Influence of oxygen on the biosynthesis of cellular fatty acids,sterols and phospholipids during alcoholic fermentation by Saccharomyces cerevisiae and Torulaspora delbrueckii

Saccharomyces cerevisiae and Torulaspora delbrueckii were grown under different O₂ availabilities on grape must. Oxygen requirements for the two yeasts were different: under anaerobic conditions, S. cerevisiae produced a higher percentage of unsaturated fatty acids, and had a greater cell yield and fermentation activity than T. delbrueckii. Addition of ergosterol (25mg/l) and oleic acid (31mg/l) caused total recovery of cellular growth and the fermentation activity of S. cerevisiae in anaerobiosis, but not of T. delbrueckii. However a short period of aeration to a 48 h culture in anaerobiosis, led to total recovery of the cellular growth and fermentation activity in both yeasts. Likewise, the effect of a short aeration period on unsaturated fatty acid biosynthesis was similar for both species.     

Use of whey lactose from dairy industry for economical kefiran production by Lactobacillus kefiranofaciens in mixed cultures with yeasts

To evaluate the feasibility of producing kefiran industrially, whey lactose, a by-product from dairy industry, was used as a low cost carbon source. Because the accumulation of lactic acid as a by-product of Lactobacillus kefiranofaciens inhibited cell growth and kefiran production, the kefir grain derived and non-derived yeasts were screened for their abilities to reduce lactic acid and promote kefiran production in a mixed culture. Six species of yeasts were examined: Torulaspora delbrueckii IFO 1626; Saccharomyces cerevisiae IFO 0216; Debaryomyces hansenii TISTR 5155; Saccharomyces exiguus TISTR 5081; Zygosaccharomyces rouxii TISTR 5044; and Saccharomyces carlsbergensis TISTR 5018. The mixed culture of L. kefiranofaciens with S. cerevisiae IFO 0216 enhanced the kefiran production best from 568 mg/L in the pure culture up to 807 and 938 mg/L in the mixed cultures under anaerobic and microaerobic conditions, respectively. The optimal conditions for kefiran production by the mixed culture were: whey lactose 4%; yeast extract 4%; initial pH of 5.5; and initial amounts of L. kefiranofaciens and S. cerevisiae IFO 0216 of 2.1×10(7) and 4.0×10(6)CFU/mL, respectively. Scaling up the mixed culture in a 2L bioreactor with dissolved oxygen control at 5% and pH control at 5.5 gave the maximum kefiran production of 2,580 mg/L in batch culture and 3,250 mg/L in fed-batch culture.     

The effect of temperature on the growth of strains of Kloeckera apiculata and Saccharomyces cerevisiae in apple juice fermentation

The influence of temperature (10°C and 25°C) on the survival and growth of Saccharomyces cerevisiae and Kloeckera apiculata was examined in mixed and pure cultures during fermentation in apple juice. The growth reached by S. cerevisiae did not seem to be affected by temperature and the presence of K. apiculata . However, the growth and survival of K. apiculata , both in single and mixed cultures, were substantially enhanced at 10°C. The highest amount of ethyl acetate was produced by K. apiculata in pure culture at 10°C. Nevertheless, this concentration was lowest when both yeasts were fermented together at 10°C and 25°C.     

Leavening ability and freeze tolerance of yeasts isolated from traditional corn and rye bread doughs.

Strains of Saccharomyces cerevisiae and Torulaspora delbrueckii isolated from traditional bread doughs displayed dough-raising capacities similar to the ones found in baker's yeasts. During storage of frozen doughs, strains of T. delbrueckii (IGC 5321, IGC 5323, and IGC 4478) presented approximately the same leavening ability for 30 days. Cell viability was not significantly affected by freezing, but when the dough was submitted to a bulk fermentation before being stored at -20 degrees C, there was a decrease in the survival ratio which depended on the yeast strain. Furthermore, the leavening ability after 4 days of storage decreased as the prefermentation period of the dough before freezing increased, except for strains IGC 5321 and IGC 5323. These two strains retained their fermentative activity after 15 days of storage and 2.5 h of prefermentation, despite showing a reduction of viable cells under the same conditions. The intracellular trehalose content was higher than 20% (wt/wt) in four of the yeasts tested: the two commercial strains of baker's yeast (S. cerevisiae IGC 5325 and IGC 5326) and the two mentioned strains of T. delbrueckii (IGC 5321 and IGC 5323). However, the strains of S. cerevisiae were clearly more susceptible to freezing damages, indicating that other factors may contribute to the freeze tolerance of these yeasts.     

