The effects of apple variety, ripening stage, and yeast strain on the volatile composition of apple cider.

This work examined the degree of influence of apple variety, apple ripening stage, and yeast strain on the volatile composition of apple cider. Four apple varieties grown in Estonia were selected for the study - Antei, Melba, Kulikovskoye, and Orlovski Sinap. The must from the apples at various stages of ripening (unripe, ripe, overripe) underwent alcoholic fermentation using commercially available yeast strains. Gas chromatography - mass spectrometry was employed to assess the differences in volatile composition between the samples. Out of the variables analyzed in this work, apple variety turned out to be the primary attribute influencing the quality and aroma properties of apple cider. The effect of yeast strain and the maturity of the fruit was variety-specific where the volatile profiles of ciders made with Melba variety were the least influenced by the ripening stage of apples and yeast strains used for the fermentation.

Assessment of bioavailable B vitamin content in food using in vitro digestibility assay and LC-MS SIDA.

Standardized analytical methods, where each B vitamin is extracted from a given sample individually using separate procedures, typically ensure that the extraction conditions provide the maximum recovery of each vitamin. However, in the human gastrointestinal tract (GIT), the extraction conditions are the same for all vitamins. Here, we present an analytically feasible extraction protocol that simulates conditions in the GIT and provides a measure of the content of bioavailable vitamins using LC-MS stable isotope dilution assay. The results show that the activities of both human gastric and duodenal juices were insufficient to liberate absorbable vitamers (AV) from pure cofactors. The use of an intestinal brush border membrane (IBBM) fraction derived from the mucosal tissue of porcine small intestine ensured at least 70% AV recovery. The rate of AV liberation, however, was strongly dependent on the cofactor, e.g., in the case of NADH, it was magnitudes higher than in the case of thiamine diphosphate. For some vitamins in some food matrices, the use of the IBBM fraction assay resulted in lower values for the content of AV than conventional vitamin determination methods. Conventional methods likely overestimate the actual bioavailability of some vitamins in these cases. Graphical abstract Assessment of bioavailable B vitamin content in food.

Impact of apple cultivar, ripening stage, fermentation type and yeast strain on phenolic composition of apple ciders.

Hydroxycinnamic acids and flavonoids in apple juices and ciders were studied using liquid chromatography. Samples were produced from four different Estonian apple cultivars using unripe, ripe and overripe apples, and six different commercial yeasts including Saccharomyces cerevisiae, Saccharomyces bayanus, and Torulaspora delbrueckii strains. Part of the samples was additionally inoculated with malolactic bacteria, Oenococcus oeni. The most notable difference among the samples was the appearance of phloretin in malolactic ciders in comparison to conventional ciders and the juices. Furthermore, the apple cultivars were significantly different in their phenolic contents and compositions. Additionally, ciders and juices made from unripe apples contained more phenolic compounds than the ripe or overripe, but the effect was dependent on cultivar. The commercial yeast strains differed in the release of free HCAs, especially p-coumaric acid, during the yeast fermentation. In ciders inoculated with S. bayanus, the content was higher than in ciders fermented with S. cerevisiae.

Diversity and Stability of Lactic Acid Bacteria in Rye Sourdoughs of Four Bakeries with Different Propagation Parameters.

We identified the lactic acid bacteria within rye sourdoughs and starters from four bakeries with different propagation parameters and tracked their dynamics for between 5-28 months after renewal. Evaluation of bacterial communities was performed using plating, denaturing gradient gel electrophoresis, and pyrosequencing of 16S rRNA gene amplicons. Lactobacillus amylovorus and Lactobacillus frumenti or Lactobacillus helveticus, Lactobacillus pontis and Lactobacillus panis prevailed in sourdoughs propagated at higher temperature, while ambient temperature combined with a short fermentation cycle selected for Lactobacillus sanfranciscensis, Lactobacillus pontis, and Lactobacillus zymae or Lactobacillus helveticus, Lactobacillus pontis and Lactobacillus zymae. The ratio of species in bakeries employing room-temperature propagation displayed a seasonal dependence. Introduction of different and controlled propagation parameters at one bakery (higher fermentation temperature, reduced inoculum size, and extended fermentation time) resulted in stabilization of the microbial community with an increased proportion of L. helveticus and L. pontis. Despite these new propagation parameters no new species were detected.

