Thomas Andlid

The potential of bifidobacteria as a source of natural folate

Aims:  To screen 19 strains of bifidobacteria for main folate forms composition in synthetic folate‐free and complex folate‐containing media.

Phytate degradation by human gut isolated Bifidobacterium pseudocatenulatum ATCC27919 and its probiotic potential

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.

Phytate degradation by micro-organisms in synthetic media and pea flour

Aims: To screen micro‐organisms for the ability to produce phytase enzyme(s) and to use promising strains for the fermentation of pea flour.

Effect of Nutrient Starvation on the Cellular Composition and Metabolic Capacity of Saccharomyces cerevisiae

ABSTRACT This investigation addresses the following question: what are the important factors for maintenance of a high catabolic capacity under various starvation conditions? Saccharomyces cerevisiae was cultured in aerobic batch cultures, and during the diauxic shift cells were transferred and subjected to 24 h of starvation. The following conditions were used: carbon starvation, nitrogen starvation in the presence of glucose or ethanol, and both carbon starvation and nitrogen starvation. During the starvation period changes in biomass composition (including protein, carbohydrate, lipid, and nucleic acid contents), metabolic activity, sugar transport kinetics, and the levels of selected enzymes were recorded. Subsequent to the starvation period the remaining catabolic capacity was measured by addition of 50 mM glucose. The results showed that the glucose transport capacity is a key factor for maintenance of high metabolic capacity in many, but not all, cases. The results for cells starved of carbon, carbon and nitrogen, or nitrogen in the presence of glucose all indicated that the metabolic capacity was indeed controlled by the glucose transport ability, perhaps with some influence of hexokinase, phosphofructokinase, aldolase, and enolase levels. However, it was also demonstrated that there was no such correlation when nitrogen starvation occurred in the presence of ethanol instead of glucose.

Biodiversity and phytase capacity of yeasts isolated from Tanzanian togwa

The focus of the present investigation was on the Tanzanian fermented food togwa as a source for dietary iron and zinc, and the potential for mineral availability improvements using selected yeasts. To establish the content of target minerals and main inhibitor for intestinal uptake, iron and zinc as well as the mineral chelating phytic acid, (IP(6) or phytate) were determined in naturally fermented togwa. Yeasts were isolated from sorghum, maize and cassava based togwa, and identified by sequencing the D1/D2 region of the LSU rRNA gene. The isolated yeasts were subsequently screened for phytase activity. The total iron content in sorghum, maize and cassava based togwa were 41.5 (+/-7.2), 85.4 (+/-31.9) and 28.6 (+/-3.8) microg/g dw (dry weight) respectively. The zinc content was 12.3 (+/-3.1), 11.0 (+/-1.1) and 6.4 (+/-4.5) microg/g dw in sorghum, maize and cassava based togwa, and the phytate content in the three varieties were 2.6+/-1.2, 4.7+/-0.8 and 0.4+/-0.4 micromol/g dw respectively. The phytate levels in the sorghum and maize based togwa are expected to substantially reduce the availability of iron. The molar ratio phytate to iron for these two varieties were estimated to be 3.5:1 and 3.1:1 respectively. In general, a phytate to iron molar ratio below 1 is needed to increase the availability of iron. Among 26 isolates, 9 different species could be distinguished: Issatchenkia orientalis, Pichia anomala, Pichia norvegensis, Pichia burtonii, Pichia guilliermondii, Kluyveromyces marxianus, Saccharomyces cerevisiae, Hanseniaspora guilliermondii and Candida glabrata. The strains were screened for phytase activity in YPD supplemented with 0.5 mM IP(6). Of 26 screened strains, the phytase activity was most prominent in strains of I. orientalis and H. guilliermondii. The strains and data constitute a basis for further improvements of iron and zinc bioavailability in togwa.

Biofortification of folates in white wheat bread by selection of yeast strain and process

We here demonstrate that folate content in yeast fermented food can be dramatically increased by using a proper (i) yeast strain and (ii) cultivation procedure for the selected strain prior to food fermentation. Folate levels were 3 to 5-fold higher in white wheat bread leavened with a Saccharomyces cerevisiae strain CBS7764, cultured in defined medium and harvested in the respiro-fermentative phase of growth prior to dough preparation (135-139 microg/100 dry matter), compared to white wheat bread leavened with commercial Bakers yeast (27-43 microg/100 g). The commercial Bakers yeast strain had been industrially produced, using a fed-batch process, thereafter compressed and stored in the refrigerator until bakings were initiated. This strategy is an attractive alternative to fortification of bread with synthetically produced folic acid. By using a high folate producing strain cultured a suitable way folate levels obtained were in accordance with folic acid content in fortified cereal products.

