; the same degradation level as with A. ficuum phytase (89%). Of the togwa-isolated yeasts, P. kudriavzevii TY13 and H. guilliermondii TY14 showed strong phytate degradation in the model-togwa; 95% or more of the initial IP(6) was degraded after 48 h. This corresponds to a remaining level of 0.4 and 0.3μmol IP(6)/g dw. Co-inoculation with L. plantarum did not increase IP(6) degradation. Moreover, fermentation with P. kudriavzevii TY13 yielded a successive increase in inorganic phosphate (P(i)), from 0.7 to 5.4 mM, suggesting a phytase production in TY13 which is fairly insensitive to P(i) repression. The study shows that phytate in a model togwa is available for yeast phytase enzymes, and addresses the importance of strain selection for effectively degrading the phytate. Certain yeasts originating from togwa seem to have developed a natural high phytase production, and P. kudriavzevii TY13 and H. guilliermondii TY14 seem particularly well adapted to phytate degradation in togwa, and is our choice for further studies and strain improvement., (Copyright © 2011 Elsevier B.V. All rights reserved.)

The growing awareness of the relationship between diet and health has led to an increasing demand for food products that support health above and beyond providing basic nutrition. Probiotics are live organisms present in foods, which yield health benefits related to their interactions with the gastrointestinal tract. Phytases are a subgroup of phosphatases that catalyse the desphosphorylation of phytate, which reduces its negative impact on mineral bioavailability, and generates lower inositol phosphates. The aims of this investigation were to (i) study the ability of the probiotic candidate Bifidobacterium pseudocatenulatum to degrade phytate in synthetic medium, to (ii) identify the lower inositol phosphates generated, to (iii) study its survival under conditions mimicking gastrointestinal passage and finally to (iv) assess adhesion of the bacteria to Caco-2 cells. The first steps of InsP(6) degradation by B. pseudocatenulatum phytate-degrading enzyme/s were preferentially initiated at the DL-6-position and 5-position of the myo-inositol ring. It suggests that the main InsP(6) degradation pathway by B. pseudocatenulatum by sequential removal of phosphate groups was D/L-Ins(1,2,3,4,5)P(5) or D/L-Ins(1,2,3,4,6)P(5); D/L-Ins(1,2,3,4)P(4); to finally Ins(1,2,3)P(3) and D/L-Ins(1,2,4)P(3)/D/L-Ins(1,3,4)P(3). This human strain also showed a notable tolerance to bile as well as a selective adhesion capacity (adhesion to control surfaces was zero), to human intestinal Caco-2 cells comparable to the commercial probiotic B. lactis. The phytate-degrading activity constitutes a novel metabolic trait which could contribute to the improvement of mineral absorption in the intestine as a nutritional probiotic feature with potential trophic effect in human gut.

Using a combination of High-Performance Ion Chromatography analysis and kinetic studies, the pathway of myo-inositol hexakisphosphate dephosphorylation by a phytase from a Malaysian waste-water bacterium was established. The data demonstrate that the phytase preferably dephosphorylates myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via D-I(1,2,3,4,5)P(5), D-I(2,3,4,5)P(4), D-I(2,3,4)P(3), D-I(2,3)P(2) to finally I(2)P. It was estimated that more than 90% of phytate hydrolysis occurs via D-I(1,2,3,4,5)P(5). Thus, the phytase from the Malaysian waste-water bacterium has to be considered a 6-phytase (E.C. 3.1.3.26). A second pathway of minor importance could be proposed which is in accordance with the results obtained from analysis of the dephosphorylation products formed by the action of the phytase under investigation on myo-inositol hexakisphosphate. It proceeds via D/L-I(1,2,4,5,6)P(5), D/L-I(1,2,4,5)P(4), D/L-I(1,2,4)P(3), D/L-I(2,4)P(2) to finally I(2)P.

Using a combination of high-performance ion chromatography analysis and kinetic studies, the pathway of myo-inositol hexakisphosphate dephosphorylation by the beta-propeller phytase of Shewanella oneidensis was established, which was then compared with that of Bacillus subtilis 168, Bacillus amyloliquefaciens ATCC 15841, and B. amyloliquefaciens 45 beta-propeller phytases. The data demonstrate that all of these beta-propeller phytases dephosphorylate myo-inositol hexakisphosphate in a stereospecific way by sequential removal of phosphate groups via d-Ins(1,2,4,5,6)P5, Ins(2,4,5,6)P4 to finally Ins(2,4,6)P3. Thus, the beta-propeller phytases prefer the hydrolysis of every second phosphate over that of adjacent ones. This finding does not support previous phytate degradation models proposed by J. Kerovuo, J. Rouvinen, and F. Hatzack (2000. Biochem. J. 352: 623-628) and R. Greiner, A. Farouk, M. Larsson Alminger, and N.G. Carlsson (2002. Can. J. Microbiol. 48: 986-994), but seems to fit with the structural model given by S. Shin, N.C. Ha, B.C. Oh, T.K. Oh, and B.H. Oh (2001. Structure, 9: 851-858).