Comparison of different extraction methods to determine free and bound forms of B-group vitamins in quinoa.

The distribution of free and bound forms of B-group vitamins (B1, B2, B3, B5, and B6) was quantified in quinoa seeds using LC-MS-TOF combined with a stable isotope dilution assay. The effectiveness of liberating thiamine, riboflavin, nicotinic acid, pantothenic acid, pyridoxal, and pyridoxine from the food matrix and cofactors was evaluated for a variety of extraction conditions, including the addition of enzymes. Phosphatase and protease inhibitors, as well as ultrafiltration, were evaluated for their ability to suppress vitamer liberation via enzymes endogenous to quinoa. Cold extraction, together with a mixture of phosphatase and protease inhibitors, is identified as the most efficient treatment to prevent the conversion of cofactors into simple vitamers. Overnight incubation at 37 °C both with and without additional hydrolytic enzyme preparations containing phosphatase and ß-glucosidase activity was almost equally effective in releasing the bound forms of the vitamers. This indicates that the endogenous enzymes within quinoa seeds have high activity. ß-Glucosidase should be used when the total pyridoxine content is to be determined, and thermal treatment followed by enzymatic treatment with phosphatase activity is recommended to liberate the bound forms of pyridoxal prior to quantification.

Evolution of bacterial consortia in spontaneously started rye sourdoughs during two months of daily propagation.

The evolution of bacterial consortia was studied in six semi-solid rye sourdoughs during long-term backslopping at different temperatures. Each rye sourdough was started spontaneously in a laboratory (dough yield 200), propagated at either 20°C or 30°C, and renewed daily at an inoculation rate of 1∶10 for 56 days. The changes in bacterial diversity over time were followed by both DGGE coupled with partial 16S rRNA gene sequencing and pyrosequencing of bar-coded 16S rRNA gene amplicons. Four species from the genus Lactobacillus (brevis, crustorum, plantarum, and paralimentarius) were detected in different combinations in all sourdoughs after 56 propagation cycles. Facultative heterofermentative lactic acid bacteria dominated in sourdoughs fermented at 30°C, while both obligate and facultative heterofermentative LAB were found to dominate in sourdoughs fermented at 20°C. After 56 propagation cycles, Kazachstania unispora (formerly Saccharomyces unisporus) was identified as the only yeast species that dominated in sourdoughs fermented at 20°C, while different combinations of strains from four yeast species (Kazachstania unispora, Saccharomyces cerevisiae, Candida krusei and Candida glabrata) were detected in sourdoughs propagated at 30°C. The evolution of bacterial communities in sourdoughs fermented at the same temperature did not follow the same time course and changes in the composition of dominant and subdominant bacterial communities occurred even after six weeks of backslopping.

Utilization of (15)N-labelled yeast hydrolysate in Lactococcus lactis IL1403 culture indicates co-consumption of peptide-bound and free amino acids with simultaneous efflux of free amino acids.

Lactococcus lactis subsp. lactis IL1403 was grown in medium containing unlabelled free amino acids and (15)N-labelled yeast hydrolysate to gain insight into the role of peptides as a source of amino acids under conditions where free amino acids are abundant. A mathematical model was composed to estimate the fluxes of free and peptide-derived amino acids into and out of the intracellular amino acid pool. We observed co-consumption of peptides and free amino acids and a considerable efflux of most free amino acids during growth. We did not observe significant differences between the peptide consumption patterns of essential and non-essential amino acids, which suggests that the incorporation of a particular amino acid is more dependent on its availability in a readily assimilated form than the organism's auxotrophy for it. For most amino acids the contribution of peptide-bound forms to the formation of biomass was initially between 30 and 60 % with the remainder originating from free amino acids. During the later stages of fermentation we observed a decrease in the utilization of peptide-bound amino acids, thus indicating that the more readily assimilated peptides are gradually exhausted from the medium during growth.