Oxygen requirements of yeasts

Type species of 75 yeast genera were examined for their ability to grow anaerobically in complex and mineral media. To define anaerobic conditions, we added a redox indicator, resazurin, to the media to determine low redox potentials. All strains tested were capable of fermenting glucose to ethanol in oxygen-limited shake-flask cultures, even those of species generally regarded as nonfermentative. However, only 23% of the yeast species tested grew under anaerobic conditions. A comparative study with a number of selected strains revealed that Saccharomyces cerevisiae stands out as a yeast capable of rapid growth at low redox potentials. Other yeasts, such as Torulaspora delbrueckii and Candida tropicalis, grew poorly mu max, 0.03 and 0.05 h-1, respectively) under anaerobic conditions in mineral medium supplemented with Tween 80 and ergosterol. The latter organisms grew rapidly under oxygen limitation and then displayed a high rate of alcoholic fermentation. It can be concluded that these yeasts have hitherto-unidentified oxygen requirements for growth.     

Changes of diversity and population of yeasts during the fermentations by pure and mixed inoculation of Saccharomyces cerevisiae strains

Mixed inoculation of Saccharomyces cerevisiae strains is used in winemaking for achieving high sensory quality of the wine. However, information on the diversity and population of yeasts during inoculated fermentation is very limited. In this study, we evaluated the effect of mixed inocula with different inoculation timing on the yeast community during fermentations of Cabernet Sauvignon. Grape must was inoculated with pure cultures of S. cerevisiae RC212 or S. cerevisiae R312, and simultaneous and sequential inoculation of both strains. Wallersterin Laboratory Nutrient (WLN) medium and sequence of the 26S rDNA D1/D2 domain were used to compare the diversity of yeast species. Five species, including Candida diversa, Hanseniaspora opuntiae, H. uvarum, Issatchenkia orientalis and I. terricola, were identified in the grape must, with Issatchenkia sp. being predominant (67.5 %). Three to four species were involved in each fermentation treatment. The fermentations by mixed inocula presented more yeast species than by pure inocula. Interdelta sequence typing was used to identify S. cerevisiae strains. Ten genotypes were identified among 322 isolated S. cerevisiae strains. Their distribution varied among different stages of fermentations and different inoculation treatments. The inoculated strains were not predominant, while indigenous genotypes I, III, and V showed strong competitiveness during fermentation. In general, this study provided information on the change of population structure and genetic diversity of yeasts in fermentations inoculated with pure and mixed S. cerevisiae strains.     

Oxygen requirements of yeasts.

Type species of 75 yeast genera were examined for their ability to grow anaerobically in complex and mineral media. To define anaerobic conditions, we added a redox indicator, resazurin, to the media to determine low redox potentials. All strains tested were capable of fermenting glucose to ethanol in oxygen-limited shake-flask cultures, even those of species generally regarded as nonfermentative. However, only 23% of the yeast species tested grew under anaerobic conditions. A comparative study with a number of selected strains revealed that Saccharomyces cerevisiae stands out as a yeast capable of rapid growth at low redox potentials. Other yeasts, such as Torulaspora delbrueckii and Candida tropicalis, grew poorly mu max, 0.03 and 0.05 h-1, respectively) under anaerobic conditions in mineral medium supplemented with Tween 80 and ergosterol. The latter organisms grew rapidly under oxygen limitation and then displayed a high rate of alcoholic fermentation. It can be concluded that these yeasts have hitherto-unidentified oxygen requirements for growth.     

