Folate-producing bifidobacteria: metabolism, genetics, and relevance.

Folate (the general term for all bioactive forms of vitamin B9) plays a crucial role in the evolutionary highly conserved one-carbon (1C) metabolism, a network including central reactions such as DNA and protein synthesis and methylation of macromolecules. Folate delivers 1C units, such as methyl and formyl, between reactants. Plants, algae, fungi, and many bacteria can naturally produce folate, whereas animals, including humans, must obtain folate from external sources. For humans, folate deficiency is, however, a widespread problem. Bifidobacteria constitute an important component of human and many animal microbiomes, providing various health advantages to the host, such as producing folate. This review focuses on bifidobacteria and folate metabolism and the current knowledge of the distribution of genes needed for complete folate biosynthesis across different bifidobacterial species. Biotechnologies based on folate-trophic probiotics aim to create fermented products enriched with folate or design probiotic supplements that can synthesize folate in the colon, improving overall health. Therefore, bifidobacteria (alone or in association with other microorganisms) may, in the future, contribute to reducing widespread folate deficiencies prevalent among vulnerable human population groups, such as older people, women at child-birth age, and people in low-income countries.

Phytate degradation in composite wheat/cassava/sorghum bread: Effects of phytase-secreting yeasts and addition of yeast extracts.

Iron deficiency anemia is highly prevalent in developing countries due to the consumption of cereal-based foods rich in phytate that chelates minerals such as iron and zinc making them unavailable for absorption by humans. The aim of the present study was to degrade phytic acid in composite flour (wheat/cassava/sorghum) bread by the addition of phytase-producing yeasts in the baking process to achieve a phytate-to-iron molar ratio <1 and a phytate-to-zinc molar ratio <15, ratios needed to achieve an enhanced absorption by humans. The high-phytase (HP)-producing yeasts were two Saccharomyces cerevisiae (YD80 and BY80) that have been genetically modified by a directed mutagenesis strategy, and Pichia kudriavzevii TY13 isolated from a Tanzanian lactic fermented maize gruel (togwa) and selected as naturally HP yeast. To further improve the phytase production by the yeasts, four different brands of phytase-promoting yeast extracts were added in the baking process. In addition, two yeast varieties were preincubated for 1 h at 30°C to initiate phytase biosynthesis. The phytate content was measured by high-performance ion chromatography (HPIC) and the mineral content by ion chromatography (HPIC). The results showed that all three HP yeasts improved the phytate degradation compared with the composite bread with no added HP yeast. The composite bread with preincubated S. cerevisiae BY80 or P. kudriavzevii TY13 plus Bacto yeast extract resulted in the lowest phytate content (0.08 µmol/g), which means a 99% reduction compared with the phytate content in the composite flour. With added yeast extracts from three of the four yeast extract brands in the baking process, all composite breads had a phytate reduction after 2-h fermentation corresponding to a phytate: iron molar ratio between 1.0 and 0.3 and a phytate: zinc molar ratio <3 suggesting a much-enhanced bioavailability of these minerals.

Expanding the genetic toolbox of Rhodotorula toruloides by identification and validation of six novel promoters induced or repressed under nitrogen starvation.

BACKGROUND: The non-conventional yeast Rhodotorula toruloides is an emerging host organism in biotechnology by merit of its natural capacity to accumulate high levels of carotenoids and intracellular storage lipids from a variety of carbon sources. While the number of genetic engineering strategies that employ R. toruloides is increasing, the lack of genetic tools available for modification of this yeast is still limiting strain development. For instance, several strong, constitutive R. toruloides promoters have been characterized, but to date, only five inducible promoters have been identified. Although nitrogen-limited cultivation conditions are commonly used to induce lipid accumulation in this yeast, no promoters regulated by nitrogen starvation have been described for R. toruloides. RESULTS: In this study, we used a combination of genomics and transcriptomics methods to identify novel R. toruloides promoter sequences that are either inducible or repressible by nitrogen starvation. RNA sequencing was used to assess gene expression in the recently isolated strain R. toruloides BOT-A2 during exponential growth and during nitrogen starvation, when cultivated with either glucose or xylose as the carbon source. The genome of BOT-A2 was sequenced using a combination of long- and short-read sequencing and annotated with support of the RNAseq data. Differential expression analysis was used to identify genes with a |log2 fold change|≥ 2 when comparing their expression during nitrogen depletion to that during exponential growth. The promoter regions from 16 of these genes were evaluated for their ability to drive the expression of a fluorescent reporter gene. Three promoters that were clearly upregulated under nitrogen starvation and three that were downregulated were selected and further characterized. One promoter, derived from gene RTBOTA2_003877, was found to function like an on-off switch, as it was only upregulated under full nitrogen depletion and downregulated in the presence of the nitrogen source. CONCLUSIONS: Six new R. toruloides promoters that were either upregulated or downregulated under nitrogen-starvation were identified. These substantially contribute to the available promoters when engineering this organism and are foreseen to be particularly useful for future engineering strategies requiring specific regulation of target genes in accordance with nitrogen availability.