Comparison of different extraction methods for simultaneous determination of B complex vitamins in nutritional yeast using LC/MS-TOF and stable isotope dilution assay.

The application of LC/MS-TOF method combined with stable isotope dilution assay was studied for determination of thiamine, riboflavin, nicotinamide, nicotinic acid, pantothenic acid, pyridoxal, and pyridoxine in food. Nutritional yeast powder was used as a model food matrix. Acid extraction was compared with various enzymatic treatments in ammonium formate buffer to find a suitable method for the conversion of more complex vitamers into the same forms as the used isotope-labeled internal standards. The enzyme preparations α-amylase, takadiastase, ß-glucosidase, and acid phosphatase were all able to liberate thiamine and riboflavin. The diastatic enzyme preparations α-amylase and takadiastase also expressed proteolytic side activities resulting in the formation of small peptides which interfered with the mass spectra of thiamine and riboflavin. Liberation of nicotinamide and pantothenic acid from NAD(+) and CoA, respectively, could not be achieved with any of the studied enzyme preparations. Hydrochloric acid extraction at 121 °C for 30 min was found to be destructive to pantothenic acid, but increased the liberation of pyridoxal.

Structural changes of starch during baking and staling of rye bread.

Rye sourdough breads go stale more slowly than wheat breads. To understand the peculiarities of bread staling, rye sourdough bread, wheat bread, and a number of starches were studied using wide-angle X-ray diffraction, nuclear magnetic resonance ((13)C CP MAS NMR, (1)H NMR, (31)P NMR), polarized light microscopy, rheological methods, microcalorimetry, and measurement of water activity. The degree of crystallinity of starch in breads decreased with hydration and baking to 3% and increased during 11 days of storage to 21% in rye sourdough bread and to 26% in wheat bread. (13)C NMR spectra show that the chemical structures of rye and wheat amylopectin and amylose contents are very similar; differences were found in the starch phospholipid fraction characterized by (31)P NMR. The (13)C CP MAS NMR spectra demonstrate that starch in rye sourdough breads crystallize in different forms than in wheat bread. It is proposed that different proportions of water incorporation into the crystalline structure of starch during staling and changes in starch fine structure cause the different rates of staling of rye and wheat bread.

YAP1 over-expression in Saccharomyces cerevisiae enhances glutathione accumulation at its biosynthesis and substrate availability levels.

Microbiological production of glutathione using genetically engineered yeast strains has a potential to satisfy the increasing industrial demand of this tripeptide. In the present work accumulation of glutathione in response to YAP1 over-expression in Saccharomyces cerevisiae was studied. The over-expression resulted in intracellular glutathione level over two times higher than in the parent strain. Transcript analyses revealed that, in addition to the genes encoding enzymes in the glutathione biosynthesis pathway (GSH1 and GSH2), the expression levels of the genes in the cysteine biosynthesis pathway (CYS3 and CYS4) were also significantly higher in the YAP1 over-expressed strain. This suggests that YAP1 over-expression affects glutathione accumulation at both its biosynthesis and substrate availability levels.

Metabolic changes underlying the higher accumulation of glutathione in Saccharomyces cerevisiae mutants.

Molecular mechanisms leading to glutathione (GSH) over-accumulation in a Saccharomyces cerevisiae strain produced by UV irradiation-induced random mutagenesis were studied. The mutant accumulated GSH but also cysteine and γ-glutamylcysteine in concentrations that were several fold higher than in its wild-type parent strain under all studied cultivation conditions (chemostat, fed-batch, and turbidostat). Transcript analyses along with shotgun proteome quantification indicated a difference in the expression of a number of genes and proteins, the most pronounced of which were several fold higher expression of CYS3, but also that of GSH1 and its transcriptional activator YAP1. This together with the higher intracellular cysteine concentration is most likely the primary factor underlying GSH over-accumulation in the mutant. Comparative sequencing of GSH1 and the fed-batch experiments with continuous cysteine addition demonstrated that the feedback inhibition of Gsh1p by GSH was still operational in the mutant.