Mixed culture of killer and sensitive Saccharomyces cerevisiae strains in batch and continuous fermentations

This paper presents a kinetic study of the dynamics of the population of two Saccharomyces cerevisiae strains (designated K1 and 522D) in mixed culture. These two strains are commonly used in wine making. The K1 strain (killer yeast) secretes a glycoprotein (killer toxin) which causes the death of the 522D strain (sensitive yeast). Initially, the mixed cultures were realized in batch fermentations. Initial concentrations of killer yeast were 5 and 10% of the total population. The influence of the killer strain on the sensitive cultures was measured in comparison with a reference fermentation. The reference fermentation was inoculated only with the sensitive strain. Results show that an initial concentration of 10% of killer strain affects the microbial population balance and the rate of ethanol production. However the fermentation was only slightly disturbed when the proportion of killer to sensitive yeast at the beginning of mixed culture was 5%. To achieve total displacement by the killer yeast at low concentrations, the mixed cultures were carried out in a continuous system. The results obtained in continuous fermentations with the same strains have shown that a level of contamination as low as 0.8% of killer strain was sufficient to completely displace the original sensitive population after 150 h incubation.     

Mixed culture of killer and sensitive Saccharomyces cerevisiae strains in batch and continuous fermentations

This paper presents a kinetic study of the dynamics of the population of two Saccharomyces cerevisiae strains (designated K1 and 522D) in mixed culture. These two strains are commonly used in wine making. The K1 strain (killer yeast) secretes a glycoprotein (killer toxin) which causes the death of the 522D strain (sensitive yeast). Initially, the mixed cultures were realized in batch fermentations. Initial concentrations of killer yeast were 5 and 10% of the total population. The influence of the killer strain on the sensitive cultures was measured in comparison with a reference fermentation. The reference fermentation was inoculated only with the sensitive strain. Results show that an initial concentration of 10% of killer strain affects the microbial population balance and the rate of ethanol production. However the fermentation was only slightly disturbed when the proportion of killer to sensitive yeast at the beginning of mixed culture was 5%. To achieve total displacement by the killer yeast at low concentrations, the mixed cultures were carried out in a continuous system. The results obtained in continuous fermentations with the same strains have shown that a level of contamination as low as 0.8% of killer strain was sufficient to completely displace the original sensitive population after 150 h incubation.     

Effect of the addition of inert cellulose substrates to grape must on Saccharomyces cerevisiae diversity and the evolution of alcoholic fermentation

AIMS: To study the addition of cellulose-based adjuvant as a resource to offset the negative effects produced by grape juice clarification during alcoholic fermentations. METHODS AND RESULTS: The effect of the addition of two kinds of inert cellulose substrates in white wine vinification was investigated in two different musts. In one of these musts, stuck fermentations were detected. One of the types of cellulose examined had a fining effect, which caused a decrease in the number of viable yeasts in the medium and altered the distribution and frequency of the clones, which performed the fermentation. The other cellulose substrate made the medium cloudier but did not alter the distribution of yeasts in comparison with the control. CONCLUSIONS: The behaviour of the inert cellulose substrates on vinification depends on its physical characteristics and its capacity for making the must cloudy. SIGNIFICANCE AND IMPACT OF THE STUDY: The addition of inert cellulose substrates in white wine vinification improves the fermentation process and the quality of wines obtained. This effect is more noticeable in difficult fermentations. One variety of cellulose showed an inhibitory effect on Torulaspora delbrueckii yeasts.     

Isolation, taxonomic identification and hydrogen peroxide production by Lactobacillus delbrueckii subsp. lactis T31, isolated from Mongolian yoghurt: inhibitory activity on food-borne pathogens

