Secretion of non-cell-bound phytase by the yeast Pichia kudriavzevii TY13.

AIMS: Mineral deficiencies cause several health problems in the world, especially for populations consuming cereal-based diets rich in the anti-nutrient phytate. Our aim was to characterize the phytate-degrading capacity of the yeast Pichia kudriavzevii TY13 and its secretion of phytase. METHODS AND RESULTS: The phytase activity in cell-free supernatants from cultures with 100% intact cells was 35-190 mU ml(-1) depending on the media. The Km was 0.28 mmol l(-1) and the specific phytase activity 0.32 U mg(-1) total protein. The phytase activity and secretion of extracellular non-cell-bound phytase was affected by the medium phosphate concentrations. Further, addition of yeast extract had a clearly inducing effect, resulting in over 60% of the cultures total phytase activity as non-cell-bound. CONCLUSIONS: Our study reveals that it is possible to achieve high extracellular phytase activity from the yeast P. kudriavzevii TY13 by proper composition of the growth medium. SIGNIFICANCE AND IMPACT OF THE STUDY: TY13 could be a promising future starter culture for fermented foods with improved mineral bioavailability. Using strains that secrete phytase to the food matrix may significantly improve the phytate degradation by facilitating the enzyme-to-substrate interaction. The secreted non-cell-bound phytase activities by TY13 could further be advantageous in industrial production of phytase.

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. METHODS AND RESULTS: HPLC was used to analyse deconjugated folates extracted from bacterial biomass. Most strains had a total folate content above 4000 µg per 100 g dry matter (DM). The highest value of 9295 µg per 100 g DM was found in Bifidobacterium catenulatum ATCC 27539 and the lowest in Bifidobacterium animalis ssp. animalis ATCC 25527 containing 220 µg per 100 g DM. Ten strains grew in a synthetic folate-free medium (FFM), showing folate autotrophy and suggesting folate auxotrophy of the remaining nine. In the autotrophic strains, a consistently higher folate level was found in FFM as compared to a more complex folate-containing medium, suggesting reduced requirements for folates in the presence of growth factors otherwise requiring folates for synthesis. The contents of total folate, 5-CH(3) -H(4) folate and H(4) folate were strain dependent. 5-CH(3) -H(4) folate dominated in most strains. CONCLUSIONS: Our results show that bifidobacteria folate content and composition is dynamic, is strain specific and depends on the medium. Suitable selection of the growth conditions can result in high levels of folate per cell unit biomass. SIGNIFICANCE AND IMPACT OF THE STUDY: This suggests that certain bifidobacteria may contribute to the folate intake, either directly in foods, such as fermented dairy products, or in the intestine as folate-trophic probiotics or part of the natural microbiota.

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. METHODS AND RESULTS: Two methods using the indirect estimation of phytate degradation were evaluated and both shown to be inadequate. A third method, measuring the inositol phosphate (IP3-IP6) content directly during fermentation, was used instead of the indirect estimations of phytate degradation. In synthetic media, some strains required customized conditions, with no accessible phosphorus sources other than phytate, to express phytase activity. The repression of phytase synthesis by inorganic phosphorus was not detected during fermentation with pea flour as substrate and seemed to be less significant with a higher composition complexity of the substrate. None of the tested lactic acid bacteria strains showed phytase activity. CONCLUSIONS: The methodology for the phytase screening procedure was shown to be critical. Some of the screening methods and media used in previous publications were found to be inadequate. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper highlights the pitfalls and difficulties in the evaluation of phytase production by micro-organisms. The study is of great importance for future studies in this area.

Inositol hexaphosphate hydrolysis by Baker's yeast. Capacity, kinetics, and degradation products.

Phytases hydrolyze myo-inositol 1,2,3,4,5,6-hexaphosphate (IP(6)), yielding lower inositol phosphates and inorganic orthophosphate. Two commercial strains of baker's yeast (Saccharomyces cerevisiae), Y(1) and Y(2), were able to express phytase activity. This was determined by the capacity to grow in a synthetic medium with IP(6) as the sole phosphorus source. IP(6) hydrolysis was rapid for both strains, and after 24 h, all IP(6) was degraded. Control cultures contained inorganic orthophosphate (P(i)) and no IP(6). Growth rate in IP(6) medium was for both strains essentially identical to growth in P(i) medium, indicating a well-adapted metabolism for utilization of phosphorus from IP(6). There was some difference in growth yield (milligrams of biomass per milligram of glucose) between the two strains: 0.95 (Y(1)) and 1.35 (Y(2)) in IP(6) medium and 1.03 and 1. 35, respectively, in P(i) medium. The phytases were of the 3-phytase type, forming mainly DL-Ins(1,2,4,5,6)P(5), DL-Ins(1,2,5,6)P(4), and DL-Ins(1,2,6)P(3).

Characterization of Saccharomyces cerevisiae CBS 7764 isolated from rainbow trout intestine.

