Formation by Yeast of the HEMF (4-hydroxy-2(or 5)-ethyl-5(or 2)-methyl-3(2H)-furanone) Aroma Component in Miso with Aging

Two kinds of miso, one with added precultured yeast and the other without, were compared with respect to the changes in the concentration of HEMF formed and the number of yeast cells in the process of aging. In miso without added yeast, the HEMF concentration increased with the increasing number of existing yeast cells. In miso without yeast aged for 21 days after the miso mash, 0.06 ppm HEMF was detected when the cell number was 2.2 × 10³ cell/g. In yeast-added miso aged for 7 days after the miso mash, no HEMF was detected, although the number of yeast cells was 1.6 × 10⁶ cell/g. In yeast-added miso aged for 14 days after the miso mash, HEMF was first detected. The pH levels of miso without yeast and with added yeast when HEMF was first detected were 5.59 and 5.57, respectively. It is suggested that the formation of HEMF in miso containing a high concentration of reducing sugar and salt was related to the growth of yeast and started when the pH level fell to less than 5.6.     

Use of mixed cultures for the fermentation of cereal-based substrates with potential probiotic properties

The production of a new cereal-based probiotic foods with suitable aroma, flavor and pH using mixed culture fermentation has been investigated. This required the selection of suitable types of cereal grains and probiotic microorganisms. In a medium of 5% (w/v) malt suspension the effects of yeast presence on the fermentation of a lactic acid bacterium (LAB), Lactobacillus reuteri, was studied. With different inoculum ratios between the yeast and the LAB, the characteristics of the fermentation broth including pH and the contents of free amino nitrogen (FAN), reducing sugar, lactic acid and ethanol were investigated. It was found that LAB growth was enhanced by the introduction of the yeast. In mixed culture broth pH was lowered and the production of lactic acid and ethanol were increased in comparison against pure LAB culture.     

Improved ethanol tolerance of Saccharomyces cerevisiae in mixed cultures with Kluyveromyces lactis on high-sugar fermentation

The influence of non-Saccharomyces yeast, Kluyveromyces lactis, on metabolite formation and the ethanol tolerance of Saccharomyces cerevisiae in mixed cultures was examined on synthetic minimal medium containing 20% glucose. In the late stage of fermentation after the complete death of K. lactis, S. cerevisiae in mixed cultures was more ethanol-tolerant than that in pure culture. The chronological life span of S. cerevisiae was shorter in pure culture than mixed cultures. The yeast cells of the late stationary phase both in pure and mixed cultures had a low buoyant density with no significant difference in the non-quiescence state between both cultures. In mixed cultures, the glycerol contents increased and the alanine contents decreased when compared with the pure culture of S. cerevisiae. The distinctive intracellular amino acid pool concerning its amino acid concentrations and its amino acid composition was observed in yeast cells with different ethanol tolerance in the death phase. Co-cultivation of K. lactis seems to prompt S. cerevisiae to be ethanol tolerant by forming opportune metabolites such as glycerol and alanine and/or changing the intracellular amino acid pool.     

Environmental Determination of Selective Significance or Neutrality of Amylase Variants in DROSOPHILA MELANOGASTER

Strains homozygous at the amylase locus were derived from a polymorphic laboratory population of Drosophila melanogaster. The Amy ^4,6 strain has higher enzyme activity than the Amy¹ strain.——Maltose has the same nutritional value as starch.——The effect of starch in pure culture depends on the yeast level. At low yeast level increasing starch increases survival, at high yeast level increasing starch increases mean dry weight. The strains do not differ in survival or mean dry weight in pure culture.——In mixed cultures at 50% input of Amy ^4,6 and Amy¹ as larvae the percentage Amy^4,6 in adults increases with increasing starch at low yeast levels, but equals input frequency at high yeast levels. No increase in percentage Amy^4,6 in adults is present with increasing maltose at low yeast levels in mixed culture. The increase in percentage Amy^4,6 with increasing starch must be due to selection on the amylase locus working by competition for food in the larval stage. The single locus selection coefficient is determined by the environment and can reach quite high values.——Viability selection in the presence of starch is in the direction indicated by the enzyme activities.     