Functional genome annotation and transcriptome analysis of Pseudozyma hubeiensis BOT-O, an oleaginous yeast that utilizes glucose and xylose at equal rates.

Pseudozyma hubeiensis is a basidiomycete yeast that has the highly desirable traits for lignocellulose valorisation of being equally efficient at utilization of glucose and xylose, and capable of their co-utilization. The species has previously mainly been studied for its capacity to produce secreted biosurfactants in the form of mannosylerythritol lipids, but it is also an oleaginous species capable of accumulating high levels of triacylglycerol storage lipids during nutrient starvation. In this study, we aimed to further characterize the oleaginous nature of P. hubeiensis by evaluating metabolism and gene expression responses during storage lipid formation conditions with glucose or xylose as a carbon source. The genome of the recently isolated P. hubeiensis BOT-O strain was sequenced using MinION long-read sequencing and resulted in the most contiguous P. hubeiensis assembly to date with 18.95 Mb in 31 contigs. Using transcriptome data as experimental support, we generated the first mRNA-supported P. hubeiensis genome annotation and identified 6540 genes. 80% of the predicted genes were assigned functional annotations based on protein homology to other yeasts. Based on the annotation, key metabolic pathways in BOT-O were reconstructed, including pathways for storage lipids, mannosylerythritol lipids and xylose assimilation. BOT-O was confirmed to consume glucose and xylose at equal rates, but during mixed glucose-xylose cultivation glucose was found to be taken up faster. Differential expression analysis revealed that only a total of 122 genes were significantly differentially expressed at a cut-off of |log2 fold change| ≥ 2 when comparing cultivation on xylose with glucose, during exponential growth and during nitrogen-starvation. Of these 122 genes, a core-set of 24 genes was identified that were differentially expressed at all time points. Nitrogen-starvation resulted in a larger transcriptional effect, with a total of 1179 genes with significant expression changes at the designated fold change cut-off compared with exponential growth on either glucose or xylose.

Screening of xylose utilizing and high lipid producing yeast strains as a potential candidate for industrial application.

BACKGROUND: Sustainable production of oil for food, feed, fuels and other lipid-based chemicals is essential to meet the demand of the increasing human population. Consequently, novel and sustainable resources such as lignocellulosic hydrolysates and processes involving these must be explored. In this paper we screened for naturally-occurring xylose utilizing oleaginous yeasts as cell factories for lipid production, since pentose sugar catabolism plays a major role in efficient utilization of lignocellulosic feedstocks. Glycerol utilization, which is also beneficial in yeast-based oil production as glycerol is a common by-product of biodiesel production, was investigated as well. Natural yeast isolates were studied for lipid accumulation on a variety of substrates, and the highest lipid accumulating strains were further investigated in shake flask cultivations and fermenter studies on xylose and hydrolysate. RESULTS: By collecting leaves from exotic plants in greenhouses and selective cultivation on xylose, a high frequency of oleaginous yeasts was obtained (> 40%). Different cultivation conditions lead to differences in fatty acid contents and compositions, resulting in a set of strains that can be used to select candidate production strains for different purposes. In this study, the most prominent strains were identified as Pseudozyma hubeiensis BOT-O and Rhodosporidium toruloides BOT-A2. The fatty acid levels per cell dry weight after cultivation in a nitrogen limited medium with either glucose, xylose or glycerol as carbon source, respectively, were 46.8, 43.2 and 38.9% for P. hubeiensis BOT-O, and 40.4, 27.3 and 42.1% for BOT-A2. Furthermore, BOT-A2 accumulated 45.1% fatty acids per cell dry weight in a natural plant hydrolysate, and P. hubeiensis BOT-O showed simultaneous glucose and xylose consumption with similar growth rates on both carbon sources. The fatty acid analysis demonstrated both long chain and poly-unsaturated fatty acids, depending on strain and medium. CONCLUSIONS: We found various natural yeast isolates with high lipid production capabilities and the ability to grow not only on glucose, but also xylose, glycerol and natural plant hydrolysate. R. toruloides BOT-A2 and P. hubeiensis BOT-O specifically showed great potential as production strains with high levels of storage lipids and comparable growth to that on glucose on various other substrates, especially compared to currently used lipid production strains. In BOT-O, glucose repression was not detected, making it particularly desirable for utilization of plant waste hydrolysates. Furthermore, the isolated strains were shown to produce oils with fatty acid profiles similar to that of various plant oils, making them interesting for future applications in fuel, food or feed production.