AIMS: The aim of this work was to isolate lactic acid bacteria (LAB) strains from Mongolian tarag (a traditionally homemade yoghurt) displaying antimicrobial activities against food-borne pathogens, identify inhibitory substances and study the kinetics of their production. METHODS AND RESULTS: Inhibitory substance-producing bacterial strains were isolated from tarag. From 300 bacterial clones, 31 were able to inhibit the growth of the indicator strain Lactobacillus bulgaricus 340. One of the most active strains was identified as Lactobacillus delbrueckii subsp. lactis strain T31 by using cluster analysis of amplified fragment length polymorphism (AFLP) DNA fingerprints. The antimicrobial substance was inactivated by catalase, demonstrating the production of hydrogen peroxide (H(2)O(2)). Production of H(2)O(2) was studied under aerated and nonaerated culture conditions. The amount of H(2)O(2) in the culture supernatant increased during bacterial growth and reached a maximum (5.12 mmol l(-1)) at the early stationary phase under aerated conditions (agitated cultures). H(2)O(2) was not detected in the culture performed without agitation. In mixed cultures performed in milk with either Lact. delbrueckii subsp. lactis T31 in the presence of Escherichia coli, or Lact. delbrueckii subsp. lactis T31 in the presence of Listeria innocua under aerated and nonaerated conditions, a significant decrease in pathogen count was observed in aerated cultures. SIGNIFICANCE AND IMPACT OF THE STUDY: The significant decrease in Listeria viability observed in aerated mixed cultures of Lact. delbrueckii subsp. lactis T31 is mainly because of H(2)O(2) production. Lactobacillus delbrueckii subsp. lactis T31 could be used as a protective culture in food industries or as a probiotic to prevent intestinal and urogenital infections.     

Scaling-up in industrial winemaking using low electric current as an alternative to sulfur dioxide addition

AIMS: To better understand the outcome of employing low electric current (LEC) technology as a new preservation and alternative in wine technology, and to contribute to its development. It is used in industrial-scale winemaking with commercial yeast (Saccharomyces cerevisiae) during the grape must fermentation. METHODS AND RESULTS: LEC (200 mA, time 16 days) was applied to fresh grape must as an alternative method to the usual sulfur dioxide addition used in the industrial process; two tanks, each 30,000 l, were employed for parallel fermentations. The results show that LEC decreased the survival time and increased the death rate of apiculate yeasts, whereas it did not affect the growth and survival of S. cerevisiae. A comparison was made of the main chemical and sensory parameters of the wines obtained. CONCLUSIONS: The results have demonstrated that the low-voltage treatment had a positive effect on the grape juice fermentation (yeast microflora) during the early stages of winemaking. SIGINIFICANCE AND IMPACT OF THE STUDY: These results could be of significant importance in developing, for 'biological wine', new winemaking technologies for an innovative control process of yeast fermentation.     

Nitrogen availability of grape juice limits killer yeast growth and fermentation activity during mixed-culture fermentation with sensitive commercial yeast strains.

The competition between selected or commercial killer strains of type K2 and sensitive commercial strains of Saccharomyces cerevisiae was studied under various conditions in sterile grape juice fermentations. The focus of this study was the effect of yeast inoculation levels and the role of assimilable nitrogen nutrition on killer activity. A study of the consumption of free amino nitrogen (FAN) by pure and mixed cultures of killer and sensitive cells showed no differences between the profiles of nitrogen assimilation in all cases, and FAN was practically depleted in the first 2 days of fermentation. The effect of the addition of assimilable nitrogen and the size of inoculum was examined in mixed killer and sensitive strain competitions. Stuck and sluggish wine fermentations were observed to depend on nitrogen availability when the ratio of killer to sensitive cells was low (1:10 to 1:100). A relationship between the initial assimilable nitrogen content of must and the proportion of killer cells during fermentation was shown. An indirect relationship was found between inoculum size and the percentage of killer cells: a smaller inoculum resulted in a higher proportion of killer cells in grape juice fermentations. In all cases, wines obtained with pure-culture fermentations were preferred to mixed-culture fermentations by sensory analysis. The reasons why killer cells do not finish fermentation under competitive conditions with sensitive cells are discussed.     