Torulopsis mogii sp.n.

The new speciesTorulopsis mogii is proposed as a substitute for the seven following, invalidly describedTorulopsis species:T. miso β, T. miso β var. 2,T. miso γ, T. miso γ var. 1,T. miso ζ, T. miso η by Mogi (1938, 1942) andT. osloensis by Dietrichson (1954).     

In Situ Analysis of Metabolic Characteristics Reveals the Key Yeast in the Spontaneous and Solid-State Fermentation Process of Chinese Light-Style Liquor

The in situ metabolic characteristics of the yeasts involved in spontaneous fermentation process of Chinese light-style liquor are poorly understood. The covariation between metabolic profiles and yeast communities in Chinese light-style liquor was modeled using the partial least square (PLS) regression method. The diversity of yeast species was evaluated by sequence analysis of the 26S ribosomal DNA (rDNA) D1/D2 domains of cultivable yeasts, and the volatile compounds in fermented grains were analyzed by gas chromatography (GC)-mass spectrometry (MS). Eight yeast species and 58 volatile compounds were identified, respectively. The modulation of 16 of these volatile compounds was associated with variations in the yeast population (goodness of prediction [Q²] > 20%). The results showed that Pichia anomala was responsible for the characteristic aroma of Chinese liquor, through the regulation of several important volatile compounds, such as ethyl lactate, octanoic acid, and ethyl tetradecanoate. Correspondingly, almost all of the compounds associated with P. anomala were detected in a pure culture of this yeast. In contrast to the PLS regression results, however, ethyl lactate and ethyl isobutyrate were not detected in the same pure culture, which indicated that some metabolites could be generated by P. anomala only when it existed in a community with other yeast species. Furthermore, different yeast communities provided different volatile patterns in the fermented grains, which resulted in distinct flavor profiles in the resulting liquors. This study could help identify the key yeast species involved in spontaneous fermentation and provide a deeper understanding of the role of individual yeast species in the community.     

Large-scale purification of the proton pumping pyrophosphatase from Thermotoga maritima: A “Hot-Solve” method for isolation of recombinant thermophilic membrane proteins

Although several proton-pumping pyrophosphatases (H⁺-PPases) have been overexpressed in heterologous systems, purification of these recombinant integral membrane proteins in large amounts in order to study their structure-function relationships has proven to be a very difficult task. In this study we report a new method for large-scale production of pure and stable thermophilic H⁺-PPase from Thermotoga maritima. Following overexpression in yeast, a “Hot-Solve” procedure based on high-temperature solubilization and metal-affinity chromatography was used to obtain a highly purified detergent-solubilized TVP fraction with a yield around 1.5 mg of protein per litre of yeast culture. Electron microscopy showed the monodispersity of the purified protein and single particle analysis provided the first direct evidence of a dimeric structure for H⁺-PPases. We propose that the method developed could be useful for large-scale purification of other recombinant thermophilic membrane proteins.     

A model for continuous fermentations with amylolytic yeasts

A two-stage, associative fermentation process is more effective for continuous yeast biomass production from starch than a single-stage mixed culture fermentation process. By operating two stages, competition for the same growth limiting substrate is reduced leading to efficient starch utilization. In this article, a mathematical model has been proposed for continuous, two-stage fermentation with a pure culture, amylolytic yeast in the first stage and a mixed culture second stage with a faster growing, nonamylolytic yeast. The model parameters were determined for Saccharomycopsis fibuligera and Candida utilis in continuous, single-stage, pure cultures. In the two-stage model, the effects of changes in dilution rate on biomass, amylase, reducing sugar, and starch concentration, and ratio of stage volumes on microbial composition are discussed and compared with experimental data.     