Extracts of Digested Berries Increase the Survival of Saccharomyces cerevisiae during H2O2 Induced Oxidative Stress.

Many studies suggest anthocyanins may prevent the development of several diseases. However, anthocyanin bioactivity against cellular stress is not fully understood. This study aimed to evaluate the protective effect of berry anthocyanins on stressed cells using Saccharomyces cerevisiae. The impact of in vitro gastrointestinal digestion on anthocyanin profiles was also assessed. Bilberry and blackcurrant had higher anthocyanin levels than raspberry and strawberry, but digestion reduced the detected anthocyanins by approximately 90%. Yeast cells with and without digested or nondigested anthocyanin extracts were exposed to H2O2 and examined for survival. In the presence of anthocyanins, particularly from digested strawberry, a significant increase in cell survival was observed, suggesting that the type and levels of anthocyanins are important factors, but they also need to undergo gastrointestinal (GI) structural modifications to induce cell defence. Results also showed that cells need to be exposed to anthocyanins before the stress was applied, suggesting induction of a cellular defence system by anthocyanins or their derivatives rather than by a direct antioxidative effect on H2O2. Overall, data showed that exposure of severely stressed yeast cells to digested berry extracts improved cell survival. The findings also showed the importance of considering gastrointestinal digestion when evaluating anthocyanins' biological activity.

Isolation, identification, and selection of strains as candidate probiotics and starters for fermentation of Swedish legumes.

BACKGROUND: The non-dairy sector is growing, fermented alternatives to dairy are sparse. Adapted starter cultures to substituting raw materials needs to be developed. OBJECTIVE: Aims of this study were to isolate, identify, and phenotypically characterize lactic acid bacteria (LAB) that inhabit Swedish legumes, and assess properties necessary for selecting strains with the ability to ferment a bean beverage and with potential health beneficial properties. DESIGN: Isolates of presumed LAB were obtained from legumes collected at Öland, Sweden. Strain diversity was assessed by repetitive polymerase chain reaction (rep-PCR). The strains were identified using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS). Species belonging to Enterococcus were predominant along with Pediococcus and closely related Bacillus. Strains were tested for tolerance to low pH, phenol, and bile as well as their bile salt hydrolase (BSH) activity. In addition, Enterococcus strains were tested for antibiotic resistance, and Pediococcus strains for their ability to ferment a bean beverage. RESULTS: From the 25 strains characterized, five were found resistant to low pH, bile, and phenol, suggesting that they can survive a passage through the gastrointestinal tract (GIT) and hence potentially exert beneficial effects in the host. These are suggested for further investigation on specific host-beneficial properties. Two of these, belonging to Pediococcus pentosaceus, were able to ferment a bean beverage without any added nutrients, indicating that the Pediococcus strains are well adapted to the bean substrate. One of the P. pentosaceus strains were also able to markedly improve the reduction of phytate by the phytase-producing yeast strain Pichia kudriavzevii TY1322 during co-fermentation as well as increase the final cell count of the yeast strain. CONCLUSION: Strain isolation and characterization performed in this study aids in selecting starter cultures for legume fermentation. Nutritional properties can be improved by co-fermentation with yeast indicating that novel nutritious fermented non-dairy products could be developed.

Effects of Pulsed Electric Field-Assisted Osmotic Dehydration and Edible Coating on the Recovery of Anthocyanins from In Vitro Digested Berries.