Kinetic study and mathematical modelling of killer and sensitive S. Cerevisiae strains growing in mixed culture

This paper presents a kinetic study of two yeasts growing in pure and mixed batch cultures. Two winemaking strains were used: S. cerevisiae K1 possessing the K2 killer character and S. cerevisiae 522D sensitive to the K2 killer toxin. Initially the kinetics of growth of the two strains were analysed in pure culture. In this case, the kinetic profiles of biomass production have shown that the growth rate of the K1 strain is slightly superior to the 522D strain. During the fermentation, the viability for both populations was higher than 90%. Fermentations in mixed culture with an initial percentage in killer strain of 5 and 10% with respect to the total population were carried out. The results showed a more important decrease in the percentage of total viable yeasts when the initial concentration of killer yeast increased. However, the kinetic profiles of total biomass (killer plus sensitive yeasts) were very similar for both fermentations. A mathematical model was proposed to simulate the microbial growth of the killer and sensitive strain developing in pure and mixed cultures. This mathematical model consists in three main reactions: the evolution of the killer toxin in the culture medium, the duplication and the mortality rates for each microbial population. The results of the simulation appeared in agreement with the experimental data.     

Effect of sodium chloride on bakers' yeast growing in gelatin

In recent years, industrial fermentation researchers have shifted their attention from liquid to solid and semisolid culture conditions. We converted liquid cultures to the semisolid mode by adding high levels of gelatin. Previous studies on liquid cultures have revealed the inhibitory activity of mineral salts, such as NaCl, on the fermentation of sugars by yeasts. We made a kinetic study of the effects of 1 to 5% (wt/vol) NaCl on the alcoholic fermentations of glucose by Saccharomyces cerevisiae in a growth medium containing 16% gelatin. Our results showed that the effect of high salt content on semisolid culture is essentially the same as the effect on liquid culture; i.e., as the salt content increased, the following occurred: (i) the growth of yeasts decreased, (ii) the lag period of the yeast biomass curve lengthened, (iii) the sugar intake was lowered, (iv) the yield of ethanol was reduced, and (v) the production of glycerol was increased. We observed a new relationship correlating the area of kinetic hysteresis with ethanol production rate, acetaldehyde concentration, and the initial NaCl concentration.     

On enhancing productivity of bioethanol with multiple species

The present work is initiated to investigate whether a defined culture comprising a mixture of three yeast species, Kluyveromyces marxianus, Saccharomyces cerevisiae, and Pichia stipitis can ferment a mixture of sugars to produce bioethanol at rates higher than those achieved by pure cultures of the same. For this purpose, we develop models of single species based on the hybrid cybernetic model framework, and simulate fermentations in the mixed culture by combining individual models. An underlying assumption is that the behavior of each species is determined only by the common environment independently of the presence and metabolism of other species. Model performance is thoroughly assessed using the experimental data available in the literature. The dynamic behavior of mixed cultures in mixed culture experiments are accurately predicted by the model reflecting faithfully the simultaneous/sequential uptake patterns of mixed substrates. This model is then used to investigate performance of various possible reactor configurations. With the foregoing species of organisms, mixed culture itself does not lead to a significant increase of bioethanol productivity. Rather, the model shows that substantial improvement is acquired by sequential use of different, properly chosen organisms during fermentation. Thus, the successive use of K. marxianus and P. stipitis is shown to increase bioethanol productivity up to about 58% in comparison to fermentation by single species alone.     

Mixed cultures of Serratia marcescens and Kluyveromyces fragilis for simultaneous protease production and COD removal of whey

AIMS: The aim of this study was to investigate the behaviour of a Serratia marcescens-Kluyveromyces fragilis mixed culture in whey, with the objective of proteases production and organic waste reduction. METHODS AND RESULTS: Discontinuous aerobic fermentations in whey were carried out using individual pure cultures and mixed cultures of S. marcescens and K. fragilis. Cell growth, protease production, lactose and proteins consumption and COD/TOC reduction were monitored. Lactose and protein content of the fermenting medium was almost depleted in the mixed cultures, achieving a reduction in the organic content much higher than in both pure cultures. Interestingly, proteolytic activity in the mixed cultures was similar to that obtained for S. marcescens in pure culture. In addition, protease stability was increased in the mixed cultures. Kinetic models were developed fitting well with the experimental results. CONCLUSIONS: Mixed cultures were found to maintain the achievements of each individual fermentation, yielding a high and stable production of proteases and a significant reduction of COD/TOC. SIGNIFICANCE AND IMPACT OF THE STUDY: Mixed cultures tested in this work have shown a synergistic effect with possible industrial applications. These results lead to a gain in the chain value for enzyme production with an environmentally friendly operation.     