Interactions between Kluyveromyces marxianus and Saccharomyces cerevisiae in tequila must type medium fermentation

Traditional tequila fermentation is a complex microbial process performed by different indigenous yeast species. Usually, they are classified in two families: Saccharomyces and Non-Saccharomyces species. Using mixed starter cultures of several yeasts genera and species is nowadays considered to be beneficial to enhance the sensorial characteristics of the final products (taste, odor). However, microbial interactions occurring in such fermentations need to be better understood to improve the process. In this work, we focussed on a Saccharomyces cerevisiae/Kluyveromyces marxianus yeast couple. Indirect interactions due to excreted metabolites, thanks to the use of a specific membrane bioreactor, and direct interaction due to cell-to-cell contact have been explored. Comparison of pure and mixed cultures was done in each case. Mixed cultures in direct contact showed that both yeast were affected but Saccharomyces rapidly dominated the cultures whereas Kluyveromyces almost disappeared. In mixed cultures with indirect contact the growth of Kluyveromyces was decreased compared to its pure culture but its concentration could be maintained whereas the growth of Saccharomyces was enhanced. The loss of viability of Kluyveromyces could not be attributed only to ethanol. The sugar consumption and ethanol production in both cases were similar. Thus the interaction phenomena between the two yeasts are different in direct and indirect contact, Kluyveromyces being always much more affected than Saccharomyces.     

Complete Growth Inhibition of Bacillus subtilis by Nisin-Producing Lactococci in Fermented Soybeans

The growth inhibition by nisin-producing lactococci against Bacillus subtilis and its application to soybean miso fermentation were investigated. Lactococcus lactis subsp. lactis IFO12007 (nisin-producing, salt-intolerant) rapidly proliferated to more than 10⁹ cells/g in cooked soybeans without any excessive pH decrease. In spite of the mild decrease in pH, the growth of B. subtilis was completely inhibited; no living cells were detected in a soybean sample inoculated with 10⁶ cells/g and incubated for 24 to 72 h. This Lc. lactis was applied to soybean miso fermentation as a starter culture. It produced high nisin activity (1.28×10⁵ AU/g) in cooked soybean, resulting in the complete growth inhibition of B. subtilis, which had been inoculated at the beginning of the koji fermentation, throughout the process of miso production. Over-acidification, which is undesirable for miso quality, was successfully prevented simply by adding salt which killed the salt-intolerant Lc. lactis. Furthermore, the nisin activity in miso disappeared with aging.     

SIMILARITY OF THE KINETICS OF INVERTASE ACTION IN VIVO AND IN VITRO

1. A method is given whereby the course of hydrolysis of sucrose by live yeast cells may be followed with precision equal to that found when invertase solutions prepared from autolyzed yeast are used to cause inversion. 2. The practical value of the equation of Nelson and Hitchcock as a means of following the course of enzymic hydrolysis of sucrose is hereby extended. 3. The inversion of sucrose by live yeast cells and by extracted invertase has been quantitatively compared. 4. The course of hydrolysis of sucrose by the invertase of Fleischmann''s yeast has been found to be identical in vivo and in vitro.     

Selenium-enriched Candida utilis: Efficient preparation with l-methionine and antioxidant capacity in rats

Selenium-enriched Candida utilis has attracted much attention due to its expanding application in food and feed additives. The objective of this study was to efficiently prepare selenium-enriched C. utilis and to investigate the effects of the prepared yeast on antioxidant capacity in rats. A batch culture of selenium-enriched C. utilis was first carried out, and the addition of sodium selenite (Na₂SeO₃) after all glucose had been consumed was found to favor higher intracellular glutathione and organic selenium content. Moreover, l-methionine boosted yeast cell growth and glutathione biosynthesis, and prevented glutathione from leaking to the extracellular space that can be caused by Na₂SeO₃. We therefore developed a two-stage culture strategy involving supplementation with l-methionine and Na₂SeO₃ at separate culture phases to improve the performance of selenized C. utilis. Using this two-stage culture strategy, intracellular glutathione content reached 18.6 mg/g and 15.5 mg/g, respectively, in batch and fed-batch systems, and organic selenium content reached 905.2 μg/g and 984.7 μg/g, respectively. The effects of selenium-enriched C. utilis on the activities of antioxidant related enzymes in rats were investigated, and the prepared selenium-enriched C. utilis was shown to be an optimal dietary supplement for enhancing antioxidant capacity in rats.     