Berry fruits, such as strawberries and blueberries, are rich sources of anthocyanins. Several studies have been made on the impact of non-thermal treatments on safety, shelf-life and nutritional characteristics of such products, but the effects of these processes on anthocyanin stability during digestion in the gastrointestinal tract are still not completely clear. The aim of this study was to assess the recovery of anthocyanins after simulated gastrointestinal digestion of (1) strawberry samples, pre-treated with pulsed electric field (PEF) at 100 or 200 V·cm-1, prior to osmotic dehydration (OD), and (2) blueberry samples coated with chitosan and procyanidin. After digestion, a significantly higher content of cyanidin-3-O-glucoside and malvidin-3-O-glucoside was quantified by LC-MS/MS in processed strawberry and blueberry samples, compared with the controls. The highest recovery of cyanidin-3-O-glucoside was detected in digested strawberry samples osmotically dehydrated with trehalose. The recovery of malvidin-3-O-glucoside was highest in digested blueberries coated with chitosan and stored for 14 days, compared with untreated samples or samples coated with chitosan and procyanidin. Our study shows the potential of mild PEF treatments combined with OD, or the use of edible coating, to obtain shelf-stable products without substantially affecting the composition or the stability of anthocyanins during digestion in the upper gastrointestinal tract.

Tailoring bilberry powder functionality through processing: Effects of drying and fractionation on the stability of total polyphenols and anthocyanins.

Bilberries are a rich natural source of phenolic compounds, especially anthocyanins. The press cake obtained during the processing of bilberry juice is a potential source of phytochemicals. The objective of this study was to evaluate different drying techniques and the fractionation of bilberry press cake powder toward obtaining phenolic-rich ingredients for incorporation into value-added food products. The derived powders were dispersed in water and dairy cream, to investigate the effects of drying and fractionation on the dispersibility and solubility of phenolic compounds. The drying techniques, hot air drying and microwave drying, applied on bilberry press cake reduced the content of total phenolics and anthocyanins. The degradation was, however, consistently small and similar for both techniques. The major anthocyanins detected in the samples were stable during drying and fractionation treatments. Fractionation of the press cake powder affected the total apparent phenolic content and composition of the different fractions. The highest phenolic content (55.33 ± 0.06 mg g-1 DW) and highest anthocyanin content (28.15 ± 0.47 mg g-1 DW) were found in the fractions with the smallest particle size (<500 µm), with delphinidin-3-O-galactoside being the most abundant anthocyanin. Dispersibility of all dried powder samples was higher in dairy cream than water, and the highest level of anthocyanins was measured in samples from the powder with the smallest particle size (<500 µm), dispersed in cream. The application of drying, milling and fractionation was found to be a promising approach to transform bilberry press cake into stable and deliverable ingredients that can be used for fortification of food products with high levels of phenolic compounds.

Appetite and Subsequent Food Intake Were Unaffected by the Amount of Sourdough and Rye in Soft Bread-A Randomized Cross-Over Breakfast Study.

Sourdough fermented bread has been suggested to have beneficial health effects, in part mediated by increased satiety in the postprandial phase, but only limited research has been conducted to verify this. The current study aimed to investigate the effect of the amounts of sourdough and rye in soft bread on postprandial appetite. On 6 occasions, 23 healthy volunteers consumed 5 different test breads, with varying amount of rye and sourdough, and a yeast-fermented refined wheat control bread as part of a breakfast meal. The sourdough ranged between 9⁻51% of dough weight and rye content between 35⁻48% of flour weight. Appetite was recorded using visual analogue scales from immediately before breakfast and every 30 min the following 4 h. An ad libitum lunch was served 4 h after the breakfast meal, from which voluntary energy intake was measured. While some of the test breads resulted in lower hunger ratings and increased sense of fullness compared to the refined wheat bread, there were no differences between the test breads. The content of rye in the test breads differed within a narrow range, which might explain the lack of a consistent effect of rye on appetite. Microstructural examination of the test breads showed an increased aggregation of proteins in the breads with high content of sourdough, indicating additional changes to the breads, beyond change in pH, which may counteract the potential effect of decreased pH in the bread on appetite. In conclusion, our study does not support an effect of sourdough on appetite and ad libitum food intake.

Bifidobacterium callitrichidarum sp. nov. from the faeces of the emperor tamarin (Saguinus imperator).