Microsatellite PCR profiling of Saccharomyces cerevisiae strains during wine fermentation

AIMS: Use of microsatellite PCR to monitor populations of Saccharomyces cerevisiae strains during fermentation of grape juice. METHOD AND RESULTS: Six commercial wine strains of S. cerevisiae were screened for polymorphism at the SC8132X locus using a modified rapid PCR identification technique. The strains formed four distinct polymorphic groups that could be readily distinguished from one another. Fermentations inoculated with mixtures of three strains polymorphic at the SC8132X locus were monitored until sugar utilization was complete, and all exhibited a changing population structure throughout the fermentation. CONCLUSIONS: Rapid population quantification demonstrated that wine fermentations are dynamic and do not necessarily reflect the initial yeast population structure. One or more yeast strains were found to dominate at different stages of the fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The population structure of S. cerevisiae during mixed culture wine fermentation is dynamic and could modify the chemical composition and flavour profile of wine.     

Characterization, ecological distribution, and population dynamics of Saccharomyces sensu stricto killer yeasts in the spontaneous grape must fermentations of southwestern Spain

Killer yeasts secrete protein toxins that are lethal to sensitive strains of the same or related yeast species. Among the four types of Saccharomyces killer yeasts already described (K1, K2, K28, and Klus), we found K2 and Klus killer yeasts in spontaneous wine fermentations from southwestern Spain. Both phenotypes were encoded by medium-size double-stranded RNA (dsRNA) viruses, Saccharomyces cerevisiae virus (ScV)-M2 and ScV-Mlus, whose genome sizes ranged from 1.3 to 1.75 kb and from 2.1 to 2.3 kb, respectively. The K2 yeasts were found in all the wine-producing subareas for all the vintages analyzed, while the Klus yeasts were found in the warmer subareas and mostly in the warmer ripening/harvest seasons. The middle-size isotypes of the M2 dsRNA were the most frequent among K2 yeasts, probably because they encoded the most intense K2 killer phenotype. However, the smallest isotype of the Mlus dsRNA was the most frequent for Klus yeasts, although it encoded the least intense Klus killer phenotype. The killer yeasts were present in most (59.5%) spontaneous fermentations. Most were K2, with Klus being the minority. The proportion of killer yeasts increased during fermentation, while the proportion of sensitive yeasts decreased. The fermentation speed, malic acid, and wine organoleptic quality decreased in those fermentations where the killer yeasts replaced at least 15% of a dominant population of sensitive yeasts, while volatile acidity and lactic acid increased, and the amount of bacteria in the tumultuous and the end fermentation stages also increased in an unusual way.     

Yeast biodiversity and dynamics during sweet wine production as determined by molecular methods

In this study we investigated yeast biodiversity and dynamics during the production of a sweet wine obtained from dried grapes. Two wineries were selected in the Collio region and grapes, grape juices and wines during fermentations were analyzed by culture-dependent methods (plating on WLN medium) and culture-independent methods (PCR-DGGE). Moreover, the capability of the Saccharomyces cerevisiae starter cultures to take over the fermentation was assessed by RAPD-PCR. On WLN agar several species of non-Saccharomyces yeasts (Hanseniaspora, Metschnikowia, Pichia, Candida, Torulaspora and Debaryomyces), but also strains of S. cerevisiae, were isolated. After inoculation of the starter cultures, only colonies typical of S. cerevisiae were observed. Using PCR-DGGE, the great biodiversity of moulds on the grapes was underlined, both at the DNA and RNA level, while the yeast contribution started to become important only in the musts. Here, bands belonging to species of Candida zemplinina and Hanseniaspora uvarum were visible. Lastly, when the S. cerevisiae isolates were compared by RAPD-PCR, it was determined that only in one of the fermentations followed, the inoculated strain conducted the alcoholic fermentation. In the second fermentation, the starter culture was not able to promptly implant and other populations of S. cerevisiae could be isolated, most likely contributing to the final characteristics of the sweet wine produced.