Giardia lamblia: isolation and axenic cultivation.

Giardia lamblia trophozoites have been axenically cultured for more than a year. Initially, organisms were established in a complex liquid medium in the presence of the host's intestinal fungi; subcultures were made of these protozoa-fungus mixtures. G. lamblia trophozoites, free of yeast, were obtained by inoculating a protozoafungus culture in one arm of a U-tube, then later removing, from the other arm of the tube, Giardia trophozoites that had migrated across the base. Medium was changed at 2- or 3-day intervals; numerous subcultures were made. Tests for the possible presence of other organisms in these axenic cultures were negative. Trophozoite cultures remained viable, after freezing in the presence of glycerol, for 14 months. This is the first reported axenic culture of this common human intestinal parasite and pathogen; its study in pure culture is now possible.     

Enhanced formation of isoamyl alcohol in Zygosaccharomyces rouxii due to elimination of feedback inhibition of α-isopropylmalate synthase

Abstract Mutants of the 'miso' yeast, Zygosaccharomyces rouxii , that produced a large amount of isoamyl alcohol, an important flavour in miso fermentation, were isolated from 5,5,5-trifluoro-dl-leucine-resistant mutants, an analogue of l-leucine. One of the mutants, M21-10, produced a three-fold higher level of isoamyl alcohol than the wild-type strain MY21 in miso fermentation. The activity of α-isopropylmalate synthase, one of the enzymes used for l-leucine synthesis, in the mutant M21-10 was not inhibited by the addition of l-leucine, a feedback inhibitor.     

Yeast Diversity in the Extreme Acidic Environments of the Iberian Pyrite Belt

In the Iberian Pyrite Belt (IPB), acid rock drainage gives rise to aquatic habitats with low pH and high concentrations of heavy metals, a situation that causes important environmental problems. We investigated the occurrence and diversity of yeasts in two localities of the IPB: São Domingos (Portugal) and Rio Tinto (Spain). Yeast isolation was performed on conventional culture media (MYP), acidified (pH 3) media (MYP3), and on media prepared with water from the study sites (MYPw). The main goal of the study was to determine the structure of the yeast community; a combination of molecular methods was used for accurate species identifications. Our results showed that the largest fraction of the yeast community was recovered on MYPw rather than on MYP and MYP3. Twenty-seven yeast species were detected, 48% of which might represent undescribed taxa. Among these, an undescribed species of the genus Cryptococcus required low pH for growth, a property that has not been observed before in yeasts. The communities of S. Domingos and R. Tinto showed a considerable resemblance, and eight yeast species were simultaneously found in both localities. Taking into consideration the physicochemical parameters studied, we propose a hierarchic organization of the yeast community in terms of high-, intermediate-, or low-stress conditions of the environment. According to this ranking, the acidophile yeast Cryptococcus sp. 5 is considered the most tolerant species, followed by Cryptococcus sp. 3 and Lecytophora sp. Species occurring in situations of intermediate environmental stress were Candida fluviatilis, Rhodosporidium toruloides, Williopsis californica, and three unidentified yeasts belonging to Rhodotorula and Cryptococcus.     