A wild-type Saccharomyces cerevisiae, strain CBS 7764, isolated from the intestine of rainbow trout, was analyzed with respect to general growth parameters and global protein expression. Characterization of this strain was of interest since previous data show non-typical S. cerevisiae cell surface properties and because data suggest a probiotic potential of CBS 7764. The heat production rate (dQ/dt), monitored by microcalorimetry, showed that the typical growth phases resulting from diauxic growth on glucose were present in the fish isolate. However, CBS 7764 differentiated from a reference strain by becoming limited in the respiratory phase as demonstrated by a plateau in the dQ/dt signal. The global protein expression, as studied by two-dimensional gel electrophoresis (2D-PAGE), revealed a large degree of resemblance of the fish isolate to the reference strain, however, also clear qualitative and quantitative expression differences were detected; e.g. 14% of the proteins differed in expression level by a factor of at least 2. In addition, the fish isolate expressed 12 unique proteins. The heat shock proteins, which for other organisms have been identified as important in mucosal colonization, were generally expressed to a higher level in CBS 7764.

Yeasts isolated from the intestine of rainbow trout adhere to and grow in intestinal mucus.

The yeast strains Saccharomyces cerevisiae CBS 7764 and Debaryomyces hansenii Hf1 (CBS 8339), isolated from the intestine of rainbow trout, were studied with respect to adhesion to and growth in fish intestinal mucus. The level of adhesion was dependent on the physiologic state of the yeast culture. Growing cells of both strains adhered more strongly than nongrowing cells. This correlates with a previously shown shift in cell surface hydrophobicity of these yeasts. In addition, forces other than hydrophobic interactions may participate, as all strains tested adhered more strongly to the membrane lipid phosphatidylserine than to phosphatidylcholine and phosphatidylethanolamine. Debaryomyces hansenii Hf1 also adhered to the most hydrophobic of the neutral lipids present in mucus, while no adhesion was observed to the other neutral lipids or to the hydrophilic silica gel, again suggesting hydrophobic interactions. Finally, the fish-isolated yeasts grew rapidly in isolated fish intestinal mucus as the sole source of energy and nutrients.

Adhesion of yeast isolated from fish gut to crude intestinal mucus of rainbow trout, Salmo gairdneri.

Two yeast strains, Saccharomyces cerevisiae CBS 7764 and Debaryomyces hansenii Hfl CBS 8339, with a high capacity to colonize fish intestine were used in this study. The ability to adhere to crude mucus obtained from fish intestine was demonstrated for both strains. Scatchard analysis of the binding indicated a positive cooperativity for D. hansenii Hfl and absence of cooperativity for S. cerevisiae CBS 7764. In neither of the strains was adhesion extensively affected by reducing the hydrophobic interaction with p-nitrophenol, or by enhancing the hydrophobic interaction with ammonium sulfate. The adhesion was heat sensitive but resistant to trypsin treatment. We conclude that adhesion is mediated partly by specific mechanisms and partly by cell surface hydrophobicity.

Yeast colonizing the intestine of rainbow trout (Salmo gairdneri) and turbot (Scophtalmus maximus).

Yeast were isolated from the intestine of farmed rainbow trout (Salmo gairdneri), turbot (Scophtalmus maximus), and free-living flat-fish (Pleuronectes platessa and P. flesus). The average number of viable yeasts recovered from farmed rainbow trout was 3.0 × 10(3) and 0.5 × 10(2) cells per gram homogenized intestine for white and red-pigmented yeasts, respectively. The dominant species were Debaryomyces hansenii, Saccharomyces cerevisiae, Rhodotorula rubra, and R. glutinis. In 5 of 10 free-lving marine fish, > 100 viable yeast cells per gram intestinal mucus were recovered. Red-pigmented yeasts dominated and composed >90% of the isolates. Colonization experiments were performed by inoculating rainbow trout and turbot with fish-specific, isolated yeast strains and by examining the microbial intestinal colonization at intervals. Inoculation of experimental fish with pure cultures of R. glutinis and D. hansenii HF1 yielded colonization at a level several orders of magnitude higher than before the inoculation. Up to 3.8 × 10(4), 3.1 × 10(6), and 2.3 × 10(9) viable yeast cells per gram intestine or feces were recovered in three separate colonization experiments. The high level of colonizing yeasts persisted for several weeks. The concentrations of yeasts in the tank water never exceeded 10(3) viable cells per milliliter. No traces of fish sickness as a result of high yeast colonization were recorded during any of the colonization experiments. For periods of the experiments, the concentration of aerobic bacteria in the fish intestine was lower than the intestinal yeast concentration. Scanning electron microscopy studies demonstrated a close association of the yeasts with the intestinal mucosa. The mucosal colonization was further demonstrated by separating intestinal content, mucus, and tissue. All compartments were colonized by >10(3) viable yeast cells per gram. No bacteria were detected on the micrographs, indicating that their affinity for the intestinal mucosa was less than that of the yeasts.

The expression of potential colonization factors of yeasts isolated from fish during different growth conditions.

Three strains, Rhodotorula rubra, Rhodotorula glutinis, and Candida zeylanoides, isolated from fish, were tested for the expression of putative tissue colonization factors. All strains were able to bind collagen type I, fibronectin, and laminin to various degrees after growing on various solid and broth media, while the binding to collagen type IV was sparse under all conditions tested. For the three strains tested, a very low cell surface hydrophobicity was shown for growth on various solid and broth media. Mostly, the strains also expressed a negatively charged surface. Extracellular protease activity using different substrates was shown for all three strains. Furthermore, two properties related to iron scavenging, i.e., binding of lactoferrin and production of siderophores, were also tested. For the three strains a capacity to bind lactoferrin as well as a capacity to excrete siderophores were demonstrated. Since these different properties have been correlated to virulence and to the capacity of colonization in other organisms, we address the question of whether the expression of these properties in yeasts could contribute to colonization in fish.