Three Gram-stain-positive, non-spore-forming, microaerophilic and fructose-6-phosphate phosphoketolase positive strains were isolated from a faecal sample of an adult subject of the emperor tamarin (Saguinus imperator). Given that the isolates revealed identical BOX PCR profiles, strain TRI 5T was selected as a representative and characterized further. Comparative analysis of 16S rRNA gene sequence similarity revealed that strain TRI 5T was closely related to Bifidobacterium saguini DSM 23967T (96.4 %) and to Bifidobacterium longum subsp. longum ATCC 15708 (96.2 %). Multilocus sequence analyses of five housekeeping genes showed the close phylogenetic relatedness of this strain to Bifidobacterium breve DSM 20213T (hsp60 94.1 %), Bifidobacterium saguini DSM 23967T (clpC 91 %), Bifidobacterium avesanii DSM 100685T (dnaG 80.3 %), Bifidobacterium longumsubsp. infantis ATCC 15697T (dnaJ 85.3 %) and Bifidobacterium longumsubsp. longum ATCC 15708 (rpoB 93 %), respectively. The peptidoglycan type was A3ß, with an interpeptide bridge comprising l-Orn (Lys) - l-Ser - l-Ala - l-Thr - l-Ala. The DNA G+C content of strain TRI 5T was 60.9 mol%. Based on the data provided, strain TRI 5T represents a novel species of the genus Bifidobacterium for which the name Bifidobacteriumcallitrichidarum sp. nov. is proposed. The type strain is TRI 5T (=DSM 103152T=JCM 31790T).

Strain improvement of Pichia kudriavzevii TY13 for raised phytase production and reduced phosphate repression.

In this work, we present the development and characterization of a strain of Pichia kudriavzevii (TY1322), with highly improved phytate-degrading capacity. The mutant strain TY1322 shows a biomass-specific phytate degradation of 1.26 mmol g-1  h-1 after 8 h of cultivation in a high-phosphate medium, which is about 8 times higher compared with the wild-type strain. Strain TY1322 was able to grow at low pH (pH 2), at high temperature (46°C) and in the presence of ox bile (2% w/v), indicating this strain's ability to survive passage through the gastrointestinal tract. The purified phytase showed two pH optima, at pH 3.5 and 5.5, and one temperature optimum at 55°C. The lower pH optimum of 3.5 matches the reported pH of the pig stomach, meaning that TY1322 and/or its phytase is highly suitable for use in feed production. Furthermore, P. kudriavzevii TY1322 tolerates ethanol up to 6% (v/v) and shows high osmotic stress tolerance. Owing to the phenotypic characteristics and non-genetically modified organisms nature of TY1322, this strain show great potential for future uses in (i) cereal fermentations for increased mineral bioavailability, and (ii) feed production to increase the phosphate bioavailability for monogastric animals to reduce the need for artificial phosphate fortification.

Isolation, Identification and Characterization of Yeasts from Fermented Goat Milk of the Yaghnob Valley in Tajikistan.

The geographically isolated region of the Yaghnob Valley, Tajikistan, has allowed its inhabitants to maintain a unique culture and lifestyle. Their fermented goat milk constitutes one of the staple foods for the Yaghnob population, and is produced by backslopping, i.e., using the previous fermentation batch to inoculate the new one. This study addresses the yeast composition of the fermented milk, assessing genotypic, and phenotypic properties. The 52 isolates included in this study revealed small species diversity, belonging to Kluyveromyces marxianus, Pichia fermentans, Saccharomyces cerevisiae, and one Kazachstania unispora. The K. marxianus strains showed two different genotypes, one of which never described previously. The two genetically different groups also differed significantly in several phenotypic characteristics, such as tolerance toward high temperatures, low pH, and presence of acid. Microsatellite analysis of the S. cerevisiae strains from this study, compared to 350 previously described strains, attributed the Yaghnobi S. cerevisiae to two different ancestry origins, both distinct from the wine and beer strains, and similar to strains isolated from human and insects feces, suggesting a peculiar origin of these strains, and the existence of a gut reservoir for S. cerevisiae. Our work constitutes a foundation for strain selection for future applications as starter cultures in food fermentations. This work is the first ever on yeast diversity from fermented milk of the previously unexplored area of the Yaghnob Valley.