For the first time a dual pathway for dephosphorylation of myo-inositol hexakisphosphate by a histidine acid phytase was established. The phytate-degrading enzyme of Klebsiella terrigena degrades myo-inositol hexakisphosphate by stepwise dephosphorylation, preferably via D-Ins(1,2,4,5,6)P5, D-Ins(1,2,5,6)P4, D-Ins(1,2,6)P3, D-Ins(1,2)P2 and alternatively via D-Ins(1,2,4,5,6)P5, Ins(2,4,5,6)P4, D-Ins(2,4,5)P3, D-Ins(2,4)P2 to finally Ins(2)P. It was estimated that more than 98% of phytate hydrolysis occurs via D-Ins(1,2,4,5,6)P5. Therefore, the phytate-degrading enzyme from K. terrigena has to be considered a 3-phytase (EC 3.1.3.8). A second dual pathway of minor importance could be proposed that is in accordance with the results obtained by analysis of the dephosphorylation products formed by the action of the phytate-degrading enzyme of K. terrigena on myo-inositol hexakisphosphate. It proceeds preferably via D-Ins(1,2,3,5,6)P5, D-Ins(1,2,3,6)P4, Ins(1,2,3)P3, D-Ins(2,3)P2 and alternatively via D-Ins(1,2,3,5,6)P5, D-Ins(2,3,5,6)P4, D-Ins(2,3,5)P3, D-Ins(2,3)P2 to finally Ins(2)P. D-Ins(2,3,5,6)P4, D-Ins(2,3,5)P3, and D-Ins(2,4)P2 were reported for the first time as intermediates of enzymatic phytate dephosphorylation. A role of the phytate-degrading enzyme from K. terrigena in phytate breakdown could not be ruled out. Because of its cytoplasmatic localization and the suggestions for substrate recognition, D-Ins(1,3,4,5,6)P5 might be the natural substrate of this enzyme and, therefore, may play a role in microbial pathogenesis or cellular myo-inositol phosphate metabolism.

Using a combination of high-performance ion chromatography analysis and kinetic studies, the pathway of dephosphorylation of myo-inositol hexakisphosphate by the phytases purified from faba bean and lupine seeds, respectively, was established. The data demonstrate that the legume seed phytases under investigation dephosphorylate myo-inositol hexakisphosphate in a stereospecific way. The phytase from faba bean seeds and the phytase LP2 from lupine seeds degrade phytate by sequential removal of phosphate groups via D-Ins(1,2,3,5,6)P(5), D-Ins(1,2,5,6)P(4), D-Ins(1,2,6)P(3), and D-Ins(1,2)P(2) to finally Ins(2)P, whereas the phytases LP11 and LP12 from lupine seeds generate the final degradation product Ins(2)P via D-Ins(1,2,4,5,6)P(5), D-Ins(1,2,5,6)P(4), D-Ins(1,2,6)P(3), and D-Ins(1,2)P(2).

The pathway of dephosphorylation of myo-inositol hexakisphosphate by the phytate-degrading enzymes of Bacillus subtilis 168, Bacillus amyloliquefaciens ATCC 15841, and Bacillus amyloliquefaciens 45 was established using a combination of high-performance ion chromatography analysis and kinetic studies. The data demonstrate that all the Bacillus phytate-degrading enzymes under investigation dephosphorylate myo-inositol hexakisphosphate by sequential removal of phosphate groups via two independent routes; the routes proceed via D-Ins(1,2,4,5,6)P5 to Ins(2,4,5,6)P4 to finally Ins(2,4,6)P3 or D-Ins(2,5,6)P3 and via D-Ins(1,2,4,5,6)P5 to D-Ins(1,2,5,6)P4 to finally D-Ins(1,2,6)P3. The resulting myo-inositol trisphosphate D-Ins(1,2,6)P3 was degraded via D-Ins(2,6)P2 to finally Ins(2)P after prolonged incubation times in combination with increased enzyme concentration.