Lactic acid fermentation stimulated iron absorption by Caco-2 cells is associated with increased soluble iron content in carrot juice

An in vitro digestion/Caco-2 cell model was applied to explore the impact of lactic acid (LA) fermentation by Lactobacillus pentosus FSC1 and Leuconostoc mesenteroides FSC2 on the Fe bioavailability of carrot juice. The redox state of Fe in fermented carrot juice was also assessed as a crucial factor for absorption. LA fermentation was shown to improve mineral solubility to different extents at simulated physiological conditions: Mn (2-fold); Fe (1.5-1.7-fold); Zn (1.2-fold); Cu (1-fold). Soluble Fe2+ was increased about 16-fold by LA fermentation, and about one third of the Fe2+ remained soluble after in vitro digestion (about 4-5-fold higher than in fresh juice). Data on cell-line studies showed a 4-fold increase in the efficiency of Fe uptake, but not in transepithelial transfer by Caco2 cells, as a result of fermentation. The increases in Fe2+ level and the efficiency of cellular Fe uptake were strain-dependent. To sum up the effect on both Fe solubility and cellular uptake efficiency, the amount of cellularly absorbed Fe from Ln. mesenteroides FSC2-fermented juice was about 20 % higher than that from L. pentosus FSC1-fermented juice (22.7 v. 19.2 microg/l juice per mg protein). To conclude, LA fermentation enhanced Fe absorption by Caco-2 cells from carrot juice because of increases in not only Fe solubility after digestion, but also the efficiency of cellular Fe uptake. The fermentation-improved efficiency of Fe uptake was possibly due to the increased level of soluble Fe2+ rather than a being a strain-specific event.

Growth rate and medium composition strongly affect folate content in Saccharomyces cerevisiae

Folate content in a Saccharomyces cerevisiae strain was monitored during aerobic batch fermentation in synthetic growth medium, yeast peptone dextrose medium, and a molasses based medium. During growth in the synthetic medium large differences in intracellular folate content was observed at different phases. Specific folate levels, expressed per unit biomass, were highest during respiro-fermentative growth (120 microg/g) and decreased during the respiratory and stationary phases. Thus, the physiological state of the cells clearly affects the folate content. This was confirmed in chemostat cultures where total intracellular folate content increased linearly with increasing growth rate (r(2)=0.998), indicating high growth rate i.e. respiro-fermentative growth to be most favourable to obtain high specific folate content. In complex media however, much lower folate content (15-40 microg/g) was found throughout the batch growth. Only minor growth-phase related differences were detected. This shows the impact of cultivation medium on folate content in yeast. To further investigate which components that influence folate content, batch experiments in synthetic medium with addition of specific components were performed. Adding a raw mixture of peptides and amino acids (peptone) decreased folate levels extensively (90%) whereas adding amino acids one-by-one only had minor effects on the intracellular folate content. Furthermore, supplementing synthetic medium with pABA, folate or nucleotides did not change the intracellular folate content. This work constitutes the first steps towards an optimised process for production of natural folates for fortification purposes, as well as an effort to gain fundamental understanding of folate requirements in yeast in relation to environmental conditions.

Production of folates by yeasts in Tanzanian fermented togwa.

We have investigated the impact of different yeasts and fermentation time on folate content and composition in a fermented maize-based porridge, called togwa, consumed in rural areas in Tanzania. The yeasts studied, originally isolated from indigenous togwa, belong to Issatchenkia orientalis, Pichia anomala, Saccharomyces cerevisiae, Klyveromyces marxianus and Candida glabrata. The main folate forms found, detected and quantified by HPLC during the fermentations were 5-methyl-tetrahydrofolate (5-CH(3)-H(4)folate) and tetrahydrofolate (H(4)folate). The content of H(4)folate, per unit togwa, remained fairly stable at a low level throughout the experiment for all strains, whereas the 5-CH(3)-H(4)folate concentration was highly dependent on yeast strain as well as on fermentation time. The highest folate concentration was found after 46 h of fermentation with C. glabrata (TY26) (6.91+/-0.14 microg 100 mL(-1)), corresponding to a 23-fold increase compared with unfermented togwa. The cell concentration per se could not predict the togwa folate level, as shown by the much higher specific folate content (g folate CFU(-1)) in the S. cerevisiae strain (TY08) compared with the other species tested. This study provides useful data when trying to maximize folate content in togwa as well as in other yeast-fermented products.

Degradation of phytate by Pichia kudriavzevii TY13 and Hanseniaspora guilliermondii TY14 in Tanzanian togwa.

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.