Biosynthesis and cellular content of folate in bifidobacteria across host species with different diets.

BACKGROUND: Bifidobacteria, one of the most common bacteria of the intestinal tract, help establish balance in the gut microbiota and confer health benefits to the host. One beneficial property is folate biosynthesis, which is dependent on species and strains. It is unclear whether the diversity in folate biosynthesis is due to the adaptation of the bifidobacteria to the host diet or whether it is related to the phylogeny of the animal host. To date, folate production has been studied in the bifidobacteria of omnivorous, and a few herbivorous, non-primate hosts and humans, but not in carnivores, non-human primates and insects. In our study we screened folate content and composition in bifidobacteria isolated from carnivores (dog and cheetah), Hominoidea omnivorous non-human primates (chimpanzee and orangutan) and nectarivorous insects (honey bee). RESULTS: Bifidobacterium pseudolongum subsp. globosum, a species typically found in non-primates, was isolated from dog and cheetah, and Bifidobacterium adolescentis and Bifidobacterium dentium, species typically found in humans, were respectively obtained from orangutan and chimpanzee. Evidence of folate biosynthesis was found in bifidobacteria isolated from non-human primates, but not from the bifidobacteria of carnivores and honey-bee. On comparing species from different hosts, such as poultry and herbivorous/omnivorous non-primates, it would appear that folate production is characteristic of primate (human and non-human) bifidobacteria but not of non-primate. Isolates from orangutan and chimpanzee had a high total folate content, the mean values being 7792 µg/100 g dry matter (DM) for chimpanzee and 8368 µg/100 g DM for orangutan. The tetrahydrofolate (H4folate) and 5-methyl-tetrahydrofolate (5-CH3-H4folate) distribution varied in the bifidobacteria of the different animal species, but remained similar in the strains of the same species: B. dentium CHZ9 contained the least 5-CH3-H4folate (3749 µ/100 g DM), while B. adolescentis ORG10 contained the most (8210 µg/100 g DM). CONCLUSION: Our data suggest a correlation between phylogenetic lineage and capacity of folate production by bifidobacteria, rather than with dietary type of the host.

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.

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.

Blocking peptides decrease tissue transglutaminase processing of gliadin in vitro.

Tissue transglutaminase (tTG) plays an important role in celiac disease pathology as it catalyzes deamidation and cross-linking of specific gluten peptides and converts them into potent epitopes recognized by intestinal T-cells. We investigated whether synthetic peptides with high affinity to gliadin could alter tTG activity on gliadin and whole gluten digest. The immobilized substrates were incubated with synthetic peptides identified by the phage display technique and a control peptide with no affinity to gliadin. Transglutaminase activity was measured with time resolved fluorescence. The mean tTG activity, compared to that of the control without the peptides, was reduced by 31, 33, and 36% for three selected gliadin-binding peptides, and 30% for the peptide pool (P < or = 0.001-0.004) when gliadin was the substrate. Finally, substrate specificity experiments suggested that avenin was processed in a manner similar that used for gliadin during in vitro assays with tTG. The results showed that the blocking peptides efficiently reduced tTG processing of gliadin in vitro, and this strategy will be further investigated as an alternative therapy for celiac disease.

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.

Impaired uptake of beta-carotene by Caco-2 human intestinal cells in the presence of iron.

At present, there are conflicting data regarding whether or not beta-carotene has a positive effect on iron absorption. This study was undertaken to evaluate possible interactions involved in the uptake of beta-carotene and iron in a human intestinal cell model (Caco-2). The Caco-2 cells were incubated with test solutions containing different amounts of ferrous chloride (10-50 microM) and beta-carotene (0.3-2.5 microM) incorporated in synthetic micelles. In the absence of iron, cellular accumulation of beta-carotene from synthetic micelles was proportional (r (2)=0.97, P <0.001) to the beta-carotene concentration in the test solution. However, with addition of ferrous chloride (30 microM), the beta-carotene uptake was significantly reduced (P <0.05), on average by 22%. There was also an inverse relationship between the beta-carotene uptake and iron concentration in the test solution (r (2)=0.93, P <0.05). Iron provided in physiological amounts inhibited the uptake of beta-carotene in the in vitro Caco-2 cell system.

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 Baker's yeast (27-43 microg/100 g). The commercial Baker's 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.