Glutathione accumulation in ethanol-stat fed-batch culture of Saccharomyces cerevisiae with a switch to cysteine feeding.

Shot-wise supplementation of cysteine to a yeast culture is a common means of promoting glutathione (GSH) production. In the present work, we study the accumulation kinetics of cysteine, gamma-glutamylcysteine, and GSH and the expression of genes involved in GSH and sulfur metabolism in ethanol-stat fed-batch cultures as a result of switching to a medium enriched with cysteine and glycine. Supplementation in this fashion resulted in a rapid but short-term increase in the rate of GSH synthesis, while the expression of GSH1 decreased. Expression of GSH1 and GSH synthesis rate were observed to revert close to the base level after a few hours. These results indicate that, under such conditions, the control of GSH synthesis at higher concentrations occurred at the enzymatic, rather than the transcriptional level. The incorporation of cysteine into GSH was limited to approximately 40% of the theoretical yield, due to its requirement as a source of sulfur for protein synthesis under conditions whereby the sulfate assimilation pathway is down-regulated. This was supported by the expression profiles of genes involved in cysteine and homocysteine interconversion.

Effect of stress pretreatment on survival of probiotic bacteria in gastrointestinal tract simulator.

The effect of stress pretreatment on survival of probiotic Lactobacillus acidophilus La-5, Lactobacillus rhamnosus GG, and Lactobacillus fermentum ME-3 cultures was investigated in the single bioreactor gastrointestinal tract simulator (GITS). The cultures were pregrown in pH-auxostat, subjected to temperature, acid, or bile stress treatment, fast frozen in liquid nitrogen (LN(2)), and tested for survival in GITS. After LN(2) freezing the colony forming ability of L. rhamnosus GG and L. fermentum ME-3 nonstressed and stressed cells was well retained (average survival of 75.4 +/- 18.3% and 88.0 +/- 7.2%, respectively). L. acidophilus La-5 strain showed good survival of auxostat nonstressed cells after fast freezing (94.2 +/- 15.0), however the survival of stress pretreated cells was considerably lower (30.8 +/- 8.5%). All LN(2) frozen auxostat cultures survived well in the acid phase of the GIT simulation (survival 81 +/- 21%); however, after the bile phase, the colony formation ability of L. acidophilus La-5, L. rhamnosus GG, and L. fermentum ME-3 decreased by approximately 1.4 +/- 0.2, 3.8 +/- 0.3, and 3.5 +/- 1.2 logarithmic units, respectively. No statistically relevant positive effect of stress pretreatments on survival of LN(2) frozen L. acidophilus La-5, L. rhamnosus GG, and L. fermentum ME-3 in GITS was observed.

Bidirectionality and compartmentation of metabolic fluxes are revealed in the dynamics of isotopomer networks.

Isotope labeling is one of the few methods of revealing the in vivo bidirectionality and compartmentalization of metabolic fluxes within metabolic networks. We argue that a shift from steady state to dynamic isotopomer analysis is required to deal with these cellular complexities and provide a review of dynamic studies of compartmentalized energy fluxes in eukaryotic cells including cardiac muscle, plants, and astrocytes. Knowledge of complex metabolic behaviour on a molecular level is prerequisite for the intelligent design of genetically modified organisms able to realize their potential of revolutionizing food, energy, and pharmaceutical production. We describe techniques to explore the bidirectionality and compartmentalization of metabolic fluxes using information contained in the isotopic transient, and discuss the integration of kinetic models with MFA. The flux parameters of an example metabolic network were optimized to examine the compartmentalization of metabolites and and the bidirectionality of fluxes in the TCA cycle of Saccharomyces uvarum for steady-state respiratory growth.

The response of the yeast Saccharomyces cerevisiae to sudden vs. gradual changes in environmental stress monitored by expression of the stress response protein Hsp12p.