Flavor Components of Miso: Basic Fraction

Basic flavor components were isolated from various types of miso and determined qualitatively and quantitatively by gas chromatography and mass spectrometry. 12 pyrazines, 3 pyridines, N-methylpyrrolidone, 2-acethylpyrrole and benzothiazole were identified. All but tetramethylpyrazine had not been reported previously for their presence in miso. Concentrations of the basic components, except for pyridines, varied considerably, depending on the type of miso. The factors which influenced the levels of these components were primarily a soy bean cooking condition and a koji ratio. The flavor contribution of alkylpyrazines to each type of miso is also discussed on the basis of the additivity of odorous mixture. Alkylpyrazines were judged to contribute to miso flavor, particularly in the soy bean miso.     

Elicitation of sanguinarine production in two-phase cultures of Eschscholtzia californica

Large increases in sanguinarine production were observed in suspension cultured Eschscholtzia californica cells treated with elicitors prepared from yeast extract, Colletotrichum lindemuthianum and Verticillum dahliae. Compounded silicone fluid, an ideal accumulation phase for the two-phase culture of E. californica, accumulated a large amount of sanguinarine produced in a specific manner and increased sanguinarine production. Elicitation in combination with two-phase culture additionally increased net sanguinarine production, as well as the sanguinarine concentration in the accumulation phase.     

Influence of mixed culture conditions on yeast-wall hydrolytic activity of Bacillus circulans WL-12 and on extractability of astaxanthin from the yeast Phaffia rhodozyma

O kagbue , R.N. & L ewis , M.J. 1985. Influence of mixed culture conditions on yeast-wall hydrolytic activity of Bacillus circulans WL-12 and on extractability of astaxanthin from the yeast Phaffia rhodozyma. Journal of Applied Bacteriology 59 , 243–255.
In mixed culture Bacillus circulans WL-12 hydrolysed cell walls of Phaffia rhodozyma and rendered astaxanthin extractable from the yeast. pH control was critical to survival and lytic activity of the bacillus; the optimum range was 6.2–6.8. The optimum range of temperature was 20–24C. Glucose (1–2%) was efficient in minimizing catabolite repression of the lytic enzyme complex of the bacillus. Slow-feeding of glucose improved ultimate yields of lytic enzyme but did not acclerate yeast cell wall modification. A relatively high inoculum level of B. circulans accelerated modification of P. rhodozyma in the mixed culture: when the bacterial inoculum was four times that of the yeast, over 80% of total astaxanthin was extractable in 48 h. High bacterial inoculum size also stimulated yeast autolysis and necessitated early harvest of the mixed culture. Results obtained in shake flasks were duplicated in 5-litre fermentors and suggest that the mixed culture has potential industrial value for producing a biomass containing biologically-available astaxanthin. Extractability of astaxanthin was also achieved when mixed culture filtrate was incubated with pure cultured Phaffia cells. When suitably fortified with nutrients, the filtrate also supported simultaneous yeast growth and modification of the yeast cell walls. A scheme incorporating mixed culture with B. circulans WL-12 and re-use of culture filtrate has been proposed for enzymatic processing of Phaffia rhodozyma for inclusion in animal diets.     

Comparative study of nickel resistance of pure culture and co-culture of Acidithiobacillus thiooxidans and Leptospirillum ferriphilum

The effect of Ni²⁺ on the growth and functional gene expression of the pure culture and co-culture of Acidithiobacillus thiooxidans and Leptospirillum ferriphilum has been studied. Compared with the pure culture, the co-culture showed a stronger sulfur and ferrous ion oxidation activity. At 100 mM, A. thiooxidans in co-culture grew faster and had 48 h shorter lag phases. The cell number of A. thiooxidans in co-culture was about 5 times higher than that in pure culture. The existence of A. thiooxidans in co-culture activated the expression of some metal resistance genes in L. ferriphilum at least 16 h in advance. A. thiooxidans in co-culture tends to chose more efficient pathways to transport nickel ion, ensuring the export of heavy metal was faster and more effective than that in pure culture. All the data indicated that there were synergetic interactions between iron- and sulfur-oxidizing bacteria under the stress of Ni²⁺.