The response of the yeast Saccharomyces cerevisiae to sudden vs. gradual changes in different environmental stress conditions during both respiratory growth and aerobic fermentative growth in the presence of excess glucose was investigated by monitoring the level and rate of expression of the stress response protein Hsp12p using the fluorescent fusion construct Hsp12p-Gfp2p. The initial expression level and the rate of Hsp12p synthesis was significantly greater under glucose-limited conditions in the chemostat (D<0.14 h(-1)) compared with when excess glucose was present in the auxostat. Decreasing the dilution rate and the glucose concentration further in the A-stat resulted in increased Hsp12p expression, which was more marked when a rapid rather than a gradual change was affected. Common stress factors such as NaCl, ethanol and elevated temperature caused stress responses in both D-stat and auxo-accelerostat culture. The magnitude of the stress response depended on the stress factor, cultivation conditions as well as the rate of change of the stress factor. The rate of Hsp12p synthesis increased due to all applied stresses, with the observed increase between 2 and 20 times lower when the stress was applied gradually rather than rapidly. The results suggested that the Hsp12p expression rate is a good indicator of applied stress in S. cerevisiae.

Single bioreactor gastrointestinal tract simulator for study of survival of probiotic bacteria.

The aim of the present study was to design an in vitro model system to evaluate the probiotic potential of food. A single bioreactor system-gastrointestinal tract simulator (GITS) was chosen for process simulation on account of its considerable simplicity compared to multi-vessel systems used in previous studies. The bioreactor was evaluated by studying the viability of four known probiotic bacteria (Lactobacillus acidophilus La-5, Lactobacillus johnsonii NCC 533, Lactobacillus casei strain Shirota, and Lactobacillus rhamnosus GG) as a function of their physiological state. L. acidophilus and L. johnsonii survived in GITS better when introduced at an early stationary or exponential phase compared to being previously stored for 2 weeks at 4 degrees C. These two species were more resistant to bile salts and survived better than L. casei and L. rhamnosus GG. The latter two species gave large losses (up to 6 log) in plate counts independent of growth state due to the bile. However, experiments with some commercial probiotic products containing Lb. GG bacteria showed much better survival compared with model food (modified deMan-Rogosa-Sharpe growth medium), thus demonstrating the influence of the food matrix on the viability of bacteria. The study demonstrated that GITS can be successfully used for evaluation of viability of probiotic bacteria and functionality of probiotic food.

Growth characteristics of Saccharomyces cerevisiae S288C in changing environmental conditions: auxo-accelerostat study.

The effect of individual environmental conditions (pH, pO(2), temperature, salinity, concentration of ethanol, propanol, tryptone and yeast extract) on the specific growth rate as well as ethanol and glycerol production rate of Saccharomyces cerevisiae S288C was mapped during the fermentative growth in aerobic auxo-accelerostat cultures. The obtained steady-state values of the glycerol to ethanol formation ratio (0.1 mol mol(-1)) corresponding to those predicted from the stoichiometric model of fermentative yeast growth showed that the complete repression of respiration was obtained in auxostat culture and that the model is suitable for calculation of Y(ATP) and Q(ATP) values for the aerobic fermentative growth. Smooth decrease in the culture pH and dissolved oxygen concentration (pO2) down to the critical values of 2.3 and 0.8%, respectively, resulted in decrease in growth yield (Y(ATP)) and specific growth rate, however the specific ATP production rate (Q(ATP)) stayed almost constant. Increase in the concentration of biomass (>0.8 g dwt l(-1)), propanol (>2 g l(-1)) or NaCl (>15 g l(-1)) lead at first to the decrease in the specific growth rate and Q(ATP), while Y(ATP) was affected only at higher concentrations. The observed decrease in Q(ATP) was caused by indirect rather than direct inhibition of glycolysis. The increase in tryptone concentration resulted in an increase in the specific growth rate from 0.44 to 0.62 h(-1) and Y(ATP) from 12.5 to 18.5 mol ATP g dwt(-1). This study demonstrates that the auxo-accelerostat method, besides being an efficient tool for obtaining the culture characteristics, provides also decent conditions for the experiments elucidating the control mechanisms of cell growth.

Study of the toxic effect of short- and medium-chain monocarboxylic acids on the growth of Saccharomyces cerevisiae using the CO2-auxo-accelerostat fermentation system.

The effect of aliphatic monocarboxylic acids (formic, acetic, propionic, valeric, octanoic and decanoic acids) on the growth and metabolic activity of Saccharomyces cerevisiae S288C was studied, using continuous cultivation method - CO(2)-auxo-accelerostat with smooth increase in the concentration of added monocarboxylic acids. Slow increase in the concentration of these acids resulted in the rapid decrease in the growth yield (Y(ATP)) and specific growth rate (micro), however, the specific ATP production rate (Q(ATP)) increased or stayed almost constant. On the other hand, Q(ATP) decreased if the concentration of formic, acetic or decanoic acids was increased rapidly. The toxic effect of aliphatic monocarboxylic acids on the growth of S. cerevisiae was characterized and quantified from the respective dose-effect curves as the IC(50) value (mM) using two different endpoints: a decrease of 50% in the specific growth rate (IC(50 micro)) and a decrease of 50% in the growth yield based on ATP production (IC(50YATP)). The concentrations of formic, acetic, propionic, valeric, octanoic and decanoic acids causing the 50% reduction in the specific growth rate (IC(50 micro)) were, respectively, 18.1, 47.1, 33.6, 2.3, 0.16 and 0.07 mM. The IC(50 micro) values were notably lower (up to 5-fold) in case of a more rapid increase in the concentration of acid in the medium. The results of the CO(2)-auxo-accelerostat experiments show that the toxic effect depends not only on the nature of the monocarboxylic acid (lipophilicity) but also on the rate at which its concentration changes in the growth environment.

Application of 13C-[2] - and 13C-[1,2] acetate in metabolic labelling studies of yeast and insect cells.

The advantage of using 13C-labelled glucose in metabolic studies is that it is an important carbon and energy source for almost all biotechnologically and medically important organisms. On the other hand, the disadvantage is its relatively high cost in the labelling experiments. Looking for cheaper alternatives we found that 13C-[2] acetate or 13C-[1,2] acetate is a prospective compound for such experiments. Acetate is well incorporated by many organisms, including mammalian and insect cell cultures as preferred source of acetyl-CoA. Our experimental results using 13C NMR demonstrated that acetate was efficiently incorporated into glutamate and alanine secreted by the insect cell culture. Using D-stat culture of Saccharomyces uvarum on glucose/13C-acetate mineral media we demonstrated that the labelling patterns of proteinogenic amino acids can be well predicted on the basis of specific substrate consumption rates using the modified scheme of yeast metabolism and stoichiometric modelling. According to this scheme aspartate and alanine in S. uvarum under the experimental conditions used is synthesised in the mitochondria. Synthesis of alanine in the mitochondria was also demonstrated for Spodoptera frugiperda. For both organisms malic enzyme was also operative. For S. uvarum it was shown that the activity of malic enzyme is sufficient for supporting the mitochondrial biosynthetic reactions with NADPH.

Modification of A-stat for the characterization of microorganisms.

Two novel modifications of continuous culture with gradual change of dilution rate (A-stat): D-stat and auxo-accelerostat were evaluated in the studies of the effect of changing individual environmental parameters (T, pH, pO(2), substrate concentration, etc.) on growth characteristics of different microorganisms. Common for those cultivation methods is that one environmental parameter is programmed to change with constant change rate (change-stat) while the others are kept constant or in the range not affecting the growth characteristics. The environment response growth curves were obtained starting with chemostat (in A-stat and D-stat) or auxostat (in auxo-accelerostat) steady-state cultures followed by change of set-point value of the desired cultivation parameter. Physiological studies of Saccharomyces sp. and Lactococcus lactis were combined with validation of the different modifications of the A-stat method based on well-known cultivation techniques: chemostat, pH-auxostat, pO(2)-auxostat CO(2)-auxostat and fed-batch. The auxo-accelerostat was shown to be very efficient for cell characterization and dynamic studies in growth environments with excess of essential substrates. Choosing the rate of change of environmental parameters was shown to be critical in comparative physiological studies of microorganisms.